Canine Serum Alkaline Phosphatase Isoenzymes Detected by Polyacrylamide Gel Disk Electrophoresis

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1 FULL PAPER Internal Medicine Canine Serum Alkaline Phosphatase Isoenzymes Detected by Polyacrylamide Gel Disk Electrophoresis Hiroshi ITOH 1), Tomoko KAKUTA 1), Go GENDA 1), Iwao SAKONJU 1) and Katsuaki TAKASE 1) 1) Department of Veterinary Surgery, School of Veterinary Medicine and Animal Sciences, Kitasato University, Towada, Aomori , Japan (Received 9 February 2001/Accepted 12 September 2001) ABSTRACT. Serum alkaline phosphatase (ALP) isoenzymes were studied in normal dogs using a commercially available polyacrylamide gel disk electrophoresis kit (PAG/disk kit). Serum samples taken from the dogs were incubated with neuraminidase, after which most showed ALP isoenzymes as two characteristic stained bands. To determine the origin of each band, ALP isoenzymes of serum and tissue extracts (liver, intestine and bone) were characterized by heating, wheat germ agglutinin (WGA) and levamisole treatments. The results suggested that the band detected on the anode was liver ALP (LALP) and that the band detected on the cathode represented bone ALP (BALP), and both were corticosteroid-induced ALP (CALP). The percentage of each ALP isoenzyme to total ALP activity was estimated by densitometry. The percentage of BALP was the highest in young dogs (age<1 year, 64.7% ), and this value decreased with age. In contrast, the percentage of LALP in young dogs (22.2%) was much lower than that in middle-aged dogs (ages 1 year to 7 years, 59.3%) and old dogs (ages>7 years, 50.4%). The present results suggested that a commercially available PAG/disk kit is capable of detecting three serum ALP isoenzymes in dogs, and further that it may have clinical applications in the evaluation of ALP isoenzymes in veterinary medicine. KEY WORDS: alkaline phosphatase isoenzyme, Alkphor kit, canine serum, polyacrylamide gel disk electrophoresis. J. Vet. Med. Sci. 64(1): 35 39, 2002 The clinical evaluation of serum alkaline phosphatase (ALP) isoenzymes is important in the evaluation of the extent of lesion invasion in the organ from which the ALP isoenzyme originates. Three ALP isoenzymes, namely liver ALP (LALP), bone ALP (BALP), and corticosteroidinduced ALP (CALP) are present in the dog [1,4,9,15], whereas intestinal ALP (IALP) and placental ALP (PALP) which are found in human serum, are absent from dog serum [6]. Analysis of ALP isoenzymes in canine serum has been carried out by heat inactivation [3, 5, 14, 19], chemical inhibition [9, 13], conventional electrophoresis [1, 4, 6, 18], immunochemical electrophoresis and affinity electrophoresis with lectin [8, 18]. Electrophoresis on cellulose acetate membrane is widely used in clinical practice due to its simplicity and reproducibility. However, it is difficult to distinguish LALP and CALP using this technique as their electrophoresis patterns are too similar [1, 18, 19]. On the other hand, polyacrylamide gel disk electrophoresis (PAG/ disk electrophoresis), in particular, has an excellent separating ability due to the molecular sieve effect created by the non-sequential gel concentration and ionic buffer strength [2]. However, PAG/disk electrophoresis has not yet been used as a routine clinical test because it involves a time-consuming procedure. In the present study, we analyzed serum ALP isoenzymes in normal dogs (separation of each ALP isoenzyme and determination of the percentage of each isoenzyme to total ALP activity) in order to assess/ investigate a method of measuring serum ALP isoenzymes using a commercially available PAG/disk kit without the need for complicated procedures. MATERIALS AND METHODS Experimental animals: Forty mongrel dogs aged 6 months to 13 years were divided into 3 groups according to age. The young group consisted of 14 dogs aged <1 year (average 0.8 years), the middle-aged group of 13 dogs were aged 1 to 7 years (average 3.5 years), and the old group of 13 dogs were aged > 7 years (average 9.8 years). All dogs were healthy and had no abnormal findings in blood biochemistry. Dog sera and tissue extracts: Serum samples were analyzed immediately after sample collection or stored at 35 C for a week. Parenchymal tissues of liver and cancellous tissues of the iliac crest were collected from six adult mongrel dogs after euthanasia. Small intestine mucosa and the renal cortex were collected from an adult mongrel dog. The placenta was collected from an adult mongrel dog immediately after delivery. Each tissue was suspended in physiological saline (2 ml/g) and homogenized at 12,000 rpm for 5 to 8 min before electrophoresis. All extracts were treated with Triton X-100 (final concentration, 0.2%, Polyscience Inc., U. S. A.) and phosphatidylinositol-phospholipase (final concentration, 0.02 U/ml, Funakoshi Co., Ltd., Japan). Neuraminidase treatment: Neuraminidase (1 U/0.35 ml, from Vibrio Cholerae, Sigma-Aldrich Japan Co., Ltd., Japan) was added to the serum at a final concentration of 0.2 U/ml. The mixture was incubated at 37 C for 15 min in a water bath and then cooled in ice water. Measurement of ALP activity: ALP activity was assayed using an ALP kit (Unimate, Nippon Roche K. K., Japan) by a spectrophotometer (7010, Hitachi, Tokyo, Japan).

