CLP 410 Clinical Biochemistry Notes 2023 PDF

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE CLINICAL BIOCHEMISTRY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2023  CLINICAL BIOCH...

UNIVERSITY OF PRETORIA FACULTY OF VETERINARY SCIENCE CLINICAL BIOCHEMISTRY CLINICAL PATHOLOGY 410 Department of Companion Animal Clinical Studies Copyright reserved 2023  CLINICAL BIOCHEMISTRY Recommended text books (copies in Clinical Pathology reserved study collection in library). 1. Thrall, Weiser, Allison & Campbell. Veterinary Hematology and Clinical Chemistry (2nd Ed). Wiley- Blackwell, 2012. 2. Stockham & Scott. Fundamentals of Veterinary Clinical Pathology (2nd Ed). Blackwell Publishing, 2008. 3. Latimer. Duncan & Prasse’s Veterinary Laboratory Medicine – Clinical Pathology (5th Ed). Wiley- Blackwell, 2011. CLINICAL ENZYMOLOGY INTRODUCTION TO ENZYMOLOGY Different organs, tissues, or cells contain different enzymes and in some cases, only a few organs or tissues contain a given enzyme. These “tissue-specific” enzymes tend to be the most diagnostically useful. Increased serum enzyme activity results when an increased quantity of the enzyme passes into the blood, either because of leakage from injured cells or because of increased production. Increased serum enzyme activity suggests either injury to the cells of origin or stimulation of the cells to produce increased quantities of the enzyme. Diagnostic enzymology is a means of locating where tissue injury or stimulation of increased enzyme production has occurred. It is important to note that enzymology does not provide information about tissue function! In the body, enzymes catalyse biochemical reactions by converting a substrate into a product. Enzyme concentrations are not measured directly, but the serum activity of an enzyme is considered to be directly proportional to its concentration. Currently, enzyme activities are reported in terms of units per liter (U/L), with a unit defined as the quantity of enzyme that catalyses the reaction of 1 μmol of substrate per minute. Basic concepts and information that must be considered to properly interpret the results of serum enzyme assays include:  the difference between leakage enzymes and induced enzymes;  the duration of enzyme activity after passage into the blood (i.e., the enzyme’s biologic half-life in the blood);  the tissue specificity of enzymes;  the proper handling and storage of serum for enzyme assays. Leakage versus induced enzymes Increased serum enzyme activities can result from either leakage or induction. Enzymes can be released when cell injury alters cell membranes; enzymes that pass into the extracellular space and then into the serum by this mechanism are termed leakage enzymes (Fig. 1). However, although membranes of fatally injured cells can certainly leak enzymes as they degrade, sub-lethally injured cells may release membrane blebs that later rupture, resulting in increased serum enzyme activity. Induction involves the increased production of an enzyme by cells that normally produce the enzyme in smaller quantities. This increased production is induced by some type of stimulus, and it results in increased release of the enzyme from the cells and increased activity of the enzyme in serum. Enzymes that pass into the serum by this mechanism are termed induced enzymes (Fig. 2). 2 Figure 1: Leakage enzymes escape from the cell because of altered plasma membranes. Some leakage enzymes, such as AST, are also present in the organelles. More severe damage is required to cause leakage from these organelles. Source: Thrall, Weiser, Allison & Campbell. Veterinary Hematology and Clinical Chemistry (2nd Ed). Wiley-Blackwell, 2012 Figure 2: Increased serum activities of induced enzymes result, in part, from increased production of these enzymes, with a subsequent increase in secretion. This increased production is caused by some type of inducer. Source: Thrall, Weiser, Allison & Campbell. Veterinary Hematology and Clinical Chemistry (2nd Ed). Wiley-Blackwell, 2012 3 Leakage enzymes are present in the cytosol, organelles, or both, and escape following sublethal or lethal (i.e., necrosis) cell injury. Increased serum enzyme activities can be detected within hours of the injury. By contrast, induced enzymes are attached to cell membranes. Increased serum activity of these enzymes depends primarily on increased production and develops more slowly (i.e., days rather than hours). The concept of leakage versus induced enzymes is important, but the difference is not entirely clear-cut in clinical situations. For example, acute hepatocyte injury can result in loss of leakage enzymes, but enzyme production may be up-regulated in the subsequent reparative process of hepatic regeneration, resulting in a slower decline of serum enzyme activity than expected based on the enzyme half-life. The release of membrane blebs containing membrane-bound enzymes may cause rapid but generally mild increases in serum activity of those “inducible” enzymes. Release of induced enzymes can also occur secondary to less acute membrane alterations. In general, there are five mechanisms by which serum enzyme activity may increase: (1) increased release from damaged cells, (2) induction of enzyme synthesis, (3) cell proliferation (hyperplastic or neoplastic), (4) decreased enzyme clearance, and (5) ingestion and absorption. Enzyme half-life After leakage or secretion from cells, the enzymes eventually are degraded and/or excreted from the body. Some enzyme molecules also lose their activity in the serum over time. The rate at which the loss of activity, degradation, or excretion occurs determines the length of time during which the enzyme activity is detectable in the serum after leakage or secretion. The disappearance rate of enzyme activity typically is measured as the biologic half-life of the enzyme, which is the time required for one-half of that enzyme’s activity to disappear from the serum. Knowledge of the average biologic half-life of an enzyme is helpful when assessing how recently leakage or increased production has occurred and whether these processes are continuing. Tissue specificity It is important to know from which tissue the enzyme most likely originated. The tissue specificity is a function of:  The presence or absence of the enzyme in the tissue.  The concentration of the enzyme in tissues. An enzyme can be present in many tissues, but have high concentration in only one or a few.  Where the enzyme goes after leakage or secretion. Enzymes that are detected in serum have either leaked or been secreted into the extracellular spaces and then passed into the serum. Some tissues may have high enzyme concentration, but leaked or secreted enzymes are not readily accessible to blood. The ideal diagnostic enzyme would be specific for only one tissue. However, almost no diagnostic enzymes are found in only one tissue; however, some are found in only a few tissues. The magnitude of the serum enzyme activity is not a reliable indicator of the type or degree of tissue injury. In the case of leakage enzymes it is tempting to assume that higher enzyme activities are indicative of more severe tissue injury. However, keep in mind that dead cells release all of their enzymes, and they produce no additional enzymes (i.e., the serum enzyme activity might eventually return to values within the reference interval). Sub-lethally injured cells, however, lose only a portion of their enzyme content and continue to produce enzymes (possibly at an increased rate). Such cells can ultimately release more enzyme than can dead cells. In other words, necrosis can result in increased enzyme activity, but diffuse, sublethal injury to the same tissue can result in even greater serum enzyme activity. The magnitude of increase also doesn’t differentiate reversible damage from irreversible damage, or local damage from diffuse damage. The relative magnitude of increased serum enzyme activity is often referred to in terms of the fold increase above the upper reference limit (URL); for example, 3× URL signifies a 3-fold increase above the upper reference limit. Isoenzymes and isoforms Enzymes that have different polypeptide structure (i.e., produced by different genes) but catalyse the same chemical reaction are called isoenzymes. Post-translational modification of isoenzymes leads to the production of several isoforms. Isoforms differ in catalytic sites and activity, immunogenicity, and 4 electrophoretic mobility. A good example is alkaline phosphatase (ALP), which has 2 isoenzymes: Intestinal ALP isoenzyme which produces the corticosteroid-ALP (C-ALP) and intestinal-ALP (I-ALP) isoforms, and tissue non-specific-ALP isoenzyme that produces the remaining isoforms of ALP, including liver-ALP (L-ALP), bone-ALP (B-ALP), and placental-ALP. Another example is lactate dehydrogenase (LDH) that has 5 isoenzymes that are tetramers of either heart subunits (H) or muscle subunits (M): LDH-1 = HHHH, LDH-2 = HHHM, LDH-3 = HHMM, LDH-4 = HMMM, and LDH-5 = MMMM. ALP isoform measurement: Differentiation of isoforms can be accomplished by electrophoretic (affinity agarose electrophoresis, immunoassays, cellulose acetate electrophoresis or isoelectric focusing on agarose) or differential inhibition methods using levamisole (inhibits L-ALP and B-ALP especially), heat (inactivates L-ALP and B-ALP) and wheat germ lectin (precipitates B-ALP and C-ALP). LDH isoenzymes can be semi-quantified using agarose electrophoresis. Sample handling Serum enzyme concentration is determined by measuring serum activity and is not directly measured in serum, like for example urea, creatinine, and electrolytes. Routine assays measure enzyme activity by detecting how fast a substrate is consumed or how fast a product forms. It is then assumed that the activity is proportional to the serum concentration. If serum samples are not properly handled the enzyme activity can be altered leading to erroneous results. It is important to note that serum enzymes are proteins that are subject to degradation or denaturation by heat, changes in pH, variable inherent stability, and exposure to various chemicals, any of which can result in loss of enzyme activity. Proper sample handling essential. A clot tube from a fasted animal is preferred (ruminants are not fasted). Harvest serum within 1 h of blood collection but allow time for clot formation and retraction. Prolonged contact with the clot will allow some enzymes to escape from erythrocytes (e.g., AST and LDH), which can also directly contribute to the increased serum enzyme activity. Most enzymes are stable in refrigerated, separated serum for 24 hours. The degree of degradation that occurs after 24 hours varies considerably depending upon the particular enzyme. Haemolysis, lipaemia and hyperbilirubinaemia should be avoided because of the potential to interfere with spectrophotometric assays. LIVER Hepatocyte injury Alanine aminotransferase (ALT) Physiology: ALT is mostly located in the cytoplasm (with small amounts in mitochondria). It catalyses a reversible reaction that is involved in the deamination of alanine to form pyruvate, which can enter the gluconeogenesis pathway or the Krebs cycle. ALT is found in the liver, muscle (cardiac and skeletal myocytes), kidneys, and erythrocytes (in some species). It is useful as a specific indicator of hepatocellular injury in dogs and cats. ALT is not a useful indicator of liver disease in large animals, and pigs, due to low enzyme activity in liver tissue of these species. Following acute hepatic injury, serum enzyme activity peaks at about 48 hours and then begins to decrease. Increases in the enzyme occur due to cell damage (increased membrane permeability or necrosis) and induction (increased synthesis). Serum half-life is 2-3 days in dogs and < 24 hours (about 3-4 hours) in cats. Increased serum ALT activity:  Artefact: Intravascular or in vitro haemolysis may cause increased levels in the cat or pig. Cats have a high RBC to plasma ALT ratio. In contrast, haemolysis (intravascular or in vitro) has a minimal effect on ALT in cattle, horses, and dogs.  Drugs: Anticonvulsants (primidone, phenobarbitone, dilantin) can increase ALT activity up to 4 x normal, most likely secondary to hepatocellular necrosis. Corticosteroids increase ALT to approximately 2-3 x normal, also due to glucocorticoid-induced hepatopathy. Any drugs that can cause hepatotoxicity can result in increased ALT activity, e.g. tetracycline in cats. 5   Pathophysiological: o Liver disease: Hepatocyte damage (reversible or irreversible) may occur because of a variety of insults (inflammation, hypoxia, toxicants, trauma, metabolic, nutritional, etc.). ALT may be released from hepatocytes also during reparative stages of liver disease. Bile duct obstruction or other causes of cholestasis may increase ALT activity due to the toxic effects of retained bile salts on hepatocytes. o Muscle disease: In large animals, ALT activity will increase with muscle injury, but it is not more useful than aspartate transaminase (AST) in this regard. In small animals with severe muscle injury (ischemic myopathy in cats, muscular dystrophy in dogs, where CK activity is usually > 10,000 U/L), ALT will increase with creatine kinase (CK) and AST activities. Note that the increases in ALT activity are usually less than increases in AST activity in primary muscle disease. Aspartate Aminotransferase (AST) Physiology: AST is located in the cytoplasm and mitochondria as different isoenzymes (AST isoenzyme differentiation is not performed in veterinary medicine). It catalyses a reversible reaction involved in the deamination of aspartate to form oxaloacetate, which can enter the Krebs cycle. AST is useful as an indicator of liver and/or muscle injury in large and small animals. AST is not organ specific. Skeletal muscle contains the highest concentration, followed by liver and cardiac muscle. Erythrocytes contain enough to raise activity in serum when intravascular or in vitro haemolysis occurs. Enzyme leakage from erythrocytes (without overt haemolysis) in aged/ stored samples can also result in elevated enzyme activity. The enzyme half-life is about 22 hours in the dog, 77 minutes in the cat, 7-8 days in horses and around 1 day in cattle. Increased serum AST activity:  Artefact: Intravascular or in vitro haemolysis or leakage from erythrocytes.  Physiological: In horses, exercise can increase serum activity as much as 30%. In early training, resting levels are 50-100% greater than resting levels of horses not in training.  Drugs: Anticonvulsants may cause an increase in AST activity, which is thought to be secondary to hepatocellular injury in dogs. Corticosteroids generally do not result in increased AST activity, unless they cause hepatocellular injury (in dogs).  Pathophysiological: o Muscle damage: Muscle trauma (including “downer” animals), rhabdomyolysis, white muscle disease (vitamin E-selenium deficiency), and infectious myositis (black leg or Clostridial myositis), and muscular dystrophy may result in marked increases. Serum CK activity will also increase. Note that as AST has a longer half-life than CK (esp. in horses), increases in AST persist for longer than increases in CK activity. Therefore, in chronic muscle disease, AST may be increased, whilst CK activity may be normal. When there is active muscle disease, both CK and AST activities are usually increased (and CK will decline more rapidly as the injury resolves due to the shorter half-life). In dogs, the degree of increase in AST is proportionally higher than that of ALT activity with muscle injury potentially helping to discriminate between hepatic and muscle sources of ALT activity increases in dogs with severe muscle injury. Canine AST is also has a shorter half-life than canine ALT, so AST might provide a better indication of active hepatocyte damage. o Liver disease: AST activity will increase in liver disease that causes hepatic injury as for ALT. It can be used as an indicator of hepatocyte damage in dogs and cats. Increased activity seen with hepatocellular injury often are not as high as those seen with muscle damage. CK activity are normal unless there is concomitant muscle disease. Glutamate Dehydrogenase (GLDH) Physiology: Glutamate dehydrogenase is a mitochondrial enzyme that catalyses the conversion of glutamate to 2- oxoglutarate. GLDH is found in many tissues in the body, including hepatocytes, kidney, intestine, muscle, and salivary gland. Most of serum GLDH originates from hepatocytes (in health and disease states). Increases in GLDH activity are used primarily to reflect leakage from damaged or necrotic hepatocytes. Due to its cellular location, injury needs to be sufficiently severe to damage mitochondria. 6 GLDH is a useful enzyme for hepatocellular injury in large animals and exotic species (birds, amphibians, reptiles). Low activity of GLDH are seen in health in small animals and horses, whereas healthy cattle and alpacas may have higher activity. The half-life of GLDH is about 8 hours in the dog, 12- 24 hours in the horse and 14 hours in cattle. Increased serum GLDH activity:  Pathophysiological: o Liver injury: GLDH is a sensitive and specific marker of liver disease in all animals, including non-mammalian species. Activity generally peaked at 1-2 days and then decreased to reference intervals within 4-9 days. It is however not specific as to the cause of liver injury. Sorbitol Dehydrogenase (SDH) Physiology: SDH is a cytosolic enzyme with high concentrations in hepatocytes. It catalyses the conversion of fructose to sorbitol. An increase in SDH activity is generally considered as liver specific in all species. Increased activity is an indicator of hepatocellular damage, which may be reversible or irreversible. Increases in activity occur within 24 hours of liver injury. The half-life is reported to be 5 hours in the dog, 3-4 hours in the cat and 12-24 hours in the horse. Not commonly used due to severe in vitro instability of the enzyme, as well as lack of availability of convenient methodology in veterinary medicine. Cholestasis Alkaline Phosphatase (ALP) Physiology: ALP is a non-specific metallo-enzyme which hydrolyses many types of phosphate esters at an alkaline pH in the presence of zinc and magnesium ions. ALP has 2 isoenzymes and several isoforms (refer to “Isoenzymes and isoforms” within the section of Enzymology). ALP is a cell membrane bound protein and is present in many tissues.  Liver: Hepatocytes and the epithelium of biliary tract are the source of the liver-ALP (L-ALP) isoform (all species) and corticosteroid-ALP (C-ALP) isoform, which is unique in the dog. ALP expression is normally restricted to the canalicular membrane of hepatocytes, but can be induced (secondary to cholestasis typically but also corticosteroids) on the sinusoidal membrane (where it can be readily liberated into blood).  Bone: This isoform is produced by osteoblasts and will increase in serum in association with osteoblastic activity (young animals, certain bone disorders).  Intestinal, renal, mammary, placental tissues: These are not usually important sources of increased serum ALP activity, though some placental isoform is present in serum of late pregnant queens and mares. Mildly increased activity of ALP is seen in dogs with various types of mammary tumours. The half-life of ALP varies depending on the source (isoform) and species. Isoforms with longer half- lives contribute more to normal and increased levels of serum or plasma ALP, i.e. increased levels of isoforms with very short half-life (minutes versus days) are unlikely to be observed on a single blood sample (since the peak may be missed and values will rapidly decrease). Isoform Species Half-life Liver-ALP Canine 2 – 3 days Feline 6 hours Steroid-ALP Canine 2 – 3 days Intestinal, kidney, placental ALP Canine/ equine < 6 minutes Feline < 2minutes Bone-ALP Canine 2 – 3 days The main use of ALP is as a sensitive indicator of cholestasis in the dog (ALP activity may be increased before icterus appears), however it is non-specific. In the cat however, ALP is a specific indicator of liver 7 disease, whereas in large animals, the enzyme is not very useful as it is insensitive, cholestatic disorders are infrequent, and reference intervals are quite broad. Test interpretation: Routine measurement of ALP gives total serum activity (all isoforms) without specificity as to source. In healthy animals, L-ALP is the predominant isoform in blood, followed by B-ALP. The proportion of B-ALP is higher in young animals. The C-ALP isoform contributes only a small amount to total serum ALP activity. The proportion of C-ALP increases with age in dogs. Isoform measurement is most commonly applied to canine samples to distinguish L-ALP and C-ALP isoforms in cases with increased total serum ALP activity of uncertain cause (to identify whether total ALP is increased due to liver disease or endogenous corticosteroids). However, this is not a very reliable test for this purpose, because any chronic disease (including that affecting the liver) can result in endogenous corticosteroid release (chronic stress), which would increase C-ALP. Increased serum ALP activity: Increases in serum ALP activity are usually due to the liver, bone and corticosteroid-inducible isoforms (only in dogs). Therefore high ALP activity is attributed to cholestasis, increased osteoblastic activity (not osteolysis) and steroids. Increased ALP in serum or plasma due to increases in other isoforms are rare, likely because the enzyme gets released into the lumen of organs or has a very short half-life.  Drugs or hormones: o C-ALP (dogs only): This isoform is induced by endogenous or exogenous corticosteroids. o L-ALP induction by drugs: Anticonvulsants (e.g. phenobarbital, primidone) possibly due to drug-induced hepatic injury. Corticosteroids will affect ALP to variable degrees in dogs, depending on the formulation or dose. The increase in L-ALP after corticosteroids has been shown to be due to induction of synthesis.  Physiological: o Age: ALP activity in young, growing animals of all species may be 2-10 times higher than adults, due to the increased B-ALP isoform. o Breed of dog: Some Siberian Huskies pups have benign (transient) familial hyperphosphatasaemia. This is due primarily to the bone isoform and doesn’t have any clinical effects. High ALP activities have also been reported in Scottish Terriers, potentially secondary to underlying subclinical adrenal dysfunction, which may be a genetic-based defect in this breed. o Endogenous corticosteroid release (chronic stress): Initially, the ALP increase is due to the L-ALP isoform; the C-ALP isoform takes several days (up to 10) to be induced. This is only in dogs.  