Bruno Sopko

      Introduction Myocardial infarction Coagulation (Blood clotting) Liver function tests Dyslipidemy Endocrinology

  ◦ ◦ Markers Detection Methods Biochemical Immunochemical

        Introduction Troponins Creatine kinase LDH AST Myoglobin Ischemia Modified Albumin Glycogen phosphorylase BB

◦ ◦ ◦ ◦ Diagnosis id based on detection of the markers released to blood circulation by damaged tissue. Concentration changes in time depends on: Localization in the cell Relative molecular mass Excretion rate Blood flow in the damaged area - tissue

   cTnT and cTnI are not present in blood and theirs AA composition is unique (release to bloodstrem 3-6 hours after the onset) Immunochemical methods GLORIA (Gold Labelled Optically Read Immuno-Assay; variation of ELISA)

 CK M(muscle) CK B (brain) subunits (CK-MB) Enzyme kinetics (subunit M antibodies halve the rate)  Immunochemical method: CK-MB mass

Specific LDH 1 1.

2.

Estimation of LDH activity Electrophoretical LDH determination 1 concentration

Release by mitchondria during necrosis 1.

2.

ASP -> oxalacetate Oxalacetate + NADH + H + -> malate + NAD +

Early occurrence, non-specific Immunochemical method

Test is based on the estimation of Co 2+ binding capacity. Albumin healthy individual HSA (human serum albumin) binds more of cobalt ions than Ischemia Modified Albumin IMA. Adding Cobalt ions to the sample, part of the Cobalt ions reacts with the Albumin and part remains dissolved. The dissolved part is then detected by the addition of dithioerythrol, which forms colored complex with Co ions.

IMA binds less Co thus resultin in higher concentration of the above mentioned complex.

 After 4 hours  ELISA

     Introduction Prothrombin Time (PT) Activated partial thromboplastin time (aPTT) Thrombin time (TT) ◦ ◦ ◦ ◦ Fibrinogen Clauss method Modified PT method Immunological methods Gravimetrical method

   Detection of the coagulation problems (vitamin K) and estimation of the anticoagulant dosage (Warfarin) Plasma + citrate + coagulation factor III Value is ratio of time consumed divided by standard coagulation time.

  Detection of coagulation problems, preoperative screening, heparin dosage estimation Plasma + citrate + phosphatidylethanolamine + kaolin clay

 Essay of the final step in coagulation cascade, conversion of fibrinogenu to fibrin after adding of thrombin. Usual time is up to 20 seconds. Prolongation can be caused by fibrin malfunction, hypofibrinogenemia or afibrinogenemia or by presence of FDP  Plasma + citrate + thrombin

    Clauss method – thrombin surplus Modified PT method – series of sample dilutions Immunological methods - ELISA, total concentration, not function Gravimetrical methods – estimation of the clot weight, time consuming

   ◦ ◦ ◦ ◦ ◦ Laboratory indicators of liver cells harm ALT AST LDH Laboratory indicators of bile congestion ALP GGT Bilirubin ◦ ◦ ◦ ◦ Laboratory indicators of liver proteosynthetic function Albumin Total protein Prothrombin time

    ALT in liver only, AST non-specific AST/ALT – de Rittis index ( > 2 indicates alcoholic liver disorders) LDH 4 LDH 5 and LDH 5 liver specific – liver tumor

Alkaline phosphatase:

p-Nitrophenyl phosphate → p-nitrophenol + PO

4 3 Gama-glutamyltransferase:

γ–glutamyl-p-nitroannilin → p-nitroannilin

Bilirubin:

Bilirubin + sulphanilic acid+ NaNO

2

azobilirubin →

Albumin: reaction with bromcresol dyes Total protein: biuret reaction PT: coagulation

     Introduction Total cholesterol Triacylglycerols HDL cholesterol LDL cholesterol

Lipoprotein Chylomicron VLDL IDL LDL HDL2 HDL3 Lp(a) Remnant CM Source Intestine Liver VLDL catabolite IDL catabolite Liver, Intestine, VLDL and CM catabolite Liver CM catabolite Diameter (nm) Density (g/ml) 90-5000 < 0,94 30-90 25-35 20-25 90-120 50-90 250-350 >500 0,93 – 1,006 1,006 1,019 1,019 – 1,063 1,063 – 1,125 1,125 1,210 1,050 – 1,100 0,93 – 1,006 Composition Protein (%) 1 - 2 Lipid (%) 98 - 99 Main lipid 7 - 10 11 21 33 57 74 6 - 8 90 - 93 89 79 67 43 26 92 - 94 Apolipoproteins Triacylglycerol Triacylglycerol Triacylglycerol , cholesterol cholesterol Cholesterol, phospholipids Cholesterol A-I, A-II, A-IV,1 B-48, C-I, C-II, C III, E B-100, C-I, C-II, C-III B-100, E B-100 A-I, A-II A-I, A-II Cholesterol Cholesterol, Triacylglycerol B-100 a apo(a) B-48, E

     Isolated hypercholesterolemia - total cholesterol concentration is increased, triglycerides concentration is normal Mixed hyperlipidemia – both, total cholesterol concentration, and triglycerides concentration are increased Isolated hypertriglyceridemia – increased triglycerides concentration, normal total cholesterol concentration Dyslipidemia of the metabolic syndrom Secondary dyslipidemia

1.

