Veterinary Research Communications, 15 (1991) 421-426 Copyright 8 Kluwer Academic Publishers bv - Printed in the Netherlands
THE ENZYMATIC PROFILE OF URINE AND PLASMA IN BOVINE URINARY BLADDER CANCER (ENZOOTIC BOVINE HAEMATURIA) R.K. DAWRA, O.P. SHARMA, L. KRISHNA AND J. VAID Laboratories of Biochemistry and Disease Investigation, Indian Veterinary Research Institute, Regional Station, Palampur, Kangra Valley, H.P. 176 061, India
ABSTRACT Dawra, R.K., Sharma, O.P., Krishna, L. and Vaid, J., 1991. The enzymatic profile of urine and plasma in bovine urinary bladder cancer (enzootic bovine haematuria). Veterimny Research Communications, 15 (6), 421-426
The enzymatic profile of urine and plasma in field cases of bovine bladder cancer was studied. Urinary lactate dehydrogenase activity was significantly altered along with the isoenzyme pattern. Activity of alkaline phosphatase and fl-glucuronidase was decreased in the affected animals. No significant changes were observed in acid phosphatase, or arylsulphatase A and B activity. In plasma, lactate dehydrogenase activity was elevated without any change in the isoenzyme pattern. No significant changes were observed in the other plasma enzymes studied or in the sialic acid concentration. Keyword:
acid phosphatase, bladder, cancer, cattle, haematuria, lactate dehydrogenase, urine
INTRODUCTION Bovine urinary bladder cancer is common in isolated pockets of the upland areas of the world (Pamukcu, 1963; Vaid et al., 1981). The disease is commonly known as enzootic bovine haematuria (EBH). In these areas cattle and buffaloes start passing blood in urine after attaining the age of 4 or 5 years. This is the overt sign of the malady (Rosenberger, 1971). The disease runs a chronic course with several intermissions when the signs disappear altogether and the animal appears normal (Datta, 1952). The main seat of lesions is the urinary bladder (Pamukcu, 1955). The tumours in the urinary bladder are epithelial or mesenchymal in origin or of mixed types, which often show evidence of malignancy and metastasis to regional lymph nodes and occasionally to some distant organs (Pamukcu, 1955; Dawra ef al., 1989). No consensus exists about the aetiology of disease, though chronic ingestion of bracken fern (Pteridiunt aquilinunt) and/or latent infection with bovine papilloma virus have been implicated (Hopkins, 1986). The usual therapy suggested for bladder cancer in humans, using chemotherapeutic agents, radiation or surgery, cannot be used in these cattle for economic reasons and because of the large number of animals involved. Diagnosis of the disease at an early stage might be helpful in controlling it by moving the animals to disease free regions. The only diagnostic test available so far is the presence of erythrocytes in urine, which may be absent during remission (Vaid et al., 1981). Studies on the enzymes and chemical constituents in urine, serum or tissues have been used for the early diagnosis and prognosis of various types of
422
cancers in humans (Bergmeyer, 1974a). The urinary and blood enzymes and chemical constituents have not previously been investigated in detail in enzootic bovine haematuria. Such information could be of value in the early detection and prognosis of the malady. We report here the enzymatic and chemical profile of urine and blood plasma from bovine cases suffering from urinary bladder cancer.
MATERIALS
AND METHODS
Animals
Animals that had a clinical history of enzootic bovine haematuria and were in the active stage of disease were bled in the field. The blood was collected in heparinized vials and plasma was prepared by centrifugation. This was transported in liquid nitrogen to the laboratory and thawed just before use. Of the 30 animals used for plasma studies, 10 were purchased for follow-up studies and transported to the research laboratory where they were subjected to detailed necropsy and histopathological examination upon death in the natural course of the malady. Comparable controls were taken from a disease-free locality. For studies on urine, animals that had a clinical history of the disease but were not passing blood were selected. Eight-hourly urine samples were collected directly on ice from affected and control animals and centrifuged at 10 000 g for 20 min at 4°C. The supernatant was concentrated 20 times by ultrafiltration using a 10 000 MW cut-off membrane filter (Amicon UM-10). The concentrated urine was used for the enzyme estimations (Mattenheimer, 1974). The chemical constituents were measured before ultrafiltration. The sediments were resuspended in a small quantity of urine for microscopic examination. All the urine samples were negative for erythrocytes, free from bacterial contamination, and were either negative or contained only 2-3 pus cells per high-power field. Occasional samples contained epithelial cells.
Chemical estimations
Creatinine was estimated in the urine using an alkaline picrate reagent (Oser, 1965) and protein by the method of Lowry et al. (1951). Sialic acid in plasma was assayed by the procedure of Shamberger (1984) using Ehrlich’s reagent.
Enzyme assays
Lactate dehydrogenase (EC 1.1.1.27) (LDH), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), glutamic oxaloacetate transaminase (EC 2.6.1.1), glutamic pyruvate transaminase (EC 2.6.1.2), &gl ucuronidase (EC 3.2.1.31), y-glutamyltranspeptidase (EC 2.3.2.2), leucine aminopeptidase (EC 3.4.11.1) and acetylcholinesterase (EC 3.1.1.7) were determined in plasma or urine by spectrophotometric or calorimetric methods as described by Bergmeyer (1974). Arylsulphatase A (EC 3.1.6.1) and B (EC 3.1.6.1) activities were measured in urine by the method of Baum et al. (1959). The data for the urinary enzymes have been expressed as units/g
423
creatinine. LDH isoenzymes in plasma and concentrated urine were determined after electrophoresis on cellulose acetate strips (Sharma er al., 1982). The isoenzyme patterns were assessed on the basis of the relative staining intensity of the different bands. Group comparisons were made by Student’s r-test (Snedecor and Cochran, 1961).
Patltological studies
A detailed necropsy was performed and pieces of urinary bladder, kidneys, iliac lymph nodes, liver, spleen and lungs along with visible or suspected tumours in the urinary bladder were collected in 10% formalin for histopathological examination. Paraffin-embedded tissues were cut into sections (5-6 pm) and stained with haematoxylin and eosin (Culling, 1957).
Cltertticals
Phenolphthalein glucuronide, r-glutamyl-p-nitroanilide, L-leucinamide hydrochloride and nitrocatechol sulphate were obtained from Sigma Chemical Company, St. Louis, MO, USA. Nicotine adenine dinucleotide (NADH), p-nitrophenyl phosphate, phenazine methosulphate (PMS) and Nitro blue tetrazolium (NBT) were procured from Sisco Research Laboratory, Bombay, India. Cellulose acetate strips were purchased from Oxoid, UK. Other chemicals were of analytical grade.
RESULTS All the 9 animals used for urine studies showed transitional cell carcinoma of the urinary bladder. In one animal there were metastatic lesions of adenocarcinoma in the liver and ileac lymph node. In addition, plexiform haemangioma was present in two animals, while in another two cystic glandularis was seen. Similar lesions have been reported from EBH-affected animals by Pamukcu (1963). Table I shows the values of the different enzymes and chemical constituents in urine and plasma from EBH-affected and control animals. Lactate dehydrogenase activity was significantly higher in the urine from EBH-affected animals. The isoenzyme pattern revealed elevated LDH2 and LDH4 in the urine from the affected animals while LDHl and LDH2 were predominant in urine from normal animals. The &glucuronidase and alkaline phosphatase activities in the urine from the affected animals were significantly lower than in that from the control group (Table I). No significant changes were observed in the acid phosphatase and arylphosphatase A and B activities. The urinary protein and creatinine concentrations were considerably raised in urine from the affected animals as compared to that from the control animals (Table I), but there was no significant difference in the protein/creatinine ratio. The lactate dehydrogenase activity was also significantly raised in the plasma of EBH-affected animals (Table I) while the activities of other enzymes and the sialic acid concentration were not significantly different from those of the control group.
424
The absence of changes in plasma glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activities is in agreement with earlier observations (Singh et al., 1973).
