1. Clinical Chemistry Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653 Travlos@niehs.nih.gov

2. Clinical Chemistry The analysis of individual constituents, proteins, enzymes, nutrients, waste products, metabolites, hormones, etc. in blood or body fluids that provides information regarding the function or integrity of a tissue, organ or organ system While almost anything may be analyzed, the efficacy of a test depends on its specificity and sensitivity to detect pathological change

3. Analytical Procedures/Methods Too numerous to cover Photometry Fluorometry Nephelometry Electrophoresis Isotopic immunoassay Chromatography Spectrometry

4. Considerations for Blood Collection Whole blood collected in a container without anticoagulant Samples from indwelling catheters are usually acceptable Allow blood to clot for 30 to 60 minutes Separate serum for red cells into a clean plastic container Glucose Enzyme leakage

5. Sources of Variation Diet NIH-07 v NTP 2000 Fasting Glucose Diurnal variation Hormones Analytical Methods & Sample Collection/Handling Techniques Cholinesterase Creatine Kinase In vitro Hemolysis Urine Collection/Handling

6. Diet: NIH-07 v. NTP-2000 AnalyteNIH-07NTP-2000 ALT (IU/L) Males 56.5 90.0 Females 47.5 77.0 BUN (mg/dL) Males 20.0 15.0 Females 20.5 14.8 Switching diets resulted in an approximately 60% increase in control animal serum ALT activity and a 26% decrease in serum BUN concentration.

10. Assay Variation: AChE (IU/L) Propargyl AlcoholControl64 ppm Males 1071 778

11. Assay Variation: AChE (IU/L) Propargyl AlcoholControl64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition

12. Assay Variation: AChE (IU/L) Propargyl AlcoholControl64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assay BTC assay Untreated 0.1 mM 1.0 mM 10.0 mM Assays: male rat serum; 2.5 hour incubation; performed in duplicate

13. Assay Variation: AChE (IU/L) Propargyl AlcoholControl64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assay BTC assay Untreated 876 272 0.1 mM 795 289 1.0 mM 825 299 10.0 mM 836 262 Assays: male rat serum; 2.5 hour incubation; performed in duplicate

14. Troponin

15. 0.00 0.05 0.10 0.15 0.20 0.25 0 5 10 15 20 Comparison of cTn Measurement in the Comparison of cTn Measurement in the Beagle cTnI (ng/mL) cTnT (ng/mL) NegLowMedHigh Roche Elecsys 2010 Dog Troponin EIA DPC Immulite OCD Vitros ECi Dade Dimension RxL Beckman Access Bayer Advia Centaur Tosoh AIA 600 II Abbott Architect

16. 0.00 0.25 0.50 0.75 1.00 0 5 10 15 20 25 Comparison of cTn Measurement in the Cynomolgus Monkey cTnI (ng/mL) cTnT (ng/mL) NegLowMedHigh Roche Elecsys 2010 Monkey Troponin EIA DPC Immulite OCD Vitros ECi Dade Dimension RxL Beckman Access Bayer Advia Centaur Tosoh AIA 600 II Abbott Architect

17. Comparison of cTn Measurement in the Sprague Dawley Rat cTnI (ng/mL) cTnT (ng/mL) NegLowMedHigh 0 1 2 3 4 5 6 0 5 10 15 20 25 30 Roche Elecsys 2010 Rat Troponin EIA DPC Immulite OCD Vitros ECi Dade Dimension RxL Beckman Access Bayer Advia Centaur Tosoh AIA 600 II Abbott Architect

18. NTP Core Clinical Chemistry Profile Protein Total protein Albumin Muscle Creatine Kinase Kidney Urea Nitrogen Creatinine Liver Alanine Aminotransferase Sorbitol Dehydrogenase Alkaline Phosphatase Total Bile Acids

19. Evaluation of Liver Alanine Aminotransferase (ALT, SGPT) Greatest activity - hepatocytes; also found in skeletal/cardiac muscle Biological half-life - varies (~48-60 hours) Sample stability - stabile at room, refrigerated and frozen temperatures Can be induced (eg., glucocorticoids) Increased - hepatocellular injury, induction, muscle injury Decreased - enzyme inhibition (cyclosporin) Sorbitol Dehydrogenase (SDH) Greatest activity - hepatocytes; also found in testes Biological half-life - short (≤6 hours ) Sample stability - not as stabile; in rats, stabile refrigerated (~2 days) Not known to be induced Only known cause for serum increase - hepatocellular injury or leakage

20. Evaluation of Liver - cont. Aspartate Aminotransferase (AST, SGOT) Greatest activity - found in numerous tissues (not specific for liver injury) Biological half-life - short (~15-24 hours) Sample stability - stabile at room, refrigerated and frozen temperatures Red blood cells contain significant amounts (hemolysis - falsely elevates) Used in past to detect hepatocellular injury (still used for large animals); used for muscle injury Alkaline Phosphatase (ALP) Greatest activity - liver, bone intestine, kidney, placenta Biological half-life - isoenzymes of different tissues highly variable Sample stability - stabile in serum; not in urine Can be induced (eg., glucocorticoids, phenobarbital, dieldrin) Increased - cholestasis, drug induction, increased osteoblastic activity, cancer Decreased - decreased food intake (rats)

