ANATOMY AND PHYSIOLOGY OF THE HEART Functions of the heart Generating blood pressure -contractions of the heart generate

blood pressure, which is required for blood flow through the blood vessels Routing blood -the heart separates the pulmonary and systemic circulations, which ensures the flow of oxygenated blood to tissues Ensuring one-way blood flow -the valves of the heart ensure a one-way flow of blood through the heart and blood vessels Regulating blood supply -changes in the rate and force of heart contraction match blood flow to the changing metabolic needs of the tissues during rest, exercise, and changes in body position Size, form, and location of the heart The heart is approximately the size of a closed fist. The adult heart is shaped like a blunt cone. The blunt, rounded point is the apex and the larger, flat part at the opposite end of the cone is the base. The heart is located in the thoracic cavity between the two pleural cavities which surround the lungs. Anatomy of the heart PERICARDIUM The pericardial cavity is formed by the pericardium or pericardial sac. Fibrous pericardium- tough, fibrous connective tissue outer layer Serous pericardium -inner layer of flat epithelial cells with a thin layer of connective tissue. Parietal pericardium-portion of serous pericardium lining the fibrous pericardium Visceral pericardium-covers the heart surface Pericardial fluid-produced by serous pericardium; reduces friction as the heart moves. EXTERNAL ANATOMY Right and left atria-located at the base of the heart Right and left ventricles-extends from the base of the heart to the apex Coronary sulcus-surrounds the heart separating the atria and ventricles Six veins that carry blood to the heart: Superior vena cava & Inferior vena cava carry blood from the body to the right atrium Four pulmonary veins- carry blood from the lungs to the left atrium INTERNAL ANATOMY Right and left atria -functions primarily as reservoirs, where blood returning from veins collects before it enters the ventricles Right atrium -receives blood through three major openings: superior vena cava, inferior vena cava and coronary sinus Left atrium -receives blood through the four pulmonary veins which drains blood from the lungs Right and left ventricles -are the major pumping chambers. they eject blood into the arteries and force it to flow through the circulatory system. They are separated from each other by the muscular interventricular septum. Heart valves Atrioventricular / AV valves -allow blood to flow from the atria into the ventricles but prevent it from flowing back into the atria. -located between the right atrium and right ventricle; has 3 cusps and is called tricuspid valves.

-the AV valve between the left atrium and left ventricle has two cusps and is called bicuspid valve or mitral valve. Route of blood flow Right atrium> tricuspid valve> right ventricle> pulmonary semilunar valves> pulmonary trunk> pulmonary arteries> lung tissue> pulmonary veins> left atrium> bicuspid valve> left ventricle> aortic semilunar valve> aorta> coronary arteries> heart tissue> coronary sinus> back to right atrium Cardiac Enzymes Cardiac Profile assesses the function of the hearts muscle and the increased level of enzymes following a myocardial infarction. The cardiac enzymes include the following: 1. Aspartate aminotransferase (AST) 2. Lactate dehydrogenase (LD) 3. Creatine Kinase (CK) ASPARTATE AMINOTRANSFERASE (AST) -also called Serum Glutamate Oxaloacetate Transaminase (SGOT) -found in all tissue, especially the heart, liver, and skeletal muscles -it catalyzes the transfer of the amino group of aspartic acid to alpha-ketoglutaric acid to form oxaloacetic acid and glutamic acid Reaction catalyzed: Amino group Alpha-keto group Oxaloacetate & In aspartic acid in alpha-ketoglutaric acid Glutamate Considerations in AST assays -Serum is the best specimen -Hemolyzed samples must be avoided -Alcohol lowers AST values -Muscle trauma like intramuscular injections, exercise, or surgical operation can significantly increase AST levels Clinical significance Myocardial infarction -In myocardial infarction, AST levels are usually 4-10 times the upper limit of normal -These develop within 4-6 hours after the onset of pain -Peak on the 24th 36th hour -Usually normalize on the 4th or 5th day Muscular dystrophy Hepatocellular disorders Skeletal muscle disorders Acute pancreatitis Increased levels of AST may be seen in: Chronic alcohol abuse Drug hepatoxicity Pulmonary infarction Pericarditis Acute hepatitis Skeletal muscle disorders Decreased levels of AST may be seen in: Pregnant women Falsely elevated results Bilirubin

Aceto-acetatae N-acetyl compounds P-aminophenol Sulfathiozole Isoniazid Methyldopa L-dopa Ascorbic acid

Substances that may inhibit AST activity Mercury Cyanide fluoride LACTATE DEHYDROGENASE (LDH) -Catalyzes the reversible oxidation of lactate to pyruvate -Used to indicate AMI -Is a cytoplasmic enzyme found in most cells of the body, including the heart -Not specific for the diagnosis of cardiac disease Distribution of LD isoenzymes: LD1 and LD2 Fast moving fractions and are heat-stable Found mostly in the myocardium and erythrocytes Also found in the renal cortex LD3

