gamma-glutamyltransferase (GGT) and glutamate dehydrogenase (GDH) and serum concentrations of total and conjugated bilirubin.The majority of samples ...
The Isoelectric Focusing Properties of Serum Alkaline Phosphatase in Disease and Following Prednisolone and Phenylbutazone Administration in the Horse Roger S. Ellison and Robert M. Jacobs
ABSTRACT This study was undertaken to ascertain if the isoelectric focusing pattern of serum alkaline phosphatase (AP) from sick horses with high activity is useful for determining its tissue origin. The effect of oral prednisolone and phenylbutazone therapy on this enzyme in healthy horses was also investigated. The sick horses were divided into three groups: hepatic, intestinal and miscellaneous. All sera had approximately thirteen bands of AP activity when focused on agarose gels with a pH gradient of 3.5 to 9.5. All the horses in the liver disease group had greater than 65% of enzyme activity in bands 3 to 7 (counted from the anode) whereas the other two groups had at least 30% and up to 80% of activity in bands 8 to 13. This was true even in the several cases of primary intestinal disease that had additional biochemical evidence of liver damage. All bands were heat sensitive indicating that little if any AP was of small intestinal or renal origin. Oral prednisolone and phenylbutazone for 20 and 12 days respectively had no affect on serum AP activity or isoelectric pattern. We concluded that the AP in bands 3 to 7 is of liver origin but the origin of bands 8 to 13 remains undetermined although small intestinal or renal origin is unlikely. Isoelectric focusing of serum AP shows promise in
differentiating cases of primary from secondary liver disease but further studies are required correlating serum patterns and tissue patterns in animals with diseases.
RESUME Cette etude a pour objectif de verifier se l'electrofocalisation des phosphatases alcalines seriques (PA) provenant de chevaux malades est une technique utile pour en determiner leur origine tissulaire. Les effets d'une therapie orale de prednisolone et de phenylbutazone sur des chevaux sains sont egalement rapportes. Les chevaux malades etaient divises en trois groupes; problemes hepatiques, probl'emes intestinaux et ceux avec diverses pathologies. A l'electrophorese sur gel d'agarose avec un gradiant de pH de 3,5 a 9,5 il fut possible d'identifier environ 13 bandes. Chez le groupe presentant des problemes hepatiques, plus de 65% de l'activite PA est concentree dans les bandes 3 a 7 (a partir de l'anode) tandis que chez les autres groupes de 30% a plus de 80% de cette activite est concentree dans les bandes 8 a 13. Cette observation est demeuree valable dans les quelques cas de pathologies intestinales primaires ou l'on rencontrait des evidences biochimiques de dommages hepatiques. Toutes les bandes etaient thermosen-
sibles, indication que peu ou pas de PA etaient produites soit par lintestin grele ou les reins. L'administration orale de prednisolone et de phenylbutazone pour une duree de 12 et 20 jours n'a pas eu d'effets sur l'activite serique des PA ni sur la cartographie electrophoretique. Nous concluons que les PA des bands 3 a 7 sont d'origine hepatique et que celle des bandes 8 a 13 quoiqu"incertaine n'est probablement pas d'origine intestinale ou renale. La technique a l'etude serait interessante dans le sens ou elle permettrait de differencier les pathologies hepatiques primaires et secondaires. Cependant, des etudes supplementaires sont necessaires pour etablir une correlation entre les catographies seriques et tissulaires chez les animaux malades. INTRODUCTION
Alkaline phosphatase [EC 3.1.3.1; orthophosphoric-monoester phosphohydrolase (alkaline optimum)] is a ubiquitous enzyme that is found in most body tissues of humans, animals and birds (1,2-4). Horses have high activities in the small intestine, pancreas, liver, kidney, bone and neutrophils and low activities in skeletal and cardiac muscle, placenta, brain and lymphocytes (3,5-7). As in other species the alkaline phosphatase (AP) from each equine tissue has
Department of Pathology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Present address of Dr. R.S. Ellison: Ruakura Animal Health Laboratory, Private Bag, Hamilton, New Zealand. Reprint requests to Dr. R.M. Jacobs. This research was funded by a grant from Agriculture Canada, Dean's Discretionary Fund. Submitted August 19, 1988.
