Clinical Chemistry

Hemolysis

Hemolysis may result when a blood sample is drawn into a moist syringe, mixed too vigorously after sample collection, forced through a needle when being transferred to a tube, or frozen as a whole blood sample. A syringe must be completely dry before it is used because water in the syringe may cause hemolysis. The needle from a syringe should be removed before blood is transferred to a tube. Forcing blood through a small needle opening may rupture cells. When transferring a blood sample to a tube, the veterinary technician should expel the blood slowly from the syringe without causing bubbles to form. Hemolysis can also result when excess alcohol is used to clean the skin and not allowed to dry before beginning the blood collection procedure.

Hemolysis, regardless of cause, can greatly alter the makeup of a serum or plasma sample. For example, fluid from ruptured blood cells can dilute the sample, resulting in falsely lower concentrations of constituents than are actually present in the animal. Certain constituents normally not found in high concentrations in serum or plasma escape from ruptured blood cells, causing falsely elevated concentrations in the sample. Hemolysis may elevate levels of potassium, organic phosphorus, and certain enzymes in the blood. Hemolysis also interferes with lipase activity and bilirubin determinations. Therefore, plasma or serum is frequently the preferred sample over whole blood, and serum is frequently preferred over plasma.

Chemical Contamination

Sterile tubes are not necessary for collection of blood samples for routine chemical assays. However, the tubes must be chemically pure. Detergents must be completely rinsed from reusable tubes so that the detergents do not interfere with test results.

Improper Labeling

Serious errors may result if a tube containing the sample is not labeled immediately after the sample is collected. The tube should be labeled with the date, time of collection, patient’s name, and clinic number. The veterinary technician should double check the sample identification with the request form, if one is used, as the sample is prepared and the test is run.

Improper Sample Handling

Ideally, all chemical measurements should be completed within an hour of sample collection, which is not always feasible. In this case, samples must be properly handled and stored so that levels of their chemical constituents approximate those in the patient’s body at the time of collection. Samples must not be allowed to become too warm. Heat may be detrimental to a sample, destroying some chemicals and activating others, such as enzymes. If a serum or plasma sample has been frozen, it must be thoroughly mixed after thawing to avoid concentration gradients.

Patient Influences

If practical, a sample should be obtained from a fasting animal. The blood glucose level can be elevated and the inorganic phosphorus level decreased immediately after a meal. Also, postprandial (after-eating) lipemia results in a turbid or cloudy plasma or serum. Kidney assays are also affected due to the transient increase in GFR after eating. Water intake need not be restricted before obtaining a blood sample.

Alanine Aminotransferase

ALT was formerly known as serum glutamic pyruvic transaminase (SGPT). In dogs, cats, and primates, the major source of ALT is the hepatocyte, where the enzyme is found free in the cytoplasm. ALT is considered a liver-specific enzyme in these species. Horses, ruminants, pigs, and birds do not have enough ALT in the hepatocytes for this enzyme to be considered liver specific. Other sources of ALT are renal cells, cardiac muscle, skeletal muscle, and the pancreas. Damage to these tissues may also result in increased serum ALT levels. Administration of corticosteroids or anticonvulsant medications may also lead to increases in serum ALT. ALT is used as a screening test for liver disease because it is not precise enough to identify specific liver diseases. No correlation exists between the blood levels of the enzyme and the severity of hepatic damage. Increases in ALT are usually seen within 12 hours of hepatocyte damage and peak levels are seen in 24 to 48 hours. The serum levels will return to reference ranges within a few weeks unless a chronic liver insult is present.

Aspartate Aminotransferase

AST was formerly known as serum glutamic oxaloacetic transaminase (SGOT). AST is present in hepatocytes, both free in the cytoplasm and bound to the mitochondrial membrane. More severe liver damage is required to release the membrane-bound AST. AST levels tend to rise more slowly than do ALT levels and return to normal levels within a day, provided chronic liver insult is not present. AST is found in significant amounts in many other tissues, including erythrocytes, cardiac muscle, skeletal muscle, the kidneys, and the pancreas. An increased blood level of AST may indicate nonspecific liver damage or may be caused by strenuous exercise or intramuscular injection. The most common causes of increased blood levels of AST are hepatic disease, muscle inflammation or necrosis, and spontaneous or artifactual hemolysis. If the AST level is elevated, the serum or plasma sample should be examined for hemolysis. Creatine kinase activity should also be assessed to rule out muscle damage before attributing an AST increase to liver damage.

Sorbitol Dehydrogenase

The primary source of SDH is the hepatocyte. Smaller amounts of the enzyme are found in the kidney, small intestine, skeletal muscle, and erythrocytes. SDH is present in the hepatocytes of all common domestic species but is especially useful for evaluating liver damage in large animals such as sheep, goats, swine, horses, and cattle. Large animal hepatocytes do not contain diagnostic levels of ALT, so SDH offers a liver-specific diagnostic test. The plasma level of SDH rises quickly with hepatocellular damage or necrosis. SDH assay can be used in all species to detect hepatocellular damage or necrosis, thus eliminating the need for other tests, such as the ALT assay. The disadvantage of SDH analysis is that SDH is unstable in serum and its activity declines within a few hours. If testing is delayed, samples should be frozen. SDH tests are not readily available to the average veterinary laboratory. Samples sent to outside laboratories should be packed in ice for transport.

Glutamate Dehydrogenase

GLDH is a mitochondrial-bound enzyme found in high concentrations in the hepatocytes of cattle, sheep, and goats. An increase in this enzyme is indicative of hepatocyte damage or necrosis in cattle and sheep. GLDH could be the enzyme of choice for evaluating ruminant and avian liver function, but no standardized test method has been developed for use in a veterinary practice laboratory.

Alkaline Phosphatase

Alkaline phosphatase (AP) is present as isoenzymes in many tissues, particularly osteoblasts in bone; chondroblasts in cartilage, intestine, and placenta; and cells of the hepatobiliary system in the liver. The isoenzymes of AP tend to remain in circulation for approximately 2 to 3 days, with the exception of the intestinal isoenzyme that circulates for just a few hours. A corticosteroid isoenzyme of AP has been identified in dogs with exposure to increased endogenous or exogenous glucocorticoids. Because AP occurs as isoenzymes in these various tissues, the source of an isoenzyme or location of the damaged tissue may be determined by electrophoresis and other tests performed in commercial or research laboratories.

In young animals, most AP comes from osteoblasts and chondroblasts because of active bone development. In older animals, nearly all circulating AP comes from the liver as bone development stabilizes. The assays used for AP in a practice laboratory determines the total blood AP concentration. AP concentrations are most often used to detect cholestasis in adult dogs and cats. Because of wide fluctuations in normal blood AP levels in cattle and sheep, this test is not as useful for detecting cholestasis in these species.

