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)

METHODS

a.PHOSPHOTUNGSTIC ACID

URIC ACID + H3PW13O40 ALLANTOIN BLUE

b. ENZYMATIC

URIC ACID + URICASE PEROXIDE + ALLANTOIN

-Measure decrease in absorbance at 290nm of Uric Acid

OR – Use o-dianisidine + Peroxide Red Color

G. CREATININE

Better method for Kidney Function. No dietary effects.Difficulty-only very low concentrations(approx. 1 mg%) in serum

BASIC EQUILIBRIUM:

CREATINE + ACID CREATININE + BASE CREATINE

NORMAL: Men= 0.6 to 1.2mg%; Women= 0.50 to 1 mg%

If Creatinine rises to 2 to 4mg% -Moderate to severe kidney damage

ASSAY: JAFFE METHOD

Alkaline picrate + Creatinine Red Color at 500 nm

To do Creatine add Acid Creatinine, assay as above

H. PROTEIN

Proteins provide a Nutritive role

If protein level changes=disease of kidney

Normal rate is 6 to 8mg% in serum

ASSAYl. TOTAL PROTEIN BY FOLIN-CIOCALTEAU REAGENT

ADD PHOSPHOTUNGSTIC ACID, OXIDIZE THE –OH GROUP IN TYROSINE RING(COLOR CHANGE FROM YELLOW TO BLUE

2. KJELDAHL METHOD : PROTEIN NH3 on digestion with acid/catalyst.Distill NH3 into Boric Acid H2BO3-(titrate with Acid)

3. BIURET METHOD: Cu(II) + PROTEIN BLUE COLOR (530nm) DUE TO REACTION OF Cu(II) WITH C=O and =NH GROUPS OF PROTEIN

CALLED BIURET BECAUSE OF ANALOGY OF Cu(II) REACTION WITH BIURET

I. ALBUMIN -ONE OF KEY PROTEINS IN BODY

ASSAY: ALBUMIN + METHYL ORANGE OR HYDROXYAZOBENZENE COLOR at 540nm.

NORMAL LEVELS: 3.8 to 4.7 g%.

J. FIBRINOGEN

A PLASMA PROTEIN FACTOR NECESSARY FOR COAGULATION.

NORMAL: 200 to 400 ng%

GOES TO 700 ng% IN TUBERCULOSIS, PNEUMONIA

ASSAY – CLOT WITH THROMBIN AND MEASURE DIFFRACTION OF LIGHT

K. AMINO ACIDS -3 ARE IMPORTANT IN CLINICAL:

1. PHENYLALANINE = BENZENE-CH2CHCOOH

NH2

2. TYROSINE = HO-BENZENE-CH2CHCOOH

NH2

3. TRYPTOPHAN = INDOLE-CH2CHCOOH

NH2

TOTAL AMINO ACIDS = 2.5 to 4.5mg% in Serum(Large Increase indicates severe liver damage)

ASSAY: NINHYDRIN + AMINOACID DEEP RED COLOR

L. l-PHENYLALANINE

MOST IMPORTANT AMINO ACID – PHENYLKETONURIA (PKU) TEST=MEASURES BUILDUP OF PHENYLALANINE IN NEWBORN BABIES. BUILD UP DUE TO LACK OF ENZYME l-PHENYLALANINE HYDROXYLASE l-TYROSINE, AN IMPORTANT STEP IN METABOLISM OF BODY.

BUILD UP LEADS TO MENTAL RETARDATION IN BRAIN=ONE OF KEY CAUSES OF MENTAL RETARDATION IN NEWBORNS.

ASSAY: PHENYLALANINE + Fe(III) GREEN COLOR

OR PHENYLALANINE + NINHYDRIN + LEUCYLALANINE FLUORESCENCE(EXCITATION 365nm,EMISSION 525nm)

INFANTS: 2.1mg% AT BIRTH, 4.5mg% 3-4 DAYS,

20-30mg% 10 days=SERIOUS

M. CHOLESTEROL/TRIGLYCERIDES/HDL/LDL TESTS

IMPORTANCE=DIAGNOSIS OF POSSIBILITY OF MYOCARDIAL INFARCTION.

SOME CHOLESTEROL NECESSARY FOR BODY FUNCTIONING. BODY SYNTHESIZES 2 gram a day(3X level that is taken in):Acetate radicals + Amino Acids,Carbohydrates and Fatty Acids Cholesterol

ATTEMPTS TO LOWER CHOLESTEROL WITHOUT LIMIT OF DIETARY INTAKE = UNSUCCESSFUL

PROBLEM: CHOLESTEROL IS INSOLUBLE AND CANNOT ENTER BLOOD STREAM BY CROSSING GI TRACT WITHOUT LIPOPROTEINS WHICH SOLUBILIZE CHOLESTEROL.THESE ARE IMPORTANT HDL AND LDL MOLECULES. TRIGLYCERIDES ARE FATTY MOLECULES THAT AFFECT MI BY BUIDING UP IN BLOODSTREAM.