2 36 H. ITOH ET AL. Heat inactivation: Tissue extracts were heated at 56 C for 10 min in a water bath and then cooled on ice to determine remaining ALP activity [10]. Wheat germ aggultinin precipitation (WGA): Sera or tissue extracts (liver, intestine and bone) were mixed with the same volume of WGA (5 mg/ml in deionized water, Honen Co., Ltd., Japan). The mixture was incubated at 37 C for 30 min and centrifuged at 3,000 rpm for 10 min. ALP activity in the supernatant was determined, and the inactivation rate was calculated as follows [12]: Inactivation rate (%) = {(Total ALP Supernatant ALP2) / Total ALP} 100 Levamisole and L-phenylalanine inhibition: Levamisole (Funakosi Co., Ltd., Japan) and L-phenylalanine (Wako Pure Chemical Industries, Ltd., Japan) were added to the ALP assay buffer at final concentrations of 4.2 mm and 5.0 mm, respectively. The inactivation rate was calculated after each treatment and CALP activity was calculated as follows [7, 9]: Fig. 1. PAG/disk electrophoresis of serum ALP isoenzyme in normal dogs. Single or double band zymograms are observed (a, b, c). After neuraminidase treatment, samples with single or double band zymograms showed two separated bands (a, b, c ). In the sera of most young dogs, an atypical ALP isoenzyme band (c, ) was observed. CALP activity value = ALP activity determined by addition of levamisole 100/58 In addition, levamisole was added to the electrophoretic substrate solution at a concentration of 4.2 mm and incubated in staining solution. PAG/disk electrophoresis: PAG/disk electrophoresis was performed using a commercially available kit (AlkPhor System, Jokoh Co., Ltd., Japan) according to the manufacturer s instructions. ALP activity was determined by a spectrophotometer (EDC, Helena Laboratories, Co., Ltd., Japan) at 610 nm. Two gel tubes were used for electrophoreses. One tube was stained normally and the other was stained in the presence of levamisole. Statistical analysis: Results were analyzed statistically for variance (ANOVA) and by Student s t-test (P< 0.05). RESULTS PAG/disk electrophoresis patterns by neuraminidase treatment: According to PAG/disk electrophoresis, serum ALP zymograms in normal dogs had a single band (16/40; 40%) or double bands (24/40; 60%) (Fig. 1a, b, c). An atypical ALP isoenzyme band was observed in the serum of most young dogs (Fig. 1c). After neuraminidase treatment, most serum samples originally showing a single band ALP zymogram had changed to a two band zymogram (14/16; 88%), and the separation of these bands was also improved (Fig. 1a, b, c ). Densitometric analysis of ALP isoenzymes demonstrated that neuraminidase treatment increased the percentage of cathodal bands (+6.3%) and decreased that of the anodal bands ( 4.3%). The atypical ALP isoenzyme band that was observed in the serum of most young dogs (Fig. 1c) disappeared after heating at 56 C for 10 min (Fig. 3d ). However, this band Fig. 2. Each ALP isoenzyme band from various tissue extracts was observed in order of electrophoretic mobilities from the anode as follows: liver (a), bone (b), kidney (c), placenta (d), and intestine (e). was not affected by neuraminidase or PI-PLC treatments (data not shown). The electrophoretic mobilities of ALP from tissue extracts, in descending order from the anode were liver, bone, kidney, placenta and intestine (Fig. 2 a-e). Effect of inhibitors and inactivation of ALP activity: The effects of heating and various inhibitors on ALP isozymes derived from liver, bone or intestine are shown in Table 1. BALP was highly sensitive to heating (56 C for 10 min), WGA, and levamisole treatment, and LALP activity was almost completely abolished by levamisole treatment. In contrast, these isoenzymes were not affected by L-phenylalanine treatment (less than 10%). IALP, which has been reported to have properties similar to CALP in terms of heat stability, and sensitivity to WGA and levamisole treatment