Gamma Glutamyltranspeptidase

Gamma glutamyltransferase (GGT or γGT) is sometimes referred to as gamma glutamyltranspeptidase. GGT is found in many tissues, including renal epithelium, mammary epithelium (particularly during lactation), and biliary epithelium, but its primary source is the liver. Cattle, horses, sheep, goats, and birds have higher blood GGT activity than dogs and cats. Other sources of GGT include the kidneys, pancreas, intestine, and muscle cells. The blood GGT level is elevated with liver disease, especially with obstructive liver disease.

Bilirubin

Bilirubin is an insoluble molecule derived from the breakdown of hemoglobin by macrophages in the spleen. The molecule is bound to albumin and transported to the liver. The hepatic cells metabolize and conjugate the bilirubin to the molecule bilirubin glucuronide. This molecule is then secreted from the hepatocytes and becomes a component of bile. Bacteria within the gastrointestinal system act on the bilirubin glucuronide and produce a group of compounds collectively referred to as urobilinogen. Urobilinogen is broken down to urobilin before being excreted in feces. Bilirubin glucuronide and urobilinogen may also be absorbed directly into the blood and excreted by the kidneys (Fig. 3-1).

Measurements of the circulating levels of these various populations of bilirubin can help pinpoint the cause of jaundice. Differences in the relative solubility of each of these molecules allow them to be individually quantified. In most animals, the prehepatic (bound to albumin) bilirubin comprises approximately two thirds of the total bilirubin in serum. Increases in this population indicate problems with uptake (hepatic damage). Increases in conjugated bilirubin indicate bile duct obstruction.

Assays can directly measure total bilirubin (conjugated bilirubin plus unconjugated bilirubin) and conjugated bilirubin. Conjugated bilirubin is sometimes referred to as direct bilirubin because test methods directly measure the amount of conjugated bilirubin in the sample. Unconjugated bilirubin is sometimes referred to as indirect bilirubin because its concentration is indirectly calculated by subtracting the conjugated bilirubin concentration from the total bilirubin concentration of the sample.

Bilirubin is assayed to determine the cause of jaundice, evaluate liver function, and check the patency of bile ducts. Blood levels of conjugated (direct) bilirubin are elevated with hepatocellular damage or bile duct injury or obstruction. Blood levels of unconjugated (indirect) bilirubin are elevated with excessive erythrocyte destruction or defects in the transport mechanism that allow bilirubin to enter hepatocytes for conjugation.

Bile Acids

Bile acids serve many functions. They aid in fat absorption (enabling the formation of micelles in the gastrointestinal system) and modulate cholesterol levels by bile acid synthesis. Bile acids are synthesized by hepatocytes from cholesterol and are conjugated with glycine or taurine. Conjugated bile acids are secreted across the canalicular membrane and reach the duodenum by way of the biliary system. The gallbladder stores bile acids (except in the horse) until contraction associated with feeding. When bile acids reach the ileum, they are transported to the portal circulation and travel back to the liver. Ninety percent to 95% of the bile acids are actively resorbed in the ileum. The remaining 5% to 10% is excreted in the feces. The reabsorbed bile acids are carried to the liver where they are reconjugated and excreted as part of the enterohepatic circulation of bile acids (Fig. 3-2).

Spillover bile acids that escape from enterohepatic circulation may be detected in normal animals; serum concentrations of bile acids correlate portal concentrations. As a result, postprandial serum bile acid (SBA) concentrations are higher than fasting concentrations. Any process that impairs the hepatocellular, biliary, or portal enterohepatic circulation of bile acids results in elevated SBA levels. The great advantage of SBA determinations as a liver function test is that they evaluate the major anatomic component of the hepatobiliary system and are stable in vitro.

The SBA level is normally elevated after a meal because the gallbladder has contracted and released increased amounts of bile into the duodenum. Paired serum samples performed after 12 hours of fasting and 2 hours postprandial are needed to perform the test. The difference in the bile acid concentration of the samples is reported. In horses, a single sample is tested. Inadequate fasting or spontaneous gallbladder contraction can increase fasting bile acid levels. Exposing the patient to even the aroma of food can result in spontaneous gallbladder contraction. Prolonged fasting and diarrhea can decrease bile acids.

Elevated SBA levels usually indicate liver diseases such as congenital portosystemic shunts, chronic hepatitis, hepatic cirrhosis, cholestasis, or neoplasms. Bile acid levels are unspecific regarding the type of liver problem that exists and are therefore used as a screening test for liver disease. Bile acid levels may detect liver problems before an animal becomes icteric. They also may be used to follow the progress of liver disease during treatment. Increased bile acid concentrations can also result from extrahepatic diseases that secondarily affect the liver. Decreased bile acid concentration may be seen in intestinal malabsorptive diseases. In horses, increased bile acid concentrations can be the result of hepatobiliary disease or decreased feed intake. The reference ranges for bile acids in cows are widely variable. Bile acid testing is not a sensitive indicator of disease in cows.

Bile acids may be determined by several methods; the most commonly used is an enzymatic method. The 3-hydroxy bile acids react with 3-hydroxisteroid dehydrogenase and then with diformazan. Color generation is measured by end point spectrophotometry. Lipemic postprandial samples must be cleared by centrifugation to avoid interference with spectrophotometry. A bile acid test that uses immunologic methods (enzyme-linked immunosorbent assay) is now available for use in the veterinary clinic.

Cholesterol

Cholesterol is a plasma lipoprotein produced primarily in the liver, as well as ingested in food. Cholestasis caused an increase in serum cholesterol in some species. However, large differences exist in lipoprotein profiles of different species, and the clearance of lipoproteins is not well characterized in most veterinary species. A number of automated analyzers are available that provide cholesterol and other lipoprotein values. Hyperlipidemia is often secondary to other conditions (Box 3-2). Primary hyperlipidemia is rare and associated with inherited conditions in some breeds.

Cholesterol assay is sometimes used as a screening test for hypothyroidism. Thyroid hormone controls synthesis and destruction of cholesterol in the body. Insufficient thyroid hormone, or hypothyroidism, results in hypercholesterolemia because the rate of cholesterol destruction is relatively slower than the rate of synthesis. Other diseases associated with hypercholesterolemia include hyperadrenocorticism, diabetes mellitus, and nephrotic syndrome. Dietary causes of hypercholesterolemia are rare but may include high-fat diets or postprandial lipemia.

