Fructosamine Measurement in Diabetic Dogs and Cats

Hunter E. Bates, DVM; Perry J. Bain, DVM, PhD; Paula M. Krimer, DVM, DVSc; and Kenneth S. Latimer, DVM, PhD

Class of 2003 (Bates) and Department of Pathology (Bain, Krimer, Latimer), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388

Introduction

Patients with diabetes mellitus may be defined as having a disturbance in the oxidation and use of glucose for energy production. This may be secondary to decreased production or release of insulin by beta cells in the pancreatic islets of Langerhans, or due to an abnormal tissue response to the presence of insulin. This disease is relatively common in both dogs and cats and is treated with insulin injections and dietary restriction. Regulation of insulin administration is important in controlling diabetes. Fructosamine is formed by the glycosylation of circulating proteins and is related to circulating glucose concentration. Fructosamine measurement can aid in the diagnosis of diabetes mellitus and in the assessment of blood glucose control in treated diabetic patients.

Clinical Signs

Animals with diabetes mellitus may exhibit various clinical signs including polyuria, polydipsia, weight loss, and cataract formation.⁶ Diabetes mellitus causes glucosuria when the renal tubular transport maximum for glucose resorption is exceeded due to high hyperglycemia. Ketonuria also may occur if a negative energy balance exists.⁷ Diabetics may have recurrent infections of the skin, urinary tract, and respiratory tract.⁶ Pollakiuria (abnormally frequent passage of urine) may be observed with diabetes related urinary tract infections. Clinical signs of diabetes mellitus may be confused with similar signs of other or concomitant diseases such as renal failure, hyperthyroidism, hyperadrenocorticism, and other endocrine diseases.⁶

Biochemical Detection and Evaluation of Diabetes Mellitus

Several biochemical findings are used in veterinary medicine to diagnose and monitor diabetes. These include fasting hyperglycemia with glucosuria, increased glycosylated hemoglobin concentration, and elevated fructosamine (glycosylated protein) concentration. As with any disease, the evaluation of the patient’s clinical signs is important in making an accurate diagnosis. Once diagnosed, diabetic patients must be monitored to determine the proper insulin dose and the degree of blood glucose control. Serial blood glucose measurements, as well as measurement of glycosylated hemoglobin and fructosamine concentrations, may be used for long term monitoring of diabetic animals.

Diagnosis of Diabetes by Fasting Hyperglycemia

Appropriate clinical signs and the presence of fasting hyperglycemia with concurrent glucosuria are used commonly to diagnose diabetes mellitus in dogs and cats.⁷ Fasting hyperglycemia is present in diabetes mellitus and distinguishes this disease from primary renal glucosuria due to acute tubular nephrosis or Fanconi syndrome.⁷

To detect persistent hyperglycemia, the blood glucose concentration is determined on the patient following a 12-24 hour fast. Blood glucose may be quantified in the hospital using glucose reagent test strips and reference intervals derived by the manufacturer for the dogs and cats.⁷ Glucose measurements also may be done using hand-held glucose monitoring devices (Fig. 1). However, the values obtained using whole blood may be 10-15 mg/dl lower than the serum glucose value. Therefore, the accuracy of these devices should be confirmed before they are used routinely in a practice setting.⁶ If neither of the above are practical, glucose may be submitted to a diagnostic laboratory either in a clot tube (red top) or in a tube with sodium fluoride anticoagulant (NaFl gray top tube). If using a clot tube, the blood should be withdrawn from the patient into the collection tube, allowed to coagulate, and then centrifuged to separate the serum. The serum then should be placed into a new tube within 30 minutes and refrigerated until submitted to the diagnostic laboratory. This procedure will minimize the glucose metabolism by red blood cells that may cause a false decrease in glucose concentration (pseudohypoglycemia)⁶ If a gray-top NaFl tube is used, the fluoride will inhibit glucose metabolism. The entire blood sample may then be refrigerated until submitted to the laboratory, and separating the serum from the blood cells is unnecessary.

Figure 1. A portable glucose meter (Bayer Glucometer Elite®).

Once fasting hyperglycemia has been confirmed, the urine should be tested for the presence of glucose (glucosuria). Ideally, a urine sample should be collected at the same time as a blood sample for glucose measurement, or within 4 hours of confirmed hyperglycemia. Glucosuria may help differentiate diabetes mellitus from stress-induced hyperglycemia. Although stress or excitement may increase blood glucose concentration, it usually will not cause glucosuria except in extremely stressed or excited cats.⁷ Urine glucose concentration may be measured using a urine dipstick or with Clinitest^® tablets (please see clerkship paper Urinalysis Dipstick Interpretation for more information on the detection of glucosuria). Both the dipstick and tablet tests indicate the presence of glucose with a color change. Persistent, marked hyperglycemia in addition to glucosuria usually indicates the presence of diabetes mellitus.⁷

Blood Glucose Curve

Insulin regulation of blood glucose concentration may be evaluated in diabetic patients by monitoring serial blood glucose concentrations over a 12-24 hour period. This process is used to construct a blood glucose curve. Because this test requires 12 to 24 hours, the diabetic dog or cat should be hospitalized during this testing period. The patient should follow its normal diet, medication and exercise routine. During the test period, the patient should be exercised as it would at home. A baseline glucose measurement is performed at the time of insulin injection and then every 1-2 hours thereafter for the next 12-24 hours. Each glucose value (Y axis) is plotted against time after insulin injection (X axis) to create a glucose curve (Fig. 2). Blood glucose concentrations can be measured using the same techniques as with fasting glucose (see above).⁶ Based on the results of the blood glucose curve, insulin dosage and timing are adjusted to bring blood glucose into the ideal range (e.g,. if the blood glucose concentration is too high, the insulin dose is increased). A new blood glucose curve should be repeated several weeks after any change in insulin type, manufacturer, or dosage.

Figure 2. Theoretical blood glucose curve of a well-regulated diabetic over a 24-hour period given one insulin injection daily (arrow = insulin administration at 0 hours), and fed twice daily (star = feeding at 0 hours and 10 hours). Green area = ideal range.

Limitations of Fasting and Serial Glucose Measurements

Blood glucose measurement is the most common technique to diagnose and monitor diabetic patients, but this test has limitations that sometimes make it unreliable. A single blood glucose measurement indicates the quantity of glucose in the blood at a given point in time, but a single value cannot demonstrate dynamic changes or trends in blood glucose values over a longer time period.⁴ Therefore, the single measured blood glucose value cannot differentiate a diabetic patient with transient epinephrine- or corticosteroid-related hyperglycemia from a patient with an inadequate insulin dosage. This can be a potential problem in any animal that is stressed or frightened, but is particularly noteworthy in fractious cats. In addition, a patient that will not eat normally in the veterinary hospital may become hypoglycemic after insulin is given. This can induce a rebound hyperglycemia later in the day that will skew the glucose curve.⁶ Besides stress and decreased appetite, diurnal variation and other medications also may affect blood glucose concentration.⁴ Glucose concentration also is influenced by exercise, other illnesses, and counter-regulatory hormone secretions.⁶ Repeated blood glucose measurements are time consuming and require the patient to remain in the hospital over an extended period of time. Therefore, some owners may feel reluctant to leave their pet with the veterinarian or may have concerns regarding the cost of this diagnostic procedure.⁶

Glycosylated Hemoglobin in the Diagnosis and Monitoring of Diabetes Mellitus

Glycosylated (glycated) hemoglobin concentration can be used as a screening test for diabetes mellitus, as well as for the monitoring of glycemic control in treated diabetic patients. Glycosylated hemoglobin is produced by the nonenzymatic, irreversible binding of glucose to hemoglobin in erythrocytes.⁶ As blood glucose concentration increases, the rate of hemoglobin glycosylation also increases.⁶ The specific fraction of glycosylated hemoglobin that is measured is HbA_1c. This is reported as a percentage of hemoglobin that is in the glycosylated form. The glycosylation of hemoglobin is a gradual process and is not affected by acute or transient hyperglycemia in dogs.⁵ Glycosylated hemoglobin reflects glycemic control for the previous 2-3 months.⁵ Therefore, glycosylated hemoglobin is a viable alternative to fasting glucose measurements because it is unaffected by stress related or post-prandial hyperglycemia. Glycosylated hemoglobin determination also is useful in long term monitoring of diabetic patients over the previous 2-3 months. Because the binding of glucose and hemoglobin is irreversible, the affected senescent erythrocytes must be degraded before the glycosylated hemoglobin value will decrease. Erythrocyte lifespan is approximately 70 to 110 days in cats and dogs, respectively.²

Glycosylated hemoglobin is not used routinely to monitor diabetic dogs and cats, as this test is not widely available for these species. Due to the relatively long life span of erythrocytes (~70 days in cats and ~110 days in dogs), glycosylated hemoglobin is not the most effective test to diagnose and monitor diabetes in veterinary medicine. Hyperglycemia must be present for at least 3 weeks before increased HbA_1c values are detected in the patient’s blood.⁵ Therefore, glycosylated hemoglobin is less effective for short term monitoring of diabetes compared to fructosamine measurement.⁸ Because hemoglobin concentration also affects glycosylated hemoglobin levels, it should also be noted that HbA_1c values may be increased or decreased due to polycythemia or anemia, respectively.⁷