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Can J Vet Res 1990; 54: 126-131
measurable differences reflecting variations in primary structure (isoenzymes) or following posttranslational modifications of a common gene product (isoforms) (5-1 1). Total serum AP activity is reportedly increased in horses with cholestatic liver disease (12) whereas acute hepatocellular necrosis has little effect (13-15). Horses with such unrelated diseases as enteritis and enteric accidents, respiratory diseases, and acute purulent infections may also have high values (10,16). There have been several attempts in the horse to improve the diagnostic specificity of total serum AP activity by defining the different properties of the tissue forms and measuring their contribution in sera (5,6,8-11,16,17). However, because of only small differences between the tissue forms, most of the techniques have lacked sufficient sensitivity and specificity to definitively separate multiple forms of the isoenzymes and isoforms in sera. Consequently there is no consensus as to the tissue origin of serum AP in different disease conditions. Several drugs affect serum AP in a variable manner. In people, hypolipemic and oral contraceptive drugs decrease the liver AP component in serum whereas antiepileptic drugs increase the total serum AP activity up to 70% (18). In dogs, glucocorticoids cause a unique response by inducing a new hepatic isoform that appears to be a sialated version of the intestinal isoenzyme (19-21). By comparison, the increase in serum AP activity following anticonvulsant and adrenocorticotrophic hormone therapy reported in dogs appears to be due solely to an increase in the normal liver component (22,23). High oral doses of phenylbutazone given to ponies increased serum AP activity fourfold (24) whereas the effect of glucocorticoids on serum AP activity in horses has not been reported. Isoelectric focusing (IEF) is a sensitive, high resolution technique able to separate closely related proteins according to differences in their isoelectric points (pl) (25). Its value in separating AP isoenzymes and isoforms has been documented in dogs and humans (26-31). In the preceeding paper, the IEF patterns have been described for a number of
equine tissues and for sera from healthy foals of various ages and from mares at two stages of pregnancy (7). This is a report of the IEF patterns of sera from horses with high total serum AP activity and with a variety of diseases. It also documents the effect of therapeutic doses of oral phenylbutazone and prednisolone on total serum activity and IEF patterns. MATERIALS AND METHODS HOSPITALIZED HORSES WITH HIGH SERUM ALKALINE PHOSPHATASE ACTIVITY
Sera from 43 hospitalized adult horses with high total serum AP activity were stored at -70°C for IEF. The AP activity was determined by measuring the rate of hydrolysis of p-nitrophenylphosphate in an automated biochemistry analyzer (Dacos, Coulter Electronics, Hialeah, Florida). The same instrumentation was used to measure the activities of gamma-glutamyltransferase (GGT) and glutamate dehydrogenase (GDH) and serum concentrations of total and conjugated bilirubin. The majority of samples were collected on the first day of hospitalization with multiple subsequent samples from some horses. These horses were divided into the following three primary disease groups as determined at necropsy or surgery or, in cases of respiratory disease, from clinical, radiographic and bronchoalveolar lavage findings.
(a) Twenty horses with gastrointestinal disease included cecocolic impaction (three), colonic torsion and/ or displacement (five), intestinal perforation and peritonitis (three), small intestinal strangulation (three), acute colitis (two), duodenal obstruction (one), salmonellosis (one) and enteritis of undetermined cause (two). b) Twenty horses with miscellaneous diseases consisting of respiratory disease (ten), genital tract disease (two), skin disease (three), hyperadrenocorticism (one), renal disease and cardioskeletal myopathy (one), septicemia and embolic nephritis (one), multiple facial fractures (one), and adverse drug reaction (one). (c) Three horses with primary liver disease; two were associated with choleliths and the cause of the third case was undetermined. EXPERIMENTAL BILE DUCT LIGATION
The common bile duct of five yearling ponies was ligated and serum samples collected as described elsewhere (12). Briefly, the common bile duct was exposed and double ligated through a midline abdominal incision under general anesthesia. Blood samples were collected before (base line) and on days 1, 2, 3, 5, 7 and 10 after ligation. Complete ligation was confirmed at necropsy after euthanasia on day 10. Alkaline phosphatase activity was measured by the hydrolysis of thymolpthalein monophosphate (Boehringer Mannheim, Indianapolis, Indiana). Aliquotes of serum were isoelectric focused (as below).