Cholesterol by itself does not cause the grossly lipemic plasma seen after eating; triglycerides also are usually present. Administration of corticosteroids also may cause an elevated blood cholesterol concentration. Fluoride and oxalate anticoagulants may elevate enzymatic method results.

Other Tests of Liver Function

Although not commonly performed in the veterinary practice, several additional tests are available at reference laboratories and research facilities. The tests are based on the ability of the liver to excrete waste and foreign substances and include the dye excretion tests, ammonia tolerance test, and caffeine clearance test.

Dye Excretion

The two available dye excretion tests are the bromsulfophthalein (BSP) excretion and indocyanine green (ICG) excretion tests. Both require administration of a dye that binds to a protein in serum. The dyes are taken up by the hepatocytes and excreted into the bile. The disappearance of the dye from the plasma requires functional hepatocytes, adequate hepatic blood flow, and bile flow. The dye used for BSP is no longer used in human medicine, and its availability is limited. The overall complexity and expense of the testing have relegated their use primarily to research facilities.

Ammonia Tolerance

Ammonia is produced by enteric microflora and during amino acid metabolism and transported to the liver through the portal circulation. Enzymes in the liver convert the ammonia to urea for excretion. Any condition that reduces the uptake of ammonia or conversion of ammonia to urea can lead to increased plasma ammonia concentration. However, normal compensatory mechanisms of the liver may result in normal fasting plasma ammonia concentrations. Impaired ammonia uptake is best identified with an ammonia tolerance test. A fasting sample is collected to provide baseline date. Patients identified with increased plasma ammonia in fasting samples should not undergo the tolerance test because of the high risk of nervous system damage. After collection of the fasting sample, ammonium chloride is administered either rectally or orally by stomach tube or in a gelatin capsule. An additional sample is collected 30 minutes later. In patients with adequate hepatic function, the postadministration results may be the same as the fasting sample or moderately increased. Patients with urea cycle enzyme deficiencies (arginosuccinate synthetase) and abnormal portal blood flow, particularly congenital portovascular anomalies, often demonstrate a threefold to tenfold increase in plasma ammonia concentrations above the baseline. The test’s chief limitation is in the handling of blood samples. Samples must be collected in ammonia-free heparin and the blood must be centrifuged immediately. The plasma should be placed on ice and analyzed within 30 minutes of collection or frozen at 220° C. Ammonia levels are stable in frozen plasma for a few days. Whole blood cannot be tested because its ammonia content increases with storage. Although the test can dramatically elevate the blood ammonia level, it does not cause nor worsen neurologic signs. Occasional vomiting with oral administration of the ammonia chloride is the only problem.

Caffeine Clearance

This is a specific assay of hepatic microsomal function. Demethylation of caffeine depends only on the specific P448 microsomal system of healthy hepatocytes; therefore it accurately reflects aberrations in hepatocellular function. This test is now used in human medicine; few experimental studies have been performed in canine species.

Caffeine sodium benzoate is dissolved in 2 ml sterile water (50:50 wt/wt), and 7 mg of caffeine/kg body weight are then injected intravenously. Plasma samples are collected from 15 to 480 minutes, and the caffeine concentration (in milligrams per milliliter) is measured by automated enzyme immunoassay. Plasma caffeine clearance and half-life are calculated from those values.

Normal half-life values in dogs are 6 ± 0.6 hours, and clearance is 1.7 ± 0.1 ml/min per kilogram of body weight. Elimination of caffeine is prolonged in hepatic insufficiency, with a clearance of 0.8 ± 0.1 ml/min per kilogram of body weight.

Creatinine Clearance Tests

Single-Injection Inulin Clearance

Inulin is excreted entirely by glomerular filtration, without tubular secretion, reabsorption, or catabolism. As a result, inulin clearance tests that use a constant infusion rate and quantitative urine sampling may be considered the best method to evaluate GFR. Single-injection inulin clearance is a simpler method that alternatively may be used. After a 12-hour fast (free access to water is permitted during the test), inulin is injected intravenously at a dosage of 100 mg/kg or 3 g/m² (body surface calculation gives more accurate results); serum samples are then obtained at 20, 40, 80, and 120 minutes. Total inulin clearance is calculated from the decrease of serum inulin concentration by using a two-compartment model. Normal dogs present a GFR of 83.5 to 144.3 ml/min per square meter of body surface area.

Sodium Sulfanilate

Sodium sulfanilate is removed only by glomerular filtration in dogs; its disappearance from the plasma is an index of glomerular filtration. The test can detect unilateral nephrectomy and diminished renal function in dogs before azotemia develops. The half-life of sodium sulfanilate is prolonged up to five times in horses with glomerulonephritis. Although the test also is performed in cats, the mode of sodium sulfanilate excretion is not confirmed in this species. This test is no longer widely used.

Phenolsulfonphthalein Clearance

Phenolsulfonphthalein is an organic dye excreted by the renal tubules. Nonetheless, its clearance is accepted as a measure of renal blood flow because this usually limits its efflux more than tubular secretion rates. Phenolsulfonphthalein clearance is decreased only when more than two thirds of the nephrons are nonfunctional or when renal perfusion is compromised. This test is no longer widely used.

Other Estimates of Glomerular Filtration Rate and Effective Renal Plasma Flow

These methods are performed at reference and research centers and require specialized equipment. The double-isotope method involves injection of a tracer solution and uses a gamma camera for nuclear imaging of kidneys at serial time intervals. The nonisotope method is similar, but after injection of contrast media serial blood samples are collected and analyzed with x-ray fluorescent laboratory assays.

Amylase

The primary source of amylase is the pancreas, but it is also produced in the salivary glands and small intestine. Increases in serum amylase are nearly always caused by pancreatic disease, especially when accompanied by increased lipase levels. The rise in blood amylase level is not always directly proportional to the severity of pancreatitis. Serial determinations provide the most information.

Amylase functions to break down starches and glycogen in sugars, such as maltose and residual glucose. Increased levels of amylase appear in blood during acute pancreatitis, flare-ups of chronic pancreatitis, or obstruction of the pancreatic ducts. Enteritis, intestinal obstruction, or intestinal perforation may also result in increased serum amylase from increased absorption of intestinal amylase into the bloodstream. In addition, because amylase is excreted by the kidneys, a decrease in GFR for any reason can lead to increased serum amylase. Serum amylase activity greater than three times the reference range usually suggests pancreatitis.

Two amylase test methods are available: the saccharogenic method and the amyloclastic method. The saccharogenic method measures production of reducing sugars as amylase catalyzes the breakdown of starch. The amyloclastic method measures the disappearance of starch as it is broken down to reduce sugars through amylase activity. Calcium-binding anticoagulants, such as EDTA, should not be used because amylase requires the presence of calcium for activity. The presence of lipemia may reduce amylase activity. The saccharogenic method is not ideal for canine samples because maltase in canine samples may artificially elevate assay results. Normal canine and feline amylase values can be up to 10 times higher than those in human beings. Therefore samples may have to be diluted if tests designed for human samples are used.

Lipase

Nearly all serum lipase is derived from the pancreas. The function of lipase is to break down the long-chain fatty acids of lipids. Excess lipase is normally filtered through the kidneys, so lipase levels tend to remain normal in the early stages of pancreatic disease. Gradual increases are seen as disease progresses. With chronic, progressive pancreatic disease, damaged pancreatic cells are replaced with connective tissue that cannot produce enzyme. As this occurs, a gradual decrease in both amylase and lipase levels are seen.

Test methods for determination of lipase levels usually are based on hydrolysis of an olive oil emulsion into fatty acids using the lipase present in patient serum. The quantity of sodium hydroxide required to neutralize the fatty acids is directly proportional to lipase activity in the sample. Newer tests for lipase are available from some reference laboratories capable of detecting canine lipase by using immunologic methods.

Lipase assay may be more sensitive for detecting pancreatitis than is amylase assay. The degree of lipase activity, like amylase activity, is not directly proportional to the severity of pancreatitis. Determinations of blood lipase and amylase activities usually are requested at the same time to evaluate the pancreas.

Increased lipase activity is also seen with renal and hepatic dysfunction, although the exact mechanisms for this are unclear. Steroid administration is correlated with increased lipase activity with no concurrent change in amylase activity.

Amylase and Lipase in Peritoneal Fluid

Comparison of amylase and lipase activity in peritoneal fluid with serum may provide additional diagnostic information. A finding of higher amylase and lipase activity in peritoneal fluid than in serum strongly suggest pancreatitis provided intestinal perforation has first been ruled out.

Trypsin

Trypsin is a proteolytic enzyme that aids digestion by catalyzing the reaction that breaks down the proteins of ingested food. Trypsin activity is more readily detectable in feces than in blood. For this reason, most trypsin analyses are done on fecal samples. Trypsin is normally found in feces, and its absence is abnormal.

Two fecal test methods are used in the laboratory: the test tube method and the x-ray film test. The test tube method involves mixing fresh feces with a gelatin solution. The test solution does not become a gel if trypsin is present in the sample to break down the protein (gelatin). If trypsin is absent, the solution becomes a gel. The x-ray film test uses the gelatin coating on undeveloped x-ray film to test for the presence of trypsin. A strip of x-ray film is placed in a slurry of feces and bicarbonate solution. If trypsin is present in the fecal sample, the gelatin coating is removed from the film upon rinsing with water. If no trypsin is present, the gelatin coating remains on the film after rinsing. The test tube method is considered more accurate than the x-ray film test in evaluating fecal trypsin proteolytic activity.

Only fresh feces should be used. Fecal trypsin activity may be decreased if the patient has recently eaten raw egg whites, soybeans, lima beans, heavy metals, citrate, fluoride, or some organic phosphorous compounds. Calcium, magnesium, cobalt, and manganese in the feces may increase trypsin activity. Proteolytic bacteria in the fecal sample may result in false-positive or apparently normal results, especially in older samples.

Serum Trypsinlike Immunoreactivity

Serum trypsinlike immunoreactivity (TLI) is a radioimmunoassay that uses antibodies to trypsin. The test can detect both trypsinogen and trypsin. The antibodies are species specific. Trypsin and trypsinogen are produced only in the pancreas. With pancreatic injury, trypsinogen is released into the extracellular space and converted to trypsin, which diffuses into the bloodstream. The test is available only for the dog and cat.

TLI provides a sensitive and specific test for diagnosis of exocrine pancreatic insufficiency in dogs. Dogs with exocrine pancreatic insufficiency (EPI) have a serum TLI of less than 2.5 mg/L. Normal dogs have a range of 5 to 35 mg/L. Dogs with other causes of malassimilation may have normal serum TLI. Dogs with chronic pancreatitis may have normal TLI values or between 2.5 and 5 mg/L. Normal cats have 14 to 82 mg/L TLI, whereas cats with EPI have less than 8.5 mg/L.

Serum TLI decreases in parallel with functional pancreatic mass. The inflammation associated with acute and probably chronic pancreatitis may enhance leakage of trypsinogen and trypsin from the pancreas and increase TLI. Also, decreased GFR increases TLI (trypsinogen is a small molecule that easily passes into the glomerular filter). Serum TLI is an important indicator of functional pancreatic mass. It is most informative if coupled with N-benzoyl-l-tyrosyl-p-aminobenzoic acid (BTPABA) and fecal fat results to characterize and diagnose malassimilation.

Serum TLI increases after eating (especially proteins) but values remain within reference intervals. In addition, pancreatic enzyme (exogenous) supplementation does not alter TLI. Therefore food should be withheld for at least 3 hours and preferably 12 hours before taking a blood sample. The blood is coagulated at room temperature and the serum stored at 20° C until assay.

Serum Pancreatic Lipase Immunoreactivity

Serum feline pancreatic lipase immunoreactivity (fPLI) is specific for pancreatitis, and its use is now recommended instead of the previously validated serum feline trypsinlike immunoreactivity test as a serum test to diagnose cats with symptoms of pancreatitis.

Glucose

Regulation of blood glucose levels is complex. Glucagon, thyroxine, growth hormone, epinephrine, and glucocorticoids are all agents favoring hyperglycemia. They boost blood glucose levels by encouraging glycogenolysis, gluconeogenesis, and/or lipolysis while discouraging glucose entry into cells. Insulin is the hypoglycemic hormone. Promoting glucose flux into its target cells, it also triggers anabolism, a process that converts glucose to other substances. This regulatory effect prevents the blood glucose concentration from exceeding the renal threshold and the spilling of glucose into the urine.

The pancreatic islets respond directly to blood glucose concentrations and release insulin (from the beta cells) or glucagon (from the alpha cells) as needed. Glucagon release also directly stimulates insulin release. Epinephrine is under direct sympathetic neural control; hyperglycemia is one aspect of the classic “flight or fight” state. The other hormones mentioned respond to hypothalamic/pituitary command. At any point in time, most of these agents are acting, shifting the blood glucose concentration up or down.

Because only insulin lowers blood glucose levels, aberrations of insulin action have the most obvious clinical effects. Hypofunction (diabetes mellitus) or hyperfunction (hyperinsulinism) can occur.