Diagnosis of Diabetes Mellitus Using Fructosamine (Glycosylated Proteins)

Fructosamine measurement also may be used to diagnose and monitor diabetes mellitus in dogs and cats. It is a highly sensitive and specific laboratory test to distinguish hyperglycemic, non-diabetic patients from diabetics with chronic hyperglycemia.¹ Fructosamines are stable complexes of carbohydrates and proteins that are produced by an irreversible, nonenzymatic glycosylation of protein (Figs. 3 and 4).¹¹ Glucose has a greater affinity for albumin in dogs and for globulins in cats.⁹ Determination of fructosamine concentration in the blood is performed using an adapted, commercially available, automated, colorimetric nitroblue tetrazolium technique. This laboratory test is fast, reproducible, inexpensive, easily automated, requires minimal labor, and provides greater precision than other tests.⁸

Figure 3. With normoglycemia, a relatively small amount of serum protein is glycosylated.	Figure 4. With persistent hyperglycemia, increased protein glycosylation occurs.

A single measure of fructosamine indicates the average glucose concentration over the previous 1-2 weeks.⁵ Fructosamine measurement may be used to assist in the diagnosis of diabetes mellitus as well as to monitor the effectiveness of insulin therapy in diabetic patients. There is very little evidence that fructosamine values are significantly influenced by acute or transient hyperglycemia. Thus, fructosamine concentration may be used to assist in the diagnosis and monitoring of diabetic patients without interference from transient hyperglycemia.⁵ This is especially beneficial in cats that are highly affected by stress-induced hyperglycemia.⁹ Quantitative measurement of fructosamine depends on the level and duration of serum glucose concentration and the rate of turnover of specific plasma proteins in the patient.³ In dogs, the half-life of albumin is approximately 8 days.² Therefore, fructosamine values will change faster than glycosylated hemoglobin values in response to changes in blood glucose concentration.

Reference Intervals for Fructosamine in Dogs and Cats

Reference intervals for fructosamine concentration have been proposed in many publications since this test was introduced for use in diabetic veterinary patients, and these reference intervals vary greatly from manuscript to manuscript. This variation in fructosamine reference intervals is most likely due to different populations of animals, different methods of performing the assay, different statistical procedures, and different environmental conditions and feeding protocols.¹¹ For example, the clinical pathology laboratory used by the University of Georgia Veterinary Medical Teaching Hospital uses fructosamine reference intervals of 175-400 µmol/L in cats and 258-343 µmol/L in dogs. It is important to know that reference intervals are variable between laboratories and that each laboratory should establish their own reference intervals. Reference intervals are not affected by the age or sex of the animal.^1,11

Limitations of Fructosamine Measurement in Monitoring of Glycemic Control

While fructosamine levels provide a useful tool for the evaluation of overall control of diabetes mellitus and long-term glucose regulation, this test is unable to detect short-term or transient abnormalities in blood glucose values. For instance, a patient may have an average blood glucose level within the reference interval over a period of 1-2 weeks preceding the test, but still have transient daily episodes of hypoglycemia and/or hyperglycemia. Serial measurements of blood and/or urine glucose are necessary for the detection of these short-term alterations, and are useful in establishing an initial protocol for the feeding and medication of a diabetic patient. Fructosamine levels are more useful for the evaluation of longer-term control, as well as owner compliance with the administration of insulin.

Effects of Selected Conditions on Fructosamine Values

Diabetes mellitus: As stated previously, the presence of persistent hyperglycemia, as in diabetes mellitus, will increase fructosamine concentrations above the reference interval.⁴ Increased fructosamine values are due to higher quantities of glucose in the blood, resulting in increased glycosylation of proteins. Fructosamine measurements also are useful to monitor the regulation of diabetes mellitus in treated animals.

Acute hyperglycemia: Fructosamine concentration is not significantly affected by acute or transient hyperglycemia.⁵ Fructosamine concentration only increases with chronic hyperglycemia. Thus, determination of fructosamine concentration will differentiate patients with diabetes mellitus from patients that only have transient, epinephrine-induced hyperglycemia.⁵

Insulinoma: An insulinoma is a tumor of the insulin-producing beta cells of the pancreatic islets of Langerhans. These neoplasms are associated with hyperinsulinism and subsequent hypoglycemia (decreased blood glucose concentration) in the blood. Because of the resulting chronic hypoglycemia, fructosamine concentration also is decreased in the blood of affected animals.⁴ Insulinoma may be diagnosed by measurement of baseline or fasting glucose and insulin levels. During fasting, a healthy patient’s insulin concentration should decrease and its glucose concentration should remain within the reference interval. Animals with insulinoma have an inappropriately increased insulin concentration with concurrent hypoglycemia. With an insulinoma or other insulin-secreting tumor, there is no regulation of the insulin secretion and the patient may become severely hypoglycemic.⁴ A single decreased fructosamine value may be suggestive of an insulinoma and may aid in the diagnosis of these neoplasms.⁴ Insulinoma is not the only cause of decreased fructosamine concentration; therefore, serum glucose and insulin levels should also be evaluated. Biopsy and histopathologic evaluation of the tumor may be required to provide a definitive diagnosis of insulinoma.

Feline hyperthyroidism: Hyperthyroid cats may have decreased fructosamine levels, despite having normal serum protein levels.¹⁰ It has been proposed that the decreased glycosylated protein levels can be explained by an increased protein turnover rate (decreased protein half-life) due to increased thyroid hormone levels.¹⁰ In hyperthyroid cats with decreased fructosamine concentrations that were treated with radioactive iodine (¹³¹I, a common treatment for hyperthyroidism in referral centers), the fructosamine concentration increased and throxine (T4) concentration decreased following treatment.³ Thus, the rise in fructosamine concentration after ¹³¹I treatment is thought to be due to normalized or decreased protein turnover.³ Serum globulin concentrations also increase after treatment with ¹³¹I and globulins have a greater affinity for glucose in cats.^3,9 Because hyperthyroidism decreases fructosamine concentration, serum fructosamine levels should be used with caution in the diagnosis and monitoring of diabetes mellitus in cats with underlying hyperthyroidism.³

Hyperproteinemia: The presence of hyperalbuminemia (e.g., from dehydration) in dogs may increase fructosamine concentration, but this increase is not significantly different from that of normoproteinemic dogs.⁹ It appears that increased protein concentration in the cat does not contribute appreciably to elevated fructosamine concentration.⁹ Therefore, hyperproteinemia may be a sign of another disease process, but does not affect fructosamine concentration in patients that do not have diabetes mellitus.

Hypoproteinemia: It has been shown that albumin and fructosamine concentrations are highly correlated in dogs.⁴ Therefore, dogs with hypoalbuminemia also have decreased fructosamine concentration.⁹ Because there is less albumin in these patients to be glycosylated, the fructosamine results will be falsely decreased. In cats, there is a correlation between globulin and fructosamine concentrations.⁹ Thus, hypoglobulinemia will result in a decreased fructosamine concentration. It is important to consider the protein concentration when interpreting fructosamine concentration in hypoproteinemic patients. Correction methods have been proposed in the cases of hypoproteinemia, and one should consult the laboratory that performed the fructosamine test as to whether a correction has been done or is necessary.

References

1. Coppo JA, Coppo NB: Serum fructosamine: A reference interval for a heterogeneous canine population. Vet Res Commun 21:471-476, 1997.

2. Duncan JR, Prasse KW, Mahaffey EA (eds): Veterinary Laboratory Medicine: Clinical Pathology, 3^rd ed. Ames, Iowa State University Press, 1994, pp. 9, 112.

3. Graham PA, Mooney CT, Murray M: Serum fructosamine concentrations in hyperthyroid cats. Res Vet Sci 67:171-175, 1999.

4. Loste A, Marca MC, Perez M, Unzueta A: Clinical value of fructosamine measurements in non-healthy dogs. Vet Res Commun 25:109-115, 2001.

5. Marca MC, Loste A, Ramos JJ: Effect of acute hyperglycaemia on the serum fructosamine and blood glycated haemoglobin concentrations in canine samples. Vet Res Commun 24:11-16, 2000.

6. Miller E: Long-term monitoring of the diabetic dog and cat. Clinical signs, serial blood glucose determinations, urine glucose, and glycated blood proteins. Vet Clin N Am: Small Anim Pract 24:571-585, 1995.

7. Nelson R: Diabetes Mellitus. In: Ettinger SJ, Feldman EC (eds): Textbook of   Veterinary Internal Medicine. Diseases of the Dog and Cat, 5th ed. Philadelphia, W.B. Saunders Co., 2000, p. 1443.

8. Plier ML, Grindem CB, MacWilliams PS, Stevens JB: Serum fructosamine concentration in nondiabetic and diabetic cats. Vet Clin Pathol 27: 34-39, 1998.

9. Reusch CE, Haberer B: Evaluation of fructosamine in dogs and cats with hypo- or hyperproteinaemia, azotaemia, hyperlipidaemia and hyperbilirubinaemia. Vet Rec 148:370-376, 2001.

10. Reusch C, Tomsa K: Serum fructosamine concentration in cats with overt hyperthyroidism. J Am Vet Med Assoc 215:1297-1300, 1999.

11. Thoresen SI, Bredal WP: Determination of a reference range for fructosamine in feline serum samples. Vet Res Commun 19:353-361, 1995.

Acknowledgement

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