TABLE I. Selected serum biochemistry analytes of hospitalized horses with intestinal, miscellaneous and liver diseases
Group Intestinal (n = 20) Miscellaneous
(n = 20) Liver
(n = 3)
ALP
437[237,850]b (20)c 435[236,1502]
GGT 49[4,367] (5) 24[8,92]
Analytesa GDH
F BIL
C BIL
76[0,924] (8) 10[1,103]
42[6,143] (8) 31[2,131]
22[6,141] (4) 11[4,22]
(20)
(1)
(1)
(3)
(0)
1343[510,2780]
939[90,2200]
223[14,422]
89[54,108]
104[75,150]
(3)
(3)
(2)
(3)
(3)
Adult Reference 10-22 10-40 0-30 8-50 90-275 Interval aALP -alkaline phosphatase (IU/L); GGT -gamma-glutamyltransferase (IU/L); GDH glutamate dehydrogenase (IU/ L); F BIL -free bilirublin (,umol/ L); C BIL -conjugated bilirubin (,umol/ L). Enzyme activities were measured at 370C bThe values are expressed as means with the range in brackets cThe number of horses with levels above the reference interval: The reference interval was established by sampling 60 Thoroughbred mares that were open, early or late pregnant
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ORAL PREDNISOLONE AND PHENYLBUTAZONE STUDY
Ten healthy horses (seven mares and three geldings) at least 3 yr old weighing 400 to 550 kg were randomized into two groups. Group 1 horses were dosed orally with a thick suspension of prednisolone at 2.5 mg/ kg/day in two divided doses for seven days, 1.25 mg/kg once daily for seven days and 1.25 mg/ kg every second day for six days. Group 2 horses were dosed with a similar volume of water. Blood samples were collected from both groups on days 0, 2, 4, 6, 8, 10, 12, 14, 21, 28 and 36 and the AP activity determined by measuring the rate of hydrolysis of p-nitrophenylphosphate at 405 nm in a Gilford spectrophotomter (Sigma Diagnostics, St Louis, Missouri). Two months following the completion of this experiment the group 2 horses were dosed orally every 12 h with a suspension of phenylbutazone to give daily doses of 8.8 mg/ kg on day 1, 4.4 mg/ kg on days 2-6 and 2.2 mg/kg on days 7-12. Group 1 horses were given a similar volume of water. Blood samples were collected and AP measured as for the first experiment. Selected samples from both experiments were stored at -70°C for IEF.
St Louis, Missouri) and % inhibition calculated. The pre and postheated samples were then isoelectric focused in neighboring lanes. STATISTICAL ANALYSIS
RESULTS HOSPITALIZED HORSES WITH HIGH SERUM AP ACTIVITY
The three horses with primary liver disease had mean serum GGT and conjugated bilirubin levels significantly greater than the intestinal and miscellaneous disease groups and serum AP activity significantly greater than the miscellaneous group (p < 0. 05) (Table I). The mean serum GDH and free bilirubin levels of the liver and intestinal disease groups were significantly higher than the miscellaneous disease group. A member of the liver disease group had the highest serum alkaline phosphatase activity at twelve times the upper reference limit. Half of all cases had serum AP activities greater than 150% of the upper reference limit and of these, 43% and 57% also had high
The t-test for unpaired samples was used to evaluate the effect of prednisolone and phenylbutazone on serum AP activity and the IEF densitometer results for the groups of hospitalized horses. The statistical significance of the change in serum AP activity and IEF banding pattern following bile duct ligation was determined by the paired t-test. Between group differences in serum biochemical results of the hospitalized horses were assessed using the Kruskal-Wallis one-way analysis of variance by ranks method and, if significant (p < 0.05) individual groups were compared by the method of Daniel (32). serum gamma-glutamyltransferase
ISOELECTRIC FOCUSING
Serum samples were isoelectric focused on commercially available 1 mm agarose gels as described previously (7) (Resolve-ALP, Isolab, Ohio). Isoelectric point markers were run on selected gels using standard procedures to determine isoelectric points of specific serum bands (Pharmacia AB, Uppsala, Sweden). Alkaline phosphatase activity in each band was visualized with napthylphosphate substrate (Resolve-ALP, Isolab, Ohio) and quantitated with a Corning densitometer (Ciba-Corning Canada Inc., Ontario). Bands were numbered consecutively from anode to cathode. HEAT TREATMENT OF SERA
To determine if the IEF bands differed in their heat sensitivity, selected sera from clinical cases were placed in a 560 C water bath for exactly Fig. 1. A representative IEF gel of sera from two miscellaneous (lanes 1 and 2), two intestinal (lanes 15 min and immediately cooled on ice. 3 and 4) and two liver disease cases (lanes 5 and 6). Lane 1 was serum from a mare with postparturient trauma and laminitis and lane 2 was serum from an adult with bacterial Total AP activity before and after heat uterovaginal bronchopneumonia. The horse whose serum was tested in lane 3 had 2 m of necrotic ileum entrapped treatment was determined as des- in the epiploic foramen and lane 4 was serum from a case of Salmonella enteritis. Lanes 5 and 6 were cribed previously (Sigma Diagnostics, sera from cases of bile duct occlusion from cholelith formation.