The blood glucose level is used as an indicator of carbohydrate metabolism in the body and may also be used as a measure of endocrine function of the pancreas. The blood glucose level reflects the net balance between glucose production, such as dietary intake and conversion from other carbohydrates, and glucose utilization, which is expended energy and conversion to other products. It also may reflect the balance between blood insulin and glucagon levels.

Glucose utilization depends on the amount of insulin and glucagon produced by the pancreas. As the insulin level increases, so does the rate of glucose utilization, resulting in decreased blood glucose levels. Glucagon acts as a stabilizer to prevent blood glucose levels from becoming too low. As the insulin level decreases (as in diabetes mellitus), so does glucose utilization, resulting in increased blood glucose concentration.

Many tests are available for blood glucose. Some of these react only with glucose, whereas others may quantitate all sugars in the blood. End point and kinetic assays are available. The kinetic enzymatic assays tend to be the most accurate and precise. Samples must be taken from a properly fasted animal. Serum and plasma for glucose testing must be separated from the erythrocytes immediately after blood collection. Glucose levels may drop 10% an hour if the sample of plasma is left in contact with erythrocytes at room temperature. Even the use of an SST may not be adequate to prevent this. Mature erythrocytes use glucose for energy and, in a blood sample, they may decrease the glucose level enough to give false-normal results if the original sample had an elevated glucose level. If the sample originally had a normal glucose level, erythrocytes may use enough glucose to decrease the level to below normal or to zero. If the plasma cannot be removed immediately, the anticoagulant of choice is sodium fluoride at 6 to 10 mg/ml of blood. Sodium fluoride may be used as a glucose preservative with EDTA at 2.5 mg/ml of blood. Refrigeration slows glucose utilization by erythrocytes.

Fructosamine

Glucose can bind a variety of structures, including proteins. Fructosamine represents the irreversible reaction of glucose bound to protein, particularly albumin. When glucose concentrations are persistently elevated in blood as in diabetes mellitus, increased binding of glucose to serum proteins occurs. The finding of increased fructosamine indicates a persistent hyperglycemia. Because the half-life of albumin in dogs and cats is 1 to 2 weeks, fructosamine provides an indication of the average serum glucose over that period. Fructosamine levels respond more rapidly to alterations in serum glucose than does glycosylated hemoglobin. However, serum fructosamine may be artifactually reduced in patients with hypoproteinemia.

Glycosylated Hemoglobin

Glycosylated hemoglobin represents the irreversible reaction of hemoglobin bound to glucose. The finding of increased glycosylated hemoglobin indicates a persistent hyperglycemia. The test result is a reflection of the average glucose concentration over the lifespan of an erythrocyte—3 to 4 months in dogs and 2 to 3 months in cats. Patients that are anemic may have artifactually reduced levels of glycosylated hemoglobin.

β-Hydroxybutyrate

Ketone bodies can also be detected in plasma. The ketone produced in greatest abundance in ketoacidotic patients is β-hydroxybutyrate. However, many tests for serum ketones only detect acetone. Tests for β-hydroxybutyrate that use enzymatic, colorimetric methods are now becoming available for use in the veterinary clinic.

Glucose Tolerance

Glucose tolerance tests directly challenge the pancreas with a glucose load and measure insulin’s effect by evaluation of blood or urine glucose concentrations. If adequate insulin is released and its target cells have healthy receptors, the artificially elevated blood glucose level peaks 30 minutes after ingestion and begins to drop, reaching normal value within 2 hours, and no glucose appears in the urine. A normal glucose blood level at 2 hours postprandial may rule out diabetes mellitus. Prolonged hyperglycemia and glucosuria are consistent with diabetes mellitus. Profound hypoglycemia after challenge may indicate a glucose-responsive, hyperactive beta-cell tumor of the pancreas. This test may be simplified by determining a single 2-hour postprandial glucose.

Oral glucose tolerance is affected by abnormal intestinal function, such as enteritis or hypermotility, and excitement (as from gastric intubation); an intravenous glucose tolerance test is preferred. The intravenous test is the only practical option for ruminants. With the intravenous glucose tolerance test (Procedure 3-3), a challenge glucose load is injected after a 12- to 16-hour fast (except in ruminants). Blood glucose is subsequently checked and its progress mapped as a tolerance curve. Results are standardized as disappearance half-lives or glucose turnover rates expressed as percent per minute:

Decreased glucose tolerance (increased half-life, decreased turnover rate) occurs in diabetes mellitus and less consistently in hyperthyroidism, hyperadrenocorticism, hyperpituitarism, and severe liver disease. Increased glucose tolerance (decreased half-life, increased turnover rate) is observed with hypothyroidism, hypoadrenocorticism, hypopituitarism, and hyperinsulinism. However, results may be erroneous. Normal animals on low-carbohydrate diets may manifest “diabetic curves.” The effect of this can be minimized by providing the animal with 2 to 3 days of high-carbohydrate meals before testing. The intravenous glucose tolerance test results are so variable in normal horses, depending on diet and fasting, that they are not useful.

Glucose tolerance tests are usually unnecessary to obtain a diagnosis of diabetes mellitus. Persistent hyperglycemia and glucosuria, frequently with a history of polyuria, polydipsia, polyphagia, and weight loss, are sufficient to diagnose diabetes mellitus. The test may be of value in detecting hyperinsulinism because most beta-cell tumors of the pancreas are not rapidly responsive to glucose. They may even cause diabetic glucose tolerance curves because insulin-antagonist hormones are released as a result of the initial hypoglycemia. Patient stress and chemical restraint also affect glucose tolerance test results. Serum glucose measurements themselves may be erroneously low if blood samples are not subjected to anticoagulants and are allowed to sit at room temperature. However, the test is still used.

The best use for the glucose tolerance test is in animals with borderline hyperglycemia without persistent glucosuria. However, this test is not cost-effective for the owner and may not result in significant therapeutic change. This dilemma is most often seen in cats in which high renal thresholds for glucose and stress-induced hyperglycemia are common and misleading. Extra information may be obtained from the intravenous glucose tolerance test if immunoreactive insulin concentrations are followed simultaneously. This protocol may differentiate diabetes mellitus resulting from absolute lack of insulin (type 1) from that resulting from target-cell insensitivity (type 2) or inappropriate slow insulin release (type 3).

