Clinical Chemistry

Gregory S. Travlos, DVM, DACVPNational Institute of Environmental Health Sciences

Research Triangle Park, NC 27709919-541-0653

Travlos@niehs.nih.gov

AbbreviationsAChE = acetylcholinesterase

ALB = albumin

ALP = alkaline phosphatase

ALT = alanine aminotransferase

AST = aspartate aminotransferase

BA or TBA = total bile acids

Bili or Bil (T or D) = total or direct bilirubin

BUN or UN = urea nitrogen

BuChE = butyrylcholinesterase

Chol = cholesterol

CK or CPK = creatine kinase

Cre or Creat = creatinine

Glu = glucose

Glob = globulin

LDH = Lactate dehydrogenase

NAG = n-acetyl-glucoaminidase

SDH = sorbitol dehydrogenase

Tn or cTn = troponin (I or T); c = cardiac

T pro = total protein

Trig = triglycerides

Clinical ChemistryThe 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

Analytical Procedures/MethodsToo numerous to cover

• Photometry• Fluorometry • Luminometry • Spectrometry • Nephelometry• Electrophoresis• Chromatography • Isotopic (RIA/IRMA) and non-isotopic (ELISA/EIA)

immunoassay

Considerations for Blood CollectionSerum: whole blood collected in a container without

anticoagulant• Plasma is acceptable for some assays (heparin)• Samples from indwelling catheters are usually acceptable

For serum: allow blood to clot for 30 to 60 minutes

Separate serum for red cells into a clean plastic container• Avoid artifact due to prolonged storage on red cells (e.g., LDH)

Sources of VariationDiet

• NIH-07 vs. NTP 2000

= NTP 2000

= NIH-07

Diet: NIH-07 v. NTP-2000Analyte NIH-07 NTP-2000ALT (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.

• NIH-07 v NTP 2000

Fasting• Glucose• Alkaline phosphatase

• NIH-07 v NTP 2000

Diurnal variation• Hormones

Estradiol Values in Cycling Rats and Mice

Early Proest Late Proest Estrus Metestrus Diestrus Diestrus 2

Estradiol (ng/mL)

Circadian Rhythm of Serum Melatonin in Female F344 Rats

06:00 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30 00:30

Serum Melatonin after Five 1-Minute Light Exposures in Female F344 Rats

06:00 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30 00:30

12/12 control

1-day exposure

• NIH-07 v NTP 2000

Analytical Methods & Sample Collection/Handling Techniques• Animal handling - rough handling: increased creatine kinase release• In vitro Hemolysis - assay interference: bilirubin • Method selection - troponin

Troponin

Comparison of cTn Measurement in the Comparison of cTn Measurement in the Sprague Dawley RatSprague Dawley Rat

NegNeg LowLow MedMed HighHigh00

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

0.000.00

0.050.05

0.100.10

0.150.15

0.200.20

0.250.25

Comparison of cTn Measurement in the Comparison of cTn Measurement in the BeaglecT

NegNeg LowLow MedMed HighHigh

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

• NIH-07 v NTP 2000

Analytical Methods & Sample Collection/Handling Techniques• Animal handling - rough handling: increased creatine kinase release• In vitro Hemolysis - assay interference: bilirubin • Method selection - troponin• Multiple factors - cholinesterase

Cholinesterase AssayTwo types of cholinesterase

acetylcholinesterase (AChE; RBCs)butyrylcholinesterase (BuChE; liver)

For rat plasma: AChE:BuChE ratio approximately 1.2:1

Spectrophotometric assay: but different assays use different substratesdinitrobenzoic acidacetylcholinepropionylthiocholinebutyrylthiocholine

And the different cholinesterases have varying affinities for the different substrates

There are species differencesFor RBC and serum: human > dog > rat; platelets: rat > human

