UNIVERSIDAD POLITÉCNICA DE MADRID FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DEL DEPORTE - INEF Evolution and treatment of vitamin B12 deficiency as a risk factor for (cognitive and functional) neurodegenerative diseases in institutionalized elderly Evolución y tratamiento de la deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores institucionalizadas TESIS DOCTORAL ULRIKE ALBERS Licenciada en Ciencia y Tecnología de los Alimentos 2012
Transcript
UNIVERSIDAD POLITÉCNICA DE MADRID
FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICAY DEL DEPORTE - INEF
Evolution and treatment of vitamin B12 deficiency as a risk factor for (cognitive andfunctional) neurodegenerative diseases in institutionalized elderly
Evolución y tratamiento de la deficiencia de vitamina B12 como factor de riesgo deenfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores
institucionalizadas
TESIS DOCTORAL
ULRIKE ALBERS
Licenciada en Ciencia y Tecnología de los Alimentos
2012
DEPARTAMENTO DE SALUD Y RENDIMIENTO HUMANO
FACULTAD DE CIENCIAS DE LA ACTIVIDAD FÍSICA Y DELDEPORTE - INEF
Evolution and treatment of vitamin B12 deficiency as a risk factor for (cognitive andfunctional) neurodegenerative diseases in institutionalized elderly
Evolución y tratamiento de la deficiencia de vitamina B12 como factor de riesgo deenfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores
institucionalizadas
Ulrike Albers
Licenciada en Ciencia y Tecnología de los Alimentos
Directores:
María Marcela González Gross, Doctora en Farmacia, Profesora Titular de laUniversidad Politécnica de Madrid
Agustín Meléndez Ortega, Doctor en Ciencias de la Actividad Física y del Deporte,Profesor “Ad honorem” de la Universidad Politécnica de Madrid
Madrid, 2012
Photo: Foto Krummen, Lingen
Ulrike Albers, 2012
Edita: Universidad Politécnica, Av. Ramiro Maeztu 7, 28040 Madrid
Imprime: MuzArt, Impresión Digital; c/ Gaztambide, 56; 28015 Madrid
ISBN-10: 84-96398-59-5
ISBN-13: 978-84-96398-59-7
Tribunal nombrado por el Magfco. y Excmo. Sr. Rector de la Universidad Politécnicade Madrid, el día 20 de junio de 2012.
PRESIDENTE: D. Luis A. Moreno Aznar, PhD
VOCAL: Dña Mary Ward, PhD
VOCAL: Dña Ángeles Carbajal Azcona, PhD
VOCAL: Dña Christina Breidenassel, PhD
SECRETARIA: Dña Ma del Carmen González Chamorro, PhD
Realizado el acto de defensa y lectura de la Tesis el día 20 de junio de 2012 en Madrid.
Calificación:
EL PRESIDENTE LOS VOCALES
LA SECRETARIA
On ne reçoit pas la sagesse, il faut la découvrir soi-même après un trajet quepersonne ne peut faire pour nous, ne peut nous épargner, car elle est un point de
vue sur les choses.
A la recherche du temps perdu, Marcel Proust
A Pilar Carazo Salmerón
This PhD Thesis has been possible thanks to the following grants and funding:
Project I
Project title
Vitamin B12 deficiency as a risk factor for neurodegenerative diseases. Longitudi-nal study for the evaluation of changes in the vitamin B12 status in Spanish elderly.(P061100509); funded by Axis-Shield Diagnostics Ltd. and Abbott Diagnostics. S.A.
Project II
Project title
Vitamin B12 deficiency as a risk factor for neurodegenerative diseases. Effect ofa cobalamin supplement during one month. (P051100649); supported by WhitehallMuch GmbH and Abbott Diagnostics. S.A.
Ulrike Albers has been financially supported by the University Politécnica de Madridwith a predoctoral scholarship (2008-2011).
Grants from the Universidad Politécnica de Madrid to do internships in the followinginstitutions.
• Instituto de Medicina Molecular, Lisbon, Portugal. 20th of September-20th De-cember 2009.
• Centre d’Investigations Cliniques CHU-Inserm, CIC 9301. Lille, France. 2ndOctober-31st of December 2010.
• School of Biomedical Sciences, University of Ulster, Coleraine, UK. 18th June-18th September 2011.
PUBLICATONS AND PRESENTATIONS FROM THE THESIS
Refereed Conference Proceedings and Abstracts
Albers U, Pedrero R, Moran P, Cañada D, Meléndez A, González-Gross M. One yearfollow-up of biochemical and cobalamin status among Spanish institutionalized el-derly. 11th European Nutrition Conference (FENS). Madrid, Spain. 26-29.10.2011.Ann Nutr Metab 2011, 58(suppl 3): 48.
González-Gross M, Pedrero-Chamizo R, Albers U. Interrelación entre la condiciónfísica, el estado nutricional vitamínico y el estado cognitivo y su influencia sobre la cal-idad de vida en mayores. 4° Congreso internacional de Actividad Física Deportivapara Mayores. Málaga, Spain. 4-6.03.2011. Proceedings ISBN 978-84-777858980.pp. 71-86.
Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-GrossM. Plurimedication among Spanish institutionalized elderly, a fact that should be con-sidered when examining their nutritional status. II World Congress of Public HealthNutrition and I Latin American Congress of Community Nutrition. Porto, Portu-gal. 23-25.09.2010. Public Health Nutr 2010, 13(9A): 248.
Pedrero-Chamizo R, Albers U, Valtueña J, Casajús JA, Castillo MJ, González -GrossM. Strength training is essential in elderly women. 5th World Conference on Womenand Sport. Sydney, Australia. 20-23.05.2010. Abstract Book ISBN 978-0-9806738-0-7, 2010.
Pedrero-Chamizo R, Albers U, Valtueña J, Moreno S, Pietrzik K, Castillo MJ González-Gross M. Relation Between Physical Strength and Cognitive Function in Spanish In-stitutionalized Elderly. 3rd International Congress on Physical Activity and PublicHealth (ICPAPH). Toronto, Canada. 5-8.05.2010.
Pedrero Chamizo R, Albers U, Morencos E, Pastor JC, Meléndez A, Castillo MJ,González–Gross M. Valoración del estado nutricional en mayores institucionalizadosa través de diferentes marcadores. II Congreso de la FESNAD. Barcelona. Spain.3-5.03.2010.
Albers U, Pedrero-Chamizo R, Meléndez A, Castillo MJ, Pietrzik K, González-GrossM. How effective can a 500 µg oral supplement of vitamin B12 for just 28 days befor institutionalized elderly? XIXth IAGG World Congress of Gerontology andGeriatrics. Paris, France. 5-9.07.2009
Pedrero-Chamizo R, Albers U, Valtueña J, Cañada D, Jathe R, Meléndez A, CastilloMJ, González-Gross M: Evolución de los niveles de fuerza en población mayor insti-tucionalizada de la Comunidad de Madrid. III Jornadas Nacionales de Medicina delDeporte. Zaragoza, Spain. 29-30.05.2009.
Pedrero-Chamizo R, Albers U, Valtueña J, Jiménez-Pavón D, Meléndez A, González-Gross M. Valores de referencia en fuerza de prensión manual en población mayorinstitucionalizada y de vida independiente. III Congreso Internacional de ActividadFísico Deportiva para Mayores. Málaga, Spain. 12-14.03.2009
Pedrero-Chamizo R, Albers U, Piera M, Jiménez-Pavón D, Meléndez A, Castillo MJ,González-Gross M. Gender, age and cognitive score effects on strength in Spanishinstitutionalized elderly. 13th Annual Congress of the European College of SportSciences. Estoril, Portugal. 9-12.07.2008
Pedrero-Chamizo R, Albers U, Valtueña J, Pietrzik K, Meléndez A, Castillo MJ,González-Gross M. Vitamin B12 deficiency. Effects on physical strength and cog-nitive health in Spanish institutionalized elderly. 6th international Conference onNutrition and Fitness. Athens, Greece. 15-17.05.2008
Albers, U, Pedrero R, Díaz V, Tobaruela JL, Pietrzik K, Castillo MJ, Meléndez A,González-Gross M. Gender differences in vitamin B12 status and functional fitnessparameters in Spanish institutionalized elderly. The 6th World Congress on TheAging Male. Tampa, Florida, United States, 21-24.02.2008.
Pedrero R, Albers U, Jiménez-Pavón D, Cupeiro R, Meléndez A, Castillo MJ, Gutiér-rez A, González-Gross M. Valoración funcional en personas mayores institucional-izadas. XII Congreso Nacional de la Federación Española de Medicina del De-porte. Sevilla, Spain. 24-27.10.2007.
Albers U, Meléndez A, Pietrzik K, Tobaruela JL, Castillo MJ, González-Gross M.Effectiveness of oral Vitamin B12 supplementation in Spanish institutionalized elderly.7th International Conference VITAMINS - Nutrition and Diagnostics. Prague,Czech Republic. 19-21.09.2007.
Albers U, Díaz V, Peinado A, Alvarez M, Sola R, Pietrzik K, Caballero J, MeléndezA, Castillo MJ, González-Gross M. Physical strength and Vitamin B12 in relation tocognitive score in Spanish institutionalized elderly. 10th European Nutrition Con-ference Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 119-120.
Refereed Journal Publications
Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-GrossM. La polimedicación en mayores institucionalizados. Su efecto sobre los niveles devitamina B12, folato y homocisteína. Nutr Hosp. 2012;27(1): 296-300.
Albers U, Pedrero R, Meléndez A, Pietrzik K, Castillo MJ, González-Gross M. Ef-fectiveness of a 28 days oral cyanocobalamin supplementation on vitamin B status inSpanish institutionalized elderly. (accepted in Int J Vitam Nutr Res)
16 Characteristics of the study population . . . . . . . . . . . . . . . 64
17 Wilcoxon test (z) for Pre and Post mean values for sCbl and itsabsolute and relative change . . . . . . . . . . . . . . . . . . . . . 66
18 t-test for Pre and Post mean values (geometric and arithmetic) forholoTC, serum and RBC folate, tHcy, MCV, HCT, Hband erythrocytes and their absolute and relative changes . . . . . 67
V
19 Summary table One-Way ANOVA with repeated measures andone fixed factor (serum cobalamin cluster) for mean serum cobal-amin (sCbl), holo transcobalamin (holoTC) and total homocys-teine (tHcy) concentrations . . . . . . . . . . . . . . . . . . . . . . 69
20 Number and percentage of elderly people who take one of the ATCgroup of medication . . . . . . . . . . . . . . . . . . . . . . . . . . 72
21 Concentrations for the biochemical parameters by population . . 73
5 Study design for longitudinal study and distribution of drop outs in thecourse of follow-up . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6 Study design for intervention study . . . . . . . . . . . . . . . . . . . 38
7 Individual changes of serum cobalamin and HoloTC values after a 28day supplementation with 500 µg oral cyanocabalamin . . . . . . . . 65
8 Individual changes of serum tHcy values after a 28 day supplementa-tion with 500 µg oral cyanocabalamin . . . . . . . . . . . . . . . . . 68
9 Change from pre to posttreatment mean serum cobalamin (sCbl) con-centrations by clusters of initial sCbl concentrations . . . . . . . . . . 69
10 Change from pre to posttreatment mean holo transcobalamin (holoTC)concentrations by clusters of initial serum cobalamin (sCbl) concentra-tions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
11 Change from pre to posttreatment mean total homocysteine (tHcy)concentrations by clusters of initial serum cobalamin (sCbl) concen-trations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
12 Percentages of participants outside and inside of the reference intervalof tHcy, B12, HoloTC and folate separated by taking or not-takingmedications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
VII
Nomenclature
AD Alzheimer’s Disease
ADL Activity of Daily Living
ATC Anatomical, Therapeutic, Chemical classification system
CBC Complete Blood Count
CNS Central Nervous System
COM Centre of Mass
EDAD Encuesta sobre Discapacidad, Autonomía personal y Situaciones de Depen-dencia
Exernet Study Estudio multi-centrico para la Evaluación de los Niveles de CondiciónFísica y su relación con estilos de vida saludables en población mayor Españolano institucionalizada
FFB Fat Free Body Mass
HCT Haematocrit
IBP International Biological Programme
INE National Institute for Statistics (in Spanish, Instituto Nacional de Estadística)
ISAK International Society for Advancement of Kinanthropometry
MCV Mean Corpuscular Volume
MMSE Mini Mental State Examination
NHP Nursing Home Placement
PA Pernicious Anaemia
PAR Psychological Assessment Resources
VD Vascular Dementia
W-H ratio Waist-to-Hip ratio
WHO World Health Organisation
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GLOSSARY
Ageing in place Having the mental and physical capability of living in one’s ownhome in old age
Arm strength Number of performed repetitions of the arm curl test
Elderly Term used according to the WHO definition for a person aged over 65 years
Grip strength Results of the dinamometry of the movement of the hand grip strength
Leg strength Number of performed repetitions of the “30 second chair stand” test
Person with disability Person with problems with one of the 44 activities of daily liv-ing according to the Encuesta sobre Discapacidad, Autonomía personal y Situa-ciones de Dependencia (EDAD) of the year 2008
ABSTRACT OF THE THESIS
Prevalence of vitamin B12 deficiency is very common in elderly people and can reachvalues as high as 40.5% of the population. It can be the result of the interaction amongseveral factors. Vitamin B12 deficiencies have been associated with neurological,cognitive deterioration, haematological abnormalities and cardiovascular diseases thathave an important influence on the health of the elderly and their quality of life. It isnecessary to approach the problems arisen from the lack of data relative to them. Themain objective of this thesis was to analyse the evolution of vitamin B12 status andrelated parameters, lipid and haematological profiles and their relationship to healthrisk factors, and to functional and cognitive status over one year and to determine theeffect of an oral supplementation of 500 µg of cyanocobalamin for a short period of 28days. An additional objective was to analyze the possible effects of medicine intakeson vitamin B status.
Three studies were performed: a) a one year longitudinal follow-up with four mea-sure points; b) an intervention study providing an oral liquid supplement of 500 µg ofcyanocobalamin for a 28 days period; and c) analysis of the possible effect of medica-tion intake on vitamin B status using the ATC classification of medicines.
The participants for these studies were recruited from nursing homes for the elderlyin the Region of Madrid. Sixty elders (mean age 84 ± 7y, 19 men and 41 women)were recruited for Study I and 64 elders (mean age 82 ± 7y, 24 men and 40 women)for Study II. For Study III, baseline data from the initially recruited participants of thefirst two studies were used. An informed consent was obtained from all participants ortheir mentors. The studies were approved by the Ethical Committee of the Universityof Granada.
Blood samples were obtained at each examination date and were analyzed for serumcobalamin, holoTC, serum and RBC folate and total homocysteine according to labora-tory standard procedures. The haematological parameters analyzed were haematocrit,haemoglobin and MCV. For the lipid profile TG, total cholesterol, LDL- and HDL-cholesterol were analyzed. Anthropometric measures (BMI, skinfolds [triceps andsubscapular], waist girth and waist to hip ratio), functional tests (hand grip, arm andleg strength tests, static balance) and MMSE were obtained or administered by trainedpersonal. The vitamin B12 supplement of Study II was administered with breakfastand the medication intake was taken from the residents’ anamnesis.
Data were analyzed by parametric and non-parametric statistics depending on the ob-tained data. Comparisons were done using the appropriate ANOVAs or non-parametrictests. Pearsons’ partial correlations with the variable “time” as control were used todefine the association of the analyzed parameters.
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The results showed that: A) Over one year, in relationship to vitamin B status, serumcobalamin decreased, serum folate and mean corpuscular volumen increased signifi-cantly and total homocysteine concentrations were stable. Regarding blood lipid pro-file, triglycerides increased and HDL-cholesterol decreased significantly. Regardingselected anthropometric measurements, waist circumference increased significantly.No significant changes were observed for the rest of parameters. B) Prevalence ofhyperhomocysteinemia was high in the elderly studied, ranging from 60% to 90 %over the year depending on the cut-off used for the classification. LDL-cholesterolvalues were high, especially among women, and showed a tendency to increase overthe year. Results of the balance test showed a deficiency and a tendency to decrease;this indicates that the population studied is at high risk for falls. Lower extremitymuscular function was deficient and showed a tendency to decrease. A highly sig-nificant relationship was observed between the skinfold of the triceps and blood lipidprofile. C) Low cobalamin concentrations correlated significantly with low MMSEscores in the elderly studied. No correlations were observed between vitamin B12 sta-tus and functional parameters. D) Regarding vitamin B12 status, holo-transcobalaminseems to be more sensitive for diagnosis; 5-10% of the elderly had a deficiency usingserum cobalamin as a criterion, and 45-52% had a deficiency when using serum holo-transcobalamin as a criterion. E) 500 µg of cyanocobalamin administered orally dur-ing 28 days significantly improved vitamin B12 status and significantly decreased totalhomocysteine concentrations in institutionalized elderly. No effect of the interventionwas observed on functional and cognitive parameters. F) The relative change (%) ofimprovement of vitamin B12 status was higher when using serum holo-transcobalaminas a criterion than serum cobalamin. G) Antiaenemic drug intake normalized cobal-amin, urologic drugs and corticosteroids serum folate, and psychoanalepticsholo-transcobalamin levels. Drugs treating pulmonary obstruction increased total ho-mocysteine concentration significantly. H) The daily mean drug intake was 5.1. Fifty-nine percent of the elderly took medication belonging to 5 or more different ATCgroups. The most prevalent were psycholeptic (53%), antiacid (53%) and antithrom-botic (47%) drugs.
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WHAT IS KNOWN ON THE TOPIC? WHAT DOES THIS PHD THESIS ADD?
Data unknown about the evolution of vitamin B status over a one year period in institutionalized elderly.
Serum cobalamin decreased, serum folate and mean corpuscular volume increased significantly and total homocysteine concentrations were stable over a one year period in institutionalized elderly.
Data unknown about the evolution of upper and lower extremity functions and balance capacity in institutionalized elderly over a one year period.
Results of the balance test showed a deficiency and a tendency to decrease indicating that the population studied is at high risk for falls. Lower extremity muscular function was deficient and showed a tendency to decrease and no correlations were found between vitamin B12 status and the functional tests.
High oral administered doses of 1 mg and above and a dose of 500 µg for a treatment period of eight weeks are effective for improving vitamin B status.
500 µg of cyanocobalamin administered orally during four weeks significantly improved vitamin B12 status and significantly decreased total homocysteine concentrations. No effect of the intervention was observed on functional and cognitive parameters.
Polypharmacy is common in the elderly, but information is scarce about the type and quantities taken.
The studied group of elderly people took a total of 858 medications of 292 different brands, which is equivalent to a daily average of 5 medications per person.
It is known that certain medications (such as metformin, H2-antagonists and proton pump inhibitors) affect vitamin B status.
Antianaemic drug intake normalized cobalamin, urologic drug and corticosteroids serum folate, and psychoanaleptics holo-transcobalamin concentrations. Drugs treating pulmonary obstruction increased total homocysteine concentration significantly.
Holo-transcobalamin is discussed in the literature as being a more sensitive marker for diagnosing vitamin B12 deficiency compared to serum cobalamin levels.
The relative change of improvement of vitamin B12 status was higher when using serum holo-transcobalamin as the main outcome. When choosing it as the criterion for deficiency, a higher prevalence (45-52%) compared to serum cobalamin (5-10%) was observed.
RESUMEN DE LA TESIS
La prevalencia de deficiencia de la vitamina B12 es muy común en las personas may-ores y el porcentaje puede llegar a ser de hasta un 40,5% de la población. La deficienciade vitamina B12 ha sido asociada con el deterioro cognitivo y neurológico, anormal-idades hematológicas, y enfermedades cardiovasculares que tienen una influencia im-portante sobre la salud del anciano y su calidad de la vida. Es necesario abordar losproblemas que surgen debido a la escasez de datos relativos a ellos, especialmente enancianos institucionalizados. El objetivo principal de esta tesis fue describir la evolu-ción del estado de vitamina B12 y sus parámetros relacionados, de los perfiles lipídicosy hematológicos y sus relaciones con factores de riesgo para la salud, y con su estadofuncional y cognitivo a lo largo de un año, y el efecto de una suplementación oral de500 µg de cianocobalamina durante un periodo de 28 días sobre estos parámetros ymarcadores de riesgo. Un objetivo adicional fue analizar los posibles efectos de latoma de medicamentos sobre el estado de las vitaminas B12 y folato.
Se realizaron tres estudios: a) un seguimiento longitudinal a lo largo de un año concuatro puntos de medición; b) un estudio de intervención suministrando un suplementooral líquido de 500 µg de cianocobalamina durante un período de 28 días y c) unanálisis del posible efecto de la ingesta de medicación sobre el estado de vitamina Butilizando el sistema ATC de clasificación.
Los participantes de los tres estudios fueron reclutados en residencias para personasmayores de la Comunidad Autónoma de Madrid. Sesenta personas (edad media 84± 7a, 19 hombres y 41 mujeres) mayores fueron reclutados para el estudio I y 64personas mayores (edad media 82 ± 7a, 24 hombres y 40 mujeres) para el estudioII. El estudio III, se realizó con los datos de los participantes reclutados inicialmentepara el primer punto de los exámenes del estudio I y II. Se obtuvo un consentimientoinformado de todos los participantes o de sus tutores. Fue aprobado por el ComitéÉtico de la Universidad de Granada.
Las muestras sanguíneas se obtuvieron en cada punto de los exámenes y fueron anal-izadas para la cobalamina, HoloTC, homocisteina y folato (todos en suero), RBC fo-lato siguiendo procedimientos estándares de laboratorio. Los parámetros hematológi-cos analizados fueron el hematocrito, la hemoglobina y el VCM. Para el nivel lipídicose examinaron los TG, el colesterol total, y el LDL y HDL-colesterol. Las medidasantropométricas (IMC, panículos [tríceps y subescapular], la circunferencia de la cin-tura, y la ratio cintura/cadera), las pruebas funcionales (prensión manual, fuerza de losbrazos y de las piernas, equilibrio estático) y el MMSE de Folstein fueron obtenidaso administradas por un personal previamente entrenado. El suplemento de vitaminaB12 del estudio II fue suministrado con el desayuno y el historial sobre la ingesta de
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medicación fue recogido de las anamnesis de los residentes.
Los datos fueron analizados por pruebas parámetricas y no-paramétricas dependiendode los datos obtenidos. Las comparaciones se hicieron utilizando los ANOVAs o prue-bas no paramétricas. Se calcularon las correlaciones parciales de Pearson con la vari-able “tiempo” como control para describir las asociaciones entre los parámetros anal-izados.
Los resultados mostraron que: A) A lo largo de un año, relativo al estado de la vita-mina B, los valores de la cobalamina sérica disminuyeron, el folato sérico y el vol-umen corpuscular medio aumentaron de forma significativa y las concentraciones dehomocisteína permanecieron estables. B) La prevalencia de hiperhomocisteinemia fueelevada en los ancianos estudiados, entre el 60% y el 90% a lo largo del año dependi-endo del punto de corte utilizado para la clasificación. Los valores de LDL-colesterolfueron elevados, especialmente en las mujeres, y mostraron una tendencia a aumentara lo largo de un año. Los resultados de la prueba del equilibrio estático mostraron unadeficiencia y tendencia a disminuir; esto es indicativo de que la población estudiadapresentaba un alto riesgo de caídas. La función muscular de las extremidades inferi-ores fue deficiente y mostró tendencia a empeorar. Se observó una relación altamentesignificativa entre los panículos del tríceps y el perfil lipídico. C) Las concentracionesbajas de cobalamina se correlacionaron de forma significativa con una menor pun-tuación del MMSE en los ancianos estudiados. No se observó ninguna correlaciónentre el estado de la vitamina B12 y los parámetros funcionales. D) En relación conel estado de la vitamina B12, la holo-transcobalamina parece ser más sensible para eldiagnóstico: 5-10% de los ancianos presentaban deficiencia utilizando la cobalaminaen suero como criterio, y 45-52% presentaban deficiencia cuando se utilizaba la holo-transcobalamina como criterio. E) La administración de 500 µg de cianocobalaminadurante 28 días mejoró significativamente el estado de la vitamina B12 y disminuyósignificativamente las concentraciones de la homocisteína total en los ancianos. No seobservaron efectos de la intervención sobre los parámetros funcionales y cognitivos.F) El cambio relativo de mejora en el estado de la vitamina B12 fue superior cuandose utilizaba la holo-transcobalamina como criterio en vez de utilizar la cobalamina. G)La toma de medicamentos antianémicos normalizó los niveles de cobalamina, los cor-ticoesteroides y los farmácos urológicos normalizaron los niveles de folato sérico y lospsicoanalépticos los niveles de holo-transcobalamina. Los fármacos contra la obstruc-ción pulmonar aumentaron de forma significativa los niveles de homocisteína. H) Latoma media diaria fue de 5.1 medicamentos. Cincuenta y nueve por ciento de los an-cianos tomaban medicación perteneciente a 5 o más grupos ATC diferentes. Los másutilizados fueron los psicolépticos (53%), antiácidos (53%) y antitrombóticos (47%).
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¿QUE SE CONOCE DE ESTE TEMA? ¿QUE AÑADE ESTA TESIS?Se desconocen los datos de la evolución de la vitamina B a lo largo de un período de un año en personas mayores institucionalizadas.
La cobalamina sérica disminuyó, el folato sérico y el volumen corpuscular medio aumentaron significativamente y las concentraciones totales de homocisteína se mantuvieron estables a lo largo de un año en personas mayores institucionalizadas.
Se desconocen los datos relativos a la evolución de la función de las extremidades superiores e inferiores y la capacidad de equilibrio a lo largo de un año en las personas mayores institucionalizadas.
Los resultados de la prueba de equilibrio mostraron una deficiencia y una tendencia a disminuir lo que indica que la población estudiada tiene un alto riesgo de caídas. La función muscular de las extremidades inferiores resultó ser deficiente y mostró una tendencia a disminuir y no se encontraron correlaciones entre el estatus de la vitamina B12 y las pruebas funcionales.
La administración oral de altas dosis de 1 mg o más, y una dosis de 500 µg durante un período de tratamiento de ocho semanas es efectivo para mejorar el estatus de lavitamina B.
500 µg de cianocobalamina administrados oralmente durante cuatro semanas mejoraron significativamente el estatus de la vitamina B12 y disminuyeron de forma significativa la concentración de homocisteína total. No se observó ningún efecto de la intervención sobre los parámetros funcionales y cognitivos.
La polifarmacia es común en las personas mayores, pero la información relativa a los tipos de medicamentos tomados es escasa.
El grupo estudiado de personas mayores tomaba un total de 858 medicamentos de 292 marcas diferentes, lo que es equivalente a un promedio de 5 medicamentos diarios por persona.
Se conoce que ciertas medicinas (como la metformina, los antagonistas-H2, y los inhibidores de la bomba protónica) afectan al estatus de la vitamina B.
La toma de medicamentos antianémicos normalizaba la cobalamina, los urológicos y los corticoides el folato sérico, y los psicoanalépticos las concentraciones de holo-transcobalamina. Los medicamentos para la obstrucción pulmonar aumentaban significativamente la concentración de homocisteína total.
La literatura discute si la holo-transcobalamina es un marcador más sensible para diagnosticar la deficiencia de la vitamina B12 en comparación a los niveles séricos de cobalamina.
El cambio relativo de la mejora del estatus de la vitamina B12 fue mayor utilizando holo-transcobalamina como resultado principal. Cuando se escogió este criterio para la deficiencia, se observó una prevalencia mayor (45-52%) que con la cobalamina sérica(5-10%).