Insulin Tolerance

The insulin tolerance test also probes the causes of diabetes mellitus. Specifically, it checks the responsiveness of target cells to challenge with regular crystalline (short-acting) insulin 0.1 IU/kg subcutaneously or intramuscularly. Serum glucose levels are measured in blood samples obtained before insulin injection (fasting blood glucose) and every 30 minutes after injection for 3 hours. If the serum glucose level fails to drop to 50% of the fasting concentration within 30 minutes of insulin injection (insulin resistance), the insulin receptors are unresponsive or insulin action is being severely antagonized. The latter may occur in hyperadrenocorticism and acromegaly. Insulin resistance profoundly influences prognostic and therapeutic decisions. If the insulin-induced hypoglycemia persists for 2 hours (hypoglycemia unresponsiveness), hyperinsulinism, hypopituitarism, or hypoadrenocorticism should be suspected. Because the test may cause this hypoglycemia, with possible weakness and convulsions, a glucose solution should always be on hand for rapid intravenous administration.

Glucagon Tolerance

The main indications for the glucagon tolerance test are repeated normal or borderline results with the amended insulin/glucose ratio test (see subsequent discussion) or lack of an insulin assay. The glucagon tolerance test gives another assessment of hyperinsulinism. Glucagon stimulates the pancreatic beta cells directly and indirectly to increase the blood insulin level. In normal animals, glucagon injection (0.03 mg/kg intravenously up to a total of 1.0 mg in dogs and 0.5 mg in cats) transiently elevates the blood glucose level to greater than 135 mg/dl. In normal animals, this concentration level is greater than 135 mg/dl. In normal animals, this concentration returns to fasting concentrations. In normal cats, peak insulin occurs at 15 minutes, declining to basal concentration at 60 minutes. Type 1 diabetic cats present a flat insulin response. If the animal has a pancre-atic beta-cell tumor, the serum glucose level peak is lower than normal and is followed within 1 hour by hypoglycemia (serum glucose is less than 60 mg/dl) because excessive insulin is secreted by the stimulated neoplasm.

To perform the test, the patient is fasted until the serum glucose level dips below 90 mg/dl (usually less than 10 hours). Glucagon is injected, and sodium fluoride anticoagulated blood samples are obtained before glucagon injection and 1, 3, 5, 15, 30, 45, 60, and 120 minutes after injection to monitor the glucose response. Unfortunately, the test is insensitive and may cause hypoglycemia convulsions up to 4 hours later. Patients must be fed immediately after the test and observed for hours.

Insulin/Glucose Ratio

The cause of hyperinsulinism may be assessed by taking simultaneous measurements of serum glucose and insulin levels in a fasting animal. Hypoglycemia normally inhibits insulin secretion. Pancreatic beta-cell tumors, hyperactive and unresponsive to glucose, secrete an abundance of insulin inappropriate to the prevailing blood glucose concentration. Although fasting serum insulin concentrations are often normal in hyperinsulinism, ratios of insulin to glucose concentrations are usually aberrant.

The absolute ratio of insulin to glucose can be amended to increase diagnostic accuracy. The amended insulin/glucose ratio (AIGR) subtracts 30 from the serum glucose concentration. At a serum glucose level of 30 mg/dl or less, insulin is normally undetectable, so this discriminant puts the zero of both the glucose and insulin scale at the same physiologic place. Because abnormally high insulin concentrations are more obvious at low serum glucose concentrations, the AIGR is most valuable in animals with a confirmed hypoglycemia of less than 60 mg/dl. Insulin and glucose have to be measured from the same serum sample. Then serial determinations may be performed to select an insulin concentration in hypoglycemia. However, the test is not totally dependable. If the results are unconvincing, the procedure should be repeated or other tests tried. Specifically, diagnostic imaging and insulinlike growth factor tests should be tried to rule out or confirm paraneoplastic hypoglycemia.

Miscellaneous Tests of Insulin Release

When results of a glucagon response test or AIGR are equivocal, glucose, epinephrine, leucine, tolbutamide, or calcium challenges may be attempted. These substances, like glucagon, may provoke a hyperinsulinemic response from pancreatic islet cell tumors, resulting in decreased serum glucose levels. However, tumors vary in their sensitivity to these agents and false-negative results (no response) can occur. These tests are also dangerous because they can precipitate severe, prolonged hypoglycemia.

ACTH Stimulation Test

Animals with suspected hypoadrenocorticism (Addison’s disease) or hyperadrenocorticism (Cushing’s disease) may be evaluated with an ACTH response test. In addition, the test is indicated to distinguish among iatrogenic and spontaneous hyperadrenocorticism (Procedure 3-4). It also may test the efficacy of mitotane, ketoconazole, or metyrapone therapy. The ACTH stimulation test evaluates the degree of adrenal gland response to administration of exogenous ACTH. Degree of response to stimulation by glucocorticoid should be in proportion to the glands’ size and development. Hyperplastic adrenal glands have exaggerated responses, whereas hypoplastic adrenal glands show diminished responses. The test can detect these abnormalities but not reveal their ultimate cause. The ACTH response test is a screening test. Adrenal glands that are hyperactive from neoplasia may be insensitive to ACTH. Nonetheless, current figures indicate that the test is more than 80% accurate in diagnosing adrenocortical hyperfunction in the dog and more than 50% in the cat.

Dexamethasone Suppression

Dexamethasone suppression tests evaluate the adrenal glands differently by using the adrenal feedback loops. The low-dosage test confirms or replaces the ACTH response test for hyperadrenocorticism (Cushing’s disease). The high-dosage test goes further, differentiating pituitary from adrenal causes of hyperadrenocorticism (Procedure 3-5). In cats, only a high-dose dexamethasone suppression test is suitable.

Dexamethasone, a potent glucocorticoid, suppresses ACTH release from the normal pituitary gland, resulting in a drop in plasma cortisol concentration. Hyperadrenocorticism of any etiology is usually resistant to suppression from small dexamethasone doses because a diseased pituitary gland is abnormally insensitive to the drug and continues elaborating excessive ACTH, although 35% of dogs with pituitary-dependent hyperadrenocorticism have a 4-hour post-dexamethasone cortisol level lower than 1 mg/dl or 50% of the baseline concentration. Neoplastic adrenal glands are autonomously secreting cortisol independent of endogenous ACTH control. The excessive cortisol production suppresses secretion of ACTH by the normal pituitary gland through negative feedback inhibition. Small doses of dexamethasone do not affect plasma cortisol measurements. However, such doses may complicate test results and may differentiate only normal animals from those with hyperadrenocorticism.

With larger dexamethasone doses, more differences appear. The sensitivity of a diseased pituitary gland to dexamethasone is incomplete; large dexamethasone doses overcome it and the abnormally high plasma ACTH and cortisol concentrations fall. Abnormal adrenal glands, however, continue to secrete cortisol autonomously. Thus plasma cortisol concentrations unresponsive to all dexamethasone doses are probably caused by primary adrenal gland disease. Suppression by large but not small doses suggests pituitary gland disease. The test has 73% accuracy in differentiating pituitary from adrenal causes in dogs and 75% sensitivity to diagnose hyperadrenocorticism in cats.

