Chapter 9 Specific Glandular Funcion Tests

 

 

 

                     

9.1 Exocrine Pancreas

9.1.1 Acinar Secretions, Contents and Characteristics

1. Proteases

• a. Trypsinogen
  • 1) Enterokinase, produced by the duodenal mucosa, activates trypsinogen to trypsin in the intestine.
  • 2) Splits whole or partially digested proteins.
• b. Chymotrypsinogen and procarboxypeptidase A and B
  • 1) Proenzymes which are activated by trypsin
  • 2) Activated enzymes split whole or partially digested proteins.

2. Lipase

• a. Lipolytic enzyme secreted in the active form
• b. Activity is enhanced by bile and is optimum at an alkaline pH.

3. Amylase

• a. Amylolytic enzyme secreted in the active form
• b. Hydrolyzes 1,4 glycoside linkages forming disaccharides and monosaccharides.

9.1.2 Laboratory Detection of Pancreatic Inflammation and Necrosis

1. Serum amylase

• a. Significance of increased serum amylase activity
  • 1) Increased serum amylase is usually caused by enzyme leakage from the pancreatic cells directly into the peripheral blood or into the blood via lymphatics. Leakage is caused by altered permeability usually associated with necrosis.
  • 2) Serum amylase is excreted by the kidneys and therefore is increased in renal failure.
  • 3) Serum amylase may be nonspecifically increased in certain non-pancreatic diseases such as salivary lesions, prostatitis, and other intraabdominal diseases.

2. Serum lipase

• a. Significance of increased serum lipase activity
  • 1) Increased serum lipase activity occurs in pancreatic necrosis.
  • 2) Lipase is excreted by the kidneys and therefore is increased in renal failure. Since renal insufficiency in the dog can cause an increase in amylase and/or lipase, values for these enzymes greater than three- to four-fold are supportive of acute pancreatitis when there is concurrent renal insufficiency.
  • 3) Serum lipase may remain high for a longer period than amylase following pancreatic necrosis.
  • 4) The administration of glucocorticoids can cause a mild increase in plasma lipase activity and a decrease in plasma amylase activity without causing clinical or histologic evidence of pancreatitis in the dog.
  • 5) An exploratory laparotomy can result in a mild increase in plasma lipase activity without evidence of pancreatitis.

3. Other findings:

• a. Neutrophilic leukocytosis with a left shift, lymphopenia, eosinopenia and occasionally monocytosis.
• b. Hyperlipemia
  • 1) Diabetes mellitus may be a sequela to severe pancreatic necrosis and cause hyperlipemia.
  • 2) Lipoprotein lipase, a plasma lipemia-clearing enzyme produced by the pancreas, is lost during pancreatic necrosis.
• c. High PCV and plasma protein concentration resulting from fluid shifts.
• d. Prerenal azotemia.
• e. Serosanguineous peritoneal effusion characterized by lipid droplets, erythrocytes, and neutrophils (nonseptic exudate).
• f. Hypocalcemia caused by fatty acids combining with ionized calcium forming insoluble soaps.

4. Pancreatitis:

Although pancreatitis occurs in the cat, very little is known regarding the clinical disease process or its diagnosis. Experimental pancreatitis in the cat has been shown to cause a minimal increase (two-fold) in lipase and a decrease in amylase. The normal range for amylase and lipase is lower for the cat than the dog. In the cat, a lipase or amylase value greater than two-fold increase supports the differential consideration of pancreatitis.

9.1.3 Laboratory Detection of Pancreatic Exocrine Insufficiency

Numerous serum and fecal tests have been developed for assessment of exocrine pancreatic function. Because of the pragmatic limitations in adapting many of these tests for clinical use or because of unreliable results, the following tests must be used with discretion.