128
natural or experimental liver disease. Any variation in the banding pattern between intestinal and miscellaneous groups could largely be explained by differences in the total AP activity. In addition, the IEF pattern of sera from horses with intestinal or miscellaneous diseases was similar irrespective of GGT or GDH activities. The one horse with pituitary dependent hyperadrenocorticism had a banding pattern comparable to other horses in the miscellaneous group. ORAL PREDNISOLONE AND PHENYLBUTAZONE STUDY
Neither prednisolone nor phenylbutazone produced a significant effect on the total serum AP activity or the IEF banding pattern (Table II). HEAT TREATMENT EXPERIMENT
Fig. 2. An IEF gel of the serum from two horses with experimental bile duct ligation. Samples were taken on day 0 (lanes 1 and 4), day 5 (lanes 2 and 5) and day 10 (lanes 3 and 6).
(GGT) and glutamate dehydrogenase levels (GDH) respectively. Of the horses with serum AP activities less than 150% of the upper reference limit, serum GGT and GDH activities were high in 4.5% and 9% respectively. Seventy-six percent of horses with serum AP activities greater than 150% of the upper reference limit had a primary diagnosis of either liver or intestinal disease. BILE DUCT LIGATION EXPERIMENT
Total serum AP activity increased significantly in the bile duct ligated horses to reach a mean of three times the presurgical level by day 10 (12). IEF RESULTS
The bands identified in the hospitalized and experimental horses had the same pl as those found in normal sera (7). In an occasional serum there were one or two additional faint bands in the pl range 6.4-7.0. There were differences among groups in the relative enzyme activity of bands (Fig. 1). Seventy-five percent of the AP activity in the horses with
experimental or natural cholestasis was confined to bands 3 to 7 (counted from the anode). The percentage of the total serum AP activity in these bands increased following bile duct ligation from 55% on day 0 to 78% on day 10 (p < 0.05). This accounted for most of the increased activity in serum (Figs. 2 and 3). In contrast, these bands made up just 36% of the total AP activity in the intestinal and miscellaneous groups (p < 0.05). The other main difference in band intensity between groups was in bands 8 to 13. In comparison with liver disease, these bands were very prominent in the sera of those horses with intestinal or miscellaneous diseases with high total serum AP activity (greater than 150% of upper reference limit) (Figs. 1 and 2). Overall these bands accounted for 13% of the total serum activity in the liver group and 44% in the other two groups (p < 0.05). The relative intensity of these bands increased as the total AP activity increased in horses with intestinal or miscellaneous diseases whereas this did not occur in cases of
Heating sera to 560 C for 15 min decreased the mean serum AP activity by 92% (range 86-95%). The IEF banding patterns were very faint in the heated samples with all bands similarly decreased.
4
5
7
8 I
11
(2
Fig. 3. A representantive densitometry trace of serum AP activity analyzed by IEF: (top) a horse before (narrow line) and ten days after (bold line) bile duct ligation; (bottom) a horse with a 180 degree torsion of the large colon that was surgically corrected.
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TABLE II. Serum alkaline phosphatase activity of horses given prednisolone and phenylbutazone Prednisolone Phenylbutazone Day Treateda Control Diffb Treated Control Diff 111 ± 13 0 124± 19 13 105±26 101 9 4 2 128± 15 114± 12 14 98±21 97±5 1 4 117± 13 117± 10 0 96±22 95±6 1 6 123± 14 122± 16 1 101 7 104±22 3 10 124± 11 118± 14 6 104±27 99±7 5 14 130±34 109±9 21 97±6 10 107±28 21 138±73 102±5 36 109±26 105 1 4 111± 33 94± 13 36 17 aValues are expressed as mean ± standard deviation bDifference of mean values between treated and control groups. There were no significant differences
DISCUSSION
Alkaline phosphatase is anchored to the hepatocyte cell membrane by covalent bonding to the glycosylated phospholipid, phosphatidylinositol (33). It is released from cell membranes in vitro by the action of bile acids, and phosphatidylinositolspecific phospholipase C (33,34). n-Butanol is believed to cause its release by activating lysosomal phosphatidyl-specific phospholipase C (35). The mechanism by which AP is released into serum in cholestasis is not fully understood but involves solubilization from the membranes by bile acids with subsequent leakage through tight junctions (34,36). This process results in more than one hepatic isoform in the sera of some people (37) and horses (16) with liver disease. In this study we have confirmed the presence of multiple isoforms in the sera of horses with primary liver disease and suggest, based on their very similar isoelectric points, that they are minor modifications of a single molecule. In addition, from comparisons with normal sera (7), there appears to be a shift in the main liver isoform from band 1 in normal adult sera to bands 3-7 in cholestatic sera. The serum banding pattern in horses with cholestasis was remarkably similar to normal foals which are believed to have high levels of the bone isoform. This confirms the close similarity between the bone and liver AP isoforms noted by others (5,6,10,11,27,28) and suggests that IEF of serum AP is not superior to other techniques for distinguishing horses with liver disease from those with bone disease. 130
In horses with serum AP activities greater than 150% of the upper reference limit there was sufficient contrast in the relative intensity of bands 3-7 and 8-13 to distinguish cases of primary cholestasis from intestinal or miscellaneous diseases. This occurred even in cases of the latter two groups where secondary liver disease was demonstrated by an increase in serum GGT and GDH activities. Consequently IEF appears to have clinical application in those horses with high serum AP activity of uncertain origin especially to differentiate primary cholestatic from secondary liver disease. The reasons for the similar banding patterns in the intestinal and miscellaneous groups remains undetermined. Although the most intense serum bands had similar isoelectric points to those from the small intestine (7) their heat sensitivity confirmed the work of others that this was an unlikely source (8,10). It has been suggested that the liver is the source of the AP in these cases (16). In some of our horses there is little doubt that the liver did contribute to the serum AP as individual horses had very high serum GGT, GDH and conjugated bilirubin levels. However, the reason for the different IEF pattern between horses with primary and secondary cholestatic liver disease cannot be readily explained. One possible explanation is that the liver isoform is modified in serum by a factor common to the intestinal and miscellaneous groups but absent in the primary liver disease group. Inflammatory or bacterial proteases and glycosylases may be such factors as most horses with intestinal and
miscellaneous diseases had an obvious inflammatory focus or site for bacterial invasion. Alternatively, the form of AP released into the circulation from the liver may be different in extrahepatic compared with intrahepatic cholestasis. All but one of the horses with primary liver disease had confirmed extrahepatic cholestasis whereas the cholestasis was most likely intrahepatic in the secondary cases as there was no evidence of bile duct occlusion in these animals. A comparative study of the IEF properties of AP extracted from livers with extrahepatic versus intrahepatic cholestasis may help to evaluate this possibility. The increase in serum AP activity in dogs given glucocorticoids results from release of the normal hepatic isoform and induction of a novel hepatic isoform (steroid-induced isoform) (19-21). This occurs within 6-14 days of prednisolone treatment at 2 mg/kg (19,38). Isoelectric focusing is a sensitive method of separating these isoforms (30,3 1). Our failure to demonstrate any increase in total serum AP or change in the banding pattern in the horse with similar dose rates suggests glucocorticoid therapy is not an important consideration when evaluating the cause for high serum AP in this species, especially as the dose rate used was greater than recommended (39). The absence of a steroid-induced isoform in the horse is supported by the unremarkable serum activity and banding pattern in the one horse with hyperadrenocorticism. The ability of phenylbutazone to induce an increase in serum AP activity is dose dependent. A dose rate of 10 mg/kg/day for 14 days caused a two to fivefold increase in serum AP activity and toxicity signs in ponies (24). Approximately half this dose rate is now recommended and the results of this and other studies indicates this does not cause an increase in serum activity of AP or other liver enzymes (40,41).
ACKNOWLEDGMENTS We are very grateful for the generosity of Dr W. Hoffmann of The University of Illinois for supplying us with the serum samples from the bile duct ligated horses.
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