There can be sex differencesFor rat: female > male

Serum AChE (IU/L)Propargyl Alcohol Control 64 ppm

Males 1071 778

Serum AChE (IU/L)Propargyl Alcohol Control 64 ppm

Males 1071 778

Suggested an approximate 30% enzyme inhibition

Serum ChE (IU/L)Propargyl Alcohol Control 64 ppm

Males 1071 778

PTC assay BTC assayUntreated0.1 mM1.0 mM10.0 mM

Assays: normal male rat serum; 2.5 hour incubation; performed in duplicate

Serum ChE (IU/L)Propargyl Alcohol Control 64 ppm

Males 1071 778

PTC assay BTC assay

Untreated 876 272 0.1 mM 795 289 1.0 mM 825 299 10.0 mM 836 262

Assays: normal male rat serum; 2.5 hour incubation; performed in duplicate

NTP Core Clinical Chemistry ProfileLiver

• Alanine Aminotransferase• Sorbitol Dehydrogenase• Alkaline Phosphatase• Total Bile Acids

Kidney• Urea Nitrogen• Creatinine

Protein• Total protein• Albumin

Muscle• Creatine Kinase

Evaluation of LiverAlanine 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

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)

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

Liver Case ExamplesRef Value 1

ALT 30-55 IU/L 34

SDH 10-20 IU/L 16

ALP 250-350 IU/L 157

TBA 25-35 µmol/L 31

Tbili 0.1-0.5 mg/dL 0.2

Dbili 0.05-0.2 mg/dL 0.1

ALT 30-55 IU/L 34

SDH 10-20 IU/L 16

ALP 250-350 IU/L 157

Tbili 0.1-0.5 mg/dL 0.2

Dbili 0.05-0.2 mg/dL 0.1

• Case 1: Decreased ALP

ALT 30-55 IU/L 34

SDH 10-20 IU/L 16

ALP 250-350 IU/L 157

Tbili 0.1-0.5 mg/dL 0.2

Dbili 0.05-0.2 mg/dL 0.1

• Case 1: Decreased ALP - decreased food intake?