Found in a number of tissues, predominantly in the white blood cells and brain

LD4 and LD5 Slow moving and are heat labile Found mostly in the liver and skeletal muscle

The relative concentration in normal serum is LD2, LD3, LD4 and LD5 in decreasing order Techniques in measuring LD isoenzymes: Physical Electrophoresis Selective absorption on diethylaminoethyl cellulose(DEAE) Solvent precipitation technique Heat denaturation at 65C for 30 mins Chemical Substrate-product relationship Coenzyme affinity Differential chemical inhibition of LD activity Immunological Tests

Considerations in LD assays: Red cells contain 150 times more LDH than serum, therefore hemolysis must be avoided LDH has its poorest stability at 0C

Clinical Significance In myocardial infarction, LD increases 3-12 hours after the onset of pain Peaks at 48-60 hours and remain elevated for 10-14 days In MI, LD1 is higher than LD2, thus called flipped LD pattern Increased levels of LD may be seen in: Megaloblastic anemia Pulmonary infarction Granulocyte leukemia Hodgekins disease Hemolytic anemia Infectious mononucleosis Progressive muscular dystrophy (PMD) CREATINE KINASE (CK) -Is a cytosolic enzyme involved in the transfer of energy in muscle metabolism -Catalyzes the reversible phosphorylation of creatine by ATP -Is a dimer comprised of two subunits, resulting in three CK isoenzymes The B, or brain form The M, or muscle form Three isoenzymes isolated after electrophoresis: 1. CK-BB (CK1) isoenzyme Is of brain origin aand only found in the blood if the blood-brain barrier has been breached 2. 3. CK-MM (CK3) isoenzyme Accounts for most of the CK activity in skeletal muscle CK-MB (CK2) isoenzyme Has the most specificity for cardiac muscle It accounts for only 3-20% of total CK activity in the heart Is a valuable tool for the diagnosis of AMI because of its relatively high specificity for cardiac injury Established as the benchmark and gold standard for other cardiac markers Heart yields about 40% CK2 and 60% CK3, while brain tissue yields 90% CK1 and 10% CK3

Considerations in CK assays: -CK is light sensitive and anticoagulants like oxalates and fluorides inhibit its action -CK in serum is very unstable and rapidly loss during storage -Laked specimens are not used since it contains cellular products and intermediate like adenylate kinase, ATP and G-6-Phosphate whhich affect the assay -Exercise and intramuscular injections causes CK elevations Clinical Significance -In myocardial infarction, CK will rise 4-6 hours after the onset of pain -Peaks at 18-30 hours and returns to normal on the third day -CK is the most specific indicator for myocardial infarction (MI) Increased levels of CK may be seen in: Progressive muscular dystrophy Polymyositis Acute psychosis Alcoholic myopathy Delirium tremens

Hypothyroidism Malignant hyperthermia Acute cerebrovascular disease Trichinosis and dermatomyositis

Normal Value: a. Male 25-90 IU/mL b. Female 10-70 IU/mL

CARDIAC PROFILE TEST ENZYMES  Creatinine Kinase MB(CK-MB)  Lactate Dehydrogenase(LDH 1 and 2)  Aspartate Aminotransferase(AST)/Serum Glutamate Oxaloacetate Transaminase(SGOT)  Alanine Aminotransferase(ALT)/ Serum Pyruvate Transaminase(SGPT) LIPID PROFILE  CHOLESTEROL  TRIGLYCERIDE  HDL  LDL Creatinine Kinase Enzymatic Methods for CK Rosalki and Hess y most widely used method y Reverse reaction y pH=6.8 y decrease in absorbance at 340nm

ATP + glucose

G-6-Phosphate + ADP

G-6-Phosphate + NADPH Tanzer and Gilvarg y Forward reaction y pH=9.8

6-phosphogluconolactone + NADP

ADP + PEP

ATP + pyruvate

Pyruvate + NADH

pyruvate + NAD

Colorimetric Method for CK ATP and creatine incubated w/ the specimen and the reaction is stopped w/ the addition of acid, producing phosphocreatine w/c is acid labile. This then dissociates into creatine and free phosphate ions which can be measured by CK activity. Sax and Moore Method (Fluorometric method) y Dissociating agent: ninhydrin solution y product: fluorophore Hughes Method y Dissociating agent: diacetyl and alpha naphthol y End color: pink Clinical Significance CK will rise 406 hours after the onset of pain, peaks at 18-30 hours and returns to normal on the third day. Most specific indicator of myocardial infarction. Lactate Dehydrogenase Measuring LD isoenzymes Physical: electrophoresis LD 1 and 2: fast moving fractions and are heat stable Chemical: coenzyme affinity Immunologic Tests Clinical Significance LD increases 8-12 hours after the onset of pain, peaks at 48-60 hours and remains elevated for 10-14 days. Here, LD 1 is higher than LD 2 thus called FLIPPED LD pattern. Aspartate Aminotransferase(AST) Serum Glutamate Oxaloacetate Transaminase(SGOT) Reitmann Frankel Method The oxaloacetate formed under fixed conditions is determined by the reddish brown hydrozone. It produces with 2,4 DNPH in alkaline medium. Karmen Method The oxaloacetate formed is reacted with malic dehydrogenase in the presence of NADH producing malic acid with the subsequent oxidation of NADH to NAD+. The decrease in absorbance reflects the enzymatic activity of AST(inverse colorimetry) Babson et al Method The oxaloacetate formed is treated with diazonium salt to produce a violet colored product. Clinical Significance AST levels are usually 4-10 times the upper limit of normal. These develop within 4-6 hours after th th th th the onset of pain and peak on the 24 -36 hour. These usually normalize on the 4 or 5 day. Alanine Aminotransferase(ALT) Serum Pyruvate Transaminase(SGPT)

Reitman and Frankel Method Walker et al Method Based on the coupled enzyme reaction. The pyruvate formed is reacted with lactate dehydrogenase producing lactic acid with the subsequent oxidation of NADH to NAD+. The decrease in absorbance reflects the enzymatic activity of ALT(inverse colorimetry). Henry Pollard Method CHOLESTEROL COLORIMETRIC METHOD y Acetic anhydride-sulfuric acid(Liebermann Burchard) Development of a blue green color when CHCl3 solution of cholesterol is reacted with a mixture of concentrated H2SO4 and acetic anhydride. This reaction is sensitive to time, temperature and light. y Ferric-chloride-sulfuric acid(Salkowski) y p-toluene sulfonic acid Total Cholesterol Abell-Kendall Simple reference method and probably the widely used for estimation of total cholesterol. This involves saponification of cholesterol esters by alcohol-KOH, extraction of cholesterol by petroleum ether and color development with acetic anhydride H2SO4. This avoids interference by bilirubin, protein and hemoglobin. Schoenheimer Sperry Method The cholesterol is extracted with alcohol-ether. The free cholesterol is then determined by precipitating it with diditonin and determining the cholesterol in the precipitate. The total cholesterol is determined by hydrolyzing the esterified cholesterol by KOH and the precipitation all the cholesterol with digitonin. Cholesterol Esters 0.5% digitonin is added to an alcohol-ether which precipitates the free cholesterol and digitonide. Petroleum ether is used to extract esters. An aliquot of the extract is evaporated to dryness and the cholesterol esters are extracted with CHCl and the color is developed by the Leibermann-Burchard reaction by addition of acetic anhydride and H2SO4. Free Cholesterol Total Cholesterol-Cholesterol ester = Free cholesterol Enzymatic Methods Probably the simplest yet most specific and accurate methods for the estimation of cholesterol are the enzymatic methods whereby cholesterol esters are hydrolyzed with cholesterol oxidase obtained from Nocardia species. H2O2 is formed and quantified colorimetrically. Cholesterol esters Cholesterol + O2
Cholesterol esterase Cholesterol oxidase

Cholesterol + Fatty acid Cholest-4-en-3-one + H2O2

Normal values: range varies according to age Total Cholesterol: 150-250mg% Cholesterol esters: 60-75% of the total cholesterol

TRIGLYCERIDES Tri-Con Method(Giegel, Ham and Clema) Triglycerides are extracted which involves partition of serum lipids between polar and non-polar liquids. Triglycerides remain in the non-polar upper phase while phospholipids, glucose, glycerol and other polar materials remain in the lower phase. Glycerol is liberated from triglycerides by treatment with a transesterifying reagent at room temperature. The released glycerol is oxidized to formaldehyde with periodate and the formaldehyde reacts with acetylacetone and ammonium ions to produce lutidine. The lutidine has a maximum absorbance at 415mu. Zilversmith and van Handel The triglycerides are saponified with alcoholic KOH which results in splitting the triglycerides into fatty acids and glycerol. Periodate solution oxidizes the glycerol into HCHO. The reaction is stopped with arsenic. Reaction of aldehyde with chromotrophic acid results in a colored solution. Enzymatic Method Agent Triglyceride Test Triglycerides are completely hydrolyzed to get free glycerol and free fatty acids by amicrobial lipase. The liberated free glycerol is then determined ezymatically. The disappearance of NADH is observed at 340nm is a stoichiometric measure of the glycerol present which is related to the triglyceride content of the sample. LIPOPROTEINS Beta Centrifugation Most widely used which is a combined ultracentrifugal precipitation method. Plasma is ultracentrifuged at density 1.006 the cholesterol content of the bottom fraction is determined as well as HDL. Precipitation Method/Indirect Beta quantification VLDL and DL are precipitated from plasma by a sulfated polysaccharide such as heparin in the presence of metal ions, phosphotungstic acid/magnesium, dextram sulfate/ magnesium and the amount of HDL is estimated. Friedewald Formula Total Cholesterol= VLDL-cholesterol +LDL-cholesterol+HDL-cholesterol VLDL-C= Triglyceride/ 5 LDL-C= Total Cholesterol Triglyceride/5 HDL-c

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