J. mem. Defic. Res. (1985) 29, 233-240

A DOUBLE BLIND STUDY OF VITAMIN Bf, INDOWN'S SYNDROME INFANTS. P A R T I -CLINICAL AND BIOCHEMICAL RESULTS

MARY COLEMAN

Department of Paediatrics, Georgetown University School of Medicine, Washington, DC,USA

SUZANNE SOBEL,HEMMIGEN. BHAGAVAN

Children's Brain Research Clinicy Washington, DC, USA

DAVID COURSIN

Department of Paediatrics, Georgetown University School of Medicine

ANNE MARQUARDT, MARIE GUAYand CARL HUNT

Children's Brain Research Clinic, Washington, DC

INTRODUCTION

In 1965 it was found that serotonin (5-hydroxytryptamine or 5-HT), a metabolite withneurotransmission as one of its functions, was significantly depressed in the blood ofretarded children with Down's syndrome (DS) (Rosner et a/., 1965; Tu & Zellweger,1965). Because of the importance of 5-HT in both the embryological (Lauder &Krebs, 1978) and infant's central nervous system (Essman, 1978), our research groupundertook a study of the effects of the elevation of the amine by administration of itsprecursor amino acid, 5-hydroxytryptophan (5-HTP) in DS infants (Coleman,1973a). The development of the infantile spasms syndrome in 14% of the patientsreceiving 5-HTP brought these studies to a halt (Coleman, 1971).

However, other ways of raising 5-HT in this patient group were explored and themost promising appeared to be the administration of vitamin B^ (B^), the co-enzymeof the second step of 5-HT metabolism (Coleman, 1973b). B^ is already established asa treatment of value in several other diseases of the central nervous system (Weiner,1976).

B(, is the generic name for all 2-methylpyridine derivatives with biological activity,including an alcohol (pyridoxol), an amine (pyridoxamine) and an aldehyde(pyridoxal). Pyridoxa!-5-phosphate (P5P) is the major co-enzyme metabolite of thisgroup of compounds and is involved in a large number of transaminations,dccarboxylations, deaminations and other reactions involving most brain amino acids

Correspondence: Mary Coleman MD, 2525 Belmoni Road, NW, Washington. DC 20008, USA.Received 24 October 1984

233

234 M. COLEMAN et al.

as well as lipid, nucleic acid and glycogen metabolism. Of particular interest in DSpatients is the role of P5P in the development of myelin. In rats madepyridoxine-deficient during the period of development of the central nervous system,myelin is both qualitatively and quantitatively different from normal rats (Stephens &Dakshinamurti, 1975). Phospholipids are strikingly reduced in such animals (Kurtz& Kanfer, 1973). The deficiency of fatty acids, a major portion of brain lipids, has acrucial role in determining the properties of membranes (Chauhan & Dakshinamurti,1979). In one study, abnormal myelin (Banik et al., 1975) was noted in DS brains. Inanother study, a depression of serum phospholipids was noted. The authorsspeculated that one factor could be the relative B^ deficiency in the tissues of thepatients (Davidenkova et al., 1974).

Also, there is much evidence beyond 5-HT studies to suggest that DS patients areBft deficient (Bhagavan et al., 1973). Leukocytes and platelets have been shown tohave less PLP (Coburn & Seidenberg, 1969; Mahuren & Coburn, 1974). Indirectstudies using deoxypyridoxine administration by the McCoy group suggests that DSindividuals have a smaller body pool of B,, compared to controls (McCoy & Chung,1964; McCoy, Anast & Naylor, 1964; McCoy & England, 1968). The reduction oftaurine metabolism in DS patients is another possible indirect piece of evidence for B^deficiency (Goodman, King & Thomas, 1964; Boullin, Airaksinen & Paasonen,1975). An enzyme, dopamine-B-hydroxylase, that utilizes pyridoxine as a co-enzyme,also is low in DS patients (Coleman et al., 1974). This paper (Part 1) describes theresults of the clinical portion of the evaluation; the following paper (Part 2) reports onthe effects of B^ on the auditory evoked potentials of the infants enrolled in this study.

P A T I E N T S A N D M E T H O D S

In the DS clinic, the double blind 3-year trial of B(, or placebo was planned for 20patients. During the first year of the protocol, two patients with congenital cardiacdisease died, and one was able to be replaced before the protocol was closed. Thus,the double blind study ended up with 19 patients. All infants, if they were under 8weeks of age, were admitted who applied for a clinic appointment during the timeperiod of the protocol. During this period, several families declined to participate in adouble blind protocol and did not remain members of the clinic population. Thus, theparents of patients in this study did not represent an unselected population of DSparents, but rather those parents willing to have their children be part of a researchproject. All patients in the clinic were required to enroll in an infant stimulation orinfant learning programme, in addition to the research protocol.