Pathophysiological: o Hepatobiliary disease: Increases in ALP (primarily the L-ALP isoform) is used as an indicator of cholestasis (intra- or extrahepatic) in animals. In cats, ALP is a specific but insensitive marker of hepatobiliary disease. Increases in ALP do occur in hepatobiliary disease in cats, but the increase is less reliable and of lower magnitude compared to the situation in dogs. Small changes in ALP should be investigated in the cat due to the lower concentration of ALP in the cytoplasm of feline hepatocytes and the short half-life of this enzyme in the cat.  Structural cholestasis is due to a physical impediment to bile flow, which can be intrahepatic or extrahepatic (or a combination of both). Extrahepatic cholestasis (bile duct obstruction) causes very dramatic increases in ALP, while intrahepatic cholestasis (localised or generalised cholestasis from hepatocyte swelling) will also induce ALP. Causes of intrahepatic cholestasis include neoplasia, hepatic lipidosis (lipidosis is the cause of the most dramatic increases in ALP in cats, often without concurrent elevations in GGT), acute hepatocellular injury, and peri-portal fibrosis and inflammation.  Functional cholestasis or sepsis-associated cholestasis: A pathophysiological state associated with endotoxaemia. There is a decreased bile flow due to cytokine-mediated downregulation or inhibition of transporters responsible for 8  excreting bile salts or conjugated bilirubin into bile. It has been reported in dogs with E. coli infections.  Neoplasia: Neoplasia: In primary liver cancer (hepatocellular/biliary), marked increases in ALP are possible (due to L-ALP or C-ALP in dogs). Metastatic neoplasia to the liver often increases ALP due to localised cholestasis.  Acute hepatocellular injury: Mild to moderate increases in ALP activity are attributed to intrahepatic cholestasis associated with hepatocellular swelling rather than hepatocellular injury per se. o Hyperadrenocorticism in dogs: Levels vary from moderate to marked (up to 100 -fold) and are frequently due to induction of the C-ALP isoform (although L-ALP increases are also seen) in dogs. However, chronic endogenous stress due to any underlying disease may increase C-ALP and total serum ALP (up to 2-3 x normal). o Adrenal dysfunction in dogs: Adrenal disease, resulting in elevated sex hormones or cortisol secretion, is associated with increased ALP activity (as high as 22,000/uL) in Scottish Terriers. o Non-hepatic neoplasia: Benign or malignant mammary tumours in dogs have been associated with mild increases in ALP activity (due to ↑ B-ALP, L-ALP and C-ALP), irrespective of metastasis or the presence of osseous metaplasia. Dogs with osteosarcoma (tumour of osteoblasts) can have high ALP serum activities, and is a negative prognostic indicator. An activity > 120 U/L being associated with a poor prognosis in some studies. o Increased, non-neoplastic, osteoblastic activity: The osteoblastic activity in response to hormones (parathyroid hormone, thyroxine) may increase total serum ALP due to the B- ALP isoform. This can be seen in dogs with primary and secondary hyperparathyroidism (2-3x increase) and healing fractures (mild increases expected) and hyperthyroidism in cats (mostly B-Alp and to lesser extent L-ALP). Gamma Glutamyl Transferase (GGT) Physiology: The enzyme GGT cleaves C-terminal glutamyl groups from amino acids and transfers them to another peptide or to an amino acid. It is important in glutathione metabolism, amino acid absorption and protection against oxidant injury. GGT is primarily membrane bound and found in many tissues. However, the main source of serum activity is the liver (primarily biliary epithelium, small amounts present on canalicular and sinusoidal surfaces of hepatocytes). The highest concentrations are found in kidney (proximal renal tubules - GGT is shed into urine, rather than blood), pancreas and gastrointestinal tract (GGT does not increase in serum in disorders involving these tissues - it is usually shed into the lumen of these organs) and mammary glands (in dog, cattle, sheep and goats; GGT is excreted into milk, particularly with colostrum). It has a half-life of 72-96 hours (equine). GGT is used mainly as a sensitive indicator of cholestasis (interpretation, in general, is similar to ALP), but it also reflects biliary hyperplasia. Increased serum GGT activity:  Drugs: Mild increases may be seen with anticonvulsant or chronic exogenous corticosteroid therapy. Increases in GGT occur as soon as 5 days after corticosteroid administration in dogs but are usually mild.  Physiological: o Neonates: Colostrum in most species, except for horses, contains high GGT concentrations. Increases in GGT occur within 24 hours of suckling and are a sensitive indicator of passive transfer. o Breed: Donkeys have 2-3 x GGT levels of horses.  Pathophysiological: Increases in GGT occur secondary to biliary hyperplasia (increased pressure within the biliary system stimulates hyperplasia) or induction of synthesis. o Hepatobiliary disease: Small animals: GGT is a sensitive indicator of biliary hyperplasia and cholestasis. In cats, GGT increases may precede increases in ALP (except with hepatic lipidosis) and is a more sensitive indicator of liver disease in this species. 9  Large animals: GGT is a sensitive test for biliary hyperplasia and cholestasis (which is relatively uncommon in large animals). GGT is considered a better marker of biliary tract disorders in large animals than ALP. - Horses: GGT activity has been reported to be increased in horses with cholangiohepatitis (e.g. secondary to cholelithiasis or proximal enteritis) or gastrointestinal problems, including proximal enteritis and colonic displacement. Acute hepatocellular necrosis (experimental) may also cause a mild increases (< 4 x baseline) in GGT activity. - Cattle: Experimentally GGT activity is increased in acute hepatic necrosis (most likely due to secondary cholestasis). Disorders associated with increased GGT activity include bile duct obstruction, cholangitis, cholecystitis, copper toxicosis, hepatic mycotoxicosis, and fascioliasis. o Renal disease: Cellular injury to the proximal renal tubular epithelial cells causes GGT to be shed into the urine and not into blood. The urinary GGT to creatinine ratio has been studied as an early indicator of renal tubular injury, especially due to aminoglycoside toxicity. PANCREAS Amylase activity Physiology: Amylase is a calcium dependent enzyme which hydrolyses complex carbohydrates at alpha 1,4-linkages to form maltose and glucose. Amylase is filtered by renal tubules and inactivated by tubular epithelium. There are four different isoenzymes of amylase in the dog: isoenzyme 3 is found in the pancreas (>50%), whereas isoenzyme 4 is found in all tissues. Tissue sources include the pancreas, intestine, ovaries and testes and salivary gland (pigs). Amylase values peak at 12-48 hours and are normal within 8-14 days after a bout of pancreatitis in dogs. It is rare to observe increased amylase in cats with pancreatitis. Increased serum amylase activity (hyperamylasaemia):  Pathophysiological: o Pancreatitis: The increase and decrease of serum amylase tends to parallel that of lipase. Increases of 3–5 times normal may be interpreted as suggestive of pancreatitis. o Chronic renal insufficiency. o Decreased glomerular filtration rate (GFR): This can cause increased amylase (up to 2- 3 times normal) in the absence of significant pancreatic disease. o Intestinal disease/obstruction: Moderate elevations in amylase are possible. Lipase activity Physiology: Lipases hydrolyse triglycerides. There are several forms of lipase: pancreatic lipase, colipase and lipoprotein lipase. The lipase molecule is small enough to pass through the glomerular filtration barrier to be inactivated or excreted. In dogs, lipase activity increases within 24 hours and peaks (at a higher level than amylase) at 2-5 days. Assay methods: Enzymatic assays that measure serum lipase activity detect lipase from pancreas as well as other tissues. Thus, increases in serum lipase activity are not specific for pancreatic injury. Most commonly colorimetric methods which measures the rate of dye formation yielded from the breakdown of long chain fatty acids (such as 1,2-o-dilauryl-rac-glycero-3-glutaric acid or 1,2-diglyceride) are used. The addition of a detergent and colipase to the 1,2-o-dilauryl-rac-glycero-3-glutaric acid- (6’-methylresorufin) ester (DGGR) assay increase the specificity of this assay for pancreatic lipase. Test interpretation: Measurement of lipase is most commonly performed to diagnose pancreatitis. The utility of measuring serum lipase activity to detect pancreatitis varies between species. Serum lipase activity is frequently normal in cats with spontaneous pancreatitis, and therefore is not considered useful for the diagnosis of 10 pancreatitis in this species. In addition, it is also not considered helpful to diagnose pancreatitis in horses and cattle. Reported causes of increased lipase activity appear to be substrate dependent. The older 1,2 diglyceride-based assays appear less sensitive than the newer DGGR-based assay. Increased serum lipase activity (hyperlipasaemia):  Drugs: Corticosteroids are reported to increase lipase activity with the 1,2-diglyceride substrate. Typically, < 2 x normal.  Pathophysiological: o Acute pancreatitis: Injury and destruction of pancreatic acinar tissue results in the escape of pancreatic enzymes into the pancreas and peritoneal cavity. The enzymes enter the blood by way of lymphatics or capillaries with subsequent increase in serum or plasma activity. Increases of 3 x normal (using the 1,2-diglyceride assay) in dogs and cats supports a diagnosis of pancreatitis more strongly than more moderate increases, which may be accounted for by non-pancreatic causes. o Gastrointestinal disease: DGGR assay – limited data available, however with the 1,2- diglyceride assay, peritonitis, gastritis, bowel obstruction, and visceral manipulation (laparotomy) may increase lipase activity by 2-3 x normal. o Hepatic disease: Hepatic disease (necrosis and fatty degeneration) and neoplasia may increase activities if the 1,2-diglyceride assay is used. o Decreased glomerular filtration rate (GFR): Increases of up to 4 x normal activity may be seen in patients with decreased GFR (pre-renal, renal or post-renal azotaemia) if the 1,2- diglyceride assay is used. Using the DGGR assay, the lipase activity is not always increased with sever azotaemia in dogs. Pancreatic lipase immunoreactivity (PLI) These tests are species-specific immunoassays that use species-specific antibodies to measure serum concentrations of lipase originating specifically from the pancreas. These canine and feline assays (Spec cPL™ and Spec fPL™) are commercially available at IDEXX Laboratories. There is also a rapid in-clinic test available for cPLI (SNAP® cPL™, IDEXX Laboratories). In dogs, the sensitivity of cPLI for the detection of pancreatitis is 65–82%, depending upon disease severity, with a specificity >95%. A few studies suggest that cPLI concentrations are minimally increased with renal failure and not affected by prednisone administration. In cats, the sensitivity of fPLI for the detection of pancreatitis is 54–100%, depending upon disease severity, with a specificity of 91%. These assays are most reliable for detection of moderate to severe pancreatitis. Serum trypsin-like immunoreactivity (TLI) Physiology: Trypsinogen is synthesised only by the pancreas, and it is converted to the active proteolytic enzyme, trypsin, in the small intestine. In health, a small amount of trypsinogen leaks into the extracellular space and then diffuses via the lymphatics into the blood. Therefore, a normal serum TLI concentration is a good indicator of adequate pancreatic trypsinogen production. Assay methods: The TLI assay uses species-specific antibodies to detect both trypsinogen and trypsin in serum. TLI assays are available for dogs and cats, and have been used experimentally in horses (only the canine assay is available in RSA). Animals should be fasted for a minimum of 12 hours prior to collection of a blood sample. Increased serum TLI:  Pancreatitis: Increased serum TLI is expected with pancreatitis due to leakage from damaged acinar cells. o The sensitivity of increased serum TLI concentration for diagnosis of pancreatitis in dogs and cats is 33–36%. Specificity has been reported between 65–90%. Therefor serum TLI concentration is now principally applied to diagnosis of pancreatic exocrine insufficiency (EPI). 