Clinical Chemistry

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 Analyte NIH-07 NTP-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 Alcohol Control 64 ppm Males 1071 778

11. Assay Variation: AChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition

12. Assay Variation: AChE (IU/L) Propargyl Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assayBTC 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 Alcohol Control 64 ppm Males 1071 778 Suggested an approximate 30% enzyme inhibition PTC assayBTC 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. Abbott Architect Tosoh AIA 600 II Bayer Advia Centaur Beckman Access Dade Dimension RxL OCD Vitros ECi DPC Immulite Dog Troponin EIA Roche Elecsys 2010 Comparison of cTn Measurement in the Beagle 20 0.25 0.20 15 0.15 cTnI (ng/mL) cTnT (ng/mL) 10 0.10 5 0.05 0 0.00 Neg Low Med High

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

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

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 Value 1 2 3 ALT 30-55 IU/L 34 130 450 SDH 10-20 IU/L 16 13 63 ALP 250-350 IU/L 157 321 279 TBA 25-35 µmol/L 31 27 43 Tbili 0.1-0.5 mg/dL 0.2 0.3 0.3 Dbili 0.05-0.2 mg/dL 0.1 0.1 0.1

25. Liver Case Examples Ref Value 4 5 6 ALT 30-55 IU/L 44 51 87 SDH 10-20 IU/L 18 20 28 ALP 250-350 IU/L 257 301 987 TBA 25-35 µmol/L 31 13 104 Tbili 0.1-0.5 mg/dL 9.3 0.3 4.7 Dbili 0.05-0.2 mg/dL 0.3 0.1 3.1

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 Analyte Control 1200 ppm SG 1.017 1.013 Volume (mL) 12.2 26.8 Creat (mg/dL) 68.4 34.0 Gluc (mg/dL) 8.0 5.0 Prot (mg/dL) 65.0 54.0 AST (IU/L) 6 26 LDH (IU/L) 27 54 NAG (IU/L) 10 9

34. PGMBE Urinalysis: converted data Analyte Control 1200 ppm Gluc (ug/mg creat) 117 147 Prot (ug/mg creat) 950 1588 AST (mU/mg creat) 9 76 LDH (mU/mg creat) 39 159 NAG (mU/mg creat) 15 26

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