21. Evaluation of Liver - cont. Bilirubin, direct (conjugated) and total (Dbili & Tbili) Breakdown product of hemoglobin Liver removes unconjugated bilirubin (insoluble) from plasma, conjugates it (glucuronide - renders bilirubin water soluble) and secreted into bile Sample stability - stabile serum and urine Increased - Retention-type (hemolysis, decreased hepatic uptake); Regurgitation-type (cholestasis) Bile Acids (TBA) Produced by liver - cholic and chenodeoxycholic (primary bile acids) Taurine or glycine conjugated and secreted into bile Intestinal bacterial modification produces deoxycholic and lithocholic acids Increased - cholestasis, decreased hepatic uptake/conjugation, hepatic injury Decreased - altered enterohepatic recirculation

24. Liver Case Examples Ref Value123 ALT 30-55 IU/L34130450 SDH 10-20 IU/L161363 ALP 250-350 IU/L157321279 TBA 25-35 µmol/L312743 Tbili 0.1-0.5 mg/dL0.20.30.3 Dbili 0.05-0.2 mg/dL0.10.10.1

25. Liver Case Examples Ref Value456 ALT 30-55 IU/L445187 SDH 10-20 IU/L182028 ALP 250-350 IU/L257301987 TBA 25-35 µmol/L3113104 Tbili 0.1-0.5 mg/dL9.30.34.7 Dbili 0.05-0.2 mg/dL0.30.13.1

26. Evaluation of Kidney Need ~75% of nephrons non-functional for alterations in serum markers to occur Urea Nitrogen (UN, BUN) Method of ammonia excretion Liver converts ammonia to urea; kidney excretes urea Sample stability - stabile serum and urine Increased - renal and non-renal causes Decreased - hepatic insufficiency Creatinine (Cre, Creat) Waste product of muscle metabolism Excreted by kidney Sample stability - stabile serum and urine Increased - renal injury Decreased - decreased muscle mass

27. Evaluation of Kidney - cont. Urine indicators Urine contains most constituents found in plasma (except molecules >70,000 daltons) But concentration varies due to water conserving ability of kidney When interpreting data must account for kidney’s concentrating ability (per time or per mg creatinine basis) Sample stability - concentrated salt solution (some enzymes are not stabile in urine) Urine specific gravity - estimates concentrating ability; alterations when 66% of nephrons affected Chemical constituents - creatinine, glucose, protein, ALP, LDH, AST, NAG, glucuronidase, electrolytes

28. Proteinuria Detection of protein in urine (plasma, genitourinary) In general: >20 mg/kg/day Persistent

29. Types Functional - reversible Stress Exercise Fever/exposure to temp extremes Seizures Congestion of kidneys Glomerular overload - Hyperproteinemia Glomerular - may result in hypoalbuminemia Tubular overload - Hgb, Mgb, Bence-Jones Tubular - defective resorption

30. Methods Tougher to do in urine v. serum Small quantities Sample-to-sample variation Origin of protein Protein degradation products Sample: Fresh or refrigerated Screening (dipstick) - uncentrifuged Quantitative or semiquantitative - centrifuged

31. Methods - cont. Dipstick Screening - based on pH dyes Albumin gives stronger results Spectrophotometric Quantitative - timed collection Toluene Ur prot/Ur creatinine ratios SSATT - semiquantitative Bence Jones - heat precipitation

32. Reference Values Dog <20mg/kg/day 0.67 - 0.96 mg prot/mg creat F344 rats (adult male) ~141 mg/dL (67 - 213 mg/dL) ~5.5 mg/16 hr ~0.87 mg prot/mg creat (0.68 - 1.01 mg prot/mg creat) F344 rats (adult female) 10 mg/dL (7 - 16 mg/dL) ~0.7 mg/16 hr ~0.11 mg prot/mg creat (0.09 - 0.13 mg prot/mg creat)

33. PGMBE Urinalysis: raw data AnalyteControl1200 ppm SG1.0171.013 Volume (mL) 12.226.8 Creat (mg/dL)68.434.0 Gluc (mg/dL)8.05.0 Prot (mg/dL)65.054.0 AST (IU/L)626 LDH (IU/L)2754 NAG (IU/L)109

34. PGMBE Urinalysis: converted data AnalyteControl1200 ppm Gluc (ug/mg creat)117147 Prot (ug/mg creat)9501588 AST (mU/mg creat)976 LDH (mU/mg creat)39159 NAG (mU/mg creat)1526

36. Other Markers Proteins Total Albumin Globulin Carbohydrate Metabolism Glucose Lipid Metabolism Cholesterol Triglycerides Muscle Creatine Kinase or Phosphokinase (CK, CPK) - total and isoenzymes Troponin T and I

37. Other Markers Electrolytes Sodium Potassium Chloride Bicarbonate Calcium Phosphorus Hormones Insulin Thyroxine (T4) Triiodothyronine (T3) Thyroid Stimulating Hormone (TSH) Estradiol (E2) Progesterone (P10) Testosterone