1. Microscopic fecal examination

• a. Sudan III or IV stained smears
  • 1) Neutral fats appear as large orange or red droplets. Fatty acids and soaps do not stain.
  • 2) After acidification (acetic acid) and heating of a stained smear, the fatty acids will melt into droplets which stain strongly with the Sudan stains.
  • 3) Pancreatogenous steatorrhea, caused by a deficiency of pancreatic lipase, is characterized by many sudanophilic globules in unheated, nonacidified feces.
  • 4) Enterogenous steatorrhea, malabsorption (malassimilation), requires that the feces be heated with acetic acid before Sudan staining is prominent. Protease activity should be present (gelatin digestion test).
• b. Lugol's stained smears
  • 1) Large blackish-blue structures with a blue-green fringe are starch granules (amylorrhea).
  • 2) Increased numbers indicate amylase deficiency.
• c. Muscle fibers may be observed in unstained or Lugol's stained smears.
  • 1) Undigested fibers have blunt ends, and cross striations are visible (creatorrhea).
  • 2) Their presence in feces from animals on a meat diet indicates deficiency in protein digestion.

2. Total fecal fat

• a. The test is performed on a 24-hour stool sample. A portion is submitted to a laboratory after the total fecal weight is determined.
• b. Normal amount of fat excreted is less than 7 grams/24 hours. Greater amounts indicate steatorrhea which may be caused by lipase deficiency, bile insufficiency, or intestinal malabsorption.

3. Gelatin digestion tests

• a. These tests detect protease action in feces, i.e., trypsin.
• b. Procedures
  • 1) Film test. A strip of x-ray film is placed in a tube containing a mixture of 1 part feces in 9 parts 5% sodium bicarbonate and incubated 1 hour @ 37oC or 2-1/2 hours at room temperature.
  • 2) Tube test. One ml of the above feces-bicarbonate mixture is mixed with 2 ml of melted 7.5% gelatin and incubated 1 hour @ 37oC then allowed to cool. The tube test is more sensitive than the film test.
• c. Digestion of the emulsion or liquification of the gelatin indicates the presence of proteases. Failure to digest the emulsion or solidification of gelatin indicates protease deficiency.
• d. A negative test (absence of fecal proteases) should be repeated because of daily fluctuation of protease levels in feces and the possible occurrence of false negatives. False negatives (absence of gelatin digestion) may occur because of trypsin inhibitors or complete utilization of the enzyme during the digestion process in the intestinal tract.
• e. False positive tests (digestion of gelatin) may be caused by bacterial proteases.

4. Fat absorption test (plasma turbidity test).

Lipid is normally present in plasma but not in sufficient concentration to cause turbidity; but when excess amounts are present, plasma becomes turbid (hyperlipemia).

• a. Hyperlipemia
  • 1) Postprandial hyperlipemia occurs in a normal animal 1-3 hours following a high fat meal.
  • 2) Fasting hyperlipemia is caused by mobilization of body fat rather than absorption.
• b. Procedure for fat absorption test
  • 1) Fast the animal 12 hours and determine if the plasma is turbid (fasting hyperlipemia). If plasma is clear, proceed with the test.
  • 2) Orally administer corn oil (3 ml/kg) and examine the plasma for turbidity 2-3 hours later.
• c. Interpretation
  • 1) The presence of plasma turbidity (positive test) indicates fat is being absorbed.
  • 2) The absence of plasma turbidity (negative test) indicates a lack of absorption of fat. Causes are:
    • a) Exocrine pancreatic insufficiency due to lack of digestion of lipid by lipase.
    • b) Bile insufficiency causing faulty emulsification of fat and reduced lipase digestion.
    • c) Intestinal malabsorption.
• d. If the fat absorption test is negative, pancreatic enzymes are added to the fat meal and incubated 30 minutes before feeding. The fat absorption test is then repeated.
  • 1) Plasma turbidity indicates pancreatic insufficiency.
  • 2) A continued negative test suggests intestinal malabsorption, bile insufficiency, or a false negative test caused by gastric inactivation of pancreatic enzymes added to the fat meal or delayed gastric emptying.
• e. If malabsorption is suspected, a D-Xylose Absorption Test can be conducted.
  • 1) 0.5 g/kg body weight of D-Xylose is administered orally as a 5 or 10% aqueous solution.
  • 2) Blood samples are taken at 0, 30, 60, 90, and 120 minutes after ingestion.
    • a) Normal values:
      • -- > 45 mg/dl at peak in dogs
      • -- > 15 mg/dl at peak in cats
    • b) Decreased values occur with:
      • - intestinal malabsorption disease
      • - delayed gastric emptying
      • - decreased intestinal blood flow
      • - edema or ascites
      • - intestinal bacterial overgrowth

