Transcript BIOANALYTICAL/CLINICAL ANALYSIS

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
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)
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
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
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
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)
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
I.
TRANSAMINASES
A.
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
Serum
1
45
1 (ALL UNITS/mL)
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
ASSAY METHODS
A. 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
C. NIELSEN/LUDVIGSEN/ROSALKI
Creatine Pi + ADP + CK(pH7) Creatine + ATP
ATP + GLUCOSE + HEXOKINASE GLUCOSE –Pi
GLUCOSE Pi + NADP + DehydrogenaseNADPH
Measure Absorbance at 340nm)
SUBSTRATES ANALYSIS
MOST IMPORTANT:
GLUCOSE,UREA,CREATININE,CHOLESTEROL, LPHENYLALANINE,HDL,LDL,TRIGLYCERIDES,ET
C
A. GLUCOSE
STARCH
METABOLISM:
+ AMYLASE
SUCROSE + INVERTASE  GLUCOSEGlucose 6 Pi
+ FRUCTOSEFructose 1,6 diPi
DiHydroxyAcetone
Pyruvate Citrate Cycle
Acetate Cycle
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
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:
1. 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
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.
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
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)
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
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)
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)