Measurement of certain plasma and serum enzymes in the horse

The study of the use of serum or plasma enzymes in the horse is reviewed.

Analyses of such enzymes in the horse have been performed mainly for the detection and monitoring of abnormalities which affect liver tissue and muscle fibres. Examples of muscle disorders are post-anaesthetic forelimb lameness and the clinical myopathy syndrome which varies from a mild “tying-up” to a severe paralytic myoglobinuria (EPM). Measurements have also been made during training periods in an endeavour to assess the effects of exercise on muscle tissue. One of the benefits of such a study could be the prediction of the period of optimum performance.

The two enzymes most frequently used for. muscle studies have been glutamic oxaloacetic transaminase (GOT) (aspartate aminotransferase) and creatine Phosphokinase (CPK). The former, while not muscle specific and less sensitive than CPK to small amounts of muscle tissue damage, has a slower clearance rate from blood than CPK. Determination of both enzymes together has some advantages in determining the prognosis in cases with severe muscle damage. Later studies have included aldolase, which is a sensitive skeletal muscle specific enzyme and lactic dehydrogenase (LDH), which is widely distributed by various tissues at high concentrations. Most of the work on serum LDH has centred on its separation into 5 isoenzymes, and on relating the isoenzyme pattern to the various tissues. More studies on this, and on the isoenzymes of CPK, are required.

Apart from cost, one of the major problems in enzymology has been lack of a standardisation of laboratory methods which has led to a wide range of published normal values. Reliance on such values may lead to difficulties in interpretation, particularly when serum changes are small.     

A Comparison of Serum Creatine Phosphokinase and Serum Glutamic Oxalacetic Transaminase in Skeletal Muscle Necrosis

Rabbits were poisoned by the intraperitoneal injection of plasmocid dihydroiodide at a dosage of 80 mg. per kg. body weight. Animals were euthanatized 1, 2, 5, 7, 9, 12, 24 and 48 hours after plasmocid injection and serum and diaphragmatic muscle collected. The serum was examined for its serum glutamic oxalacetic transaminase (SGOT) and creatine phosphokinase (CPK) concentrations. The diaphragm was sectioned and examined for the progression of morphologic alterations. It was found that SGOT and CPK concentrations were similar in their time and duration of elevation and that the serum CPK elevations closely paralleled the occurrence and degree of morphologic muscle degeneration. It appears that CPK is the most specific clinical biochemical test for the detection of muscle necrosis available at this time.     

Effect of nickel deficiency on biochemical variables in serum, liver, heart and kidneys of goats.

Nickel deficiency was induced in 2- to 4-year-old goats by feeding 0.1 mg Ni/kg dry matter with a semisynthetic diet. The control group consumed 5.0 mg Ni/kg d.m. Activity of several enzymes (SDH, LDH, HBDH, AST, ALT, ALD, CK, CHE) was determined in the serum, liver, heart and kidneys. Serum urea-N level was also measured and transmission electron microscopic (TEM) examinations were performed. Signs characteristic of nickel deficiency (retarded growth, increased mortality of dam and offspring, parakeratosis of the skin) appeared in the low-nickel group. The activity of SDH and ALD, as well as the level of urea-N was significantly lower in the serum of Ni-deficient animals than in the control. Ni-deficient animals also had significantly lower enzyme activities in the heart (SDH, HBDH, AST, ALT, ALD and CK), liver (SDH and CHE) and kidneys (HBDH and CK). Electron micrographs showed degeneration of cardiac and skeletal muscle in the Ni-deficient animals. Ni deficiency elicited changes primarily in the heart and these resulted in depressed activity of several enzymes.     