INTRODUCTION
1 INTRODUCTION
1.1 Introduction to the Thesis
Worldwide, the proportion of people aged 60+ is increasing. As mortality and fertilitydecrease, median ages are increasing in nearly all countries, although the rate and paceof this rise vary considerably among the world regions [170]. Europe is considered tobe one of the continents that will age fast over future decades. Specifically, Spain isamong the top 20 oldest countries today with 22.2% ranking 18 on the list of those withthe greatest proportion of the elderly over 60y [249]. The highest number of elderlypeople can be found in the autonomous regions of Andalucía, Cataluña and Madrid.
At present, Europe’s baby boom generation of the 50s and 60s are already reachingage 50 and 60; in the near future, this cohort will enter the elderly group. In 2001, forthe first time the proportion of elderly 65y+ exceeded (inflection point) the populationyounger than 15; today, in Spain, the proportions are 17% and 15%, respectively [3][2] reflecting the present demographic shift to an old-age structure of the population.Spanish projections estimate the population of 65y+ to be 31.9% of the total population[3] in the year 2049 which corresponds to an absolute number of 15 million of elderlypeople.
Since 1950, the ongoing increase in life expectancy can be attributed largely to im-provements in survival after age 60 [254]. Old people, and even very old people, area more and more important part of reality in Spain. Among various other Europeancountries and Japan, Spain leads the list of countries with the highest life expectancies:Spanish women live 85 years on average and male life expectancy is currently set at79 years [2]. Among the EU27 countries, life expectancies for Spanish women andmen over 65y are with 22.4y and 18.3y, respectively, the second highest after France[3]. The National Institute for Statistics (INE) predicts 87y for Spanish women and81 for Spanish men in 2030 and although calculated with slower declines in mortalitythan in the past, the United Nations predicts a life expectancy of 84.5y for the WesternEuropean and 81y for the total European population in 2050 [250]. According to somedemographic projections, women and men in Europe will survive to become nonage-narians and centenarians. In the countries with the top levels of life expectancy, femalelife expectancy has increased for 160 years at a constant rate of nearly three monthsper year [195]. On average, women live longer than men; however, life expectancy hasalso risen linearly for men, although less rapidly.
Improvements in survival that have led to increasing life expectancy are mainly at-tributed to the complex interaction of progress in income, nutrition, education, betterliving standards, sanitation, medicine and public health efforts [216] [195]. The possi-
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bility of reaching an older age is raising the issues of whether longer lives will be livedin good health or disability. It raises questions such as how healthy will one be dur-ing the gained years and will one have to face longer periods of disability and frailty.There is some evidence that one is living longer because one is staying healthy longer.Increasing life expectancy does not necessarily mean that the period of disability willbe longer. In effect, it appears that this period at the end of life is becoming somewhatshorter [104]. The proportion of people aged over 80 (considered as the thresholdvalue for disability) will increment rapidly in all European countries over the comingdecades.
Age is the main factor that raises the risk of needing long-term care, which is of specialimportance to the age groups of 80+ or 85+. In Spain, in 2008, 7,632,925 people intotal (4,394,624 women and 3,238,301 men) were over 65 years and 2,123,785 peoplein total (1,377,425 women and 746,360 men) were over 80 years [146]. At the begin-ning of 2011 the population of elderly people consisted of 8,092,853 people of whomthe octogenarians were the group with the highest growth rate [3].
The rate of disability increases with age. According to the Survey on Disability,Personal Autonomy and Situtions of Dependency (Encuesta sobre Discapacidad, Au-tonomía personal y Situaciones de Dependencia (EDAD) [148]) from the year 2008;at age 80, more than half of the Spanish population experienced problems performingdaily living activities [3]. But over the last decade, the age group of 80+ has shown adifferent tendency than the people between 65-75y. Whereas the percentage of peoplesuffering some kind of disability in the age group of 65-75y decreased from 37.4% in1999 to 31.2% in 2008 the percentage for the age group 80+ increased from 21.3% in1999 to 26.7% of the total number of people suffering some kind of disability [148].From a quantitative point of view, problems of mobility are the basis of the most fre-quent problems of elderly dependent people reflected in difficulties performing dailyliving activities (in terms of household managing) followed by tasks of personal hy-giene [83]. Increasing age does not have to result in a commensurate increase in caredemand or long-term care expenditure, but a considerable increase in the numbers ofthose who need care over the following decades.
Following the approach of Doblhammer and Ziegler [74] from the Rostock Centerfor the Study of Demographic Change there are two scenarios: an optimistic healthy-years-of-life scenario, which assumes that the years gained in life expectancy will beyears without disability; and a pessimistic constant-caring scenario, which assumesthat the years added will be years of disability. In any case, the greater number ofoldest-old will imply a greater need for long term care but supposedly not reach thehigh values forecast under the pessimistic constant-caring scenario. At the same time,the proportion of middle-aged people who can provide care will decrease.
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Today, nearly two-thirds of elderly Spanish people in need of care have some kind offamily support, whereas about 22% live in a single household [83]. The number ofelderly who will move to an institution is likely to rise over the coming years, partly asa result of individual preferences and partly because of a diminishing supply of privatecare, especially after 2030. At that time, the baby boomers (born between 1957 and1977) will begin to need long-term care. Spain being a Southern European countrywill confront particular challenges [74]. At present, the rate of institutionalization isnot very high because the family tradition is strong and the old are cared for by youngerfamily members. The ratio of the number of people 85y+ relative to 100 of the peoplebetween 45-65y (ratio of family support) has increased progressively and is estimatedto keep on increasing so that family resources available for old people’s care to secureadequate service in response to changing needs are decreasing [3]. Most elderly peopleprefer to remain in their homes because they are able to maintain the integrity of theirsocial network, to preserve their environmental landmarks and to enjoy a higher qualityof life [169].
Findings of a recent systematic review by Luppa at al. [169] suggested that predictorsof nursing home placement (NHP) in the general population of developed countriesare mainly based on underlying cognitive and/or functional impairment, and associatedlack of support and assistance in daily living. Also, a meta-analysis of 2007 by Gaugleret al. [108] showed that the strongest predictors of admission to a nursing home in theU.S. were three or more Activities of Daily Living (ADLs) dependencies, cognitiveimpairment, and prior nursing home use.
In fact, two of the most common diseases affecting the elderly are cardiovascular dis-eases and cognitive impairments. Dementia is the most common cause of cognitiveimpairment [37]. Two pathologically distinct subtypes of dementia, vascular dementia(VD) and Alzheimer’s disease (AD), constitute the vast majority of cases [1]. AD isthe most common cause of dementia in the aged. The age-adjusted prevalence of ADincreases exponentially after age 65 and the estimation is that 50% of women will beaffected after 85 years [24]. VD is the second most frequent cause of dementia in theelderly after AD [88]. Stroke is a major cause of VD [51] [182].
Cognitive impairment has in most cases a multifactorial origin. Among the nutrition-related risk factors there are hypercholesterolaemia and its association with hyperten-sion, low levels of antioxidants, amino acids, folate, vitamin B6 and vitamin B12 andhigh levels of homocysteine [110]. Hyperhomocysteinaemia is a risk factor for vascu-lar disease and is associated with AD. Low levels of methionine, an essential aminoacid that depends on a normal functioning of the one-carbon metabolism in which ho-mocysteine is an intermediate product have been observed in plasma samples in ADpatients in comparison to control subjects [89].
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Cigolle et al. [55] found that about 32% of individuals aged 90+ had reported cognitiveimpairment in the U.S.A. The prevalence and proportion of severe stages of cognitiveimpairment increase with age [127]. Since the disease has a great impact on the qual-ity of life of patient and caregiver alike, the need not only for adequate treatment butalso for prevention is high. As pyridoxine, cobalamin and folate play an importantrole in the nervous system by interacting as coenzymes in the metabolism of methio-nine [231][186] research on the status of these vitamins is required to give furtherinsights. The elderly represent a vulnerable group in terms of vitamin B supply, espe-cially with regard to cobalamin deficiency. Age-related declines in vitamin absorptionand impairment of liberating cobalamin from proteins, as well as an age-related in-crease in autoimmunity against intrinsic factor or gastric parietal cells are causes ofdeficiency which are common in the elderly [186]. Elevated homocysteine values as aconsequence of vitamin B12 and/or folate deficiencies are also considered as an inde-pendent risk factor for cardiovascular diseases, another leading cause of morbidity andmortality in the elderly.
Aging-related motor disability is another major health concern for the elderly. Themechanisms underlying impaired motor performance in old age are complex as thecentral and peripheral nervous systems as well as the muscle tissue itself are involved[162]. There is general consensus that the loss of muscle mass and strength due toaging has a negative impact on motor performance and on the ability to recuperatefrom falls, leading to a higher risk of fractures and dependency [162]. Thus, preventingfalls and gait instability is a very important safety concern, and various programs toimprove motor performance among the elderly are commonly built around measuringand addressing specific impairments, such as impaired balance capacity and reducedstrength of arms and legs.
It is widely accepted that keeping a good level of physical activity is a powerful meansof obtaining long term benefits on muscle function, to reduce the frequency of falls,and to maintain independence and a high quality of life in older persons. To be able toperform ADLs keeps the elderly person independent, a very important issue, not onlyconcerning “ageing in place” but also once transferred to a residential setting. In orderto perform ADLs sufficient arm strength is needed to move things around and opencans, jars, etc., and leg strength for moving around and transfers. A high percentageof elderly people have to utilize all their muscle power in order to rise from a chair sothat a small additional impairment in muscle function may dramatically change theirlives from independent to dependent [90][139].
Power handgrip strength is recorded by clinicians as a quick and viable measure ofimpairment and function which can serve as a useful evaluation of hand strength status[69]. Hand grip strength was highly predictive of functional limitations and disability
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25 years later among healthy 45 to 68y old men concluding that good muscle strengthin midlife may protect people from old age disability by providing a greater safetymargin above the threshold of disability [205].
1.2 Significance of the Thesis
The present thesis addresses different aspects of the set of problems which haveemerged as a consequence of the huge increase in the elderly population. Althoughageing is a biological process and not a disease, it is recognized that the elderly presenta greater facility for getting ill and for suffering disabilities; with advancing age peopleare more likely to develop chronic diseases that require medical care and sometimesreach a high degree of frailty and multiple diseases.
Lack of specific data related to the functional, haematological, biochemical and cog-nitive status and medication intake of the elderly and the way in which this populationgroup responds to treatments and medication make it necessary to address these aspectsfor their clarification.
The functional importance of the group of B vitamins in relation to the problems ofarteriosclerosis and anaemia, and the combined factors in relation to the deteriorationof the nervous system and even more precisely in relation to cognitive impairment anddementias has to be stressed. The problems which have emerged as a consequenceof the possible interplay are characteristic of this new situation. Bringing insight isessential for Public Health Policies.
The future of Europe is reliant on its ageing population which among other aspects willhave impact on health care costs. The rising proportion of old and very old people willhave major long-term impact at an individual level as well as at society level [255].Ageing populations are a challenge, but at the same time they provide an opportunityto think about how to live a longer, healthier life [255]. More people reach advancedage in which they could suffer some kind of difficulties.
The situation is even more serious for the elderly who have been institutionalized inmost of the cases due to their delicate health status or because of the need for specialassistance and in some cases because of lack of direct family support. Health problemsare important especially in advanced age and are forecast to increase due to the higherlongevity. The significance of the present thesis which is presented in three sections istherefore to study these problems to get a better understanding of the ageing process.Study I focusses on the evolution of vitamin B12 status, lipid profile, anthropometric,functional and cognitive status over a one year study period. Study II describes a 28day intervention with a 500 µg cyanocobalamin supplementation and Study III centres
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on the effects of polypharmacy on the vitamin B12, folate and homocysteine status; allin relation to an institutionalized elderly Spanish population.
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REVIEW OF RELATED LITERATURE
2 REVIEW OF RELATED LITERATURE
As was presented in the previous chapter, there is certain evidence that deficiencies ofthe vitamins of the vitamin B complex are risk factors for cardiovascular diseases andcognitive impairment especially affecting the population of elderly people.
Despite the impressive ways vitamins function individually, oftentimes it is difficult toknow which vitamin is responsible for a given effect because the nutrients are inter-connected; the presence or absence of one influence the absorption, metabolism andexcretion of another. In this context the interaction between folate and vitamin B12and/or the relationship between riboflavin and B6 could be mentioned. Deficiency ofone nutrient can alter the action or create a deficiency of another [263]. Within thevitamins of the vitamin B complex, vitamin B12 is the vitamin whose absorption ismost negatively affected as a consequence of different pathologies, a characteristicespecially of the elderly.
The main focus of the present review is on the functions, metabolism, problems ofdetection of deficiency and the consequences of their deficiencies. Maintaining func-tional integrity of the upper and lower extremities is necessary for performing a seriesof labours and tasks of everyday life and this integrity might be affected by the abovementioned deficiencies. The following review focusses as well on the effects of thesedeficiencies on the function of balance and the upper and lower extremities.
Figure 1: Chemical structure of vitamin B12
2.1 Definition of Vitamin B12 and Food Sources
Vitamin B12 is part of the vitamin B complex, a group of water-soluble nutrients. Itis the biggest and has a molecular weight of 1355.4 Da. The term B12 is the generic
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descriptor for all corrinoids; those are compounds having a corrin ring surroundinga central cobalt (Co) that is linked to a lower as well as an upper ligand. The lowerligand is a benzimidazole group which is bound to the corrin ring by means of a ribose-phosphatase group. The upper ligand is what distinguishes one chemical form fromanother.
In the two different coenzymatically active cobalamin derivatives, the upper ligand iseither a methyl or a 5-deoxyadenosyl group (Figure 1)[201]. Both are built in sep-arated cell compartments; methylcobalamin in the cytosol and adenosylcobalamin inthe mitochondria.
In nature, two other forms of vitamin B12 also exist: hydroxycobalamin and aqua-cobalamin, where hydroxyl and water are bound respectively to the cobalt. Cyanocobal-amin, the synthetic form of vitamin B12, has cyanide bound to the cobalt and is com-monly used for supplements and fortified foods. Hydroxy-, aqua- and cyanocobalaminare enzymatically activated to the methyl- or deoxyadenosylcobalamins in all mam-malian cells. Corrinoids other than cobalamins, i.e. cobinamide have been shown tobe present in stools and in the circulation, but no physiological functions in humanshave been demonstrated so far [7][125].
2.2 Dietary Sources and Availability
Vitamin B12 cannot be synthesized by humans and therefore must be supplied viathe diet. It is produced by most microorganisms [225], including bacteria and algae.The vitamin B12 produced by microorganisms gains access to the human food chainvia incorporation into food of animal origin. Gastrointestinal fermentation aids thegrowth of these microorganisms. The vitamin is absorbed and concentrated mainlyin animal tissues especially in the liver, the main store of this vitamin. Therefore,animal-origin food is the only natural dietary source for providing large amounts ofvitamin B12 (see Table 1) [272]. Products derived from herbivorous animals, suchas milk, meat, and eggs, are also important dietary sources except if the animal camefrom geochemically deficient cobalt regions [181]. Vitamin B12 intake from eggs isgenerally high, because they are a highly consumed food item [102] although it isgenerally poorly absorbed (<9%) compared to other animal-origin foods [76]. Plantfoods usually do not contain vitamin B12 unless they were in contact with vitaminB12-containing substances, exposed to vitamin B12 synthesizing bacteria, or fortifiedwith vitamin B12 [18]. Certain plant foods such as dried green and purple lavers (nori)contain substantial amounts of vitamin B12, whereas other consumable algae containnone or only traces of vitamin B12. However, most of the consumable blue-green algaemainly contain pseudovitamin B12, which is inactive in humans [262][261].
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Table 1: Sources of vitamin B12 in foods (modified from [61])
2.3 Requirements for the Daily Intake of Cobalamin in the Elderly
The vitamin B12 requirements have been determined via different methodic approaches.On the one hand, by means of curative tests, the amount of vitamin B12, necessary toreverse an already existing megaloblastic anaemia, on the other hand the requirementwas calculated by means of the storage quantity and the turnover rate. Apart from that,the vitamin B12 values of healthy and deficient subjects provide an indication of thepreferable quantity of supply.
Under the condition of entire bioavailability, the minimial requirements for humans canbe achieved with a daily intake of less than 1 µg vitamin B12. As with higher intakethe utilisation rate of vitamin B12 decreases (IF-dependent reaction), the recommendedvitamin B12 intakes vary between 1.4 and 3.0 µg/d due to the selection of different CV(10-20%) and health indicators (maintenance of haematological status or basal losses)[75].
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Spain participated as one of thirteen countries in the European Nutrition and HealthReport 2004 and also in 2009; the assessment of the nutritional average cobalamin in-take of European elderly showed that the intake was 6.2 and 7.4 µg for Spanish womenand men between 65 and 75 years respectively in 2004 and was the highest cobalaminintake from all participating countries [87]. In the European Nutrition and Health Re-port of 2009 the intake was 3.5± 0.5 µg for the elderly population over 64 years. Thus,the intake lies above the reference value of 1.4 µg/day recommended by the ScienitificCommittee on Food [62] and also above 3 µg/day recommended by DACH 2000. Astudy which analyzed the dietary intake of an elderly institutionalized population inLeón (Spain) revealed that the average intakes of folate and vitamin B12 exceededRDA, (103% and 144%) [106]. However, 45.83% of males and 5.97% of femalesshowed deficiencies in vitamin B12 and 53.91% of the subjects showed deficiencies infolic acid intake [106]. Because of the usual dietary habits in South Europe, vitaminB12 is generally ingested in amounts exceeding the requirements. In line with otherSouthern European Countries, in Spain, the proportion of animal products in total en-ergy supply greatly increased over the past four decades (from 13% in the year 1961to 28% in 2003, respectively) whereas the proportion of vegetable products in total en-ergy supply decreased (from 87% in 1961 to 72% in 2003, respectively) [78]. Althoughthere is agreement upon the fact that vitamin B12 is not one of the critical nutrients interms of requirement coverage via dietary intake, a longtime coverage of requirementsthrough the depletion of human stores is not possible when suffering certain diseasessuch as gastrointestinal alterations.
2.4 Absorption, Storage and Excretion of Vitamin B12
2.4.1 Absorption and Storage
Cobalamins cannot be produced by higher organisms so that humans depend on thedietary intake. Mammals possess a complex pathway for absorption, transportationand cellular uptake of cobalamin (Figure 2). There are two pathways for vitamin B12absorption; an intrinsic factor (IF) dependent absorption and passive diffusion.
IF Dependent Absorption
IF-dependent absorption is an active process, which needs a normally functioningstomach, presence of IF, pancreatic enzymes, and an intact terminal ileum. VitaminB12 in food is attached to protein and is liberated from dietary protein in the stomachvia the acid milieu (high concentration of hydrochloric acid) and pepsin. Once re-leased, free vitamin B12 binds to R protein in the stomach. R protein (or R-binder) is a
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haptocorrin present in saliva, gastric juice, bile, intestinal juice, and serum which pro-tect vitamin B12 from denaturation in the stomach. In the alkaline milieu of the smallintestine, pancreatic enzymes degrade the R protein liberating vitamin B12 which thenbuilds a complex with IF (60-kDa glycoprotein produced by gastric parietal cells af-ter stimulation by food). The vitamin B12-IF complex is stable and enters the ileum.For the B12-IF complex to attach to the receptor site, ionic calcium and a pH greaterthan 6.0 are needed. Vitamin B12 is transported into the mucosal enterocytes againsta concentration gradient, so it is an active process and saturable as well. Various stud-ies indicate that the normal capacity of this system is to absorb about 1.5-2.0 µg ofvitamin B12 per meal due to saturation of the ileal receptors [226]. After a few hoursof recuperation, the system can absorb an alike amount of vitamin B12 as a subse-quent event [226]. Inside the enterocytes, the IF is degraded and the liberated Cbl linksto transcobalamin (TC) [202] [188]. The TC-Cbl complex is then released into theplasma, where it is endocytosed via membrane receptors of the target cells.
Passive Absorption
Vitamin B12 can also be absorbed passively via diffusion in a non-IF mediated man-ner. This has been demonstrated by the fact that patients who suffered from PerniciousAnaemia and therefore had no IF function absorbed a proportion of an administereddose. The rate of the passive diffusion is apparently directly correlated to the quantityadministered. Chanarin [43] [44] concluded that once the saturation of the IF-mediatedactive absorption system at about 1.5-2.0 µg per meal or test dose is reached furthercobalamin is absorbed by passive diffusion. Urinary excretion tests have been usedto determine the quantity absorbed by diffusion and calculated the rate to be approxi-mately 1%.
Vitamin B12 Binding Proteins
Three circulating plasma vitamin B12 binding proteins exist: namely transcobalamin(TC) I, TC II, and TC III. TC I links to around 80% of the circulating vitamin B12,while TC II links to less than 20%. Nevertheless, vitamin B12 enters cells throughoutthe body mainly linked to TC II. TC II links to 7%–20% of the endogenous cobalamin[81] [121] and mediates 33%–99% of the total plasma vitamin B12 clearance [6] [120].Although TC I links 80%–90% of the endogenous cobalamin, TC I mediates less than1% of the total cellular uptake of vitamin B12 from plasma [5] [6] [95] [114] [123][149] [229] [230].
Estimates of total-body vitamin B12 stores vary from 2.0 to 5.0 mg [4] [115] [210],of which approximately 60% are stored in the liver, 30% in the muscles and the restin other tissues. The mean biological half time of the storage are 485 days. Each day0.143% are eliminated. For the maintenance of high vitamin B12 stores of 5 mg, a
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Figure 2: Absorption, transport and cellular uptake of cobalamins in mammals modified after [204]
daily intake of 7.2 µg B12 and for the maintenance of stores of 2 mg, a daily intake ofaround 2.9 µg is needed.
2.4.2 Enterohepatic Circulation and Excretion of Vitamin B12
In hepatocytes, vitamin B12 is added to bile that flows to the small intestine. Asindicated before, vitamin B12 attaches to IF and thereafter two thirds are reabsorbed[115] and returned to the liver in the portal blood. Although both Green et al. [116] andTeo et al. [244] suggested that bile enhances vitamin B12 absorption, the enterohepaticcirculation of vitamin B12 is dependent on the presence of intrinsic factor. In theabsence of intrinsic factor, all the vitamin B12 from the bile is excreted into the stoolinstead of being recirculated. Individuals with pernicious anaemia, with total absenceof IF, develop vitamin B12 deficiency rapidly, in approximately 1–3 years, comparedwith having vitamin B12 deficiency due to other causes [9] [95] [155].
The vitamin B12 which is not absorbed from the bile is excreted in the feces (aver-age loss about 0.4 µg/day), thus making bile the principal way of eliminating the bodyof excessive vitamin B12 and removing potentially hazardous vitamin B12 analogues[14] [119]. Vitamin B12 in the feces comes from non-absorbed dietary vitamin B12 orvitamin B12 from bile, desquamated cells, gastric and intestinal secretions, and vita-min B12 synthesized by intestinal bacteria. After high dose supplementation amountsexceed plasma vitamin B12 binding capacity, vitamin B12 is also excreted throughurine. Other routes of vitamin B12 loss are through the skin and other body secre-tions. The amount of vitamin B12 excreted from the body (turnover rate) is fixed at0.1%–0.2% of total body stores daily, regardless of the pool size [9] [28] [29] [133][211] [210].
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Figure 3: Biochemical pathway of homocysteine
Analyses by Heinrich et al. in the late 60s [128] of the last century showed that theoral intake of 0.1-500 µg amount of 60Co-cyanocobalamin was retained quantitativelyin the whole body and was exactly the same amount of the intestinal resorbed 60Co-cyanocobalamin quantity. When the retention capacity is exceeded, the proportion ofvitamin B12 eliminated with urine increases with rising application rates.
2.5 Functions of Vitamin B12
Vitamin B12 functions as a cofactor for two enzymes: methionine synthase (EC 2.1.1.13),which is involved in methionine biosynthesis, and L-methylmalonyl coenzyme A (CoA)mutase (EC 5.4.99.2), which is involved in amino acid and odd-chain fatty acidmetabolism in mammalian cells [46] [92].
Function of Methylcobalamin
After having bound to a specific receptor in the presence of Ca2+ cobalamin entersthe cells as a complex with transcobalamin via endocytosis. There, transcobalamin isdecomposed proteolytically and cobalamin is liberated into the cytosol as trivalent Cbl(Cbl3+) and afterwards it is reduced to Cbl2+. The latter is bound to the apomethion-insynthase, begins its enzymatical activity and functions as a methyl-transmitter in thesynthesis of methionine from homocysteine, in which methyl-THF is the real methyldonor (Figure 3).
Function of Adenosylcobalamin
Adenosylcobalamin is formed in the mitochondria from cobalamin after reduction ofCbl2+ to Cbl1+(reductive system within the mitochondria) through transfer of adenosyl
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Figure 4: Methylmalonyl CoA mutase
from ATP with seperation of triphosphate (see Eq 1).
Cbl1++AT P→ Adenosylcobalamin+PPP (1)
Adenosylcobalamin participates in two intramolecular transmission processes:
1. Transfer of methymalonyl-CoA to Succinyl-CoA by methylmalonyl CoA mutase(Figure 4). With the help of this reaction propionic acid, which is formed dur-ing the degradation of odd-numbered fatty acids or the amino acids methionine,threonine and isoleucine, and methylmalonyl-CoA, which is formed during thedegradation of valine, gain access to the citric acid cycle.
2. Reversible transfer of leucine to 2-aminoisocapronacid (ß-leucine) by the L-αleucine mutase. In this reaction, the aminoacid group moves from C-atom 2 toC-atom 3 in exchange with a hydrogen atom.
2.6 Prevalence of Vitamin B12 Deficiency in the Elderly
Vitamin B12 concentrations decline with age, whereas metabolite levels, namely ho-mocysteine and methylmalonic acid increase indicating an impairment of vitamin B12status. The increased prevalence of vitamin B12 deficiency in this population groupis due to different factors which include the presence of pernicious anemia (type Aatrophic gastritis) and type B atrophic gastritis. The prevalence of both conditionsincreases with age. The prevalence of subdeficient vitamin B12 levels in the elderlywhich can be found in the literature differs from 3.0% to 40.5%, depending on thediagnostic criteria chosen [20] [21] [27] [31] [39] [48] [65] [79] [80] [47] [107] [118][124] [154] [173] [177] [192] [199] [270] [269].
2.6.1 Factors Contributing to Poor Vitamin B12 Status with Ageing
Pernicious Anaemia
Pernicious anaemia (PA) is the most common cause of clinically apparent vitaminB12 deficiency in North American and European populations. PA is the final stage of
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autoimmune gastritis, type A chronic atrophic gastritis or gastric atrophy, in which thefundus as well as the body of the stomach are involved. These are the places wherethe acid-secreting parietal cells and pepsinogen-secreting zymogenic cells are located.When suffering pernicious anaemia, autoantibodies against parietal cells cause loss ofgastric parietal cells whose continuous destruction from the gastric mucosa results inimpaired IF secretion. Also, blocking antibodies in the gastric juice can link to thevitamin B12 binding site of IF to prevent the formation of the vitamin B12–IF complex.In another less common form of PA, the antibodies allow vitamin B12 to bind to theintrinsic factor but prevent the absorption of the intrinsic factor–vitamin B12 complexby the ileal receptors. Thus, in pernicious anemia, vitamin B12 deficiency develops byseveral mechanisms [247]. Achlorhydria, low serum pepsinogen I concentrations, andhigh serum gastrin concentrations caused by hyperplasia of gastrin-producing cells arefound in type A gastritis.
In addition to causing malabsorption of vitamin B12 from the diet, PA also results inan inability to reabsorb the vitamin B12 secreted into the bile (~ 0.3 and 0.5 mg/day)within the enterohepatic circulation of vitamin B12. This results in an even moresignificant negative balance for the vitamin. Like most autoimmune diseases, the in-cidence of PA increases markedly with age. The mean age at diagnosis of perniciousanemia is 60 years. The female-to-male ratio is approximately 1:5. In Caucasians, theprevalence of the disease rises with increasing age, peaking after age 65 [44].
Atrophic Gastritis and Food-Bound Vitamin B12 Malabsorption
Type B chronic atrophic gastritis mainly involves the gastric antrum and is associatedto Helicobacter pylori infection. The gastric antrum is initially affected, but later on thegastritis extends to the body of the stomach. Subclinical vitamin B12 deficiency withaging is caused primarily by type B atrophic gastritis accompanied by low acid-pepsinproduction and food-bound vitamin B12 malabsorption.
In addition, hypochlorhydria leads to intestinal bacterial overgrowth, which interfereswith vitamin B12 absorption. Therefore, malabsorption of protein-bound vitamin B12is caused by both mechanisms in individuals with atrophic gastritis and leads to adecreased vitamin B12 status [159] [222] [242]. However, the absorption rate of crys-talline vitamin B12 does not decrease in type B atrophic gastritis, as intrinsic factorcontinues to be produced in sufficient amounts [77] [179].
Alteration with ageing in the functional and structural integrity of the vitamin B12binding proteins resulting in compromised TC II–B12 delivery system has also beensuggested to be a factor in reducing vitamin B12 status in the body [173]. There areonly a few cases known of an inborn deficiency of the transport protein Transcobal-amin II. This disease manifests itself in the first weeks of life and leads to – despite
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often physiological serum cobalamin levels - severe megaloblastic anaemia. Very highadministrations of hydroxycobalamin result in the relief of symptoms.
2.6.2 Other Causes and Effects of Vitamin B12 Deficiency in the Elderly
Poor vitamin B12 dietary intake is not very common in the elderly. Because of thehigh vitamin B12 stores and the low turn-over rate, vitamin B12-hypo- or rather avi-taminosis only occur after longterm malnutriton and/or undernourishment or when thegastrintestinal tract absorption is disturbed for a long time (food-bound vitamin B12malabsorption) and therefore the stores are depleted. Impaired gastric acid produc-tion caused by type B atrophic gastritis accompanied by bacterial overgrowth is theunderlying mechanism of food-bound vitamin B12 malabsorption in this population.
Type A atrophic gastritis (pernicious anemia) and total or rather partial gastrectomycause deficient intrinsic factor, leading to vitamin B12 malabsorption. Other, infre-quent causes of vitamin B12 malabsorption in the elderly are malabsorptive statusafter intestinal bypass, exocrinic pancreatic insufficiency, terminal ileal disease i.e.in Morbus Crohn, disturbed cleavage of the vitamin B12-haptocorrin-complex in theduodenum, lymphoma, radiation enteritis, intestinal tuberculosis, infestation with Di-phyllobothrium latum, severe celiac disease, and tropical sprue, Blind-Loop syndrome,extensive parasite decay (for example fish tape worm), as well as a selective inherentB12 resorption disturbance (Imerslund-Gnasbeck-syndrome).
Vitamin-Pharmaka-Interactions
Beside the mentioned changes of the stomach physiology, a series of drugs (such ascolchicine), alcohol or dependence-causing substances impair vitamin B12metabolism. The set of problems of the vitamin-pharmaceutical-interactions are of-ten neglected, usually discovered by chance, and often long after the drug entered themarket. These interactions are even more complex in polypharmacy therapy, oftenfound among old, multimorbid people.
Drugs which increase the gastric pH may reduce the absorption of vitamin B12 andtherefore produce deficiency by impairing the release of protein-bound vitamin B12[221] [239]; this is well-reported for the proton pump inhibitor omeprazole [172] [33]and also for histamine-2 receptor antagonists [103] [220]. However, whereas the ab-sorption of protein-bound vitamin B12 is decreased, the absoprtion of cristalline vita-min B12 remains uninfluenced, a fact that is of practical relevance.
Other drugs, which negatively influence vitamin B12 absorption are the lipid-loweringcholestyramine, the antibiotika chloramphenicol, clioquinol and neomycin as well asthe antidiabeticum metformin [70]. In addition, nitrous oxide rapidly oxidises cobalt
18
bound to methylcobalamin and therefore inactivates methionine synthase resulting inhigher homoysteine levels. Elderly people with impaired vitamin B12 and/or folatelevels may be notably vulnerable to nitrous anaestesia, which is reported in severalcase reports.
2.7 Clinical and Subclinical Manifestations of Vitamin B12 Deficiency in the El-derly
Vitamin B12 deficiency is characterized by the following clinical picture.
Table 2: Clinical Manifestiations of Vitamin B12 Deficiency
HematologicMegaloblastic anemiaPancytopenia (leucopenia, thrombocytopenia)NeurologicParesthesiasPeripheral neuropathyCombined systems disease (demyelination of dorsal columns and corticospinal tract)PsychiatricIrritability, personality changeMild memory impairment, dementiaDepressionPsychosisCardiovascularPossible increased risk of myocardial infarction and stroke
2.7.1 Neurologic Effects of Vitamin B12 Deficiency
Neurologic complications are found in 75%–90% of individuals with clinically appar-ent vitamin B12 deficiency. The presence of neurologic findings caused by vitamin B12
deficiency is negatively correlated with the degree of anaemia, which means that indi-viduals presenting severe anaemia showed fewer or no neurologic manifestations andvice versa [126][223]. In contrast to what was previously thought that neurologicalas well as the psychiatric complications only occur at a later stage of vitamin B12 de-ficiency, these disturbances can predate haematological anomolies by several monthsor even years or even occur in the absence of hematologic complications. The spinalcord, brain, optic nerves and peripheral nerves may all be affected by vitamin B12(cobalamin) deficiency, giving rise to one of the classic neurologic syndromes.
The neuro-psychological symptoms can be differentiated into subacute combined de-generation of spinal cord, encephalopathy (perniziosa psychosis) and polyneuropathies.
The spinal cord is usually affected first and often exclusively. The term subacutecombined degenereation (SCD) is customarily reserved for the spinal cord lesion of
19
vitamin B12 deficiency and serves to distinguish it from other types of spinal corddiseases that happen to involve the posterior and lateral columns (loosely referred toas combined system disease). Whether the peripheral neuropathy is a primary com-ponent of the disease or is secondary to damage of the fibers of entry in the spinalcord has been debated, but the available pathologic evidence favors the former. Thesymptoms are symmetric parestisias, acrodistal disturbances, sensory disturbances inthe extremities (paresthesia or numbness), motor disturbances alone, especially gaitataxia as well as elevated reflexes and spasticity. The nervous system involvementon subacute combined degeneration is roughly symmetrical and sensory disturbancesprecede the motor ones; predominantly motor involvement from the beginning and adefinite asymmetry of motor or sensory findings maintained over a period of weeksor months should always cast doubt on the diagnosis. Mental signs are said to befrequent, ranging from irriability, apathy, somnolence, suspiciousness and emotionalinstability to a marked confusional or depressive psychosis or intellectual deteriora-tion. The psychiatric symptoms are acute disorientation in combination with apathy,hallucinations, paranoia and frank psychosis, violent behavior and changes in person-ality are not common, but vitamin B12 deficiency should be considered as a possiblecause of these symptoms [117] [241][126] [223][273] . Anosmia, fecal and urinary in-continence, leg weakness, impaired manual dexterity, and impotence are less frequentsymptoms. Rare symptoms are orthostatic lightheadedness, diminished taste, paranoidpsychosis, and diminished visual acuity [126]. In 0.5% of cases, visual impairmentwas found, which might be related to optic atrophy and retrobulbar neuritis or pseudo-tumor cerebri [238].
2.7.2 Vitamin B12 and Cognitive Impairment
Cognitive decline may occur, ranging from concentration impairment to loss of mem-ory, disorientation, and frank dementia, including or not mood changes. Dementia in-creases at an remarkably rate and becoming a public health problem impacting healthcare expenditure. It is well known that age is a risk factor for dementia [42]; andthe prevalence increases with advancing age [37]. In Europe, prevalence of dementiaamong elders of 65+ is approximately around 10% [110]. Preservation of cognitiveability well into old age is important not only in terms of promoting adequate healthstatus but also regarding the postponement of the onset of dementia and decreasingits progress. Among the non-genetic and thus modifiable risk factors, increased ho-mocysteine levels in addition to poor folate, vitamin B12 and B6 status, all involvedin the control of homocysteine, have been found to correlate with impaired cognitiveperformance [212][259]. The prevalence of high total serum homocysteine dependson age and hyperhomocysteinemia is common in the elderly [113]. Poor status of
20
B vitamins (folate, vitamin B6 and vitamin B12) may play a role in pathogenesis ofcognitive impairment in the elderly via hyperhomocysteinemia. Both folate and vi-tamin B12 are needed for the methylation of homocysteine to methionine and in there-methylation and synthesis of S-adenosylmethionine, a major methyl donor in thecentral nervous system. Thus current research suggests that hyperhomocysteinemiacaused by an impaired monocarbon metabolism may contribute to cognitive impair-ment and Alzheimer’s disease [59] and can be considered a sensitive marker of cogni-tive impairment. On the other hand, raised plasma homocysteine concentrations are asensitive marker for cobalamin and folate deficiency. Findings suggest that decreasedvitamin B12-dependent transmethylation reactions might be involved in the pathogen-esis of dementia [243]. Hence, increased homocysteine levels in association with lowlevels of folate, vitamin B6 and vitamin B12, show significant correlation with de-creased performance on cognitive tests [212][259]. For these reasons, B vitamin sup-plementation has been shown to prevent or reverse cognitive decline. However, theeffect of vitamin B12 oral supplementation shows heterogeneous results and is foundto be related to the dose, route of administration and duration of treatment. A few trialshave been conducted to assess the effect of vitamin B12 supplementation on cognitivefunction in humans [142][84], with duration of supplementation varying from 4 wk to6 months. For most of the cognitive tests performed, no improvement was observed forvitamin B12. Only a few cognitive tests showed statistically significant improvement[142]. At least two studies are available in the literature showing statistically signifi-cant worsening of cognitive functions after vitamin B12 supplementation [142][84].
Cognitive impairment is multifactorial in origin sharing modifiable and non-modifiablerisk factors. As there are no curative treatments for it, the only possibility is that treat-ment may delay or slow down disease progression. It is highly appropriate to detectsuch modifiable risk factors like hyperhomocysteinemia, vitamin B12 and folate defi-ciency, etc. In the absence of curative treatment for dementia, vitamin B12 and folatemay be relevant to the clinical course of dementia and should be considered for thera-peutic intervention. However, more therapeutic research is needed. Clinical trials mustbe initiated in high risk populations to determine whether, lowering of blood homocys-teine levels by vitamin B supplementation reduces the risk of cognitive impairment, theclinical onset of dementia. Longitudinal studies should also be undertaken to explorethe association between nutrition status and cognitive impairment.
2.7.3 Haematologic Effects of Deficiency
Vitamin B12 deficiency leads inevitably to disturbances of vitamin B12 metabolismprocesses because of insufficient amounts of active coenzymes (adenosyl- and methyl-cobalamin) which again results in impaired transmethylation processes by what the
21
close-linked metabolic pathways of folate, methionine and purinenucleotides can beaffected (methyltrap-hypothesis). Main symptoms are specific morphologic changesin blood and bone marrow.
Erythropoiesis reacts quickly and sensitively to the blocked nucleic acid metabolismbecause of its high turnover rate. The disturbed DNS replication influences the nucleusmaduration, whereas the development of the cytoplasma nearly takes course normallyresulting in oversized forms (megalocytes or rather pre-stages of erythrocytes [mega-loblasts]) as well as the hyperchromic macrocytic megaloblastic anaemia (perniciousanaemia, Morbus Addison, Morbus Biermer). In the periphery more or less changedcells can be verified. The mean corpuscular volume (MCV > 110 fl; macrocytosis) andthe mean haemoglobin level of erythrocytes (MCH > 40 pg; hyperchromasy) are ele-vated. The megaloblastic changes of erythropoiesis in the bone marrow can be causedby cobalamin and folate deficiency. A morphologic differential diagnosis is not possi-ble. Granulopoiesis and thromboisis can be affected as well.
Megaloblastic anaemia can be rarely traced to inadequate dietary intake. Atrophy ofparietal cells of the stomach mucosa is common with a consequent achlorhydria. Auto-antibodies against the parietal cells and against the intrinsic factor (IF) as well as IFdeficiency due to a gastrectomy are the main causes for pernicious anaemia, the classsi-cal vitamin B12 deficiency disease. Serum cobalamin levels decrease under 200 pg/mlin the manifest state. The clinical symptoms of pernicious anaemia are skin palenessand mucosa, Hunter’s Glossitis, frailty, fatigue, lack of drive and vertigo.
2.7.4 Cardiovascular Effects of Deficiency
Cardiovascular diseases are a problem in the elderly and one of the most importantcauses for mortality. Among the risk factors, high levels of total cholesterol LDL-cholesterol and triglycerides and low levels of HDL-cholesterol, hypertension andmore recently high levels of homocysteine have to be mentioned. Regarding the lat-ter, studies have shown that elevated homocysteine is a risk factor for cardiovascular,cerebrovascular and peripheral arterial disease [141][30][17]. Wald et al. [258] foundthat for every 5-µmol/L increase in serum homocysteine concentration, the risk of is-chemic heart disease increased 20% to 30%. Among the causes of elevated plasmahomocysteine we can point out the deficiency of the B vitamins and renal impairment.
Several possible mechanisms for the association between homocysteine and atheroscle-rosis have been demonstrated in experimental models. These include stimulation ofsmooth muscle growth, reduction in endothelial cell growth, impaired endothelial cellrelaxation, decreased synthesis of high-density lipoprotein, promotion of autoimmuneresponse, and accumulation of inflammatory monocytes in atherosclerotic plaques
22
[17][271][268].
Hcy is considered as a risk factor but recently in the literature there has been a diver-gence of opinion. Studies were performed for investigating if the risk for cardiovascu-lar diseases onset can be reduced by lowering elevated homocysteine levels. Althoughthe studies suggest that higher B-vitamin intake correlates with less risk for vasculardisease and its sequelae, there is uncertainty as to whether it is folic acid, vitamin B6,or vitamin B12 that is responsible, and also whether supplements would provide thesame protective benefit as the presence of these nutrients in a varied diet.
Those studies, of primary and secondary prevention nature, have had mixed results.Some trials of homocysteine-lowering therapy taking folic acid or other B vitaminsshowed positive results whereas other randomized controlled trials of supplementationshowed no evidence. In addition, a recent Cochrane Database review of eight random-ized controlled trials in patients at low risk receiving B-complex vitamins did not find alower risk of myocardial infarction (fatal or nonfatal), stroke, or death from any causein patients [174]. However, most patients for these trials were recruited regardless ofhomocysteine levels; most of them had normal baseline plasma homocysteine levels.
2.7.5 Cognitive Impairment and the Function of the Neuromuscular System
The cognitive system plays an important role in helping to appropriately interpret theincoming sensations and plan the subsequent motor response. This system, whichcomprises the processes of attention, memory storage, and intelligence, provides uswith the collective ability to anticipate or adapt our actions in answer to changing taskdemands and the environment.
Age-related impairment of the sensory and motor systems negatively affects older peo-ple’s balance and mobility. The same effect was observed for changes in the cognitivesystem. In fact, as has been stated above at least 10% of all people over the age of65 and 50% of those older than 80 have some form of cognitive impairment, rangingfrom mild deficits to dementia. Adverse alterations of the processes of attention, mem-ory, and intelligence are most likely to affect in older people’s ability to anticipate andadapt to changes in the environment.
Any decline in cognition or attention will severely compromise one’s ability to ac-curately perceive what type of response is needed and implement the response, orresponses, once selected. Not only has it been well documented that older adultswith cognitive impairment showed an accelerated impairment of function followingan acute illness or hospitalization, but they also experience many more falls than theirnot-cognitively impaired peers.
23
Balance can be defined as the process by which one controls the body’s center of mass(COM) in terms of the base of support independently if it is stationary or moving.Although one often considers that standing upright in space is a static balance task andleaning through space or walking are dynamic balance tasks, it has to be rememberedthat keeping a stable upright position involves as well the active contraction of differentmuscle groups to control the position of the COM against the destabilizing energy ofgravity. There is evidence to indicate that the ability to maintain balance easily, nomatter whether static or dynamic depends upon the function of the mechanisms inthe semicircular canals; the kinesthetic sensations in the muscles, tendons, and joints;visual perception while the body is in motion; and the ability to coordinate these threesources of stimuli [150]. Balance is an important ability which one needs during dailyactivities (i.e. walking and standing, as well as in most games and sports).
Multiple systems contribute to our ability to maintain balance in standing and mov-ing environments. First, the various sensory systems (i.e. vision, somatosensory andvestibular) give one information arising from the surrounding environment and result-ing from one’s own actions. This information is of special interest for successful goal-directed action planning as well as the subconscious or automatic adjustments neededto maintain a given position in space or respond rapidly to a change in task or environ-mental demands.
Between the ages of 50 and 70 muscle strength has been shown to decline by around30% with even higher declines experienced after age 80. This decline is thought tobe largely the consequence of a decreased size and number of muscle fibers. Physicalinactivity also contributes to the loss of muscle strength, particularly in antigravity orpostural muscles required for good upright posture. Posture refers to the biomechanicalalignment of the individual body parts as well as the orientation of the body to theenvironment.
A good posture is crucial for good balance. When one is standing quietly in space,one’s goal is to align each body part vertically and thereby to spend the least amount ofinternal energy needed to maintain an upright and stable position relative to gravity. Tocounteract the energy of gravity, a number of muscles are active during quiet standing.
Anticipatory postural control refers to those actions that can be planned beforehand,whereas reactive postural control referes to those situations that cannot be planned inadvance of actions being required. Reactive postural control is needed when we haveto respond quickly to an event we did not expect.
Mobility is the ability to transfer oneself independently and safely from one place toanother. Adequate levels of mobility are required for many different types of activitieswe perform in our daily lives. These may include transfers, e.g. rising from a chair,
24
climbing or descending stairs, walking or running and other types of recreational ac-tivities (e.g. gardening, sport, dancing). As already mentioned in chapter 1, impairedmobility is another major concern for the elderly and to keep their independence andto perform ADL sufficient strength in hands, arms and legs must retained. The pro-grammes to improve mobility are commonly built around measuring and addressingspecific impairments, such as balance capacity and the reduced strength of arms andlegs. Data from our research group have found how strength values and MMSE werepositively associated with hand grip strength, and arm and leg strength (p < .01) (inreview in Ageing and Mental Health). Structural and functional changes that occurwithin the Central Nervous System (CNS) with advancing age appear to have the mostprofound and observable effect on motor function as a whole. And at a behaviourallevel, these cumulative changes as a consequence of the ageing nervous system [thatcan be aggravated as a consequence of vitamin B12 deficits] appear to manifest them-selves in a reduced ability to perform a variety of complex movements that requirespeed and accuracy, balance, strength, or coordination and are necessary to maintainindependence and to be able to perform utilitarian or basic ADLs.
Handgrip strength is recorded by clinicians as a quick and viable measure of handimpairment and function that provides a useful evaluation of the hand status [69]. Poorhand strength as measured by handgrip is a predictor of disability in older people. Thehandgrip strength test was highly predictive of functional limitations and disability 25years later among 45 to 68y old men concluding that good muscle strength in midlifemay protect people from old age disability by providing a greater safety margin abovethe disability threshold [205][180].
When multiple systems become impaired, the quality of the interaction between the im-paired systems declines and results in observable motor dysfunction. Adverse changesoccurring in the visual system make it more difficult to use visual information for bal-ance, information from the somatosensory and vestibular systems could be used tocompensate for the impaired visual system in most situations this would not be thecase when these systems are also impaired as in the case of a general nervous systemdeterioration.
2.8 Laboratory Diagnosis of Vitamin B Deficiency
Most people with suspected vitamin B12 deficiency do not present anaemia as the clas-sical sign, but do show vague non specific symptoms such as neurologic or intestinalcomplaints. That´s why several laboratory tests should be considered.
25
Tabl
e3:
Ove
rvie
wof
diff
eren
tcut
-off
sus
edto
defin
evi
tam
inB
12an
dfo
late
defic
ienc
yin
sele
cted
Stud
ies,
sort
edby
vita
min
B12
nA
geB
12bc
Hol
oTC
bcM
MA
bctH
cybc
sFol
ate
bcR
BC
fola
tebc
No.
Ref
eren
ces
City
(Cou
ntry
)a(a
ll;m
,f)
(y)
(pm
ol/L
)(p
mol
/L)
(µm
ol/L
)(µ
mol
/L)
(nm
ol/L
)(n
mol
/L)
1Jo
oste
net
al.1
993
[154
](B
E,D
E,N
L)
350;
135,
215
61-9
7d
103
0.24
713
.95.
42
Nau
rath
etal
.199
5[1
87]
13ce
ntre
s(B
E)
285;
105,
180
65-9
6d
103
0.24
713
.95.
43
Bjo
rkeg
ren
and
Svar
dsud
d20
01[2
5]Sk
utsk
är(S
E)
224;
94,1
3078
(77.
2-7
8.9)
e11
50.
3715
3.7
275
4R
ayet
al.2
000
[206
]To
ront
o(C
A)
711;
403,
308
58f
120
1521
55
Gor
inge
etal
.200
6[1
12]
Car
diff
(GB
)49
<75
125
380.
4715
6B
oret
al.2
006
[32]
Cop
enha
gen
(DK
)98
;0,9
841
-75
d13
050
0.28
11.9
7H
errm
ann
etal
.200
0[1
31]
Saar
land
(DE
)23
2;79
,153
69(1
9-1
02)g
132
0.27
115
6.4
8L
loyd
-Wri
ghte
tal.
2003
[166
]O
xfor
d(G
B)
195;
195,
044
(18
-78)
g13
550
0.28
126.
8L
ondo
n(G
B)
9C
arm
elet
al.1
999
[41]
Los
Ang
eles
(US)
591;
345,
246
>60
140
0.37
17.1
(m)
5.7
16.8
(f)
10Fl
icke
reta
l.20
06[9
8]Pe
rth
(AU
)29
9≥
7514
015
11Fi
glin
etal
.200
3[9
6]Te
l-H
asho
mer
(IS)
1271
147
0.24
1511
12A
ndre
set
al.2
006
[10]
Stra
sbou
rg(F
R)
201;
57,1
4467
±6
h14
813
6.8
13vo
nC
aste
l-D
unw
oody
etal
.200
5[2
57]
Flor
ida
(US)
359;
0,35
920
-30
d14
835
14L
ewer
inet
al.2
003
[163
]G
othe
nbur
g(S
E)
209
76(7
0-9
3)i
148
0.34
168.
615
Mill
eret
al.2
006
[183
]Sa
cram
ento
(US)
1789
;751
,103
8>
6014
835
0.35
136.
836
3
16M
orri
set
al.2
002
[185
](U
S)11
45≥
6514
80.
37
17So
laet
al.2
006
[111
]G
rana
da(E
S)21
8;82
,136
79.2
(60
-105
)g14
845
0.3
156
397
18So
lom
on20
05[2
36]
New
Hea
ven
(US)
304
28-8
2d
148
0.37
613
.619
Fene
chet
al.1
997
[91]
Ade
laid
e(A
U)
64;6
4,0
50-7
0d
150
106.
831
720
Loi
kas
etal
.200
3[1
68]
Lie
to(F
I)33
3≥
6515
037
0.45
1921
Nils
son
etal
.200
4[1
91]
Lun
d(S
E)
209;
91,1
1873
±11
h15
040
0.41
19.9
722
Ref
sum
etal
.200
1[2
08]
Puna
(IN
D)
205;
169,
3527
-55
d15
035
0.26
155
23R
iggs
etal
.199
6[2
12]
Bos
ton
(US)
70;7
0,0
54-8
1d
150
146.
824
Smul
ders
etal
.200
6[2
34]
Am
ster
dam
(NL
)1;
1,0
5115
00.
3518
6.5
25G
ambl
eet
al.2
005
[105
]A
raih
azar
(BD
)16
50;6
77,9
7320
-65
d15
111
.4(m
)9
10.4
(f)
26H
errm
ann
etal
.200
3[1
30]
Saar
land
(DE
)54
5;25
1,29
457
(18
-92)
g15
635
0.27
112
727
Hir
sch
etal
.200
2[1
37]
Sant
iago
(CL
)10
8;43
,65
74.4
±3.
7h
165
146.
8
26
Tabl
e4:
(Con
tinua
tion)
Ove
rvie
wof
diff
eren
tcut
-off
sus
edto
defin
evi
tam
inB
12an
dfo
late
defic
ienc
yin
sele
cted
Stud
ies,
sort
edby
vita
min
B12
nA
geB
12bc
Hol
oTC
bcM
MA
bctH
cybc
sFol
ate
bcR
BC
fola
tebc
No.
Ref
eren
ces
City
(Cou
ntry
)a(a
ll;m
,f)
(y)
(pm
ol/L
)(p
mol
/L)
(µm
ol/L
)(µ
mol
/L)
(nm
ol/L
)(n
mol
/L)
28H
olle
land
etal
.199
9[1
38]
Osl
o(N
O)
224
18-9
0d
170
0.37
615
5
29C
hen
etal
.200
5[4
5](U
S)an
d(E
S)62
56f
180
300.
279
14.9
(m)
14.5
(f)
30St
otte
tal.
2005
[240
]L
eide
n(N
L)
185;
88,9
7≥
6518
412
4G
lasg
ow(G
B)
31Fl
ood
etal
.200
6[9
9]Sy
dney
(AU
)29
63≥
5018
515
6.8
32O
beid
etal
.200
4[1
94]
Hom
burg
(DE
)22
8;72
,156
>65
196
290.
2814
.111
.1
33C
lark
eet
al.2
003
[57]
Oxf
ord
City
(GB
)15
02;6
18,8
84≥
6520
00.
3515
34C
lark
eet
al.2
004
[56]
(GB
)35
11≥
6520
020
35H
vas
and
Nex
o20
05[1
44]
Aar
hus
(DK
)93
7;34
9,58
872
(19
-102
)i20
040
0.28
11.9
350
36H
vas
and
Nex
o20
03[1
43]
Aar
hus
(DK
)14
3;51
,92
72(2
4-9
0)i
200
500.
2915
350
37L
indg
ren
etal
.199
9[1
65]
Got
henb
urg
(SE
)10
1;35
,66
52(1
8-8
0)i
200
350.
413
38N
exo
etal
.200
2[1
89]
Hau
kela
nd(N
O)
90;6
9,21
38-8
0d
200
400.
2739
Ref
sum
and
Smith
2003
[207
]O
xfor
d(G
B)
116;
43,7
373
f20
040
0.3
1415
40K
oehl
eret
al.1
996
[157
]A
lbuq
uerq
ue(U
S)10
0;50
,50
68-9
6d
221
0.27
116
.25
41Pe
nnyp
acke
reta
l.19
92[1
99]
Den
ver(
US)
152
65-9
9d
221
0.37
621
.642
Raj
anet
al.2
002
[203
]Se
attle
(US)
315;
203,
112
65-1
00d
221
0.27
113
.97.
