EFFECT OF TIME AND TEMPERATURE ON THE STORAGE STABILITY OF HEPATOBILIARY ENZYME ACTIVITIES IN CATTLE SERUM

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1 Indian J. Anim. Res., 48 (2) : , 214 DOI1.5958/j AGRICULTURAL RESEARCH COMMUNICATION CENTRE EFFECT OF TIME AND TEMPERATURE ON THE STORAGE STABILITY OF HEPATOBILIARY ENZYME ACTIVITIES IN CATTLE SERUM P. D. Divya 1 and K.K. Jayavardhanan 2 College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur India Received: Accepted: ABSTRACT The present research was conducted to choose an ideal storage condition for cattle sera samples meant for the assay of hepatobiliary enzymes viz., Alanine aminotransferases(alt), Aspartate aminotransferases (AST), Alkaline phosphatase (ALP) and Gamma glutamyltransferases (GGT). The study was performed in adult healthy female crossbred cattle, during the period of April to October 28. Stabilities of enzymes in serum stored at room temperature (22 to 27 ºC), 4 ºC and 2 ºC were compared with the enzyme activities of fresh sera samples (day ) on day 1, 2, 5, 8, 11 and 14. Alanine aminotransferase was found to be sufficiently stable up to the study period of 14 days at 4 ºC, whereas it was unstable at room temperature and 2 ºC. Aspartate aminotransferase was stable both at 4 ºC and 2 ºC up to14 days whereas at room temperature stable only up to 2 days. Alkaline phosphatase showed great variation upon storage as compared to other hepatobiliary enzymes and it is suggested that its estimation should be performed in fresh serum samples to get an accurate result. Cattle serum showed a lesser stability for GGT activity at 2 ºC as compared to 4 ºC where the observed stability period was 8 days and 11 days, respectively and at room temperature the enzyme was stable only for one day. From these results it is therefore advisable to consider stability of each serum hepatobiliary enzymes for different animals separately before preserving sera samples to get more valid and reliable result. Key words: Crossbred cattle, Hepatobiliary enzymes, Room temperature, Storage stability. INTRODUCTION The measurement of serum enzymes is an important tool for disease diagnosis in veterinary and human clinical practice. The routinely used enzymes to evaluate hepatic damage in animals includes ALT, AST, ALP, GGT, Sorbitol dehydrogenase (SDH), Lactate dehydrogenase (LDH), Ornithine carbamoyl transferase (OCT) and 5 ' Nucleotidase (NTP) (Kaneko et al., 28). Refrigeration and freezing preserve most of the enzymes but some deteriorate even when frozen. Many investigations have been undertaken on the stability of enzymes, in vitro, but the results are widely divergent (Kaplan and Pesce, 1989). In veterinary medicine, to date not much studies have been published on the stability of biochemical markers especially serum hepatobiliary enzymes, which are routinely analysed for clinical diagnostic use. So it is of primary importance to re examine the storage stability of these enzymes. Hence, the present study is taken up to evaluate the effect of storage time and temperature on the measured activities of the hepatobiliary enzymes in the sera samples of cattle under various storage condition viz at room temperature (22 to 27 ºC), 4ºC and 2 ºC for a period of two weeks. MATERIALS AND METHODS Eight adult healthy female crossbred cattle of age 3 to 7 years maintained at University Livestock Farm, College of Veterinary and Animal Sciences, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur were selected randomly for the study. Blood samples were collected by jugular venipuncture using sterile needles (18 gauge) directly 1 Corresponding author s drdivyavet1@gmail.com. 2 Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences, Pookot, Wayanad , India.