Enzyme histochemistry on muscle biopsies as an aid in the diagnosis of diseases of the equine neuromuscular system: a study of six cases

Muscle biopsies from six horses with clinical histories of muscle atrophy, muscle tremors, myopathic symptoms, unsteadiness of pelvic limbs and progressive ataxia were examined. Muscle biopsies were studied with enzyme histochemical techniques to evaluate the diagnostic values of these methods in cases suspected of suffering from neuromuscular disorders. Hypertrophy, atrophy, fibre splitting, waxy degeneration, phagocytosis and necrosis were seen in haematoxylin eosin stained sections of the different cases. Fibre type predominance and fibre type grouping were seen in the calcium ion stimulated myosine ATP-ase (Ca-ATP-ase) stained sections of some cases. 'Moth-eaten fibres' were demonstrated in three cases by staining with NADH: nitro blue tetrazolium oxidoreductase (NADH-TR), succinate dehydrogenase (SDH), NADH dependent malate dehydrogenase, cytochrome c oxidase and by lactate dehydrogenase. The catabolic enzymes, acid phosphatase (ACP) and 5'-nucleotidase were active in cases with fibre phagocytosis. The oxidative part of the pentose phosphate pathway in myopathic tissue seemed to be important in three cases, demonstrated by the increased activity of glucose-6-phosphate dehydrogenase (GPDH) and 6-phosphogluconate dehydrogenase (PGDH). The important feature of diseased horse muscle was that the pathohistochemical changes were exactly the same as in diseased skeletal muscles of humans. The application of tissue saving enzyme histochemical techniques can be recommended in the study of muscle tissue from horses suffering from suspected neuromuscular disorders.     

Growth performance, meat quality and activities of glycolytic enzymes in the blood and muscle tissue of calves infected with Sarcocystis cruzi

Growth performance and the pattern of glycolytic enzymes in the blood plasma were assessed during experimental Sarcocystis cruzi infection (1 x 10(5) sporocysts per calf) in six calves; five calves served as noninfected controls. At slaughter (68 or 88 days post infection), carcass weight, dressing percentages and several parameters of meat quality (pH, color brightness, rigor, water absorbing capacity, water binding capacity) were recorded. Moreover, enzyme activities were measured in muscle homogenates. Weight gain was significantly impaired by the infection. Activities of lactate dehydrogenase (LDH) and aldolase (ALD) significantly increased in the blood plasma of the infected calves during the chronic stage of the disease, while glucose-6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase (ICDH) were not significantly altered. This was accompanied by a significant decrease of enzyme activities in the Musculus longissimus dorsi (LDH, ALD), in the diaphragmatic musculature (ALD, G6PDH) and in the heart (LDH, ALD). Activities of LDH, ALD, ICDH and G6PDH were visualized by enzyme histochemistry within the developing sarcosporidial cysts. However, isoenzymes of parasite origin could not be demonstrated by agar-gel electrophoresis of muscle homogenates or blood plasma. It is concluded that sarcocystiosis of even moderate severity alters the performance of calves but not meat quality. Leakage of glycolytic enzymes from the affected muscles is the probable cause of increased plasma enzyme activities. Although these enzymes are also synthesized by the parasite, the contribution of parasite-derived enzymes to the observed changes of enzyme patterns remains in question.     

Some enzyme interrelationships in serum and organs of Egyptian camel

In 7 normal healthy Egyptian one-humped camels aged 3-4 years, the relationships were studied between enzyme activities of lactic dehydrogenase (LDH), creatine phosphokinase (CPK), alkaline phosphatase (ALP), acid phosphatase (ACP), and cholonesterase (CHE) of serum and organs as well as between ACP and ALP and between LDH and CPK. Liver, heart, kidney, and spleen tissue samples as well as serum were analysed for total enzyme activity. The following results were obtained: --The heart was the organ with the highest activities of ALP, LDH, and CPK, and it contained high values of CHE, whereas the lowest activities of all enzymes were recorded from serum. The spleen exhibited of the highest activity of ACP. --Each of the serum enzymes ALP, LDH, and CHE were in strong inverse relationship with the corresponding enzymes in the liver. Strong inverse relationships existed also between serum LDH and kidney LDH as well as between CPK in serum and heart. --Direct relationships were remarkable serum LDH and that of spleen and heart as well as between serum ACP and that of liver and heart. --Interrelationships were inverse between ACP and ALP in liver, kidney, and heart, but weak direct interrelationships were characteristic between the 2 enzymes in serum and spleen. --LDH was inversely interrelated with CPK in serum and heart.     