ALL OF THESE ARE IMPORTANT AND MUST BE ASSAYED.

l. CHOLESTEROL

NORMAL 150 to 180 mg%

at up to 850mg% =GOOD CHANCES OF MI

Cholesterol is removed by liver Bile Acids(Hydroxylation Reaction)

METHODS OF ANALYSIS:

a. ENZYMATIC:

TOTAL CHOLESTEROL + ESTERASE CHOLESTEROL + OXIDASE PEROXIDE + o-DIANISIDINE (RED COLOR)

OR CAN MEASURE IN NOVA OR YELLOWSPRINGS INSTRUMENT WITH IMMOBILIZED ENZYMES IN BIOSENSOR

b. LIEBERMAN-BURCHARD METHOD

CHOLESTEROL + SULFURIC ACID GREEN COLOR(Bis Chlosteradienyl Monosulfonic Acid)

c. CHOLESTEROL INSTRUMENT(SEE NEXT PICTURE)

2. TRIGLYCERIDES

TRIGLYCERIDES= ESTERS OF HIGH M.W., CONSTITUTE 95% OF FAT STORED IN TISSUES.

TERMED POLYUNSATURATED FATA

UNDERGO DIGESTION IN DUODENDUM AND ILEUM.

THEY ARE HYDROLYZED TO GLYCEROL + FATTY ACIDS BY LIPASES. DANGER: IN BLOOD STREAM, RESTRICT FLOW OF BLOOD SINCE THEY ARE INSOLUBLE.

THE BAD LDL AND VLDL LIPOPROTEINS CAUSE ELEVATED TRIGLYCERIDES AND HENCE INCREASED POSSIBILITY OF MI.

THE GOOD HDL CAUSES ONLY NORMAL LEVELS-LOWER MI

NORMAL 40-160 mg% Male; 35-135 mg% Female.ABOVE 200 BAD

ASSAY: ADD LIPASEFATTY ACIDS(MEASURE WITH DYE)

3. LIPOPROTEINS

- TRANSPORT INSOLUBLE LIPIDS

- PLAY KEY ROLE IN CHOLESTEROL/TRIGLYCERIDE METABOLISM.

a. GOOD = HDL(HIGH DENSITY LIPOPROTEIN)

- Important in removing Cholesterol from Tissues,thereby reducing the amount of cholesterol stored in body

- Involved in returning cholesterol from arteries to liver for removal as bile acids(Reverse Cholesterol transport)

- Plays a major role as scavenger of lipid and apolipid proteins

MEASUREMENT: USE ANTIBODY TO HDL

Higher Value the better(low risk of MI)->35 mg% best

b. BAD = Low Density Lipoproten(LDL)and Very Low Density Lipoprotein(VLDL)

-Causes Increased Uptake of Cholesterol by allowing Transport Across GI Tract. Cholesterol deposits in arterial walls leading to premature athersclerosis.

- Patients with Genetic Disorder Familial Hypercholesterolemia(FH) have a gene that codes fopr the LDL receptor.

HIGH LDL and VLDL = GRAVE RISK

Range of LDL 0 to 150mg%. Above 150 BAD

K. THYROID GLAND = Size of Butterfly, Synthesizes Hormones

TEST FOR FUNCTIONING CALLED PBI(Protein Bound Iodine- Av PBI = 3.5 to 8.0 microgram %)

T3 and T4 Important-Assayed by Immunology,old method RadioImmunoassay,Now Enzyme Immunoassay with Antibodies

AUTOMATION

a. First Machine called AUTOANALYZER. Developed by Leonard Skeggs at Oak Ridge Labs in 1960’s.

ONE OF BIG ADVANCES IN CLINICAL ANALYSIS

TWO BIG FACTORS IN INSTRUMENT

- USE OF DIALYZER MEMBRANE TO SEPARATE 2 GROUPS(WATER SOLUBLE-DIFFUSABLE-AND NON DIFFUSABLE)

- USE OF AIR BUBBLES TO SEPARATE SAMPLES FOR RAPID INTRODUCTION INTO INSTRUMENT

ORIGINAL INSTRUMENT(SMA) 60 samples/hour with 12 tests

Then New SMAC – Computer Linked 24 assays with 240/hour

See Next View

SAMPLE DIALYZER Non-Diffusable: Total Protein(Folin Calcateau Reagent) Albumin(Dye) Billirubin(dye) Alkaline Phosphatase(Color) LDH,CK,Transaminases etc DIFFUSABLE: Electrolyes(Na+,K+,Ca++,Cl-)-ISE’s Urea(Diaceyl Reaction) Glucose (Enzymatic) Bicarbonate(Acid Base) l-Phenylalanine(dye) Cholesterol(LB Reaction)RESULTS DISPLACED AUTOMATICALLY AS SHOWN.2. DUPONT AUTOMATIC CLINICAL ANALYZER:

SAMPLE CRUSHER INCUBATE READ PACKINJECTED INTO PACK AS CUVETTE

EACH TEST HAD SEPARATE COMPUTER LABELED PACK.ALL PACKS LOADED ON CONVEYER BELT. TEST RESULTS COMPUTER REPORTED. USUAL METHODS USED.

