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Clinical Chemistry
Gregory S. Travlos, DVM, DACVPNational Institute of Environmental Health Sciences
Research Triangle Park, NC 27709919-541-0653
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 • Nephelometry• Electrophoresis• Isotopic immunoassay• Chromatography• Spectrometry
Considerations for Blood CollectionWhole 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
Sources of VariationDiet
• 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
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.
Estradiol Values in Cycling Rats and Mice
0
10
20
30
40
50
60
Early Proest Late Proest Estrus Metestrus Diestrus Diestrus 2
Estradiol (ng/mL)
F344
SD
B6
CD-1
Serum Melatonin after Five 1-Minute Light Exposures in Female F344 Rats
0.0
50.0
100.0
150.0
200.0
250.0
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
Time
pg/mL
12/12 control
1-day exposure
Assay Variation: AChE (IU/L)Propargyl Alcohol Control 64 ppm
Males 1071 778
Assay Variation: AChE (IU/L)Propargyl Alcohol Control 64 ppm
Males 1071 778
Suggested an approximate 30% enzyme inhibition
Assay Variation: AChE (IU/L)Propargyl Alcohol Control 64 ppm
Males 1071 778
Suggested an approximate 30% enzyme inhibition
PTC assay BTC assayUntreated0.1 mM1.0 mM10.0 mM
Assays: male rat serum; 2.5 hour incubation; performed in duplicate
Assay Variation: AChE (IU/L)Propargyl Alcohol Control 64 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
Troponin
0.000.00
0.050.05
0.100.10
0.150.15
0.200.20
0.250.25
00
55
1010
1515
2020
Comparison of cTn Measurement in the Comparison of cTn Measurement in the BeaglecT
nI (
ng
/mL
)cT
nI (
ng
/mL
)
cTn
T (
ng
/mL
)cT
nT
(n
g/m
L)
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
0.000.00
0.250.25
0.500.50
0.750.75
1.001.00
00
55
1010
1515
2020
2525
Comparison of cTn Measurement in the Cynomolgus Comparison of cTn Measurement in the Cynomolgus MonkeyMonkey
cTn
I (n
g/m
L)
cTn
I (n
g/m
L)
cTn
T (
ng
/mL
)cT
nT
(n
g/m
L)
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
Comparison of cTn Measurement in the Comparison of cTn Measurement in the Sprague Dawley RatSprague Dawley Rat
cTn
IcT
nI (
ng
/mL
)(n
g/m
L)
cTn
T (
ng
/mL
)cT
nT
(n
g/m
L)
NegNeg LowLow MedMed HighHigh00
11
22
33
44
55
66
00
55
1010
1515
2020
2525
3030
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
NTP Core Clinical Chemistry ProfileProtein
• Total protein• Albumin
Muscle• Creatine Kinase
Kidney• Urea Nitrogen• Creatinine
Liver • Alanine Aminotransferase• Sorbitol Dehydrogenase• Alkaline Phosphatase• Total Bile Acids
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 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
Liver Case ExamplesRef 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
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 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, electrolytes
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)
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
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
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
