Observations on γ-glutamyl transferase, 5′-nucleotidase and leucine aminopeptidase activities in the plasma of the horse

Research in Veterinary Science 1989, 46, 301-306

Observations on y-glutamyl transferase,S' -nucleotidase and leucine aminopeptidase activities in the plasma of the horse H. J, WEST, Department of Veterinary Clinical Science, University of Liverpool, Leahurst, Neston, S Wirral L64 7TE

In 18 horses there was no effect of age or sex on plasma activities of y-glutamyl transferase (y-GT), 5' -nucleotldase (5' -NT) and leucine aminopeptidase (LAP). All the enzymes were equally stable after storage for one month at - 20°C and there was no significant difference between their activities in serum and plasma in clinically normal horses. The pattern of release of y-GT, 5' -NT and LAP into plasma was studied in 114 horses which had a variety of orthopaedic, gastrointestinal, cardiovascular and hepatic (necrosis, lipidosis, neoplasia and cirrhosis) conditions. A definitive diagnosis of hepatic disease was established by histological examination of the liver. y-GT and 5' -NT were leaked into plasma in hepatic disease and y-GT was the more sensitive indicator of liver damage. There was some evidence that y·GT and 5' -NT plasma activities may increase in hepatic necrosis as well as in biliary obstruction. LAP was insensitive and not hepatic specific in the horse. ALKALINE phosphatase (ALP) is widely distributed in tissue; of horses and there is a wide range of normal activity in plasma (Gerber 1969). Increased plasma activities have been observed in a variety of conditions. For this reason, alternative enzymes have been sought which are more reliable indicators of hepatobiliary disease, particularly of cholestasis. Iditol (ID) and glutamate dehydrogenase (GD) are highly concentrated in the livers of horses and are specific for hepatic necrosis (Freedland et al 1965, Ikeda et al 1976). Arginase was similar in specificity to ID and GD but less sensitive in horses (Ford and Gopinath 1974, Aller et al 1981) as an indicator of hepatic necrosis. Aspartate aminotransferase (AST), total lactate dehydrogenase (LDH) and isocitrate dehydrogenase (!CD) are not liver specific but may be of use diagnostically to measure the extent of hepatic necrosis if all tissues other than the liver are known to be free of pathology (Cornelius et al 1959, Cornelius 1961, Freedland et a11965, Gerber 1969). Naftalin et al (1969) showed that y-glutamyl transferase (y-GT) was associated with the biliary epithelium in man, sheep, cattle and pigs. In the horse, the renal cortex was the richest source of y-GT, then the pancreas and liver (Rico et al 1977, Ford and Adam

1981). There was no release of y-GT into the serum of sheep poisoned with mercuric chloride (Ford 1974). Shaw (1976) and Robinson and Trafford (1977) demonstrated a marked urinary y-GT excretion in sheep with mercuric chloride nephrotoxicity. y-GT was a sensitive indicator of bile duct damage in acute fascioliasis in cattle (Simesen et alI973), sporidesmin toxicity in sheep (Ford 1974, Towers and Stratton 1978, Ford and Evans 1985), bile duct ligation in sheep, cattle and horses (Ford 1974, Ford and Gopinath 1976, Ford and Evans 1985), poisoning with carbon disulphide in calves and horses (Gopinath and Ford 1976) pretreated with phenobarbitone and in poisoning due to Senecio jacobaea in horses (Craig et al 1978, Giles 1983). Following hepatic damage in horses caused by carbon tetrachloride (Ikeda et al 1976, Noonan 1981) and aflatoxin B I poisoning (Aller et al 1981) y-GT was released into serum. Aller et al (1981) and Noonan (1981) felt that this may represent hepatocellular necrosis in horses as well as bile duct obstruction. 5 I -nucleotidase (5' -NT) was described by Wachstein and Meisel (1957) as being located in the hepatic sinusoids and biliary canaliculi of the rat, mouse, guinea pig, rabbit, dog and man. Studies of rat liver by Essner et al (1958) and Wootton et al (1977) demonstrated intense 5 I ·NT activity in the plasma membranes limiting the bile canaliculi and liver sinusoids. In horses (Ford and Adam 1981) and sheep (Ford and Evans 1985) 5 -NT activity was present in a wide range of tissues. In horse liver 5 -NT activity was low compared with that of glutamate dehydrogenase activity being mainly seen at the sinusoidal border of the hepatic cells, particularly in the central zone of lobules with slight activity in the bile duct epithelium and wall of portal blood vessels (Ford and Adam 1981). 5' -NT was released into plasma in fascioliasis of cattle (Rowlands and Clampitt 1979) and in sheep during fascioliasis after ligation of the bile duct but not during sporidesmin toxicity or from renal lesions in sheep (Ford and Evans 1985). The study by Ford and Adam (1981) in horses suggests that damage to blood vessels in a variety of organs could lead to leakage of 5 I -NT into plasma. Leucine aminopeptidase (LAP) has been found in