3. OTHER INSTRUMENTS

a. PERKIN ELMER CLINICAL ANALYZER – PROGRAMMED FOR 35 TESTS, 300/HOUR:

GLUCOSE = HEXOKINASE/NADH; UREA = NITROPRUSSIDE REACTION; CREATININE=JAFFE REACTION; CHLORIDE = Hg++ + Fe+++ + SCN- ; PHOSPHATE = MOLYBDATE; URIC ACID = URICASE;TOTAL PROTEIN = Cu++ COMPLEX; ALBUMIN= COMPLEX; LDH and CK = NADH PRODUCTION; TRANSAMINASES = NADH; Ca,K and Na by ISE’s; ETC

b. MASS SPEC CENTER OF USPHS –WORLDWIDE DATA BASE FOR POISONS. ANYTHING CAN BE IDENTIFIED=CHILD?

c. DADE STRATUS – DRUGS, CLINICAL METABOLITES(LIKE PSA, T3,T4 ETC BY AUTOMATED IMMUNOASSAY=LATER)

d. BLOOD GAS ANALYZER= RESPIRATORY SYSTEM BY pH, BICARBONATE, FREE CO2 ETC) = VERY BIG IN CLINICAL LAB

e. NOVA INSTRUMENT FOR ELECTROLYTES/GLUCOSE/UREA/CHOLESTEROL/LACTATE

- USES ISE’s ON WHOLE BLOOD FOR ELECTROLYTES

- BIOSENSORS FOR GLUCOSE, UREA, CHOLESTEROL, LACTATE

f. TODAY BIGGEST ARE (1)NOVA (2)BLOOD GAS (3) LARGE CLINICAL ANALYZERS LIKE OLYMPUS AND ROCHE(FORMERLY BOEHRINGER ANALYZER). FOUND IN ALL CLINICAL LABS

CLINICAL LAB IS BIGGEST EMPLOYER OF ANALYTICAL CHEMIST. WHY? Over 10,000 Hospitals Worldwide each with lab(Some very big) plus private labs that do only blood work worldwide=estimate at least 5000 large ones.Sommerville N.J.eg.

IMMUNOASSAYS

ANTIGEN + ANTIBODY(LINKED TO ENZYME) COMPLEX. CALLED ENZYME IMMUNOASSAY = REPLACES OLD RIA OF ROSEMOND YALLOW WHO WON NOBEL PRIZE FOR THIS WORK in 1950’s

ACTIVITY OF ENZYME

CONC OF ANTIGEN

CONCEPT: ON BINDING TO ANTIGEN, THE ANTIBODY-ENZYME COMPLEX LOSES ACTIVITY BECAUSE BINDING SITE OF ENZYME IS COVERED BY ANTIGEN. HENCE ACTIVITY DECREASES WITH TIME AND ACTIVITY IS RELATED TO ANTIGEN CONCENTRATION>

LARGE USEAGE IN CLINICAL ANALYSIS TODAY:

l. PSA(PROSTATE SPECIFIC ANTIGEN). USE KIT WITH ANTIBODY TO PSA LABELED WITH ENZYME. LOOK AT COLOR – RELATE TO PSA ACTIVITY.

2. ISOENZYMES. THERE ARE SPECIFIC ANTIBODIES TO LDH 1,2 and LDH 4,5, TO CK MB,

3. LIPOROTEINS – HDL, LDL and now VLDL

4. THYROID FUNCTION – T3 and T4

DADE STRATUS MACHINE FOR IMMUNOASSAY:

SERUM PACKAGE FOR EACH IMMUNOASSAY CRUSH MIX ADD SUBSTRATE READ FLUORESCENCE

In reagent pack we have Antibody for each test labeled to Alkaline Phosphatase. Add substrate(naphthol phosphate),read fluorescence

Labeled Pack for I

IMMUNOASSAY IS IMPORTANT IN

- FOOD .ASSAY FOR BACTERIA(E.COLI,SALMONELLA, LISTERIA)

-ENVIRONMENTAL( ALL PESTICIDES DONE IN KITS TODAY.CHEAPER, NO NEED FOR CLEAN UP)

- CLINICAL – IMMUNOASSAY IS THE SECOND GENERATION TEST

NEXT STEP WILL BE RECEPTORS DIRECTLY

but LACK OF LARGE SUPPLY WILL HINDER THIS

ALREADY DONE BY PHARMACEUTICAL COMPANIES FOR DRUG DEVELOPMENT

REVIEW

MUST KNOW:

l. SIGNIFICANE/METHODOLOGY FOR

4 SUBSTRATE AND 4 ENZYMES

GRADED ON RECOGNIZING MOST IMPORTANT

2. ISOENZYMES – WHAT ARE THEY, WHAT ARE THEY USED FOR, EXAMPLES

3. AUTOMATION IN CLINICAL LAB – HISTORY, TYPES OF INSTRUMENTS, WHAT DO THEY DO

4. FUTURE – WHERE ARE WE GOING??

5. VALIDATION

6. SET UP OF CLINICAL LAB

7. HISTORY OF CLINICAL ANALYSIS(IMPORTANT MARKER)