Bruno Sopko. Introduction Myocardial infarction Coagulation (Blood clotting) Liver function...
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Transcript of Bruno Sopko. Introduction Myocardial infarction Coagulation (Blood clotting) Liver function...
Biochemical Screening Methods
Bruno Sopko
Introduction Myocardial infarction Coagulation (Blood clotting) Liver function tests Dyslipidemy Endocrinology
Content
Markers Detection Methods
◦ Biochemical◦ Immunochemical
Introduction
Introduction Troponins Creatine kinase LDH AST Myoglobin Ischemia Modified Albumin Glycogen phosphorylase BB
Myocardial infarction
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
Myocardial infarction - Introduction
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)
Myocardial infarction - Troponins
CK M(muscle) CK B (brain) subunits (CK-MB) Enzyme kinetics (subunit M antibodies halve
the rate)
Immunochemical method: CK-MB mass
Myocardial infarction – Creatine kinase
Specific LDH1
1. Estimation of LDH activity 2. Electrophoretical LDH1 concentration
determination
Myocardial infarction - LDH
Release by mitchondria during necrosis
1. ASP -> oxalacetate2. Oxalacetate + NADH + H+-> malate +
NAD+
Myocardial infarction – AST
Early occurrence, non-specific
Immunochemical method
Myocardial infarction – Myoglobin
Test is based on the estimation of Co2+ 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.
Myocardial infarction – Ischemia Modified Albumin
After 4 hours
ELISA
Myocardial infarction – Glycogen phosphorylase BB
Introduction Prothrombin Time (PT) Activated partial thromboplastin time (aPTT) Thrombin time (TT) Fibrinogen
◦ Clauss method◦ Modified PT method◦ Immunological methods◦ Gravimetrical method
Coagulation (Blood Clotting)
Coagulation - Introduction
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.
Coagulation – PT (Quick’s test)
Detection of coagulation problems, preoperative screening, heparin dosage estimation
Plasma + citrate + phosphatidylethanolamine + kaolin clay
Coagulation - aPTT
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
Coagulation - TT
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
Coagulation - Fibrinogen
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
Liver function tests
ALT in liver only, AST non-specific AST/ALT – de Rittis index ( > 2 indicates
alcoholic liver disorders) LDH4 and LDH5 liver specific LDH5 – liver tumor
Liver function tests – AST, ALT, LDH
Alkaline phosphatase:p-Nitrophenyl phosphate → p-nitrophenol + PO4 3-
Gama-glutamyltransferase:γ–glutamyl-p-nitroannilin → p-nitroannilin
Bilirubin:Bilirubin + sulphanilic acid+ NaNO2 →
azobilirubin
Liver function tests – ALP, GGT, Bilirubin
Albumin:reaction with bromcresol dyes
Total protein:biuret reaction
PT:coagulation
Liver function tests – Albumin, total protein, PT
Introduction Total cholesterol Triacylglycerols HDL cholesterol LDL cholesterol
Dyslipidemia
Lipoprotein Source Diameter (nm)
Density (g/ml)
Composition Main lipid Apolipoproteins
Protein (%)
Lipid (%)
Chylomicron Intestine 90-5000 < 0,94 1 - 2 98 - 99 Triacylglycerol A-I, A-II, A-IV,1 B-48, C-I, C-II, C-III,E
VLDL Liver 30-90 0,93 – 1,006
7 - 10 90 - 93 Triacylglycerol B-100, C-I, C-II, C-III
IDL VLDL catabolite
25-35 1,006-1,019
11 89 Triacylglycerol, cholesterol
B-100, E
LDL IDL catabolite
20-25 1,019 – 1,063
21 79 cholesterol B-100
HDL2 Liver, Intestine, VLDL and CM catabolite
90-120 1,063 – 1,125
33 67 Cholesterol, phospholipids
A-I, A-II
HDL3 50-90 1,125-1,210
57 43 Cholesterol A-I, A-II
Lp(a) Liver 250-350 1,050 – 1,100
74 26 Cholesterol B-100 a apo(a)
Remnant CM CM catabolite
>500 0,93 – 1,006
6 - 8 92 - 94 Cholesterol, Triacylglycerol
B-48, E
Dyslipidemia - Introduction
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
Dyslipidemia – Introduction, classification
1. Hydrolysis of the cholesterol esters to free cholesterol and fatty acids by cholesterol esterase enzyme
2. Oxidation of the free cholesterol to 4-cholesten-3-on with simultaneous production of hydrogen peroxide by cholesterol oxidase
3. Quantification of hydrogen peroxide by oxidative copulation of 4-aminoantipyrin and phenol by peroxidase enzyme
Dyslipidemia – Total cholesterol
1. Lipoprotein lipase catalyses the hydrolysis of triacylglycerols to glycerol and fatty acids
2. 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.
3. Hydrogen peroxide is detected by the same reaction as in case of cholesterol
Dyslipidemia – Triacylglycerols
1. Blocking of non-HDL particles by utilization of the anti-ApoB (VLDL, LDL, CM) antibodies
2. The same reactions as in case of total cholesterol
Dyslipidemia – HDL cholesterol
Indirect estimation – Friedewald’s formula:
LDL-cholesterol = total cholesterol – HDL-cholesterol – TG/2.2
Direct estimation1. By using specific detergents and other reagents,
te non-LDL cholesterol is blocked2. Estimation follows the same route as in case of
total cholesterol
Dyslipidemia – LDL cholesterol
Introduction Somatotropic hormone (STH) Prolactin Adrenocorticotropic hormone (ACTH) Thyroid-stimulating hormone (TSH) Follicle-stimulating hormone(FSH) Luteinizing hormone (LH) T4 thyroxine (tetraiodothyronine), T3
triiodothyronine Parathyroid hormone Cortisol Diabetes mellitus
Endocrinology
Estimation of hormone concentration Surveillance of the metabolism Dynamic tests
◦ Stimulating◦ Suppressive
Endocrinology – Introduction:Laboratory diagnostics
Stimulates growth of the muscles and connective tissues
Promotes lipolysis Reduces liver uptake of glucose Increases ions retention
IRMA method (Immunoradiometric analysis)
Endocrinology - Growth hormone (STH)
Complicated secretions regulation Hyperprolactinemia is pathological,
hypoprolactinemia not detected
IRMA or ELISA method
Endocrinology - prolactin
Adrenocorticotropic hormone controls the secretion of glukocorticoids and androgens in adrenal cortex
Electroluminiscence (ECLIA)◦ chemiluminiscence is electricaly initiated◦ tripropylamine (TPA) - transfers e- to Ru
Endocrinology - ACTH
Thyroid-stimulating hormone
ELISA
Endocrinology - TSH
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
Endocrinology – FSH, LH
Endocrinology – T4, T3
Regulation of metabolism (increase) and thus increase of O2 uptake, followed by increase of the heat energy
Stimulation of RNA synthesis and proteosynthesis
T4 is prohormone of T3
T3 a T4 has negative effect to TSH
ELISA
Endocrinology – T4, T3
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
Endocrinology - Parathyroid hormone
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 Ca2+ uptake in GIT
Endocrinology - Cortisol
In saliva ELISA
Endocrinology - Cortisol
Indirect screening – concentration of blood Glc
2 types◦ absolute insulin deficiency→ diabetes mellitus of
the 1st Type◦ Cells response to insulin is lower - diabetes
mellitus of the 2nd Type
Endocrinology - Diabetes mellitus
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
Literature: