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Veterinarian Technician June 2007 (Vol 28, No 6) Focus: Endocrine Disorders

Laboratory Tests for Diagnosis and Management of Diabetes

by Margi Sirois, EdD, MS, RVT, Elaine Anthony, MA, CVT

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    Key Points

    • Tests that measure the levels of glucose in blood and urine are essential to the diagnosis and management of diabetes mellitus.
    • Because blood glucose levels are affected by several factors, proper patient and sample preparation are important.
    • Early diagnosis and control of diabetes can help reduce patient morbidity and mortality.

    Diabetes mel­litus is a chronic meta­bolic disorder characterized by persistently high blood glucose levels (hyperglycemia). It may be congenital or acquired. In small animals, the acquired form is more common and usually occurs in middle-aged dogs and cats. Regardless of the etiology, patients with diabetes mellitus have excess glucose in their blood and low levels of glucose in most body cells. Patients receiving insulin for diabetes require periodic testing to monitor disease progression and to evaluate the effectiveness of therapy and client compliance.

    Diabetes mellitus is usually diagnosed based on the patient's history and clinical signs and the results of several blood and urine tests. Numerous conditions, including pancreatic trauma, neoplasia, and infection, may contribute to the onset of the disease (see box).1 Common clinical signs of diabetes mellitus include polydipsia, polyuria, and polyphagia. Weight loss, restlessness, ataxia, disorientation, and weakness may also occur. Patients that develop ketoacidosis may present with tachycardia, seizures, and Kussmaul's respiration (rapid, deep, labored breathing).

    Most tests used in the diagnosis of diabetes measure the level of glucose in the patient's blood. Normal blood glucose levels vary among species, but individual healthy animals tend to have relatively stable glucose levels because the body's normal homeostatic mechanisms, such as insulin release, maintain glucose within a very narrow range.a Although excessive carbohydrate intake, strenuous exercise, stress, and various disease conditions can affect plasma glucose, daily fluctuations are usually maintained within 10% to 20% of the normal range in an individual.2 In animals with diabetes, however, the presence of fasting hyperglycemia with concurrent glucosuria is common. Lipemia — the buildup of chylomicrons and other triglycerides in the blood — is also common in diabetic patients. Lipemia can interfere with numerous test methodologies, especially photometric assays for glucose and other clinical chemistry parameters.3

    Diagnostic Testing

    Once the initial diagnosis has been established, testing in diabetic animals generally involves a blood glucose curve, urinalysis, clinical chemistry profile, and hematology profile.1 A thorough workup is also normally conducted to identify other diseases that result from diabetes (e.g., cataracts, urinary tract infection, hepatomegaly) and to develop an initial therapeutic plan.

    Sample Collection Considerations

    Glucose testing is often the first test conducted in diagnosing suspected diabetes. Animals undergoing glucose testing must be properly fasted (8 to 12 hours) before a blood sample is taken because mild, transient hyperglycemia is normal after consuming a meal.

    Although some tests use whole blood to obtain a glucose measurement, serum or plasma is preferred for laboratory testing. The serum or plasma must be separated from the erythrocytes immediately after blood collection because mature erythrocytes use glucose for energy. If not removed, erythrocytes can decrease the glucose level in the sample by as much as 10% per hour,3,4 leading to false normal or low results when glucose is measured. Even the use of a serum separator tube may not be adequate to prevent this. Fluoride provides the best glucose preservation of all the anticoagulants. When glucose measurements are critical, the sample should be collected into a sodium fluoride anticoagulant tube.3

    Blood glucose in diabetic patients may be as high as 10 times the normal level. The glucose test provides an indication of the plasma glucose level at the time the sample was taken. A single glucose test is usually not sufficient to definitively diagnose diabetes because results can be influenced by several factors. Cats in particular often exhibit hyperglycemia resulting from the stress of transport to the veterinary clinic or from restraint during blood collection. Persistent hyperglycemia and glucosuria with a history of polyuria, polydipsia, polyphagia, and weight loss are strong indicators of diabetes mellitus. If hyperglycemia is evident on the blood glucose test without concurrent glucosuria, or if clinical signs are ambiguous, additional testing is usually conducted.

    Measuring Blood Glucose Levels

    There are many tests for measuring blood glucose levels. Some react only with glucose, whereas others quantify all sugars in the blood. The enzymatic assays (i.e., glucose oxidase, hexokinase) tend to be the most accurate and precise.3 Most dipstick tests for measuring urine glucose levels also use enzymatic methods. Handheld glucose meters are available for monitoring blood glucose levels in diabetic patients. These tend to be reasonably accurate if used correctly. Many of these meters, however, are configured for human blood samples and calibrated for capillary blood; therefore, their reference ranges are different than those for venous blood.5 Although these meters vary, each clinic should establish normal ranges for species and sample type. Glucose meters designed for veterinary use are now available.

    Glucose Tolerance Test

    The glucose tolerance test involves administration of glucose followed by serial glucose measurements. The test may be used as part of the initial workup in a patient with suspected diabetes or to aid in determining the therapeutic dose of insulin. In nondiabetic animals, the blood glucose level peaks 30 minutes after administration of glucose and returns to normal within 2 hours, and no glucose appears in the urine.3 A normal blood glucose level at 2 hours after glucose administration may rule out diabetes mellitus.

    The preferred method of glucose administration for the glucose tolerance test is intravenous.3 Oral glucose tolerance tests are affected by abnormal intestinal function, such as enteritis or hypermotility, and excitement (as from gastric intubation).3 The oral glucose tolerance test requires a pretest fasting blood sample followed by oral glucose administration. Serial blood glucose measurements are then determined over the next 3 to 4 hours. Several protocols may be used for the IV glucose tolerance test. The most common protocol requires the patient to be fasted for 12 to 16 hours and an initial blood sample taken. The glucose level of this baseline sample should be approximately 70 mg/dl. Healthy animals on low-carbohydrate diets may have lower results. These animals should be given high-carbohydrate meals for several days before testing.

    After the baseline sample is collected, glucose is administered intravenously to the fasted patient over a 30-second period at a rate of 1.0 g/kg. Blood samples are taken at 5, 15, 25, 35, 45, and 60 minutes after glucose infusion, using sodium fluoride as an anticoagulant. An additional blood sample is collected after 120 minutes for feline patients. Because of the need for repeated venipuncture, it may be useful to place an IV catheter before the glucose infusion. The glucose can be administered via the catheter and blood samples drawn from the catheter.

    The glucose level of each sample is plotted on semilogarithmic graph paper and the glucose half-life (time required for glucose levels to decrease by 50%) is determined. The postinfusion blood glucose level should decrease to approximately 160 mg/dl in 30 to 60 minutes and return to the baseline value in 120 to 180 minutes. An increased half-life indicates decreased glucose tolerance and suggests diabetes mellitus. Increased glucose half-life may also be seen with hyperthyroidism, hyperadrenocorticism, hyperpituitarism, and severe liver disease. A decreased half-life indicates increased glucose tolerance and is observed with hypothyroidism, hypoadrenocorticism, hypopituitarism, and hyperinsulinism.3

    Glucose Curve

    A glucose curve, or insulin tolerance test, is a visual representation of the patient's blood glucose level over the course of a day. The primary use of a glucose curve is to aid in establishing the insulin dose and dosing interval for patients with newly diagnosed diabetes. The patient is usually fed its normal meal rather than an IV glucose solution. A blood sample is taken, the glucose value determined, and the patient given a dose of insulin. Ideally, blood glucose measurements are then taken every 1.5 to 2 hours for 12 hours. This may not be practical in some veterinary clinics. An alternative method involves taking blood glucose measurements every hour until the lowest point on the blood glucose curve is reached. An additional sample is then taken each hour for the next 2 hours to verify that the low point on the curve has been reached. Once these data are obtained, the insulin dose, dosing interval, and type of insulin used can be evaluated.

    In patients with well-regulated diabetes, the highest blood glucose value will be the sample taken just before the insulin was given, and the concentration will be less than 300 mg/dl (see figure). The time from the insulin injection until the lowest glucose level is reached is between 5 and 8 hours. The lowest point should be greater than 100 mg/dl, and the midpoint (the value halfway between the lowest and highest glucose concentrations) should be between 150 and 250 mg/dl.

    If the lowest point is reached in less than 5 hours, the patient's dosing interval is usually decreased (insulin is given more often) or a longer-acting insulin is used. If the elapsed time to the lowest point is greater than 8 hours, the insulin dosing interval is usually increased. If the lowest point falls below 100 mg/dl or the midpoint is below 150 mg/dl, the patient's insulin dose is usually decreased (see figure).