343
John
son
etal
.200
3[1
51]
Geo
rgia
(US)
103;
21,8
276
±8
h(6
0-9
5)d
258
0.27
113
.96.
829
544
Lin
denb
aum
etal
.199
4[1
64]
New
Yor
k(U
S)54
8;20
0,34
877
.5(6
7-9
6)i
258
0.37
621
.311
.3
45R
ober
tson
etal
.200
5[2
19]
Lon
don
(GB
)42
1;21
5,20
666
(37
-90)
g25
80.
271
1446
Stab
lere
tal.
1999
[237
]B
altim
ore
(US)
762;
0,76
2≥
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27
2.8.1 Indicators of Haematologic Status
Hemoglobin, hematocrit, red blood cell count, and mean corpuscular volume [122] areall suitable measurements. However, the 120-day red blood cell survival time responsetime explains the slow response time of these indices. Therefore, affirming solely withthese indices the diagnosis for vitamin B12 deficiency is not sufficient to identify asubclinical vitamin B12 deficiency. Hypersegmented neutrophils occur before macro-cytosis [246]; however, the sensitivity of this finding has recently been questioned [40].
2.8.2 Serum or Plasma Cobalamin Levels
Concentrations of serum or plasma cobalamin are suitable initial diagnostic tests forvitamin B12 deficiency because they are widely available, cheap and because of thefamiliarity with the test due to its time on the market. Serum and plasma cobalamindetermine both the fraction available for the cells that is linked to transcobalamin (20-30%) and the fraction that is linked to haptocorrin (70-80%). Currently and commonlyused assays for measuring serum or plasma cobalamin concentrations are run on au-tomatic analyzers and based on the competition between labelled vitamin B12 andcobalamin of the sample for binding to IF. The design of the assay and consequentlythe interval of reference vary depending on the manufacturer. Therefore, it is advisableto use the reference interval from the manufacturer. Decreased values considered asvalues well below the lower level of the interval of reference most often indicate vita-min B12 deficiency except for pregnant women and patients with decreased haptorrinlevels.
2.8.3 Holotranscobalamin (HoloTC, Active B12)
Holotranscobalamin refers to the fraction of cobalamin linked to transcobalamin andthereby the part of the circulating cobalamin that is responsible for receptor-mediateduptake of vitamin B12 into cells. It is synthesized by the liver and binds only a smallfraction of plasma vitamin B12 (7%–20%) to form the transcobalamin-vitamin B12complex. Two different types for determining holoTC are commonly used, one thatmeasures holoTC by a transcobalamin specific ELISA after removal of unsaturatedtranscobalamin [189] and one that directly measures transcobalamin saturated withcobalamin [34]. Only the latter method is available on an automatic platform. From atheoretical point of view holoTC (active B12) should be the first marker to show alter-ations in vitamin B12 deficiency at early stages. So far methods for measurement ofholoTC are available only on one automated analyzer consequently the clinical expe-rience from the use of holoTC measurements in routine clinical use is scarce.
28
2.8.4 Serum Methylmalonic Acid
Methylmalonic acid (MMA) values rise when vitamin B12 is subadequate and tend torise in the elderly [153]. The metabolite is currently considered as the most specificmarker of vitamin B12 deficiency [117]. The metabolite is measured via chromato-graphic methods such as GC-MS, but till now, there is no method available for analzingit on an automatic system. Consequently, analysis of MMA is not widely accessibleand is costly. The interpretation of MMA in serum has to take into consideration thatMMA is correlated to kidney function and thereby to serum creatinine. The upper limitfor the reference interval has been indicated as values between 0.28 and 0.55 umol/I.The uncertainty concerning the exact value does not relate to methodological issuesbut rather to the reference populations included for the establishment of the interval.Borderline elevations in serum methylmalonic acid levels will not respond to cobal-amin therapy in the presence of renal failure [184], although Lindenbaum et al. [164]reported that moderate renal dysfunction in the absence of renal failure did not affectmethylmalonic acid values as strongly as did inadequate vitamin B12 status.
2.8.5 Serum Homocysteine Concentration
Serum homocysteine levels show a strong inverse association with folate plasma lev-els, but there is also an inverse association (albeit weaker) with vitamin B12 and B6plasma levels. Inadequate plasma concentrations of one or more of the above three Bvitamins appear to account for 67% of cases of high homocysteine levels (more than 14pmol/liter) in an elderly population. Because hyperhomocysteinemia is also observedin renal insufficiency or hypovolemia, serum creatinine is useful for interpretation.Because elevated serum homocysteine concentrations are not specific for vitamin B12deficiency, it is of limited usefulness for evaluation of vitamin B12 status [117].
2.9 Oral Supplements as Alternatives to Conventional Vitamin B12 Treatment
It is indifferent for the intracellular metabolism in which form cobalamin is offeredto the cells (cyano-, hydroxy-, or mehthyl cobalamin, because the ligands are splitoff during the liberation from the lysosomes and the reduction process). Preformedmethylcobalamin as well has to be remethylated after liberation from the lysosomesand reduced to Cbl2+ [50] [158]. Therefore, metyhlcobalamin does not have the edgeover hydroxy- or cyanocobalamin. Due to the small resorption of cyanocobalamin ofbetween 1 and 3%, parenteral administration is usually preferred. However, an oraltherapy can be worthwile for some defined patient groups with hypersensitivity forintramuscular administration and with elevated bleeding tendency (haemophilia, oralanticoagulants) that showed good compliance.
29
Adverse reactions
Pure oral as well as parenteral vitamin B12 is also in high dosage highly compatible andpracticallly without adverse reactions. Although, cobalamin as a drug has been on themarket for more than 50 years and as is regularly prescribed in high amounts, adversereactions are described only in a few single cases. The mayoritiy of adverse reactionnotations (mainly allergies) are attributed to parenteral induced sensibilisation, whichprobably is not caused by the molecule itself, but most likely is caused by associatedmaterial (trace elements of the biosynthesis, culture media, streptomycin, liver extract)or rather by additives (preservatives). After parenteral administration single cases ofacne, as well as anaphylactic reactions were also described which are most likely alsoattributable to galenic dependent substances
30
HYPOTHESES AND OBJECTIVES
3 HYPOTHESES AND OBJECTIVES
The hypotheses and objectives were classified according to the three Studies of theThesis.
3.1 Hypotheses of Study I
The main experimental hypothesis of this study was that a period of one year would besufficient to show a worsening of the vitamin, haematological and lipid profile as wellas of the functional and cognitive status of institutionalized elderly.
A secondary experimental hypothesis was that the known association among severalparameters found in younger subjects would be confirmed in this population group.
To test these hypotheses, null hypotheses were established for the statistical analyses.
3.2 Objectives of Study I
In this context, in order to contrast these hypotheses, the following objectives werespecified:
1. To analyze the evolution of vitamin B profile, selected haematological parameters,lipid profile, selected anthropometric measurements, functional and cognitive statusover one year in institutionalized elderly
2. To quantify the prevalence of health-related risk factors over one year in institution-alized elderly
3. To study the relationship between cobalamin status and cognitive and functionalcondition in institutionalized elderly
4. To study the diagnostic efficacy of holo-transcobalamin compared to other biomark-ers regarding vitamin B12 status
3.3 Hypotheses of Study II
The experimental hypothesis was that an intervention with an oral supplement (pro-viding 500 µg cyanocabalamin) for 28 days would be effective to improve vitamin Bstatus in institutionalized elderly.
The statistical hypothesis was formulated as a null hypothesis that there were not anydifferences between pre and post supplementation values.
33
3.4 Objectives of Study II
In order to test this hypothesis the following objectives were specified:
5. To analyze the effect of 500 µg oral cyanocobalamin during 28 days on vitamin B12status and related parameters in institutionalized elderly
6. To investigate the effect of the intervention on holo-transcobalamin as a marker ofvitamin B12 status
3.5 Hypotheses of Study III
The main experimental hypothesis was that the medication intake affects values ofvitamin B status and homocysteine levels.
The null hypothesis was that there were not statistical differences in the percentageof people with adequate or low (in the case of homocysteine elevated) values and thepercentage of people taking or not-taking certain groups of medication.
3.6 Objectives of Study III
To contrast this hypothesis the following objectives were established:
7. To analyze if drug intake affects vitamin B12, folate and homocysteine status ininstitutionalized elderly
8. To assess the number and type of medications which are taken routinely by Spanishinstitutionalized elderly
34
METHODOLOGY
4 METHODOLOGY
For the present thesis three different studies were performed in four old people’s homesin the Region of Madrid. The first one (I) was a longitudinal descriptive study, the sec-ond one (II) was an intervention study with an oral supplement of 500 µg cyanocoabal-amin, and the last one (III) was a descriptive study about drug use and its associationwith vitamin B status.
Selection of the old people’s home
Selection of the old people’s homes was not random. With the support of the “Aso-ciación de Familiares de Alzheimer (AFAL, Spain)”, we got in contact with twelve oldpeople’s homes, both private and public, in the Region of Madrid. In order to reducethe influence of confounders, the minimal number of residences was aimed for, to se-lect the desired number of subjects. After visiting all of them, explaining the aims ofthe project and asking for their willingness to participate, finally four were selectedto be included in the studies. Inclusion criteria for the old people’s homes were: geo-graphical distribution, urban/rural area, private or public ownership, and willingness ofthe medical staff to support the study in situ. From the four residences finally selected,one was of private ownership and the other three of public ownership, all four coveringdifferent metropolitan areas of Madrid.
Sample size
For both the intervention and the longitudinal study, the sample size was calculatedto be 60, on the basis of the parameter with most variance, which for these studieswas holotranscobalamin. In the case of the longitudinal study, at least 20 additionalsubjects were recruited bearing in mind the possible experimental mortality, in orderto guarantee the final number.
Participants
From the selected residences, volunteers of both sexes were recruited to participatein one of the studies. Inclusion criteria were to be older than 60 years, not clinicallydiagnosed with dementia and having signed the informed written consent. Exclusioncriteria were vitamin B12 and folate supplement intake, neuropathy and Hunter’s glos-sitis. The concomitant intake of medication related to affect Cbl absorption (such asmetformin, H2-antagonists, and proton pump inhibitors) was not considered as an ex-clusion criterion if the medication had been started at least three months before thestudy and was taken continuously by the study participant for the whole duration ofthe intervention period.
37
4.1 Study Designs
4.1.1 Study I (Longitudinal)
The study design was a prospective longitudinal study (Figure 5). The study popula-tion was visited four times, the first one to establish reference baseline data and threecontrols at four months intervals over a one year period. The information gatheredduring data collection was identical at each examination point.
Figure 5: Study design for longitudinal study and distribution of drop outs in the course of follow-up
4.1.2 Study II (Intervention)
The study design was an intervention study with pre- post comparisons (Figure 6) .The study population was examined before and after the treatment period of 28 days.
Figure 6: Study design for intervention study
4.1.3 Study III (Polypharmacy)
The study design was a descriptive cross-sectional study with a posteriori comparisons.
38
4.2 Ethical Aspects
All three studies were performed in the framework of a broader study dealing withthe early diagnosis and treatment of vitamin B12 deficiency in Spanish elderly. Thestudy proposal was sent to the Human Research Review Committee of the Universityof Granada for its approval and was checked for respecting people’s rights and dig-nity. The Board reviewed the proposals to ensure that ethical issues would be handledcorrectly and that the study procedures met all the legal requirements necessary to con-duct research and approved the proposal. The study was performed in accordance withthe Helsinki Declaration of 1964 and later amendments, the last one revised in 2000in Edinburgh, with the Convention on Human Rights and Biomedicine of Oviedo in1997. A written informed consent was obtained from all study participants, familyrepresentatives or guardian if participants were unable to provide consent.
4.3 Materials and Equipment
4.3.1 Equipment
The materials and equipment used to collect the data and analysis are shown in Table5. Medication profile forms (for chronic as well as acute diseases) were extracted fromthe local data management systems.
4.3.2 Procedures
Phlebotomy and Blood Distribution
4.3.2.1 Blood analyses Blood was collected following established venipuncture pro-tocol and procedure from the participants after an overnight fast in situ at the old peo-ple’s homes. Blood specimens were collected into Vacutainer Tubes containing EDTAas anticoagulants or gel for serum. The EDTA tubes were used for whole blood countand red blood cell folate (RBC folate) and the gel tubes were immediately placed onice. After blood clotting, they were centrifuged at 3000 g for 15 min, aliquoted andfrozen at -86° C until analysis in the biochemical laboratory of the Faculty for SportSciences, UPM, Madrid (Registered Lab number 242, from Madrid Community labo-ratory network [Red de Laboratorios de la Comunidad de Madrid]).
39
Tabl
e5:
Equ
ipm
entu
sed
fora
naly
ses
Type
ofan
alys
esE
quip
emen
t/M
ater
ials
Man
ufac
ture
r/B
atte
ryFo
rth
ebl
ood
anal
yses
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lebo
tom
yV
acut
aine
r®Sy
stem
Bec
ton,
Dic
kins
onan
dC
ompa
ny,F
rank
lin20
Lak
es,N
JV
itam
inB
12st
atus
Aut
oana
lyze
rAxS
YM
Abb
ottP
ark,
Illin
ois,
USA
)B
ioch
emic
alpr
ofile
Hita
chi9
12R
oche
Dia
gnos
tics,
Man
nhei
m,G
erm
any
Com
plet
eB
lood
Cou
ndB
eckm
anC
oulte
rMax
M/H
MX
IZA
SA,M
adri
d,Sp
ain
For
the
anth
ropo
met
ric
mea
sure
men
ts:
Wei
ght
Wei
ghin
gsc
ale
Seca
709
Seca
Ham
burg
,Ger
man
yK
nee
Hei
ght
Ant
hrop
omet
erG
PM,S
witz
erla
ndSk
info
ldth
ickn
ess
Skin
fold
calip
erH
olta
inL
td.,
Cry
myc
h,U
KG
irth
sA
nthr
opom
etri
cta
peFo
rco
gniti
vest
atus
cogn
itive
para
met
ers
MM
SEqu
estio
nnai
re™
Offi
cial
tran
slat
ion
toSp
anis
hof
PAR
Inc.
Lut
z,Fl
orid
a,U
SAW
rist
wat
chPe
ncil
orba
llpen
Som
ebl
ank
pape
rPi
ece
ofpa
perw
ithC
LO
SEY
OU
RE
YE
Sw
ritte
nin
larg
ele
tters
For
the
func
tiona
ltes
ts:
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dgr
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reng
thD
igita
lhan
dgr
ipdy
nam
omet
erTa
keiT
KK
5101
Toky
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pan
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dyst
reng
thD
umbb
ells
(2kg
forw
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and
4kg
form
en),
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wat
chR
ikli
Jone
sL
ower
body
stre
ngth
Stan
dard
ized
chai
r(H
eigh
t=43
.18
cm),
Stop
wat
chR
ikli
Jone
sSt
atic
bala
nce
Stop
wat
chE
urofi
t
40
Vitamin B12 Status Serum cobalamin: Total sCbl concentrations were determinedby a Microparticle Enzyme Immunoassay (MEIA; Abbott AxSYM, Abbott Park, USA,total CV ≤ 11%) [64] [266].
Serum holoTC: Serum holoTC was analysed by a two-step sandwich MEIA (AbbottAxSYM, total CV ≤ 10%) [248] [197][34].
Serum folate (sFolate): Serum and RBC folate were measured by an ion-captureimmunoassay (ICIA; Abbott AxSYM, Abbott Park, USA, total CV ≤ 19 %) [233].RBC folate was determined in whole blood from EDTA-coated tubes.
Serum tHcy: Serum tHcy was determined by a fluorescence polarization immunoas-say (FPIA; Abbott AxSYM, Abbott Park, USA, total CV ≤ 6%) [200].
The samples for sCbl, holoTC, sFolate, RBC folate, and tHcy of each subject weremeasured within one run to avoid between-run variation.
For project II, the samples for sCbl, holoTC, sFolate, RBC folate, and tHcy of eachparticipant were measured within one run to avoid between-run variation.
Haematological Parameters Complete blood count (CBC) was performed withinfour hours after extraction and was obtained by an automated haematology analyser(Beckman Coulter MaxM, IZASA, Madrid, Spain). The CVs for the analyses were thefollowing: haemoglobin (Hb, CV≤ 1.5%), haematocrit (HCT), mean corpuscular vol-ume (MCV, CV ≤ 2.0%). The analyses were conducted by a local clinical laboratory(CLIMESA, Gabinete Medico Conde-Duque, Madrid, Spain).
Biochemical Status Basic clinical chemistry variables including lipid profile and cre-atinine, were obtained by standard spectrophotometric assays on a Hitachi 912 (RocheDiagnostics, Mannheim, Germany) and were conducted by a local clinical laboratory(CLIMESA, Gabinete Medico Conde-Duque, Madrid Spain).
4.3.2.2 Anthropometric Measurements Weight: The participant stood in light cloth-ing in the center of a scale platform. Body weight was recorded to the nearest tenth ofa kilogram.
Height Estimated from Knee Height
To estimate height, knee-heel height was measured to the nearest 1 mm using a GPMspreading anthropometer (Siber-Hegner GPM, Switzerland, range 0-2100mm) at theleft side in a sitting position, lower legs relaxed, with the knee flexed at a 90° angle.Standing height was calculated by the equations proposed by Chumlea and Guo [53].
41
Table 6: Analysed variables and methods used
Variable Index measured Sample/MethodB12 vitamin B12 Serum
total cholesterol SerumLDL-cholesterol SerumHDL-cholesterol Serum
Body composition Height Estimation from Knee HeightSF-thickness Anthropometry (SF caliper)
Functional tests Hand grip strength DynamometryStrength of arm flexors Arm curl testsLeg strength 30-Second Chair StandBalance Stork Stand
Cognitive status MMSE Folstein Questionnaire
Girths
Girths were measured using a anthropometric tape of inextensible material and weremeasured in the following anatomical locations: waist (obvious narrowing between therib and the iliac crest), hip (horizontal level of greatest gluteal protuberance).
Skinfolds
Skinfold measurements were measured with a Holtain skinfold caliper (Crosswell,Crymych, UK, pressure 10 g/mm²). The laboratory manual of the International So-ciety for Advancement of Kinanthropometry (ISAK) served as a compendium for ex-amination but the left side has been chosen following the recommendations by theInternational Biological Programs [176] [134]. All examiners were previously trained.Anatomical landmarks were marked for reference. For each location three measure-ments were taken and their means were used as representative values.
The skinfold measurements were asssessed in the following anatomical locations:
- Biceps (vertical fold, anterior aspect of the arm over the belly of the biceps muscle,1 cm above the level used to mark the triceps site, arm held freely to the side of thebody)
- Triceps (vertical fold, posterior midline of the upper arm halfway between theacromion (shoulder) and olecranon (elbow), arm held freely to the side of the body)
In the above mentioned anatomical locations, the skinfolds were taken avoiding in-cluding muscle tissue. The hand pressure was maintained constant when measuring,and the reading was taken after two or three seconds after having situated the caliper.For greater precision, the whole measurement was repeated three times [60].
42
4.3.2.3 Cognitive Status Cognitive functioning was assessed using the official trans-lation of the Mini Mental State Examination™ (MMSE) [101] [Psychological Assess-ment Resources (PAR), Inc., Lutz, FL]. MMSE questionnaire was administered in apersonal interview by an expert in a quite room. The data and final score were recordedon the working sheet (see Appendix C). After having got the patient’s confidence withsome introductory ice-breaking questions, each question was read as it was exactlywritten. If the patient had not understood acoustically, the question was repeated up toa maximum of three times without any additional explaination. During the interview,any hints for the patient were forbidden as well as any indication that they answeredincorrectly or to correct the patient if they had answered incorrectly.
The seven categories, representing different cognitive domains were scored as follows:
1. Orientation to time (5 points)
2. Orientation to place (5 points)
3. Registration of three words (3 points)
Names of three unrelated objects were presented clearly and slowly, about one secondfor each name. After all three names were said, the patient was asked to repeat them.
Scoring: The first repetition determines scoring (0-3) but the words can be repeateduntil the patient can repeat all three, up to six trials. If all three are not eventuallylearned, recall cannot be meaningfully tested but should nevertheless be attempted.
4. Attention and calculation (5 points)
5. Recall of three words (3 points)
6. Language (8 points: name items (2 points); repeat (1 point); understanding (3points); reading (1 point); and writing (1 point).
7. Visual construction (1 point)
The patient had to draw two five-sided figures intersected by a four-sided figure.
For total scoring, the points obtained for each item were summed and recorded on thelast page of the questionnaire. If the patient did not respond to the items, 0 wasscored. The maximum possible score was 30.
4.3.2.4 Functional Tests Strength was evaluated by the following tests.
Handgrip strength: It was assessed with a handgrip dynamometer (Takei TKK 5101,Tokyo, Japan, range 5-100 kg, precision .1 kg). Due to the disability of some of theparticipants to maintain the upright position without any help, the handgrip strengthwas performed in a sitting position performing the test with the elbow in extension and
43
in a vertical position. Two trials were recorded on both hands with at least one minutebreak between them and the average value was included in the statistical analysis.
Body strength
Body strength was assessed by two items from the Senior Fitness Test (SFT) of Rikliand Jones [215]: the upper limb (arm curl) and the lower limb (30-Second ChairStand).
Upper-body strength [215]The strength of the upper extremities was assessed by the “arm curl” test. The partici-pant sat in a an upright position on a chair of standard size (43.18 cm seat height) withfeet flat on the floor holding a dumbbell in his/her hand down at the side, perpendicularto the floor, in a handshake grip. Instead of the 5 pounds (2.27 kg) of load for womenand 8 pounds (3.63 kg) of load for men suggested in the battery, the load were ad-justed to “kg”, the International System (SI) unit routinely used in Spain, resulting inan adaption to the load of 2 kg for women and 4 kg for men. The participants were in-structed to perform the biceps curls with his/her dominant arm and had 30 seconds forperforming the greatest possible number of repetitions. The palm had to rotate up dur-ing the curl-up phase and then should return to a handshake position at extension. Theupper arm had to remain still throughout the test. The participants got the instructionsand made an attempt to see if the execution was correct. Participants were encouragedverbally to improve their motivation. The test was repeated twice with the dominantarm, leaving a break of about 3 min for recuperation between each performance. Theaverage of both trials was calculated and included in the statistical analyses.
Lower body strength [215]
The strength of the lower extremities was assessed by the “30-Second Chair Stand”.Participants who were able to perform the test sat on a straight back chair (43.18 cmseat height) with a solid seat and feet flat on the floor and arms across chest; then theywere asked to rise to a full stand and to sit down again to a fully seated position. Theparticipants got the instructions and performed one complete extension and flexion inorder to check if the execution was correct. The participants repeated the action athis/her own tempo for 30 s. The final test score were the number of times that theparticipant got to a full stand from the seated position with arms folded within 30 s.This test was only performed once.
Static balance (Stork Stand)
Static balance was evaluated by the stork stand test on the foot of the dominant leg.The participant stood on the dominant leg with opened eyes, and placed the other footon the inside of the supporting knee and placing the hands on the hips. Upon a givensignal, the participant tried to maintain balance as long as possible without placing the
44
non supporting foot on the floor or touching it. The final score was the greatest numberof seconds during which the participant kept balance on three trials. Only the highestscore was recorded. Participants who failed at the first attempt were retested.
4.4 Delimitations of the Studies
Common for all the three Studies:
The samples chosen were limited to Spanish elderly volunteers aged over 60 residingin old people’s homes in the Madrid Autonomous Community.
Owing to the impossibility to control the medication taken by the participants and itsspecificity drug intake was recorded.
The diet given in the old people’s homes was kept as usual.
Serum vitamin B12, serum folate and RBC folate, holoTC, the metabolite serum ho-mocysteine, full blood count and biochemical parameters were analyzed with the sameequipment which is considered of high precision.
Functional evaluation was assessed by hand grip strength and additionally by the “ArmCurl Test” of the Rikli Jones battery and the “30-Second Chair Stand” of the SFTbattery and static balance was assessed by the Stork Stand test of the Eurofit battery.
All tests were analyzed with the same equipment which is considered of high precision.Staff was trained beforehand to perform the tests.
Specific to the Studies:
In study I, the analyzed parameters were examined at regular four months intervalsfor determining the evolution and avoiding possible variations due to seasons as apossible time varying covariate. The data used for statistical analyses were the dataof those participants for whom data of the key outcomes were complete for all fourexamination points. No significant differences were found between the means of thestudied key outcomes of the persons whose data were complete for all four examinationpoints and the participants lost during follow-up.
In study II, the treatment of the intervention was set at 500 µg cyanocobalamin givenas an oral daily supplement. The analysis of the effectiveness of the intervention wasassessed by means of a factorial pre post test in which the participants were their owncontrol.
In study III, the medication intake was recorded from the local data management sys-tem. The medication was classified according to the Anatomical Therapeutic Chemical(ATC) Classification System. The statistic analysis of the possible effects of the med-ication intake on the status of vitamin B was based on the known and unknown effectof the medication type.
45
4.5 Justification of the Chosen Tests
Height Estimated from Knee Height
Accurate and valid measurements of standing height are particularly difficult to ob-tain from elderly people because of the difficulty for most of them to stand erect andof bowing of the legs [54] [253] [26]. Suffering chronic diseases, such as osteoporo-sis, arthritis and Parkinson-like disorders, which influence the neuromuscular system,aggravate this problem. Body height decreases in the course of senescence [16] [72][140] [161] [68] due to pathologic changes in the spine including increased kyphosis(curvature of the spine), collapsed disks and vertebral bodies (compression of vertrebaldisc space) [52].
Body Mass Index (BMI)
Body mass index is a commonly used measurement to estimate obesity. In 1977, theWHO proposed a classification system for overweight and obesity and this classifica-tion system has been utilized all around the world since the year 2000. Overweight isdefined as values between 25.0 and 29.9 and obesity for values over 30. Overweightand obesity are associated with an overall death rate whose relationship is curvilinear.A major risk increases when BMI exceeds 30 although BMI values between 25 and 30are associated with an increase risk for many diseases among them heart disease andhypertension [265]. Despite these assumptions, several authors think that BMI is notan accurate index of body composition. However, it has been and it still is an indicatorfrequently used to control and evaluate body weight and body composition in HealthSciences.
Skinfolds
According to Lohman et al. [167] the subscapular skinfold thickness is a measure ofsubcutaneous adipose tissue and skin thickness on the posterior aspect of the torso.It is an important measure of nutritional status and, in combination with other skin-fold measurements, it is a useful predictor of total body fat, blood pressure and bloodlipids. It has been recommended in combination with the tricipital skinfold by theInternational Biological Programme (IBP) and the WHO as a relatively new tool.
Changes in the proportions and densities of the Fat Free Body (FFB) Mass componentdue to ageing limit the usefullness of the two component model for estimating rela-tive body fatness. Relative mineral content of the FFB decreases approximately 1%per year between 50 and 70 years of age, and because of these changes the density ofthe FFB of the elderly, particularly elderly women, changes and the prediction equa-tions based on the models using series of Brozeck’s equations tend to overestimatethe relative body fat [135]. Because of the lack of skinfold cross-validaded equations
46
to estimate body density for elderly people the WHO has recommended to use twoskinfolds, subscapular and tricipital [264].
Waist-to-Hip ratio
The Waist-to-Hip ratio (W-H ratio) is the circumference of the waist divided by thecircumference of the hips and has been used as a simple method for determining bodyfat distribution [36].
Waist
Waist circumference can be chosen singly as an indicator of health risk because ab-dominal obesity is the primary concern. Furthermore, the proportion of mortality at-tributable to large waist circumference among never smoking men was found to bethree-fold the proportion attributable to high BMI [256]. The Expert Panel on the Iden-tification, Evaluation and Treatment of Overweight and Obesity in Adults provided aclassification of disease risk based upon both BMI and waist circumference [86]. Fur-thermore, a newer risk stratification scheme for adults based on waist circumferencehas been proposed [35]. Assessments for risk stratification to evaluate chronic diseaserisk are recommended to include a minimum of either waist circumference or BMI butpreferably both.
Functional Tests
The chosen functional tests were easy-to-perform tests that are considered not be in-fluenced by learning; however, the time period between the examination dates wereconsidered to be sufficient for wiping out possible effects of learning.
Handgrip Strength
There are different protocols for examining hand grip strength [196] [94]. The maindifferences are participant’s position at the moment of test performance (lying, sittingor standing) and the arm position, especially the one of the elbow (flexed or extended)when performing the test. There is evidence that better results are obtained if the testis performed in a standing position [175][245][147]. However, as some of the partic-ipants were uncapable of staying in an upright position the test was performed in asitting position. Regarding elbow position, there is divergence of opinion. Some au-thors showed that better results were obtained when the elbow was flexed [71]; others,when the elbow was in extension [19] [82] [198], although also some authors showedthat the results were similar [190] [93].
Lower Body Strength
The test, “30- Second Chair Stand” was used to measure lower body strength. Rikliand Jones demonstrated that this test is a valid and reliable test for measuring proximallower limb (LL) strength in older adults [213] [66] [214] [152].
47
Static Balance
Regarding the population studied the chosen static balance test, stork stand test, wasused as a factor in general motor ability tests [196] [150].
The Mini-Mental State Examination (MMSE)
MMSE by Folstein [101] is a screening tool for quantitatively estimating the sever-ity of cognitive impairment and for documenting cognitive changes. It is widely usedbecause of its easy and fast administration (5-10 minutes). In addition, this question-naire is recommended by the National Institute of Neurological and CommunicativeDisorders and Stroke and the Alzheimer’s Disease and Related Disorders Association(NINCDS-ADRDA) to document the clinical diagnosis of probable Alzheimer’s Dis-ease.
4.6 Limitations of the Studies
In addition to limitations arising from the above delimitations the following ones canbe pointed out:
Common for all the three Studies:
The results can be applied to elderly people with similar characteristics to the studiedpopulation.
The results can be applied to the context of institutions where the evaluations wereperformed.
Specific for the Studies:
Regarding study I, there were 36 dropouts due to the special characteristics of thestudy group such as death, change of residence or dropped during following-up, buttheir values were similar to the ones who finished the whole evaluation period andfulfilling the calculated sample size of 60 elders with complete main outcome data.
Regarding study II, the used dosis was sufficient for demonstrating significant differ-ences after a 28 days supplementation. Nevertheless, further investigation is neededfor determining the effectiveness of supplements or rather treatments of various dosesand/or different durations. The time lapse of one month between the two examinationdates was unlikely to be the reason to explain the observed differences due to matura-tion and or history.
Regarding study III, the complexity and diversity of the medications taken by the par-ticipants and the possible interactions among them and on the vitamin B status makesit difficult to make definitive conclusions of the effects.
48
4.7 Statistical Analysis
For the statistical analysis, continuous variables were checked for Gaussian normaldistribution with the Kolmogorov-Smirnov test and whenever a normal distributioncould not be achieved with the raw data, logarithmic transformation was applied forsignificance testing. Statistical analyses were performed using SPSS for Windows(ver.19.0, SPSS Inc). All tests were performed two-tailed and a P value ≤ .05 wasconsidered statistically significant.
Cutoff values for each biomarker were based on a literature report [183] and were asfollows: vitamin B12 < 148 pmol/L, holoTC < 35 pmol/L, tHcy > 13 µmol/L, serumfolate < 15.8 nmol/L (< 7 ng/mL) and RBC folate < 362.4 nmol/L (< 160 ng/mL).
For study I, statistical analyses were carried out with data from those participants whohad complete data (n=60). Differences of means of each analysed parameter over time(four examination dates) were evaluated by using a general lineal mixed model forrepeated measures with time as repeated factor and gender as fixed factor. Correlationbetween variables was evaluated by using partial Pearsons’ Correlations coefficientswith time as control.
For study II, geometric and arithmetic means with standard deviations of pre- and post-supplementation concentrations were computed for tables. Besides, the trimmed meanwas also calculated to check the degree of variation. Differences between means orrather medians were analysed with a t-test for paired samples and Wilcoxon matched-pairs signed-ranks test in the case of sCbl. Since there is still no consensus on cut-offvalues to define Cbl deficiency, neither for serum cobalamin nor for holoTC values, andgiven that the distribution of cobalamin concentration values was not normal, partici-pants were grouped in clusters (k-means clustering) according to the initial cobalaminstatus in order to achieve maximum homogeneity within the groups. GLM repeatedmeasures analysis with “sCbl clusters” as a fixed factor and post-hoc Bonferroni wereused in order to assess the interaction of the initial sCbl status on the effect of thesupplementation. Results were reported as arithmetic means.
For study III, the reference central intervals (95%), established by the manufacturersof the inmunoassays were used to divide the participants in two groups, (adequateor deficient level of the according parameter). The values were as follows: HoloTC(19.1-119.3 pmoL/L), Hcy (4.72-14.05 µmoL/L), serum folate (7.2-15.4 ng/mL) andCbl (208-963.5 pg/mL).
The medications was classified into 39 groups following the Anatomical TherapeuticChemical (ATC) Classification System [63] and a dichotomous variable (medicationintake / no medication intake) was created for the comparisons. In order to examine the
49
possible influence of the variables of medication intake on biochemical variables con-tingence tables of the chi square test applying the exact test of Fisher for dichotomousvariables were used.
50
RESULTS
5 RESULTS
5.1 Study I (Longitudinal)
Regarding disposition of the study sample, 98 elders were eligible for participationafter screening. Thirty-eight were lost to follow-up, 36 because they moved out ofstudy area or died and were therefore excluded for the final analysis due to missingkey outcome information. Another two were excluded because they started folate orrather cobalamin supplementation during the observation time period so that the finalanalytic sample consisted of sixty elders fulfilling the established sample size. Themean (SD) age of the study group was 84 (7) years, and 68% were women. The meanweight at baseline (month 1) was 70.0 kg (11.0) for men and 59.3 kg (12.9) for women,respectively. Men presented a BMI of 27.9 kg/m2 (4.1) and women of 28.3 kg/m2 (5.2)(Table 7).
Tables 8 - 12 show the averages (means or medians) of the analyzed parameters atbaseline and at the other three examination dates (5th ,9th,13th month). The results areclassified according to the following catagories: vitamin B status, selected haemato-logical variables, lipid profile, anthropometric parameters, functional tests and MMSE.
Parameters related to vitamin B status
Descriptive data for selected vitamin parameters over one year period are shown inTable 8. Median serum cobalamin, serum folate as well as serum RBC folate andserum creatinine values were within the normal range on all four examination dates.Median values for holoTC were in the normal range except at month 9 (median: 42.8pmol/L). In contrast, median serum homocysteine values were elevated on all fourexamination dates, independently of whether 13 or 15 µmol/L (= the most commonlyfound cut-offs revising the literature) were used as decision criteria.
Despite median values for serum cobalamin of the overall study population beingwithin the normal range, 10% of the study population showed deficient levels (de-
53
fined as < 200 pg/mL) which remained stable over the year. When holoTC is takenas criterion for deficient vitamin B status (defined as < 45 pmol/L), these prevalenceswere higher ranging between 45 and 52%.
Whereas the prevalence for deficient RBC folate levels showed quite big oscilations(4%, 7%, 25%, 14%), the prevalence taking serum folate as the criterion was higher,but more stable (43%, 40%, 38%, 37%). Prevalence for elevated Hcy levels was veryhigh, when taking 15 µmol/L prevalence was found between 60 and 70% and whentaking 13 µmol/L even around 82% to 90%. Seventeen per cent of men and between17 and 27% of women showed renal alterations (creatinine values < 1.1 for womenand < 1.4 for men mg/dL).
Regarding the pattern of these parameters, two parameters showed significant changesover the studied time period: serum folate levels increased from 6.2 to 7.0 ng/mL(F=2.86, df=3, p<.05) whereas serum cobalamin values decreased continuously from388.3 to 319.1 pg/mL (F=3.43, df = 2.35, p<.05).
Selected haematological variables
The evolution of the haematological parameters are summarized in Table 9. Meanhaematocrit and haemoglobin values as well as median MCV values of the study groupwere within their corresponding normal ranges. Statistical analysis revealed statisticaldifferences for the intrasubject effect time as well as for the between gender effect forthe parameter haematocrit (Ftime=4.21, df = 2.51, p<.01 and Fgender= 20.25, df = 1,p<.001) and also for the parameter haemoglobin (Ftime= 4.09, df = 2.53, p<.01 andFgender= 22.96, df = 1, p<.001), whereas it was only significant for MCV after time(F=19.8, df=3, p≤.001).
Lipid profile
The evolution of the parameters related to the lipid profile are shown in Table 10.Overall, TG values increased significantly from 92 to 106 mg/dL (F = 9.53, df = 3,p<.001) and HDL-cholesterol decreased significantly from 49.8 to 47.1 mg/dL (F =3.21, df =3, p<.05). The values for total cholesterol and LDL cholesterol showed aslight increase although not significant, but showed significant differences betweengender (Fgender = 7.30, df = 1, p<.001 and Fgender = 4.77, df = 1, p<.05, respectively).
Anthropometric measurements
The evolution of the anthropometric measurements are summarized in Table 11. Meanvalues for BMI and median values of skinfold thickness of the triceps and waist-to-hip ratio did not show significant changes over the studied year. Mean values forsubscapular skinfold thickness oscillated significantly over the year (F = 3.29, df =2.48, p<.05) and the mean values of waist girths increased from 84.8 to 87.3 cm (F =
54
Tabl
e8:
Evo
lutio
nof
para
met
ers
rela
ted
tovi
tam
inB
stat
usov
era
one
year
peri
od
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF
dfSe
rum
coba
lam
in(p
g/m
L)a,
c,d
6038
8.3
(283
.0-4
80.3
)37
3.8
(300
.0-4
75.1
)34
1.4
(272
.3-4
42.1
)31
9.1
(276
.2-4
86.5
)3.
43*
2.35
%ab
norm
alb
105
1010
Hol
oTC
(pm
ol/L
)a,c,
d60
46.0
(30.
7-57
.9)
46.2
(34.
9-66
.2)
42.8
(34.
2-57
.0)
47.7
(34.
4-60
.7)
1.64
2.49
e
%ab
norm
alb
4845
5247
Seru
mfo
late
(ng/
mL
) a,c
,d
606.
2(4
.8-7
.4)
6.7
(5.2
-9.6
)6.
8(5
.3-9
.5)
7.0
(5.2
-8.4
)2.
86*
3
%ab
norm
al43
4038
37R
BC
fola
te(n
g/m
L)a,
c,d
5727
6.8
(224
.5-3
41.6
)27
7.1
(227
.8-3
08.6
)24
8.9
(173
.7-3
37.0
)27
1.3
(192
.5-3
59.3
)%
abno
rmal
b4
725
14tH
cy(µ
mol
/L)a,
c,d
6017
.1(1
3.9-
21.6
)16
.9(1
4.7-
23.1
)17
.2(1
4.1-
27.0
)17
.0(1
4.0-
24.4
)0.
902.
40e
%ab
norm
alb
63(>
15)
70(>
15)
60(>
15)
68(>
15)
87(>
13)
88(>
13)
90(>
13)
82(>
13)
Cre
atin
ine
(mg/
dL)a,
c,d
590.
8(0
.7-1
-1)
0.8
(0.7
-1.1
)0.
8(0
.6-1
.0)
0.8
(0.7
-1.2
)3.
12*
2.66
e
Men
181.
0(0
.78-
1.30
)0.
95(0
.80-
1.30
)0.
85(0
.78-
1.03
)1.
0(0
.80-
1.15
)3.
061
%ab
norm
alb
1717
1717
Wom
en41
0.8
(0.6
0-1.
10)
0.7
(0.7
0-1.
00)
0.8
(0.6
0-1.
00)
0.7
(0.7
0-1.
20)
%ab
norm
alb
1720
2027
a Med
ian
Q2
(int
erqu
artil
era
nge;
Q1-
Q3)
b Cut
-off
valu
esw
ere
the
follo
win
g:se
rum
coba
lam
in<
200
pg/m
L;H
oloT
C<
45pm
ol/L
;ser
umfo
late
<6
ng/m
L,R
BC
fola
te<
175
ng/m
L;t
Hcy
>15
/13
µmol
/L;c
reat
inin
e<
1.1
forw
omen
and
<1.
4m
g/dL
form
en.
c GL
Mfo
rrep
eate
dm
easu
rem
ents
with
epsi
lon
corr
ectio
nby
Gre
enho
use-
Gei
sser
whe
neve
rMau
chy
test
sre
sults
viol
ated
sphe
rity
.d da
talg
10tr
ansf
orm
edfo
rmea
nco
mpa
riso
nse df
corr
ecte
d(e
psilo
n)ac
cord
ing
toG
reen
hous
e-G
eiss
er*p≤
0.05
**p≤
0.01
***p≤
0.00
1Si
gnifi
cant
resu
ltsar
em
arke
dw
ithbo
ldty
pefa
ce.
55
Tabl
e9:
Evo
lutio
nof
rela
ted
haem
atol
ogic
para
met
ers
over
aon
eye
arpe
riod
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF/
Chi
-squ
are
dfH
aem
atoc
rit(
%)a,
d57
38.3
5.1
39.7
4.8
39.8
5.2
39.0
4.5
4.21
**2.
51f
Men
1841
.95.
243
.53.
343
.24.
641
.64.
120
.25##
#1
%ab
norm
alc
38.9
16.7
22.2
27.8
Wom
en39
36.7
4.1
40.0
4.4
38.3
4.7
37.8
4.3
%ab
norm
alc
35.9
25.6
25.6
25.6
Hae
mog
lobi
n(g
/dL
)a,d
5712
.71.
613
.11.
513
.00
1.6
12.7
1.6
4.09
**2.
53f
Men
1813
.71.
614
.31.
214
.11.
513
.81.
322
.96##
#1
%ab
norm
alc
38.9
22.2
22.2
27.8
Wom
en39
12.2
1.3
12.6
1.4
12.4
1.4
12.2
1.5
%ab
norm
alc
35.9
20.5
28.2
38.5
MC
V(f
L)b,
d,e
5790
.1(8
7.8-
93.8
)89
.8(8
8.1-
93.6
)90
.8(8
8.9-
93,7
)91
.2(8
8.7-
95.1
)19
.79*
**3
%ab
norm
alc
5.3
5.3
5.3
3.5
a Mea
n(S
D)
b Med
ian
Q2
(int
erqu
artil
era
nge;
Q1-
Q3)
c Cut
-off
valu
esw
ere
the
follo
win
g:M
CV
<80
fL;f
orm
enH
gb<
13.5
,for
wom
enH
gb<
11.5
g/dL
d GL
Mfo
rrep
eate
dm
easu
rem
ents
with
epsi
lon
corr
ectio
nby
Gre
enho
use-
Gei
sser
whe
neve
rMau
chy
test
sre
sults
viol
ated
sphe
rity
.e da
talg
10tr
ansf
orm
edfo
rmea
nco
mpa
riso
nsf df
corr
ecte
d(e
psilo
n)ac
cord
ing
toG
reen
hous
e-G
eiss
erg C
hi-s
quar
eof
non-
para
met
ric
test
fork
grou
psFr
iedm
an*:
Intr
asub
ject
effe
ct(t
ime)
:*p≤
0.05
**p≤
0.01
***p≤
0.00
1# :B
etw
een
gend
eref
fect
:# p≤0.
05##
p≤0.
01##
# p≤0.
001
56
Tabl
e10
:Evo
lutio
nof
lipid
profi
leov
era
one
year
peri
od
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF
dfT
G(m
g/dL
)b59
92(6
7-12
1)98
(75-
129)
110
(87-
139)
106
(83-
132)
9.53
***
3%
abno
rmal
f5(
B);
5(H
)5(
B);
5(H
)10
(B);
10(H
)14
(B);
8(H
)M
en18
95(6
2.5-
109.
3)10
1(7
4.3-
126.
8)10
5(7
8.0-
129.
8)10
1(8
9.3-
119.
3)1.
871
%ab
norm
alf
6(B
);0(
H)
0(B
);0(
H)
0(B
);0(
H)
11(B
);0(
H)
Wom
en41
92(7
0.0-
129.
0)96
(76.
5-13
3.5)
113
(78.
4-16
3.5)
112
(81.
5-15
0.5)
%ab
norm
alf
5(B
);7(
H)
7(B
);7(
H)
15(B
);15
(H)
15(B
);12
(H)
Tota
lcho
l(m
g/dL
)b59
189.
00(1
68-2
07)
195.
00(1
79-2
15)
198.
00(1
75-2
18)
196.
00(1
69-2
13)
1.20
3%
abno
rmal
f24
(B);
5(H
)31
(B);
15(H
)37
(B);
8(H
)29
(B);
12(H
)M
en18
171.
5(1
56.8
-197
.5)
177.
9(1
17.9
-200
.5)
174.
4(1
39.8
-206
.8)
184.
3(1
66.0
-197
.5)
7.30
##1
%ab
norm
alf
11(B
);0(
H)
22(B
);6(
H)
33(B
);0
(H)
6(B
);11
(H)
Wom
en41
193.
0(1
74.0
-220
.5)
201.
0(1
81.5
-236
.5)
201.
0(1
80.0
-225
.0)
200.
0(1
74.0
-214
.0)
%ab
norm
alf
29(B
);7(
H)
34(B
);20
(H)
39(B
);12
(H)
39(B
);12
(H)
LD
L-c
hol(
mg/
dL)a
5911
9.2
29.4
124.
930
.112
4.3
34.3
123.
331
.11.
063
%ab
norm
al25
(B);
8(H
)29
(B);
15(H
)37
(B);
8(H
)29
(B);
10(H
)M
en18
107.
923
.711
2.1
26.9
109.
029
.811
7.4
25.9
4.77
#1
%ab
norm
alf
17(B
);0(
H)
17(B
);6(
H)
28(B
);0(
H)
17(B
);11
(H)
Wom
en41
124.
230
.513
0.6
30.0
131.
034
.312
5.8
33.1
%ab
norm
alf
29(B
);12
(H)
34(B
);20
(H)
41(B
);12
(H)
34(B
);10
(H)
HD
L-c
hol(
mg/
dL)a
5949
.811
.949
.611
.746
.411
.447
.113
.03.
21*
3%
abno
rmal
f19
2429
34M
en18
47.4
13.4
45.8
10.5
44.7
13.9
45.8
16.2
1.15
1%
abno
rmal
f17
3344
44W
omen
4150
.811
.151
.311
.947
.210
.347
.711
.5%
abno
rmal
f20
2022
29a M
ean
(SD
)b M
edia
n(i
nner
quar
tile
P25-
P75)
c Cut
-off
valu
esw
ere
the
follo
win
g:A
bbre
viat
ions
:TG
Trig
lyce
ride
s;(B
)bor
derl
ine;
(H)h
igh;
chol
chol
este
rol
d data
lg10
tran
sfor
med
form
ean
com
pari
sons
e GL
Mfo
rrep
eate
dm
easu
rem
ents
with
epsi
lon
corr
ectio
nby
Gre
enho
use-
Gei
sser
whe
neve
rMau
chy
test
sre
sults
viol
ated
sphe
rity
.f T
G15
0-19
9m
g/dL
bord
erlin
ean
d>
200
mg/
dLhi
gh;t
otal
chol
este
rol2
00-2
39m
g/dL
bord
erlin
ean
d>
240
mg/
dLhi
gh;L
DL
130-
159
mg/
dLbo
rder
line
high
and
160-
189
mg/
dLhi
gh;H
DL
<40
mg/
dLhi
ghri
sk*:
Intr
asub
ject
effe
ct(t
ime)
:*p≤
0.05
**p≤
0.01
***p≤
0.00
1# :B
etw
een
gend
eref
fect
:# p≤0.
05##
p≤0.
01##
# p≤0.
001
57
9.42, df = 2.21, p<.001). As expected, the latter values were significantly different formen and women (Fgender = 29.22, df = 1, p<.001).
Functional tests
Table 12 summarizes the evolution of the functional parameters over a one year period.There were no changes over time in grip strength of both hands and in arm strength.However, as expected, the values of right and left hand grip strength and the strengthof the dominant arm were significantly different for genders (Fgender = 54.41, F gender
= 70.01, F gender = 17.47, respectively, all df = 1, all p <.001).
MMSE
The evolution of the MMSE scoring is shown in Table 13. Mean MMSE scoring val-ues did not change significantly over time, but gender differences were found womenhaving lower values (Fgender = 11.30, df = 1, p<.001).
Correlations
Table 14 illustrates the partial (controlled for 4 time measures) Pearsons’ Correlationcoefficients and significance levels for the analyzed parameters.
Serum vitamin B12 was correlated positively with HoloTC and RBC folate (p≤.001)and MMSE scores (p≤.01) and negatively with tHcy (all p≤.001). HoloTC was corre-lated positively with RBC folate (p≤.01) and negatively with tHcy (p≤.05). Serum fo-late was correlated positively with RBC folate and negatively with tHcy (both p≤.001)and RBC folate was correlated negatively with tHcy (p≤.001).
Age correlated negatively with serum vitamin B12 and MMSE (both p≤.001) andHoloTC (p≤ .05) and positively with tHcy (p≤.001).
Creatinine values were correlated positively with age and HoloTC (both p≤.01) andtHcy (both p≤.001) and negatively with serum folate (p≤.001).
As expected, the right hand grip strength correlated positively with left hand gripstrength and arm strength (both p≤.001); and with the MMSE scoring (p≤.001) andnegatively with age (p<.01). The left hand grip strength correlated negatively with age(p≤.001) and positively with creatinine (p≤0.05), arm strength and MMSE scoring(both p≤.001). Arm strength correlated negatively with age (p≤.05) and positivelywith MMSE p≤.001).
Correlation between body composition and lipid profile
BMI was positively correlated to waist circumference, triceps and subscapular skin-fold (p≤.001). Waist circumference was positively correlated with Waist-to-Hip ratio,triceps and subscapular skinfolds (all p≤.001) and negatively correlated with HDL-cholesterol (p≤.01). Waist-to-Hip ratio was negatively correlated to triceps skinfold
58
Tabl
e11
:Evo
lutio
nof
anth
ropo
met
ric
para
met
ers
over
aon
eye
arpe
riod
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF
dfB
MIa (k
g/m
²)36
27.4
4.4
27.8
4.3
27.7
4.6
27.6
5.0
0.82
3M
en13
27.7
3.4
28.1
3.4
28.6
3.4
28.5
3.8
0.38
1W
omen
2327
.25.
027
.64.
727
.35.
127
.15.
6SF
tric
eps
(mm
)b47
12.9
(9.3
-18.
0)14
.5(1
1.2-
21.0
)14
.4(1
0.9-
20.3
)13
.5(1
1.1-
18.2
)2.
782.
25f
Men
1511
.9(7
.5-1
5.5)
12.1
(9.1
-16.
1)12
.6(9
.2-1
4.1)
11.8
(9.8
-13.
5)3.
111
Wom
en32
13.7
(11.
1-20
.5)
16.0
(12.
5-22
.8)
16.5
(11.
3-23
.3)
16.2
(11.
3-21
.0)
SFsu
bsca
pula
r(m
m)a
4116
.57.
818
.68.
617
.88.
816
.47.
83.
29*
2.48
f
Men
1418
.97.
220
.18.
218
.97.
618
.36.
91.
061
Wom
en27
15.3
7.9
17.9
8.9
17.2
9.4
15.4
8.1
Wai
st(c
m)a
3784
.810
.685
.610
.587
.710
.487
.311
.69.
42**
*2.
21f
Men
1593
.17.
194
.37.
995
.38.
096
.69.
629
.22##
#1
Wom
en22
79.2
8.9
79.7
7.5
82.5
8.5
80.9
8.0
W-H
ratio
b34
0.86
(0.8
0-0.
95)
0.87
(0.7
8-0.
97)
0.89
(0.8
1-0.
97)
0.87
(0.8
0-0.
96)
2.06
3M
en15
0.95
(0.9
1-0.
98)
0.98
(0.9
5-0.
99)
0.97
(0.9
4-1.
01)
0.97
(0.9
3-1.
01)
70.2
6###
1W
omen
190.
80(0
.77-
0.84
)0.
80(0
.77-
0.83
)0.
82(0
.77-
0.86
)0.
81(0
.79-
0.86
)a M
ean
(SD
)b M
edia
nQ
2(i
nter
quar
tile
rang
e;Q
1-Q
3)c C
ut-o
ffva
lues
wer
eth
efo
llow
ing:
d data
lg10
tran
sfor
med
form
ean
com
pari
sons
e GL
Mfo
rrep
eate
dm
easu
rem
ents
f dfco
rrec
ted
(eps
ilon)
acco
rdin
gto
Gre
enho
use-
Gei
sser
*:In
tras
ubje
ctef
fect
(tim
e):*
p≤0.
05**
p≤0.
01**
*p≤
0.00
1# :B
etw
een
gend
eref
fect
:# p≤0.
05##
p≤0.
01##
# p≤0.
001
Abb
revi
atio
ns:S
F:sk
info
ld;W
-Hra
tio:W
aist
-to-
Hip
ratio
59
Tabl
e12
:Evo
lutio
nof
func
tiona
ltes
tsov
era
one
year
peri
od
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF
dfFu
nctio
nalt
ests
Rig
htha
ndgr
ipst
reng
th(k
g)b
4411
.59
(7.6
-20.
4)11
.75
(7.9
-22.
3)10
.5(8
.0-2
1.7)
11.8
(8.1
-19.
4)0.
142.
20f
Men
1525
.55
(18.
7-29
.2)
24.6
(21.
8-26
.2)
24.8
(19.
9-29
.8)
24.3
(19.
1-30
.3)
54.4
1###
1W
omen
298.
9(7
.4-1
2.9)
9.1
(7.0
-12.
9)9.
2(7
.2-1
2.2)
9.5
(7.0
-12.
3)L
efth
and
grip
stre
ngth
(kg)
b45
10.6
5(7
.1-1
8.2)
10.0
0(7
.6-1
5.2)
10.2
(6.5
-13.
3)10
.5(6
.6-1
4.3)
2.18
2.41
f
Men
1523
.4(1
6.8-
29.1
)20
.9(1
2.6-
24.6
)21
.1(1
1.5-
24.9
)22
.25
(13.
6-24
.8)
70.0
1###
1W
omen
308.
38(6
.5-1
1.2)
8.88
(6.4
-10.
7)7.
38(6
.0-1
1.0)
7.9
(4.9
-10.
8)A
rmst
reng
th(R
ept.
Nr.)
a46
9.77
6.0
10.6
56.
410
.77
6.5
10.3
36.
11.
463
Men
(Loa
d4
kg)
1613
.13
5.8
14.8
44.
415
.56
5.4
14.2
85.
0017
.47##
#1
Wom
en(L
oad
2kg
)30
7.98
5.4
8.42
6.2
8.22
5.6
8.22
4.6
Leg
stre
ngth
(Rep
t.N
r.)b
395.
00(0
-9)
5.00
(0-1
1)4.
00(0
-11)
0.00
(0-1
0)M
en11
7.00
(0-1
1)10
.00
(0-1
4)11
.00
(0-1
1)2.
00(0
-12)
Wom
en28
5.00
(0-8
)5.
00(0
-10)
4.00
(0-1
0)0.
00(0
-9)
Bal
ance
(sec
)b25
1.87
(0-3
.25)
1.66
(0-2
.71)
1.40
(0.4
3-2.
00)
1.10
(0.4
2-1.
88)
Men
102.
13(0
-6.1
6)2.
18(0
.65-
3.69
)1.
97(1
.50-
3.06
)1.
56(1
.05-
5.87
)W
omen
151.
00(0
-2.5
5)1.
00(0
.2.1
0)0.
83(0
-1.4
4)0.
72(0
-1.5
9)a A
rith
met
icm
ean
±SD
b Med
ian
Q2
(int
erqu
artil
era
nge;
Q1-
Q3)
c Arm
volu
me
load
=Se
txN
rrep
etiti
ons
xL
oad
d data
lg10
tran
sfor
med
form
ean
com
pari
sons
e GL
Mfo
rrep
eate
dm
easu
rem
ents
f dfco
rrec
ted
(eps
ilon)
acco
rdin
gto
Gre
enho
use-
Gei
sser
# :Bet
wee
nge
nder
effe
ct:# p≤
0.05
##p≤
0.01
### p≤
0.00
1
60
Tabl
e13
:Evo
lutio
nof
MM
SEsc
ores
over
aon
eye
arpe
riod
Mon
th1
Mon
th5
Mon
th9
Mon
th13
NA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nA
vera
geD
ispe
rsio
nF
dfM
MSE
(Sco
re)a
4618
7.3
187.
818
7.8
188.
0M
en17
235.
522
7.1
226.
722
7.8
0.98
3W
omen
2916
7.0
167.
215
7.2
157.
711
.30##
1a A
rith
met
icm
ean
±SD
# :Bet
wee
nge
nder
effe
ct:# p≤
0.05
##p≤
0.01
### p≤
0.00
1
61
Tabl
e14
:Par
tialP
ears
ons
Cor
rela
tions
(with
time
asco
ntro
l)co
effic
ient
san
dsi
gnfic
ance
leve
lfor
sele
cted
bloo
dpa
ram
eter
s
Age
B12
aH
oloT
Ca
sFol
atea
RB
Cfo
late
atH
cya
Cre
atin
inea
Rig
htha
nda
Lef
than
daA
rmM
MSE
Age
1B
12a
-.235
***
1H
oloT
Ca
-.136
*.6
46**
*1
sFol
atea
.039
.105
-.047
1R
BC
Fola
tea
-.064
.221
***
.172
**.4
55**
*1
tHcy
a.3
49**
*-.2
87**
*.1
52*
-.436
***
-.317
***
1C
reat
inin
ea.1
84**
.073
.199
**-.
231*
**-.0
80.5
43**
*1
Rig
htha
nda
-.212
**-.0
36.0
37-.0
37.0
19.0
16.1
041
Lef
than
da-.2
37**
*.0
60.1
03-.1
37.0
07-.0
59.1
68*
.895
***
1A
rm-.1
37*
.079
.117
.012
.128
-.039
.077
.676
***
.617
***
1M
MSE
-.318
***
.206
**.0
47.0
38.0
72-.0
85-.0
45.4
88**
*.4
76**
*.5
61**
*1
*p≤
0.05
**p≤
0.01
***p≤
0.00
1a da
talg
10tr
ansf
orm
ed
62
Tabl
e15
:Par
tialP
ears
ons
Cor
rela
tions
(with
time
asco
ntro
l)co
effic
ient
san
dsi
gnfic
ance
leve
lbet
wee
nan
thro
pom
etri
cpa
ram
eter
san
dlip
idpr
ofile
BM
IW
aist
W-H
ratio
aTr
icep
saSF
subs
capu
lar
LD
L(m
g/dL
)H
DL
(mg/
dL)
Cho
lest
erol
aT
Ga
BM
I1
Wai
st.5
61**
*1
W-H
ratio
a.0
53.5
98**
*1
Tric
epsa
.523
***
.259
***
-.352
***
1SF
subs
capu
lar
.442
***
.500
***
.007
.465
***
1L
DL
(mg/
dL)
.005
.020
-.155
*.1
68*
.049
1H
DL
(mg/
dL)
-.052
-.194
**-.2
54**
*.1
16.0
43.0
331.
000
Cho
lest
erol
a-.0
24-.0
69-.2
78**
*.2
27**
.072
.927
***
.276
***
1T
Ga
.046
-.044
-.188
**.1
68**
.078
.339
***
-.341
***
.400
***
1*p≤
0.05
**p≤
0.01
***p≤
0.00
1a da
talg
10tr
ansf
orm
ed
63
(p≤.001) and negatively correlated with LDL-cholesterol (p≤.05), and TG (p≤.01)and HDL and total cholesterol (both p≤.001). Triceps skinfold thickness was posi-tively correlated with subscapular skinfold (p≤.001), total cholesterol and TG (bothp≤.01) and LDL- cholesterol (p≤.05). LDL-cholesterol was positively correlated withtotal cholesterol and TG values (both p≤.001) and HDL-cholesterol was positivelycorrelated with total cholesterol and negatively with TG values (both p≤.001). Totalcholesterol was positively correlated with TG values (p≤.001).
5.2 Study II (Intervention)
The characteristics of the whole study population are summarised in Table 16. Thestudy group comprised 64 participants, 40 (63%) women and 24 (37%) men, with amean age of 82 (range 63-93 y). At baseline, 13 (20%) participants of the study pop-ulation showed serum cobalamin values lower than 200 pmol/l and 23 (36%) serumholo transcobalamin levels lower than 40 pmol/l. Anaemia, defined as haemoglobinconcentrations < 13 g/dl in men and < 12 g/dl in women was present in 28% of thestudy group. Only one participant had MCV levels above 100 fl. Folate deficiency,defined as serum folate≤ 6 ng/ml was present in 12 participants (19%). For each anal-ysed parameter the trimmed mean at 5% was calculated and no significant differenceswere found between their means and trimmed means. Creatinine values were withinreference range.
Mean serum cobalamin levels improved from 308.4 to 558.3 pmol/l (P < .001, Table17); the individual absolute changes for serum cobalamin, HoloTC and tHcy are shownin Figure 7 and 8. After supplementation, from the above mentioned 13 subjects withinadequate serum cobalamin levels 11 (85%) reached normal levels (defined as > 200pmol/l) and from the 23 with inadequate holoTC levels, 18 (78%) reached normalvalues (defined as > 40 pmol/l).
The quick cluster analysis delineated four clusters according to initial sCbl values.The “low” sCbl cluster consisted of eight subjects who had serum cobalamin values of
64
Figure 7: Individual changes of serum cobalamin and HoloTC values after a 28 day supplementationwith 500 µg oral cyanocabalamin
65
Tabl
e17
:Wilc
oxon
test
(z)f
orPr
ean
dPo
stm
ean
valu
esfo
rsC
blan
dits
abso
lute
and
rela
tive
chan
ge
Vari
able
sN
Pre
Post
Wilc
oxon
(z)
Cha
nge
(%)
sCbl
(pm
ol/l)
6430
8.4
(132
.9)
558.
3(26
2.2)
-6.9
5***
249.
9(17
2.8)
88.6
7(58
.6)
***
p<
0.00
1V
alue
sar
epr
esen
ted
asm
ean
(SD
)
66
Tabl
e18
:t-
test
for
Pre
and
Post
mea
nva
lues
(geo
met
ric
and
arith
met
ic)
for
holo
TC
,se
rum
and
RB
Cfo
late
,tH
cy,
MC
V,H
CT,
Hb
and
eryt
hroc
ytes
and
thei
rabs
olut
ean
dre
lativ
ech
ange
s
Vari
able
sN
Pre
GM
Pre
Post
GM
Post
tC
hang
e(%
)H
oloT
C(p
mol
/l)64
45.7
753
.8(3
2.5)
84.4
995
.7(4
7.2)
-14.
11**
*41
.8(2
8.0)
97.3
(85.
5)sF
olat
e(n
mol
/l)64
19.3
820
.97(
8.6)
19.2
121
.02(
9.1)
0.31
0.04
(4.8
)1.
5(21
.3)
RB
CFo
late
(nm
ol/l)
6477
9.7
859.
2(41
7.7)
863.
894
5.3(
434)
-2.5
5*86
.1(3
34.8
)15
.8(3
7.4)
tHcy
(µm
ol/l)
64-
15.1
(4.5
)-
13.1
(3.5
)8.
55**
*-2
.0(1
.9)
-11.
9(10
.9)
MC
V(fl
)64
-89
.69(
5.57
)-
89.3
5(5.
64)
1.74
-0.3
4(1.
56)
-0.3
7(1.
71)
HC
T(%
)64
-41
.4(5
.15)
-41
.03(
4.96
)1.
38-0
.37(
2.14
)-0
.72(
5.13
)H
b(g
/dl)
6413
.213
.33(
1.66
)13
.513
.56(
1.59
)-2
.69*
*0.
23(0
.70)
1.91
(5.4
2)E
ryth
rocy
tes
(1012
/l)64
-4.
62(0
.54)
-4.
60(0
.53)
0.76
-0.0
2(0.
24)
-0.3
6(5.
24)
*p
<.0
5**
p<
.01
***
p<
.001
67
Figure 8: Individual changes of serum tHcy values after a 28 day supplementation with 500 µg oralcyanocabalamin
126.8 (25.3) pmol/l, the “medium” cluster consisted of 17 with mean serum cobalaminlevels of 220.3 (26.6) pmol/l. The “high” cluster comprised 30 and the “very high”nine participants with mean serum cobalamin concentrations of 332.6 (38.2) and 555.5(107.3) pmol/l, respectively. The “low” sCbl cluster had serum holo transcobalaminvalues of 21.4 (15.2) pmol/l, the “medium” cluster of 46.0 (16.7), and the “high” and“very high” of 54.4 (21.6) and 95.8 (52.3) pmol/l, respectively. The “low” sCbl clusterhad serum tHcy values of 18.5 (6.6) µmol/l, the “medium” cluster of 15.1 (3.7), andthe “high” and “very high” of 13.8 (3.8) and 16.2 (4.9) µmol/l, respectively.
There was a significant increase from pre- to posttreatment sCbl values (308.4 to 558.3pmol/l, z = -6.95, df = 63, P < .001, Table 17). Similarly, holoTC concentrationsrose significantly (53.8 to 95.7 pmol/l, t = -14.11, df = 63, P < .001, Table 18) as aresult of the treatment. Cobalamin supplementation lowered significantly mean tHcyconcentrations (15.1 to 13.1 µmol/l, t = 8.55, df = 63, P < .001, Table 18). RBC folateincreased significantly from pre to postsupplementation (859.2 to 945.3 nmol/l, t = -2.55, df = 55, P < .05, Table 18). The haemoglobin values increased significantly (13.3to 13.6 g/dl, t = -2.69, df = 63, P < .01). No significant changes were found for sFolate,MCV, HCT and erythrocyte values (Table 18).
The comparison of the pre and post sCbl, holoTC and tHcy values within each clustershowed significant changes (all P < .01); sCbl and holoTC concentrations increasedwhereas tHcy values decreased in each cluster (Figures 9,10,11). Regarding tHcy, the
68
Table 19: Summary table One-Way ANOVA with repeated measures and one fixed factor (serum cobal-amin cluster) for mean serum cobalamin (sCbl), holo transcobalamin (holoTC) and total homocysteine(tHcy) concentrations
“low” and “very high” sCbl clusters showed pre as well as posttreatment tHcy levelswith a high variation and the “very high” sCbl cluster had higher initial tHcy concentra-tions than the “high” and “medium” group but without statistical significance. Whenapplying the GLM model for repeated measurements with the initial sCbl as a fixedfactor in order to compare the effect of the supplement according to the pretreatmentsCbl status, only the increase of sCbl depended on the initial status (F = 4.61, P < .01).The effect of the supplement on holoTC and tHcy was not significantly affected by theinitial sCbl status (F = 0.46 and F = 2.0, respectively, both P > .05, Table 19).
Figure 9: Change from pre to posttreatment mean serum cobalamin (sCbl) concentrations by clusters ofinitial sCbl concentrations
69
Figure 10: Change from pre to posttreatment mean holo transcobalamin (holoTC) concentrations byclusters of initial serum cobalamin (sCbl) concentrations
Figure 11: Change from pre to posttreatment mean total homocysteine (tHcy) concentrations by clustersof initial serum cobalamin (sCbl) concentrations
70
5.3 Study III (Polypharmacy)
Study participants were the participants from the first two projects at baseline, con-sisting of 167 elderly people from four old people’s homes of the Madrid Area. Thepopulation studied took a total of 858 medications of 292 different brands, which isequivalent to an average of 5.13 medications per resident. More than half of the res-idents (59%) took medications belonging to five or more different ATC groups. Themost frequent groups were the psycholeptics (ATC group N05), taken by 83 residents(53%), antiacids (A02), taken by 82 residents (53%), antithrombotics (B01) 47% andpsychoanaleptics (N06) 39%. Table 20 shows the complete results of the groups ofmedications taken. Table 21 shows the values of the biochemical variables both for thetotal population and for the population in which some of these variables were outsidethe reference interval. A deficit of vitamin B12 was found in 10.7%, of serum folate in52.1%, and of holo-TC in 8% of the residents. There was mild hyperhomocysteinemiain 65.9% (average Hcy = 19.31 µmol/L). The influence of the variables of medica-tion on the biochemical variables was significant for vitamin B12 and the anti anaemiamedications (group ATC B03, p<0.05). The percentage of subjects with a vitamin B12deficit was greater for the group which did not take anti anaemia medications. Hcy in-creased significantly with the administration of medications for obstructive pulmonarydisorders (R03, p<0.05), the holo-TC deficit was lesser when psychoanaleptic medica-tions were taken (N06, p<0.05) and the percentage of elderly people who had a deficitof serum folate was lower if they took urological preparations (G04, p<0.05) or sys-temic corticosteroids (H02, p<0.05). The pattern of the percentages was the same forHcy, vitamin B12 and holo-TC, while serum folate showed an inverse pattern betweenthe taking or not of medications (Figure 12).
71
Tabl
e20
:Num
bera
ndpe
rcen
tage
ofel
derl
ype
ople
who
take
one
ofth
eA
TC
grou
pof
med
icat
ion
AT
Cco
de*
Defi
nitio
nn
%A
TC
code
*D
efini
tion
n%
N05
Psyc
hole
ptic
s89
53C
07B
etab
lock
ing
agen
ts11
7A
02D
rugs
fora
cid
rela
ted
diso
rder
s88
53M
05D
rugs
fort
reat
men
tofb
one
dise
ases
106
B01
Ant
ithro
mbo
ticag
ents
7847
C02
Ant
ihyp
erte
nsiv
es9
5N
06Ps
ycho
anal
eptic
s65
39H
03T
hyro
idth
erap
y9
5C
09A
gent
sac
ting
onth
ere
nin-
angi
oten
sin
syst
em62
37C
05V
asop
rote
ctiv
es7
4C
03D
iure
ctic
s56
34H
02C
ortic
oste
roid
sfo
rsys
tem
icus
e7
4N
02A
nalg
esic
s53
32C
04Pe
riph
eral
vaso
dila
tors
64
A06
Lax
ativ
es43
26M
04A
ntig
outp
repa
ratio
ns6
4C
01C
ardi
acT
hera
py36
22L
02E
ndoc
rine
ther
apy
53
A12
Min
eral
supp
lem
ents
2515
A03
Dru
gsfo
rfun
ctio
nalg
astr
oint
estin
aldi
sord
ers
42
C08
Cal
cium
chan
nelb
lock
ers
2414
J01
Ant
ibac
teri
als
fors
yste
mic
use
32
R03
Dru
gsfo
robs
truc
tive
airw
aydi
seas
es23
14A
05B
ilean
dliv
erth
erap
y2
1B
03A
ntia
nem
icpr
epar
atio
ns18
11N
07O
ther
nerv
ous
syst
emdr
ugs
21
A10
Dru
gsus
edin
diab
etes
1710
R05
Cou
ghan
dco
ldpr
epar
atio
ns2
1C
10L
ipid
mod
ifyi
ngag
ents
1710
A11
Vita
min
s1
1G
04U
rolo
gica
ls17
10D
03Pr
epar
tions
fort
reat
men
tofw
ound
san
dul
cers
11
N03
Ant
iepi
lept
ics
1710
D06
Ant
ibio
tics
and
chem
othe
rape
utic
sfo
rder
mat
olog
ical
use
11
S01
Oph
thal
mol
ogic
als
1710
G02
Oth
ergy
neco
logi
cals
11
N04
Ant
ipar
kins
on’s
drug
s13
8R
06A
ntih
ista
min
esfo
rsys
tem
atic
use
11
M01
Ant
i-in
flam
mat
ory
and
antir
heum
atic
prod
ucts
127
*Ana
tom
ical
The
rape
utic
Che
mic
al(A
TC
)Cla
ssifi
catio
nSy
stem
72
Tabl
e21
:Con
cent
ratio
nsfo
rthe
bioc
hem
ical
para
met
ers
bypo
pula
tion
Glo
balp
opul
atio
nPo
pula
tion
with
conc
entr
atio
nsou
tsid
eof
the
refe
renc
ein
terv
alM
edia
np2
5p7
5C
ondi
tion*
n%
Med
ian
p25
p75
tHcy
(µm
ol/L
)15
.91
13.2
20.2
9>1
4.05
110
65.9
19.3
116
.03
21.9
8Se
rum
fola
te(n
g/m
L)
6.9
5.3
9.4
<7.2
087
52.1
5.3
4.45
6.3
Hol
oTC
(pm
ol/L
)46
29.5
362
.26
<19.
113
7.8
11.7
67.
4517
.43
B12
(pg/
mL
)39
0.35
288.
9249
9.92
<208
1810
.715
8.2
139.
417
8.8
*Min
imum
orm
axim
umco
ncen
trat
ion
from
whi
chth
eva
riab
lew
asde
fined
asbe
ing
outs
ide
ofth
eno
rmal
ityin
terv
al
73
Figure 12: Percentages of participants outside and inside of the reference interval of tHcy, B12, HoloTCand folate separated by taking or not-taking medications
74
DISCUSSION
6 DISCUSSION
6.1 Study I (Longitudinal)
The lack of data related to the evolution of risk factors for cardiosvascular diseasesand cognitive impairment which especially affect institutionalized people of advancedage makes it necessary to evaluate these parameters in this population for determiningtheir evolution. To the best of our knowledge, this is the first study observing vitaminB status over a one-year period in Spanish insitutionalized elderly. The present discus-sion is framed in this context. After the data analysis, it could be observed, that themajority of the parameters showed a stable evolution over the studied year.
Regarding vitamin B status it has to be stressed that serum cobalamin values decreasedsignificantly and continuously over the studied period of one year. The percentageof people showing deficient serum vitamin B12 levels was the same on three of thefour examination dates (10%) and even lower in one control (5%, at month 9). Thepercentage of deficiency found was similar to the ones found by other investigatorswhich ranged from around 5% [154][193][171] to 19.71% [260] using a similar cutoffvalue as in the present thesis. When holoTC is chosen as the criterion for vitamin Bdeficiency (defined as < 45pmol/L), the percentages found were higher; nearly halfof the participants studied showed deficient levels (46% in the beginning and 48% inthe end of the study) which were higher than the ones found by other investigatorsfor independently-living elderly (25%-34% depending on the age groups [58]). Thevalues of holoTC found in this study were lower than those found by Hin et. al. [136]and even more than half as low as the ones found by Kivelto et al. [156]. In thiscontext, it has to be stressed that both cut-off points are under discussion. Differentcut-off points have been suggested ranging from 103 to 300 pmol/L for serum vitaminB12 (see Table 3), leading to divergence about the number of prevalences found in theliterature.
Regarding folate status, a notable percentage (37-43%) of participants showed lowserum folate concentrations (defined as < 6 ng/mL), but only a small percentage showedfolate deficiency when RBC folate was chosen as the criterion, but the latter showed agreater variation (between 4% at month 1 and 25% at month 9). Keeping in mind thatRBC folate reflects folate stores, it can be stated that folate status showed a tendencyto worsen over the study period.
Both B12 and folate deficiencies are associated with the appearance of macrocytic-megaloblastic anaemia and immunological irregularities [22][129]. In the presentstudy, anemia (HCT < 40% in men and HCT < 35% in women) was present in 22 and16 participants from the first to the fourth examination time point, respectively. The
77
anemia was typically normocytic (MCV between 80 –100 fL) throughout the study ex-cept for one person. Mean Hgb concentrations were at the lower end of the referencerange (men: 13.5-18.0 g/dl; women: 11.5-16.0 g/dL), and did not change during thestudy and were slightly lower to the ones found by other investigators [156] [136].
No significant changes were observed for median serum tHcy concentrations through-out the study duration but remained with a median value of around 17 µmol/L above thecommonly two used cutoff points of 13 and 15 µmol/L. These levels were lower thanthe ones found by Kivipelto et al. [156] for community-living elderly although otherinvestigators [136] found lower tHcy levels in free-living elderly. Total Hcy concentra-tions were significantly inversely correlated with serum and RBC folate, respectivelyas reported in others studies [154][164] [156] as well as with HoloTC and serum vita-min B12 [156]. In this study, prevalence of moderate hyperhomocysteinemia definedby serum tHcy of 14 –30 µmol/L did not increase over time. Of the B vitamins, folateintake generally seems to have the greatest influence on Hcy concentrations, althoughvitamin B12 may be more important for elderly people who suffer from cobalaminmalabsorption [232]. The hypothesis that a negative cobalamin balance, occurringprior to a fall in serum cobalamin, may have resulted in a different pattern of tHcyvalues over the observation period could not be confirmed including in the GLM forHcy groups with low and normal initial holoTC values (cut off point 45 pmol/L) as afixed factor (data not shown).
Other risk factors for cardiovascular diseases
Among the risk factors for cardiovascular diseases are an unfavourable lipid profile,overweight and high percentage of body fat and its distribution.
Lipid profile
TG levels of the overall group increased whereas HDL-cholesterol decreased, both sig-nificantly; but the changes occurred within the corresponding normal reference values.However, subdividing the group by gender, the values for women showed an incrementwhereas the values for men were stable. Total cholesterol and LDL-cholesterol re-mained stable over time, but gender differences were found with women having highervalues. Whereas in the case of total cholesterol these slight variations occurred withinthe normal range, LDL-cholesterol levels surpassed the lower threshold for high bor-derline values according to the ACM’s Guidelines for Exercise Testing and Prescription[8] at month 5 and 9 .
Body composition
Among middle aged adults (50-65y), overweight and obesity are important publichealth problems in various countries. For elderly people, 65y+ the health risk of over-weight is unclear; in fact, data indicate that moderate overweight at advanced ages is
78
related to lower mortality [12] [209]. Among those 80y+, thinness and loss of leanbody mass may be a more significant problem than overweight. A U-shaped relation-ship between BMI and mortality has been reported for Finnish men [217]. Lowestmortality occurred at a somewhat higher BMI among men over 75 years of age thanamong younger men, but the curve showed excess mortality at the tails of the distri-bution. Among thin men mortality from cardiovascular causes increased with BMI inthe younger cohorts but not in those aged 55-90. A U-shaped relationship betweenBMI and mortality was also apparent in young women, but was more uncertain forolder women, who showed little variation and mortality with BMI. Overweight did notreduce life expectancy in Finnish women aged 65-79 years [218]. Indeed, a modestdegree of overweight seemed to be protective against death. The most favourable BMIwas 27-31, a considerably higher figure than was observed in Norway. A follow-upstudy in Finland of 95 men and 431 women over 85 years of age showed that a lowBMI was a more important predictor of risk of death than a high BMI [132]. High-est 5-year mortality was reported in the group with BMI < 20.0 and the lowest in thegroup with BMI greater than 30. It was concluded that overweight ceases to be a riskfactor for death in this age group. Both overweight and thinness appear to carry riskfor mortality, but in the elderly thinness carries a greater risk than overweight. Weightchange, especially involuntary weight loss, also poses considerable risk, which mayinterfere with the interpretation of the data from large studies. In our study, the medianvalues of BMI for men remained stable (between 28 and 29); same was observed forwomen (BMI values between 27 and 28), which are considered to be normal for thisage group according to the criteria used by the WHO Expert Committee [264]. Com-paring the results to the ones found by Camiña Fernández et al. [38], the values forthe women correspond to the category of good to average whereas the values for menwere a little higher than the average. As BMI was stable during the studied year, therisk for cardiovascular diseases and stroke seem to be not affected by this parameter.
Skinfold thickness (triceps and subscapular)
Skinfolds are considered a good measure of subcutaneous fat in different group of sub-jects but older subjects of the same gender and body density have proportionally lesssubcutaneous fat than their younger counterparts [160]. They also have a higher pro-portion of internal fat and the proportion of fat located internally decreases as overallfat increases. The sum of several skinfolds has been proposed to estimate body fat butbiological variation in fat distribution is affected by age, gender and degree of fatness[134]. Skinfold thickness of the triceps remained stable (between 12 and 13 mm formen and between 14 and 17 mm for women, respectively). Subscapular skinfold thick-ness varied between 18 and 22 mm for men and 15 and 18 mm for women, respectively.The subscapular skinfold is considered a useful predictor of lipid profile; however, in
79
our study, triceps skinfold but not the subscapular skinfold correlated positively withthe values of total cholesterol and TG but not with HDL or LDL-cholesterol. The val-ues registered in this study can be used for comparisons among groups with similarcharacteristics to the ones of the present study.
Waist and Waist-to-Hip ratio
Health risk increases with W-H ratio levels, and standards for risk range with age andgender. Health risk is very high for young men when W-H ratio exceeds 0.95 and foryoung women when WHR exceeds 0.86, whereas for people 60 to 69 years old, thecut-offs for the same risk classification are greater than 1.03 for men and greater than0.90 for women [100] [135]. In our study, the median as well as the whole interquar-tile section were in the normal range and were similar to the ones found in the ExernetStudy by Ara et al. [13] (0.96 for men and 0.89 for women, respectively). Waist cir-cumferences increased 4 cm for both genders (from 93 cm to 97 cm for men and from79 cm to 83 cm for women over the year). For both genders those values were smallerthan the ones found in the Exernet study whose P50 values were 98.8 cm for men and94.3 cm for women, respectively [13], although the change of waist circumferences inmen over the studied year entered in the category of increased mortality (WC between94 and 102 cm) revealed by the results of the Rotterdam Study, a population-basedcohort study with a mean follow-up of 5.4 y [256].
Functional tests and MMSE
Presenting a deteriorated vitamin status can lead to functional implications of clinicalimportance. Since physical impairment is one reason for long-term institutionalization,impaired strength may be expected for elderly people residing in old people’s homes.
Poor hand strength as measured by hand-grip is a predictor of disability in older people[109]. In the present study, median right and left hand grip were stable for men andwomen with higher values in men compared to women over the entire duration of thestudy. The range of the right and left hand grip strength of our male participants (21and 23 kg for left hand and between 24 and 26 kg for right hand strength, respectively)was similar to the average category of hand strength reported by Camiña Fernándezet al. [38] (20.7-30.6 kg) for elderly active people aged over 80 participating in aprogramme of physical activity. The range of the right and left hand grip strength ofour female participants (between 9 and 10 kg for right hand and between 7 and 9 kgfor the left hand grip strength) was smaller than the lowest category of hand strengthreported by Camiña Fernández et al. [38] (<12.4 kg).
Arm strength remained stable over the year. The mean value for men which rangedbetween 13 and 16 Nr. of repetitions were the same as the ones of the age group 80+from the Exernet study group [13] whereas the women in this study only performed
80
half of the repetitions of the women of the age group 80+ of the Exernet study group[13] (8 compared to 15 Nr of repetitions, respectively). The values for both genderwere notably lower than the ones found in the study performed by Vallejo et al. [251]in which Spanish male participants performed 25.0 ± 4.8 (3.63kg load) and the femaleparticipants 20.1 ± 3.6 (2.27kg load) Nr. of repetitions, respectively.
Regarding leg strength, men as well as women experienced a notably decrease frommonth 9 to month 13. The values of the present study were lower compared to theones found within the Exernet study [13] (14 (=P50) for both gender for the age group80+) and also to the ones found by Vallejo et al. [251] (men 18.27 ± 4.94; women14.86 ± 1.98 for an elderly group of 33 people, 22 women and 11 men with a mean ageof 67.37 ± 4.79y). The latter group studied was younger and both mentioned elderlygroups studied were active; however, the great differences are still indicating very poorstatus of leg strength which might be due to lack of use because of a sedentary life andthe unnecessary use of wheel chairs. It has to be stressed that several participants werenot able to perform the test and that the results of the subjects who performed wereextremely low which might indicate that this test is not appropriate when evaluatingthis population group.
In terms of balance, male participants performed the balance test with a median averageof 2 sec, whereas the female participants were able to stand on one foot for 1 sec. Thoseresults were lower than the values found for the group aged over 80 of active Spanishelderly people who participated in the EXERNET [13] study (P50 for men 6.2 secand for women 5.6 sec, respectively). As mentioned before for leg strength a highpercentage of participants was not able to perform the test which again might indicatethat this test is not appropriate as a measurement for this study group.
Mean MMSE scores showed a stable pattern over the year of examination with higherscoring for men compared to women (between 22 and 23). In the present study, apositive correlation was found between the MMSE scores and serum vitamin B12but not for holoTC concentrations, nor with serum folate or RBC folate, nor betweenMMSE scores and serum tHcy concentrations. This was in agreement with some stud-ies [235][23] [224] that found no association between folate status and cognitive statealthough on the other hand, Riggs et al. [212] and Gottfries et al. [113] showed thatserum Hcy concentrations can be considered an early and sensitive marker for cogni-tive impairment. Lack of association between serum tHcy concentrations and MMSEscores in the present study may be related to the length of the study duration and to thesmall number of study population.
81
6.2 Study II (Intervention)
The main results of the study were that Hcy values decreased significantly whereasserum cobalamin and holoTC values increased significantly after only 4 weeks of daily500 µg cyanocobalamin supplementation. The observed effect improves the results ofa former investigation of a dose-finding trial conducted by Eussen et al. [85] who con-cluded that when comparing a daily oral dosage of 500 µg vs 1000 µg cyanocobalaminadministered for eight weeks that 500 µg was the lowest oral dose associated with amaximum increase of holoTC levels in mild vitamin B12 deficient subjects, deficiencydefined as serum vitamin B12 levels of 100-300 pmol/l. In the study by Eussen et al.[85] they did not analyse a period of 4 weeks as was done in our study. The increase inthe subjects included in the “low” cluster in our study was slightly higher than the onepresented by the group in the dose-finding trial by Eussen et al. [85] which receivedthe same amount of oral cyanocobalamin (186 vs 182 pmol/l) but during double oftime. In addition, the increase of the “medium” cluster even reached the same effect asthe group receiving 1000 µg/d of Eussen´s et al. dose-finding trial [85] for eight weeksand the milk trial in the study performed by Dhonukshe-Rutten [73], who administereda milk drink enriched with 1000 µg crystalline Cbl or the same amount containing cap-sules for 12 weeks (249 vs 248 vs 250 pmol/l) to mildly cobalamin deficient elderlydefined as serum vitamin B12 levels of 100-300 pmol/l.
Highly significant increases were also observed for holoTC values, both for the wholestudy group and for each cluster. The increase of the “low” and “medium” clusterswere smaller (36 vs 45 vs 49 pmol/l) than the one presented by the 500 µg oral Cblreceiving group in the dose-finding trial [85].
Regarding tHcy, mean values of the “medium” cluster decreased less than in the 500µg oral Cbl administered subgroup in the dose-finding trial [85] (-1.5 vs -1.9 µmol/l)and the capsule trial (-1.8 µmol/l) of the above named study performed by Dhonukshe-Rutten [73]. In contrast, the “low” cluster showed a higher response of -3.4 µmol/l.But it has to be mentioned that both, pre and posttreatment values, presented a greatvariation. The “very high” sCbl cluster had higher pretreatment tHcy concentrationsthan the ones for the “high” and even “medium” group. It has to be mentioned thatin our study, high tHcy values were mostly independent of Cbl status. In relationshipto folate, folate deficiency according to serum folate levels was present in 12 sub-jects (18.5%) of the whole study population. Only two subjects had tHcy values < 13µmol/L. This seems to indicate that other factors beyond Cbl and folate status such aslifestyle factors [67] are affecting tHcy concentrations in this population group. Fur-thermore, that cluster consisted of few people and showed a big variance and pretreat-ment value did not reach statistical significance compared to the pretreatment values
82
of the other clusters.
The oral vitamin B12 supplementation did not significantly change mean sFolate. RBCfolate levels increased (P < .05) in the whole study population. These findings are sup-ported by a double-blind, placebo-controlled trial [84] where oral vitamin B12 wassupplemented alone or together with folic acid. In the latter study, mildly vitamin B12deficient subjects (n = 195) were randomly assigned to receive 1000 µg vitamin B12,1000 µg vitamin B12 in combination with 400 µg folic acid, or a placebo. After12 weeks of supplementation, mean RBC folate values increased significantly in the vi-tamin B12 group and in the vitamin B12 plus folic acid group. In the above-mentionedstudy performed by Dhonukshe-Rutten et al. [73] after 12 weeks both intervention setsalso presented an increase of RBC folate, but the increase reached statistical signifi-cance only in the milk trial. After having performed the quick cluster analysis, in ourstudy the significant increase of RBC folate disappeared (data not shown).
To the best of our knowledge, only two studies included haematological parameters inthe outcome. The results of the present study are consistent with previous trials thatreported no significant changes in haematological parameters within one month of Cbladministration such as the study conducted by Seal et al. [227] who compared theeffectiveness of 10 and 50 µg oral cyanocobalamin or placebo administered daily forfour weeks in 31 older patients with deficient or borderline serum vitamin B12 levelsbetween 100 and 150 pmol/l. Hvas et al. [145] found no significant changes in bloodHb and MCV between the treatment group (n = 70) and the placebo group (n = 70) in aDanish clinical trial with participants older than 18 y. It has to be mentioned that thesestudies administered much lower doses than was the case in our study.
The results support the fact that a daily dose of 500 µg of cyanocobalamin during28 days improves vitamin B12 status in institutionalized elderly. No control groupwas included because foods provided at the nursing homes followed a regular pat-tern so dietary factors were unlikely to be important. Additionally, as has been men-tioned above, dietary vitamin B12 intake as a determinant of blood concentrations de-creases in importance during the ageing process because of increasing malabsorptionproblems[130][11].
6.3 Study III (Polypharmacy)
The first contribution of this study is a description of the type of medications which theinstitutionalized population of old people in the Region of Madrid take every day, datawhich are currently very scarce. The second is the high percentage of elderly with con-centrations which are considered pathological of mainly Hcy (hyperhomocysteinemia)and serum folate (deficit). These percentages are very similar to those found by other
83
authors in populations of institutionalized elderly [111][183] except in the case of holo-TC where the percentage of elderly with a deficit was lower than that described in otherstudies. The reason is the use of different cut off points for the deficit of holo-TC. Ashas been stated already, for this study the cut offs recommended by the immunoassaymanufacturer (19.10 pmoL/L) were used while other authors used higher cut off points,between 30 and 40 pmol/L depending on the measurement procedure used [183]. Afolate deficit can be corrected with a folate rich diet or even with folate supplements.However, hyperhomocysteinemia, even when mild, is more difficult to resolve, as it isnot always due to a lack of vitamin B12 or folate [15]. Thus the importance of car-rying out frequent follow ups of homocystein levels on this type of population, as therelation is known between hyperhomocysteinemia and the increased risk of a cardio-vascular event and other problems mentioned above. A significant influence on Hcylevels was only found for the medications for pulmonary obstruction. Several authorshave already described slight increases of Hcy in obstructive pulmonary pathologies,in particular COPD [228]. It is also known that bronchodilators derived from theo-phylline can increase Hcy levels [97]. Thus the increased level of Hcy in patients whotook this group of medications could be due to the reasons mentioned above. The antianaemia medications, formed by compounds of vitamin B12, folates, and iron or acombination of them, revealed a significant influence on the normalization of vitaminB12 levels. The levels of folate tended to normalize under the influence of the psy-choanaleptic medications, mainly the antidepressants, taken by some residents. Herethe reason for the influence is not clear. Some studies show that the risk of sufferingfrom dementia or depression increases when the levels of vitamin B12 and/or folateare low [178]. In fact it is thought that supplementation with vitamin B12 and folatecan potentiate the action of the psychoanaleptics [49]. It is in fact unknown whetheror not there is a direction relation between levels of holo-TC and depressive or cog-nitive pathologies. Lastly the mechanisms or reasons why urological preparations orsystemic corticoids could increase levels of serum folate are still unexplained. To carryout a statistical study to show the influence of a determined group of medications on abiochemical parameter in this type of population is complex. Most of the subjects takemedications from more than five different groups, which cause an interrelation of allthe medications, making it difficult to study individual relations between a medicationand the biochemical variable. As an example, although there is a relation between lev-els of Hcy and vitamin B12, medications which had a significant influence on one ofthe variables had no influence on the other. Another additional problem is the impos-sibility of working with a control group of elderly people who take no medications.
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CONCLUSIONS
7 CONCLUSIONS
The following conclusions address the topics presented in the hypotheses as well asthe objectives of this thesis.
1. Over one year, in relationship to vitamin B status, serum cobalamin decreased,serum folate and mean corpuscular volumen increased significantly and total homocys-teine concentrations were stable. Regarding blood lipid profile, triglycerides increasedand HDL-cholesterol decreased significantly. Regarding selected anthropometric mea-surements, waist circumference increased significantly. No significant changes wereobserved for the rest of the parameters.
2. Prevalence of hyperhomocysteinemia was high in the studied elderly, ranging from60% to 90 % over the year depending on the cut-off used for the classification. LDL-cholesterol values were high, especially among women, and showed a tendency toincrease over the year. Results of the balance test showed a deficiency and a ten-dency to decrease; this indicates that the population studied is at high a risk for falls.Lower extremity muscular function was deficient and showed a tendency to decrease.A highly significant relationship was observed between the skinfold of the triceps andblood lipid profile.
3. Low cobalamin concentrations correlated significantly with low MMSE scores inthe elderly studied. No correlations were observed between vitamin B12 status andfunctional parameters.
4. Regarding vitamin B12 status, holotranscobalamin seems to be more sensitive fordiagnosis; 5-10% of the elderly were deficient using serum cobalamin as a criterion,and 45-52% were deficient when using serum holotranscobalamin as a criterion.
5. 500 µg of cyanocobalamin administered orally during 28 days improved signifi-cantly vitamin B12 status and decreased significantly total homocysteine concentra-tions in institutionalized elderly. No effect of the intervention was observed on func-tional and cognitive parameters.
6. The relative change (%) of improvement of vitamin B12 status was higher whenusing serum holotranscobalamin as a criterion than when using serum cobalamin.
7. Antiaenemic drug intake normalized cobalamin, urologic drugs and corticosteroidsserum folate, and psychoanaleptics holotranscobalamin levels. Drugs against pul-monary obstruction increased total homocysteine concentration significantly.
8. The daily mean drug intake was 5.13. Fifty-nine percent of the elderly consumedmedication belonging to 5 or more ATC different groups. The most prevalent werepsycholeptic (53%), antiacid (53%) and antithrombotic (47%) drugs.
87
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8 Appendices
8.1 Case Report Form Longitudinal Study
8.2 Case Report Form Intervention Study
8.3 MMSE approved form (version date September 6, 2002)
117
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 1/14
CASE REPORT FORM
PROJECT:
- La deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores
- Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people
Date Code
Name
Date of birth Age Sex
Entry date to the residence Profession
I. STUDY START:
Yes No
The subject has signed the informed consent form
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 2/14
II. SELECTION OF SUBJECTS
a) Inclusion criteria Yes No
01 Male and female subjects aged 60 and more
02 Subclinical vitamin B12 deficiency
b) Exclusion criteria Yes No
01 Neuropathy
02 Hunter’s glossitis
03 Addison’s/Megaloblastic anaemia
04 Vitamin B12 intake (supplements)
05 Folate intake (supplements)
Date and signature of the investigator:
III. NUTRITIONAL ASSESSMENT
The subject has completed the MMSE Yes No
A
B
C
D
Comments:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Date and signature of the investigator (C):
Date and signature of the investigator (D):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 3/14
IV. PSYCHOLOGICAL ASSESSMENT
The subject has participated this part of the study Yes No
A
B
C
D
The subject has completed the MMSE Yes No
A
B
C
D
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Date and signature of the investigator (C):
Date and signature of the investigator (D):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 4/14
V. PHYSICAL EXAMINATION
The subject has participated this part of the study Yes No A B C D
A B C D 01 Fuerza y presión manual 02 Fuerza piernas 03 Fuerza brazos 04 Equilibrio (flamenco) 05 Equilibrio dinámico 06 Transferencia de fichas 07 Guardar cerillas
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Date and signature of the investigator (C):
Date and signature of the investigator (D):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 5/14
VI. ANTHROPOMETRIC DATA
The subject has participated this part of the study Yes No A B C D
A B C D
01 Weight
02 Height
03 Skin fold thickness a) Bicipital b) Tricipital c) Subscapular d) Thigh e) Calf
04 Circumferences a) Arm b) Biceps c) Thigh d) Calf e) Waist f) Hip
05 Diameter a) Wrist b) Elbow joint c) Knee joint
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Date and signature of the investigator (C):
Date and signature of the investigator (D):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 6/14
VII. ANALYTIC (SCREENING)
Blood samples have been collected Yes No A B C D
Sample
Date (dd/mm/yyyy)
Hour (hh/mm)
A B C D
Has the subject been fasting at the time of blood sampling? Yes No A B C D
If no, please indicate taken foods and/or drinks
A
B
C
D
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 7/14
VIII. ALIQUOTS
Aliquots A B C D 01 3 mL Vacutainer with EDTA (haematology) 02 5 mL Vacutainer with heparin (vitamin B6) 03 8 mL Vacutainer with gel (serum) 04 8 mL Vacutainer with gel (serum)
A B C D 01 Genotype thermolabile MTHF reductase --- --- --- 02 Plasma folate 03 Erythrocyte folate 04 Homocysteine 05 Methylmalonic acid 06 Vitamin B12 07 Holotranscobalamin II 08 Plasma vitamin B2 09 Plasma vitamin B6 10 Hematology 11 Biochemistry 12 Coagulation 13 14 Serothek
Comments:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Date and signature of the investigator (C):
Date and signature of the investigator (D):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 8/14
IX. CLINICAL DATA
Yes No 01 Cardiovascular disease 02 Thrombotic incidences 03 Diabetes 04 Alzheimer 05 Hypertension 06 Cancer 07 Others:
Comments:
X. INTESTINAL HABIT
Yes No 01 Diarrhoea 02 Constipation
Comments:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 9/14
XI. (A) CURRENT TREATMENT (Medication or supplements)
Has the subject taken any treatment for more than 7 days during the last 30 days?
Yes No
If yes, please specify when what was taken:
Start date
End date
On-going
01
02
03
04
05
06
07
08
09
10
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 10/14
XII. (B) CURRENT TREATMENT (Medication or supplements)
Has the subject taken any treatment for more than 7 days during the last 30 days?
Yes No
If yes, please specify when what was taken:
Start date
End date
On-going
01
02
03
04
05
06
07
08
09
10
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 11/14
XIII. (C) CURRENT TREATMENT (Medication or supplements)
Has the subject taken any treatment for more than 7 days during the last 30 days?
Yes No
If yes, please specify when what was taken:
Start date
End date
On-going
01
02
03
04
05
06
07
08
09
10
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 12/14
XIV. (D) CURRENT TREATMENT (Medication or supplements)
Has the subject taken any treatment for more than 7 days during the last 30 days?
Yes No
If yes, please specify when what was taken:
Start date
End date
On-going
01
02
03
04
05
06
07
08
09
10
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 13/14
XV. SERIOUS ADVERSE EVENT REPORT
Investigator aware of serious adverse event Date:
01 Death *
02 Hospitalisation **
03 Life threatening
04 Persistent or significant disability/incapacity
05 Important medical event
06 Other:
* If death:
Date of death: Probable cause of death: Autopsy performed? Yes No
** If hospitalised:
Date of hospitalisation: Date of discharge::
Description of the adverse event 01 Symptoms
02 Course
03 Diagnostic
04 Investigations
05 Results
06 Treatment
07 Other comments
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 14/14
XVI. STUDY TERMINATION
Has the subject completed all exams and questionnaires of the study?
Yes No
If no, please specify:
Date of subject’s withdrawal
Main reason for premature discontinuation:
01 Expressed personal request
02 Exclusion criteria
03 Adverse event
04 Others: please specify:
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 1/10
CASE REPORT FORM
PROJECT:
- La deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores
- Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people
Date Code
Name
Date of birth Age Sex
Entry date to the residence Profession
STUDY START:
Subject’s informed consent Yes No
The subject has signed the informed consent form
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 2/10
SELECTION OF SUBJECTS
Inclusion criteria Yes No
01 Male and female subjects aged 60 and more
02 Subclinical vitamin B12 deficiency
Exclusion criteria Yes No
01 Neuropathy
02 Hunter’s glossitis
03 Addison’s/Megaloblastic anaemia
04 Vitamin B12 intake (supplements)
05 Folate intake (supplements)
Date and signature of the investigator:
NUTRITIONAL ASSESSMENT
The subject has completed the following questionnaires:
Yes No
01 Mini Nutritional Assessment (MNA)
02 Questionnaire B12
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 3/10
PSYCHOLOGICAL ASSESSMENT
A B The subject has participated this part of the study Yes No Yes No
The subject has completed the following questionnaire A B
Yes No Yes No Mini Mental State Examination (MMSE)
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
PHYSICAL EXAMINATION
A B The subject has participated this part of the study: Yes No Yes No
A B
Yes No Yes No 01 Test of Rikli and Jones 02 Test of Groningen for adults aged over 55 years 03 Dinamometria 04 Equilibrium 05 Barthel Index [3]
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 4/10
ANTHROPOMETRIC DATA
A B The subject has participated this part of the study: Yes No Yes No
A B
Yes No Yes No
01 Weight
02 Height
03 Skin fold thickness a) Bicipital b) Tricipital c) Subscapular d) Thigh e) Calf
04 Circumferences a) Arm b) Biceps c) Thigh d) Calf e) Waist f) Hip
05 Diameter a) Wrist b) Elbow joint c) Knee joint
Incidences:
Date and signature of the investigator (A):
Date and signature of the investigator (B):
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 5/10
ANALYTIC (SCREENING)
A B Blood samples have been collected Yes No Yes No
A B Sample Date (dd/mm/yyyy) Hour (hh/mm)
A B Has the subject been fasting at the time of blood sampling?
Yes No Yes No
If no, please indicate taken foods and/or drinks
A
B
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 6/10
A B Aliquots Yes No Yes No
01 3 mL Vacutainer with EDTA (haematology) 02 5 mL Vacutainer with heparin (vitamin B6) 03 8 mL Vacutainer with gel (serum) 04 8 mL Vacutainer with gel (serum)
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 7/10
CLINICAL DATA
Yes No 01 Cardiovascular disease 02 Thrombotic incidences 03 Diabetes 04 Alzheimer 05 Hypertension 06 Cancer 07 Others:
Comments:
INTESTINAL HABIT
Yes No 01 Diarrhoea 02 Constipation
Comments:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 8/10
CURRENT TREATMENT (Medication or supplements)
Has the subject taken any treatment for more than 7 days during the last 30 days?
Yes No
If yes, please specify when what was taken: Start date
End date
On-going
01
02
03
04
05
06
07
08
09
10
Comments:
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 9/10
SERIOUS ADVERSE EVENT REPORT
Investigator aware of serious adverse event Date:
01 Death *
02 Hospitalisation **
03 Life threatening
04 Persistent or significant disability/incapacity
05 Important medical event
06 Other:
* If death:
Date of death: Probable cause of death: Autopsy performed? Yes No
** If hospitalised:
Date of hospitalisation: Date of discharge::
Description of the adverse event 01 Symptoms
02 Course
03 Diagnostic
04 Investigations
05 Results
06 Treatment
07 Other comments
Date and signature of the investigator:
Vitamin B12 deficiency as a risk factor for neurodegenerative (cognitive and functional) diseases in elderly people 10/10
STUDY TERMINATION
Has the subject completed all exams and questionnaires of the study?
Yes No
If no, please specify:
Date of subject’s withdrawal
Main reason for premature discontinuation:
01 Expressed personal request
02 Exclusion criteria
03 Adverse event
04 Others: please specify:
Comments:
Date and signature of the investigator:
DOCUMENTO FUENTE – N.º DE PROTOCOLO Iniciales del paciente: Randomización N.º Fecha: Visita: ________ Página 1 de 4
Mini-Mental State Examination (MMSE)
Instrucciones: las palabras en negrita deben leerse en voz alta y clara a la persona a la que se evalúa. Entre paréntesis aparecen las distintas alternativas para las preguntas. La prueba debe realizarse en privado y en la primera lengua del examinando. Rodee el 0 si la respuesta es incorrecta, o 1 si es correcta. Comience haciendo las siguientes preguntas:¿Tiene algún problema de memoria? ¿Puedo hacerle unas preguntas sobre su memoria?
ORIENTACIÓN EN EL TIEMPO RESPUESTA PUNTUACIÓN (marque una)
¿ En qué año estamos? ____________ 0 1
estación ____________ 0 1
mes del año ____________ 0 1
día de la semana ____________ 0 1
fecha ____________ 0 1
ORIENTACIÓN EN EL ESPACIO*
¿Dónde estamos ahora? ¿En qué ... estamos?
estado (provincia) ____________ 0 1
país (o ciudad/pueblo) ____________ 0 1
ciudad/pueblo ____________ 0 1
(o zona de la ciudad/barrio)edificio (nombre o tipo) ____________ 0 1
piso del edificio ____________ 0 1( nº habitación o dirección)
*Se pueden utilizar y anotar otras palabras de lugar que encajen en la serie y sean más precisas.
MEMORIA INMEDIATA
Escuche atentamente. Voy a decir tres palabras. Repítalas cuando yo termine. ¿Está listo?Estas son: MANZANA [pausa], CÉNTIMO [pausa], MESA [pausa]. Ahora repítame las palabras.[Repítalo 5 veces, pero puntúe sólo la primera serie]
MANZANA ____________ 0 1
CÉNTIMO ____________ 0 1
MESA ____________ 0 1
Ahora memorice estas palabras. Le pediré que las repita dentro de unos minutos.
DOCUMENTO FUENTE – N.º DE PROTOCOLO Iniciales del paciente: Randomización N.º Fecha: Visita: ________ Página 2 de 4
ATENCIÓN Y CÁLCULO [Serie 7s]*
Ahora tiene que restar 100 menos 7. Luego siga restando 7 del resultado que obtenga hasta que le diga que puede parar.
¿Cuánto es 100 menos 7? [93] ___________ 0 1
Si es necesario, diga : Continúe [86] ___________ 0 1
Si es necesario, diga : Continúe [79] ___________ 0 1
Si es necesario, diga : Continúe [72] ___________ 0 1
Si es necesario, diga : Continúe [65] ___________ 0 1
RECUERDO DIFERIDO
¿Qué tres palabras le pedí que recordara? [No dé ninguna pista]
MANZANA ___________ 0 1
CENTIMO ___________ 0 1
MESA ___________ 0 1
NOMBRAR
¿Qué es esto? (Señale un lápiz o bolígrafo) ___________ 0 1
¿Qué es esto? (Señale un reloj) ___________ 0 1
REPETICIÓN
Ahora voy a pedirle que repita lo de que yo diga. ¿Está preparado? “EL FLAN TIENE FRUTILLAS Y FRAMBUESAS”. Ahora dígalo. [Repítalo hasta 5 veces, pero puntúe sólo la primera.]
“EL FLAN TIENE FRUTILLAS Y FRAMBUESAS” ___________ 0 1
MMSE Version date: September 6, 2002
Sólo se deben hacer otras preguntas (MUNDO al revés) si la persona a la que se está evaluando se niega a realizar el ejercicio de la Serie 7. Realice y puntúe esta pregunta sólo si la persona que está siendo evaluada se niega a realizar el ejercicio de la Serie 7s.Deletree MUNDO hacia delante y luego hacia atrás.Si al deletrear hacia delante se ha equivocado corríjale, pero puntúe sólo cuando deletree hacia atrás.
Escuche con atención porque voy a pedirle que haga algo.Coja este papel con su mano derecha [pausa], dóblelo por la mitad [pausa], y póngalo en el suelo (o mesa).
COGER CON LA MANO DERECHA ___________ 0 1
DOBLAR POR LA MITAD ___________ 0 1
PONER EN EL SUELO (o MESA) ___________ 0 1
LECTURA
Por favor lea esto y haga lo que dice. [Muéstrele el impreso de estímulo]
CIERRE LOS OJOS 0 1
ESCRITURA
Por favor, escriba una frase. [Si no responde, diga: Escriba sobre el tiempo.] 0 1
Coloque el trozo de papel en blanco (sin doblar) frente a la persona que realiza el examen y dele un bolígrafo o un lápiz. Anote 1 punto si la frase es comprensible y contiene sujeto y predicado. Ignore los errores de gramática u ortografía.
DIBUJO
Por favor, copie este dibujo. [ Muestre los pentágonos del impreso de estímulo.] 0 1
Anote 1 punto si el dibujo consta de dos figuras con cinco lados que forman una intersección para formar una figura de cuatro lados.
Anote este valore en la página de puntuaciónde las evaluaciones del CRD.
Nombre del evaluador: _________________________
Firma del evaluador: ___________________________
Fecha: _______________________________________
Hora de comienzo de la evaluación: ______________ (00:00 – 23:59)
MMSE Version date: September 6, 2002
Puntuación total = ________
(Sume todos los puntos) (máx. 30)
MMSE Version date: September 6, 2002
CIERRE LOS OJOS
AGRADECIMIENTOS
Escribir una tesis doctoral no es una tarea sencilla, aunque resulte mucho más fácilcuando se cuenta con un equipo que te apoya en tu trabajo y personas que realizanparte de las diversas gestiones necesarias. Durante esta etapa son muchas las personasque me han rodeado. No solamente personas sin más, sino sobre todo, un equipohumano muy especial, al que no puedo dejar de mencionar.
Desde los últimos años de estudiante de la carrera de nutrición una mujer me ha guiado,la Profesora María Marcela González Gross y a ella le debo en gran parte el constanteempuje para seguir con mi andadura en la investigación. He aprendido mucho de ellay han sido muy gratificantes todos estos años. En realidad, no existen palabras paraagradecer todo el apoyo y confianza que ella ha puesto en mi durante todo este tiempo.He contado con su apoyo, cuento y estoy segura que contaré con él.
