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TOXICOLOGY 707
Clinical Chemistry
Dr. Greg Travlos
Fall 2006
Introduction
Clinical chemistry evaluations are commonly recommended in animal toxicology studies. Some regulatory agencies (e.g., FDA, EPA) have set guidelines for clinical pathology testing in nonclinical toxicity and safety studies. Measurement of chemical components of biological fluids (e.g., serum, plasma, urine, CSF) afford the toxicologist some advantages compared to standard histopathology evaluations. Advantages include serial sampling, detection of metabolic injury, detection of organ specific effects, assistance in establishment of the no effect level and determination of toxic mechanism.
There are practical problems that must be considered when including clinical chemistry evaluations in a study design. Choosing the appropriate endpoints for analysis, preanalytical sources of variation (e.g., age of animals used, diet, fasted v. non-fasted animals, site of blood collection, time of blood collection, sample handling/storage), blood volume required for testing (especially important when mice or very young rodents are used), species idiosyncrasies, processing large numbers of samples in a timely manner, appropriate instrumentation and methods, and establishment reference values (for the age, sex, and strain of laboratory animal) are examples of considerations that must be addressed by the laboratory investigator. Because clinical pathology testing of mice is limited by blood volume, interim, or in-life, sampling should not be performed and blood samples from mice should be collected at terminal sacrifice. Additionally, it often is not possible to perform both hematology and clinical chemistry analyses with the blood volumes obtained from mice. Therefore, if interim sampling times, or, both hematology and clinical chemistry analyses are required, additional mice should be added to the study protocol.
Much emphasis has been placed on the use of serum enzymes as markers of tissue or organ damage. Enzymes are a highly specialized class of proteins that catalyze biochemical reactions that otherwise would occur at a very low rate. They catalyze reactions by complexing with the substrates and lowering the energy of activation without altering the equilibrium constant. They are not consumed during the reaction process. They are usually reaction specific, but not substrate specific, and act within a specific cellular location (e.g., cell membrane, cytosol, mitochondria). For an enzyme to be valuable diagnostically, it must reasonably reflect pathological change in a specific tissue, organ, or group of organs and must be easily measured. Alterations in cell membrane integrity and enzyme synthesis, release, catabolism, inactivation, inhibition, and excretion have all been implicated as effectors of serum enzyme activity. Another significant factor affecting tissue release is the enzyme concentration within a particular tissue. Obviously a tissue with a greater concentration of enzyme will contribute more to the serum than tissues with little or no concentration. The magnitude of a serum enzyme activity increase is not always a reflection of the severity of damage or the tissue concentration (e.g., ALT and ALP). Differences related to species, age, and sex should also be considered. Similar considerations hold true for measurements of other variables. Additionally, some constituents (e.g., hormones) may have cyclic variations.
In 1986, the National Toxicology Program (NTP) revised its procedures for clinical pathology (hematology and clinical chemistry) evaluations in 13-week toxicity studies. Objectives of this were to standardize the approach to clinical pathology testing, produce relevant information on chemicals selected for study, and generate an extensive data base for the Fischer 344 rat and B6C3F1 mouse. Frequently, little information exists concerning the toxicity of the compounds selected for study. So a "core" of clinical chemistry tests was established, permitting evaluation of several organ systems. The use of a core removes guesswork from study design and prevents inadvertent omission of important endpoints. However, the use of a core does not preclude the inclusion of other relevant clinical pathology tests in a study.
The clinical chemistry tests in the NTP core include:
Alanine aminotransferase
Sorbitol dehydrogenase
Alkaline phosphatase
Total bile acids
Creatine kinase
Urea nitrogen
Creatinine
Total protein
Albumin
The purpose of this review is to provide general information concerning individual biochemical components and their application in a toxicology setting. Applied properly, these evaluations provide evidence of general or target-organ treatment effects at various time points, help identify possible mechanisms of action, and assist with establishment of no effect levels and dose selection for chronic studies. The relevance of the data obtained will depend on the appropriateness of time points sampled and the endpoints evaluated.
