Elevation of urinary enzyme levels in rat bladder carcinogenesis

Emile M. Youssef1, Hideki Wanibuchi, Satoru Mori, Elsayed I. Salim, Shuji Hayashi and Shoji Fukushima2

First Department of Pathology, Osaka City University Medical School,1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Urinary enzyme levels were investigated in rats administered different promoters in their diet for 32 weeks after being initiated by treatment with 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine in their drinking water for 4 weeks. All groups were composed of 10 rats each. Group 1: females treated with 3% uracil (100% carcinoma incidence). Group 2: control females kept on basal diet only (0% carcinoma incidence). Group 3: males treated with 5% sodium L-ascorbate (100% carcinoma incidence). Group 4: control males (0% carcinoma incidence). Urine was collected at the end of weeks 12, 24 and 36 and tested for lactate dehydrogenase (LDH), alkaline phosphatase, N-acetyl-ß-D-glucosaminase and aspartate aminotransferase activity. To facilitate comparison, data were related to the corresponding excreted creatinine levels. All measurements were made using a centrifugal automatic analyzer. The urine of rats with cancer lesions (groups 1 and 3) showed significant elevation in all enzyme activities at weeks 24 and/or 36 except for LDH in females (group 1). The M/H ratio of the LDH isozymes was reversed (1.10 ± 0.10) in the tested rats with carcinomas at week 36. This study thus provides evidence of a correlation between high urinary enzyme levels and cancer development in the rat bladder. Measurement of the tested enzymes might thus provide a method to detect malignant changes in bladder epithelium by direct urine analysis.

Abbreviations: AAT, aspartate aminotransferase; ALP, alkaline phosphatase; BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; HE, hematoxylin and eosin; LDH, lactate dehydrogenase; Na-AsA, sodium L-ascorbate; NAG, N-acetyl-ß-D-glucosaminase; PN, papillary or nodular.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Much attention is being devoted in the biochemistry field to the search for disease biomarkers. Recently, a number of advances have been made in the detection of cancers. Estimation of enzyme levels excreted in the urine is a non-invasive method for diagnosis of urinary bladder lesions. There are methodological problems because urine is an unfavorable medium (13) but this obstacle has recently been overcome by the development of novel assays (46). Except for very low levels due to renal tubular cell turnover, the presence of enzymes in the urine is an abnormal finding since their high molecular weights normally prevent passage through the renal glomerular membrane (7).

Lactate dehydrogenase (LDH, EC 1.1.1.27) is mainly located in the distal tubular cells of the kidney, but is also detected in blood and released by various ischemic organs (8). It has been extensively investigated for applicability as a tumor marker for human bladder carcinomas (7) and a cytochemical analysis technique for its demonstration has been described (9).

Sporadic reports related to high values of urinary alkaline phosphatase (ALP, EC 3.1.3.1) in cases of human bladder carcinomas have also been published (1013). Amador et al. (14) demonstrated an increase in urinary ALP in the absence of primary urinary tract disorders following acute hemorrhage and neoplasms of the adrenal cortex but no reports are available of animal model changes. Because this enzyme is very sensitive to inhibitors usually found in urine, the methodology is very important for its detection.

N-acetyl-ß-D-glucosaminase (NAG, EC 3.2.1.30), a lysosomal enzyme and a member of the glycosidase family, might be expected to correlate with malignancy since malignant cells can produce enzymes that catabolize glycosaminoglycans. NAG has in fact been reported to be a sensitive marker for neoplasms of the breast and digestive tract (15). High levels of aspartate aminotransferase enzyme (AAT, EC 2.6.1) are reported to be linked with liver carcinogenesis caused by N,N-dibutylnitrosamine in ACI/N rats (16). Rawson and Peto (17) also recommended that serum AAT should be considered as a prognostic factor for small cell lung cancer. To our knowledge, no reports are available concerning NAG or AAT in bladder carcinogenesis either in man or in an animal model.

The purpose of the present study was to assess levels of different urinary enzymes (LDH, ALP, NAG and AAT) during carcinogenesis in the rat bladder using our standard experimental protocol (18).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Twenty female and 20 male 5-week-old F344 rats (Charles River Japan, Hino, Shiga, Japan) were used in this study. The rats were housed five per cage in an animal room with a 12 h light/12 h dark cycle at a temperature of 22 ± 2°C and a 55 ± 5% humidity. Animals were observed daily to assess general health.

Chemicals
N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was obtained from Tokyo Kasei Kogyo (Tokyo, Japan) and administered in drinking water in opaque bottles at a concentration of 0.05%, renewed at intervals of 3 days. 2,4-Dioxypyrimidine (uracil) was obtained from Yamasa Shoyu (Chiba, Japan). Its administration was at a concentration of 3% in Oriental MF powdered basal diet (Oriental Yeast, Tokyo). Sodium L-ascorbate (Na-AsA) was purchased from Wako Pure Chemical Industries (Osaka, Japan) and administered at a concentration of 5% in the same diet.

Experimental design
F344 rats were divided into four groups, each of 10 rats. All were first treated with BBN for 4 weeks as an initiator. The subsequent treatment varied for each group until the end of the experiment at week 36. Group 1 females were treated with 3% uracil in basal diet while group 2 females were maintained on basal diet as controls. Group 3 males were treated with 5% Na-AsA in basal diet and group 4 males were maintained as untreated controls.

Pathological examination
At the beginning of week 37, all the rats were killed under ether anesthesia and their urinary bladders were inflated by intraluminal injection of 10% phosphate-buffered formalin solution. After fixation, each bladder was divided sagittally in two and each half was cut into four strips for histopathological examination. Sections of paraffin-embedded specimens were cut and stained with hematoxylin and eosin (HE) for examination under a light microscope. The kidneys were also examined both macroscopically and microscopically.

Urinalysis
At weeks 12, 24 and 36, rats were housed individually in metabolic cages without food or water for 6 h in the early morning. Urine was collected and used immediately for analytical tests. The use of metabolic cages, as described by Lethwood and Plummer (19), is warranted because rat feces are very rich in enzymes, especially ALP (20). All measurements were made using a centrifugal automatic analyzer (model nos. 7450, 7150 and 7170; Hitachi, Tokyo, Japan). Urine specimens were diluted to minimize the effect of enzyme inhibitors (21). The methods used for individual tests have been described previously elsewhere (22,23). LDH activity was measured by a spectrophotometric method using the LDH UV test and expressed in U/mg creatinine. Electrophoretic separation of LDH isozymes was performed on agarose gels as described by van der Helm (24). The relative percentage of the H form was calculated assuming 0, 25, 50, 75 and 100% for the respective LDH isoforms: M4 (LDH1), M3H (LDH2), M2H2 (LDH3), MH3 (LDH4) and H4 (LDH5). ALP activity was analyzed by the p-nitrophenyl phosphate method (25). NAG activity was determined by the 3-cresol sulfonphthaleinyl NAG, sodium salt method (26,27). AAT activity was analyzed by the coupled enzymatic reaction of malate dehydrogenase and NADH (28). Variation in the urine volume can make quantification of urinary enzymes difficult and therefore enzyme activities were also expressed relative to the creatinine excreted (29,30), assessed by Jaffe's method and expressed in mg/dl (31). Osmolarity was determined by the freezing point depression method using an Osmett A (Precision System, Sudbury, MA). The numbers of red and white blood corpuscles were also determined because increased urinary LDH activity may also occur when leukocytes or erythrocytes are present in urine. Samples contaminated by more than five cells per high power microscopic field were excluded (32). However, The resultant numbers of rats examined were 8–10/group at each analysis point.

Statistical methods
The two-sided Fisher exact probability test was used to assess urinary bladder lesion incidences. Differences found between groups were studied with the Kruskal–Wallis test for comparison of more than two groups and the Mann–Whitney test for comparison of two groups. Correlation coefficients (r values) were assessed using the StatView statistical program on a personal computer. Urinary enzyme levels were calculated using the equation urinary enzyme/urinary creatinine (2930) and M/H ratio by the equation (LDH2x1) + (LDH3x2) + (LDH4x3) + (LDH5x4) ÷ (LDH1x4) + (LDH2x3) + (LDH3x4) + (LDH4x5) (9). All values are given as means ± standard errors (SE) or the standard deviations (SD). P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IGo summarizes data for initial and final body weights and absolute bladder weights. Groups 1 and 3 both showed significant reductions in body weight in comparison with their corresponding controls, along with significant elevations in absolute bladder weights. Table IIGo shows data on the incidences of epithelial proliferative lesions of the bladder: papillary or nodular (PN) hyperplasias, papillomas and carcinomas (33). The incidences of carcinoma and papilloma lesions were significantly elevated in groups 1 and 3, with ~2.5 malignancies/bladder. All carcinomas obtained were of the transitional cell type showing varying degrees of atypia. Mitotic figures were frequent. The majority of lesions were moderately differentiated. The transitional epithelial cells in papillomas were arranged as branched finger-like processes with a delicate fibrovascular core or showed an endophytic growth pattern. Cellular irregularity was also slight in papillomas in comparison with carcinomas (Figure 1Go).


View this table:
[in this window]
[in a new window]
 
Table I. Body and bladder weights for all groups
 

View this table:
[in this window]
[in a new window]
 
Table II. Quantitative data for PN hyperplasias, papillomas and carcinomas
 


View larger version (154K):
[in this window]
[in a new window]
 
Fig. 1. HE staining of moderately differentiated transitional cell carcinoma of the female rat bladder. The tumors induced by 3% uracil promoter in the diet for 32 weeks after being initiated by treatment with 0.05% BBN in the drinking water for 4 weeks. Magnification x200.

 
Table IIIGo summarizes data for urine chemistry at the end of the experiment. Groups 1 and 3 showed hypo-osmolarity in comparison with their corresponding controls. The kidneys showed no morphological changes in any group.


View this table:
[in this window]
[in a new window]
 
Table III. Urinary chemistry data for all groups at the end of the experiment
 
Table IVGo summarizes data for all urinary enzymes tested and their sums at weeks 12, 24 and 36. No significant sex differences were evident in urinary enzyme levels for rats of the control groups (groups 2 and 4). Groups 1 and 3 demonstrated significant elevations of both carcinoma incidence and multiplicity and also different enzymes at both weeks 24 and 36 (Table IVGo). At week 24 NAG and AAT were affected while at week 36 all enzymes were changed in at least one of the groups. The sums of all enzymes in groups 1 and 3 were significantly elevated at week 36.


View this table:
[in this window]
[in a new window]
 
Table IV. Urinary enzyme activities relative to the corresponding creatinine levels for all groups at weeks 12, 24 and 36
 
At the end of the experiment, the correlation coefficients (r) for different enzymes were: group 1, r = 0.6 and 0.4 for NAG with ALP and AAT, respectively; group 3, r = 1.0, 0.6 and 0.8 for ALP with AAT, LDH and NAG, respectively; r = 0.8 and 0.8 for AAT with LDH and NAG, respectively. The r value for NAG with LDH was 0.8. No other significant correlations between groups were observed.

An analysis of LDH isozymes was also conducted (Table VGo). Three rats from each group at both weeks 12 and 36 were investigated. Although there were no statistically significant differences in terms of the percentages of each isozyme, the M/H ratio was increased in the cancer groups at the end of the experiment. In the cancer groups there were significant differences between the values at weeks 36 and 12. In the non-cancer groups the differences between weeks 36 and 12 were not significant but there was a trend towards M/H inversion at week 36 (0.90 ± 0.10). Figure 2Go illustrates a typical spectrum showing the percentages of the different LDH isozymes in group 3 at week 36.


View this table:
[in this window]
[in a new window]
 
Table V. Comparison of LDH isozymes between carcinoma- and non-carcinoma-bearing rats
 


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Profile of LDH isoenzymes in urine from a carcinoma-bearing rat at week 36. Note the predominance of LDH3, LDH4, and LDH5 over LDH1 and LDH2 (see Materials and methods). The M/H ratio was inverted with a value of 1.16.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the present study, we have demonstrated that elevation of certain enzymes in the urine may reflect malignant changes in the rat bladder epithelium. Under normal conditions, urinary enzymes may originate from four sources: blood contamination, the kidneys, glands in the urogenital tract and the covering epithelium (7). In the present study, we could exclude the possibility of leukocytes or erythrocytes as the origin. The tendency for inversion of the M/H ratio for LDH (Table VGo) further argues against a role for blood. Although ALP could be detected in normal urine due to cellular turnover in the renal tissues, this could not explain the presence of higher levels of enzymes in rats with cancers. Furthermore, there were high correlation coefficients for ALP with the other enzymes (LDH, NAG and AAT). The lack of any significant differences between the sexes for either the control or the cancer groups excludes the possibility of a seminal plasma origin for LDH (34). Finally, the enzyme content of urogenital epithelial cells is relatively low and would not be expected to be a significant source of urinary enzymes (7).

Therefore, pathological conditions must have been responsible. Bacterial infection of the kidneys, pyelonephritis and renal abscesses could contribute to abnormal levels of some urinary enzymes. Indeed, renal toxicity can be diagnosed on this basis (7,3537). However the finding for blood corpuscles and microscopic histological examination of renal tissues were negative. A previous study (38), including scanning electron microscopy, showed that no major changes in the renal pelvis are caused by either Na-AsA or uracil.

Previous studies (7,14) have suggested that tumors of the urogenital tract could contribute to the presence of LDH and ALP enzymes in the urine but activities were found to be non-specific with regard to the type of malignancy (7). Urinary LDH has been proposed as a tumor marker for human bladder carcinomas (39,40), but Fujii et al. (9) demonstrated a positive cytochemical reaction for LDH in exfoliated bladder epithelium of rats in which hyperplasia was caused by BBN administration in the drinking water for 8 weeks (9). They also found, however, that while 90% of high grade cancers (with invasion) exfoliated into the urine, this was the case for only 35% of lower grade cancers (without invasion).

The synthesis of the M and H subunits of the LDH molecule is affected by environmental conditions. M-type LDH is involved in anerobic reduction of pyruvate, while H-type LDH is activated under high oxygen tension conditions (41). In malignancies, anerobic glycolysis predominates (42) and thus, synthesis of the H-type would be expected to be reduced. In both human (43) and rat cancers (9), a clear elevation of the M/H ratio has been demonstrated in bladder cancers. This is in line with our present results. The M/H ratio was 1.10 ± 0.10 at week 36 for the animals bearing tumors, as opposed to 0.9 in the control groups. However, the early reversion shown previously by Fujii et al. (9) after only 8 weeks of BBN treatment was not evident. Further study of a larger population is necessary to determine how early this inversion can be detected during carcinogenesis.

Renal and bladder tumors of man are frequently accompanied by elevated urinary ALP (10). This has been found to be particularly the case with bladder tumors (14). Data on ALP activity levels of bladder epithelium are conflicting. While Tsuda et al. (11) described a decrease in ALP activity levels in transitional cell carcinomas of either human or rat origin and Wilson et al. (12) reported the same finding after treatment with bladder carcinogens in vivo in rats and mice, El-Sharabasy and El-Dosoky (13) found significant elevation of ALP in neoplastic bladder tissues. Histochemistry is influenced by several factors, such as type of tissue fixation, the solubility of the substrates and the length of incubation (44). In the present case, activities were measured directly in the urine using an automatic analyzer system. The question of whether anomalous findings are due to methodological differences remains to be clarified. To our knowledge, this is the first report of a correlation between NAG or AAT and bladder tumorigenesis in rats. Although glycosidase levels in general have been widely studied in sera in relation to lysosomal storage disease, they have received little attention with regard to carcinogenesis (15,45). Severini et al. (15) concluded that NAG is more promising than ß-glucuronidase, another lysosomal glycosidase, especially with regard to gastric and breast cancer. In line with previous reports for man (46) as well as rats (47), there was no sex dependence of the NAG values in the present study. Spot urine determination of the NAG/creatinine ratio was found to significantly correlate with 24 h urinary NAG excretion (48), but this source of variation could be ruled out by the standard timing of sampling. Furthermore, urinary excretion of NAG does not change in response to osmotic diuresis induced by mannitol (49). While urinary enzyme levels can be elevated by hyper-osmolarity of the urine (50), this was not the case in the present study since the ingested chemicals were associated with a drop in osmolarity. Okada et al. (16) reported that BBN does not cause any elevation of either AAT or ALP in serum and further concluded that changes in these enzymes might be related to liver carcinogenesis. This could also exclude any role for BBN treatment in enzyme elevation in our present study. Experimental investigations in rats demonstrated differences in behavior of AAT in urine as compared with other aminotransferases, such as alanine aminotransferase (51). AAT also was detected in variable amounts in all tested rats.

In conclusion, the mathematical sum of LDH, ALP, NAG and AAT activities divided by the corresponding urinary creatinine excretion in the present study could be an indicator of bladder tumor development. This non-invasive approach clearly deserves further attention and confirmation in the human case.


    Acknowledgments
 
The present study was supported by a grant-in-aid for scientific research on a priority area from the Ministry of Education, Science, Sports, and Culture of Japan.


    Notes
 
1 Present address: Department of Molecular Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan Back

2 To whom correspondence should be addressed Email fukuchan{at}med.osaka-cu.ac.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Price,R.G. (1982) Urinary enzymes, nephrotoxicity and renal disease. Toxicology, 23, 99–134.[ISI][Medline]
  2. Guder,W.G. and Heidland,A. (1986) Urine analysis. Report on the workshop conference of the German Society for Clinical Chemistry and the Society of Nephrology in Würzburg. J. Clin. Chem. Clin. Biochem., 24, 611–620.[ISI][Medline]
  3. Maruhn,D. (1979) Preparation of urine for enzyme determination by gel filtration. Curr. Probl. Clin. Biochem., 9, 22–30.[Medline]
  4. Matteucci,E., Gregori,G., Pellegrini,L., Navalesi,R. and Giampietro,O. (1991) Effects of storage time and temperature on urinary enzymes. Clin. Chem., 37, 1436–1441.[Abstract/Free Full Text]
  5. Matteucci,E., Gregori,G., Pellegrini,L., Navalesi,R. and Giampietro,O. (1991) How can storage time and temperature affect enzymic activities in urine? Enzyme, 45, 116–120.[ISI][Medline]
  6. Matteucci,E. and Giampietro,O. (1994) To store urinary enzymes: how and how long? Kidney Int. Suppl., 47, S58-S59.[Medline]
  7. Raab,W.P. (1972) Diagnostic value of urinary enzyme determinations. Clin. Chem., 18, 5–25.[Abstract/Free Full Text]
  8. Anundi,I., King,J., Owen,D.A., Schneider,H., Lemasters,J.J. and Thurman,R.G. (1987) Fructose prevents hypoxic cell death in liver. Am. J. Physiol., 253, G390–G396.[Abstract/Free Full Text]
  9. Fujii,M., Mori,H., Kato,K. and Takahashi,M. (1982) Cytochemical studies of LDH isoenzymes in experimental bladder tumors. J. Urol., 128, 1349–1352.[ISI][Medline]
  10. Dubach,U. C. and Padlina,G. (1966) Aktivitat der alkalise Phosphatase im Urin. Klin. Wochenschr., 44, 180–186.[ISI][Medline]
  11. Tsuda,H., Inoue,T., Asamoto,M. et al. (1989) Comparison of enzyme phenotypes in human bladder tumours and experimentally induced hyperplastic and neoplastic lesions of the rat urinary bladder. A combined histochemical and immunohistochemical approach. Virchows Arch. B Cell Pathol., 56, 307–316.[ISI][Medline]
  12. Wilson,P.D., Summerhayes,I.C. and Frank,L.M. (1978) Alkaline phosphatase as marker of transformation in adult mouse bladder epithelium after in vitro exposure to 7,12-dimethylbenz(a)anthracene. Cell Biol. Int. Rep., 2, 365–374.[ISI][Medline]
  13. El-Sharabasy,M.M.H. and El-Dosoky,I. (1986) Hydrolytic enzymes in normal and neoplastic human bladder tissue. Br. J. Urol., 58, 279–282.[ISI][Medline]
  14. Amador,E., Zimmerman,T.S. and Wacker,W.E.C. (1963) Urinary alkaline phosphatase activity. I. Elevated urinary LDH and alkaline phosphatase activities for the diagnosis of renal adenocarcinomas. J. Am. Med. Assoc., 185, 769–775.[ISI]
  15. Severini,G., Diana,L., Di Giovannandrea,R. and Tirelli,C. (1995) A study of serum glycosidases in cancer. J. Cancer Res. Clin. Oncol., 121, 61–63.[ISI][Medline]
  16. Okada,M., Suzuki,E. and Hashimoto,Y. (1976) Carcinogenicity of N-nitrosamines related to N-butyl-N-(4-hydroxybutyl) nitrosamine and N,N-dibutylnitrosamine in ACI/N rats. Gann, 67, 825–834.[ISI][Medline]