11   Decreased glomerular filtration rate (GFR): Trypsinogen is cleared by glomerular filtration, thus any disorder causing a decreased GFR can increase serum TLI concentration. MUSCLE Creatine Kinase (CK) Physiology: Creatine kinase (CK) is an enzyme present in highest concentrations in skeletal muscle, cardiac muscle, smooth muscle, and brain, with lesser amounts present in various organs such as intestine, liver, and spleen. It is found free in the cytoplasm of muscle cells and leaks from these cells when they are damaged. CK is considered a muscle-specific leakage enzyme and has a very short half-life. The half- life is 123 minute and 2.5 hours in horses and dogs respectively. Increased in CK activity following muscle injury occur rapidly (peaking in 6-12 hours). The CK activity returns to normal within 24-48 hours after acute, transient muscle injury in horses. Persistent or ongoing muscle injury will maintain high CK activity. In contrast, AST activity (which has a longer half-life of 24 hours in the dogs and several days in the horse) will increase more gradually after muscle injury and stays increased for a longer period of time than CK. Figure 3: Skeletal muscle contains a high concentration of CK, which catalyses the reversible reaction with creatine and creatine phosphate as an energy source of muscle metabolism. CK is released into the plasma following skeletal muscle injury and is routinely measured. Creatinine is formed within the muscle from creatine, rapidly diffuses into the plasma at a relatively constant rate, and is freely filtered by the glomeruli (kidneys). From: Veterinary Laboratory Medicine: Interpretation & Diagnosis. Meyer & Harvey, 2nd edition, 1998. Increased serum CK activity:  Artefact: Haemolysis will increase CK activity as constituents within red blood cells or in their membranes contribute to the enzymatic reaction for CK, falsely increasing activity. Inadvertent penetration of muscle (“muscle stick”) during venipuncture can cause 3- to 4-fold increases in CK activity in the sample.  Physiological: CK activity in young puppies is higher than in adults (4-fold adult levels in puppies < 1 month of age) – unknown cause.  Pathophysiological: o Muscle disease: Detection of increased activity in serum is useful as an indicator of muscle injury. High CK activity are observed in inherited muscular dystrophies, exercise- induced rhabdomyolysis, polymyositis, vitamin E-selenium deficiency, snake bite poisoning, etc. Note that animals which are recumbent (“downer” cows or post-surgical patients) will have high CK activity (up to 10 x normal) resulting from muscle injury or ischaemia. Similarly, horses and cattle after transport have moderate increases in CK activity. o Muscle catabolism: Increased CK activity can occur in critically ill anorexic cats that have diseases not directly involving muscle. Muscle catabolism to supply amino acids for protein synthesis and gluconeogenesis is thought to result in the leakage of CK from muscle cells. 12 Aspartate aminotransferase (AST) As previously mentioned, AST is present at highest concentrations in hepatocytes as well as in skeletal and cardiac muscle cells. Serum AST activity increases more slowly than serum CK activity after muscle injury. It peaks at approximately 24–36 hours after acute muscle injury, and it decreases more slowly than serum CK activity after the muscle injury ceases. The relative serum activities of both CK and AST can be used to estimate when muscle injury occurred and whether active muscle injury is ongoing (Fig. 4). An increase in only the serum CK activity (Fig. 4, line A) suggests very acute muscle injury (i.e., there has not been sufficient time since the injury occurred for the serum AST activity to increase). Increased serum activities of both AST and CK (Fig. 4, line B) suggest active or recent muscle injury. An increase in only the serum AST activity (Fig. 4, line C) suggests that muscle injury stopped more than 2 days earlier, and that the serum CK activity returned to normal as a result of the short half-life of CK. This latter combination of results also can occur with liver injury (i.e., if liver is the source of the AST, the CK activity would be normal). Figure 4: Serum activities of both AST and CK increase as a result of muscle injury, but rise and fall at different rates. Evaluation of these two enzymes together can help estimate when a muscle injury occurred and indicate whether such injury is still occurring. Source: Thrall, Weiser, Allison & Campbell. Veterinary Hematology and Clinical Chemistry (2nd Ed). Wiley-Blackwell, 2012 Alanine aminotransferase (ALT) As previously mentioned, ALT is primarily used to detect hepatocyte injury, however it is not totally liver specific. The ALT activities in skeletal and cardiac muscles are approximately 5% and 25%, respectively, of the liver ALT activity. Muscle should be considered as a potential source of increased serum ALT activity, however measuring a muscle specific enzyme, like CK, is preferable to detecting muscle damage. Lactate dehydrogenase (LDH) Physiology: Lactate dehydrogenase (LDH) is an enzyme that catalyses the conversion of lactate to pyruvate. It is not tissue-specific, being found in a variety of tissues, including liver, heart and skeletal muscle. Five LDH isoenzymes exist, which can be semi-quantified using agarose electrophoresis. Each isoenzyme is present in a limited number of tissues and, therefore, is more tissue specific than the serum total LDH activity. Increased serum LDH activity:  Artefact: Haemolysis - LDH is high in erythrocytes in dogs, cats and pigs.  Physiological: During exercise LDH activity rise to meet the increased generation of lactic acid.  Pathophysiological: 13  o Liver injury: Hepatocellular injury will result in increased LDH activity in cattle, sheep, horses and small animals. o Muscle disease in cattle, sheep and horses. o Because LDH is so non-specific and isoenzyme measurement is not routinely available, its measurement does not confer any additional information about skeletal muscle or hepatic disease in domestic animals than that provided by enzyme assays routinely used for this purpose (i.e. CK for muscle and SDH/GLDH or ALT for liver in large and small animals, respectively). PROTEINS CLASSIFICATION OF PLASMA PROTEINS Plasma proteins are usually divided into 2 major categories: Albumin (Alb) and Globulins (Glob), including fibrinogen. Other proteins, such as clotting factors, also contribute to plasma proteins. Serum differs from plasma in that it has lost fibrinogen (Fib) and some globulins in the form of the non-enzymatic coagulation factors (VIII, V and I [fibrinogen]) during clotting. Of these only Fib is present in concentrations which are measurable by routine methods. Thus the difference between Total Plasma Protein (TPP) concentration and Total Serum Protein (TSP) concentration is principally attributable to fibrinogen. i.e. TPP - TSP = Fibrinogen. Most plasma proteins are synthesised in the hepatocytes. The major exceptions are the immunoglobulins that are produced by B-lymphocytes and plasma cells. Albumin is one of the smallest of the plasma proteins and the single most abundant, accounting for approximately 75% of the oncotic pressure (colloid osmotic pressure, COP) of plasma within the vasculature, which regulates water from diffusing from the blood into the tissues. Albumin is an important carrier protein for free fatty acids, bile acids, bilirubin, calcium, hormones and drugs. The plasma T½ of albumin varies among species: 8-10 days in the dog, 2-3 weeks in cattle, and approximately 20 days in horses. Globulins are a heterogenous group of proteins that are variable in size, but usually larger than albumin. Globulins present in plasma include immunoglobulins, complement proteins, clotting factors, various enzymes and a variety of carrier proteins. Globulins typically are classified as alpha, beta or gamma on the basis of their electrophoretic mobility. Protein ratio’s A:G ratio The albumin and globulin concentration can be used to calculate the A/G (A:G) ratio, by dividing the Alb concentration by the Glob concentration. In general, the normal ratio in most species approximates 1:1. This ratio, by exaggerating the change in Alb in relation to the change in Glob, may be used as a useful and reasonably sensitive initial screening test for the detection of a number of disease states and suggest further analysis of the protein fractions. Often a particularly abnormal ratio would suggest that one should look at the composition of the major serum globulin fractions by requesting electrophoretic analysis (see below). TPP:Fib ratio TPP: Total plasma protein concentration The ratio of TPP/Fib can prove useful in differentiating hyperfibrinogenaemias of inflammation from dehydration hyperproteinaemia:  Cattle: Ratio >15 = dehydration (normofibrinogenaemia) o Ratio < 10 = inflammation (hyperfibrinogenaemia)  Equines: Ratio > 20 = dehydration (normofibrinogenaemia) o Ratio < 15 = inflammation (hyperfibrinogenaemia) Besides the application (above) of calculating Fib, TPP is not usually determined in the clinical pathology laboratory. Thus most interpretive data is based on the assay of serum proteins. 14 Serum Protein Electrophoresis Because proteins consist of long chains of amino acids, each protein molecule will have at least one amino and one carboxy terminal (any branching will obviously increase this number). Consequently, virtually all proteins have the ability to carry a charge. In a slightly alkaline environment most proteins carry a net negative charge. Therefore, if one places an aliquot of serum in an electric field, on a gel (cellulose acetate and agarose) the protein will tend to migrate toward the anode (positive pole). A mixture of proteins of varying charge would soon separate out into zones of proteins carrying a similar charge. If these proteins differ in mass as well, then the separation will be based on the combined charge and mass-induced inertia. If the surface on which this is taking place is porous, but the pore spaces are fairly small, then proteins with more complicated tertiary and quaternary structure (such as immunoglobulins) would move disproportionately slowly for their charge and mass. Consequently, such a process (called electrophoresis) will cause (under standardised conditions of pH, voltage and surface) a predictable separation of the proteins in a fluid, like serum. The gel is then stained, revealing the various protein bands, which are then scanned by a densitometer to produce an electrophoretogram. The scanning densitometers also calculate the concentration of protein in each fraction after the operator inputs the total protein concentration of that sample. Capillary zone electrophoresis (CZE) is another technique that is now more commonly used. The ultimate product from CZE is similar to agarose gel, however, the resolution of the capillary zone is greater, especially with more negatively charged molecules. Identification of protein electrophoretogram fractions Each band on the gel represents one or more proteins with similar size and charge characteristics. Albumin is the most abundant single protein in serum and forms a single distinct band in the gel. The globulins are further separated into several fractions, including alpha, beta, and gamma globulins (α globulins are right next to albumin with γ globulins being the furthest away from albumin). These are often further subdivided (alpha 1, alpha 2, alpha 3, beta 1, beta 2 etc.) but, for the purposes of this course such further subdivision does not provide much extra clinically relevant information. The alpha- and beta-globulin fractions includes most of the globulins produced by the liver. The lipoproteins and acute phase proteins of inflammation are alpha- and beta-globulins. Some immunoglobulins (IgM, IgA) may extend from the gamma region into the beta region of the electrophoretogram. Most of the immunoglobulins migrate in the gamma-globulin region. Figure 1: A serum protein electrophoretogram from a normal cat. (From: Veterinary laboratory medicine: Clinical Pathology. Duncan, Prasse & Mahaffey, 4th edition, 2003.) Use the following link: http://eclinpath.com/chemistry/proteins/electrophoretic-patterns/ for some examples of electrophoretograms. Note the units used - we use g/L (= g/dL × 10). Diagnostic information from globulin fractions: The contents of the alpha, beta, and gamma bands in serum protein electrophoresis are quite varied, but in most species the most clinically relevant ones can be summarised as follows:  Alpha (α1 and α2): acute phase proteins, e.g., haptoglobin, α1-acide glycoprotein. 15   Beta (β1 and β2): heterogeneous group of proteins which include some acute-phase proteins, lipoproteins, immunoglobulins (e.g., IgM) and other proteins, e.g. transferrin (a β1-globulin). Note that fibrinogen, a β2-globulin, should not be present in an electrophoretogram because the test should be run on serum not plasma.  Gamma (γ): Usually IgG, however IgM and IgA span the late β2-γ region.  The shape of the electrophoretogram tracing and the quantity of protein in the different fractions provides information about the underlying disease, i.e., both qualitative and quantitative interpretation required. Increases Alpha: Current or recent tissue damage Beta: Low grade early liver damage Gamma: Antigenic stimulation (>1-2 weeks) Usually not viral except for feline infectious peritonitis (FIP) in cats Decreases Alpha: Reduced functional hepatic mass (FHM) Beta: Reduced FHM Gamma: Failure of passive transfer of colostral antibodies Humoral immune-incompetence Acute phase proteins Acute phase proteins (APPs) are defined as proteins that change their serum concentration by >25% in response to inflammatory cytokines. Inflammation (caused by infection or other causes) stimulates the synthesis of certain proteins (globulins) by hepatocytes. Several cytokines, especially interleukin-6 (IL- 6), alter protein synthesis in, or protein release from, hepatocytes. Collectively these proteins are called acute phase proteins. Acute phase proteins are biomarkers for inflammation and correlate well with extent and activity of disease. The acute phase response is thought to be an innate host defence mechanism, occurring during the early stages of infection, tissue injury or immunological disorders. In addition to hepatocytes, other cells including Kupffer cells, lymphocyte subsets, blood monocytes and alveolar macrophages have been shown to synthesise these proteins. It is responsible for accumulation and activation of granulocytes and mononuclear cells, which in turn release acute phase cytokines, including interleukin (IL)-1, IL-6 and tumour necrosis factor alpha (TNF-α). During an acute phase response the serum concentration of APPs change in response to these cytokines. Some APPs will decrease (negative APP, e.g., albumin, transferrin and adiponectin), while others will increase in concentration (positive APP, e.g., C-reactive protein). Positive APPs can be divided into 3 main groups:  Major – Normally low serum concentration; increase significantly (>100-fold) during acute inflammation; peak with 24-48 hours; decline rapidly during recovery  Moderate – Only moderate increase (5-10 fold); peak 2-3 days post stimulation; decrease more slowly than major APP  Minor – Only gradual increase by 50-100% Important major and moderate positive APPs in veterinary science. Species Major APP Moderate APP C-reactive protein (CRP); serum Haptoglobin (Hp); α-glycoprotein Dog amyloid A (SAA) (AGP) Cat SAA AGP; Hp Horse* SAA Hp Cow Hp; SAA AGP CRP; Pig-Major acute phase Pig Hp; Ceruloplasmin (CP) protein (MAP) *Although routinely used to evaluate inflammation in horses, fibrinogen is considered a minor APP. 16 Analytical methods Total protein concentration:  Units: g/L (mg/dL x 10)  Refractometry: This method is used for estimating plasma protein (including fibrinogen) in EDTA plasma. The reading is actually a measurement of total solids and is only an estimate of protein concentration.  Biuret method: This is a colorimetric method used on automated chemistry analysers and is the best method. Albumin concentration:  Bromocresol green (BCG) dye-binding method. Serum is the preferred sample for albumin measurement.  Serum protein electrophoresis Total globulin concentration:  This is determined by subtraction. [Globulin] = [total serum protein] – [albumin]. The principal behind this method of quantification is the fact that all serum proteins, except albumin, are considered to be globulins.  Protein electrophoresis Fibrinogen concentration:  Heat-precipitant method  Thrombin time, von Clauss modification  Fibrinogen antigen CLINICAL EVALUATION OF PLASMA PROTEINS A/G ratio The A/G ratio is influenced by changes in Alb as well as Glob. For each change in Alb (increase, normal and decrease) there are three possible changes in Glob (increase, normal and decrease). Consequently one can set up a 3×3 tabulation as follows: ALBUMIN G Increase Normal Decrease L O Increase A/G +-N A/G low A/G v low B U Normal A/G high A/G norm A/G low L I Decrease A/G v high A/G high A/G +-N N Each of these 9 situations (yielding 3 with high, 3 with low and 3 with normal A/G ratios) can arise in a patient, and each is likely to lead to a different interpretation. It is often helpful to consider alterations in albumin and globulin concentrations (as well as total protein concentration) together for interpretation. Causes of decreased protein concentrations Decreased total protein concentrations can result from decreased concentrations of albumin, globulin, or both. Hypoalbuminaemia with hypoglobulinaemia Concurrent hypoalbuminemia and hypoglobulinaemia (i.e., non-selective hypoproteinaemia) can result from overhydration (e.g., excessive fluid therapy, excessive water intake) or from loss of both protein fractions. 17 Loss of both fractions are much more common and occurs in the following disorders:  Blood loss. Hypoproteinaemia occurs when the remaining plasma proteins are diluted by movement of extracellular fluid from the extracellular space to the intracellular space. This water shift takes time to develop and will not be evident for the first few hours following acute haemorrhage. Hypoproteinaemia due to blood loss is generally caused by external (rather than internal) haemorrhage, and may also be caused by bloodsucking parasites (external or internal).  Protein-losing enteropathy (PLE). This may result from a variety of generalised intestinal lesions, including inflammatory bowel disease, lymphangiectasia, infectious diseases, neoplasia, severe prolonged starvation, and gastrointestinal haemorrhage.  Severe skin disease. Generalised exudative skin disease or burns can result in loss of plasma proteins due to increased vascular permeability.  Effusive disease. This results in the accumulation of body cavity fluids with high protein concentration that can result in decreased serum albumin and globulin concentrations. Selective hypoalbuminaemia Decreased albumin concentration that is not accompanied by decreased globulin concentration can result from either decreased production or increased loss of albumin. If the globulin concentration is concurrently increased, total protein concentration may be within the reference interval despite hypoalbuminaemia. Decreased albumin production can occur in the following disorders:  Inflammation: Albumin is a negative acute phase protein and production is decreased during acute inflammatory diseases  Hepatic insufficiency: A marked reduction in functional hepatic mass (50 g/L), for which an underlying cause for inflammation or chronic antigenic stimulation has not been identified. Hyperalbuminaemia The primary cause of hyperalbuminaemia is dehydration. Loss of plasma water results in a relative increase in albumin. The globulin concentration may also be increased in some patients with dehydration. Rarely, induced synthesis by glucocorticoid therapy has been associated with a mild transient hyperalbuminaemia. Hyperalbuminaemia with hyperglobulinaemia Concurrent increases in albumin and globulin concentrations most commonly result from dehydration, which causes loss plasma water and a relative increase in both protein fractions. The A/G ratio will not be altered. Selective hyperglobulinaemia The significance of hyperglobulinemia depends on the magnitude and the type of globulin that is increased, which can be determined by serum protein electrophoresis. No matter what the underlying cause, mild to moderate hypoalbuminemia is often also present. Common disorders and typical electrophoretic patterns are discussed below. Increased alpha/ beta globulin concentrations: During acute inflammation, increased synthesis of acute phase proteins may cause hyperglobulinaemia, which is generally mild. The acute phase proteins are located in the alpha and beta globulin regions of the electrophoretogram. Albumin is a negative acute phase protein, therefore albumin concentrations usually decrease due to decreased hepatic production during acute inflammation. The magnitude of the decrease is usually 1 week), production of immunoglobulins and complement proteins may be increased; acute phase proteins may remain increased as well. The magnitude of the hyperglobulinaemia that occurs with chronic inflammation is variable, but can be marked in some cases (>100 g/L).  Liver disease. Chronic liver disease may lead to increased globulin production. The globulins are frequently immunoglobulins that migrate in the beta or gamma region of an electrophoretogram and can obscure the border between the beta and gamma regions, known as beta–gamma bridging. Note that beta-gamma bridging isn’t pathognomonic for hepatic disease and is frequently seen with infectious diseases as well. Monoclonal gammopathies, however, have narrow-based peaks (i.e., similar in width to the base of the albumin peak, with a slope that is as steep as or steeper than the albumin peak) on the electrophoretogram (Fig. 3), and they result from increased production of a single type of immunoglobulin by a single clone of B lymphocytes or plasma cells. Proliferation of a single clone of lymphocyte results in overproduction of its specific immunoglobulin molecule. Figure 3: An electrophoretogram and corresponding gel from a cat with a monoclonal gammopathy due to multiple myeloma. There is a narrow-based peak in the gamma region; the corresponding monoclonal band is apparent on the gel. Source: Thrall, Weiser, Allison & Campbell. Veterinary Hematology and Clinical Chemistry (2nd Ed). Wiley- Blackwell, 2012 20 Conditions typically associated with monoclonal gammopathies include:  Multiple myeloma. This is a malignant, proliferative disease of plasma cells that involves the bone marrow at multiple sites and, often, other tissues (e.g., spleen, liver). Multiple myeloma typically results from the proliferation of a single clone of plasma cells that produce a homogeneous type of protein that is referred to as paraprotein. Multiple myeloma paraproteins can be composed of entire immunoglobulin molecules or of just heavy or light chains of these molecules. Paraproteins typically are found as a monoclonal peak in the beta or gamma region.  Extramedullary plasmacytoma. Extramedullary plasmacytomas are proliferations of plasma cells originating from a site other than bone.  B cell lymphoma and chronic lymphocytic leukaemia. The immunoglobulin most commonly increased is IgM. Passive transfer failure Failure of transfer of passive immunity (FTPI) is a major cause of neonatal infectious disease and mortality in foals, calves and crias. These species are hypogammaglobulinaemic or agammaglobulinemic at birth and depend on transfer of immunoglobulins from maternal colostrum until they can produce their own immunoglobulins. This takes approximately 10-14 days in the foal and 8-16 days in the calf. Enterocytes of the neonate are capable of absorbing the large immunoglobulin molecules only for the first 24 hours after birth, and many different factors can result in decreased or absent transfer. General guidelines have been established for minimum IgG concentrations that indicate adequate passive transfer; >80 g/L for foals and >100 g/L for calves and crias. In order to assess for FTPI blood is drawn between 18 and 24 hours after birth and IgG can be measured by several methods. For llamas, 48 hours is considered optimal. IgG measurement for failure of passive transfer (FPT) in different species For diagnosis of FPT, determination of IgG is recommended within 24 to 48 hours of birth.  Total protein measurement by refractometry o Calves: A serum total protein concentration of 52 g/L correlates with an IgG concentration of 100 g/L and adequate passive transfer. Note that many sick calves with FPT are also dehydrated, resulting in relative hyperproteinaemia; using higher decision thresholds may be more appropriate for these individuals, e.g., using a serum total protein concentration of 55 g/L (sensitivity = 94% and specificity = 74%). o Crias: A concentration of > 55 g/L indicates adequate passive transfer and < 45 g/L indicates FPT. Concentration between those values can’t be accurately interpreted.  Total protein concentration using the Biuret method o Llamas: 30 umol/L), because central nervous system disease may 29 occur. For the oral ammonia tolerance test, after measuring baseline ammonium, an ammonium chloride solution (20 mg/mL) at a dosage of 100mg/ kg body weight is administrated via a stomach tube. A total dose of 3 g should not be exceeded. A blood sample (EDTA) is collected 30 minutes post-administration and immediately sent to the laboratory. A per rectum administration has been described for cases which are vomiting. For the rectal tolerance test, blood should be collected 20- and 40-minutes post-administration. Because time is critical you should contact the laboratory before engaging on the test protocol so that the laboratory management can ensure that a technologist will be on hand to carry out the assay. Interpretation of hyperammonaemia:  Increased plasma ammonium concentrations most commonly are found in animals with portosystemic shunts (either congenital shunts or acquired shunts secondary to severe hepatic cirrhosis).  Increased plasma ammonium concentrations also can occur with the loss of 60% or more of the hepatic functional mass. Ammonium biurate crystals (see urology notes) may be observed in the urine of animals with hyperammonaemia.  Rare instances of inherited or acquired urea cycle defects that may cause hyperammonaemia.  Increased ammonium production can occur in conditions, not related to the liver, such as post- prandial, urea toxicosis in cattle, strenuous exercise in dogs and horses and intestinal disease in horses. Albumin The liver is the primary site of all albumin synthesis. Hypoalbuminaemia usually is not noted until 60–80% of hepatic function is lost. Albumin has a relatively long serum T½, therefore, normoalbuminaemia is present during acute liver disease. Hypoalbuminaemia is quite common in dogs with chronic liver diseases (>60% have hypoalbuminaemia), but it does not appear to be as common in horses with chronic liver diseases (∼20% have hypoalbuminaemia). It is important to note that hypoalbuminaemia is not a specific indicator of hepatic insufficiency as many non-hepatic factors can influence the blood albumin concentration. Globulins The liver is the site of synthesis for the majority of globulins, with the exception of immunoglobulins synthesised in lymphoid tissue. Hepatic failure can result in decreased synthesis and, therefore, decreased serum concentration of these globulins. But, globulin concentration usually does not decrease as much as the albumin concentration, and so the A/G ratio commonly decreases in hepatic failure. In many cases, globulin concentration may increase with chronic liver disease, either as a result of increased acute phase protein production or immunoglobulin production. In animals with severe liver disease, the clearance of foreign proteins by the Kupffer cells of the liver is theorised to be decreased. Such foreign proteins are thought to be absorbed from the intestine and carried to the liver by the portal circulation. Thus, when Kupffer cells fail to efficiently clear these proteins on their first passage through the liver, they come in contact with the immune system in other parts of the body resulting in an immune response and hyperglobulinaemia. Glucose The liver plays a key role in glucose metabolism. The hepatocytes convert glucose to glycogen, which helps to regulate blood glucose concentration. The hepatocytes also synthesise glucose via gluconeogenesis and release stored glucose via glycogenolysis. Thus, abnormalities in glucose homeostasis may occur with reduced FHM (>70% loss of function). In animals with liver failure glucose levels may be increased (prolonged postprandial hyperglycaemia) due to decreased hepatic glucose uptake, or the glucose levels may be decreased due to reduced hepatocyte gluconeogenesis or glycogenolysis. 30 Urea Urea is synthesised in hepatocytes from ammonia. Consequently, in animals with liver failure the blood ammonia concentration increases and the urea concentration decreases. However, blood urea concentration also may decrease because of numerous other disorders. Cholesterol Bile is a major route of cholesterol excretion from the body. Therefore, interference with bile flow (i.e., cholestasis) can result in hypercholesterolaemia. Note that other non-hepatic disorders can also result in elevated serum cholesterol concentration. The liver is also a major site of cholesterol synthesis. In some forms of hepatic failure, decreased cholesterol synthesis can lead to hypocholesterolaemia. If decreased synthesis of cholesterol is the major alteration in hepatic failure, hypocholesterolaemia can result; if cholestasis is the major alteration, hypercholesterolaemia may occur. However, many animals with liver failure have normal serum cholesterol concentration. Coagulation factors The liver plays a central role in the regulation of coagulation as the sole source of synthesis for the majority of coagulation factors; it also produces anticoagulants such as antithrombin. Furthermore, the blockage of bile flow can result in decreased absorption of vitamin K leading to decreased function of the vitamin K–dependent coagulation factors (factors II, VII, IX, and X). Therefore, defects in both haemostasis and fibrinolysis may occur in animals with liver disease. 31 EXOCRINE PANCREAS AND INTESTINE Classification of malassimilation MALASSIMILATION MALDIGESTION MALABSORPTION Exocrine Pancreatic Insufficiency (EPI) Chemical  Juvenile  Toxins  Post inflammatory  Irritants Lactase deficiency Osmotic Sucrase deficiency Decreased absorption surface area Bile salts low Cell infiltration  Lymphocytic  Plasmacytic  Eosinophilic  Granulomatous Impaired drainage  Lymphatic  Venous Macroscopic faecal examination It is important to only conduct further faecal tests and/or intestinal function tests in a patient where the indications (historical, clinical and macroscopic faecal appearance) strongly suggest that such a further examination is indeed indicated. Some of the most important indications for further testing are obtained by simply doing a good macroscopic examination of the faeces. Finding Condition Colour Red Large intestine bleeding (haematochezia) Black Upper GIT bleeding (melaena) Yellow Haemolysis Green Abnormal GIT flora Acholic Bile duct obstruction Consistency Watery Large bowel diarrhoea Doughy Reduced absorptive surface area Osmotic/Absorption inhibition Steatorrhoea (see definition below) Hard Constipation Mucoid Large bowel (esp) lower colon and rectal irritation (Protozoa?) Odour Acidic High carbohydrate feeding or low digestion (EPI) Rancid High fat feeding or low digestion or absorption (malassimilation) Foreign Hair Examine coat again. Possibility of finding trichobezoars material Bones Pica/Overfeeding with bones Sand Pica or poor feeding facilities 32 Steatorrhoea Steatorrhoea means "fatty diarrhoea”. Steatorrhoea is typical:  Large volume (low frequency)  Pale (not acholic) or greyish.  Rancid and or acidic.  Fatty or greasy Microscopic faecal examination Adequate amounts of faeces are required for various tests:  1–2 g faeces is required for faecal floatation or sedimentation,  2–3 g faeces are necessary for faecal culture,  10 and up to 10 g of faeces should be used for the Baermann technique Faecal examination should preferably be performed on fresh faeces, but if there is a delay for more than 2 hours after collection, the faeces should be refrigerated at 4°C. Organisms such as Giardia and trichomonads are fragile and undergo rapid deterioration with time, refrigeration, or processing, and faecal samples that are more than 5 minutes old are inadequate for the detection of these organisms. Microscopic (unstained – wet preparation) Mix small amount of faeces with a few drops of physiological saline. A small sample should be mixed with a drop of warm saline on a glass slide with a wooden applicator stick, which is then covered with a cover glass. The smear preparation can then be examined on low power (×10) for eggs, cysts, and larvae. Other organisms may be found using higher magnification (×40). Motile organisms (protozoa, e.g., Giardia and bacteria e.g., spirochaete) seen on wet mounts can also be assessed on dry mount, stained slides, but it is important not to make dry smears too thick. Microscopic (stained – dry smear) Thin faecal smears may be stained with routine haematology stains (e.g., Wright-Giemsa or Diff-Quik stain. These smears may reveal:  Epithelial cells (as small number is normal)  Protozoa (Giardia + Pentatrichomonas) in mucus  Bacterial spectrum (should be mixed with no one type overwhelmingly predominant)  Leukocytes (neutrophils with inflammation, eosinophils with endoparasitism, large atypical lymphocytes in lymphoma)  Erythrocytes (often partially lysed)  Fungal spores or hyphae. Faecal occult blood test The test for occult blood is a simple test, which is available for in-practice use. The test detects the pseudoperoxidase activity of faecal hemoglobin and picks up minute amounts of faecal blood at concentrations as low as 20× to 50× times less than those where blood is visible grossly. A loss of 30– 50% of the blood volume into the gastrointestinal tract can occur without gross blood being visible in the faeces. The test procedure involves application of the faeces to the test paper, and when blood is present, the peroxidase activity results in the formation of a blue colour. Indications: Animals with unexplained acute or chronic diarrhoea, those with loose stools, or in cases of microcytic anaemias, where the cause of chronic blood loss is not apparent. It can also be used to monitor animals that are at risk of developing gastrointestinal haemorrhage due of treatment with ulcerogenic drugs (e.g., NSAIDs) or those with a history of gastrointestinal neoplasia. Restrictions: The faecal occult blood test is extremely sensitive, and thus false positive results may be seen with meat or fish diets which contain myoglobin and haemoglobin, and some vegetable diets including plants such as brassicas. It is important to observe strict dietary restriction for at least 3–5 days prior to performing the occult blood test as this decreases the number of false-positive results. Recommended feed 33 restrictions include meat-free, low-peroxidase diets (e.g., rice or pasta with cottage cheese or egg as a protein source). Test interpretation: A positive test result is one where there is a quick and intense blue colour change of the film. Positive results on the faecal occult blood test in the absence of grossly visible blood in the faeces suggests the possibility of upper or lower gastrointestinal tract inflammation, ulceration, or neoplasia. At least three tests for faecal occult blood should be carried out to make a definitive diagnosis. To note: The faecal occult blood test in ruminants has been reported to have a sensitivity of 77% and a specificity of 97% for abomasal ulceration. Tests of protease activity/function Serum trypsin-like immunoreactivity (TLI) concentration Also refer to the earlier trypsin-like immunoreactivity (TLI) section in “Enzymology”. Physiology: Most trypsinogen is secreted in enzyme-rich pancreatic secretions into the intestine, where it is converted to trypsin, a potent digestive protease. In health, a small amount of trypsinogen escapes the pancreas and enters the blood, in which it can be measured as TLI. Also, small amounts of trypsin may be formed in the pancreas, this trypsin may enter the blood, bind to anti-proteases, and contribute to the TLI concentration. Plasma trypsinogen and trypsin are degraded in the kidneys and by the mononuclear phagocyte system. Assay: Immunological assays that detects trypsinogen, trypsin and trypsin bound to protease inhibitors. In healthy animals, nearly all TLI is trypsinogen. Canine, feline and equine TLI concentration are measured by species-specific immunoassays (only the canine assay is available in RSA). A starved (12 hours) serum sample is preferred. Interpretation: 1. Increased TLI concentration  Acinar cell damage caused by pancreatitis – peak TLI concentration occurs 1-2 days before peak activities for amylase and lipase. Therefore, the diagnostic sensitivity for acute pancreatitis is less than for lipase.  Increased release from pancreatic acinar cells, stimulated by food intake or cholecystokinin and secretin administration.  Decreased renal clearance in disorders that cause decreased glomerular filtration rate (GFR); pre-renal, renal or post-renal disorders. 2. Decreased TLI concentration  Decreased release from pancreatic acinar cells o Chronic pancreatitis that leads to destruction of most acinar cells o Pancreatic acinar atrophy in dogs o Maldigestion due to exocrine pancreatic insufficiency (EPI)  The diagnostic sensitivity and specificity and accuracy of a fasting serum TLI concentration are very high - approaching 100% Cobalamin (Vit B12) concentration Physiology : Cobalamin enters the stomach via ingested foods. In the acidic environment, it binds with R protein (cobalophilin or haptocorrin) that is produced by the gastric mucosa. Cobalamin enters the intestine bound to R protein (R), but when it enters the alkaline environment, it detaches from R protein and binds to intrinsic factor that is secreted by the pancreatic cells (dogs and cats) and the gastric mucosa (dogs). Enteric bacteria use some of the cobalamin as it moves through the small intestine. When it reaches the ileum, the cobalamin/intrinsic factor complex binds to specific mucosal receptors involving cubam and megalin, and enters enterocytes. When cobalamin enters the portal blood, it binds to transcobalamin 2 34 (Trans), a transport protein. From the blood, cobalamin may be used in tissues, stored in the liver, or excreted in bile. Sample: Serum is the preferred sample. Falsely low concentrations may occur when cobalamin is degraded by excess exposure to light. Interpretation: 1. Increased serum cobalamin concentration  Increased serum cobalamin concentration is uncommon  Cobalamin supplementation (oral or parental)  Release from damaged hepatocytes. Hepatocytes are a storage site for cobalamin 2. Decreased serum cobalamin concentration  Cobalt deficiency in cattle  Pre-absorptive defect in dogs and cats  EPI: pancreatic atrophy, chronic pancreatitis  Intestinal bacterial overgrowth  Defective absorption of cobalamin in the ileum of dogs and cats  Ileal disease: inflammation, resection, villous atrophy  Congenital deficiency of receptor in giant schnauzers and Border collies Tests of carbohydrate assimilation Glucose absorption test in horses Physiology: The glucose absorption test is used primarily in horses that are presented because of chronic weight loss. Glucose is a simple sugar that is absorbed in the duodenum and proximal jejunum, from which it enters the portal blood. A large percentage is removed from the portal blood by the hepatocytes, and the remainder enters the peripheral blood. From the peripheral blood, glucose may enter metabolic pathways of cells or be excreted by kidneys if the renal resorptive maximum for glucose is exceeded. In horses, microbes of the large intestine digest carbohydrates and produce volatile fatty acids that are absorbed by the mucosa. Therefore, disorders of the large intestine in horses can result in clinical conditions where ingested carbohydrates are malassimilated. The glucose absorption test can be used to assess the intestinal absorption of monosaccharides in monogastric mammals; however, the same is not true for ruminants. Glucose is administered via a stomach tube, and then multiple blood samples are collected to assess the intestinal ability to absorb it. Test procedure:  The patient is starved overnight prior to the procedure  A blood sample is collected for a baseline glucose concentration  Glucose (1 g/kg body weight as a 20% solution, weight/volume) is administered via stomach tube  Blood samples are collected at 30, 60, 90, 120 and 180 minutes after glucose is administered Interpretation:  Evidence of adequate intestinal absorption is when the glucose concentration at 120 minutes is approximately twice the concentration in the baseline value.  Diseases and conditions that alter oral glucose absorption curves: o Failure to meet adequate absorption  Malabsorption causes by small intestinal mucosal disease (inflammation, neoplasia, villous atrophy)  Incomplete delivery of glucose to the intestine (delayed gastric emptying, prolonged intestinal transit time)  Rapid transit time associated with diarrhoea  Increased utilisation of glucose by hepatocytes or peripheral tissues o Peak concentrations unexpectedly high or prolonged  Diabetes mellitus 35 ELECTROLYTES AND KETONE BODIES Electrolytes - basic concepts For further in-depth reading/ your own interest, please see “Electrolyte Disorders”, AM Manning, Veterinary Clinics: Small Animal Practice, Volume 3, Issue 6, 1289 - 1321 Please also ensure that you go through the lectures on ClickUP Electrolytes include: Na+, Cl- , K+, Ca2+, PO4 and Mg2+, For the purposes of clinical pathology, electrolyte concentrations are measured in whole blood, plasma or serum, but electrolytes are present in all extra- and intracellular body fluids. Serum or plasma concentrations of electrolytes are the net result of one or more of the following four processes:  decreased or increased intake  shifts (either in vivo or in vitro) between ICF and ECF  increased renal retention  increased loss via the kidneys, alimentary tract, skin or airways (HCO3 indirectly) In health ECF is relatively rich in Na+ and Cl- but poor in K+, Ca2+, PO4 and Mg2+, while ICF is relatively rich in K+, Ca2+, PO4 and Mg2+ but poor in Na+ and Cl- (note species/breed differences in erythrocytes – high K+ in RBC of horses, pigs and cattle and Akitas and other Japanese dog breeds). The serum electrolyte concentration may not accurately reflect the balance of that particular electrolyte in the body. This is particularly true of intracellular electrolytes. Electrolytes and water enter plasma from the alimentary tract and cells and by injections or infusions. Na+, K+ and Cl- typically enter the body via oral intake of food or fluids. In vitro lysis (i.e in the sample tube) of blood cells and platelet activation may add K+ to serum/plasma. Electrolytes and water leave plasma/ECF and the body