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Total proteinTotal protein - cca 100 - 120 proteins
HalfHalf--life:life: prealbumin 12-24 hours transferrin 8 daysalbumin, 20 daysIgE 2 days
SynthesisSynthesis - liver, lymphocytes, plasmocytes (Ig, complementcomponents)
DegradaDegradationtion - excretion, endogenic catabolism
PhysicalPhysical consequencesconsequences – colloid-osmotic pressure, transporters, enzymes, antibodies, hormones andreceptors, haemostasis and coagulation, nutrition, buffer
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IndicationIndication to protein to protein investigationsinvestigations
oedemapolyuriableedingchronic renal diseasechronic diarrhoeachronic liver diseasechronic binder diseasesbone painslymphoproliferative diseasesfrequent prevalence of infectionsípathological finding - high FW, proteinuria
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RelationshipRelationship amongamong diagnosisdiagnosisand and biochemicalbiochemical teststests
depletionsdepletions of of proteinsproteinsinadequateinadequate synthesissynthesis retentionretentionof of waterwater and and saltssaltsdehydratationdehydratationacuteacute and and chronicchronicinflammationsinflammationsmonoclonalmonoclonal gammapathygammapathyanaemiaanaemia of iron of iron deficiencydeficiencyaanaemianaemia atat chronicchronic diseasesdiseasesm. m. WilsonWilsonhaemochromatosishaemochromatosismalnutritionmalnutrition
• TP, albuminELPHO
• Immunoelpho• transferrin, ferritin, soluble
Trf receptor
• ceruloplasmin, copper• transferrin, TIBC, ferritin• Trf, alb, prealb
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TotalTotal proteinprotein
DecreasingDecreasing- malabsorption- liver diseases- enhanced depletions
gut, urine, burns- enhanced catabolism
inflammation, malignitychronic diseases
- dilution- catabolism
starvation
IncreasingIncreasing++ hypergammaglobulinaemiahypergammaglobulinaemia++ hypovolaemiahypovolaemia
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AlbuminAlbumin
DecreasingDecreasing- malnutrition- malabsoption- higher consuption
in pregnancy- defective synthesis -
cirrhosis- enhanced decay- depletions- dilution- inflammation
IncreasingIncreasingdehydratationdehydratation
++ i. v. albumin i. v. albumin administrationadministration
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γγ -- globulinsglobulins
polyclonal – most frequent:
chronic hepatopathy, collagenoses, chronic
infection
monoclonal gammapathy – malignant myeloma
dysgammaglobulinaemia and aglobulinaemia
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TransferrinTransferrin
676 AA, Fe3+, MW 81 000, glycoproteinsynthesis in liver, marrow, nodes, 2-3.7 g/ltransport of iron - anaemia, malnutrition, hemochromatosisnegative acute-phase reactantCDT transferrin
anaemia of iron (inferior level at chronicdiseases with anaemia)
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FerritinFerritin
reservoir of iron, MW 450 000,increasing level at seniors
20-300 µg/l 4-200 µg/l
liver diseases, liver ca., obstructive icterus,anaemia of iron
leucaemia, inflammation, haemochromatosis
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CeruloplasminCeruloplasmin
- MW 140 000, six copper atoms, α-2 fraction- 90 % of copper in serum- higher values at women, - 0.2 – 0.6 g/l
m. Wilson, nephrotic syndrome
intrahepatal cholestasis, inflammations, AMI
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PrealbuminPrealbumin
MW 21 000 , combination with TBG, half-life1-2 days0.1-0.4 g/l
nutrition, proteosynthesis, RAF
steroids
liver diseases, necrosis, RAF
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RBPRBP - Retinol binding protein
- 50-70 mg/l, half-life 12 hours
- nutrition, deficiency of vitamin A
TBGTBG - 10-20 mg/l, advanced pregnancyand newborns
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FibrinogenFibrinogenDimer of three chains, MW 340 0002-4 g/l, half-life 2-4 days
acute-phase reactant of coagulation, inflammation, status after surgery, malignity, RA, AMI, nephrotic syndrome, obstruction of biliary tract
DIC, hyperfibrinolysis, difficult liver damage, fibrinolytic therapy
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CRP CRP
β-fraction, MW 110 000 - 140 000
Binds C polysaccharide from pneumococci
Sensitive but non-specific
physiological values under 10 mg/l (8.5)determination – supersensitive CRP
at inflammation, FW,
checking of immunosuppressive therapy
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HaptoglobinHaptoglobin0.3 – 2.0 g/l, allotypes – paternity investigation formerly
haptoglobin binds released haemoglobin 1:1at strong haemolysis zero valuesliver diseases - intravascular haemolysis, haemolytic anaemia, cirrhosis,
inflammation, operation and traumaCAVE - inflammation + haemolysis - normal value !!
