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CM3107 BIOANALYTICAL/CLINICAL ANALYSIS. Prof. George G. Guilbault Room 205, Robert Kane Building University College Cork, Ireland. CLINICAL BIOMEDICINE STUDY OF CHEMISTRY OF HUMAN SYSTEM -WHAT CHEMICALS ARE INVOLVED IN DISEASES - WHICH ONES AFFECT WELL BEING
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CM3107BIOANALYTICAL/CLINICAL ANALYSIS Prof. George G. Guilbault Room 205, Robert Kane Building University College Cork, Ireland
CLINICAL BIOMEDICINE STUDY OF CHEMISTRY OF HUMAN SYSTEM -WHAT CHEMICALS ARE INVOLVED IN DISEASES - WHICH ONES AFFECT WELL BEING - WHY IMPORTANT 2/3 OF ALL ANAL WORK IS IN THIS AREA 1 OF 2 OF ALL PERSONNEL IN THIS AREA HISTORY OF CLINICAL: l. OTTO WARBURG IN 1940 – NADH ABSORBS AT 340nm 2. 1965 OVERHAUL OF CLINICAL ANALYSIS(NEXT) 3. 1970 – SKEGGS - AUTOMATION
HIGH GLUCOSE = DIABETES- IMBALANCE OF METABOLISM SUCROSE GLUCOSE GLYCOGEN IN 1965 NATIONAL INSTITUTES OF HEALTH DID A STUDY OF THE ABILITY OF ANALYTICAL LABS TO “ GET THE CORRECT RESULT” TRUE RESULT WAS NORMAL: 90 mg/dL 30% OF LABS REPORTED HIGH(RESULT:INJECTION OF INSULIN DEATH) 40% OF LABS REPORTED NORMAL 30% OF LABS BELOW NORMAL(RESULT:INJECTION OF SUGAR LETHAL TO DIABETIC) RESULT: BIG SHAKEUP IN LABS LEADING TO BETTER RESULTS AND A MODERNIZATION OF ANALYSIS.
MOST IMPORTANT TESTS l. GLUCOSE – HIGH DIABETES( IMBALANCE OF METABOLISM) 2. UREA/CREATININE – MALFUNCTIONING OF KIDNEY 3. CHOLESTEROL/HDL/LDL/VLDL – MYOCARDIAL INFARCT(HEART) 4. URIC ACID – GOUT 5. ALKALINE PHOSPHATASE – DAMAGE TO LIVER 6. AND OF COURSE – PHARMACEUTICAL INDUSTRY BENEFITS BY PROVIDING NEW DRUGS FOR THESE DEFICIENCIES BIGGEST – DIABETES – NEW DRUG RECENTLY APPROVED FOR TYPE 2 DIABETES = JANUVIA
PURPOSE OF CLINICAL LAB=QUAL AND QUANT ANALYSIS OF BODY FLUIDS-BLOOD,URINE,SPINAL FLUID,FECES,etc UNLIKE ALL OTHER ANALYSIS HERE WE USE LIQUID NOT MASS(TAKE FEW MICROLITERS BLOOD).USE dL AS UNIT.IN IRELAND/UK USE mM,ALL OTHERS mg/dL: Mg per liter/Molecular Weight = mM
CURRENT TYPICAL VALUES IN PLASMA GLUCOSE,mg% 90 (5.0 mM) UREA,mg % 16 CREATININE,mg% 1.1 URIC ACID, mg% 4.6 TOTAL CHOLESTEROL 194 POTASSIUM,meq/liter 4.4 ALKALINE PHOSPHATASE,units 3.0 LDH, units 360
ORIGINS OF SPECIES DETERMINED STAGE 1 – TESTS ARE ON ENZYMES OR SUBSTRATES COMING FROM SPECIFICALLY DAMAGED ORGANS l. ALKALINE PHOSPHATASE – DAMAGED LIVER HIGH: OBSTRUCTIVE JAUNDICE,PADGETTS DISEASE, RICKETS 2. CHOLINESTERASE – DAMAGE TO NERVE ENDINGS HIGH: NEPHROTIC SYNDROME LOW: PESTICIDE POISONING, ANEMIA, MALNUTRITION 3. LACTATE DEHYDROGENASE(LDH) MYOCARDIAL INFARCTION, LEUKEMIA 4. GLUCOSE – DIABETES(TYPES 1 AND 2) 5. UREA/CREATININE – DISEASES OF KIDNEY
QUALITY CONTROL AND VALIDATION • VERY IMPORTANT IN CLINICAL LABS WHERE LIFE/DEATH DECISIONS ARE MADE • Reference: N. TIETZ-FUNDAMENTALS OF CLINICAL CHEMISTRY • MUST CALIBRATE INSTRUMENT TWICE DAILY WITH STANDARD BLOOD SAMPLE WITH KNOW VALIDATED VALUES • B. MUST USE REAL BLOOD SAMPLES: • l. POOLED SERUM • 2. FREEZE DRIED SERUM CONTROLS • a. DADE – MONITROL: NORMALS FOR ALL SUBSTRATES(eg GLUCOSE),METALS, ENZYMES • b. LEDERLE LABS – LEDERNORM(MOST TESTS)