An additional estimated 400 patients were given B(, in pharmacological doses.These consisted of two 'open' populations: 25 patients who received the vitamin priorto the beginning of the protocol as we learned experimentally what dose should begiven to keep the whole blood serotonin (5-HT) level elevated and what early clinicaleffects might be apparent; and the remainder of the patients who came to the clinicafter the protocol was closed and constituted a large population to check for sideeffects. This open study of the effects of the vitamin included patients started on thevitamin in all age groups up to 12 years of age. A few of these patients had been started

Bft IN DOWN'S SYNDROME — PART 1 235

on the vitamin by other physicians but transferred to the clinic to be followed there.Patients in the open populations received the vitamin for up to 8 years; dosages rangedfrom 15 mg/kg to 65 mg/kg. The largest doses occurred in older patients and werebased on attempts to normalize whole blood serotonin levels, a process which oftenbecomes increasingly more difficult with age. Although this paper is primarilyconcerned with the double blind patients, side-effect information will be includedfrom the open populations. Informed consent was obtained from the parents of thepatients.

The double blind protocol consisted of the administration oi" pharmacological dosesof Bfc (25 mg/kg for the first 6 months of age and 35 mg/kg thereafter) or placebo toeach infant once daily in the morning until they were 3 years of age. Patientassignment was by random chance. Since the patients were infants, the vitamin wasadministered in solution. The patients were brought to the clinic in most cases eighttimes a year in the first year of life and four times a year in subsequent years. After thechild's third birthday and the completion of the 3-year independent evaluations, thecode was broken for parents and professionals.

Each clinic visit consisted of a brief history and physical examination, routinelynoting on each visit the height, weight, cranial circumference, and amount of tongueprotrusion. Also, as part of each clinic visit, blood samples were obtained by venouspuncture. The heparinized whole blood was frozen immediately in dry ice for 5-HTand pyridoxal phosphate (PLP) determinations. 5-HT was analyzed within 48 hourswhile PLP was determined within 2 weeks. Platelet counts were also determinedusing the standard Coulter counter method. 5-HT was determined fluorometricallyaccording to the method of Yuwiler et al. (1970). PLP in whole blood was extractedusing perchloric acid. PLP was assayed by a sensitive radioisotopic method developedin our laboratories using 1-tyrosine apodecarboxylase with 1-tyrosine-tagged withcarbon 14 as the substrate (Sundaresan & Coursin, 1970).

In addition to the clinic visits, patients had independent evaluative procedures.Auditory and visual evoked potentials were measured in each child at 1 and 3 years ofage. (The results of the auditory evoked potential study are described in Part 2 of thispaper.) Psychological testing of each child was done at 3 years of age using the BayleyScales of Infant Development or the Stanford-Binet and the Viaeland Social MaturityScale. As an extra procedure, following after the code had been broken and thus nottechnically part of the double blind study, a repeat psychological test was done onpatients when they reached 6 years of age.

Methods of statistical analysis had to utilize those suitable for a small number. TheKurskall-Waliace one way analysis of variance and the Chi square were used in thispaper.

R E S U L T S

Parents kept all scheduled appointments with rare exceptions. When the double blindcodes were revealed, 10 patients were found to be receiving B^ and nine patients werereceiving placebo. There were six male infants and four female infants who receivedthe vitamin; there were five male infants and four female infants on placebo.

236 M. COLEMAN et al.

The administration of the vitamin elevated PLP 100 to 500 times the levels seen inplacebo patients. Thus, it was possible to determine whether all patients had taken thevitamin solution during the 3-year monitoring period. Eight parents clearly had givenadequate doses at all times when measured; one parent had given adequate dosesexcept for two occasions and the tenth parent had given the vitamin except for fouroccasions.

Laboratory results

The results of Bf, administration on PLP levels averaged 6017 ng/ml for this study.For patients on placebo, the mean level was 13 ng/ml.

The results of Bf, levels on 5-HT levels in treated and placebo patients indicatedthat during the first year of administration of the vitamin the levels of 5-HT weresignificantly elevated comparing the two groups (/=2.32, / ' -0 .04) . However, in thesecond (r=1.03) and third (/=1.7) years of the study, although the treated patientsalways averaged higher levels than placebo patients, these levels were not statisticallysignificantly elevated.

Clinical results

A comparison of the height and weight of the children at birth and at 3 years of agewas done. In order to correct for genetic factors, the height and weight of the parentswas also examined. By chance, the mothers of the control group were 5 inches talleron the average and the difference in heights between the two sets of mothers wasstatistically significant! Whether this unplanned-for variable had any meaning in thestudy is unknown, but there was no significant difference between the height andweight of the treated and control patients.

In the case of the cranial circumferences, again there was no difference betweentreated and control patients. Broken down by sex, the statistical results were: males(treatment vs. control) F - 1 . 3 8 , no difference; and females (treatment vs. controlF=14.19, P^O.IS. In both groups, the cranial circumferences were smaller thanaverage for normals.

Psychological testing at 3 years of age indicated no difference between the twogroups in any type of testing. The mental age statistics at 3 years of age were x^^ 1-6,P^0.19. Psychological testing was repeated at 6 years of age. After 3 years of age,these children were, of course, no longer part of the double blind protocol.) In thefollow-up 6-year testing, the mental age statistics were unchanged between the twogroups; x^=I-6, P&0.19. However, the treatment group was significantly higher forsocial quotient, ?=2.81, P=0.04.

An informal system of scoring the amount of tongue protrusion was developedduring the study. No statistically significant differences were found (x^=l-9,P^O.167). There was the suggestion of a slight trend favouring the pyridoxinepatients since the controls had somewhat more tongue protrusion, but it was notenough to be statistically significant.

A comparison of the health records of each child was made to determine if medicalcomplications of DS might have been a factor that altered the development of any ofthe children in the study. Number of visits to paediatricians and patterns of most

IN DOWN'S SYNDROME — PART 1 237

illnesses tended to be generally similar in both groups, although not amenable to exactstatistical analysis.

Side effects

During the double blind study, one of the infants developed the infantile spasmssyndrome. The parents elected for the child to remain in the double blind protocolwhile the patient received ACTH, to which she responded clinically. At the 3 yearcode breaking, it was discovered that the infant was on placebo. There were nodiscernable side effects in the 10 double blind patients receiving pyridoxine.

However, side effects have been identified in the open populations receiving B^,often for much longer periods of time than occurred in the double blind study. Thethree B̂ , correlated side effects were skin blisters related to the sun, vomiting, andperipheral neuropathy. By far the commonest side effect was blistering of the face andhands, mostly in the summer time, in areas when the skin was exposed to the sun(Figure I). A dermatologist described the lesions as resembling those of 'epidermoly-sis bullosa or porphyria cutanea tarda'. The lesions could be reversed by terminatingthe vitamin treatment, by the arrival of autumn or the adding of a small dose ofniacin. Twenty-one patients developed the photosensitive blisters; all patientsdeveloping blisters had been on pharmacological doses of the vitamin for a minimumof 4'/2 years.

Figure 1. 'Sun blisters' seen in patient with trisomy 21 on 25 mg/kg of vitamin B̂ daily.

238 M. COLEMAN et al.

Vomiting, accompanied by abdominal pain or irritability, was reported in sixpatients. In five of these patients it occurred with elevating an already tolerated doseof the vitamin to a higher level, in one patient it occurred with the initial ingestion ofthe treatment solution. In all cases, vomiting was controlled by decreasing oreliminating the dose. Attempts to reintroduce the vitamin or the higher dose resultedin reoccurrence of the vomiting.

Two patients developed a motor and sensory polyneuropathy after 9 years ofpyridoxine administration in doses up to 50 mg/kg. In both patients, there wasevidence of niacin deficiency as measured by the 2-PY/NIMN (l-methyl-5-carboxyl-amide-2-pyridone/N-l-methylnicotinamide) ratio in the urine. Total discontinua-tion of B(, resulted in a gradual return of clinical functioning lo these children and thecorrection of the depression of niacin metabolites without the addition of anysupplementary niacin.

D I S C U S S I O N

In this small group of patients, no statistically significant differences were foundbetween the two groups in mental age, height, weight, cranial circumference ortongue protrusion. The only statistically significant positive results were of elevatingwhole blood 5-HT the first year of the study and the effect on auditory evokedpotentials (described in Part 2). In a study of 23 DS patients receiving B5, Pueschel etal. also found no effect on mental age (Pueschel et al., 1980). Although they reported astatistically significant effect on the Vineland Social Maturity Scale (a finding we alsonoted on our 6-year follow-up), they did not fee! it was significant due to parentalfactors and 'uninterpretable negative interactions' of treatment agents.