301

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I

H. J. West

302

TABLE 1: y·GT, S'·NT and LAP activities in plasma in clinically normal horses

Description of horses Stallions Geldings All male horses Mares All horses

n 5 5 10 8 18

y-glutamyl transferase iu litre l r

17·' ± 15·8 ± 16·5 ± 17·3 ± 16'8±

1·7 1·5 1·' 2·2 1·1

5'-nucleotidase miu litre- 1 2·1 ± 0·9 1·4 ± 0·3 1·8± 0·5 1·9 ± 0·3 1·8 ± 0·3

Leucine aminopeptidase iu Iitre- 1 4·1 ± 5·3 ± 4·7 ± 3·6 ± 4·2 ±

0·6 0·7 0·5 0·3 0·3

Mean ± standard error of the mean. Average of two determinations on each sample

high activity in a number of tissues in the rat, dog, guinea pig and man (Nachlas et al 1957). Histochemically, Kowlessar et al (1961) showed that the activity of LAP was intense in the bile ducts and canaliculi and weak in parenchymal cells. Rutenburg et al (1958) and Bardawill and Chang (1963) have described the elevation of serum LAP activity in a variety of diseases of man which affect the pancreas and hepatobiliary tree. In human medicine 5' -NT and LAP serum activities are not raised in diseases of bone (Dixon and Purdom 1954, Kowlessar et al 1961) and are used to differentiate between bone and liver damage in the presence of increased serum ALP activity. LAP activity increased in serum in carbon tetrachloride poisoning in dogs (Fleisher et al 1958) but not in renal damage caused by mercuric chloride in sheep (Robinson and Trafford 1977). LAP has not been assayed previously in horses. The purpose of this investigation was to compare the alterations in plasma y-GT, 5' NT and LAP in different clinical conditions in horses with and without defined liver lesions to evaluate the clinical usefulness of these enzymes and to compare the relative stability of the enzymes on storage. Materials and methods Heparinised blood samples were collected from 18 clinically normal horses of different breeds and weights and aged from two to 14 years; mares, stallions and geldings were studied to establish a range of reference values of plasma activities of y-GT, 5' -NT and LAP. Samples were assayed in duplicate immediately following collection and centrifugation for 20 minutes or, if this was not convenient, after storage for a few days at - 20°C. The average of two determinations on each sample was used. Activities of y-GT, 5' -NT and LAP were also compared in samples of serum and plasma collected from 15 clinically normal horses of both sexes. The effect of storage on y-GT, 5' -NT and LAP activities was measured in plasma samples from 12 clinically normal horses. Heparinised blood samples were centrifuged immediately after collection and the plasma divided into three equal portions. In the first

portion enzyme activities were assayed immediately. The other two portions were rapidly frozen at - 20°C and analysed after storage for one week and one month. Changes in activity in plasma due to clinical disease were measured in 114 horses at the onset of clinical illness. The diagnosis was based on clinical examination, and a definitive diagnosis in the case of hepatic lesions was established by needle biopsy and laboratory tests including assay of y-GT, 5' -NT and LAP activities. These horses comprised 39 with orthopaedic conditions (fractures, arthritis); 19 with gastrointestinal disease (obstructions, strongylosis, grass sickness, malabsorption); seven with cardiovascular conditions (atrial fibrillation, verminous aneurysms); 18 with hepatic necrosis; seven with hepatic lipidosis; four with hepatic neoplasia as a result of metastases and nine with hepatic cirrhosis as a result of S jacobaea toxicity. The rest of the horses (11) with conditions such as lymphosarcoma, teeth problems and neurological problems are described as miscellaneous. The activity of y-GT in plasma was assayed at 25°C with the Biochemica test combination (Boehringer) by a method based on that of Szasz (1969). 5' -NT activity in plasma was measured with a kit (BDH Chemicals) based on the method of Persijn et al (1969) at 25°C. Plasma LAP activity was measured at 25°C with a kit (Boehringer) based on the method described by Nagel et al (1964). Liver biopsies were fixed in buffered formalin, embedded in paraffin wax, sectioned, and stained with haematoxylin and eosin, periodic acid Schiff with and without prior diastase incubation or a fat stain, oil red O. Results

