Clinical Chemistry

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

Sample Collection & Handling • Most chemical analyses require collection and preparation of serum samples • Whole blood or blood plasma used for some test methods or with specific types of equipment • Do not use EDTA; heparin is a good choice for clinical chemistry samples • 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 • Collect blood samples for chemical testing before beginning treatment. • Preprandial samples are preferred; postprandial samples may produce erroneous results. • Label sample tube with date and time of collection, owner’s name, patient’s name, and patient’s clinic identification number.

Serum Sample Collection • 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 • 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 • 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. • Certain constituents, normally not found in high concentrations in serum or plasma, escape from ruptured blood cells, causing falsely elevated concentrations in the sample. • Hemolysis may elevate levels of potassium, organic phosphorus, and certain enzymes in the blood • Hemolysis also interferes with lipase activity and bilirubin determinations.

Factors Influencing Results • Chemical contamination: collection tubes must be chemically pure • Improper labeling: label all tubes properly. • Patient influences: obtain samples from a fasting animal • 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 • 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 • 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 • Plasma proteins have many functions: • 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 • Total Plasma Protein • Total Serum Protein • Albumin • Globulins • Albumin/Globulin Ratio • Fibrinogen

Total Protein • Total plasma protein measurements include fibrinogen values; total serum protein determinations measure all the protein fractions except fibrinogen. • 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.

Total Protein Alterations in plasma protein concentrations occur in a variety of disease conditions, especially disease of liver and kidneys. 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. • 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. • Biuret method measures the number of molecules containing more than three peptide bonds in serum or plasma.

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

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

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

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

Hepatobiliary Assays • 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 • These functions are run by enzymaticreactions.

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

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

Hepatobiliary Assays • 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” Alanineaminotransferase (ALT) Aspartateaminotransferase (AST) Sorbitoldehydrogenase Glutamate dehydrogenase

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

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

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

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

SorbitolDehydrogenase (SDH) • Found in liver (primarily), kidney, small intestine, skeletal muscle, and RBCs • Especially useful for evaluating liver damage in large animals such as sheep, goats, swine, horses, and cattle. • Plasma level rises quickly with hepatocellular damage or necrosis. • Assays can be used in all species to detect hepatocellular damage or necrosis • 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 • Increase in this enzyme is indicative of hepatocyte damage or necrosis. • 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

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. • 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. • A corticosteroid isoenzyme of AP has been identified in dogs with exposure to increased endogenous or exogenous glucocorticoids.

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

AP (cont’d) • Assay in a practice laboratory determines the total blood concentration of AP. • Concentrations used to detect cholestasis in adult dogs and cats • 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 • Primary source is liver • Also found in renal epithelium, mammary epithelium, biliary epithelium, kidneys, pancreas, intestine, and muscle cells • Cattle, horses, sheep, goats, and birds have higher blood activity than dogs and cats • Blood level is evaluated with liver disease, especially obstructive.

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

Hepatocyte Function Tests • 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 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 (bilirubinglucuronide) and secreted in bile Bacteria in GI system act on bilirubinglucuronide to produce urobilinogen which is broken down and excreted in feces

Bilirubin (cont’d) • Some bilirubinglucuronide 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 unconjugatedbilirubin) and conjugated bilirubin

Bilirubin (cont’d) • Blood levels of conjugated (direct) bilirubin are elevated with liver damage or bile duct injury/obstruction • Blood levels of unconjugated (indirect) bilirubin are elevated with excessive erythrocyte destruction or defects in the transport mechanism that allow bilirubin to enter hepatocytes for conjugation.

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

Bile Acids (cont’d) • 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 12hours 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) • Inadequate fasting or spontaneous gallbladder contraction can increase fasting bile acids; prolonged fasting, diarrhea, and GI malabsorptiondecreases 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 • Produced primarily in the liver and ingested in food. • Some forms of hepatic failure = decreased blood cholesterol • Cholestasis causes an increase in serum cholesterol in some species because bile is a major route of cholesterol excretion from the body. • In some animals with liver failure, the serum cholesterol concentrations are normal.

Cholesterol (cont’d) • 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. • Administration of corticosteroids may also cause an elevated blood cholesterol concentration.

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. • Ammonia tolerance: any condition that reduces the uptake of ammonia or conversion of ammonia to urea can lead to increased plasma ammonia concentration. • Caffeine clearance: test used in human medicine; few experimental studies have been performed in canine species.

Kidney Assays • Kidney functions: • Conserve or eliminate water and electrolytes in times imbalance. • Excrete or conserve hydrogen ions to maintain blood pH 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. • Each nephron contains sections that function to reabsorb or secrete specific solutes. • 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) • Nephron has a specific resorptive capability for each substance called the renal threshold. • Blood returns from the kidneys to the rest of the body through the renal veins, which connect to the caudal vena cava. • Urine and blood may be analyzed to evaluate kidney function.

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

Clinical Chemistry