Clinical Chemistry Reference Laboratory Procedures for Veterinary Technicians

Clinical Chemistry Reference: Laboratory Procedures for Veterinary Technicians 5 th Ed. (Hendrix & Sirois)

Sample Collection & Handling Most chemical analyses require collection and preparation of serum samples u Whole blood or blood plasma used for some test methods or with specific types of equipment u – Do not use EDTA; heparin is a good choice for clinical chemistry samples u Most adverse influences on sample quality can be avoided by careful consideration of sample collection and handling

Sample Collection & Handling Specific blood collection protocols vary depending on patient species, volume of blood needed, method of restraint, and type of sample needed u Collect blood samples for chemical testing before beginning treatment. u Preprandial samples are preferred; postprandial samples may produce erroneous results. u Label sample tube with date and time of collection, owner’s name, patient’s name, and patient’s clinic identification number. u

Serum Sample Collection u u u u Blood should be collected from calm, fasted animal when possible Avoid hemolysis by selecting needles of the correct size. Place blood in a container that contains no anticoagulant. Allow blood to clot at room temperature for 20 to 30 minutes. Gently separate clot by “rimming” with a wooden applicator stick around the inside of the tube. Replace top and centrifuge at 2000 to 3000 rpm for 10 minutes. Remove serum with a pipette and transfer to appropriate container.

Factors Influencing Results u Hemolysis: may result when a blood sample is: – drawn into a moist syringe – mixed too vigorously after collection – forced through a needle when being transferred to a tube – Frozen as a whole blood sample u Hemolysis can also occur when excess alcohol is used to clean the skin and not allowed to dry prior to drawing blood.

Hemolysis Fluid from hemolyzed blood cells can dilute the sample, resulting in falsely lower concentrations of constituents present in the animal. u Certain constituents, normally not found in high concentrations in serum or plasma, escape from ruptured blood cells, causing falsely elevated concentrations in the sample. u Hemolysis may elevate levels of potassium, organic phosphorus, and certain enzymes in the blood u Hemolysis also interferes with lipase activity and bilirubin determinations. u

Factors Influencing Results Chemical contamination: collection tubes must be chemically pure u Improper labeling: label all tubes properly. u Patient influences: obtain samples from a fasting animal u – Postprandial samples may have increased blood glucose levels and decreased inorganic phosphorus. – Lipemia results in turbid or cloudy serum – Kidney assays affected due to increase in GFR after eating.

Factors Influencing Results u Improper Sample Handling: complete chemical analysis within 1 hour of sample collection. – Do not allow samples to become too warm. – Thoroughly mix serum or plasma that has been frozen after thawing to avoid concentration gradients.

Reference Ranges u Reference ranges are a range of values derived when a laboratory has repeatedly assayed samples from a significant number of clinically normal animals of a given species using specific test methods.

Protein Assays Plasma proteins are produced primarily by the liver, as well as reticuloendothelial tissues, lymphoid tissues, and plasma cells u Plasma proteins have many functions: u – Form the structural matrix of all cells, organs, and tissues – Maintain osmotic pressure – Serve as enzymes for biochemical reactions – Act as buffers in acid-base balance – Serve as hormones – Function in blood coagulation – Defend the body against pathogenic microorganisms – Serve as transport/carrier molecules for most constituents of plasma

Protein Assays u Total Plasma Protein u Total Serum Protein u Albumin u Globulins u Albumin/Globulin Ratio u Fibrinogen

Total Protein Total plasma protein measurements include fibrinogen values; total serum protein determinations measure all the protein fractions except fibrinogen. u Total protein concentration may be affected by altered hepatic synthesis, altered protein distribution, and altered protein breakdown or excretion, as well as dehydration or overhydration. u

Total Protein u Alterations in plasma protein concentrations occur in a variety of disease conditions, especially disease of liver and kidneys. u Age-related changes in plasma protein are also seen.

Determination of Total Protein Levels: Refractometric & Biuret Photometric Methods Refractometric method measures the refractive index of serum or plasma with a refractometer. u Refractive index of the sample is a function of the concentration of solid particles in the sample. In plasma, the primary solids are the proteins. u Biuret method measures the number of molecules containing more than three peptide bonds in serum or plasma. u

Albumin comprises 35% to 50% of the total plasma protein in most animals. u Significant hypoproteinemia is most likely caused by albumin loss. u Synthesized by hepatocytes u Liver disease, renal disease, dietary intake, and intestinal protein absorption may influence the plasma albumin level. u Major binding, transport protein in blood; responsible for maintaining osmotic pressure of plasma u

Globulins: Complex Group of Proteins Alpha globulins are synthesized in the liver and primarily transport and bind proteins (include HDL & VLDL). u Beta globulins include complement, transferrin, and ferritin u Gamma globulins (immunobulins) are synthesized by plasma cells u Concentration estimated by determining difference between total protein and albumin concentrations. u

Albumin/globulin (A/G) ratio u Alteration in the normal ratio is frequently the first indication of a protein abnormality u Determined by dividing the albumin concentration by the globulin concentration. u In dogs and horses albumin>globulin u In cats and cattle, albumin=/<globulin

Fibrinogen u 3% to 6% of the total plasma protein content u Synthesized by hepatocytes u Elevation occurs with acute inflammation or tissue damage. u Most common method of fibrinogen evaluation is the heat precipitation test (you can review this in chapter 2).

Hepatobiliary Assays u Liver functions include: – metabolism of amino acids, carbohydrates, and lipids – synthesis of albumin, cholesterol, plasma protein, and clotting factors – digestion and absorption of nutrients related to bile formation – secretion of bilirubin, or bile – elimination, such as detoxification of toxins and catabolism of certain drugs u These functions are run by enzymatic reactions.

Hepatobiliary Assays The gallbladder is closely associated with the liver, both anatomically and functionally; its primary function is as a storage site for bile. u Malfunctions in the liver or gallbladder result in CS such as jaundice, hypoalbuminemia, problems with hemostasis, hypoglycemia, hyperlipoproteinemia, and hepatoencephalopathy. u

Liver Disease vs. Liver Failure u u u Liver disease includes any process resulting in hepatocyte injury, cholestasis, or both. These include hypoxia, metabolic diseases, toxicoses, inflammation, neoplasia, trauma, and bile duct blockage. Liver failure usually results from some form of liver disease and is recognized by both failure to clear the blood of those substances normally eliminated by the liver and by failure to synthesize those substances normally produced by the liver.

Liver Disease vs. Liver Failure u Liver disease does not always result in liver failure. u Hepatic cells are capable of regeneration if damaged. u 70% to 80% of liver function must be lost before liver failure occurs. u Usually liver disease has to be greatly progressed before clinical signs appear.

Hepatobiliary Assays u Tests for liver disease or failure fall into three categories: – Serum enzyme assays that detect hepatocyte injury – Serum enzyme assays that detect cholestasis – Tests that evaluate or are indicative of liver functions.

Enzymes Released from Damaged Hepatocytes “Leakage Enzymes” Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) Sorbitol dehydrogenase Glutamate dehydrogenase

Alanine Aminotransferase (ALT) Formerly known as serum glutamic pyruvic transaminase (SGPT) u Enzyme found free in the cytoplasm of hepatocytes u Considered a liver-specific enzyme in dogs, cats, and primates u Horses, ruminants, pigs, and birds do not have enough ALT in the hepatocytes for this enzyme to be considered liver specific u Other sources of ALT are renal cells, cardiac muscle, skeletal muscle, and pancreas. u

ALT (cont’d) Administration of corticosteroid or anticonvulsant medications can lead to increases in ALT u Used as a screening test for liver disease because it is not precise enough to identify specific liver diseases u Increases are usually seen within 12 hours of hepatocyte damage and peak levels in 24 to 48 hours u Serum levels will return to reference ranges within a few weeks unless a chronic liver insult is present. u

Aspartate Aminotransferase (AST) Formerly known as serum glutamic oxaloacetic transaminase (SGOT). u Found free in the cytoplasm of hepatocytes and bound to the mitochondrial membrane. u Levels tend to rise more slowly than ALT and return to normal levels within a day if chronic liver insult is not present u Found in significant amounts in many other tissues, including RBCs, cardiac muscle, skeletal muscle, kidneys, and pancreas u

AST (cont’d) u Increased blood level may indicate nonspecific liver damage or be caused by strenuous exercise or intramuscular injection u Assess creatine kinase activity to rule out muscle damage before attributing an AST increase to liver damage.