Enzyme activities in tissues of clinically normal Large White pigs. Variations with age and sex

The activities of eight enzymes (glutamate dehydrogenase, sorbital dehydrogenase, malate dehydrogenase, lactate dehydrogenase, alpha-hydroxy butyrate dehydrogenase, gamma-glutamyl transpeptidase, alkaline phosphatase and creatine kinase) were determined in tissue homogenates of liver, kidney, spleen, lung, small intestine, cardiac muscle and skeletal muscle, from 15 Large White pigs of three different ages (1.5 weeks, 18--22 weeks and 113 weeks). The results showed that variation in tissue enzyme concentration due to differences in sex is minimal. Variation due to differences in age, however, appears to be of greater importance, particularly when considering young animals. These age differences may affect the interpretation of plasma enzyme changes due to tissue damage, and the use of additional enzyme assays as an aid to interpretation in these cases is advisable.     

Experimental paratuberculosis (Johne's disease)--studies on biochemical parameters in cattle

Ten male Holstein-Friesian calves naturally infected by Mycobacterium paratuberculosis were experimentally re-infected orally at an average of 17 days. Monthly measurements were conduced of the following activities, in the period between post infection days 160 and 400: total protein (TPR), albumin (ALB), cholesterol (CHOL), triglycerides (TRIG), Zn and Cu concentrations as well as sorbitol dehydrogenase, lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (alpha-HBDH), gamma-glutamyltransferase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), alkaline phosphatase and fructose-1,6-diphosphate aldolase (ALD). TPR, ALB, TRIG, and CHOL were reduced by day 400, in conjunction with disorders of digestion and absorption. Increased activities of CK, ALD, LDH, alpha-HBDH, AST and ALT primarily indicated damage to skeletal muscle and/or liver. Serum CK and ALD activities as well as TRIG and TPR concentrations may serve as aids to specific diagnosis of paratuberculosis, particularly in the advanced stage of the disease.     

Distribution of tissue enzymes in three species of pinnipeds.

In domestic animal medicine, changes in serum enzyme levels are routinely used as diagnostic tools to detect liver disease. Hepatic disease occurs in pinnipeds, but limited data are available on the tissue distribution of serum enzymes in marine mammals. The objectives of this study were to determine the tissue distribution of seven serum enzymes in three pinniped species. Enzymes evaluated were alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) in tissues from California sea lions (Zalophus californianus) (n = 5), harbor seals (Phoca vitulina) (n = 5), and northern elephant seals (Mirounga angustirostris) (n = 5) that stranded and then died at a rehabilitation center. Samples were evaluated in duplicate from liver, skeletal muscle, cardiac muscle, kidney, adrenal, spleen, pancreas, lung, lymph node, and intestine. Patterns of tissue enzyme distribution were similar in all species, with SDH activity highest in liver and kidney, CK activity highest in skeletal and cardiac muscle, ALP activity highest in adrenal, and GGT activity highest in the kidney. Aspartate aminotransferase and LDH activities were less specific, with high activity in multiple tissues. Tissue ALT activity was high in the liver of all species, but was also high in cardiac muscle (California sea lions), skeletal muscle (harbor seals), and kidney (elephant seals). These results suggest that concurrent analysis of SDH, ALT, and CK would provide high specificity and sensitivity for the detection of hepatic lesions, and allow differentiation of liver from skeletal muscle lesions in pinniped species.     

Serum enzyme changes in lambs with experimentally induced acute muscular dystrophy

An experiment was performed to study some serum enzyme changes taking place in artificially fed lambs made dystrophic on a skim milk ration supplemented with α-tocopherolextracted cod liver oil. In the course of two to three weeks the lambs showed highly increased serum values of aspartate- and alanine aminotransferase (AspAT = GOT and A1AT = GPT), total lactate dehydrogenase (LDH), and creatine phosphokinase (GPK). The latter enzyme proved to be the most sensitive test of myopathies, with serum values above 1000 times the normal level. A high-grade hyaline skeletal muscle degeneration was confirmed by histology. Myocardial changes were also present.Contrary to expectations, electrophoretic separation of LDH isoenzymes in the serum of the dystrophic lambs did not reflect any increase of the prevalent muscular cathodic thermolabile fraction LDH₅. The use of a so-called LDH heat stability test as an aid in clinical diagnostic work, as previously suggested, therefore does not seem to be valid in this disease. The results are discussed.     

Enzymes of glucose and fatty acid metabolism of liver, kidney, skeletal muscle, adipose tissue and mammary gland of lactating and non-lactating sheep

The effect of peak lactation on the activities of a number of enzymes of glucose and lipid metabolism of perirenal and subcutaneous adipose tissue, skeletal muscle, liver, kidney cortex and mammary parenchyma of sheep are described. Enzymes studied included hexokinase (glucose utilization), pyruvate carboxylase (gluconeogenesis), pyruvate dehydrogenase (glucose oxidation and production of acetyl CoA for fatty acid synthesis), acetyl CoA carboxylase (fatty acid synthesis) and glycerol-3-phosphate acyltransferase (fatty acid esterification). Major changes that were found include a decrease in activities of enzymes of fatty acid synthesis and esterification in adipose tissues, decreased activity of pyruvate dehydrogenase in muscle and adipose tissues and increased pyruvate carboxylase; there was no change in activities of enzyme of fatty acid esterification in liver. Activities of hexokinase, acetyl CoA carboxylase and glycerol-3-phosphate acyltransferase have been estimated per tissue; this shows the quantitative importance of limiting glucose utilization by muscle and of suppression of fatty acid synthesis in adipose tissue for efficient partitioning of nutrients for milk production.     

Clinical study on experimental Theileria annulata infections of cattle. 1. Clinical-chemical studies

The extent of liver and kidney damage in 10 young steers, infected with a stabilate of Theileria annulata, was estimated by the determination of enzymes (GOT, GPT, SDH, ALD), serum levels of bilirubin and urea. At the same time the effectiveness of some liver protecting medical agents was tested. The following results were obtained: Change in the activity of the enzymes GOT, SDH and ALD and the increase of bilirubin during the advanced course of the disease are indicative of severe tissue damage in the liver. The levels of urea were found to be in the normal range with only a few exceptions. This seems to exclude the involvement of the kidney in the disease. The group of animals receiving liver protecting medication did not show any difference compared with those animals without medication.     

Increased Serum Alanine Aminotransferase Activity Associated With Muscle Necrosis in the Dog

Serum activity of alanine aminotransferase (ALT) was consistently increased in dogs with canine X-linked muscular dystrophy (CXMD), a primary myopathy characterized by profound and on-going skeletal muscle necrosis. In order to determine whether the ALT was of liver origin, serum activity of creatine kinase (CK), aspartate aminotransferase (AST), ALT, and sorbitol dehydrogenase (SDH) obtained from dystrophic dogs was compared with enzyme activity present in clinically normal dogs. In dystrophic dogs at all ages tested, serum activity of CK, AST, and ALT was increased, and significant increases were present in dogs four weeks or older. In contrast, SDH activity in dystrophic dogs was not statistically different from values in clinically normal dogs. Ultrastructural examination of liver tissue revealed no evidence of hepatic degeneration in dystrophic dogs. It was concluded that increased serum activity of ALT in the dog may be associated with severe skeletal muscle degeneration, without concurrent hepatocellular necrosis.     