5. Serum Trypsinogen

• a. Trypsinogen is measured as trypsin-like immunoreactivity (TLI) by a radioimmunoassay.
• b. Serum trypsinogen is determined on one fasted sample and increased serum activity is associated with acute pancreatitis.
• c. Chronic renal insufficiency will also cause increased serum TLI.
• d. Decreased serum TLI activity is supportive of exocrine pancreatic insufficiency.
• e. The test is currently only useful in the dog.

6. Bentiromide Test

• a. Oral bentiromide is administered, and chymotrypsin, normally secreted by the pancreas, cleaves off the P-aminobenzoic acid (PABA).
• b. PABA levels are measured in serum at four 30-minute time points following administration of the bentiromide.
• c. In normal animals, the PABA serum concentrations will increase with time until peak concentrations are reached. In animals with exocrine pancreatic insufficiency, the PABA concentration curve remains blunted due to the decreased levels of chymotrypsin.
• d. This test is currently only useful in the dog.

9.2 Endocrine Pancreas

9.2.1 Physiologic Mechanisms

1. Regulation and function of insulin

• a. Insulin is secreted by beta cells of pancreatic islets under the stimulus of hyperglycemia.
• b. Insulin affects every organ of the body by promoting anabolic metabolism of carbohydrates, fats, proteins, and nucleic acids.
• c. Insulin potentiates the transfer of the following substances into target cells.
  • 1) Glucose and other monosaccharides.
  • 2) Some amino acids and fatty acids.
  • 3) Potassium and magnesium.
• d. Hypoglycemia and hypokalemia are the usual insulin effects noted clinically.
• e. The primary target cells of insulin are skeletal muscle cells, hepatocytes, and fat cells. Erythrocytes, neurons, and renal tubular cells (glucose reabsorption) Do NOT require insulin for glucose uptake.

2. Regulation and function of glucagon

• a. Glucagon is secreted by alpha cells of pancreatic islets under the stimulus of hypoglycemia.
• b. Glucagon promotes mobilization of energy-producing metabolites by stimulating glycogenolysis, gluconeogenesis, and lipolysis each of which causes a rise in blood glucose concentration.

3. Glucose metabolism and blood glucose concentration

• a. Factors that cause a rise in blood glucose concentration are as follows.
  • 1) Glucagon:
    • a) Inhibits insulin secretion and glycogenesis.b) Stimulates glycogenolysis, gluconeo-genesis, and lipolysis.
  • 2) Cortisone:
    • a) Inhibits glucose transport across cell membranes.
    • b) Stimulates gluconeogenesis and glyco-genesis.
  • 3) Epinephrine:
    • a) Inhibits glycogenesis.
    • b) Stimulates gluconeogenesis and glyco-genolysis.
  • 4) Digestion and intestinal absorption:
    • a) Cause a rise in blood glucose concentration for 2-4 hours postprandial in simple stomach animals.
    • b) Cause minimal or no glucose concentration rise in ruminants because most of the dietary carbohydrates are fermented in the rumen to volatile fatty acids. The primary source of glucose in ruminant metabolism is gluconeogenesis.
    • b. Insulin is the only physiologic metabolic factor that decreases blood glucose concentration.