Sorbitol Dehydrogenase (SDH) Found in liver (primarily), kidney, small intestine, skeletal muscle, and RBCs u Especially useful for evaluating liver damage in large animals such as sheep, goats, swine, horses, and cattle. u Plasma level rises quickly with hepatocellular damage or necrosis. u Assays can be used in all species to detect hepatocellular damage or necrosis u – Disadvantages: SDH is unstable in serum and tests not readily available to average vet. lab.

Glutamate Dehydrogenase (GLDH) Mitochondrial-bound enzyme found in high concentrations in the hepatocytes of cattle, sheep, and goats u Increase in this enzyme is indicative of hepatocyte damage or necrosis. u GLDH could be enzyme of choice to evaluate liver function in ruminants and avians but no standardized test method has been developed for use in a veterinary practice laboratory u

Enzymes Associated with Cholestasis “Induced Enzymes” Alkaline phosphatase (AP) Gamma glutamyltransferase (GGT)

Alkaline Phosphatase (AP) Present as isoenzymes in osteoblasts in bone, and as chondroblasts in cartilage, intestine, placenta, and cells of the hepatobilary system in the liver. u Isoenzymes of AP remain in circulation for approximately 2 to 3 days, with the exception of intestinal isoenzyme, which circulates for just a few hours. u A corticosteroid isoenzyme of AP has been identified in dogs with exposure to increased endogenous or exogenous glucocorticoids. u

AP (cont’d) u Source of an isoenzyme or location of the damaged tissue is determined by electrophoresis and other tests performed in commercial or research laboratories. u In older animals, nearly all circulating AP comes from the liver as bone development stabilizes.

AP (cont’d) u Assay in a practice laboratory determines the total blood concentration of AP. u Concentrations used to detect cholestasis in adult dogs and cats u Not a useful test for detecting cholestasis in cattle and sheep because of wide fluctuations in normal blood levels of AP in these species.

Gamma glutamyltransferase (GGT) Also called gamma glutamyltranspeptidase u Primary source is liver u Also found in renal epithelium, mammary epithelium, biliary epithelium, kidneys, pancreas, intestine, and muscle cells u Cattle, horses, sheep, goats, and birds have higher blood activity than dogs and cats u Blood level is evaluated with liver disease, especially obstructive. u

Hepatocyte Function Tests Bilirubin Bile acids Cholesterol Others (dye excretion, ammonia tolerance, caffeine clearance)

Hepatocyte Function Tests u u u Tests of liver function include measurement of serum concentrations of substances that are normally removed from the blood by the liver and then metabolized and/or excreted via the biliary system (bilirubin, bile acids, cholesterol, ammonia). In addition, these tests include measurement of the serum concentrations of blood constituents that are normally synthesized by the liver (albumin, globulins, urea, cholesterol, coagulation factors) Abnormal concentrations + evidence of liver injury = liver disease or liver failure

Bilirubin u u u RBCs phagocytized and hemoglobin is dismantled Heme portion split into iron and protoporphyrin Protoporphyrin converted to biliverdin then bilirubin Bilirubin attached to protein (albumin or globulin) and transported to liver Bilirubin conjugated to water-soluble glucuronic acid in liver (bilirubin glucuronide) and secreted in bile Bacteria in GI system act on bilirubin glucuronide to produce urobilinogen which is broken down and excreted in feces

Bilirubin (cont’d) u u Some bilirubin glucuronide is absorbed back in bloodstream rather than excreted in bile and excreted by kidneys. Unconjugated (albumin-bound) bilirubin is less water soluble and comprises ~2/3 of the total bilirubin in serum. Measurements of the circulating levels of these various populations of bilirubin can help pinpoint the cause of jaundice. Assays can directly measure total bilirubin (conjugated bilirubin plus unconjugated bilirubin) and conjugated bilirubin

Bilirubin (cont’d) Blood levels of conjugated (direct) bilirubin are elevated with liver damage or bile duct injury/obstruction u 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. u

Bile Acids Aid in fat absorption and modulate cholesterol levels u Synthesized by hepatocytes from cholesterol and conjugated with glycine or taurine u Conjugated bile acids are secreted across the canalicular membrane and reach the duodenum by the biliary system u Gallbladder stores bile acids (except in the horse) until contraction associated with feeding. u 95% are reabsorbed and travel back to liver and are reconjugated (these are what are detected in serum tests) u

Bile Acids (cont’d) u u u Any process that impairs the hepatocellular, biliary, or portal enterohepatic circulation of bile acids results in elevated serum levels. Serum 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. – Difference in concentration of the samples is reported – Only a single sample is tested in horses.

Bile Acids (cont’d) u u u Inadequate fasting or spontaneous gallbladder contraction can increase fasting bile acids; prolonged fasting, diarrhea, and GI malabsorption decreases bile acids Bile acid levels are unspecific regarding the type of liver problem that exists (used as a screening test) May detect liver problems before other CS present (icterus, jaundice, etc. ) Used to follow progress of liver disease during treatment Lipemia will interfere with chemical analysis via spectrophotometry. Bile acid test that uses immunologic methods (ELISA) is available for use in the veterinary clinic.

Cholesterol u Produced primarily in the liver and ingested in food. u Some forms of hepatic failure = decreased blood cholesterol u Cholestasis causes an increase in serum cholesterol in some species because bile is a major route of cholesterol excretion from the body. u In some animals with liver failure, the serum cholesterol concentrations are normal.

Cholesterol (cont’d) u Assay is sometimes used as a screening test for hypothyroidism – Thyroid hormone controls synthesis and destruction of cholesterol in the body Other diseases associated with hypercholesterolemia include hyperadrenocorticism, diabetes mellitus, and nephrotic syndrome. u Administration of corticosteroids may also cause an elevated blood cholesterol concentration. u

Other Tests of Liver Function Dye excretion: anaphylactic reactions have been observed in humans; therefore dye excretion method not widely used. Measurement of bile acid concentration = more specific and easier to perform. Dye excretion tests require injection of a dye. u Ammonia tolerance: any condition that reduces the uptake of ammonia or conversion of ammonia to urea can lead to increased plasma ammonia concentration. u Caffeine clearance: test used in human medicine; few experimental studies have been performed in canine species. u

Kidney Assays u Kidney functions: – Conserve or eliminate water and electrolytes in times imbalance. – Excrete or conserve hydrogen ions to maintain blood p. H within normal limits. – Conserve nutrients (eg. glucose and proteins) – Remove end products of nitrogen metabolism (urea, creatinine, allantoin) – Produce renin, erythropoietin, and prostaglandins – Aid in regulation of body temperature and platelet aggregation (prostaglandins) – Aid in vitamin D activation

Kidney Assays Kidneys receive blood from the renal arteries; blood enters the glomerulus of the nephrons where nearly all water and small dissolved solutes pass into the collecting tubules. u Each nephron contains sections that function to reabsorb or secrete specific solutes. u – Resorption of glucose occurs in the proximal convoluted tubule – Secretion and reabsorption of mineral salts occurs in the ascending limb of the loop of Henle and in the distal convoluted tubule.

Kidney Assays (cont’d) u Nephron has a specific resorptive capability for each substance called the renal threshold. u Blood returns from the kidneys to the rest of the body through the renal veins, which connect to the caudal vena cava. u Urine and blood may be analyzed to evaluate kidney function.

Kidney Assays (cont’d) u Primary serum chemistry tests for kidney function: urea nitrogen and creatinine. u Other tests are designed to evaluate the rate and efficiency of glomerular filtration.

Blood Urea Nitrogen (BUN) Also called serum urea nitrogen (SUN) u Urea is the principal end product of amino acid breakdown in mammals. u Urea passes through the glomerulus and enters the renal tubules u Approximately half the urea is reabsorbed in the tubules and the remainder excreted in the urine u – If the kidneys do not remove sufficient urea from the plasma, BUN levels increase.

BUN (cont’d) Several photometric tests are available to measure urea nitrogen u Chromatographic tests are available and tend to be less accurate. u – Use only as a quick screening test u Contamination of the blood sample with urease-producing bacteria may result in decomposition of urea and decreased BUN levels. – Staphylococcus aureus, Proteus spp. and Klebsiella spp

BUN (cont’d) u Dehydration results in increased retention of urea in the blood (azotemia) – Urea is insoluble molecule; must be excreted in a high volume of water High-protein diets and strenuous exercise may cause elevated BUN levels because of increased amino acid breakdown (not decreased glomerular filtration) u Differences in rate of protein break-down in male vs female animals as well as young vs older animals also affect BUN levels u

Serum Creatinine Formed from creatine found in skeletal muscle as part of muscle metabolism u Creatine diffuses out of muscle cells and into most body fluids, including blood u Amount of creatine metabolized to creatinine usually remains constant, as does blood level of creatinine u Total amount of creatinine is a function of the animal’s total muscle mass. u

Creatinine (cont’d) u Serum creatinine is filtered through the glomeruli and eliminated in urine – Any condition that alters glomerular filtration rate alters serum creatinine level Nearly 75% of kidney tissue must be nonfunctional before blood creatinine levels rise. u Postprandial decreases in creatinine occur from transient increase in glomerular filtration rate after a meal. u

Creatinine (cont’d) Increased serum creatinine levels are seen when there is a lack of functional glomeruli u Serum creatinine concentrations are influenced by: u – Fluid and hydration levels – Prerenal factors, such as shock – Postrenal factors, such as bladder and urethral obstructions u Used to evaluate glomerular function

BUN/Creatinine Ratio Both measurements have a wide range of reference intervals u Used in human medicine for diagnosis of renal disease u BUN and creatinine have an inverse logarithmic relation u A disproportionate increase in BUN can indicate dehydration, dietary treatment failure, or owner noncompliance with treatment regimens. u

Urine Protein/Creatinine Ratio Mathematical method that compares urine protein level with urine creatinine levels in a single urine sample u Based on the concept that the tubular concentration of urine increases urinary protein and creatine concentrations equally u 5 to 10 m. L of urine collected via cystocentesis u Sample is centrifuged and supernatant used to determine both concentrations for each sample by photometric methods. u

Water Deprivation Test u u Urine concentration test performed to determine if inappropriate diuresis is attributable to failure of the neuroendocrine pathway that releases ADH or if nephrons are not responding properly. The patient is gradually deprived of water over 3 to 5 days until there is a stimulus for endogenous ADH release. (This usually occurs at about 5% weight loss) Failure to concentrate urine over the duration of the test is indicative of insufficient ADH or unresponsive nephrons. Contraindications: dehydration, azotemia

Pancreas Assays u The pancreas has endocrine and exocrine functions. Pancreatic endocrine function involves production of glucagon and insulin. Diabetes mellitus, or a deficiency of insulin resulting in hyperglycemia, is the most common endocrine disorder of the pancreas. Pancreatic exocrine function involves the production of lipase, amylase, and trypsin. Most pancreatic disturbances occur in the exocrine function of the pancreas. Dogs seem to have a greater incidence than cats.

Pancreas Assays Exocrine pancreas: also referred to as the acinar pancreas. u Secretes enzymes necessary for digestion into the small intestine u Primary pancreatic enzymes are trypsin, amylase, and lipase u Trauma to pancreatic tissue is often associated with pancreatic duct inflammation that results in a back-up of digestive enzymes into peripheral circulation. u

Pancreas Assays Endocrine Pancreas: interspersed within the exocrine pancreatic tissue are the islets of Langerhans u Four types of islet cells present; designated as alpha, beta, delta, and PP (pancreatic polypeptide) cells. u Delta and PP cells comprise less than 1% of the islet cells and secrete somatostatin and pancreatic polypeptide, respectively. u Beta cells comprise approximately 80% of the islet cells and secrete insulin. u 20% consists of alpha cells that secrete glucagon and somatostatin. u

Pancreas Assays u Diseases of the pancreas may result in inflammation and cellular damage that causes leakage of digestive enzymes or insufficient production or secretion of enzymes. u Primary exocrine pancreas tests are amylase and lipase; trypsinlike immunoreactivity and pancreatic lipase immunoreactivity

Amylase Primary source is the pancreas, but also produced in the salivary glands and small intestine. u Amylase functions to break down starches and glycogen in sugars. u Increases in serum amylase are nearly always caused by pancreatic disease (pancreatitis), especially when accompanied by increased lipase levels u

Amylase (cont’d) u Enteritis, intestinal obstruction, or intestinal perforation may also result in increased serum amylase from increased absorption of intestinal amylase into bloodstream. u Decrease in GFR for any reason can lead to increased serum amylase because amylase is excreted by the kidneys.

Amylase (cont’d) Animals have a greater serum amylase activity level than humans (10 times greater in dog and cat) so it is recommended to dilute the serum with normal saline before testing if using tests designed for human samples. u Lipemia, hemolysis, and calcium cheleating anticoagulants will affect results. u

Lipase Nearly all serum lipase is derived from the pancreas; function of lipase is to break down fatty acids of lipids. u Excess lipase is normally filtered through the kidneys, so lipase levels tend to remain normal in the early stages of pancreatic disease. u Lipase assay is more sensitive for detecting pancreatitis than amylase assay. u Increased lipase is also seen in renal failure, hyperadrenocorticism, dexamethasone treatment, and bile tract disease. u Manual methods for testing are cumbersome, easier to use automated or SNAP test. u

Trypsinlike Immunoreactivity (TLI) u u u u Considered the test of choice, TLI is highly specific and sensitive in detecting pancreatic insufficiency in dogs. Radioimmunoassay using antibodies to trypsin that can detect both trypsinogen and trypsin Antibodies are species specific Trypsin and trypsinogen are produced only in the pancreas Serum TLI decreases in parallel with functional pancreatic mass Decreased glomerular filtration rate increases TLI Important to fast animal for 12 hours prior to collecting sample.

Serum Pancreatic Lipase Immunoreactivity (PLI) u Serum feline PLI is specific for pancreatitis and is recommended instead of the previously validated serum feline TLI to diagnose cats with symptoms of pancreatitis. u Cats must also be fasted for 12 hours prior to drawing blood sample.

Pancreas Assays u Endocrine Pancreas Tests Primary test is blood glucose; others include fructosamine, betahydroxybutyrate, glycosylated hemoglobin, serum cholesterol, and triglyceride

Glucose Pancreatic islets respond directly to blood glucose concentrations and release insulin (from the beta cells) or glucagon (from the alpha cells) as needed. u Blood glucose level is used as an indicator of carbohydrate metabolism in the body and as a measure of endocrine function of the pancreas. u Some tests for blood glucose react with only glucose, whereas others may quantify all sugars in the blood. u

Glucose (cont’d) Ideally, samples should be taken from an animal that has been fasted for 16 to 24 hours (ruminants should not be fasted) u Serum is preferred u It is essential to centrifuge sample and transfer serum to another tube immediately because blood continues to use glucose at a rate of 7% to 10% per hour if allowed to remain in contact with the serum or plasma. u

Glucose (cont’d) u Hyperglycemia may result from diabetes mellitus, or any of several nonpancreatic causes such as stress and hyperadrenocorticism (Cushing’s disease) *Diagnosis of diabetes mellitus is not made unless glycosuria accompanies hyperglycemia. u Hypoglycemia may result from malabsorption, severe liver disease, or prolonged contact of the serum or plasma with the cellular component of blood.

Fructosamine u Represents irreversible reaction of glucose bound to protein, particularly albumin. u Increased fructosamine indicates persistent hyperglycemia u Indicates average serum glucose over time period represented by the half-life of that species’ serum protein. u Serum fructosamine may be artifactually reduced in patients with hypoproteinemia.