2 13 INDIAN JOURNAL OF ANIMAL RESEARCH into clean dry sterile glass tubes without anticoagulants. Serum was harvested after 3 to 45 minutes following clot formation and by centrifugation for 1 minutes at 2 g. The clear serum was immediately assayed for the following hepatobiliary enzymes ALT, AST, ALP and GGT within an hour of serum separation to serve as basal fresh values (day ). The remaining serum were dispensed into 18 sample tubes, closed tightly and divided into three groups. One of each group was stored upright at room temperature (approximately 25 ºC), 4 ºC and 2 ºC. The stored serum aliquots from all temperature and time points were analysed together in one batch for hepatobiliary enzymes on 1, 2, 5, 8, 11 and 14 days post collection. Prior to analysis, at each designated time, the aliquots of the frozen samples were left to stand at room temperature to thaw and inverted several times to mix. The enzyme assay was performed using Days of storage Alanine aminotransferase (ALT) Ecoline Merck diagnostic kits (Merck Specialities Pvt. Ltd, Mumbai) on an automated blood analyzer ( Microlab 2). To test the significant differences in enzyme activity between storage temperatures and to assess the significant trends over time at each temperature, the data was analysed statistically using paired t test (Snedecor and Cochran, 1994). The stability of an enzyme activity under each temperature condition and time was determined by calculating the percentage change in concentrations from the mean fresh value (day ) at each timepoint for each animal. RESULTS AND DISCUSSION The time and temperature had significant effects on hepatobiliary enzyme activity in cattle sera samples during their storage (Table 1 and 2). Aspartate aminotransferase was found to be the most stable enzyme studied being stable for 14 days TABLE 1: Activity of Alanine aminotransferase and Aspartate aminotransferase in cattle sera samples (n= 8) preserved at 2227 ºC, 4 ºC and 2 ºC for 14 days Aspartate aminotransferase (AST) 2227 ºC 4 ºC 2 ºC 2227 ºC 4 ºC 2 ºC (Base line value) 15.8± ± ± ± ± ± ± 2.8 ( ) 21.8± 1.88 ( ) 23.4± 1.81* (+ 48.1) 43.2± 5.86 (31.43) 65.8± 5.4 (+ 2.8) 68.4± 3.75 (+ 8.56) ± 1.29* (+ 48.1) 21.4± 1.54 ( ) 21.2± 1.53* ( ) 34.2± 5.22* (45.71) 64.6± 4.64 (+ 2.5) 59.2±.86 (6.3) ± 1.39* (+ 63.3) 22.8± 1.7 (+ 44.3) 18.8± 1.43 ( ) 32.4± 7.12* (48.57) 65.6± 4.34 (+ 4.1) 59.6± 1.29 (5.39) ±.58* (+ 12.6) 23.± 3.22 ( ) 19.8± 3.38 ( ) 31.± 6.35* (5.79) 6.2± 5.69 (4.4) 59.8± 2.62 (5.8) ± 1.58 (+ 7.59) 23.8± 2.22 (+ 5.63) 26.6± 3.89* ( ) 28.6± 7.9* (54.6) 6.8± 4.9 (3.5) 58.4± 1.63 (7.3) ± 1.48 ( ) 17.8± 2.33 ( ) 2.± 1.55 ( ) Percentage change from initial activity in parenthesis, * P< ± 7.42* (32.69) 54.6± 5.28 (13.3) 6.6± 2.66 (3.81)

3 Days of storage Al kaline phosphatase (ALP) Vol. 48, No. 2, 214 TABLE 2: Activity of alkaline phosphatase and gamma glutamyl transferase in cattle sera samples (n= 8) preserved at 2227 ºC, 4 ºC and 2 ºC for 14 days Gamma glutamyl transferase (GGT) 2227 ºC 4 ºC 2 ºC 2227 ºC 4 ºC 2 ºC 131 (Base line value) 116.4± 116.4± 116.4± 11.± ± ± ± (+ 1.55) 134.6± ( ) 144.6± 2.3* ( ) 12.8±.58 ( ) 13.4±.4 ( ) 12.2±.66 ( ) ± 11.8 ( ) 135.4± 1.43* ( ) 187.2± 16.2 (+ 6.82) 14.±.45* ( ) 13.6±.6 ( ) 12.8±.58 ( ) ± 1.85 (+ 6.36) 138.4± 16.4* (+ 18.9) 133.6± 15. ( ) 15.±.71* ( ) 12.±.93 (+ 9.9) 13.±.56 ( ) ± 1.93 (+ 3.61) 146.± 9.88 ( ) 149.8± ( ) 16.8± 1.59* ( ) 12.±.43 (+ 9.9) 13.6±.4 ( ) ± (28.35) 149.± 14.79* (+ 28.1) 168.4± ( ) 17.6± 3.17* (+ 6..) 1.8±.8 (1.82) 14.2±.63* ( ) ± (34.7) 156.8± 17.17* ( ) 198.8± 29.16* (+ 7.79) Percentage change from initial activity in parenthesis, * P<.5 at 4 ºC as well as 2 ºC. The stability of AST activities in cattle serum revealed a steady decrease over the entire experimental period. Serum samples lost appreciable AST activity at room temperature, with only less than 5 % of the initial activity remaining on 14 th day and the enzyme was found to be totally unstable at room temperature. The decline in AST activity noticed for samples at room temperature may be due to increased degradation of enzyme active site with increased temperature. Changes in the temperature of storage alter the rate of reaction and rate of denaturation of enzymes. Intracellularly, enzymes are protected from degradation when bound to their substrates and cofactors. In serum, enzymes, substrates and cofactors are dispersed and binding is uncommon, leaving the enzymes more susceptible to degradation (Kaplan and Pesce, 1989). So if transportation of specimens is required, AST assay should be done within 24 h of venipuncture in case of cattle, 2.6± 3.71* ( ) 15.2± 1.2* ( ) 16.8± 1.24* ( ) otherwise proper storage conditions should be provided. At refrigerator and freezer, AST was seen to be highly stable up to two weeks. The results of the AST stability in freezer support the study of Benjamin (21) in bovine serum, who reported that AST was stable for as long as 38 days in freezer. Contrary to the findings of the present investigation, Benjamin (21) also reported five days stability at 4 ºC and increased activity at room temperature. However, Heins et al. (1995) reported a decreased AST activity in human serum samples stored at room temperature. To preserve sera samples of cattle for AST estimation, the present study recommends either 4 ºC or 2 ºC as the ideal storage condition. According to the results, it has been observed that, ALT was sufficiently stable for a period of two weeks at 4 ºC, but with variations in the percentage of activity. In contrast, the enzyme was found to be unstable at 2 ºC, where it showed marked increase in enzyme activities through out the investigational