3 SERUM ALP ISOENZYMES BY PAGE 37 Table 1. Effects of various inhibitors on alkaline phosphatase activity Sample n Heat WGA Levamisole L-phenylalanine liver extract ± 14.7** 68.0 ± 14.4 ** 99.1 ± ± 4.1 bone extract ± ± ± ± 4.7 intestinal extract Results are expressed as the percent inactivation of original total activity (Mean ± SD). Heat: heated at 56 C for 10 min, WGA: wheat germ agglutinin. ** liver vs bone p<0.01. Fig. 3. PAG/disk electrophoresis of the supernatant of the untreated (a: LALP, BALP) and wheat germ agglutinin (WGA) treated serum (a : LALP). To elucidate the origin of serum ALP isoenzymes bands, they were characterized by WGA, levamizole and heat treatments. In WGA treated serum, the cathodal band completely disappeared (a ). In levamisole untreated substrate solution, two bands were observed (b: LALP, BALP). In levamisole treated substrate solution, two bands were completely inhibited (b ). However, the cathodal band observed was not always inhibited by this treatment (c : CALP). In addition, the levamisole resistant cathodal bands did not disappear after heating at 56 C for 10 min (c : CALP). An atypical (d: ) and cathodal bands (BALP) disappeared after heating at 56 C for 10 min (d ), and after levamizole treatment (d ). [14, 16], was shown to be resistant to heat and levamizole treatment, but was moderately sensitive to L-phenylalanine treatment. Analysis of serum ALP isoenzyme: The results of the PAG/disk analysis of each serum ALP isoenzyme are shown in Fig. 3. When PAG/disk electrophoresis was performed using supernatants from WGA-treated serum, the cathodal band completely disappeared (Fig. 3a ). ALP activity of the anodal band was completely suppressed by levamisole treatment, but that of the cathodal band was not always inhibited by this treatment. In addition, the levamisole resistant cathodal bands did not disappear after heating at 56 C for 10 min (Fig. 3c, c ). These results indicate that the band detected on the anode was LALP (electrophoretic mobility, sensitive to levamisole and resistant to WGA treatment), that the two bands detected on the cathode were BALP (sensitive to heat, WGA, and levamisole) and CALP (resistant to heat and levamisole). The atypical band on the cathode in young dogs disappeared in response to both heating (Fig. 3d ), and levamisole treatments (Fig. 3d ). Percent of serum ALP isoenzyme: After neuraminidase treatment, all serum samples were subjected to densitometry (Table 2). The fractionation percentage of each ALP isoenzyme was determined as follows: LALP(%) = Fractionation percentage on the anode side band. BALP(%) = Fractionation percentage on the cathode side band percentage of CALP. CALP(%) = CALP activity value*/total ALP activity value 100 * (The method used to calculate CALP activity is the method of Hoffmann et al. [7]) The percentage of BALP was much higher in young dogs than in middle aged and old dogs, and this value decreased gradually with age. In contrast, the percentage of LALP was much lower in young dogs than in middle-aged and old dogs. The percentage of CALP tended to be higher in old dogs. DISCUSSION Analysis of serum ALP is an established diagnostic assessment tool in bone and liver diseases [14]. Such disorders are associated with an increase in serum total ALP as a

4 38 H. ITOH ET AL. Table 2. dogs The age dependent changes of serum ALP isoenzymes in clinically healthy Group n TALP BALP LALP CALP (IU/L) (%) (%) (%) Young (age<1) ± ± ± ± 2.5 Middle (1 age<7) ± ± ± ± 4.0 Old (7 age) ± ± ± ± 26.3 Data are represented by mean ± standard deviations. n: Number of dogs in the group; TALP: Total alkaline phosphatase activity; BALP: Bone alkaline phosphatase; LALP: Liver alkaline phosphatase; CALP: Corticosteroid-induced alkaline phosphatase. result of increased entry of bone and liver ALP isoenzymes into serum. In particular, when total ALP activity is within the normal range, quantitative rather than qualitative measurement of bone and liver phosphatase levels increases the sensitivity of isoenzyme analysis by permitting the detection of minor changes in the activities of these enzymes. Quantitative measurements are also needed for serial monitoring of the activity of tissue fractions with bone or liver disease, and the response of these to therapy. However, no simple electrophoresis technique has yet been established for the analysis of ALP in the veterinary clinical setting. Thus, the present study assessed the use