Quiero expresar mi agradecimiento a mi co-director Dr. Agustín Meléndez Ortegaque siempre ha estado a mi lado, por su motivación, por sus comentarios y críticasconstructivas que poco a poco me han permitido completar el trabajo, por sus consejos,especialmente en los momentos menos agradables y por la amistad que como personame ha demostrado a lo largo de la realización de esta tesis. Aunque no lo parezca,sí me han quedado bien claras sus historias y moralejas y ya poseo un buen “saco dehistorias” que contar.
Quiero agradecerle a los Prof. Klaus Pietrzik y Prof. Manuel Castillo su contribucióna mi formación y a los trabajos presentados en diversas ocasiones.
Gracias también a los doctores Gabriel Rodríguez Romo, Pedro Jesús Jiménez Martíny Enrique López Adán, por sus comentarios, correcciones y consejos durante la pre-lectura de la tesis que me han permitido detectar errores y conseguir una mayor claridaden su redacción.
Quiero agradecer también a Ma Jesús Morala del Campo y Blanca Clavijo Juaneda dela Asociación de Familiares de Alzheimer (AFAL) el apoyo facilitado para conseguirla participación de las residencias de ancianos seleccionadas y al personal tanto sani-tario como auxiliar de éstas, la cooperación prestada para la realización de las pruebasy la recogida de las muestras. Merecen especial mención los médicos Javier Caballero,José Luis Tobaruela, Patricia Morán y Juan Carlos Pastor de dichas residencias por sutotal apoyo y colaboración. No puedo olvidar la sincera cooperación de las personasmayores residentes que participaron de forma desinteresada e ilusionada en estos estu-dios, que me dieron un extraordinario ejemplo de dignidad.
A Gonzalo Palacios por su dedicación y colaboración y por su apoyo profesional ypersonal sincero.
149
A David, Víctor, Ana, María, Rocío, y Jara que participaron de forma activa en larecogida de datos y especialmente a Raquel por su más estrecha colaboración.
A Monika, Christina, Eva, Rebecca e Ilaria por toda su ayuda, interés y dedicación entodos estos proyectos.
Quiero expresar mi más sincero agradecimiento a Pilar Irureta-Goyena Sánchez y alpersonal de la Biblioteca de la Facultad por su ayuda y gestiones para conseguir algu-nas referencias difíciles de encontrar; igualmente, quiero agradecerle al personal delRectorado su amable trato y la información que me han proporcionado.
A Laura Barrios del Consejo Superior de Investigaciones Científicas (CSIC) por su as-esoramiento en el tratamiento estadístico de los datos y a Gádor Faura y especialmentea Paloma Navarro por las gestiones administrativas necesarias para la realización deesta tesis.
Al mismo tiempo, quiero agradecer a la Universidad Politécnica de Madrid la oportu-nidad que me ofrecieron para realizar el doctorado concediéndome la beca predoctoraly la posibilidad de la realización de mis estancias en el extranjero.
Cómo olvidar a Rosa Torres, siempre dispuesta a colaborar y con la capacidad de podertolerar bien los momentos tensos de la elaboración de la tesis debidos a cuestionestodavía sin resolver o partes del trabajo sin completar o a Ms Diane Schofield que harevisado las diferentes copias de esta tesis y los artículos, comunicaciones y postersoriginales de estos estudios.
I would like to thank all the people I got the chance to know during my internships inLisbon, Lille and Coleraine. You are all so different and also all so very important forme.
Meinem Vater Carl-Heinz für sein großes Verständnis.
Meiner Mutter Christina für ihre einzigartige Sichtweise der Dinge und meinem BruderBenedikt für seine allumfassende Liebe. Mögen sie beide in Frieden ruhen.
Me gustaría agradecer también a todos mis profesores de idiomas su gran dedicacióna la enseñanza y por haber podido mantenerme motivada, “CHAPEAU”!
A Ricardo Sola por su asesoramiento en los temas hematológicos y a Reme por apor-tarme su experiencia en los temas del laboratorio.
Quiero agradecer especialmente a Rosa Carazo, Rosa Madre y Pilar Carazo no sólo sumaravillosa hospitalidad, sino su empatía, su capacidad de tranquilizarme y ponermela cabeza donde debería estar en tiempos de emociones alteradas, sea mirando cactusesde verdad y/o en documentales o sea llevándome para pedir una tabla en Rossini; caberesaltar que con el último van incluidas lecciones de Español a un Nivel mucho másalto o elevado del Nivel Europeo C2 por parte del dueño.
150
A Kati Schulz y Eva Olsson por ser tan buenas personas y por la amistad que en estetiempo me han dispensado.
A Patri, por haber sido mi apoyo afectivo durante la última fase de la tesis y por pro-porcionarme esos fantásticos momentos que hacen que las cosas sean más fáciles.
Por último, quiero agradecer a las empresas Axis Shield LTD, Abbott Diagnostics S.A.,Whitehall-Much GmbH su apoyo y destacar especialmente la colaboración con el Dr.Thomas Schettler y el Dr. Edward Valente.
Espero que los muy individuales lazos establecidos con cada una de estas personas nose rompan jamás ahora que voy a comenzar un nuevo camino.
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CURRICULUM VITAE
EDUCATION
2008-2012: PhD student Universidad Politécnica de Madrid (UPM), Madrid, Spain01.01.2008-31.12.2011: Grant for the realization of the PhD programme funded by UniversidadPolitécnica de Madrid (UPM)01.05.2006-31.12.2007: Grant related to laboratory research funded by the I+D Contract Programmesigned by the UPM and the Madrid Community01.01.-31.04.2006: Grant within the vitamin B12 project funded by Universidad Politécnica de Madrid (UPM)30.09.-31.12.2005: Research contract within the HELENA project. University of Bonn, Germany.1999-2005: German Diploma (= 5 years) in Nutrition Sciences, University of Bonn, Germany (Rheinische Friedrich-Wilhelms –Universität)
PARTICIPATION IN RESEARCH PROJECTS
Physical activity, fitness, ageing and quality of life: functional, cognitive, social and biomedical determinants. The FELIX study (Project code: c1538b684423560a069997e544772e65)300.000 € (passed first round, without funding). 2012-2014. Marcela González-Gross
Estudio Longitudinal EXERNET: Genética y su relación con el deterioro de la composición corporal y la condición física en personas mayores de 65 años no institucionalizadas (147/2011). Ministerio de Sanidad, Política Social e Igualdad-Instituto de Mayores y Servicios Sociales (IMSERSO) 2012-2013. Marcela González-Gross/coordinador: Ignacio Ara Royo (Universidad de Castilla-La Mancha)
Determinantes de riesgo de primeros eventos cardiovasculares. Un estudio coordinado de casos y controles anidado de la cohorte PREDIMED. Antioxidantes y estrés oxidativo (PI11/01791) Instituto de Salud Carlos III. 2012-2014. Marcela González-Gross/coordinador: Pep Tur (Universidad de las Islas Baleares)
Estudio de las alteraciones neurodegenerativas en la población geriátrica debidas a la deficiencia de vitamina B12 [Research Project of the neurodegenerative changes in the geriatric population due to vitamin B12 deficiency] (GRS 309/A/08). Regional Administration for Health of the Public Health Service of Castilla y León (SACYL, Sanidad de Castilla y León). 2008-2009. Principal researchers: Belén Cantón Álvarez, Carmen de la Fuente Hontañón, Carmina Vázquez Pedrazuela
La composición del ternasco de Aragón, cualidades dietéticas y su influencia en la salud humana [Composition of the lamb meat of Aragón (GI), dietetic properties and its influence on human health]. (PI08/0022 – PET2007-07-C08). Association for lamb meat (Ternasco) of Aragón. Hospital “Obispo Polanco”, Teruel. Superior School for Gastronomy of Aragón, National Institute for Agrarian and Alimentary Investigation and Technology (INIA, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria). 2008-2009. Principal researcher: Luis Alberto Moreno Aznar. Personal researcher: Grupo GENUD (Growth, Exercise, Nutrition and Development), University school for Health Sciences, University of Zaragoza, Spain
Estudio Multi-céntrico para la Evaluación de los Niveles de Condición Física y su relación con Estilos de Vida Saludables en la población mayor española no institucionalizada [Multicenter study for the assessment of physical fitness of Spanish free-living elderly and its relation to a healthy life style]. Spanish Ministry for Labour and Social Affairs. 2008-2009. Marcela González-Gross / coordinator: Ignacio Ara Royo (University of Zaragoza)
EXERNET - Research Network in Physical Exercise and Health for Special Populations. (DEP2005-00046/ACTI). Strategic Action on Sports and Physical Activity, National Plan for I+D+i 2004-2007. Spanish Ministry of Education and Science. 2006-2008. Responsible researcher: Jose Antonio Casajús Mallen (University of Zaragoza)
Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA). 6th Framework Programme of the European Community (Contract FOOD-CT-2005-007034). 2005-2008. Marcela González-Gross/ coordinator: Luis Moreno (University of Zaragoza)
Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Longitudinal study for the evaluation of changes in the vitamin B12 status in Spanish elderly (P061100509). Axis-Shield Diagnostics Ltd. 2006- Feb 2009. Responsible researcher: Marcela González-Gross
Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Effect of a cobalamin supplement during one month. (P051100649) Whitehall Much GmbH. 2006-2007. Responisble researcher: Marcela González-Gross
Vitamin B12 deficiency as a risk factor for neurodegenerative disease. Masking of the deficiency by folic acid (pteroil-monoglutamic acid) and effect of the nutritional intervention with 5-methyl-tetrahydrofolate. EPROVA Merck GMBH. 2004-2005. Responsible researchers: Klaus Pietrzik/Manuel J. Castillo/ Marcela González-Gross
EDUCATION AND TRAINING
Accreditation course for anthropometric assessment by ISAK, Level I (16.-19.10.2007). Organized by Universidad Complutense de Madrid (UCM) in collaboration with the International Society for the Advancement of Kinanthropometry (ISAK)
Training course for the user of the AxSYM System (24.-25.05.2006). Organized by Abbott Diagnostics, Madrid, Spain
HELENA Cross-sectional Study Training Workshop. (15.-20.01.2006). Organized by E.U. Ciencias de la Salud. University of Zaragoza, Spain
Summer school “Fundamentals of Nutritional Epidemiology”. (19.-30.07.2004). Organized by German Institute of Human Nutrition (DIfE). Potsdam, Germany
PUBLICATIONS
Journal Publications
• Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. Polymedication in institutionalized elderly and its effect on vitamin B12, folate, and homocysteine levels. Nutr Hosp. 2012;27(1): 296-300.
• Albers U, Pedrero R, Meléndez A, Pietrzik K, Castillo MJ, González-Gross M. Effectiveness of a 28 days oral cyanocobalamin supplementation on vitamin B status in Spanish institutionalized elderly. (accepted in Int J Vit Nutr Res).
• Spinneker A, Egert S, González-Gross M, Breidenassel C, Albers U, Stoffel-Wagner B, Huybrechts I, Manios Y, Venneria E, Molnar D, Widhalm K, Moreno L, Stehle P. Lipid, lipoprotein and apolipoprotein profiles in European adolescents and its associations with gender, biological maturity and body-fat-The HELENA Study. Eur J Clin Nutr. 2012 Jan 18. doi: 10.1038/ejcn.2011.222. [Epub ahead of print].
• Gomez-Cabello A, Pedrero-Chamizo R, Olivares PR, Luzardo L, Juez-Bengoechea A, Mata E, Albers U, Aznar S, Villa G, Espino L, Gusi N, Gonzalez-Gross M, Casajus JA, Ara I . Prevalence of overweight and obesity in non-institutionalized people aged 65 or over from Spain: The Elderly EXERNET Multi-center Study. Obesity Reviews 2011;12(8):583-92.
• Ortega FB, Chillón P, Ruiz JR, Delgado M, Albers U, Alvarez-Granda JL, Marcos A, Moreno LA, Castillo MJ. Sleep patterns in Spanish adolescents: associations with TV watching and leisure-time physical activity. Eur J Appl Physiol 2010;110(3):563-73.
• González-Gross M, Breidenassel C, Gómez S, Ferrari M, Beghin L, Spinneker A, Díaz L, Maiani G, Demailly A, Al-Tahan J, Albers U, Wärnberg J, Stoffel-Wagner B, Libersa C, Pietrzik K, Marcos A, Stehle P, on behalf of the HELENA study group: Use of fresh blood samples to analyse multiple nutritional biomarkers in a European Multicenter Nutritional study. Experience from the HELENA cross sectional pilot study. Int J Obes 2008; 32(suppl 5): S66-S75.
• González-Gross M, Sola R, Albers U, Barrios L, Alder M, Castillo MJ, Pietrzik K: B-Vitamins and Homocysteine in Spanish institutionalized elderly. Int J Vit Nutr Res 2007;77:22-33.
• González-Gross M, Benser J, Breidenassel C, Albers U, Huybrechts I, Valtueña J, Spinneker A, Segoviano M, Widhalm K, Molnar D, Moreno LA, Stehle P, Pietrzik K on behalf of the HELENA Study group. Characterizing blood folate, vitamin B12 and vitamin B6 levels in adolescents in Europe: The HELENA Study. (submitted).
• Maroto B, Valtueña J, Albers U, Benito PJ, González-Gross M. Acute physical exercise, both of maximal and submaximal intensity, increase Homocysteine concentrations in young trained male subjects. (submitted).
• Pedrero-Chamizo R, Albers U, Tobaruela JL, Meléndez A, Castillo MJ, González-Gross M. Relation between physical strength and cognitive function in Spanish institutionalized elderly". (submitted).
Book chapters
• Meléndez Ortega A, Albers U, Pedrero-Chamizo R, López Torres O. Aspectos sociológicos del envejecimiento en Ejercicio Físico y Salud en Poblaciones Especiales in: Casajus JA, Vicente-Rodríguez G. Ejercicio físico y salud en poblaciones especiales. EXERNET. Editorial CSD. Colección ICD. ISBN 9788479492168. 2011, pp . 101-121.
• Meléndez A, Albers U: Respuesta fisiológica al ejercicio en edad infanto-juvenil. in: Redondo Figuero C, González-Gross M, Moreno LA, García-Fuentes M, eds. Actividad física, deporte, ejercicio y salud en niños y adolescentes. Editorial Everest. ISBN 9788444102597. 2010, pp 53-66.
Communications presented at congresses
• Albers U, Pedrero R, Moran P, Cañada D, Meléndez A, González-Gross M. One year follow-up of biochemical and cobalamin status among Spanish institutionalized elderly. 11th European Nutrition Conference (FENS). Madrid, Spain. 26-29.10.2011. Ann Nutr Metab 2011, 58(suppl 3): 48.
• González-Gross M, Pedrero-Chamizo R, Albers U. Interrelación entre la condición física, el estado nutricional vitamínico y el estado cognitivo y su influencia sobre la calidad de vida en mayores institucionalizados y de vida independiente. 4° Congreso internacional de Actividad Física Deportiva para Mayores. Málaga, Spain. 4-6.03.2011. Proceedings ISBN 978-84-777858980. pp. 71-86.
• Vicente-Rodríguez, G; Rey-López, JP; Mesana, MI; Poortvliet, E; Albers, U, Sjöström, M and Moreno, LA, on behalf of the HELENA study group. Bicipital and tricipital are the best predictive skinfolds for Bod Pod body density using t-distribution modeling. The HELENA Study. Child Health in Europe, The IDEFICS Study. Zaragoza, Spain. 8-9.11.2010.
• Albers U, Palacios G, Pedrero-Chamizo R, Meléndez A, Pietrzik K, González-Gross M. Plurimedication among Spanish institutionalized elderly, a fact that should be considered when examining their nutritional status. II World Congress of Public Health Nutrition and I Latin American Congress of Community Nutrition. Porto, Portugal. 23-25.09.2010. Public Health Nutr 2010, 13(9A): 248.
• Pedrero-Chamizo R, Albers U, Valtueña J, Casajús JA, Castillo MJ, González -Gross M. Strength training is essential in elderly women. 5th World Conference on Women and Sport. Sydney, Australia. 20-23.05.2010. Abstract Book ISBN 978-0-9806738-0-7. p. 209.
• Pedrero-Chamizo R, Albers U, Valtueña J, Moreno S, Pietrzik K, Castillo MJ, González-Gross M. Relation Between Physical Strength and Cognitive Function in Spanish Institutionalized Elderly. 3rd
International Congress on Physical Activity and Public Health (ICPAPH). Toronto, Canada.5-8.05.2010
• Chamizo R, Albers U, Morencos E, Pastor JC, Meléndez A, Castillo MJ, González–Gross M. Valoración del estado nutricional en mayores institucionalizados a través de diferentes marcadores. II Congreso de la FESNAD. Barcelona (Spain). 3-5.03.2010.
• Albers U, Pedrero-Chamizo R, Meléndez A, Castillo MJ, Pietrzik K, González-Gross M: How effective can a 500 µg oral supplement of vitamin B12 for just 28 days be for institutionalized elderly? XIXth IAGG World congress of gerontology and geriatrics. Paris, France. 5-9.07.2009 JNHA 2009, 13 (suppl 1): S641.
• Pedrero-Chamizo R, Albers U, Valtueña J, Cañada D, Jathe R, Meléndez A, Castillo MJ, González-Gross M. Evolución de los niveles de fuerza en población mayor institucionalizada de la Comunidad de Madrid. III Jornadas Nacionales de Medicina del Deporte. Zaragoza, Spain. 29-30.05.2009.
• Al-Tahan J, Albers U, Valtueña J, Spinneker A, Breidenassel C, Azzini E, Diaz LE, Marcos A, Gottrand F, Kunert J, Manios Y, Pietrzik K, Stehle P, González-Gross M, on behalf of the HELENA study group. Folate and vitamin B12 status in European adolescents: associations with gender, age, and maturity. Health behaviour and health status in European adolescents: The HELENA project. Pre congress of the 17th European Congress on Obesity. Amsterdam, Netherlands.6.05.2009.
• Pedrero-Chamizo R, Albers U, Valtueña J, Jiménez-Pavón D, Meléndez A, González-Gross M. Valores de referencia en fuerza de prensión manual en población mayor institucionalizada y de vida independiente. III Congreso Internacional de Actividad Físico Deportiva para Mayores. Málaga, Spain. 12-14.03.2009.
• Pedrero-Chamizo R, Albers U, Piera M, Jiménez-Pavón D, Meléndez A, Castillo MJ, González-Gross M: Gender, age and cognitive score effects on strength in Spanish institutionalized elderly. 13th
Annual Congress of the European College of Sport Sciences. Estoril, Portugal. 9-12.07.2008.• Pedrero-Chamizo R, Albers U, Valtueña J, Pietrzik K, Meléndez A, Castillo MJ, González-Gross M.
Vitamin B12 deficiency. Effects on physical strength and cognitive health in Spanish institutionalized elderly. 6th international Conference on Nutrition and Fitness. Athens, Greece.15-17.05.2008.
• Albers U, Pedrero R, Díaz V, Tobaruela JL, Pietrzik K, Castillo MJ, Meléndez A, González-Gross M. Gender differences in vitamin B12 status and functional fitness parameters in Spanish institutionalized elderly. 6th World Congress on the Aging Male. Tampa, Florida, United States, 21-24.02.2008.
• Pedrero R, Albers U, Jiménez-Pavón D, Cupeiro R, Meléndez A, Castillo MJ, Gutiérrez A, González-Gross M. Valoración funcional en personas mayores institucionalizadas. XII Congreso Nacional de la Federación Española de Medicina del Deporte. Sevilla, Spain. 24-27.10.2007.
• Albers U, Meléndez A, Pietrzik K, Tobaruela JL, Castillo MJ, González-Gross M. Effectiveness of oral Vitamin B12 supplementation in Spanish institutionalized elderly. 7th International Conference VITAMINS - Nutrition and Diagnostics. Prague, Czech Republic. 19-21.09.2007.
• Albers U, Díaz V, Peinado A, Alvarez M, Sola R, Pietrzik K, Caballero J, Meléndez A, Castillo MJ, González-Gross M. Physical strength and Vitamin B12 in relation to cognitive score in Spanish institutionalized elderly. 10th European Nutrition Conference. Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 119-120.
• Spinneker A, Albers U, González-Gross M, Sola R, Castillo MJ, Pietrzik K. Difficulty in proving the masking of cobalamin by folic acid supplementation – PGA vs 5-MTHF. 10th European Nutrition Conference. Paris, France. 10-13.07.2007. Ann Nutr Metab 2007; 51 (suppl 1): 216.
• Moran P, Albers U, Pedrero R, Sola R, Castillo MJ, González-Gross M. La deficiencia de vitamina B12 como factor de riesgo de enfermedades neurodegenerativas (cognitivas y funcionales) en las personas mayores. 49 Congreso de la Sociedad Española de Geriatría y Gerontología. Palma de Mallorca, Spain. 6-9.06.2007.
• Albers U, González-Gross M, Sola R, Castillo MJ, Pietrzik K. Einfluss von 5-MTHF- und PGA-Supplementen auf den hämatologischen Status von spanischen Heimbewohnern. 43rd Congress of the German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE). Stuttgart, Germany. 9-10.03.2006.
ORGANIZATION OF CONGRESSES
• Congress PRONAF. Madrid, Spain. 15-17.12.2011.• International Symposium HEALTH(A)WARE 2009. Physical Activity and Health Education in
European Schools. Madrid, Spain. 23.04-24.04.2009.
ATTENDANCE AT CONGRESSES, SYMPOSIA AND COURSES
• Bial forum of Neurology 2009, organized by Bial. Lisbon, Portugal. 21.11.2009. • Jornada “Doença de Alzheimer e outras formas de demência: Que políticas?” organized by Alzhei-
mer Portugal. Lisbon, Portugal. 29-30.10.2009.• International Symposium HEALTH(A)WARE 2009. Physical Activity and Health Education in
European Schools. Madrid, Spain. 23.04-24.04.2009.• II National Symposium about Women in Sports “Promotion of women in sports: programmes and
activities” organized by the Spanish National Sports Council. Madrid, Spain. 14-15.02.2009.• Annual meeting of the committees of the forum Gain Health (Gana Salud) organized by the forum
Gain Health in collaboration with the Community of Madrid. Madrid, Spain. 28-30.01.2009.• International symposium of exercise and health in special populations. Huesca. Spain.
23-24.11.2008.• HELENA Symposium organized by the European Consortium HELENA. Granada, Spain.
20-22.04.2008.• I National Symposium about Women in Sports “Promotion of women in sports: programmes and
activities” organized by the Spanish National Sports Council. Madrid, Spain. 14-15.02.2008. • V Conference of the University Complutense of Madrid (UCM) for Human Nutrition and Dietetics:
“Health and Fast Food”. Madrid, Spain. 5.11.2007.• III International University Conference for Health Science and Sports organized by the Comité
Olímpico Español (COE), Real Madrid and Atlético de Madrid, Madrid, Spain. 18-20.10.2007.• International Symposium about Physical Activity and Health organized by the General Directorate
for the Promotion of Sports, The Sports and Cultural Council of the Madrid Community, Madrid, Spain. 27-29.11.2006.
• International symposium: ”Dementia: a growing epidemic” organized by the Foundation Ramón Aceres, Madrid, Spain. 15-16.11.2006.
• Regional conference about the recent progresses in Alzheimer disease organized by the Association of the family members of Alzheimer patients (Asociación de Familiares de Enfermos de Alzheimer y otras Demencias de la Comarca de Medina del Campo, AFAMEC). Medina del Campo, Spain. 28.09.2006.
• Dietary Supplements and Health; organized by the Novartis Foundation in collaboration with the Royal Society of Medicine and The Physiological Society, London, UK. 12.05.2006.
• II Conference of nutrition, food technology and food safety; organized by the University San Pablo CEU, Madrid, Spain. 4-5.05.2006.
• 41st Annual Congress of the German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE), Freising-Weihenstephan, Germany. 11.-12.03.2004.
• Conference: Nutrition science as a multi disciplinary focusing. German Nutrition Society (Deutsche Gesellschaft für Ernährung e. V. / DGE). Bonn, Germany. 9-10.10.2003.
WORK EXPERIENCE IN LABORATORY
2005-ongoing: Biochemical laboratory of the Facultad de Ciencias de la Actividad Física y del Deporte-INEF. Universidad Politécnica de Madrid. Technics used: Axsym System (Abbott Diagnostics), automatic blood cell counter Celltac E (Nihon Kohden), ELISA for adiponectin and leptin with ELISA reader Multiscan (Thermo Fisher).
08.11-15.11.2007: Universidad de Cantabria, Santander, Spain. Technics used: determination of the methylenetetrahydrofolate gene (MTHFR) C677T polymorphism including DNA extraction, amplification by Frost, enzymatic digestion with Hinf I (GE Healthcare), electrophoresis.
01.11.2003-01.02.2004: Facultad de Medicina de la Universidad Granada, Dpto. Fisiología Médica in collaboration with the Hospital Clínico Universitario San Cecilio (Granada). Technics used: ImX (Abbott Diagnostics), blood smears coloured with Giemsa, DNA Extraction via QIAGEN kits.
04.02.-10.04.2002: Microbiologic laboratory of the Universitá degli studi de Perugia, Italy, Dipartimento de scienze degli alimenti; Sezione di microbiologia agro-alimentare. Technics used: determination of bioactive substances in various milk types, identification of lactic acid produced by bacteria (LAB), test of antibacterial activity, DNA purification of the “LAB”, determination of proteins via “Lowry method”.
05.03.-12.04.2001: Nordmilch eG Beesten, Germany. Technics used: chemical and microbiologic analyses of milk products, component analyses with infrared instruments, fat content determination via “Röse-Gottlieb”, purity grade and contents of milk and milk powder, determination of the dried extract of milk with sand at 102°C, analyses of industrial wastewater, Microbiology: method by Koch (agar plates), counting of yeast, enterobacteria and fungi.
MENTORING OF FINAL PROJECTS
• Title: ”Cognitive function and B vitamins in institutionalizedelderly“Student: Ilaria Mattoni, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik). Year: 2010.
• Title: “Вody composition and health-related aspects in institutionalized elderly”Student: Rebecca Jathe , Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. November 2010.
• Title: “Vergleichende Betrachtung von Homocystein, Lipiden und weiteren biochemischen Risikoparametern unter Alters- und Gesundheitsaspekten bei spanischen Altenheimbewohnern”Student: Eva Kiesswetter, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. February 2009.
• Title: “Experimental evaluation of changes in the vitamin B12 status in Spanish elderly and theoretical implications from Transcobalamin 776C>G polymorphisms”
Student: Christina Behrendt, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. June 2008. • Title: “Influence of oral vitamin B12 supplementation on selected haematological and biochemical
parameters in Spanish institutionalized elderly”Student: Monika Alder, Rheinische Friedrich-Wilhelms-Universität Bonn Supervisors: Marcela González-Gross and Prof. Klaus Pietrzik. Year: March 2007.
TEACHING EXPERIENCE
01.01.2010-31.12.2011, participating in lectures of
• Dietetics (Dietética, Cod. 2209, Licenciatura en Ciencia y Tecnología de los Alimentos 2º ciclo, 6 credit points)
• Nutrition and Ergogenic Aids in Sport (Nutrición y Ayudas Ergogénicas en el Deporte, Cod. 0645, Licenciatura de Ciencias de la Actividad Física y del Deporte, 4 credit points)
LANGUAGES
Mother tongue: GermanOther language(s):Spanish: Diploma of Spanish as a Foreign Language (Diploma de Español como Lengua Extranjera, DELE), Superior level (C2*) by Cervantes InstituteEnglish: Certificate in Advanced English, CAE (C1*) by University of Cambridge* Common European Framework of Reference (CEF) level