NORMAL VS ABNORMAL WHAT IS NORMAL = AVERAGE PERSON(?) -ACCEPTED AS ABOUT 80% OF POPULATION -RANGE IN WHICH 95% FIT(1 in 20 OUTSIDE) - 80 to 95% OF POPULATION ARE GREY AREA - 5% CONSIDERED ABNORMAL(See TIETZ) DADE FOR EXAMPLE CAREFULLY SCREENS DONORS FOR DISEASES, MEDICAL HISTORY. -PICKES SEVERAL HUNDRED,COLLECTS BLOOD=FORMS A POOL. FREEZE DRY BLOOD, ASSAY AND REPORT ALL VALUES(SEPARATE FOR EVERY DIFFERENT INSTRUMENT).ONLY ABOUT 5% TAKEN AS REPRESENTATIVE FOR ANALYSIS. -ABNORMAL- SPIKE WITH PURE REAGENTS.ASSAY
TYPES OF ASSAYS l. EQUILIBRIUM – LET ALL REACT, MEASURE AT EQN A + B PRODUCTS MEASURE PRODUCTS BY COLOR, FLUORESCENCE, ELECTROCHEMICAL METHOD 2. KINETICS MEASURE RATE OF REACTION AS AFFECTED BY A ABOVE AT CONSTANT B. 4 X C1 ABSORBANCE 3 X C1 2 X C1 C1 TIME
ASSAY OF ENZYMES MUST BE DONE BY KINETIC(RATE ) METHOD SINCE THEY ARE CATALYSTS ADVANTAGES: THEY ARE HIGHLY SPECIFIC AND SENSITIVE BASE EQUATIONS E + S(k1/k2) ES (k3) PRODUCTS + E Vo = Vmax[So]/Km + So where Vmax = k3[Eo] Km = k2 + k3/k1 ENZYME ACTIVITY = HOW MUCH SUBSTRATE IS CONVERTED AMBIGUITY ARISES SO USE INTERNATIONAL UNITS IN OLD SYSTEM WE HAD MANY ASSAYS WITH DIFFERENT CONDITIONS/IMPOSSIBLE TO CORRELATE THERE ARE A NUMBER OF IMPORTANT ENZYMES WE WILL LOOK AT AND ALSO ISOENZYMES.
TO SYSTEMATIZE: COMMISSION ON ENZYMES IF INTERNATIONAL UNION OF BIOCHEMISTRY IN 1961 MET IN NEW YORK: RECOMMENDATIONS OF I.U.B., ELSEVIER, 1965 ONE UNIT = AMOUNT OF ENZYME THAT CATALYZES REACTION OF ONE MICROMOLE OF SUBSTRATE PER mg ENZYME PER MINUTE AT STANDARD CONDITIONS(USUALLY T=30 C,pH OPTIMUM ETC) RECOMMEND U/mL or U/L at 30 C REMEMBER: ENZYMES ARE SECONDARY INDICATORS OF CELL OR TISSUE ACTIVITY- IF UP OR DOWN= SOMETHING WRONG.BETTER = DIRECT ASSAY OF TISSUE. THIS IS DIFFICULT:IN SECOND GENERATION TESTS WE USE (l) IMMUNOCHEMISTRY,eg PSA TEST .
ENZYMES AND DISEASES. ENZYME ORGAN OF DISEASE SPECIMEN Acid Phosphatase Prostate Serum Alkaline Phosphatase Liver, bone Serum/urine Amylase Pancreas Serum/urine Lipase Pancreas Serum/urine Lactate Dehydrogenase(LDH)Liver,Heart Serum/urine Aspartate transaminase(GOT) Liver,Heart Serum/urine Alanine transaminase(GPT) Liver,Heart Serum/urine Creatine Hinase(CK) Heart,Muscle,Brain Serum Aldolase Copper transport disease(Wilson) Serum
USE OF PROFILING SEVERAL TESTS USED TO INDICATE A SPECIFIC FUNCTIONING OF BODY WHY-COVER ALL POSSIBILITIES(AVOID LAWSUITS) l. LIVER : LDH, GOT,GPT,ALK PHOSPATASE,GAMMA GLUTAMYL TRANSPEPTIDASE(GGTP) 2. KIDNEY : UREA, CREATININE, GGTP 3.PANCREAS : LIPASE, AMYLASE,LEUCINE AMINOPEPTIDASE 4. MUSCLE: CK, LDH 5. BONE: ALKALINE PHOSPHATASE –TARTRATE INHIBITED(ISOENZYME 1)OR UREA INHIBITED(ISO 2) 6. CANCER : ALK AND ACID PHOSPHATASE,LDH,ASPARAGINASE,LAP
ISOENZYMES=MAJOR IMPROVEMENT in ASSAYS WHAT ARE THEY:SOME ENZYMES FROM AN INDIVIDUAL ORGANISM CAN EXIST IS SEVERAL DIFFERENT FORMS=ISOENZYMES DIFFERENCES- l. CHARGE = CAN BE SEPARATED BY ELECTROPHORESIS 2. STABILITY TO HEAT DENATURATION 3. REACTION TO CHEMICAL INHIBITORS(Alk Phosphatase) 4. AFFINITY FOR SUBSTRATES OR COENZYMES WHICH ONES ARE COMMON: LDH,CK,ALK AND ACID PHOSPHATASE(PERHAPS ALL BUT NOT GLUCOSE OXIDASE)
ANALYSIS OF ENZYMES • WHICH ARE MOST IMPORTANT: • ALKALINE PHOSPHATASE –SIGNIFICANCE: • LIVER DISEASE INDICATOR, OBSTRUCTIVE JAUNDICE,PADGETTS AND OTHER LIVER DISEASE INDICATORS • ASSAY: PHENYLPHOSPHATE PHENOL + Pi • PHENOL + DIAZOREAGENT RED COLOR • ACTIVATORS – Mg(II) AND Mn(II) • OR p-NITROPHENYLPHOSPHATE p-NITROPHENOL(410nm) • OR FLUOROMETRIC USING NAPTHOL ASBI PHOSPHATE GREEN FLUORESCENCE