of a simple electrophoretic procedure for the analysis of ALP isoenzymes in the serum of dogs using a commercially available PAG/disk electrophoresis kit. In general, separation of isoenzymes in dog serum ALP by cellulose acetate electrophoresis reportedly yields three isoenzymes (LALP, BALP and CALP) [1, 4]. In the present study, ALP isoenzymes in all serum samples taken from the dogs showed a single or double band ALP zymogram (Fig. 1). However, since the single band on the ALP zymogram was broad, it may represent an overlap of several ALP isoenzymes. It has been reported that partial neuraminidase digestion affects the separation of ALP isoenzymes by differentially delaying the electrophoretic mobilities of each ALP isoenzyme [11]. We believed that most samples with a single zymogram subsequently separated into two distinct bands on the zymogram after treatment of serum with neuraminidase. The present study investigated the tissue of origin of each serum ALP isoenzyme band detected by PAG/disk electrophoresis by characterizing the properties of each ALP isoenzyme from liver, bone and intestine. The results of characterization of each dog ALP isoenzyme with heat, WGA and levamizole (Table 1) agreed with the results of previous reports; BALP has been reported to be thermolabile [9], readily sedimented with WGA [12], and highly inactivated by levamisole treatment [7]. Another previous study observed that LALP is highly sensitive to levamisole, and slightly resistant to heat and WGA treatment [3]. Furthermore, IALP is reportedly more resistant to heat treatment than BALP and LALP [15, 19], and is also resistant to levamisole treatment [7]. In terms of the electrophoretic mobilities of ALP isoenzymes, CALP migrates to the anodal side of LALP [1, 3 5] or overlaps with LALP [18] in cellulose acetate electrophoresis. On the other hand, CALP has been shown to be separated on the cathodal side of LALP [15] in starch gel electrophoresis. It is possible that CALP was detected on the cathodal side of LALP at the same position as BALP by the present PAG/disk electrophoresis kit, which has a molecular sieve effect similar to starch gel electrophoresis [15]. The present PAG/disk kit detected an atypical ALP isoenzyme band on the cathodal side in most serum samples taken from young dogs (Fig. 1c). This band was heat labile (Fig. 3d ) and was not sensitive to neuraminidase or PI-PLC treatment (data not shown). Variant BALP present in the sera of 99% of normal human children [20] has also been reported to have the above characteristics. In the present study, dog serum BALP was heat labile (Table 1). Therefore, atypical ALP isoenzyme in the serum of young dogs might correspond to the variant BALP of human serum. The densitometric values of the two bands detected in the normally stained gel tube were used to compare the fractionation percentage between LALP and other ALP isoenzymes. The assessment of ALP bands when using a PAG/disk kit in the clinical setting should proceed as follows. (1) Two gel tubes are concurrently electrophoresed after treatment of the sample with neuraminidase. (2) One tube is stained normally and the other is stained with levamisole. (3) The densitometric values of the two bands detected in the normally stained gel tube are used to compare the fractionation parentage between LALP and other ALP isoenzymes. (4) The band in the gel tube stained with levamisole has been shown to represent CALP. (5) The percentage of CALP and BALP can be estimated by comparison of gel tubes stained normally with levamisole-stained tubes. The observed age-dependent changes of each ALP isoenzyme in the present serum samples were similar to those reported by Syakalima et al. [17], except that the serum of young dogs showed a much higher percentage of BALP and a much lower percentage of LALP. This disparity could be due to differences in the methods used and the ages of the dogs studied in the present study and those of others. The actual LALP activity in the WGA-treated supernatant fluid was expected to be lower than the calculated value, given that the sedimentation of LALP due to WGA [9] was not considered in the paper of Syakalima et al. [17].

