1

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