Liver Case ExamplesRef Value 1 2

ALT 30-55 IU/L 34 130

SDH 10-20 IU/L 16 13

ALP 250-350 IU/L 157 321

TBA 25-35 µmol/L 31 27

Tbili 0.1-0.5 mg/dL 0.2 0.3

Dbili 0.05-0.2 mg/dL 0.1 0.1

ALT 30-55 IU/L 34 130

SDH 10-20 IU/L 16 13

ALP 250-350 IU/L 157 321

TBA 25-35 µmol/L 31 27

Tbili 0.1-0.5 mg/dL 0.2 0.3

Dbili 0.05-0.2 mg/dL 0.1 0.1

• Case 2: Increased ALT

ALT 30-55 IU/L 34 130

SDH 10-20 IU/L 16 13

ALP 250-350 IU/L 157 321

TBA 25-35 µmol/L 31 27

Tbili 0.1-0.5 mg/dL 0.2 0.3

Dbili 0.05-0.2 mg/dL 0.1 0.1

• Case 2: Increased ALT - suspect enzyme induction

Liver Case ExamplesRef 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

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

• Case 3: Increased ALT, SDH, TBA

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

• Case 3: Increased ALT, SDH, TBA - suspect hepatocellular injury

ALT 30-55 IU/L 44

SDH 10-20 IU/L 18

ALP 250-350 IU/L 257

Tbili 0.1-0.5 mg/dL 9.3

Dbili 0.05-0.2 mg/dL 0.3

ALT 30-55 IU/L 44

SDH 10-20 IU/L 18

ALP 250-350 IU/L 257

Tbili 0.1-0.5 mg/dL 9.3

Dbili 0.05-0.2 mg/dL 0.3

• Case 4: Increased Tbili

ALT 30-55 IU/L 44

SDH 10-20 IU/L 18

ALP 250-350 IU/L 257

Tbili 0.1-0.5 mg/dL 9.3

Dbili 0.05-0.2 mg/dL 0.3

• Case 4: Increased Tbili - suspect hemolytic disease

ALT 30-55 IU/L 44 51

SDH 10-20 IU/L 18 20

ALP 250-350 IU/L 257 301

TBA 25-35 µmol/L 31 13

Tbili 0.1-0.5 mg/dL 9.3 0.3

Dbili 0.05-0.2 mg/dL 0.3 0.1

ALT 30-55 IU/L 44 51

SDH 10-20 IU/L 18 20

ALP 250-350 IU/L 257 301

TBA 25-35 µmol/L 31 13

Tbili 0.1-0.5 mg/dL 9.3 0.3

Dbili 0.05-0.2 mg/dL 0.3 0.1

• Case 5: Decreased TBA

ALT 30-55 IU/L 44 51

SDH 10-20 IU/L 18 20

ALP 250-350 IU/L 257 301

TBA 25-35 µmol/L 31 13

Tbili 0.1-0.5 mg/dL 9.3 0.3

Dbili 0.05-0.2 mg/dL 0.3 0.1

• Case 5: Decreased TBA - suspect impaired enterohepatic recirculation

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

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

• Case 6: Increased ALT, SDH, ALP, TBA, T & Dbili

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

• Case 6: Increased ALT, SDH, ALP, TBA, T & Dbili - suspect biliary obstruction

Evaluation of KidneyNeed ~75% of nephrons non-functional for alterations in serum

markers to occurUrea 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 (e.g., dehydration) 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

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, electrolytesWhen evaluating quantitative urine chemistry data, always normalize (e.g., creatinine, urine volume)

PGMBE Urinalysis: raw dataAnalyte Control 1200 ppmSG 1.017 1.013Volume (mL) 12.2 26.8Creat (mg/dL) 68.4 34.0Gluc (mg/dL) 8.0 5.0Prot (mg/dL) 65.0 54.0AST (IU/L) 6 26LDH (IU/L) 27 54NAG (IU/L) 10 9

Normalization Calculations

• Using creatinine• Assay Variable (units/vol.) / Creatinine (mg/dL)

Creatinine Normalization Calculation

• Assay Variable (units/vol.) / Creatinine (mg/dL)

• Examples• Urine glucose

• 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat

• 8.0 mg/dL / 68.4 mg/dL = 0.117 mg Glu/mg creat or 117 g/mg

• Urine LDH

• Urine LDH• (27 U/L / 68.4 mg/dL) = 0.39 U LDH/mg creat

• Urine LDH• (27 U/L / 68.4 mg/dL)/10 = 0.039 U LDH/mg creat

• Urine LDH• (27 U/L / 68.4 mg/dL)/10 = 0.039 U LDH/mg creat or 39 mU/mg

PGMBE Urinalysis: converted dataAnalyte Control 1200 ppmGluc (ug/mg creat) 117 147Prot (ug/mg creat) 950 1588AST (mU/mg creat) 9 76LDH (mU/mg creat) 39 159NAG (mU/mg creat) 15 26

Normalization Calculations

• Using creatinine• Assay Variable (units/vol.) / creatinine (mg/dL)

• Using volume• Assay Variable (units/vol.) x volume (mL/time)

Urine constituent unit conversions for the 1-chloro-2-propanol study

Conversions were performed using treatment group mean values.