A dual high-dosage dexamethasone test and ACTH response test are described in Procedure 3-6. Although combined protocol is a step saver, possibly ambiguous results necessitate more tests and expense. The ACTH response segment of the test is particularly prone to error. Because dexamethasone alters the adrenal responsiveness to ACTH (enhances or inhibits, depending on the duration of activity), the timing of the test is crucial. Normal standards must be newly established for any changes in protocol.

Corticotropin-Releasing Hormone Stimulation

This test may be indicated to differentiate among pituitary-dependent and primary hyperadrenocorticism. Plasma cortisol concentration and ACTH should not be elevated after corticotropin-releasing hormone (CRH) stimulation in dogs with adrenal-dependent Cushing’s disease.

The protocol of this test consists of obtaining a pretest sample to determine cortisol and ACTH, administering 1 mg/kg of CRH, and obtaining blood samples again 15 and 30 minutes later to evaluate cortisol and ACTH.

TSH Response

This test is used on small animals (except in cats with hyperthyroidism) and horses and provides a reliable diagnostic separation of patients with normal versus abnormal thyroid function (Box 3-6). Exogenous TSH challenge may sort out borderline cases and separate real hypothyroid patients from those with other illness or drug-depressed thyroxine concentrations and also may pinpoint the site of the lesions.

The test usually is used to explore canine hypothyroidism. After TSH is injected, thyroid response (usually serum T₄ levels, the most reliable index) is followed. An increase in the serum T₄ level occurs in normal animals. Primarily exhausted or insensitive thyroids do not respond to exogenous TSH. Indeed, endogenous TSH concentrations are already high from failing T₄ inhibition. Therefore serum T₄ level is not increased in these animals. With pituitary or brain disease, however, the thyroid glands remain responsive. Such lesions result in too little endogenous thyrotropin. Although an increase in the serum T₄ level is expected in animals with pituitary lesions, 2 to 3 days of TSH challenge may be necessary before increased serum T₄ levels are seen. The extra TSH is required to overcome chronic glandular atrophy, similar to “priming the pump.”

Glucocorticoids seem to inhibit both TSH and T₄ secretion, so euthyroidism with low serum T₃ levels only often accompanies Cushing’s disease or vigorous glucocorticoid therapy. Fortunately, the TSH and ACTH response tests may be performed simultaneously. In such animals, the glands remain responsive to TSH but the absolute values of prechallenge and postchallenge serum T₄ are low or low resting with normal post-TSH values. Feline hyperthyroidism is usually caused by functional thyroid adenomas. Oddly, with exogenous TSH challenge, little or no increase occurs in the serum T₄ level, as in canine primary hypothyroidism. This phenomenon suggests that the neoplasm either functions independently of the trophic hormone or is already manufacturing and leaking T₄ at maximum capacity. A lack of TSH responsiveness, appropriate clinical manifestations, and high baseline plasma T₄ concentrations all attest to feline hyperthyroidism.

In horses, iodine-deficiency hypothyroidism is rare because iodized salt usually is offered free choice or in feeds. Overzealous iodine supplementation with kelp meal or vitamin-mineral mixes, however, provokes hypothyroidism and goiter. Excessive use of iodine inhibits thyroid function. In the assessment of thyroid function in horses, the normal serum T₄ values are 1 to 3 mg/dl, which is lower than in other species. Hypothyroidism should be suspected only with serum T₄ concentrations of less than 0.5 mg/dl.

Rare tumors of the pars intermedia of the pituitary, compressing the anterior pituitary, may cause secondary hypothyroidism in older horses. Because pituitary damage induces a plethora of signs, the TSH response test may be especially helpful.

Thyrotropin-Releasing Hormone (TRH) Response

TRH response is used on small animals and provides a reliable diagnostic separation of patients with normal versus abnormal thyroid function. FT₄ is the fraction of thyroxine that is not bound to protein. FT₄ levels are less influenced by nonthyroidal diseases or drugs that total T₄ concentrations. Exogenous TRH challenge may sort out borderline cases and separate real hypothyroid and hyperthyroid patients from those with other illness or drug-depressed thyroxine concentrations. The test usually is used to explore canine hypothyroidism when TSH is not available. Baseline serum TT₄ and FT₄ concentrations are determined. Four hours after 0.1 mg/kg or 0.2 mg (total dose) of TRH are injected intravenously, thyroid response (serum TT₄ and FT₄ levels) is followed. An increase of the serum TT₄ concentration of 50% or 1 μg/dl (13 nmol/L) and FT₄ (1.9 times) concentration, compared with baseline concentrations, occurs in normal animals. The evaluation of FT₄ levels allows a clearer distinction between euthyroid and hypothyroid dogs when TT₄ results are equivocal. TRH response test may be used to diagnose mild to moderate feline hyperthyroidism. Baseline serum TT₄ and FT₄ concentrations are determined. Approximately 4 hours after 0.1 mg/kg of TRH are injected intravenously, serum TT₄ and FT₄ levels are determined. An increase of the serum TT₄ less than 50%, compared with baseline concentrations, occurs in hyperthyroid cats. Increases between 50% and 60% are borderline, and increases more than 60% rule out hyperthyroidism.

Triiodothyronine Suppression Test

Hyperthyroidism is common in middle-age to old cats in the United States and Great Britain. Diagnosis may be based on resting thyroid hormone concentrations. Determination of both TT₄ and FT₄ may help distinguish a nonthyroidal disease. The combination of high FT₄ value with low TT₄ is indicative of nonthyroidal illness, whereas a high FT₄ concentration and high-normal TT₄ concentration indicates hyperthyroidism. However, some cases may require a functional test to confirm or rule out the disease.

Thyroid suppression testing is based on the expected negative feedback regulation of TSH, induced by high concentrations of circulating thyroid hormone. Hyperthyroid cats should not have a normal pituitary-thyroid regulation. As a result, administration of exogenous T₃ must induce a decrease on endogenous T₄ unless feedback TSH regulation is altered.

To perform the test, a basal T₃ and T₄ determination is required. Seven T₃ doses of 25 μg orally every 8 hours are administered at home. Approximately 2 to 4 hours after the seventh dose, a blood sample is obtained for T₃ and T₄ determination. Cats with hyperthyroidism have serum T₄ concentrations higher than 1.5 μg/dl or 20 nmol/L, whereas nonhyperthyroid cats have lower values. Low posttest T₃ concentrations indicate an invalid test resulting from failure of exogenous T₃ administration.