4 132 INDIAN JOURNAL OF ANIMAL RESEARCH period. A nonsignificant change was observed after 24 h storage period at 2ºC. A similar study was reported by Davy et al. (1984) in marmoset plasma, where ALT was found to be highly unstable when kept at 15 to 2ºC for a period of 48 h and stable at 4 ºC for the same period. The enzyme was found to be stable only for one day, at room temperature, after which a marked increase in enzyme activities was observed up to five days followed by a gradual decline in activity till the end of experimental period. These findings were in accordance with the increased ALT activity at room temperature and freezer for cattle serum (Benjamin, 21). The results suggest 4ºC as the ideal storage condition for the preservation of cattle sera samples for ALT assay. Serum ALP activity showed great variations under the three storage conditions. Even though, significant differences were not found in ALP activities at room temperature, considering the great fluctuations in the percentage change in initial activity, it was considered to be unstable at room temperature. Compared to 2ºC, ALP was more stable at 4 ºC. In both the conditions, an increased ALP activity was observed with a marked increase by more than 6 % of initial activity at 2 ºC within 24 h of blood collection and up to 7 % increase after two weeks. At 4ºC, the enzyme showed significant increase in activity from the second day of storage onwards with a maximum increase of % after two weeks (P<.5). Alkaline phosphatase in human serum demonstrates a linear increase in activity depending upon the increase in temperature and time (Kaplan and Pesce, 1989). The increased ALP activities at 4 ºC and 2 ºC might be due to occurrence of several isozymes for ALP which differ in their sensitivity to temperature or due to inter individual variations or the enzyme may became more concentrated at lower temperatures. Since, cattle serum ALP activity increased significantly with decrease in storage temperature, it is suggested that the assay should be done on the day of blood collection itself. Gamma glutamyl transferase was found to be stable for a period of one day at room temperature, 11 days at 4ºC and 8 days at 2 ºC in cattle serum. Significant increase in GGT activity was observed after one day at room temperature and after 14 days, more than 8 % increase in initial activity was noticed (P<.5). The GGT activity increased significantly by about 38% at 4 ºC after 11 days of storage. At 2 ºC, the enzyme was stable for as long as 8 days after which the enzyme showed marked increase in the activity and on 14 th day about 5 % increase in initial activity was observed. In human serum, GGT was reported to be stable for 2 days at room temperature, one week at 4ºC and one month at 25ºC (Kaplan and Pesce, 1989). The present study suggests 4ºC as ideal storage condition for GGT assay in cattle serum as compared to room temperature and 2ºC. Summary of storage stability in days for the activity of ALT, AST, ALP and GGT is presented in figure 1. Thus the present study reveals significant differences in the storage stability characteristics of Days of storage AL T AST ALP* * GGT FIG.1: Sum mary of storage stability of hepatobiliary enzym es in cattle serum * * Though, not significant, AL P showed w ide variatio ns in percentage change in acti vity and therefore, not recom mended for storage as per the days m entioned ºC 4 ºC 2 ºC

5 each hepatobiliary enzyme. The investigation recommends 4ºC as the ideal storage condition for ALT and GGT assay and for AST estimation either 2ºC or 4ºC. It is suggested that ALP estimation Vol. 48, No. 2, should be performed in fresh serum samples. These differences in enzyme stability should be considered while preserving sera samples to get an accurate result. REFERENCES Benjamin, M.M. (21). Outline of Veterinary Clinical Pathology. Third edition. Kalyani Publishers, New Delhi, 351pp. Davy, C.W., Jackson, M.R. and Walker, J.W. (1984). Stabilities of some constituents of Marmoset (Callithrix jacchus) plasma under various conditions of storage. Clin. Chem., 3: Heins, M., Heil, W. and Withold, W. (1995). Storage of serum or whole blood samples, effects of time and temperature on 22 serum analytes. Eur. J. Chem. Clin. Biochem., 33: Kaneko J J, Harvey J W and Bruss M L. (28). Clinical Biochemistry of Domestic Animals. 6 th edn. Academic Press, California. Kaplan, L.A. and Pesce, A.J. (1989). Clinical Chemistry, Theory, Analysis and Correlation. Second edition. The C.V. Mosby Company, Baltimore, 1149pp. Snedecor, G.W. and Cochran, W.G. (1994). Statistical Medhods. 8 th edition. Oxford and IBH Publishing Company, Calcutta, 564 pp.

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