  17. Rawson,N.S. and Peto,J. (1990) An overview of prognostic factors in small cell lung cancer. A report from the Subcommittee for the Management of Lung Cancer of the United Kingdom Coordinating Committee on Cancer Research. Br. J. Cancer, 61, 597–604.[ISI][Medline]
  18. Fukushima,S., Shibata,M.A., Hirose,M., Kato,T., Tatematsu,M. and Ito, N. (1991) Organ-specific modification of tumor development by low-dose combination of agents in a rat wide-spectrum carcinogenesis model. Jpn. J. Cancer Res., 82, 784–792.[ISI][Medline]
  19. Leathwood,P.D. and Plummer,DT. (1969) Enzymes in rat urine. I. A metabolism cage for the complete separation of urine and feces. Enzymologia, 37, 240–250.[ISI][Medline]
  20. Plummer,D.T. and Wright,P.J. (1970) The collection of rat urine free of contamination. J. Physiol., 209 (suppl.), 16p–17p.[Medline]
  21. Plummer,D.T., Noorazar,S., Obatomi,D.K. and Haslam,J.D. (1986) Assessment of renal injury by urinary enzymes. Uremia Invest., 9, 97–102.[ISI]
  22. Malaga,C.A., Weller,R.E., Buschbom,R.L. and Ragan,H.A. (1991) Serum chemistry of the wild caught karyotype I night monkey (Aotus nancymai). Lab. Anim. Sci., 41, 143–145.[Medline]
  23. Weller,R.E., Baer,J.F., Malaga,C.A., Buschbom,R.L. and Ragan,H.A. (1992) Renal clearance and excretion of endogenous substances in the owl monkey. Am. J. Primatol., 28, 115–123.[ISI]
  24. van der Helm (1962) A simplified method of demonstrating lactic dehydrogenase isoenzymes in serum. Clin. Chim. Acta, 5, 124–128.
  25. Bessey,O.A., Lowry,O.H. and Brock,M.J. (1946) A method for the rapid determination of alkaline phosphatase with five cubic millimeters of serum. J. Biol. Chem., 164, 321–329.[Free Full Text]
  26. Stolarek,I., Howey,J.E. and Fraser,C.G. (1989) Biological variation of urinary N-acetyl-ß-D-glucosaminidase: practical and clinical implications. Clin. Chem., 35, 560–563.[Abstract/Free Full Text]
  27. Yuen,C.T., Kind,P.R., Price,R.G., Praill,P.F. and Richardson, A.C. (1984) Colorimetric assay for N-acetyl-ß-D-glucosaminidase (NAG) in pathological urine using the µ-nitrostyryl substrate: the development of a kit and the comparison of manual procedure with the automated fluorimetric method. Ann. Clin. Biochem., 21, 295–300.[ISI][Medline]
  28. Bergmeyer,H.U., Bowers,G.N.Jr., Horder,M. and Moss,D.W. (1976) Provisional recommendations on IFCC methods for the measurement of catalytic concentrations of enzymes. Part 2. IFCC method for aspartate aminotransferase. Clin. Chim. Acta, 70, F19–F29.[ISI][Medline]
  29. Bishop,S.A., Lucke,V.M., Stokes,C.R. and Gruffydd-Jones,T.J. (1991) Plasma and urine biochemical changes in cats with experimental immune complex glomerulonephritis. J. Comp. Pathol., 104, 65–76.[ISI][Medline]
  30. Jung,K., Schulze,G. and Reinholdt,C. (1986) Different diuresis-dependent excretions of urinary enzymes: N-acetyl-ß-D-glucosaminidase, alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase. Clin. Chem., 32, 529–532.[Abstract/Free Full Text]
  31. Chasson,A.L., Grady,H.T. and Stanley,M.A. (1961) Determination of creatinine by means of automatic chemical analysis. Am. J. Clin. Pathol., 5, 83–88.
  32. Riggins,R.S. and Kiser,W.S. (1963) A study of lactic dehydrogenase in urine and serum of patients with urinary tract disease. J. Urol., 90, 594–601.[ISI]