5 SERUM ALP ISOENZYMES BY PAGE 39 In the present study, dog serum CALP, which has been reported to be derived from the liver [1] was detectable only in old dogs (Table 2). The CALP percentage was also high in the serum of old dogs in the report of Syakalima et al. [17]. Determination of total serum ALP activity levels is essential for diagnosing bone, liver, and biliary tract diseases. However, the measurement of total ALP activity is not sufficient for the evaluation of the disease condition or for determining the cause of abnormal total ALP levels. The analysis of each ALP isoenzyme is clinically important for these purposes. Using the PAG/disk kit, it was possible, after biochemically processing the serum, to calculate the percentage of each ALP isoenzyme. When disease conditions associated with a high level of ALP activity are encountered, the present method, which uses a commercially available PAG/disk, might be useful in evaluating all three ALP isoenzymes in dog serum. REFERENCES 1. Dorner, J. L., Hoffmann, W. E. and Long, G. B Corticosteroid induction of an isoenzyme of alkaline phosphatase in the dog. Am. J. Vet. Res. 35: Epstein, E., Wolf, P. L., Horwitz, J. P. and Zak, B An indigogenic reaction for alkaline phosphatase in disk electrophoresis. Am. J. Clin. Pathol. 48: Farley, J. R., Hall, S. L., Ritchie, C., Herring, S., Orcutt, C. and Miller, B. E Quantitation of skeletal alkaline phosphatase isoenzyme activity in canine serum. J. Bone and Mineral Res. 7: Hoffmann, W. E. and Dorner, J. L Separation of isoenzymes of canine alkaline phosphatase by cellulose acetate electrophoresis. J. Am. Anim. Hosp. Assoc. 11: Hoffmann, W. E. and Dorner, J. L A comparison of canine normal hepatic alkaline phosphatase and variant alkaline phosphatase of serum and liver. Clin. Chim. Acta 62: Hoffmann, W. E. and Dorner, J. L Disappearance rates of intravenously injected canine alkline phosphatase isoenzymes. Am. J. Vet. Res. 38: Hoffmann, W. E., Sanecki, R. K. and Dorner, J. L A technique for automated quantification of canine glucocorticoid-induced isoenzyme of alkaline phosphatase. Vet. Clin. Pathol. 17: Kidney, B. A. and Jackson, M. L Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog. Can. J. Vet. Res. 52: Mahaffey, E. A. and Lago, M. P Comparison of techniques for quantifying alkaline phosphatase isoenzymes in canine serum. Vet. Clin. Pathol. 20: Moss, D. W. and Whiby, L. G A simplified heat-inactivation method for investigating alkaline phosphatase isoenzymes in serum. Clin. Chim. Acta 61: Moss, D. W. and Edwards, R. K Improved electrophoretic resolution of bone and liver alkaline phosphatases resulting from partial digestion with neuraminidase. Clin. Chim. Acta 143: Rosalki, S. B. and Ying Foo, A Two new methods for separating and quantifying bone and liver alkaline phosphatase isoenzymes in plasma. Clin. Chem. 30: Ruegnitz, P. C. and Schwartz, E Effects of chemical inhibition of alkaline phosphatase isoenzymes in the dog. Am. J. Vet. Res. 32: Saini, P. K. and Saini, S. K Origin of serum alkaline phosphatase in the dog. Am. J. Vet. Res. 39: Saini, P. K., Peavy, G. M., Hauser, D. E. and Saini, S. K Diagnostic evaluation of canine serum alkaline phosphatase by immunochemical means and interpretation of results. Am. J. Vet. Res. 39: Sanecki, R. K., Hoffmann, W. E., Gelberg, H. B. and Dorner, J. L Subcellular location of corticosteroid-induced alkaline phosphatase in canine hepatocytes. Vet. Pathol. 24: Syakalima, M., Takiguchi, M., Yasuda, J. and Hashimoto, A The age dependent levels of serum ALP isoenzymes and the diagnostic significance of corticosteroid-induced ALP during long-term glucocorticoid treatment. J. Vet. Med. Sci. 59: Syakalima, M., Takiguchi, M., Yasuda, J. and Hashimoto, A Separation and quantification of corticosteroid-induced, bone and liver alkaline phosphataseisoenzymes in canine serum. J. Vet. Med. A 44: Teske, E., Rothuizen, J., De Bruijne, J. J. and Mol, J. A Separation and heat stability of the corticosteroid-induced and hepatic alkaline phosphataseisoenzymes in canine plasma. J. Chromatography 369: Van Hoof, V. O., Hoylaerts, M. F., Geryl, H., Van Mullem, M., Lepoutre, L. G. and De Broe, M Age and sex distribution alkaline phosphatase isoenzymes agarose electrophoresis. Clin. Chem. 36: