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: