Lizard and Snake Biochemistry

Biochemical analysis of blood is a useful diagnostic tool. It is often necessary to prioritise the components of a profile because of the small volume of blood submitted. Consider the following tests.

Total protein

Plasma proteins include albumins (prealbumin and albumin) and globulins (alpha, beta and gamma fractions). Albumin is the principal factor in maintaining the oncotic pressure of blood. Antibodies migrate primarily in the gamma fraction. Protein electrophoresis separates the fractions. In normal reptiles, TP values generally vary between 30–80 g/l. Hypoproteinaemia and hyperproteinaemia are usually indicators of disease. Measurement of TP is an important diagnostic test but perhaps more useful is TP in association with protein electrophoresis. Ranges for protein electrophoresis are becoming available for bird and non-domestic mammal species.

Hypoproteinaemia

Hypoproteinaemia is often associated with malnutrition. Other causes include blood loss, gastrointestinal problems, chronic hepatopathies or chronic renal disease. Physiological increases, primarily a hyperglobulinaemia, may occur in healthy female reptiles in active folliculogenesis due to the presence of the yolk precursor, vitellin, in the blood. Following ovulation the protein level returns to normal. Pathological increases in TP are usually associated with either dehydration or hyperglobulinaemia related to inflammatory, primarily infectious, disease. Alpha globulins may increase with tissue necrosis and decrease with severe hepatic disease, malnutrition and malabsorption. Beta globulins may increase with kidney disease.

Protein electrophoresis

Protein electrophoresis only requires a very small amount of plasma and may give very useful diagnostic data. Presently specific information in reptiles, let alone snakes, is limited. Extrapolation from results in mammals and birds may be of dubious value but early evaluation shows it to be comparable.

Aspartate aminotranferase

Some species may have higher concentrations of Aspartate aminotransferase (AST) in liver, skeletal muscle and myocardium. AST values are usually less than 250 iu/l. Increased levels are suggestive of tissue damage, specifically liver or muscle in some species. In association with CP to rule out muscle damage it is a useful test in some species for hepatocellular damage. Generalised diseases, such as trauma, septicaemias and toxaemias, may also elevate AST due to tissues necrosis.

Creatine kinase

Creatine kinase (CK, creatine phosphokinase, CPK) is derived predominantly from skeletal muscle and heart. Normal CK values in association with non-specific tests for hepatocellular damage (AST, LDH) may indicate liver disease. Plasma CK values increase with muscle damage. CK elevations may be due to rough handling at the time of venipuncture and following the intramuscular injection of some chemotherapeutic agents, especially enrofloxacin.

Uric acid

The primary catabolic end product of protein, non-protein nitrogen and purines depends upon a reptile’s natural environment. Terrestrial reptiles excrete uric acid as the primary nitrogenous waste product (i.e. they are uricotelic). Uric acid is synthesised in the liver and excreted by renal tubular secretion. The blood level is therefore largely independent of urine flow rate (and is therefore not a sensitive indicator of dehydration in reptiles or birds). Both animal and environmental factors influence uric acid levels. The normal blood uric acid value for most reptiles is 0 to 600 µmol/l.

Hyperuricaemia

Plasma uric acid increases with renal disease but is neither a sensitive indicator of renal dysfunction, nor a specific indicator since physiological increases are common. Physiological high levels are seen in several species during hibernation, probably due to decreased tubular function at low temperatures. Healthy snakes should be resampled after a fast if a high uric acid level is seen. Increased uric acid levels are seen with gout and renal failure. The loss of about two thirds of the renal functional mass is necessary before uric acid increases and therefore rises late in the course of renal failure. Renal failure has been associated with nephrocalcinosis (associated with high dietary levels of calcium or hypervitaminosis D), visceral gout (caused by dehydration, renal failure or toxicosis) and nephrotoxic drugs (aminoglycosides can result in significant renal tubular necrosis).

Calcium

The majority of the body’s calcium is stored in bone. Blood calcium levels are kept within a tight range by the actions of parathyroid hormone, calcitonin and vitamin D. Blood levels vary between species but generally range between 2–5 mmol/l.

Hypercalcaemia

Hypocalcaemia occurs with imbalances of calcium, phosphorus and vitamin D but by comparison to other reptiles this is relatively rare in snakes. Low calcium may be seen with renal failure. Female reptiles may have increased calcium levels of two to fourfold during times of reproductive activity. Mobilisation from bone results from increased oestrogen activity and calcium levels return to normal after egg laying. Persistently high calcium (and phosphorus) may be normal in indigo snakes. Iatrogenic hypercalcaemia has been reported in captive reptiles and results from excessive dietary or parenteral calcium and vitamin D. Primary hyperparathyroidism, pseudohyperparathyroidism and osteolytic bone lesions could also cause hypercalcaemia but are unlikely to be encountered. Hypercalcaemia may lead to nephrocalcinosis and renal failure.

• Calcium:Phosphorus ratio <1 - The calcium to phosphorus ratio may reverse with kidney disease. This may be the first indication of renal failure.

Phosphorus

The metabolism of phosphorus and its blood levels are closely linked to those of calcium. Most reptiles have a range between 1.0–3.0 mmol/l. Young, growing reptiles may have higher blood phosphorus levels than adults.

Hypophosphataemia

phosphorus may be the only biochemical parameter increased in renal failure. Hyperphosphataemia occurs from excessive dietary phosphorus, excessive vitamin D3 and renal disease. It may occur with folliculogenesis. The calcium-phosphorus indices (calcium to phosphorus ratio and the calcium phosphorus solubility index) are useful in the diagnosis of renal failure. Hypophosphataemia may occur due to dietary lack of phosphorus.

Additional tests

Amylase

Amylase production and secretion is not restricted to the pancreas and may originate from several areas in the gastrointestinal tract of many reptiles. Amylase values vary between reptile species and individuals. Increased levels may be useful as a diagnostic indicator in some species but its value in snakes has to be shown.

Bile acid

Bile acid metabolism is presently poorly understood in reptiles. Bile acids have been poorly studied and are currently poor diagnostic indicators but further use and research may indicate their usefulness. Normal upper limits of bile acids levels in reptiles vary between 10 and 70 µmol/l. Bile acid levels in reptiles may be indicators of reduced hepatic function.

Electrolytes

Sodium, potassium and chloride levels are presently evaluated in a similar manner to mammals. There is a wide variation in normal levels among and within species. High potassium levels generally carry a poor prognosis.

• Sodium - Sodium of normal reptiles generally varies between 120 and 170 mmol/l and varies between species. Hypernatraemia will occur with dehydration (inadequate uptake or excessive loss of fluid). Hyponatraemia will occur with gastrointestinal loss (diarrhoea).
• Potassium - Potassium is generally in the range of 2 to 8 mmol/l. Hypokalaemia in reptiles will occur from inadequate intake or excessive loss (diarrhoea). In mammals hyperkalaemia may occur with excessive potassium intake, decreased secretion or shift from intracellular to extracellular fluid (e.g. severe acidosis).
• Chloride - Chloride varies in the range of 100 to 150mmol/l. Hypercholoraemia is associated with dehydration and possibly renal failure.

Glucose

Both animal and environmental factors affect levels. Animal factors include species and nutritional status, and environmental factors include ambient temperature and season. Response to environmental cues tends to be species-specific. Glucose values are generally between 3–16 mmol/l. Glucose values in reptiles are presently considered of limited value because any changes tend to be non-specific and not sensitive. For instance, hypoglycaemia has been associated with starvation, malnutrition, high protein diets, severe liver disease, endocrinopathies and septicaemia. Hypoglycaemia has also been reported to cause tremors, loss of righting reflex, torpor and non-responsive pupils in some reptiles. Other problems, such as hypocalcaemia, are far more common though. Hyperglycaemia may occur with iatrogenic glucocorticoid use or excess delivery of glucose. Diabetes mellitus should also be considered.

Lactate dehydrogenase

Lactate dehydrogenase (LDH) has a wide distribution in reptile tissues and elevations suggest tissue damage. In some species elevations over 1000 iu/l are considered significant. An increased value for LDH indicates tissue damage.

Not commonly used

• Alanine aminotransferase - Elevated blood values of alanine aminotransferase (ALT), formerly glutamic-pyruvic transaminase, GPT or SGPT, are not specific for one tissue and are not a reliable indicator of liver or muscle damage. ALT is not usually part of routine biochemistry.
• Alkaline phosphatase - Alkaline phosphatase (AP, ALP) is widely distributed in tissue and generally at low levels. Low tissue activity and lack of tissue specificity limit the use of AP as a diagnostic indicator in reptiles.
• Bilirubin - Assessment of bilirubin is not a useful diagnostic test since the major bile pigment of reptiles is biliverdin. Bilirubin cannot be detected or occurs at low concentrations. Plasma elevations of biliverdin may be useful in the diagnosis of liver-associated disease.
• Creatinine - Normal plasma creatinine values for reptiles are very low and are a poor diagnostic indicator in reptiles.
• Gamma glutamyl transferase (GGT) - Gamma glutamyl transferase (GGT) activity in plasma and tissue is low or undetectable and GGT appears to be of little diagnostic use.
• Urea - Since reptiles are primarily uricotelic, blood urea of most reptiles is low. It is generally considered a poor diagnostic indicator for renal disease in snakes.
• Cholesterol - Cholesterol is a useful diagnostic parameter in mammalian biochemistry and is used in human medicine as an indicator for heart disease. Although vascular disease has been observed in reptiles, the relationship between cholesterol levels and disease has not been established in reptiles.
• Triglycerides - Most of the lipid in the blood of reptiles is in the form of triglycerides (but carotenoid pigments may be present in significant levels in many squamates). Levels vary significantly between species and change slowly with starvation and other environmental stressors. Triglyceride is not presently a useful diagnostic parameter in reptiles because there is little information relating level to disease states.