or are regulated mainly via the kidneys and the alimentary tract, but as mentioned, also the respiratory tract and the skin. Electrolytes may enter extravascular sites such as the peritoneal and pleural cavities. Water and electrolytes may also be absorbed from third space fluids if not removed by centesis. Drinking water after the loss of ECF will dilute the remaining ECF. Pathological states that alter electrolyte concentrations can create acid base abnormalities. Sodium Sodium homeostasis Most sodium is found extracellularly. Normal extracellular fluid volume is maintained by decreasing or increasing renal sodium excretion. ECF volume expansion increases sodium excretion and ECF volume depletion decreases sodium excretion. Most disorders of sodium concentration are a result of abnormal water handling and not a decreased or increased number of sodium molecules (i.e. changes in sodium are usually relative and not absolute). Plasma sodium concentration is the major determinant of plasma osmolality, with potassium, glucose and urea. Antidiuretic hormone (ADH) and thirst are the two major physiologic mechanisms for the control of plasma osmolality. Hyperosmolality (i.e. increased Na concentration) and decreased effective circulating volume will stimulate the release of ADH and the sensation of thirst, thereby initiating water reabsorption from the glomerular filtrate, and stimulating free water intake. This will result in a decrease in osmolality and an increase in circulating volume. The renin-angiotensin-aldosterone system fine-tunes effective circulating volume, while ADH maintains plasma osmolality. The maintenance of 36 circulating volume is prioritised over normal plasma osmolality, so that patients with poor circulating volume will be thirsty and have ADH released, regardless of osmolality. This can lead to hypo-osmolality (and therefore hyponatraemia) in patients with poor circulating volume. An example of this is the hyponatraemia seen in patients with chronic congestive heart failure (low circulating volume due to decreased cardiac output but osmolality normal – thirst/ ADH – drink water – increase in volume but decrease in osmolality to become hypoosmolar). Plasma sodium concentration is different to and independent of total body sodium content. It reflects the amount of sodium relative to the volume of water in the body and it is not a reflection of total body sodium. If patients have increased total body sodium, more fluid is held in the interstitial space and the animal looks overhydrated, independent of plasma sodium levels. If patients have decreased total body sodium, less fluid is held in the interstitial space and the animal looks dehydrated, independent of plasma sodium levels. Plasma sodium levels should be evaluated together with the hydration status of the patient. Hypernatraemia Common causes for hypernatraemia (which is due to body water imbalance with decreased water intake or increased loss of water compared to electrolytes) in veterinary practice include water that is not available to the patient, panting, fever, gastrointestinal causes (vomiting, diarrhoea, small intestinal obstruction) and renal causes (osmotic diuresis such as with mannitol infusion, hyperglycaemia, non-osmotic diuresis such as with furosemide administration, chronic renal failure, nonoliguric renal failure, postobstructive diuresis). Hyponatraemia Common causes for hyponatraemia (which is due to increases in body water or increased loss of sodium compared to water) in veterinary practice include  hyperglycaemia (high plasma glucose – increased plasma osmolality – shifting of water from ICF to plasma to compensate – sodium diluted)  severe liver disease causing ascites (ascites – loss of fluid from circulation – hypovolaemia – RAAS activation – retention of H2O)  congestive heart failure (decreased cardiac output – activation of RAAS)  hypotonic fluid infusion, vomiting and diarrhoea (isotonic loss of ECF – hypovolaemia – animal drinks (pure) H2O – plasma sodium diluted) 37   hypoadrenocorticism (no aldosterone – no reabsorption of Na in distal renal tubule)  diuretic (thiazide or loop diuretics) administration (with low plasma osmolality and hypovolaemia). Clinically significant hyponatraemia does not commonly occur in critically ill small animals. Normonatraemia Normonatraemia does not necessarily mean that the sodium balance is normal. Causes of normonatraemia in dehydrated or oedematous animals include vomiting, diarrhoea, renal disease, diuretic therapy, osmotic diuresis, sweating in horses, congestive heart failure, hepatic cirrhosis, nephrotic syndrome. Potassium Potassium homeostasis Potassium is the most abundant intracellular cation, with 95% or more of total body potassium in the intracellular fluid (ICF) compartment, and 60-75% of this in skeletal muscle cells. The remaining 5% of the body’s potassium is in the extracellular fluid (ECF) compartment. Normal serum potassium concentration is required for normal neuromuscular function. The body maintains potassium balance by matching the quantity excreted with the quantity ingested via the gastrointestinal tract. Over the short term the potassium concentration is maintained by shifting potassium into liver and muscle cells under the influence of insulin and β2-adrenergic catecholamines. The kidneys regulate long term balance of potassium. Aldosterone is the most important hormone affecting renal potassium excretion. Hyperkalaemia and ECF volume contraction are the chief stimuli for the secretion of aldosterone. Hypokalaemia Disturbances in potassium are very frequently encountered and are immediately life- threatening. Veterinarians are sensitised to the detrimental effects of hyperkalaemia, but the 38 adverse effects of hypokalaemia are nearly as harmful. Cardiac arrhythmias will develop with severe hypokalaemia, as with hyperkalaemia. Hypokalaemia occurs when the serum potassium concentration is less than 3.6 mmol/L (reference interval 3.6-5.1 mmol/L). Hypokalaemia arises from decreased intake, translocation of potassium from ECF to ICF and excessive loss via either the gastrointestinal tract or the kidneys. Decreased intake alone is unlikely to cause hypokalaemia, but it might be a contributing factor. Translocation of potassium into cells may occur with alkalaemia and insulin and catecholamine release. Most patients with hypokalaemia have increased potassium losses. Conditions that are important and commonly cause hypokalaemia include vomiting of gastric contents, small intestinal diarrhoea, chronic renal failure, hypokalaemic renal failure and post- obstructive diuresis in cats, inappropriate fluid therapy, diuresis due to diabetes mellitus and treatment with loop diuretics or glucose-containing fluids. Most dogs with chronic renal failure have normal serum potassium levels. Hyperkalaemia Hyperkalaemia occurs when the serum potassium concentration is more than 5.1 mmol/L (reference interval 3.6-5.1 mmol/L). Serum concentrations of >7.5 mmol/L are life-threatening. Hyperkalaemia arises from increased intake or administration, translocation of potassium from ICF to ECF and decreased renal excretion or from pseudohyperkalaemia. Conditions that are important and commonly cause hyperkalaemia include urethral obstruction, anuric or oliguric renal failure and selected gastrointestinal diseases (trichuriasis, salmonella, perforated duodenal ulcer). Less common but important conditions include ruptured bladder and hypoadrenocorticism. Also consider body cavity effusions and chylothorax with repeated pleural drainage (rare), metabolic acidosis, severe tissue injury and in vitro artefacts causing pseudohyperkalaemia, such as severe thrombocytosis and haemolysis, especially in species/breeds with high K in RBCs and neonates, contamination with EDTA, severe leukocytosis (>100x109/l, rare). 39 Sodium to potassium ratio (Na+:K+ ratio) Calculating this ratio might make it easier to detect electrolyte abnormalities. Sodium and potassium might change concurrently in the same animal. The ratio is obtained by dividing serum or plasma Na+ with serum or plasma K+.  The normal ratio is 40:1 to 27:1 A lowered Na+:K+ ratio can be caused by hypoadrenocorticism (Addison's disease) – but a low ratio is not unique to this disease. A low percentage of dogs with primary hypoadrenocorticism have normal Na:K ratios and dogs with secondary hypoadrenocorticism (due to low ACTH) have normal Na+:K+ ratios. A lowered ratio is also seen with diarrhoea in cats, dogs and horses, and in cats and dogs with acute renal failure, urinary tract obstruction, diabetes mellitus with ketonuria, pleural and peritoneal effusions and many other conditions. Chloride Chloride homeostasis Chloride is the major anion in the ECF, constituting about two thirds of the anions in the ECF. It is an important anion in the renal regulation of acid base balance. If evaluating an abnormal chloride serum level, sodium levels and acid base must also be determined. Hypochloraemia This usually occurs when there is hyponatraemia, a loss of chloride causing a metabolic alkalosis or certain types of metabolic acidosis. Conditions that are important and commonly cause hypochloraemia include conditions causing loss of sodium, loss or sequestration of HCl (due to vomiting of stomach contents, displaced abomasum, pyloric obstruction, bovine haemorrhagic bowel syndrome), bovine renal failure, ketoacidosis, lactic acidosis, and renal loss due to thiazide or loop diuretic therapy. Hyperchloraemia This usually occurs when there is hypernatremia or a decreased serum HCO3, as with hyperchloraemic metabolic acidosis. Conditions that are important and commonly cause hyperchloraemia include conditions causing hypernatraemia (e.g. water deprivation, panting, 40 hyperventilation, fever, osmotic diuresis) and excess gain of chloride through administration of fluids and other therapies containing chloride. A loss of HCO3 through the GIT or kidney causes a hyperchloraemic metabolic acidosis. Calcium Calcium homeostasis Calcium exists in three forms in serum: around 50% as ionised (iCa2+, free), 5-10% complexed (cCa2+) or chelated (bound to phosphate, bicarbonate, sulphate, citrate, lactate) and 40-45%% protein bound, the majority to albumin. iCa2+ is the biologically active form and is tightly regulated hormonally. PTH is responsible for minute-to-minute control of serum iCa2+, while calcitriol (Vit D/1,25-dihydroxycholecalciferol) is responsible for the day to day control. Calcitriol increases serum calcium and phosphate levels by increased absorption from the GIT. 41 If there is mild hypocalcaemia, a marked increase in PTH secretion occurs. This stimulates renal calcium reabsorption, the excretion of phosphorus and increased resorption from bone. PTH therefore increases serum calcium levels and decreases serum phosphate levels. After several hours, increased PTH secretion stimulates the secretion of calcitriol, which causes increased absorption of calcium and phosphorus from the intestines, at the level of the enterocyte. It promotes bone formation and has an effect on osteoclast differentiation. It also promotes calcium and phosphorus reabsorption in the kidneys. Calcitonin is produced in the thyroid gland, in response to hypercalcaemia and after a ca