Gastrin Secretion

The diagnosis of Zollinger-Ellison syndrome (gastrinoma) may be required after identification of gastrointestinal ulceration and amine precursor uptake and decarboxylation, neoplasia (prevalently located in the pancreas), and the assessment of high serum gastrin concentrations. However, some cases may require provocative tests to document the excessive gastrin secretion. Stimulation of gastrin secretion may be accomplished by test meal, intravenous calcium infusion, or intravenous secretin infusion. Serum samples for gastrin determination must be obtained before, 2, 5, 15, and 30 minutes after intravenous administration of 2 to 4 IU/kg of secretin. A positive test is considered if twofold increase gastrin concentration occurs 2 and/or 5 minutes after administration.

Fecal Occult Blood

Blood loss into the gut is another cause of protein-losing gastroenteropathy. Dramatic bleeding is evident as black feces (melena) or frank fecal blood (hematochezia). Less-obvious, subtle bleeding is a significant sign of GI ulcers, neoplasia, or parasitism. Chronic, low-level bleeding may lead to iron-deficiency anemia.

Useful reagents to detect insidious bleeding include orthotoluidine (Occultest, Ames, Iowa) and benzidine (Hemoccult, Beckman Coulter Inc., Fullerton, Calif.). Impregnated strips or tablets are oxidized to a colored product by hemoglobin peroxidase activity in the feces. Both reagents are so sensitive that they respond to dietary hemoglobin and myoglobin; therefore the patient’s diet must be meat free for 3 days before the test. A cottage cheese and rice diet, 50% of each, is recommended for the patient before the test is performed. This precaution is less pertinent to herbivores, but the technician must check that the diet has not been supplemented with meat or bone meal. Another test for fecal occult blood is the guaiac test. The test is less sensitive but also less affected by diet. However, the reagents used for the test are not commonly found in the veterinary clinic.

Fecal Proteolytic Activity

The assay for fecal proteolytic activity was found to be effective for the evaluation of exocrine pancreatic insufficiency in cats and other species. Fecal proteolytic activity can be determined colorimetrically by using an azocasein or azoalbumin substrate or the radial enzyme diffusion method. Dogs with EPI may seldom have normal fecal proteolytic activity. A reliable radial enzyme diffusion requires fecal samples of three different days. Normal values of diameter of digestion halo are 10.7 ± 2.7 mm in dogs and 9.6 ± 3.4 mm in cats.

Fecal α₁-Protease Inhibitor (α₁-Antitrypsin)

This plasma protease resists intraluminal proteolytic degradation. Detection of α₁-protease inhibitor may be accomplished by radial immunodiffusion or enzyme-linked immunosorbent assay in dogs. The assay is species specific. Documentation of protein-losing enteropathy or gastrointestinal hemorrhage is appropriate.

The best method for diagnosing protein-losing enteropathy is quantitative loss of chromium-52-labeled albumin. The test is based on intravenous administration of radioactive chromium, which links to circulating plasma albumin; albumin loss is proportional to radioisotope quantity (radioactivity) in feces.

Lipid Absorption

Lipid absorption relies on bile, pancreatic lipase, and a healthy intestinal mucosa. Associated lesions cause steatorrhea and prevent the normal hyperlipemia that follows a fatty meal. Lipemia, as judged by plasma turbidity, is then a gauge of GI function.

The patient must be fasted for 12 hours and a baseline blood sample must be drawn and centrifuged. If fasting lipemia is encountered (e.g., in diabetes mellitus, starvation, feline steatitis, hypothyroidism, hyperadrenocorticism, liver/biliary disease, familial hyperlipemia of Schnauzers, white muscle disease of sheep, or hyperlipemia of ponies, among others), the test must be abandoned. If the plasma is clear, either corn oil (3 ml/kg) or peanut oil (2 ml/kg) is administered orally. Blood samples are drawn at hourly intervals for 4 hours. This plasma should be cloudy. Clear follow-up samples indicate disturbed absorptive function, but the site of disturbance may not be known. Some argue that this test reveals nothing more than the simple discovery of steatorrhea.

A variation of this method may better define the lesion. The test is repeated on another day; however, the oil is preincubated at room temperature for 20 minutes with pancreatic enzymes. A cloudy postchallenge plasma implies a pancreatic enzyme deficiency. If it is clear, inadequate bile production or intestinal malassimilation must be considered. The fat absorption test may yield false-negative results (no fat absorption) with delayed gastric emptying (fat induces this), gastric inactivation of added pancreatic enzymes, or enteritis.

Slight lipemia before oil administration indicates increased levels of low lipoproteins (mainly triglycerides and cholesterol). The enzyme lipoprotein lipase usually clears plasma of triglycerides; the activity of this enzyme is enhanced by insulin, thyroid hormones, glucagon, and heparin. Determination of triglyceride concentrations of slightly lipemic plasma may reveal increased triglyceride concentration, which could be attributed to disease of hepatic, pancreatic, renal, or endocrine origin. Slight postprandial lipemia also may be seen in some canine patients with malabsorptive syndromes.

Monosaccharide Absorption Tests

These tests more specifically probe intestinal function. Again, the agent is given orally; blood concentrations are the measure of absorption.

Serum Folate and Cobalamin

Serum concentrations of folate and cobalamin may be assessed by radioimmunoassay. Both concentrations tend to be decreased in malabsorption. Folate is absorbed in the proximal intestine, whereas cobalamin is absorbed in the ileum. Bacterial overgrowth also may alter these concentrations; folate synthesis is increased in bacterial overgrowth, whereas some bacteria may decrease the cobalamin availability.

Mucin Clot Test

Synovial fluid mucin forms a clot when added to acetic acid. The nature of the resultant clot reflects the quality and concentration of hyaluronic acid. The following is one method. To perform the test, 1 ml nonanticoagulated synovial fluid is added to a 7-N glacial acetic acid diluted 0.1:4. The synovial fluid/acetic acid solution is gently mixed and allowed to stand at room temperature for 1 hour before evaluating for the presence of a clot. The mucin clot generally is graded as good (large, compact, ropy clot in a clear solution), fair (soft clot in a slightly turbid solution), fair-poor (friable clot in a cloudy solution), or poor (no actual clot, but some large flecks in a turbid solution). Clot assessment is enhanced by gently shaking the tube. Good clots remain ropy, whereas poor clots fragment. If only a few drops of synovial fluid are obtained at arthrocentesis, an abbreviated mucin clot test may be performed. If available after preparation of a cytologic smear (and possibly total nucleated cell count), a drop of non-EDTA-preserved fluid is placed on a clean microscope slide. Three drops of diluted acetic acid are added and mixed. The resultant clot is graded after approximately 1 minute. Assessment may be easier against a dark background.