Laboratory evaluation of liver:
Hepatic impairment is well recognized as a toxicological problem and chemicals can cause a variety of functional and/or structural injuries. There are several types of laboratory tests that can be used to evaluate hepatic integrity/function. Analytical methods include, serum activities of enzymes of hepatic importance, tests of hepatic metabolism, and hepatic clearance/function tests. It would be recommended that at least two appropriate estimators of hepatocellular (e.g., alanine aminotransferase, sorbitol dehydrogenase, or total bile acids) and hepatobilliary (e.g., alkaline phosphatase, 5'-nucleotidase, or total bile acids) health be performed when evaluating the liver.
A). Serum enzymes of hepatocellular injury:
1. Alanine aminotransferase (ALT):
Alanine aminotransferase is a cytosolic enzyme that catalyzes the reaction:
L-alanine + a-ketoglutarate pyruvate + glutamate
Activity of ALT is found normally in serum and CSF, but not urine. It is stable at room, refrigerated, and frozen temperatures. Depending on the species, ALT has a biological half-life of approximately 48-60 hours. Cyclosporin decreases serum and liver tissue ALT activity as a result of ALT inhibition due to a drug metabolite. If vitamin B6 is added to the assay, ALT activity will be partially restored.
The greatest tissue activity in laboratory rodents, rabbits, primates, dogs, cats, and man is found in the liver. In these species increased serum activity is used to indicate hepatocellular injury (leakage). Additionally, certain drugs can cause increases in liver ALT without causing histopathological hepatocellular change. For example, in rats, glucocorticoids can induce increased liver ALT activity, in a dose-related fashion, by as much as 13-fold.
ALT is also found in low concentrations in skeletal and cardiac muscle of several laboratory animal species, including rats. In some laboratory animal species, for example the laboratory pig, ALT is found in higher concentrations in cardic and skeletal muscle than it is in liver. Thus, it is possible that serum ALT activity may increase related to muscle injury. For example, dogs were treated with CCL4 (liver necrosis) and plasmocid-dihyroiodide (muscle necrosis). Treatment with CCL4 caused increases in serum activity of ALT, AST, and SDH. Treatment with plasmocid-dihyroiodide caused increases in serum activity of ALT and AST only.
2. Sorbitol dehydrogenase (SDH):
This is a liver-specific cytosolic enzyme that catalyzes the reaction:
D-sorbitol D-fructose
The enzyme is found in appreciable amounts in hepatocytes and testes. Hepatocellular damage, however, appears to be the only cause for increased serum activity. It is a diagnostically useful enzyme in all species for hepatocellular injury. SDH has a short circulating half-life (<6 hours). Thus, SDH may be useful for demonstrating an acute hepatic injury but may not be useful for long term monitoring. Metal chelating anticoagulants, such as EDTA, decrease SDH activity . In some species, SDH is not very stable in stored serum. In laboratory rodents, however, SDH is stable at refigerated temperatures for more than 2 days.
3. Aspartate aminotransferase (AST):
Aspartate aminotransferases catalyze the reaction:
L-aspartate + 2-oxoglutarate oxaloacetate + glutamate
There are two isoenzymes of AST, one is located in the cytosol and the other is mitochondrial. AST is present in significant amounts in a wide variety of soft tissues, thus, precluding its use as marker of organ-specific injury. In general, increases of serum AST activity would be used as an indicator of soft tissue damage. Serum AST activity has been used as an indicator of hepatocellular injury/leakage. But, because AST is not tissue specific, the utility of AST as a diagnostic aid for hepatocellular damage has given way to more organ-specific serum indicators (e.g., SDH, ALT). AST activity in serum is stable and serum samples can be held at least 3 days at room temperature, 7 days at refrigerated temperatures, 30 days frozen. Red blood cells contain significant amounts of AST and red cell leakage or hemolysis can elevate serum AST activity.
4. Lactate dehydrogenase (LDH):
LDH is a cytosolic enzyme which catalyzes the reaction:
pyruvate L-lactate
LDH exists as a tetramer of 2 protimers (designated H and M). There are 5 isoenzymes LDH1 (LDH-H4); LDH2 (LDH-H3M1); LDH3 (LDH-H2M2); LDH4 (LDH-H1M3); LDH5 (LDH-M4). LDH is found in most tissues in varying amounts and increases of serum LDH activity would be used as an indicator of soft tissue damage; isoenzyme profiles may have some utility to help identify tissue-specific injury. Red blood cells contain approximately 150-fold greater LDH activity than in serum. For most species, the LDH isoenzyme profile of red cells is similar to that of cardiac muscle (predominantly LDH1 and LDH2). However, in the rat, the LDH isoenzyme profile of red blood cells is similar to that of skeletal muscle (predominantly LDH5). Thus, care must be taken when collecting and handling the specimen to avoid hemolysis. LDH4 and LDH5 are cold labile and freezing should be avoided. Oxalate anticoagulants inhibit LDH activity.
