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B. ACID PHOSPHATASE SIGNIFICANCE- PROSTATE CANCER, IN FORENSIC FOR ASSAULT CASES Use same substrates as above but at pH

B. ACID PHOSPHATASE SIGNIFICANCE- PROSTATE CANCER, IN FORENSIC FOR ASSAULT CASES Use same substrates as above but at pH 5 C. AMYLASE ENZYME THAT CAUSES HYDROLYSIS OF C-O(ESTERS) C-N (PEPTIDES)

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B. ACID PHOSPHATASE SIGNIFICANCE- PROSTATE CANCER, IN FORENSIC FOR ASSAULT CASES Use same substrates as above but at pH

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  1. B. ACID PHOSPHATASE SIGNIFICANCE- PROSTATE CANCER, IN FORENSIC FOR ASSAULT CASES Use same substrates as above but at pH 5 C. AMYLASE ENZYME THAT CAUSES HYDROLYSIS OF C-O(ESTERS) C-N (PEPTIDES) P-O(PHOSPHATES) SIGNIFICANCE: ACUTE PANCREATITIS, GASTRIC ULCERS, MUMPS – NORMAL VALUE 550 U RISING TO 4000 in PANCREATITIS, 700 in MUMPS, 1000 ULCERS SOMOGI METHOD:STARCH + AMYLASE  REDUCING SUGAR ADD FEHLINGS SOLUTION  BLUE COLOR

  2. D. LIPASE Is a high molecular weight hydrolyzing enzyme, acting only on insoluble interfaces(such as oils). Pancreatitic indicator, like amylase. Most common assay is Cherry Crandall method: OLIVE OIL + LIPASE(24 hrs,37 C)  FATTY ACID(Titrate) Seligman proposed Beta Naphthyl Laurate Beta Naphtol Beta Naphthol + 0-Dianisidine  Red Dye(Measure Absorbance) Guilbault: Use Olive Oil(natural substrate) but measure acid produced with 4-Methyl Umbelliferone(fluorescence changes due to pH changes can be measured in minutes rather than 24 hours). E. CHOLINESTERASE(Indicates:Pesticide Poisoning) TWO TYPES: I-TRUE OR RED BLOOD CELL and II-PSEUDO (Liver,Heart,Brain and Serum)

  3. CURRENT ASSAY: BENZOYL CHOLINE(PSEUDO SUBSTRATE) Beta-METHYLCHOLINE(TRUE CHOLINESTERASE) Hydrolysis to free acid and choline derivative Titrate H+ = MICHEL METHOD C-N-C-C-O-CO-CH3 or Phenyl Choline + Acetic Acid C OR HYDOXAMATE METHOD – de la Huerga: CH3COO-Choline + NH2OH  CH3CONHOH CH3CONHOH + Fe(III)  Red Color IF 30-50% Decrease in Activity of ChE = Acute Hepatitis 80-100% decrease =Pesticide Poisoning

  4. F. LACTATE DEHYDROGENASE LACTATE + NAD (pH 8.8-9.8)  Pyruvate + NADH(340nm) MW = 140,000 Exists in 5 isoenzymes(2 types of M(Muscle) and H(Heart)peptide chains).SO=USEFUL FOR HEART AND MUSCLE ASSAYS. LDH Present in All Tissues Liver- 260,000 U/g Heart – 240,000 U/g Muscle - 133,000 U/g Serum – 200 to 430 U/g IF INCREASE IN SERUM EITHER MUSCLE OR HEART SO LOOK AT ISOENZYME : 1,2 =Heart; 4,5 = Muscle In myocardial infarct, for example, 2000 Units in serum

  5. INCREASE STARTS 48 to 72 hours after, stays high for 10-14 days. IN EMERGENCY ROOM, GOT(Transaminase ) better. Get increase in 6 to 12 hours, back to normal in 5 days. IF > 2000 U in serum, end is near LIVER DISEASE – GOES TO 4000 IF TOXIC JAUNDICE ANALYSIS: VALEE METHOD: NADH PRODUCTION AT HIGH pH HENRY METHOD : DECREASE OF NADH(REVERSE REACTION AT LOW pH) CABAUD AND WROBLEWSKI: PYRUVATE + 1,4 DINITRO, 2 AMINO BENZENE  RED COLOR AT 440 to 525 nm G. ALDOLASE Present in all Cells, Indicates Muscle Disease and Myocardial Infarction

  6. ASSAY: D-Fructose-1,6 DiPhosphate + Aldolase(pH 7 to 9.6)  D-Glyceraldehyde + DHAP Values: Normal 11 to 17 mU/mL 5 to 10 fold increase in muscle disease 50 X higher in muscular dystrophy METHODS: l.SIBLEY :GLYCERALDEHYDE + DHAP + 2,4 DINITROPHENYLHYDRAZINE  HYDRAZONE(Measure at 540nm) 2. MEYERHOFF AND LOHMAN DHAP + BASE  PHOSPHATE  HETEROPOLY BLUE(610nm)

  7. H. LEUCINE AMINOPEPTIDASE A Proteolytic enzyme in Tissue, Plasma, Urine ASSAY R3 – NH-CO-NHR1-NH2 + LAP  AMMONIA ,AMINE OR PEPTIDE + AMINOACID(l-LEUCINE or l-ALANINE) SUBSTRATES ARE l-LEUCYL AMIDE,l-LEUCYLGLYCERINE or L-LEUCYLGLYCYLGLYCINE USES; JAUNDICE, CANCER OF PANCREAS METHODS: L-leucyl Beta Naphthylamide Beta Naphthyl Amine Azo Dye Enzyme is specific for Aminopeptidases=No Reaction Trypsin,Chymotrypsin or Pepsin

  8. TRANSAMINASES • ASPRTATE TRANSAMINASE(GLUTAMATE OXALOACETATE TRANFERASE)(GOT) • L-Aspartate + Alpha Oxoglutarate + GOT Oxaloacetate + l-Glutamate • B. ALANINE TRANSAMINASE(GLUTAMATE PYRUVATE TRANSFERASE)(GPT) • L-Alanine + Alpa-Oxoglutarate + GPT Pyruvate + Glutamate • ORGAN GOT GPT • Heart 7800 450 • Liver 7100 2850 • Lung 500 45 • Serum 1 1 (ALL UNITS/mL)

  9. ASSAY EITHER GOT or GPT REACTION  L-GLUTAMATE L-GLUTAMATE + NAD + Glutamate Dehydrogenase NADH Measure Absorbance at 340 nm. Either GOT or GPT Analyzed J. CREATINE (PHOSPHO) KINASE (CK) EXISTS IN 3 ISOENZYMES: CK-MM INDICATES DISEASE OF MUSCLE CK-MB INDICATES HEART DISEASES(MYOCARDIAL INFARCTION CK-BB INDICATES DEATH OF BRAIN ONE OF MOST IMPORTANT ENZYMES REACTION: Creatine + ATP + CK(pH 9) creatine phosphate + ADP

  10. ASSAY METHODS • GEL ELECTROPHORESIS-SEPARATE 3 ISOENZYMES, ADD STAIN TO GIVE FLUORESCENCE OR COLOR • B. SAX-MOORE; • CREATINE + NINHYDRIN + BASE FLUORESCENCE • REACTION RUN IN REVERSE AT pH 7.4 • NIELSEN/LUDVIGSEN/ROSALKI • Creatine Pi + ADP + CK(pH7) Creatine + ATP • ATP + GLUCOSE + HEXOKINASE GLUCOSE –Pi • GLUCOSE Pi + NADP + DehydrogenaseNADPH • Measure Absorbance at 340nm)

  11. SUBSTRATES ANALYSIS • MOST IMPORTANT: GLUCOSE,UREA,CREATININE,CHOLESTEROL, L-PHENYLALANINE,HDL,LDL,TRIGLYCERIDES,ETC • GLUCOSE STARCH • METABOLISM: + AMYLASE • SUCROSE + INVERTASE  GLUCOSEGlucose 6 Pi • + FRUCTOSEFructose 1,6 diPi • DiHydroxyAcetone • Pyruvate Citrate Cycle • Acetate Cycle

  12. FASTING STATE: GLYCOGENOLYSIS(GLUCOSE MADE FROM GLYCOGEN) NORMAL = GLYCOGENESIS (PRODUCE GLYCOGEN)PLUS LIPOGENESIS (PRODUCE FAT) DIABETES = GLUCOSE BUILDS UP DUE TO LACK OF INSULIN AND REPRESSION OF LIPOGENESIS AND OF GLYCOGENESIS TYPE 1 – GENETIC, MORE SERIOUS,INJECTIONS NECESSARY TYPE 2 – MOST OF PEOPLE GET, LACK OF INSULIN DEVELOPS INSULIN ADDED TO TYPE 1(AT LAST STAGES TYPE 2= DRUGS GLUCOBAY/GLUCOPHARGE AT FIRST-ADSORBS GLUCOSE) INSULIN NORMALLY PRODUCED IN PANCREAS: -PROMOTES GLYCOGENESIS AND LIPOGENESIS - INCREASES PERMEABILITY

  13. NORMAL STATE 90-120mg% in blood • In diabetes goes up to 150-400mg% • In hypoglycemia or hyperinsulism – can be as low as 50mg% • Caused by tumor which secretes insulin at high levels • ASSAY METHODS: • MOST COMMON INVOLVES GLUCOSE OXIDASE=Works on • Beta-D-Glucose(Glucose is 36% alpha,64% beta • -add mutarotase100% beta) • GLUCOSE + O2 + Glucose Oxidase  Gluconic Acid + Peroxide • Oxidation occurs at aldehyde (position #1) of glucose • a.Electrochemical is most common.Measure peroxide by oxidation