HHemopexinemopexin
0.5 – 1.0 g/l significant haemolysis (not affected by
inflammation markedly)
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αα11--AcidAcid--glycoproteinglycoprotein == orosomucoidorosomucoid
45 % saccharides (sialic acid)
Vague function
0.5 – 1.2 g/l, MW 44 000
RAF, latter coming than CRP (monitoring of process –
relation CRP and orosomucoid)
inflammation, perinatal infection, chronic inflammations, malignities, SLE
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αα22--MMacroglobulinacroglobulin• 1.5–3.0 g/l, MW 725 000
- protease inhibitor, inhibitor of coagulation factors
nephrotic syndrome, cirrhosis,
DIC, acute pancreatitis
αα11--AntitrypsinAntitrypsininhibitor of serine proteases, 1.0 – 2.0 g/l, MW 53 000Different genetic variations e.g. ZZ (10% of activityinflammations, tumours, hepatopathiesPrimary pulmonary emphysema, neonatal hepatitis, cystic fibrosis, exudativeenteropathy
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IInternter--αα--trypsintrypsin inhibitorinhibitor
inhibition of proteasesinflammation
αα11--AntichymotrypsinAntichymotrypsinserine protease with cathepsin G, 26 % of saccharides0.3 – 0.6 g/l
inflammation, RAF
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ββ22--MicroglobulinMicroglobulin• non-glycated polypeptide 100 AA, MW 11 000 • 0.8 – 2.4 mg/la component of HLA system- Tumour marker – NHL, CLL, myeloma- tubular damage
LysozymeLysozyme
basic protein, MW 14 500production in lymphocytes,bacteriolysis, 5 - 10 mg/lβ-2 fraction
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AmyloidAmyloid
Serum amyloid A (small lipoproteins)fibrillae of amyloid AL - L chains
- secondary chains of AAneuraminic acid
protein precursor SAA – source of amyloid deposits in organs
ranges 50-300 mg/l
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ComponentsComponents of of complementcomplement
• Determinations of C3 and C4 components – most often
• decrease – a consumption atimmunocomplex production
• increase - load
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New parameters of inflammationNew parameters of inflammation
• Elastase from granulocytes (60-110 µg/l)– response to bacterial inflammation
• Procalcitonin (up to 0.5 µg/l)• 116 AA, MW 13 000
– stimulation by bacterial and mycotic infection • Neopterin (2-7 nmol/l)
– produced in macrophages after stimulation INF-γ– excreted by T lymphocytes– a criterion of cell immunity
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CytokinesCytokines
• Low molecular proteins• Autocrine, paracrine, endocrine effect• short half-time s-min• Interleukins, interferons, colonies
stimulated factors (CSF), growth factors• IL-6, TNF, IL-8, srIL2
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Electrophoresis of proteinsElectrophoresis of proteins
FFractionsractions – albumin 55 - 68α-1 1.6 - 5.6α-2 5.9 - 11.1β 7.9 - 13.9γ 11.4 - 18.4
inflammation α-fractionchronic inflammation, DM β-fractionold age: albumin, α-2 fraction,
β-fraction
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Protein components on cellulose acetateProtein components on cellulose acetate
albuminα1-lipoproteinα 1-acid-glycoproteinα1-antitrypsinα2-macroglobulinhaptoglobinpre-β-lipoproteintransferrinβ-lipoproteincomplementIgAIgMIgG
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NormalNormalsample sample
Electrophoresis Electrophoresis with highwith highresolutionresolution
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OLYMPUS HITE SYSTEMOLYMPUS HITE SYSTEM
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Results of capillary electrophoresisResults of capillary electrophoresisnormal serum
biclonal PPPP component M
Electrophoresis of serum proteins
Electrophoresis of serum proteins Electrophoresis of serum proteinsFractions Range [%]
Fractions Range [%]
Fractions Range [%] Range g/l
Range g/l
Range g/l
TP :
TP : TP :
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The most important The most important gammapathiesgammapathies
Normal findingPolyclonal gammapathy
Monoclonal gammapathy
A-gamma-globulinaemia
Globulins
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EELLEECCTTRROOPPHHOORREEOOGGRRAAMMS
acute pyelonephritis (4th day) pyogenic bronchitis(late stage)
bronchiectasis rheumatoidal arthritis(acute outbreak)
S
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Reference Reference samplesample Monoclonal increaseMonoclonal increase
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Deficit of Deficit of antitrypsinantitrypsin NephroticNephrotic syndromesyndrome
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CirrhosisCirrhosisInflammationInflammation
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Rare fractions in ELPHORare fractions in ELPHO
bisalbuminaemiahyper-α2-macro-globulinaemiahyperlipoproteinaemia(type IV, III)haemoglobin, myoglobinfibrinogendenatured proteinshigh rhematoid factor,uraemic serumhigh lysozyme
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Diagnostics of Diagnostics of nephrologicalnephrological diseasesdiseases
• Dipstick tests for urine – Haematuria, glucose, ketone bodies, bilirubin, nitrites,
leukocytes• Microscopic examination of urine
– Erythrocytes, leukocytes, tubular cells, casts– Hamburger’s sediment
• Renal functional tests - Creatinine, urea, other routine parameters– GFR – collection, algorithms (Cocroft and Gault, other),
cystatin C- Tubular function – wastes, β-2-microglobulin
• Examination of proteinuria– Microalbuminuria (up to 30 mg/d, up to 2,5 g/mol creat)
• Immunological assays
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GlomerularGlomerular filtrationfiltrationDecrease of GFR at chronic renal diseases does not correspond to
number of destroyed glomeruli – hypertrophic changes of residual nephrons
Filtration in one Filtration in one glomerulusglomerulus –– SNGFSNGF
SNGF = SNGF = KiKi x (Px (P--ππ))
KKii –– filtration coefficientfiltration coefficientKKii = k x S = k x S (k (k –– effective hydraulic permeability, effective hydraulic permeability,
S S –– filtration surface filtration surface aarea) rea) P P –– mean mean transcapillarytranscapillary hydraulic gradienthydraulic gradientππ –– mean difference of mean difference of oncoticoncotic pressurepressure
• GF = N x SNGF
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GFR IGFR I
Measurements of exogenous substances– Inulin or polyfructosans– iohexol– 51 Cr-EDTA– 99mTc - DTPA– 125I-iothalamat
– Stabilized plasma concentration– Measurement of renal clearence– Fick principle
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GFR IIGFR II
• Clearence of endogenous creatinine• Serum concentration of creatinine• Equation Cockroft and Gault
– M - (140 - age) x weight /49 x S - creat– F – x 0,85
possible age 20-80yvery good correlation of GFR and calculation
• Equation Levey• Equation MDRD Study• Equation Schwartz – child
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GlomerularGlomerular filtrationfiltration
S-creatinine [µmol/l]
Clcreat
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CystatinCystatin CC
CystatinCystatin familyfamily proteinprotein122 AA, 13 122 AA, 13 kDakDaproductproduct of of „„housekeepinghousekeeping““ gene gene expressedexpressed in in
allall nucleatednucleated cellscellsproductionproduction in in constantconstant rateratefreelyfreely filtratedfiltratednot not secretedsecretedbutbut tubularytubulary reabsorbedreabsorbed and and catabolisedcatabolisednot not returnreturn to to bloodblood flowflow
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CystatinCystatin CC
SerumSerum/plasma /plasma cystatincystatin C C –– markermarker for GFRfor GFR-- pediatricpediatric patientspatients-- elderlyelderly patientspatients-- patientspatients receivingreceiving chemotherapychemotherapy-- transplantedtransplanted patientspatients-- practicalypracticaly independent of independent of ageage and sex, and sex,
musclemuscle massmass-- immunochemistryimmunochemistry assayassay
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Diagnostic algorithm in patients with Diagnostic algorithm in patients with proteinuriaproteinuria
Proteinuria (Pu) - positive dipstick examination
quant. Pu > 2 g/24 h quant. Pu < 2g/24 h quant. Pu < 150 mg/24 h
glomerular Pu glomer., tubular, or prerenal Pu noobservations
serum tests – assessment of proteins in urine
albumin albumin + IgG light chains Ig, Hb, β2-microglobulinmyoglobin
↑ S-β2-m N S-β2-m
selective selective PuPu nonselectivenonselective PuPu prerenalprerenal PuPu tubular tubular PuPu
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StrategyStrategy for for genitourinarygenitourinary disordersdisorders
Albumin, total protein, Albumin, total protein, DisturbedDisturbedExclusionExclusion αα11--microglobulin, microglobulin, leukleukoo--, , eryerytrocytestrocytes renal functionrenal function
(by test strip)(by test strip) unlikelyunlikely
DifferentiationDifferentiation ≥≥ 1 Parameter positive1 Parameter positive
ProteinProtein--, , AlbuminuriaAlbuminuria LeukocyturiaLeukocyturia HematuriaHematuria
NAG, NAG, IgGIgG SedimentSediment EryEry. differentiation . differentiation αα22--macroglobulin macroglobulin MiMiccrobiologyrobiology αα22--macroglobulinmacroglobulin
ProteinuriaProteinuria ⋅⋅ Uper and lowerUper and lower HeHemmaturiaaturia⋅⋅glomerularglomerular seleselecctivetive urinary tract infectionurinary tract infection ⋅⋅glomerularglomerular⋅⋅tubulotubulo--interstitialinterstitial ⋅⋅ContaminationContamination tubulotubulo--interstitialinterstitial⋅⋅ postrepostrennalal ⋅⋅ postrenalpostrenal
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ClasificationClasification of of albuminuriaalbuminuriaand and αα11--microglobulinmicroglobulin
Albumin in urine mg/l mg/24h mg/g creatinine
g/mol creatinine
Physiological level < 20 < 30 < 20 < 2.5
Microalbuminuria 20 - 200 30 - 300 20 - 300 2.5 - 25
Albuminuria > 200 > 300 > 300 > 25
α1−Microglobulin < 12 < 20 < 14 < 0.5
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Types and markers of Types and markers of proteinuriaproteinuria
Form Causes Marker
Selective glomerular Diabetes Hypertension Early stage
Albumin, transferin
Non-selective glomerular postural proteinuria, glomerulopathies fever, exercise
Albumin, IgG
Tubular Bact.Pyelonephritis Interstitial nephritis Toxic nephropaties
α1 microglobulin, β2 microglobulin
Mixed DM, hypertensive nephropathy Burns, chronic pyelonephritis
Albumin, total protein
Prerenal Intravascular hemolysis, rhabdomyolysis, myeloma
Hemoglobin, myoglobin, Ig- light chains
Postrenal Postrenal hematuria (stones, tumors) IgG/albumin, α2 macroglobulin/ albumin
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ProteinuriaProteinuria
semi-quantitative, quantitativeGBM up to 150 000 MW, GBM up to 150 000 MW, norm 1norm 1220 mg/0 mg/ddtubulointerstitial disorders generally < 1.0 g/dtubular tubular proteinuriaproteinuria - β2-microglobulinselective - albumin & transferrin without Ig; MW < 100000nonselective – if immunoglobulins MW > 100 000 are presentedglomerularglomerular proteinuriaproteinuria - selective and non-selectiveType of Type of proteinuriaproteinuria -- index of index of sectivitysectivity:: IgG m
IgG sTrf mTrf s
< 0,1 selective, > 0.2 non-selective0,1 – 0,2 moderately selective
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Detection of free radicals damageDetection of free radicals damageDirect measurementsDirect measurements
electron spin resonance (ESR)
radical trapping
pulse radiolysis
chemiluminiscence
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Detection of free radicals damageDetection of free radicals damageIndirect measurementsIndirect measurements
AAntioxidantntioxidant systemssystems– Individual markers
(enzymes, substrates, trace elements)
– Total antioxidant capacity
AuAutoantibodiestoantibodies
AGEsAGEs aandnd AOPPAOPP
Markers from radical Markers from radical reactionsreactions
• malondialdehyde• conjugated diens• lipid hydroperoxides• aldehydes (e.g. 4-HNE)• pentane, ethane• damaged DNA base• protein alteration• modified amino-acids• oxLDL