Tx. Grps. Body Wght. Ur. Vol. Ur. Gluc. Ur. Prot. Ur. Gluc. Ur. Prot. Ur. Gluc. Ur. Prot. ppm g mL/16 hr mg/dL mg/dL mg/16 hr mg/16 hr mg/100 g/16 hr mg/100 g/16 hr

Males (day 15):0 193 5.6 24 73 1.34 4.09 0.70 2.1233 191 8 23 62 1.84 4.96 0.96 2.60

100 198 6.6 22 60 1.45 3.96 0.73 2.00330 192 5 29 53 1.45 2.65 0.76 1.381000 189 4.5 33 63 1.49 2.84 0.79 1.503300 155 0.9 89 81 0.80 0.73 0.52 0.47

Males (Wk 13):0 383 5.9 31 68 1.83 4.01 0.48 1.0533 372 6.8 25 69 1.70 4.69 0.46 1.26

100 373 6.2 27 70 1.67 4.34 0.45 1.16330 378 5.6 29 81 1.62 4.54 0.43 1.201000 373 4.7 33 80 1.55 3.76 0.42 1.013300 312 2.7 73 95 1.97 2.57 0.63 0.82

Volume Normalization Calculation

• Assay Variable (units/vol.) x Volume (mL/time)

• Example• Urine glucose

• 24.0 mg/dL x 5.6 mL/16 hr x dL/100 mL = 1.34 mg Glu/16 hr

Urine constituent unit conversions for the 1-chloro-2-propanol study

Conversions were performed using treatment group mean values.

Tx. Grps. Body Wght. Ur. Vol. Ur. Gluc. Ur. Prot. Ur. Gluc. Ur. Prot. Ur. Gluc. Ur. Prot. ppm g mL/16 hr mg/dL mg/dL mg/16 hr mg/16 hr mg/100 g/16 hr mg/100 g/16 hr

Males (day 15):0 193 5.6 24 73 1.34 4.09 0.70 2.1233 191 8 23 62 1.84 4.96 0.96 2.60

100 198 6.6 22 60 1.45 3.96 0.73 2.00330 192 5 29 53 1.45 2.65 0.76 1.381000 189 4.5 33 63 1.49 2.84 0.79 1.503300 155 0.9 89 81 0.80 0.73 0.52 0.47

Males (Wk 13):0 383 5.9 31 68 1.83 4.01 0.48 1.0533 372 6.8 25 69 1.70 4.69 0.46 1.26

100 373 6.2 27 70 1.67 4.34 0.45 1.16330 378 5.6 29 81 1.62 4.54 0.43 1.201000 373 4.7 33 80 1.55 3.76 0.42 1.013300 312 2.7 73 95 1.97 2.57 0.63 0.82

Other MarkersProteins

• Total• Albumin• Globulin

Carbohydrate Metabolism • Glucose

Lipid Metabolism • Cholesterol• Triglycerides

Muscle• Creatine Kinase or Phosphokinase (CK, CPK) - total and isoenzymes• Troponin T and I

Other MarkersElectrolytes

• Sodium• Potassium• Chloride• Bicarbonate• Calcium• Phosphorus

Hormones • Insulin• Thyroxine (T4)• Triiodothyronine (T3)• Thyroid Stimulating Hormone (TSH)• Estradiol (E2)• Progesterone (P10)• Testosterone

0.000.00

0.250.25

0.500.50

0.750.75

1.001.00

Comparison of cTn Measurement in the Cynomolgus Comparison of cTn Measurement in the Cynomolgus MonkeyMonkey

NegNeg LowLow MedMed HighHigh

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

ProteinuriaDetection of protein in urine (plasma,

genitourinary)In general:

>20 mg/kg/dayPersistent

TypesFunctional - reversible

• Stress• Exercise • Fever/exposure to temp extremes

• Seizures• Congestion of kidneys

Glomerular overload - HyperproteinemiaGlomerular - may result in hypoalbuminemiaTubular overload - Hgb, Mgb, Bence-JonesTubular - defective resorption

MethodsTougher 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

Methods - cont.Dipstick

• Screening - based on pH dyes• Albumin gives stronger results

Spectrophotometric• Quantitative - timed collection • Toluene• Ur prot/Ur creatinine ratios

SSATT - semiquantitativeBence Jones - heat precipitation

Reference ValuesDog

• <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)

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CLINICAL CHEMISTRY CHAPTER 12