Normal plasma y-GT, 5' -NT and LAP activities Table 1 shows that there was no difference in plasma y-GT, 5' -NT and LAP activities between sexes nor was there any effect of age in adult horses. The overall mean figures (± SEM) for all horses were: y-GT 16, 8 ± 1. 1 iu litre - I; 5' -NT 1·8 ± O·3 miu litre - I and LAP 4·2 ± 0'3 iu litre : '.

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There was no significant difference between y-GT, 5' -NT or LAP activities in serum and plasma. The mean values for serum and plasma, respectively, were y-GT 16'9 ± 1·09 and 17·1 ± 1'14 iu litre-I; for 5 '-NT I ·9 ± O·34 and I ·8 ± O·43 miu litre - I and LAP 3' 9 ± 0'30 and 3'9 ± 0'31 iu litre-I.

Effect on plasma activity of y-GT, 5' -NT and l.AP of storage at -20 o e There was no significant decline in the activities of y-GT, 5 - NT or LAP in the plasma after one week's or one month's storage at - 20o e. Activity in fresh plasma and after one month's storage respectively was for j-or 12·9± 1'82and 14'2± 1'26iulitre- i , for 5'-NT 2'5 ± 0·27 miu litre-I and 2'5 ± 0·27 miu

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litre - I and for litre-I.

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Activity of y-GT, 5 I-NT and LAP in plasma of horses with different clinical conditions Figs I and 2 show that there were no significant alterations in y-GT and 5 -NT plasma activities outside the normal range in orthopaedic and cardiovascular conditions and in a variety of miscellaneous conditions not affecting the liver, except for three horses in the orthopaedic group which had y-GT above the normal range and one in the miscellaneous group which had 5' -NT above normal. y-GT plasma activity was slightly elevated in four out of seven horses with gastrointestinal disease (grass sickness), see Fig I. Fig 3 shows that there were significant elevations in I

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The horses with hepatic cirrhosis (ragwort toxicity)

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activities of 956 and 778 iu litre ~ I also had of 177 and 78 miu litre - J, respectively. Similarly the horses with highest y-GT activities with hepatic necrosis, lipidosis and neoplasia also had the highest 5' -NT activities (Figs I and 2). The horses with the highest plasma y-GT and 5' -NT activities had particularly severe clinical signs and in horses with hepatic necrosis that recovered, these values declined to normal on clinical recovery whereas in animals that subsequently died, these values remained high. LAP plasma activities were highest in horses with hepatic lipidosis and to a lesser extent in horses with hepatic necrosis (Fig 3). These raised LAP activities were associated with high y-GT and 5' -NT plasma activities (Fig 1 and 2). The LAP plasma activities were only high in hepatic necrosis in horses that eventually died (Fig 3). None of the horses with hepatic lipidosis recovered and six out of eight had high LAP plasma activities (Fig 3). y-OT

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LAP plasma activities outside the normal range in orthopaedic, gastrointestinal and cardiovascular diseases but not in a variety of other conditions not affecting the liver. y-GT activity in plasma was elevated in most cases of hepatic necrosis, in all cases of hepatic cirrhosis (Fig I) and in most cases of hepatic lipidosis and neoplasia (hepatic metastases). Fig 2 shows that plasma 5' -NT activity was elevated in most but not all cases of hepatic disease. There was little release of LAP into plasma in hepatic disease except in hepatic lipidosis (Fig 3). In hepatic disease, y-GT was the most sensitive indicator of the enzymes studied. In six of the horses the activity of LAP in plasma was below the normal range (Fig 3) and a similar observation was made for y-GT in five orthopaedic conditions and one gastrointestinal case (Fig I).