Enzymes Sériques pour le Diagnostic de la Nécrose Hépatique Aigüe Expérimentale

The activity of certain serum enzymes ornithine carbamyl transferase (OCT), serum isocitric dehydrogenase (SIC-D), total serum lactic dehydrogenase (LDH) and its isoenzymes (LD₁ and LD₅) was evaluated as a mean of assessing experimental hepatic necrosis on dogs treated with CCl₄. Measurement of activity levels of these enzymes, seldom carried out in veterinary clinical pathology, was made together with tests commonly used in our laboratories: serum glutamic pyruvic transaminase (SGPT), serum glutamic oxalacetic transaminase (SGOT) and serum alkaline phosphatase (SAP), cholesterol, bilirubin and prothrombin time. Measurement of the level of OCT was useful in the diagnosis of liver necrosis. The SIC-D level was important during the first four days of the experiment, but on subsequent days, the enzymatic activity was practically normal. Because of the wide variations of LDH serum levels in normal animals and since many factors influence its activity, the measurement of this enzyme and its isoenzymes was not a good index in the diagnosis of canine liver necrosis. The evaluation of cholesterol and bilirubin was judged of secondary importance because these metabolites are not specific to hepatic problems.     

The potential use of plasma glutamate dehydrogenase activity for the evaluation of hepatic disease in the cockatiel (Nymphicus hollandicus)

A reference interval for plasma glutamate dehydrogenase (GD) (E.C. 1.4.1.3) activity, of 0-8 IU/L, was established for the cockatiel (Nymphicus hollandicus). An automated modification of a commercial manual assay was used. This enzyme is considered liver specific in humans and numerous domestic animals, due to its organ distribution. A similar distribution was found in cockatiels in this study. Maximal enzyme activity was recovered from liver and kidney homogenate supernatants. Minimal activity was detected in skeletal muscle preparations. These results suggest a potential use for plasma GD activity in the evaluation of hepatocelluar injury/necrosis in cockatiels.     

Biochemical evaluation of mitochondrial respiratory chain enzymes in canine skeletal muscle

OBJECTIVE: To perform respiratory chain enzymatic activity assays on canine skeletal muscle biopsy specimens and establish reference range values of skeletal muscle enzyme activities for dogs. SAMPLE POPULATION: Biopsy specimens from the vastus lateralis muscle were obtained from 24 dogs (8 sexually intact males and 14 sexually intact females) ranging from 15 months to 6 years of age. PROCEDURE: Mean values of citrate synthase, cytochrome-c oxidase, succinate dehydrogenase, succinate dehydrogenase-cytochrome-c reductase, nicotinamide adenine dinucleotide (NADH) dehydrogenase, and NADH dehydrogenase-cytochrome-c reductase activities were established by use of 6 standard spectrophotometric assays for respiratory chain enzyme analysis. RESULTS: Compared with published data for skeletal muscle enzyme activities in humans, skeletal muscle enzyme activities in dogs were 2- to 4-fold higher. Additionally, citrate synthase activity, a marker for mitochondrial volume, was positively correlated with age in dogs, suggesting that mitochondrial volume increases with age, although no apparent change in respiratory chain enzymatic activity with an increase in age was found. CONCLUSIONS AND CLINICAL RELEVANCE: Reference range values for skeletal muscle enzyme activities of dogs are needed to accurately interpret results of respiratory chain enzymatic activity assays. During investigation of metabolic myopathies, if skeletal muscle biopsy specimens are evaluated for respiratory chain enzyme kinetics, they should be performed and evaluated in concert with skeletal muscle biopsy specimens from clinically normal animals of the same species.     

Masseter myodegeneration as a cause of trismus or dysphagia in adult horses

The medical records of eight horses with histological evidence of myodegeneration of the masseter muscles were examined. While they were alive their most common clinical signs had included difficulty in eating or opening their mouths, weight loss, difficulty in moving, and noticeable atrophy of the masseter muscles. The serum activities of muscle enzymes were abnormally high in all of the horses. Whole blood and/or liver selenium and vitamin E concentrations were less than the reference ranges in some of the horses. The lesions varied with the stage of the disease and consisted of swelling and discoloration, or muscle atrophy and fibrosis. Histologically, the muscle changes ranged from acute to subacute degeneration, with regenerative changes accompanying ongoing degeneration, to chronic degeneration with fibrotic replacement of muscle tissue. There were changes in the masseter muscle of all the horses, but some had widespread lesions in skeletal muscle, and a few also had myocardial lesions.     