B). Laboratory evaluation of cholestasis:
1. Alkaline phosphatase (ALP):
Alkaline phosphatase is a group of nonspecific enzymes that hydrolyze phosphate esters including ATP at an alkaline pH. ALP is ubiquitous and is found in most cells of the body. Intestine and kidney have the highest amounts of ALP but tissue concentration is generally not related to serum activity in disease (e.g., liver). ALP is primarily a membrane enzyme and is usually found on brush borders of secretory/absorptive epithelium. It is also found in significant amounts in bone and placenta. There are isoenzymes of ALP related to the expression of different genes. In man and some nonhuman primates, there are three genes coding for ALP; one for placental ALP, one for intestinal ALP, and one for others (liver, bone, kidney). In other mammals, including rats, there are just two genes coding for ALP; one for intestinal and one for the others. Isoforms identified for various tissues (for example, liver versus bone) are antigenically similar because they are products of the same gene. The isoforms result from a posttranslational modification of the molecule but are enzymatically similar. The isoenzymes and isoforms can be identified immunologically and by varying sensitivities to heat inactivation and inhibitors (e.g., levamisol, L-phenylalanine).
In the normal rat, circulating ALP is primarily the intestinal and bone fractions. In man normal ALP activity arises from bone, liver, and intestine; in pregnant women placenta is a major contributor. In general, young animals have higher serum ALP activity than adult animals due to increased bone activity. In rats, conditions that cause a decreased food intake cause a decrease in serum ALP activity due decreased contribution of the intestinal isoenzyme.
Serum ALP activity is used as marker of hepatic injury, specifically cholestasis (e.g., bile duct ligation). ALP activity can increase with active bone disease (e.g., bone neoplasia). However, increases related to bone disease are usually mild. The diagnostic value of ALP is affected by the variable composition of serum ALP and the effects of diet and age.
2. 5'-Nucleotidase (5-NT):
5'-Nucleotidase is a membrane enzyme which catalyzes the hydrolysis of nucleoside 5-monophosphates. 5-NT is found in many body tissues with the highest activities found in kidney and intestinal mucosa. Diagnostic utility of 5-NT have focused on the serum increases in 5-NT activity associated with hepatobilliary disease (e.g., cholestasis). While most tissues contain 5-NT, the specificity this enzyme exhibits appears to be related to the observation that a detergent or bile acids are required for enzyme solubilization and release from membranes. Thus, biliary disease (particularly involving cholestasis) would provide optimum conditions for enzyme solubilization and release.
3. Total bile acids:
Bile acids are synthesized in the liver from cholesterol. In most species, synthesis of cholic and chenodeoxycholic acids (known as primary bile acids) predominate. In the pig, chenodeoxycholic acid is hydroxylated in the liver to form hyocholic acid. The primary bile acids are predominantly taurine (rat and dog) or glycine (rabbit) conjugated, and released into the small intestine via the bile. In the intestine, they play an important role in the digestion and absorption of lipids and lipid-soluble vitamins. The daily need is greater than hepatic synthesis and demands are met by a significant enterohepatic recirculation of bile acids. Enteric bacteria modify the primary bile acids to secondary bile acids. For example, cholic acid deoxycholic acid and chenodeoxycholic acid lithocholic acid. Lithocholic acid is relatively insoluble and not reabsorbed; deoxycholic acid is reabsorbed in the large intestine and returned to the liver where it is either rehydroxylated to cholic acid and secreted or secreted as conjugated deoxycholic acid.
Bile acid concentrations are sensitive indicators of cholestasis. Serum total bile acid concentrations can also be affected by mechanisms other than cholestasis (e.g., altered enterohepatic circulation or impaired hepatic function). Non-cholestatic liver injury can elevate circulating bile acid concentrations. In contrast, serum ALP activity increases minimally in response to hepatocellular damage. It has been demonstrated that increases in total bile acid concentration were useful for detecting early development of chloroform and carbon tetrachloride induced liver injury in male rats; ALP activity was not affected by either hepatotoxicant.