One useful result of this study has been a documentation of side effects of largedoses of Bfe over long periods of time to DS individuals (Bhagavan et al., 1975;Coburn et al., 1983). In general, it appears to be relatively safe compared to previousagents used, such as 5-HTP. The major side effect, sun blisters, is reversible and ofminor consequence. In fact, it is interesting that B^ has been used in other types ofpatients as a treatment for photosensitive skin blisters (Mandel, 1963). Two patients,on a maximum dose for many years, developed a serious side effect—polyneuropathy,which appeared to be related to relative niacin deficiency. This finding is in contrastto results found in adults, however, where neuropathies can develop within a fewmonths (Schaumbcrg et al., 1983). The role of magnesium depletion as a factor in sideeffects of pharmacological doses of B(, was not explored in this study.

Alerted by the previous 5-HTP study, a watch for seizures was conducted withoutconclusive results. This result is in concordance with animal studies showing thatsusceptibility of mice to audiogenic seizures is reduced by B^ or increased by B^deficiency (Schlesinger & Uphouse, 1972). Other animal studies predicting other sideeffects of high doses of B^ administration appear not to be relevant (Driskell & Loker,1976; Black, Guirard & Snell, 1977; Cohen et al., 1973).

Because of the findings in Part 2, it is possible that further studies ofpyridoxine-administration in DS may be undertaken in the future. For any suchfuture studies, we would recommend beginning the vitamin at as early an age as

Be IN DOWN'S SYNDROME — PART I 239

possible (within the first week of life would be ideal), increasing the number ofpatients to a larger number and careful monitoring of the vitamin after 3 years of age.A separate study of the value of pyridoxine together with magnesium supplementationalso might be of interest.

S U M M A R Y

Nineteen patients with Down's syndrome participated in a double blind study of theclinical effects of pharmacological doses of vitamin B(, administration, starting under 8weeks of age and continuing until 3 years of age. Ten patients received the vitaminand nine the placebo. In Part I of this study, no statistically significant differenceswere found between the two groups in mental age, height, weight, cranialcircumference or tongue protrusion. A study of side effects conducted on a largeropen population found vitamin B(, to be relatively safe when administered over longperiods of time with photosensitive blisters as the major complication.

A C K N O W L E D G E M E N T S

The assistance of Freda Hur and Anne Shapiro was very much appreciated in thepreparation of this study.

REFERENCES

BANIK NL, DAVISON AN, PALO J & SAVOLAINEN H (1975) Biochemical studies on myelinisolated from the brains of patients with Down's syndrome. Bram 98, 2L^.

BHAGAVAN H N , COLEMAN M , ROSENFELD P & COURSIN D B (1973) Pyridoxal-5-phosphate

levels in whole blood in home-reared patients with trisomy 21. Lancet 1, 339.BHAGAVAN HN, COLEMAN M & COURSIN DB (1975) Distribution of pyridoxal-5-phosphate in

human blood between the cells and plasma: effect of oral administration of pyridoxine onthe ratio in Down's and hyperactive patients. Biochem. Med. 14, 201.

BLACK AL, GUIRARD BM & SNELL EE (1977) Increased muscle phosphorylase in rats fed highlevels of vitamin B^. J. Nutrition 107, 1962.

BouLLiN D j , AIRAKSINEN EM & PAASONEN M K (1975) Platelet taurine in Down's syndrome.Med. Biology 53, 184.

CHAUHAN MS & DAKSHINAMURTI K (1979) The elongation of fatty acids by microsomes andmitochondria from normal and pyridoxine-deficiem rat brains. Exp. Brain. Res. 36, 265.

COBURN S P & SEIDENBKRG MS (1969) Leukocyte pyridoxal phosphate and alkalinephosphatase in Down's syndrome and other retardates. Am. J. Clin. Nutr. 22, 1197.

COBURN SP, SCHALTENBRAND WE, MAHUREN J D , CLAUSMAN RJ & TOWNSEND D (1983) Effect

of megavitamin treatment on mental performance and plasma vitamin B(, concentrations inmentally retarded young adults. Am. J. Clin. Nutr. 38, 352.

COHEN PA, ScHNEtDMAN K, GINSBERG-FELLNER F , STURMAN J A , KNITTLE J & GAULL G E

(1973) High pyridoxine diet in the rat: possible implications for megavitamin therapy. J .Nutrition 103, 143.

COLEMAN M (1971) Infantile spasms induced by 5-hydroxytryptophan in patients with Down'ssyndrome. Neurology 21, 911.

COLEMAN M (1973a) A double blind trial of 5-hydroxytryptophan in trisomy 21 patients. InColeman M (ed.) Serotonin in Dovm's Syndrome. Amsterdam: North-Holland PublishingCompany.

240 M . C O L E M A N et al.

CoLEMAN M (1973b) Other methods of raising serotonin. In Coleman M (ed.) Serotonin inDown's Syndrome. Amsterdam: North-Holland Publishing Company.

COLEMAN M , CAMPBELL M , FREEDMAN L S , ROFFMAN M , HBSTEIN R P & GOLDSTEIN M (1974)

Serum dopamine-beta-hydroxylase levels in Down's syndrome. Clin. Genetics 5, 312.DAVIDENKOVA E F , AYTBAYER K A , ROSENBERG O A , BUTOMO IV, TABAKIAN V G & SHWARTZ E I

(1974) Lipid composition of blood serum in patients with Down's syndrome. Pediatria12, 30.

DRISKELL JA & LoKER SF (1976) Behavioral patterns of female rats fed high levels of vitaminBfi. Nutrition Reports International 14, 467.New York: Spectrum.

GOODMAN HO, KING J S & THOMAS J J (1964) Urinary excretion of beta-amino-isobutyric acidand taurine in mongoiism. Nature 204, 650.

KURTZ D J & KANFER J N (1973) Composition of myelin Hpids and synthesis of 3-ketodihydrosphingosine in the vitamin B(,-dericienr developing rat. J . of Neurochem. 20,963.

LAUDER J M & KREBS H (1978) Serotonin as a differentiation signal in early neurogenesis. Dev.Neurosci. 1, 15.

MAHUREN J D & COBURN S P (1974) Pyridoxal phosphate in lymphocytes, polymorphonuclearleukocytes and platelets in Down's syndrome. Am. J. Clin. Nutr. 27, 521.

MANDEL E (1963) New treatment for photosensitive skin eruptions: results obtained withvitamin B^. New York State Journal of Medicine 63, 2097.

MCCOY E E & CHUNG SI (1964) The excretion of tryptophan metabolites followingdeoxypyridoxine administration in mongoloid and non-mongoloid patients. J. Ped. 64,227.

MCCOY EE, ANAST C S & NAYLOR j j (1964) The excretion of oxalic acid followingdeoxypridoxine and tryptophan administration in mongoloid and non-mongoloid subjects.J. Ped. 65, 208.

MCCOY EE & ENGLAND J (1968) Excretion of 4-pyridoxic acid during deoxypyridoxine andpyridoxine administration to mongoloid and non-mongoloid subjects. Jf. Nutr. 96, 525.

PUESCHEL S M , REED R B , CRONK C E & GOLDSTEIN B I (1980) 5-hydroxytryptophan and

pyridoxine. Am. J. Dis. Child 134, 838.RosNER F, ONG B H , PAINE R S & MAHANAND D (1965) Blood-serotonin activity in trisomic and

translocation Down's syndrome. Lancet 1, 1191.SCHAUMBERG H, KAPLAN J, WiNDEBANK A, ViCK N, RASMUS S, PLEASURE D & BROWN M

(1983) Sensory neuropathy from pyridoxine abuse. N. Hngl. J. Med. 309, 445.ScHLESiNGER K & UPHOUSE L L (1972) Pyridoxine dependency and central nervous system

excitability. In Advances in Biochemical Psychopharmacology Vol. 4. New York: RavenPress.

STEPHENS M C & DAKSHINAMURTI K (1975) Brain lipids in pyridoxine-deficient young rats.Neurobiology 5, 262.

SuNDARESAN PR & CouRSiN DB (1970) Methods in Enzymology (Part A). New York: AcademicPress.

Tu J & ZELLWEGER H (1965) Blood serotonin deficiency in Down's syndrome. Lancet 2, 715.WEINER Wj (1976) Vitamin B^ in the pathogenesis and treatment of diseases of the central

nervous system. In Klawans HL (ed.) Clinical Neuropharmacology Vol. 1. New York:Raven Press.

YuwiLER A, PLOTKIN S , GELLER S & RiTVO ER (1970) A rapid accurate procedure fordetermination of serotonin in whole human blood. Biochem. Med. 3, 426.