TABLE I An enzymic and chemical control animals
profile
of the urine and plasma of EBH-affected
EBH-affected
and
Control
Urine: Lactate dehydrogenase (units/g creatinine)
9.63 2 5.40 (7)
3.95 2 1.89d (7)
Alkaline phosphatase (units/g creatinine)
0.36 2 0.23 (7)
0.74 2 0.47e (7)
@-Glucuronidase (units/g creatinine)
0.54 2 0.32 (7)
1.01 ” o.30c (7)
2.09 2 1.28 (5)
0.66 f 0.21C (7)
1.07 2 0.33 (8)
0.59 + 0.16a (8)
1180.70 f 258.90 (30)
927.34 + 127.81b (14)
Protein (g/L) Creatinine (g/L) Plasma: Lactate dehydrogenase
Values are mean f SD; the significance of the difference between the affected and control groups are indicated by the suffuses: ap
THE ENZYMATIC PROFILE OF URINE AND PLASMA IN BOVINE URINARY BLADDER CANCER (ENZOOTIC BOVINE HAEMATURIA) R.K. DAWRA, O.P. SHARMA, L. KRISHNA AND J. VAID Laboratories of Biochemistry and Disease Investigation, Indian Veterinary Research Institute, Regional Station, Palampur, Kangra Valley, H.P. 176 061, India
ABSTRACT Dawra, R.K., Sharma, O.P., Krishna, L. and Vaid, J., 1991. The enzymatic profile of urine and plasma in bovine urinary bladder cancer (enzootic bovine haematuria). Veterimny Research Communications, 15 (6), 421-426
The enzymatic profile of urine and plasma in field cases of bovine bladder cancer was studied. Urinary lactate dehydrogenase activity was significantly altered along with the isoenzyme pattern. Activity of alkaline phosphatase and fl-glucuronidase was decreased in the affected animals. No significant changes were observed in acid phosphatase, or arylsulphatase A and B activity. In plasma, lactate dehydrogenase activity was elevated without any change in the isoenzyme pattern. No significant changes were observed in the other plasma enzymes studied or in the sialic acid concentration. Keyword:
acid phosphatase, bladder, cancer, cattle, haematuria, lactate dehydrogenase, urine
INTRODUCTION Bovine urinary bladder cancer is common in isolated pockets of the upland areas of the world (Pamukcu, 1963; Vaid et al., 1981). The disease is commonly known as enzootic bovine haematuria (EBH). In these areas cattle and buffaloes start passing blood in urine after attaining the age of 4 or 5 years. This is the overt sign of the malady (Rosenberger, 1971). The disease runs a chronic course with several intermissions when the signs disappear altogether and the animal appears normal (Datta, 1952). The main seat of lesions is the urinary bladder (Pamukcu, 1955). The tumours in the urinary bladder are epithelial or mesenchymal in origin or of mixed types, which often show evidence of malignancy and metastasis to regional lymph nodes and occasionally to some distant organs (Pamukcu, 1955; Dawra ef al., 1989). No consensus exists about the aetiology of disease, though chronic ingestion of bracken fern (Pteridiunt aquilinunt) and/or latent infection with bovine papilloma virus have been implicated (Hopkins, 1986). The usual therapy suggested for bladder cancer in humans, using chemotherapeutic agents, radiation or surgery, cannot be used in these cattle for economic reasons and because of the large number of animals involved. Diagnosis of the disease at an early stage might be helpful in controlling it by moving the animals to disease free regions. The only diagnostic test available so far is the presence of erythrocytes in urine, which may be absent during remission (Vaid et al., 1981). Studies on the enzymes and chemical constituents in urine, serum or tissues have been used for the early diagnosis and prognosis of various types of
422
cancers in humans (Bergmeyer, 1974a). The urinary and blood enzymes and chemical constituents have not previously been investigated in detail in enzootic bovine haematuria. Such information could be of value in the early detection and prognosis of the malady. We report here the enzymatic and chemical profile of urine and blood plasma from bovine cases suffering from urinary bladder cancer.
MATERIALS
AND METHODS
Animals
Animals that had a clinical history of enzootic bovine haematuria and were in the active stage of disease were bled in the field. The blood was collected in heparinized vials and plasma was prepared by centrifugation. This was transported in liquid nitrogen to the laboratory and thawed just before use. Of the 30 animals used for plasma studies, 10 were purchased for follow-up studies and transported to the research laboratory where they were subjected to detailed necropsy and histopathological examination upon death in the natural course of the malady. Comparable controls were taken from a disease-free locality. For studies on urine, animals that had a clinical history of the disease but were not passing blood were selected. Eight-hourly urine samples were collected directly on ice from affected and control animals and centrifuged at 10 000 g for 20 min at 4°C. The supernatant was concentrated 20 times by ultrafiltration using a 10 000 MW cut-off membrane filter (Amicon UM-10). The concentrated urine was used for the enzyme estimations (Mattenheimer, 1974). The chemical constituents were measured before ultrafiltration. The sediments were resuspended in a small quantity of urine for microscopic examination. All the urine samples were negative for erythrocytes, free from bacterial contamination, and were either negative or contained only 2-3 pus cells per high-power field. Occasional samples contained epithelial cells.