    A similar test, commonly referred to as the mini-glucose curve, can aid in diagnosing diabetes mellitus. The test evaluates the responsiveness of target cells to short-acting insulin. A fasting blood glucose measurement is obtained, and the patient is administered insulin at a dose of 0.1 IU/kg SC or IM. Serum glucose levels are measured every 30 minutes for 3 hours after injection. The serum glucose level should decrease to 50% of the fasting concentration within 30 minutes of insulin injection. Failure to reach a 50% reduction in that time frame indicates insulin resistance — that is, the insulin receptors are unresponsive or insulin action is being severely antagonized. If insulin-induced hypoglycemia persists for 2 hours or longer (hypoglycemia unresponsiveness), hyperinsulinism, hypopituitarism, or hypoadrenocorticism may be present. Because this test may cause prolonged hypoglycemia, with possible weakness and convulsions, a glucose solution should always be available for rapid IV administration.3

    Patient stress can significantly affect glucose tolerance and glucose curve test results. Serum glucose measurements themselves may be erroneously low if blood samples are not mixed with appropriate anticoagulants or are allowed to sit at room temperature.3 Glucose curves are often repeated periodically to monitor the patient's level of regulation. Previously well-regulated patients that begin to exhibit clinical signs, such as polyuria and polydipsia, usually require a glucose curve in order to properly modify the therapeutic regimen.

    Fructosamine

    Glucose can bind various molecules. When it binds with plasma proteins, particularly albumin, fructosamine is formed.1 Persistent elevation of blood glucose, as in diabetes mellitus, leads to an increase in the amount of glucose bound to serum proteins; therefore, the finding of increased fructosamine indicates persistent hyperglycemia. Because the half-life of albumin in dogs and cats is 1 to 2 weeks, the fructosamine level indicates the average serum glucose level over that period6 and responds more rapidly to alterations in serum glucose than glycosylated hemoglobin. Serum fructosamine may be artificially reduced in patients with hypoproteinemia.

    The fructosamine test is a useful indicator of the level of regulation of diabetic patients because it is not affected by stress. However, this test cannot evaluate whether the patient had instances of hypoglycemia during the previous few weeks. There is evidence that significant daily variability in glucose concentrations can occur even in patients with well-regulated diabetes.7

    Glycosylated Hemoglobin

    Glycosylated hemoglobin is formed when glucose binds to hemoglobin. As with fructosamine, the finding of increased glycosylated hemoglobin indicates persistent hyperglycemia. Because the reaction that binds glucose and hemoglobin cannot be reversed, the level of glycosylated hemoglobin is a reflection of the average blood glucose concentration over the life span of an erythrocyte: 3 to 4 months in dogs and 2 to 3 months in cats.6 Like the fructosamine test, glycosylated hemoglobin can be a useful indicator of the level of regulation of diabetic patients but cannot evaluate whether the patient had instances of hypoglycemia during the previous few months. Patients that are anemic may have artificially reduced levels of glycosylated hemoglobin.3

    Ketone Bodies

    Ketone bodies can be detected in plasma or in urine. The ketone produced in greatest abundance in patients with ketoacidosis is b-hydroxybutyrate. However, many tests for serum and urine ketones only detect acetone and/or acetoacetate, and ketonuria often goes undetected until the patient begins to exhibit obvious clinical signs.3 The plasma tests for b-hydroxybutyrate are now becoming available for use in the veterinary clinic.

    Additional Serum Chemistry Tests

    In addition to glucose, other serum chemistry components may be altered in diabetic patients.1 As a consequence of the effects of diabetes mellitus on hepatocytes, serum alanine aminotransferase and alkaline phosphatase are often elevated. Increases in blood urea nitrogen and creatinine caused by dehydration are common in patients with ketoacidosis. Serum sodium, potassium, and chloride are all decreased in animals with poorly regulated diabetes. Lipase may be elevated if the animal has pancreatitis or the glomerular filtration rate is reduced because of dehydration. Ketone bodies can also be detected in plasma, although most in-house analyzers are not capable of conducting these tests.

    Hematology

    Hematology findings are usually within normal ranges in patients with well-regulated diabetes. Leukocytosis may occur as a result of tissue necrosis, secondary infection (e.g., urinary tract infection, bronchopneumonia), or pancreatitis. Packed cell volumes may be increased if the patient is dehydrated.6

    Urinalysis

    A complete urinalysis profile is essential for diagnosing and monitoring diabetes. Glucosuria is an early indication of poor response to therapy. Proteinuria is a common finding and is often associated with secondary infection of the urinary tract. Diabetic patients are more susceptible to bacterial and fungal infections, such as cystitis. If the patient has ketoacidosis, ketone bodies may be detected in urine.6

    Although glucosuria can indicate a poor response to therapy, insulin dosages should not be adjusted based solely on a positive result from a urine glucose test. Transient glucosuria can occur in various situations, such as excitement, excess carbohydrate intake, and corticosteroid administration.6 When insulin dosages are escalated in error, the body releases hormones that increase the amount of circulating glucose and modify the effects of insulin on body tissues. Over time, even as the body is experiencing dramatic fluctuations in blood sugar levels, the tissues develop a resistance to insulin that may require weeks of insulin withdrawal to resolve. Each time the insulin dose is increased because of a positive result on the urine glucose test, the subsequent insulin toxicity (and associated hypoglycemia) results in the initiation of protective compensatory mechanisms that cause massive transient increases in blood sugar and subsequent hyperglycemia. This is referred to as the Somogyi effect (see figure), or rebound hyperglycemia.8 The condition can be life threatening.

    Home Testing

    Clients may monitor blood glucose, urine glucose, urine ketones, and clinical signs. Various products, including handheld glucose meters and urine glucose and ketone dipstick tests, are available for these purposes. Products that develop color changes when glucosuria is present are also available for use in litterboxes. Clients must be well educated about the significance of test results and clinical signs to ensure that they contact the veterinary clinic when necessary and do not try to adjust insulin dosages themselves. Well-educated clients who understand the clinical signs that signal problems with regulating their diabetic pets can be reliable evaluators of patient status9 and an invaluable addition to the health care team.

    Conclusion

    Diagnostic testing is important to differentiate diabetes mellitus from other serious conditions that can produce the clinical signs of polyuria and polydipsia, such as kidney or liver disease, adrenal hormone or electrolyte imbalances, or uterine infections. Early diagnosis of diabetes makes treatment considerably less complicated and reduces the morbidity and mortality that is associated with progression to ketoacidosis. Client education about the importance of compliance with therapy and home monitoring is also critical. Because animals with ketoacidosis make poor candidates for anesthesia or other physiologically stressful situations, it is vital that regular examination and diagnostic testing be included as part of the diabetic patient's overall treatment plan.

    aFor a brief explanation of the interaction between glucose and insulin levels, please see the article in this issue entitled "Understanding Common Endocrine Tests."

    1. Sodikoff C: Laboratory Profiles of Small Animal Diseases: A Guide to Laboratory Diagnosis, ed 3. St. Louis, Mosby, 2001, pp 1-56, 163-258, 352-567.

    2. Sirois M: Clinical chemistry and serology, in Sirois M (ed): Principles and Practice of Veterinary Technology, ed 2. St. Louis, Mosby, 2004, pp 227-247.

    3. Sirois M: Clinical chemistry, in Hendrix C, Sirois M (eds): Laboratory Procedures for Veterinary Technicians, ed 5. St. Louis, Mosby, 2007, pp 74-113.

    4. Stockham S, Dolce K: Glucose, in Cowell R (ed): Veterinary Clinical Pathology Secrets. St. Louis, Elsevier, 2004, pp 191-203.

    5. Joseph RJ, Allyson K, Graves TK, et al: Evaluation of two reagent strips and three reflectance meters for rapid determination of blood glucose concentrations. J Vet Intern Med 1:170-174, 1987.

    6. Evans E, Duncan J: Proteins, lipids, and carbohydrates, in Lattimer KS, Mahaffey EA, Prasse KA (eds): Duncan and Prasse's Veterinary Laboratory Medicine: Clinical Pathology, ed 4. Ames, Iowa State Press, 2003, pp 162-192.

    7. Fleeman LM, Rand JS: Evaluation of day-to-day variability of serial blood glucose concentration curves in diabetic dogs. JAVMA 222:317-321, 2003.

    8. McMillan FD, Feldman EC: Rebound hyperglycemia following overdosing of insulin in cats with diabetes mellitus. JAVMA 188(12):1426-1430, 1986.

    9. Briggs CE, Nelson RW, Feldman EC, et al: Reliability of history and physical examination findings for assessing control of glycemia in dogs with diabetes mellitus: 53 cases (1995-1998). JAVMA 217(1):48-53, 2000.

    References »

    NEXT: Management Matters: "Never 'Just' A Receptionist"

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