Glucose Tolerance Test u Challenge the pancreas with a glucose load and measure insulin’s effect by blood or urine glucose concentrations; used to rule out diabetes mellitus u IV test is preferred over oral because oral test is affected by abnormal intestinal function such as enteritis or hypermotility, and excitement.

Glucose Tolerance Test (cont’d) u Glucose is injected after a 12 - to 16 hour fast (except in ruminants) u Blood glucose is subsequently checked and progress mapped as a tolerance curve. u Results are standardized as disappearance half-lives or glucose turnover rates expressed as percent/minute

Insulin Tolerance Test u Probes causes of diabetes mellitus u “Glucose Curve” u Serum glucose levels are measured in blood samples obtained before insulin injection (fasting blood glucose) and every 30 minutes after injection for 3 hours.

Other Endocrine Pancreas Tests u Glucagon tolerance: indicated when repeated normal or borderline results are obtained. u Insulin/glucose ratio: involves simultaneous measurements in a fasting animal. u Miscellaneous tests of insulin release: glucose, epinephrine, leucine, tolbutamide, or calcium challenges may be attempted.

Other Endocrine System Assays Adrenocortical Function Tests Thyroid Assays Pituitary Function Tests

Adrenocortical Function Tests Brain or pituitary tumors leading to secondary bilateral adrenal hyperplasia, idiopathic adrenal hyperplasia, or neoplasia may cause excessive cortisol release and hyperadrenocorticism. u Misuse of glucocorticoids is the most common cause of cortisol excess. u Hypoadrenocorticism (Addison’s disease) includes mineralocorticoid deficiency, which does not occur in iatrogenic disease from rapid withdrawal of glucocorticoids. u

Adrenocortical Function Tests (cont’d) Addison’s disease also may result from Lysodren (a medicaiton for adrenal hyperplasia) or from idiopathic causes. u Dogs with nonadrenal disease such as diabetes mellitus, liver disease, or renal disease may have false-positive results u Adrenocorticotropic hormone (ACTH) and cortisol concentrations may be a helpful diagnostic aid in differentiating primary (adrenal-dependent) from secondary (pituitary-dependent) hypoadrenocorticism. u

Adrenocortical Function Tests (cont’d) Measurements taken as baseline data and compared with data obtained from challenge to the adrenal gland with ACTH or dexamethasone. u Low to undetectable ACTH concentrations occur in secondary Addison’s disease, whereas normal (or increased) concentrations are expected in primary Addison’s disease. u Refer to pp 100 -101 in your textbook for step-by-step instructions on performing ACTH stim. and Dex. suppression tests! u

Thyroid Assays Baseline thyroxine concentrations are used diagnostically, but normal values vary dramatically u Semiquantitative immunologic tests are available to measure T 4 concentrations u TSH response test is used on small animals (except hyperthyroid cats) and horses and provides a reliable diagnostic separation of patients with normal versus abnormal thyroid function. u

Thyroid Assays (cont’d) Free T 4 test measures the fraction of thyroxine not bound to protein; levels are less influenced by nonthyroidal diseases or drugs than are total T 4 concentrations. u Triiodothyronine suppression test: based on the expected negative feedback regulation of TSH; induced by high concentrations of circulating thyroid hormone. u

Pituitary Function Tests u Diagnosis of canine acromegaly may be based on documentation of elevated growth hormone (GH).

Electrolyte Assays Electrolytes: negative ions, or anions, and positive ions, or cations, of elements found in all body fluids of all organisms. u Functions of electrolytes include maintenance of water balance, fluid osmotic pressure, and normal muscular and nervous functions. u Also function in the maintenance and activation of several enzyme systems and in acid-base regulation u Acid-base status depends on electrolytes and should be interpreted together. u

Electrolyte Assays u Sodium, potassium, chloride, and bicarbonate are the four electrolytes in plasma. u Minerals of importance are calcium, phosphate, and magnesium u These two groups together are often simply called “electrolytes”

Electrolyte Assays Calcium Inorganic Phosphorus Sodium Potassium Magnesium Chloride Bicarbonate