Chemical estimations
Creatinine was estimated in the urine using an alkaline picrate reagent (Oser, 1965) and protein by the method of Lowry et al. (1951). Sialic acid in plasma was assayed by the procedure of Shamberger (1984) using Ehrlich’s reagent.
Enzyme assays
Lactate dehydrogenase (EC 1.1.1.27) (LDH), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), glutamic oxaloacetate transaminase (EC 2.6.1.1), glutamic pyruvate transaminase (EC 2.6.1.2), &gl ucuronidase (EC 3.2.1.31), y-glutamyltranspeptidase (EC 2.3.2.2), leucine aminopeptidase (EC 3.4.11.1) and acetylcholinesterase (EC 3.1.1.7) were determined in plasma or urine by spectrophotometric or calorimetric methods as described by Bergmeyer (1974). Arylsulphatase A (EC 3.1.6.1) and B (EC 3.1.6.1) activities were measured in urine by the method of Baum et al. (1959). The data for the urinary enzymes have been expressed as units/g
423
creatinine. LDH isoenzymes in plasma and concentrated urine were determined after electrophoresis on cellulose acetate strips (Sharma er al., 1982). The isoenzyme patterns were assessed on the basis of the relative staining intensity of the different bands. Group comparisons were made by Student’s r-test (Snedecor and Cochran, 1961).
Patltological studies
A detailed necropsy was performed and pieces of urinary bladder, kidneys, iliac lymph nodes, liver, spleen and lungs along with visible or suspected tumours in the urinary bladder were collected in 10% formalin for histopathological examination. Paraffin-embedded tissues were cut into sections (5-6 pm) and stained with haematoxylin and eosin (Culling, 1957).
Cltertticals
Phenolphthalein glucuronide, r-glutamyl-p-nitroanilide, L-leucinamide hydrochloride and nitrocatechol sulphate were obtained from Sigma Chemical Company, St. Louis, MO, USA. Nicotine adenine dinucleotide (NADH), p-nitrophenyl phosphate, phenazine methosulphate (PMS) and Nitro blue tetrazolium (NBT) were procured from Sisco Research Laboratory, Bombay, India. Cellulose acetate strips were purchased from Oxoid, UK. Other chemicals were of analytical grade.
RESULTS All the 9 animals used for urine studies showed transitional cell carcinoma of the urinary bladder. In one animal there were metastatic lesions of adenocarcinoma in the liver and ileac lymph node. In addition, plexiform haemangioma was present in two animals, while in another two cystic glandularis was seen. Similar lesions have been reported from EBH-affected animals by Pamukcu (1963). Table I shows the values of the different enzymes and chemical constituents in urine and plasma from EBH-affected and control animals. Lactate dehydrogenase activity was significantly higher in the urine from EBH-affected animals. The isoenzyme pattern revealed elevated LDH2 and LDH4 in the urine from the affected animals while LDHl and LDH2 were predominant in urine from normal animals. The &glucuronidase and alkaline phosphatase activities in the urine from the affected animals were significantly lower than in that from the control group (Table I). No significant changes were observed in the acid phosphatase and arylphosphatase A and B activities. The urinary protein and creatinine concentrations were considerably raised in urine from the affected animals as compared to that from the control animals (Table I), but there was no significant difference in the protein/creatinine ratio. The lactate dehydrogenase activity was also significantly raised in the plasma of EBH-affected animals (Table I) while the activities of other enzymes and the sialic acid concentration were not significantly different from those of the control group.
424
The absence of changes in plasma glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activities is in agreement with earlier observations (Singh et al., 1973).
TABLE I An enzymic and chemical control animals
profile
of the urine and plasma of EBH-affected
EBH-affected
and
Control
Urine: Lactate dehydrogenase (units/g creatinine)
9.63 2 5.40 (7)
3.95 2 1.89d (7)
Alkaline phosphatase (units/g creatinine)
0.36 2 0.23 (7)
0.74 2 0.47e (7)
@-Glucuronidase (units/g creatinine)
0.54 2 0.32 (7)
1.01 ” o.30c (7)
2.09 2 1.28 (5)
0.66 f 0.21C (7)
1.07 2 0.33 (8)
0.59 + 0.16a (8)
1180.70 f 258.90 (30)
927.34 + 127.81b (14)
Protein (g/L) Creatinine (g/L) Plasma: Lactate dehydrogenase
Values are mean f SD; the significance of the difference between the affected and control groups are indicated by the suffuses: ap
