Total protein - cca proteins

Transcription

1 Total protein - cca proteins Half-life: life: prealbumin hours transferrin albumin, IgE 8 days 20 days 2 days Synthesis - liver, lymphocytes, plasmocytes (Ig, complement components) Degradation - excretion, endogenic catabolism Physical consequences consequences colloid-osmotic pressure, transporters, enzymes, antibodies, hormones and receptors, haemostasis and coagulation, nutrition, buffer 1

2 Indication to protein investigations oedema polyuria bleeding chronic renal disease chronic diarrhoea chronic liver disease chronic binder diseases bone pains lymphoproliferative diseases frequent prevalence of infectionsí pathological finding - high FW, proteinuria 2

3 Relationship among diagnosis and biochemical tests depletions of proteins inadequate synthesis retention of water and salts dehydratation acute and chronic inflammations monoclonal gammapathy anaemia of iron deficiency anaemia at chronic diseases m. Wilson haemochromatosis malnutrition TP, albumin ELPHO Immunoelpho transferrin, ferritin, soluble Trf receptor ceruloplasmin, copper transferrin, TIBC, ferritin Trf, alb, prealb 3

4 Total protein Decreasing - malabsorption - liver diseases - enhanced depletions gut, urine, burns - enhanced catabolism inflammation, malignity chronic diseases - dilution - catabolism starvation Increasing + hypergammaglobulinaemia + hypovolaemia 4

5 Albumin Decreasing - malnutrition - malabsoption - higher consuption in pregnancy - defective synthesis - cirrhosis - enhanced decay - depletions - dilution - inflammation Increasing dehydratation + i. v. albumin administration 5

6 γ - globulins polyclonal most frequent: chronic hepatopathy, collagenoses, chronic infection monoclonal gammapathy malignant myeloma dysgammaglobulinaemia and aglobulinaemia 6

7 Transferrin 676 AA, Fe 3+, MW , glycoprotein synthesis in liver, marrow, nodes, g/l transport of iron - anaemia, malnutrition, hemochromatosis negative acute-phase reactant CDT transferrin anaemia of iron (inferior level at chronic diseases with anaemia) 7

8 Ferritin reservoir of iron, MW , increasing level at seniors µg/l µg/l liver diseases, liver ca., obstructive icterus, anaemia of iron leucaemia, inflammation, haemochromatosis 8

9 Ceruloplasmin - MW , six copper atoms, α-2 fraction - 90 % of copper in serum - higher values at women, g/l m. Wilson, nephrotic syndrome intrahepatal cholestasis, inflammations, AMI 9

10 Prealbumin MW , combination with TBG, half-life 1-2 days g/l nutrition, proteosynthesis, RAF steroids liver diseases, necrosis, RAF 10

11 RBP - Retinol binding protein mg/l, half-life 12 hours - nutrition, deficiency of vitamin A TBG mg/l, advanced pregnancy and newborns 11

12 Fibrinogen Dimer of three chains, MW 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 12

13 CRP β-fraction, MW 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 13

14 Haptoglobin g/l, allotypes paternity investigation formerly haptoglobin binds released haemoglobin 1:1 at strong haemolysis zero values liver diseases - intravascular haemolysis, haemolytic anaemia, cirrhosis, inflammation, operation and trauma CAVE - inflammation + haemolysis - normal value!! Hemopexin g/l significant haemolysis (not affected by inflammation markedly) 14

15 α 1 -Acid-glycoprotein = orosomucoid 45 % saccharides (sialic acid) Vague function g/l, MW RAF, latter coming than CRP (monitoring of process relation CRP and orosomucoid) inflammation, perinatal infection, chronic inflammations, malignities, SLE 15

16 g/l, MW α 2 -Macroglobulin - protease inhibitor, inhibitor of coagulation factors nephrotic syndrome, cirrhosis, DIC, acute pancreatitis α 1 -Antitrypsin inhibitor of serine proteases, g/l, MW Different genetic variations e.g. ZZ (10% of activity inflammations, tumours, hepatopathies Primary pulmonary emphysema, neonatal hepatitis, cystic fibrosis, exudative enteropathy 16

17 Inter-α-trypsintrypsin inhibitor inhibition of proteases inflammation α 1 -Antichymotrypsin serine protease with cathepsin G, 26 % of saccharides g/l inflammation, RAF 17

18 β 2 -Microglobulin non-glycated polypeptide 100 AA, MW mg/l a component of HLA system - Tumour marker NHL, CLL, myeloma - tubular damage Lysozyme basic protein, MW production in lymphocytes, bacteriolysis, 5-10 mg/l β-2 fraction 18

19 Amyloid Serum amyloid A (small lipoproteins) fibrillae of amyloid AL - L chains - secondary chains of AA neuraminic acid protein precursor SAA source of amyloid deposits in organs ranges mg/l 19

20 Components of complement Determinations of C3 and C4 components most often decrease a consumption at immunocomplex production increase - load 20

21 New parameters of inflammation Elastase from granulocytes ( µg/l) response to bacterial inflammation Procalcitonin (up to 0.5 µg/l) 116 AA, MW 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 21

22 Cytokines 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 22

23 Electrophoresis of proteins Fractions albumin α α β γ inflammation α-fraction chronic inflammation, DM β-fraction old age: albumin, α-2 fraction, β-fraction 23

24 Protein components on cellulose acetate albumin α 1 -lipoprotein α 1 -acid-glycoprotein α 1 -antitrypsin α 2 -macroglobulin haptoglobin pre-β-lipoprotein transferrin β-lipoprotein complement IgA IgM IgG 24

25 Normal sample Electrophoresis with high resolution 25

26 OLYMPUS HITE SYSTEM 26

27 Results of capillary electrophoresis normal serum Electrophoresis of serum proteins Fractions Range [%] Range g/l PP component M TP : biclonal PP Electrophoresis of serum proteins Fractions Range [%] Range g/l Electrophoresis of serum proteins Fractions Range [%] Range g/l TP : TP : 27

28 The most important gammapathies Normal finding Polyclonal gammapathy Globulins Monoclonal gammapathy A-gammaglobulinaemia 28

29 O P H ELE C T R acute pyelonephritis (4 th day) pyogenic bronchitis (late stage) O RE bronchiectasis rheumatoidal arthritis (acute outbreak) OG R A M S 29

30 Reference sample Monoclonal increase 30

31 Deficit of antitrypsin Nephrotic syndrome 31

32 Inflammation Cirrhosis 32

33 Rare fractions in ELPHO bisalbuminaemia hyper-α 2 -macroglobulinaemia hyperlipoproteinaemia (type IV, III) haemoglobin, myoglobin fibrinogen denatured proteins high rhematoid factor, uraemic serum high lysozyme 33

34 Diagnostics of nephrological diseases 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 34

35 Glomerular filtration Decrease of GFR at chronic renal diseases does not correspond to number of destroyed glomeruli hypertrophic changes of residual nephrons Filtration in one glomerulus SNGF SNGF = Ki x (P-π) K i filtration coefficient K i = k x S (k effective hydraulic permeability, S filtration surface area) P mean transcapillary hydraulic gradient π mean difference of oncotic pressure GF = N x SNGF 35

36 GFR 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 36

37 GFR 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 20-80y very good correlation of GFR and calculation Equation Levey Equation MDRD Study Equation Schwartz child 37

38 Glomerular filtration S-creatinine [µmol/l] Cl creat 38

39 Cystatin C Cystatin family protein 122 AA, 13 kda product of housekeeping gene expressed in all nucleated cells production in constant rate freely filtrated not secreted but tubulary reabsorbed and catabolised not return to blood flow 39

40 Cystatin C Serum/plasma cystatin C marker for GFR - pediatric patients - elderly patients - patients receiving chemotherapy - transplanted patients - practicaly independent of age and sex, muscle mass - immunochemistry assay 40

41 Diagnostic algorithm in patients with proteinuria 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 no observations serum tests assessment of proteins in urine albumin albumin + IgG light chains Ig, Hb, β2-microglobulin myoglobin N S-β2-m S-β2-m selective Pu nonselective Pu prerenal Pu tubular Pu 41

42 Strategy for genitourinary disorders Exclusion Albumin, total protein, α1-microglobulin, leuko-, erytrocytes (by test strip) Disturbed renal function unlikely Differentiation 1 Parameter positive Protein-, Albuminuria Leukocyturia Hematuria NAG, IgG α 2 -macroglobulin Sediment Microbiology Ery.. differentiation α 2 -macroglobulin Proteinuria glomerular selective tubulo-interstitial postrenal Uper and lower urinary tract infection Contamination Hematuria glomerular tubulo-interstitial postrenal 42

43 Clasification of albuminuria and α -microglobulin 1 Albumin in urine mg/l mg/24h mg/g creatinine g/mol creatinine Physiological level < 20 < 30 < 20 < 2.5 Microalbuminuria Albuminuria > 200 > 300 > 300 > 25 α 1 Microglobulin < 12 < 20 < 14 <

44 Types and markers of proteinuria Form Causes Marker Selective glomerular Non-selective glomerular Tubular Mixed Prerenal Diabetes Hypertension Early stage postural proteinuria, glomerulopathies fever, exercise Bact.Pyelonephritis Interstitial nephritis Toxic nephropaties DM, hypertensive nephropathy Burns, chronic pyelonephritis Intravascular hemolysis, rhabdomyolysis, myeloma Albumin, transferin Albumin, IgG α1 microglobulin, β2 microglobulin Albumin, total protein Hemoglobin, myoglobin, Ig- light chains Postrenal Postrenal hematuria (stones, tumors) IgG/albumin, α2 macroglobulin/ albumin 44

45 Proteinuria semi-quantitative, quantitative GBM up to MW, norm mg/d tubulointerstitial disorders generally < 1.0 g/d tubular proteinuria - β 2 -microglobulin selective - albumin & transferrin without Ig; MW < nonselective if immunoglobulins MW > are presented glomerular proteinuria - selective and non-selective Type of proteinuria - index of sectivity: IgG m IgG s Trf m Trf s < 0,1 selective, > 0.2 non-selective 0,1 0,2 moderately selective 45

46 Detection of free radicals damage Direct measurements electron spin resonance (ESR) radical trapping pulse radiolysis chemiluminiscence 46

47 Detection of free radicals damage Indirect measurements Antioxidant systems Individual markers (enzymes, substrates, trace elements) Total antioxidant capacity Autoantibodies AGEs and AOPP Markers from radical reactions malondialdehyde conjugated diens lipid hydroperoxides aldehydes (e.g. 4-HNE) pentane, ethane damaged DNA base protein alteration modified amino-acids oxldl 47