4. Bilirubin, direct and total:
Bilirubin is a product of heme metabolism from a variety hemoproteins. The destruction of hemoglobin, myoglobin, and heme-containing enzymes leads to heme. Heme is first degraded by heme oxygenase to biliverdin (green). Further degradation by biliverdin reductase converts biliverdin to bilirubin (yellow). Most heme catabolism occurs in the spleen and liver. Plasma bilirubin concentration is directly proportional to the rate of heme turnover and inversely proportional to the rate of hepatic clearance.
In health, plasma bilirubin is primarily in the unconjugated form. It is water insoluble and requires albumin as a transport protein. Unconjugated bilirubin is cleared by hepatocytes, bound to an intracellular organic ion (carrier) and conjugated with glucuronic acid. The conjugated bilirubin is then excreted in the bile.
Increases in serum bilirubin concentrations can occur with excessive heme turnover (e.g., hemolytic disease), decreased hepatocellular uptake and conjugation (loss of hepatic mass or functional defect), and cholestasis. With cholestasis the bilirubin increase is primarily the conjugated (direct) form (>50% of the total circulating bilirubin). The hyperbilirubinemia of hemolytic disease or decreased uptake or conjugation results in excess (>50% of the total) unconjugated bilirubin. An indication of where the defect is occurring can be narrowed down by measuring total and conjugated bilirubin. The unconjugated bilirubin can be calculated by subtracting the conjugated from the total bilirubin. Bilirubin uptake is competitively inhibited by the dyes BSP or ICG, but not bile acids.
C). Laboratory evaluation of hepatic clearance/function; exogenous dyes:
Dye clearance tests can be used as a measure of hepatic function. Sulfobromophthalein (BSP) and indocyanin green (ICG) are two cholephilic dyes that are commonly used. BSP administered IV binds to albumin and is transported to the liver. Hepatocytes remove the bound dye from the circulation and conjugate it to glutathione by the action of glutathione transferase. The conjugated dye (and a portion of free dye) is excreted in the bile. An estimate of dye clearance is obtained by measuring the dye remaining in the circulation in accurately timed, post-administration blood samples.
delayed dye clearance can be seen with hepatic necrosis, fibrosis, cholestasis, and decreased hepatic blood flow. Approximately 50% of the functional liver mass must be lost before delayed clearance (increased retention) occurs. Decreased albumin concentrations cause an increased clearance.
Laboratory evaluation of kidney:
The kidneys play an important role in the regulation of water, electrolyte, and acid-base balance, and the removal of certain metabolic wastes and toxins. The functional unit (nephron) of the kidney has two functionally distinct parts: the glomerulus and the tubule system. The glomerulus acts as a semipermeable diffusion membrane while the tubule system acts on the glomerular ultrafiltrate to maintain water and solute homeostasis in the animal. Quantitative and qualitative serum and/or urine analyses are used evaluate kidney function. For general evaluation of kidney function, both serum urea nitrogen and creatinine concentrations should be performed. If, however, the compound being tested is a suspected renal toxicant, the serum testing should be supplemented with measurements of urine constituents in a timed (for rats and mice, 16-24 hour) urine collection. The urine must be collected in a metabolism cage and the urine container maintained cool during the collection period.
A). Serum indicators of renal injury:
Urea nitrogen (UN) and Creatinine (Cre):
Serum concentrations of UN and Cre are traditional screening tests for kidney function and increased serum concentrations can provide evidence of renal dysfunction (e.g., decreased glomerular filtration rate). Urea provides a mechanism for ammonia excretion. UN synthesis occurs in the liver and is excreted in the urine. Creatinine is a metabolic waste product of muscle metabolism. Muscle contains phosphocreatine which undergoes spontaneous cyclization with loss of inorganic phosphorus to form creatinine. Conversion of creatine to creatinine is a nonezymatic, irreversible process occurring at the rate of about 1.6-2% per day.
Both UN and Cre are used as estimators of the glomerular filtration rate and approximately 75% of the nephrons have to be nonfunctional before changes in serum concentration of these variables occur. Levels of serum UN can be influenced by many extrarenal causes. High protein diets, dehydration, liver function, animal health and nutritional status are factors that can affect serum UN concentration. Serum creatinine is not as affected by extrarenal factors and has been considered more sensitive than serum UN for detecting nephropathy.