9.2.2 Means of Evaluating Glucose Metabolism

1. Blood glucose concentration

• a. Procedures.
  • 1) Most methods are calorimetric.
  • 2) Assay for glucose must be completed within 20-30 minutes after sample collection because blood cells utilize glucose. Longer periods may elapse if serum or plasma is immediately separated from cells and the sample refrigerated. Sodium fluoride may be used as an anticoagulant that prevents glucose oxidation.
  • 3) A 12-hour fast is advised before determination of blood glucose concentration in dogs and cats.
  • 4) Lipemia interferes with nearly all blood glucose methods.
• b. Stages of hyperglycemia in diabetes mellitus.
  • 1) Preclinical stages may be characterized by prolonged postprandial hyperglycemia; diagnosis at this stage is rare.
  • 2) Animals may present with persistent hyperglycemia at levels below the renal threshold (140-170 mg/dl).
  • 3) Most cases of diabetes mellitus are diagnosed in the stage of glycosuria when persistent hyperglycemia exceeds the renal threshold.
  • 4) A fourth stage of diabetes mellitus is ketoacidosis characterized by hyperglycemia, glycosuria, ketonemia, ketonuria, and metabolic acidosis.  

2. Glucose tolerance test

• a. Indications.
  • 1) An oral glucose tolerance test may be used to evaluate intestinal absorption.
  • 2) Intravenous or oral tolerance tests are used to detect glucose intolerance in patients with persistent hyperglycemia below the renal threshold. Glucose tolerance tests are NOT indicated if persistent hyperglycemia exceeds the renal threshold.
  • 3) Intravenous or oral tolerance tests may be used to evaluate a hypoglycemic patient in which a functional beta cell tumor is suspected. This test has limited value because failure of tumor cells to release insulin in response to hyperglycemia is a common feature.
• b. Procedures and Interpretation.
  • 1) Oral glucose tolerance test (dog).
    • a) Give 1 g glucose/2.2 kg of body weight per os.
    • b) Blood glucose concentration should reach 160 mg/dl by 30-60 minutes and return to baseline levels by 120-180 minutes in normal animals. Vomiting or delayed gastric emptying will reduce the amount absorbed.
    • c) Diabetic animals fail to decline to baseline values by 180 minutes. Hyperadrenocorticism may yield similar results.
    • d) Animals with intestinal Malabsorption fail to reach 160 mg/dl by 60 minutes.
    • e) Hyperinsulinism (beta cell tumor) causes a lower than normal peak glucose concentration and a return to hypoglycemic values by 1-2 hours after glucose administration.
  • 2) Intravenous (low-dose) glucose tolerance test (dog).
    • a) Administer intravenously 0.25 g glucose/2.2 kg of body weight.
    • b) Blood glucose concentration should return to the baseline value by 90 minutes.
    • c) Failure to return to the baseline value by 90 minutes is consistent with diabetes mellitus.
  • 3) Feline intravenous glucose tolerance test.
    • a) Administer intravenously 0.5 g glucose/kg of body weight.
    • b) Blood glucose values should return to baseline levels by 120- 180 minutes.
    • c) Failure to return to baseline values by 180 minutes is consistent with diabetes mellitus.

3. Glucagon response test (dog)

• a. The purpose of this test is to provoke insulin release via the hyperglycemic effect of glucagon. An exaggerated insulin response is expected if the patient has a functional beta cell tumor.
• b. Procedure and Interpretation:
  • 1) After a 12 hour fast, blood glucose concentration is measured, and 0.03 mg glucagon/kg of body weight is administered intravenously.
  • 2) The early rise in blood glucose concentration in dogs with beta cell tumor may be slightly less than controls because affected dogs have small glycogen reserves.
  • 3) The finding most consistent with a functional beta cell tumor is profound hypoglycemia (less than 50 mg/dl) 45-90 minutes after administration; seizures may accompany the hypoglycemia. Control dogs have a slower decline in glucose concentration.
  • 4) Normal animals or patients with beta cell neoplasms that fail to respond to the insulinogenic effects of glucagon do not become hypoglycemic as defined above.

 9.3 Thyroid Gland

9.3.1 Physiologic Mechanisms

• 1. The thyroid gland secretes thyroxine (T4 or tetraiodothyronine) and triiodothyronine (T3) under the stimulus of thyroid stimulating hormone (TSH).
  • a. T4:T3 ratio in plasma is about 20:1.
  • b. About 60% of plasma T4 is bound to carrier proteins, whereas 40% is free. Free T4 concentration regulates pituitary TSH production
    • 1) Decreased free T4 concentration stimulates TSH production, whereas increased levels inhibit production.
    • 2) Carrier protein concentration may influence the balance of the regulatory mechanism. High carrier concentration (occurs with pregnancy, hyperestrogenism, and liver disease in man) creates more binding sites, less free T4, more TSH, and more thyroxine secretion.
• 2. The biologically active form of thyroxine is believed to be T3.
  • a. T4 to T3 conversion occurs at the target cell.
  • b. The biological activities include:
    • 1) Stimulation of basal metabolic rate.
    • 2) Promotion of basal metabolic rate.
    • 3) Stimulation of glycolysis, gluconeogenesis, lipid catabolism, and cholesterol synthesis.

9.3.2 Means of Evaluating Thyroid Function

• 1. Serum T4 concentration by competitive protein binding assay (T4CPB).
  • a. Test is specific for T4.
    • 1) Thyroid therapy should be withheld for 7 days for a valid baseline value.
    • 2) Dilantin and salycilates may compete and falsely raise the T4CPB value.
    • 3) Iodinated compounds do NOT affect the T4CPB value.
  • b. Method and Interpretation (dog):
    • 1) Collect a baseline sample for T4CPB.
    • 2) Administer 5 units TSH/10 kg of body weight.
    • 3) Collect a post-TSH sample at 12 hours for T4CPB.
    • 4) Euthyroid dogs double or triple their baseline values, whereas dogs with primary hypothyroidism have little or no change.
• 2. Serum T4 concentration by radioimmunoassay (T4RIA).
  • a. T4RIA procedure is more sensitive than T4CPB.
  • b. The interpretative aspects are the same as described for T4CPB.
• 3. Serum T3 concentration by radioimmunoassay (T3RIA).
  • a. The procedure is similar to T4CPB and T4RIA and is very sensitive.
  • b. An advantage of this test over T4 concentration is that it measures the biologically active form of thyroxine.
  • c. The interpretative aspects are the same as described for T4CPB.
• 4. Serum cholesterol.
  • a. Cholesterol methods are calorimetric.
  • b. About 2/3 of canine hypothyroid cases have hypercholesterolemia; the mechanism is unknown.
  • c. Hypercholesterolemia is not specific for hypothyroidism. 

9.4 Adrenal Cortex

A. Physiologic Mechanisms

• 1. Glucocorticoids, regulation of secretion and function.
  • a. Glucocorticoids are secreted by the zona fasciculata. Secretion is stimulated by ACTH which is released from the pituitary under the stimulus of reduced blood cortisol concentration.
  • b. The general effects of glucocorticoids are glucose sparing and hyperglycemia.
  • c. Glucocorticoids have many other actions including suppression of wound healing, inflammation, and immunologic responsiveness. Specific changes in blood leukocyte numbers are attributed to these hormones.
• 2. Mineralcorticoid, regulation of secretion and function.
  • a. Aldosterone, the mineralcorticoid, is secreted by the zona glomerulosa. Regulation is by several complex mechanisms which involve ACTH, renin, direct stimulation by rising serum K+ concentration, and possibly declining serum Na+ concentration.
  • b. The kidney is the primary target organ of aldosterone in which tubular Na+ reabsorption and tubular K+ excretion are promoted. Similar effects may occur in skin and other tissues.
• 3. The zona reticularis secretes androgens, estrogens, and proestrogens which may relate to certain clinical features of adrenocortical disease.