Discussion The plasma activity of y-GT in normal horses (Table I) agrees with the published values of Rico et al (1977), Blackmore et al (1979), Noonan (1981) and Ricketts (1981), but was lower than those of Blackmore and Kent (1977) who used a higher incubation temperature of 35°C. Any variation in results between this study and previous reports could be accounted for by variation in the type of horses, assay time and temperature and in the time of storage of samples. The normal plasma 5' -NT activity agreed with the results of Ford and Adam (1981) although their results were slightly lower. There are no previous studies on the influence of age or sex on 5' -NT and LAP plasma activities in horses. Blackmore and Kent (1977) found no significant sex effect on y-GT activities in the horse. This lack of effect is not surprising in view of studies in man which showed no sex or age effect on 5' -NT and LAP in plasma (Young 1958, Banks et al 1960). The high stability of y-GT, 5' -NT and LAP in plasma on storage at - 20°C for a month is an advantage over the more labile enzymes such as ID and lCD, particularly when coupled with reports of the stability of y-GT in sheep (Shaw 1976) and cattle (Grunder 1976) serum at room temperature. The similar activity of y-GT, 5'-NT and LAP in serum and plasma suggests that examination of either blood component would be satisfactory. The activities of y-GT and 5' -NT in plasma were specifically raised in diseases of the liver and the extent of the increase could be related to the degree of clinical illness (Figs I and 2). Of the two enzymes, y-GT was the more sensitive indicator although the normal

y-GT,

activity in plasma

was higher for y-GT

5 '-NT and LAP in horse plasma

than

for

5I -NT

and LAP. y-GT and 5' -NT were increased in intra- and extrahepatic cholestasis and also possibly as a result

of hepatic necrosis. These observations support the of y-GT in the horse (Aller et al 1981, Noonan 1981) and 5' -NT in sheep (Ford and Evans 1985) may be associated with hepatocellular necrosis as well as biliary obstruction. The hepatic specificity of 5' -NT in horses is contrary to the expectations of Ford and Adam (1981). In the horse, y-GT is an indicator of cholestasis (Ford and Gopinath 1976, Craig et at 1978, Giles 1983) although the enzyme may be elevated in plasma in hepatocellular necrosis (Aller et al 1981, Noonan 1981). Experimental evidence exists that 5' -NT leaks into plasma in cholestasis in ruminants (Rowlands and Clampitt 1979, Ford and Evans 1985) and LAP is high in plasma in cholestasis in man (Rutenburg et al 1958, Bardawill and Chang 1963). lD and GD are specific for hepatocellular necrosis in the horse (Freedland et al 1965) and are present in higher activities in liver than 5'-NT (Ford and Adam 1981). The present study indicates that y-UT, 5' -NT and LAP may be elevated in intra- and extrahepatic cholestasis. Further studies are necessary to ascertain whether these enzymes are released as a result of hepatic necrosis per se. The increase in plasma activities of y-GT (Fig 1) and LAP (Fig 3) in gastrointestinal disease may be explained by a degree of liver damage secondary to the intestinal changes. However, plasma LAP activities (Fig 3) were also elevated in some orthopaedic conditions and the enzyme was a disappointingly insensitive indicator in hepatic disorders. This may be because the enzyme is released into the tissues and only slowly leaks into the plasma or that low activities exist in the liver. The elevated plasma activities of LAP in diseases of bone (Fig 3) may be due to the specificity of the method because several enzymes in addition to classical LAP hydrolyse the substrate (Patterson et al 1963). There are reports of elevation of 5' -NT but not of LAP in a small sample of human patients with diseases of bone and other non-hepatic diseases (Kowlessar et al1961, Eshchar et al 1967, Boone et al 1974). In conclusion, y-GT and 5' -NT are equally stable and only released into plasma during hepatic disease. y-GT is more sensitive than 5' -NT. LAP proved to be much less hepatic specific and sensitive but equally as stable as y-GT and 5' -NT. The interpretation of elevated activities of plasma enzymes is dependent on their half-time of clearance. Further work is necessary to establish the half-lives of these three enzymes in the plasma of horses, and to study the mechanisms of release of y-GT, 5' -NT and LAP in tissues by histochemistry during different types of hepatic damage. It will also be necessary to measure the tissue activities

view that release into plasma

305

of LAP in horses and its liver by histochemistry.

normal

distribution in

the

Acknowledgements The author would like to thank Professor R. J. Fitzpatrick and Professor E. J. H. Ford in whose departments this work was carried out and also Mr G. Hynes for technical assistance and for help in preparation of the figures. The author is grateful to Mrs C. Roberts for typing the manuscript and to the veterinary surgeons in practice who referred the horses.

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Received November 3. 1987 Accepted June 16, 1988