Serum enzymes levels and influencing factors in three indigenous Ethiopian goat breeds

Serum enzymes were studied in 163 apparently healthy goats from three indigenous goat breeds of Ethiopia. The effect of breed, age, sex and season on alanine aminotransferase (ALT) / glutamic pyruvic transaminase (GPT), aspartate aminotransferase (AST) / glutamic oxalacetic transaminases (GOT), alkaline phosphatase (ALP) and acid phosphatase (AcP) levels was assessed. The mean serum enzymes levels of the indigenous Arsi-Bale, Central Highland and Long-eared Somali goat breeds ranged from 14.0–20.2 iu L⁻¹ for ALT/GPT, from 43.2–49.3 iu L⁻¹ for AST/GOT, from 83.7–98.8 iu L⁻¹ for ALP, and from 2.99–4.23 iu L⁻¹ for AcP, were within the normal range for goats elsewhere. Breed had significant influence on AST/GOT values. Sex had significant effect on ALT/GPT for Arsi-Bale goats with higher values in males than females. Age was significant on all serum enzymes studied in the Arsi-Bale goats and on ALP in the Central Highland goats. Season had significant influence on all serum enzymes except for ALT/GPT in the Arsi-Bale goats. The serum enzyme levels of these indigenous goat breeds can be used as normal reference values for Ethiopian goat breeds adapted to similar agro-ecology and production system.     

Value of measurements of the serum activities of creatine phosphokinase,aspartate aminotransferase and lactate dehydrogenase for predicting whether recumbent dairy cows will recover

Over a period of seven years, 262 recumbent dairy cows were investigated, and serum samples were analysed for creatine phosphokinase (CPK), lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) activities. The results from cows for which all three measurements were available, were analysed statistically by the non-parametric receiver operating characteristic (ROC) method, to evaluate the value of the serum enzyme activities for predicting a failure to recover. The sensitivity and specificity of the activity levels were calculated over all cut-off points, and ROC curves were created by plotting sensitivity as a function of 1--specificity at each cut-off point for samples grouped by the interval after the cows became recumbent. The predictive values of the tests were compared by calculating the areas under the curves, and the positive and negative predictive values of the tests were calculated and plotted against the prevalence of a failure to recover. The results from each of the enzymes were significantly better than from a random test with no discriminatory ability. The optimal cut-off points maximising the sensitivity and specificity of the tests were 2330, 2225 and 171 U/litre for CPK, LDH and AST, respectively. The predictive value of AST was significantly better than that of CPK or LDH, and measures made on the second and third day of recumbency were significantly better with optimal cut-off points of 128 and 189 U/litre, respectively.     

Tissue and plasma enzyme activities in juvenile green iguanas

OBJECTIVE: To determine activities of intracellular enzymes in 8 major organs in juvenile green iguanas and to compare tissue and plasma activities. ANIMALS: 6 green iguanas < 1 year old. PROCEDURE: Lysates of liver, kidney, epaxial muscle, heart, lung, spleen, small intestine, and pancreas were analyzed for alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate transaminase (AST), alanine transaminase (ALT), gamma-glutamyltransferase (GGT), creatine kinase (CK), glutamate dehydrogenase (GMD), and amylase (AMS) activities. RESULTS: In general, low tissue enzyme activity coincided with low plasma activity. The CK activity was high in epaxial muscle and the heart and low in all other tissues tested. The AMS activity was found exclusively in the pancreas. Moderate LDH and AST activities were found in all tissues. Low ALT and ALP activities were found in a variety of tissues. Plasma and tissue activities of GGT and GMD were low or undetectable. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that plasma CK activity may be muscle specific in iguanas, but high values may not always indicate overt muscle disease. The AMS activity may be specific for the pancreas, but the wide range of plasma activity would likely limit its diagnostic usefulness. Activities of AST and LDH may reflect tissue damage or inflammation, but probably do not reflect damage to specific tissues or organs.