B. Means of Evaluating the Adrenal Cortex

• 1. Indirect evaluation.
  • a. Glucocorticoids and aldosterone have numerous effects that cause alterations detectable by many routinely used clinical laboratory procedures.
    • 1) These effects occur when the hormones are in excess, i.e., hyperadrenocorticism, and when deficient, i.e., hypoadrenocorticism
    • 2) The routinely used procedures are:
      • a) Leukogram
      • b) Blood glucose
      • c) Blood cholesterol
      • d) Serum alkaline phosphatase
      • e) Serum Na+ and K+ 

       

Figure 9.1

Common Laboratory Tests in Adrenocortical Disease*

* N = normal; I = increased; D = decreased

** Na+:K+ ratio or less than 23:1 is probably pathognomonic for hypoadrenocorticism.

• 2. Direct evaluation
  • a. plasma cortisol concentration
    • 1) Laboratory methods for plasma cortisol concentration.
      • a) Competitive protein binding (CPB) measures cortisol.
      • b) Radioimmunoassay (RIA) measures cortisol.
      • c) Fluorometry measures cortisol plus corticosterone. Values are higher than those by CPB and RIA.
    • 2) Synthetic corticoids do not affect results from these methods.
    • 3) Normal and abnormal values overlap slightly. The usual procedure is to conduct an ACTH challenge test (dog).
      • a) Baseline plasma sample is collected at 9 A.M.
      • b) Give 1 unit ACTH/2.2 kg of body weight intramuscularly.
      • c) Collect post-ACTH sample 2 hours later. A particular time in other species is not defined but clinically normal control animals should be tested in parallel with the patient.
  • b. Low-dose Dexamethasone Supression Screening Test
    • 1) This test is helpful in confirming hyperadrenocorticism in patients in whom the ACTH test results were equivocal.
    • 2) The test is conducted by obtaining a fasting plasma sample, administering 0.01 mg/kg dexamethasone IV, and intravenously collecting a second sample for cortisol assay 6 and 8 hours later.
    • 3) Normal dogs will have an 8-hour post-dexamethasone cortisol value that is usually less than 1 ug/dl. If the postdexamethasone value is greater than 1.0 ug/dl, one has confirmed the presence of hyperadrenocorticism.
  • c. High-dose Dexamethasone Suppression Screening Test
  • 1) This test is conducted in the same fashion as the low dose test except that dexamethasone is given at the rate of 0.1 mg/kg.
  • 2) Such high doses of dexamethasone should completely suppress ACTH secretion from abnormal pituitary cells. Normal suppression is considered to be a cortisol that is less than 50% of base line. If the second value is more than 50% of the baseline, it suggests adrenal tumor or a large pituitary chromophobe adenoma. Some dogs with pituitary dependent hyperadrenocorticism do not suppress, even with a megadose of dexamethasone.
  • d. ACTH Assay
    • 1) Differentiation between primary dependent hyperadrenocorticism and adrenal neoplasia hyperadrenocorticism is best accomplished with an assay for plasma ACTH.
    • 2) Normal plasma ACTH is from 20 to 100 pg/ml. In the dog or cat, endogenous ACTH values that are less than 20 pg/ml are highly indicative of adrenal neoplasm whereas ACTH values greater than 100 pg/ml support pituitary dependent hyperadrenocorticism.
    • 3) Samples collected for this assay require special handling and must be kept cold at all times.
  • e. Interpretation of the ACTH response and dexamethasone suppression tests is summarized in Figure 2.

Figure 9.2

Adrenocortical Disorders and Associated Plasma Cortisol Findings

Plasma Cortisol Concentration*

* N = Normal; I = Increased; D = Decreased; B = Baseline