Calcium Do not use EDTA, oxalate, or citrate anticoagulants to collect calcium samples for testing because they bind with calcium and make it unavailable for assay. u 99% of the body’s calcium is in bone u Remaining calcium maintains neuromuscular excitability and tone, acts as an enzyme activator, plays a role in coagulation, and helps in transport of ions across cell membranes. u Serum calcium levels vary with serum protein and albumin levels (these should be elevated with increased serum calcium) u

Calcium (cont’d) u Hypercalcemia seen with hyperparathyroidism, excessive vitamin D intake, bony metastases u Hypocalcemia seen in malabsorption, eclampsia, pancreatic necrosis, hypoalbuminemia, gastrointestinal stasis or blockage in ruminants, postparturient lactation in cow, bitch, ewe, and mare, hypoparathyroidism

Inorganic Phosphorus u u Most phosphorus in whole blood is found within the RBCs as organic phosphorus Phosphorus in plasma and serum is inorganic phosphorus and is the phosphorus assayed in the laboratory Plasma or serum phosphorus and calcium concentrations are inversely related: as phosphorus concentrations decrease, calcium concentrations increase Hemolyzed samples should not be used because organic phosphorus liberated form ruptured RBCs may be hydrolyzed to inorganic phosphorus, which results in a falsely elevated inorganic phosphorus concentration.

Inorganic Phosphorus u Hyperphosphatemia may be seen in renal failure, anuria, excessive vitamin D intake, ethylene glycol poisoning, and hypoparathyroidism. u Hypophosphatemia may occur in primary hyperparathyroidism, malabsorption, inadequate intake, hyperinsulinism, diabetes mellitus, lymphosarcoma, hyperadrenocorticism

Sodium u Most abundant extracellular cation that plays a major role in the distribution of water and the maintenance of osmotic pressure of fluids in the body. u If sodium is retained, water is retained. u Heparin sodium should not be used as an anticoagulant because it may falsely elevate results.

Sodium (cont’d) u Hypernatremia is rare unless the animal is deprived of water. u Hyponatremia is quite common and is seen in such conditions as renal failure, vomiting, or diarrhea; use of diuretics; excessive ADH; congestive heart failure; water toxicity; or excessive administration of fluids.

Potassium Major intracelular cation; important for normal muscular function, nerve impulse transmission, and carbohydrate metabolism. u Serum levels are so low that measurement of serum potassium does not give much information about the body’s potassium levels. u Plasma is the preferred sample because platelets may release potassium during the clotting process (elevating K+ levels). u Hemolysis releases potassium into plasma (elevating K+ levels). u

Potassium (cont’d) u Hyperkalemia will be seen in adrenal cortical hypofunction, acidosis, or late-stage renal failure. u Hypokalemia will be seen in alkalosis, insulin therapy, or excess fluid loss due to diuretics, vomiting, and diarrhea.

Magnesium Functions to activate enzyme systems and involved in production and decomposition of acetylcholine u Cattle and sheep are the only domestic animals that show clinical signs related to magnesium deficiencies. u Imbalance in calcium-magnesium ratio can lead to muscle tetany in cattle and sheep u Anticoagulants other than heparin may artificially decrease results u Hemolysis may elevate the results through liberation of magnesium from RBCs u

Chloride Predominant extracellular ion. u Functions in maintenance of water distribution, osmotic pressure, and the normal anion/cation ratio. u Concentration is regulated by the kidneys u There is a close relationship between sodium and chloride levels u Hemolysis may affect test results by diluting the sample with RBC fluid u

Chloride (cont’d) u Hyperchloremia may be due to metabolic acidosis or renal tubular acidosis u Hypochloremia may be due to excessive vomiting, anorexia, malnutrition, or diabetes insipidus, or may accompany hypokalemia.

Bicarbonate Second most common anion of plasma. u Functions in the bicarbonate/carbonic acid buffer system and aids in the transport of carbon dioxide from the tissues to the lungs. u Kidney regulates bicarbonate levels in the body by excreting excesses after it has resorbed all that it needed. u Levels are frequently estimated from blood carbon dioxide levels (arterial blood = best) u Bicarbonate level is approximately 95% of the total carbon dioxide measured. u