  33. Fukushima,S., Murasaki,G., Hirose,M., Nakanishi,K., Hasegawa,R. and Ito, N. (1982) Histopathological analysis of preneoplastic changes uring N-butyl-N(4-hydroxybutyl) nitrosamine-induced urinary bladder carcinogenesis in rats. Acta Pathol. Jpn., 32, 243–250.[Medline]
  34. Rhodes,J.B. and Williams-Ashman,H.G. (1960) Oxidizing enzymes in human seminal plasma. Med. Exp., 3, 123–134.
  35. Bennett,W.M., Elzinga,L.W. and Porter,G.A. (1991) Tubulointerstitial disease and toxic nephropathy. In Brenner,B.M. and Rector,F.C. (eds) The Kidney, 4th Edn. WB Saunders, Philadelphia, PA, pp. 1430–1496.
  36. Foulkes,E.C. (1993) Functional assessment of the kidney. In Hook,J.B. and Goldstein,R.S. (eds) Toxicology of the Kidney, 2nd Edn. Raven Press, New York, NY, pp. 37–60.
  37. Guder,W.G. and Ross,B.D. (1984) Enzyme distribution along the nephron. Kidney Int., 26, 101–111.[ISI][Medline]
  38. Shibata,M.A., Asakawa,E., Hagiwara,A., Kurata,Y. and Fukushima,S. (1991) DNA synthesis and scanning electron microscopic lesions in renal pelvic epithelium of rats treated with bladder cancer promoters. Toxicol. Lett., 55, 263–272.[ISI][Medline]
  39. Lee,D.A., Cockett,A.T., Caplan,B.M. and Chiamori,N. (1966) Urinary lactic dehydrogenase activity in the diagnosis of urologic neoplasm. J. Urol., 95, 77–78.[ISI][Medline]
  40. Hemmingsen,L., Hoiby,N. and Sorensen,P.K. (1970) Protein and LDH-isoenzyme pattern of the urine from patients with diabetes mellitus determined by disc-electrophoresis. Diabetologica, 6, 512–518.[ISI][Medline]
  41. Dawson,D.M., Goodfriend,T.L. and Kaplan,N.O. (1964) Lactic dehydrogenases: functions of two types. Science, 143, 929–933.[ISI]
  42. Macbeth,R.A.L. and Bekesi,J.G. (1962) Oxygen consumption and anaerobic glycolysis of human malignant and normal tissue. Cancer Res., 22, 244–248.[ISI]
  43. Bredin,H.C., Daly,J.J. and Prout,G.R. (1975) Lactic dehydrogenase isoenzymes in human bladder cancer. J. Urol., 113, 487–490.[ISI][Medline]
  44. Herz,F., Barlebo,H. and Koss,L.G. (1974) Modulation of alkaline phosphatase activity in cell cultures derived from human urinary bladder carcinoma. Cancer Res., 34, 1943–1946.[ISI][Medline]
  45. Araki,T. (1969) ß-N-Acetylglucosaminidase, ß-galactosidase and {alpha}-mannosidase activities in human female genital cancer. Gann, 60, 373–382.[ISI][Medline]
  46. Tucker,S.M., Boyd,P.J., Thompson,A.E. and Price,R.G. (1975) Automated assay of N-acetyl-ß-glucosaminidase in normal and pathological human urine. Clin. Chim. Acta, 62, 333–339.[ISI][Medline]
  47. Nakamura,M., Miyata,K., Tamura,T. and Kawabata,T. (1981) Sex difference in urinary excretion of alanine aminopeptidase in the rat. Jpn. J. Nephrol., 23, 209–212.
  48. Grauer,G.F., Greco,D.S., Behrend,E.N., Mani,I., Fettman,M.J. and Allen,T.A. (1995) Estimation of quantitative enzymuria in dogs with gentamicin-induced nephrotoxicosis using urine enzyme/creatinine ratios from spot urine samples. J. Vet. Intern. Med., 9, 324–327.[ISI][Medline]
  49. Brouhard,B.H., Lagrone,L. and Rowe,J. (1985) Acute response of urinary N-acetyl-ß-D-glucosaminidase to mannitol infusion in the dog. Am. J. Med. Sci., 290, 11–14.[ISI][Medline]
  50. Herz,F. and Koss,L.G. (1979) Alkaline phosphatase in cultured urinary bladder cancer cells. Arch. Biochem. Biophys., 194, 30–36.[ISI][Medline]
  51. Raab,W. and Donhoffer,E. (1967) Die transaminase-Aktivitaten im Rattenharnes. Klin. Wochenschr., 45, 1254–1255.[ISI][Medline]
Received July 23, 1998; revised December 31, 1998; accepted February 5, 1999.