2.

3.

Hydrolysis of the cholesterol esters to free cholesterol and fatty acids by cholesterol esterase enzyme Oxidation of the free cholesterol to 4 cholesten-3-on with simultaneous production of hydrogen peroxide by cholesterol oxidase Quantification of hydrogen peroxide by oxidative copulation of 4-aminoantipyrin and phenol by peroxidase enzyme

1.

2.

3.

Lipoprotein lipase catalyses the hydrolysis of triacylglycerols to glycerol and fatty acids Released glycerol is modified by glycerolkinase in presence of ATP to glycerol-3-phosphate, which is subsequently oxidised by glycerol-3 phosphate oxidase to dihydroxyacetonephosphate. Hydrogen peroxide is detected by the same reaction as in case of cholesterol

1.

2.

Blocking of non-HDL particles by utilization of the anti-ApoB (VLDL, LDL, CM) antibodies The same reactions as in case of total cholesterol

 Indirect estimation –

Friedewald’s formula: LDL-cholesterol = total cholesterol – HDL cholesterol – TG/2.2

 Direct estimation 1.

By using specific detergents and other reagents, te non-LDL cholesterol is blocked 2.

Estimation follows the same route as in case of total cholesterol

           Introduction Somatotropic hormone (STH) Prolactin Adrenocorticotropic hormone (ACTH) Thyroid-stimulating hormone (TSH) Follicle-stimulating hormone(FSH) Luteinizing hormone (LH) T 4 thyroxine (tetraiodothyronine), T triiodothyronine Parathyroid hormone Cortisol Diabetes mellitus 3

   ◦ ◦ Estimation of hormone concentration Surveillance of the metabolism Dynamic tests Stimulating Suppressive

    Stimulates growth of the muscles and connective tissues Promotes lipolysis Reduces liver uptake of glucose Increases ions retention  IRMA method (Immunoradiometric analysis)

  Complicated secretions regulation Hyperprolactinemia is pathological, hypoprolactinemia not detected  IRMA or ELISA method

 Adrenocorticotropic hormone controls the secretion of glukocorticoids and androgens in adrenal cortex  ◦ ◦ Electroluminiscence (ECLIA) chemiluminiscence is electricaly initiated tripropylamine (TPA) - transfers e to Ru

 Thyroid-stimulating hormone  ELISA

 Follicle-stimulating hormone and Luteinizing hormone cooperate in controlling of the function of gonades – triggers ovulation and development of the corpus luteum in females, production of testosterone in males  ELISA

    Regulation of metabolism (increase) and thus increase of O 2 uptake, followed by increase of the heat energy Stimulation of RNA synthesis and proteosynthesis T 4 T 3 is prohormone of T 3 a T 4 has negative effect to TSH  ELISA

  secreted by the chief cells of the parathyroid glands, diagnosis of primery hyperparathyroidism, differential diagnosis of hypocalcaemias Regulates the concentration of calcium a phosphorus in blood  IRMA

    ◦ ◦ ◦ ◦ ◦ Steroid hormone, produced by the zona fasciculata of the adrenal gland Regulation of ACTH, CRH Sugar metabolism increase, protein metabolism decrease Effects: Gluconeogenesis from AA and FA Proteolysis- protein catabolism – mainly in muscles Lipolysis Decrease of the insulin receptors sensitivity Suppression of the protein anabolism immunosuppressive , antiallergenic and antiflogistic (anti-inflammatory) effects, decrease of the Ca 2+ uptake in GIT

  In saliva ELISA

  Indirect screening – concentration of blood Glc ◦ ◦ 2 types absolute insulin deficiency→ diabetes mellitus of the 1 st Type Cells response to insulin is lower - diabetes mellitus of the 2 nd Type

  R.K. Murray et al.: Harper's Illustrated Biochemistry, twenty-sixth edition, McGraw Hill Companies, 2003 Allan D. Marks, MD: Basic Medical Biochemistry a Clinical Approach, Lippincott Williams & Wilkins, 2009