  14. Most instuments like Glucose pen(home use)or Yellowsprings(industry) b. o-Dianisidine Method o-Dianisidine + Peroxide + Peroxidase  Red Color(Oxidized o-D) 2. o-Toluidine = Dubowski Method Glucose + o-Toluidine  Schiff Base(Color at 630nm) Problem: Interference from other reducing sugars like galactose, mannose, lactose. 3. BEST METHOD = LONG TERM GLUCOSE HbA1c = HEMOGLOBIN LINKED TO TERMINAL AMINOACID OF BETA CHAIN OF HEMOGLOBIN(ANTIBODY TEST) Normal Value 4.2 – 6 % OTHER SUGARS: B. GALACTOSE  GALACTOSEMIA(HIGH GALACTOSE IN INFANTS WITH INABILITY TO METABOLIZE GALACTOSE.

  15. CAN’T TOLERATE MILK SINCE LACTOSE IS METABOLIZED TO GALACTOSE)- Measure using galactose oxidaseperoxide C. ASCORBIC ACID = VITAMIN C C. ASCORBIC ACID + O2  ACID FORM OF ASCORBATE(Oxidizes rapidly in air=precautions necessary) Normal Levels 0.5 to 1.5mg% Needed for regulation of Colloidal Condition of intercellular Substances Not naturally produced = must be taken in diet COMMON ASSAY: 2,6 Dichloro Indophenol + Ascorbic Acid Colorless Max Wavelength 500nm STABILITY OF BLOOD-After blood is drawn and stands uncentrifuged there is a 7% decrease in serum glucose/hour – Glycolysis

  16. Prevented by collecting in a tube coated with NaF – stabilized glucose, inhibits coagulation. Oxalate also used. PROTEIN FREE SOLUTION(Necessary in some assays) l. FOLIN WU METHOD: Protein + Tungstic Acid  Precipitate out all proteins 2. Somogyi-Nielson Method ZnSulfate + Ba(OH)2 + Proteins  ZnProteinate + BaSulfate 3. Trichloroacetic Acid + Proteins Precipitate WHAT IS BLOOD = Complicated Mixture of Organic and Inorganic materials dissolved in water. Plasma is a mixture of particulates of cell forms in fluid If collect without anticoagulant – Native Plasma(similar to blood in your veins) If add anticoagulant = modified plasma(oxalate, citrate,heparin,EDTA prevents clots. If clotting occurs, we obtain Serum-centrifuge off precipitate,use liquid for assays.Lacks fibrinogen(3 to 6% of protein)

  17. BLOOD IS 92 to 93% Water, 7 to 8% solutes. MOST ANALYSIS IS WITH SERUM EXCEPT ELECTROLYTES(Na+,K+, Ca++) MUST BE DONE ON WHOLE BLOOD USING ION SELECTIVE ELECTRODES(OLD METHOD FLAME EMISSION MEASURES TOTAL IONS- WE NEED MEASURE OF FREE IONS ONLY) D. REDUCING SUGAR TEST a.Ferricyanide + Reducing Sugar  Ferrocyanide(measure at 420nm) b. Cu(II) + Reducing Sugar  Cu(I) + Phosphomolybdic Acid Blue COMPARISON: TRUE GLUCOSE,mg% REDUCING SUGAR,mg% 70 90 100 120 200 220 So about 20mg% due to galactose,mannone,lactose

  18. E. UREA WAS THE FIRST CLINICAL TEST, VERY RELIABLE INDICATION OF KIDNEY FUNCTIONING DIFFICULT-IS DIET DEPENDENT, SO MANY LABS PREFER CREATININE TO BE DONE ALSO SERUM- 15 to 40mg % UREA.Goes to 100mg% if failure of kidney ASSAY : a. NESSLERS REAGENT UREA + UREASE  2 NH3 NH3 + 2 HgI2-KI  H2N-Hg2I3(Red Color at 500 nm) b. INDOPHENOL METHOD NH3 + NaOCl + 2 Phenol + Nitroprusside(Na2Fe(CN)5NO  Indophenol Blue (560nm)

  19. c. DIACETYL MATHOD Because of Instability of Diacetyl, Use Diacetyl Monoxide, add Acid  Diacetyl CH3COCOCH + UREA  CH3C-C-CH3 Diacetyl N N C O Diazine yellow F. URIC ACID Test for Kidney and Renal(Gout) Function Normal- Males 2.5 to 7mg% Females – 1.5 to 6mg% In gout goes to 10mg%(Reflection of Nucleic Acid Breakdown)

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