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Research Communication

Stability of Vitamin B-6–Dependent Aminotransferase Activity in Frozen Packed Erythrocytes Is Dependent on Storage Temperature^{1 ,2}

Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376

³To whom correspondence should be addressed. E-mail: shultz{at}wsu.edu.

	ABSTRACT

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Pyridoxal 5'-phosphate (PLP) stimulation of erythrocyte alanine and aspartate aminotransferase (EALT, EAST) activities is a frequently used functional measure of vitamin B-6 status. Stability of enzyme activities and activity coefficients (AC, stimulated ÷ unstimulated) was assessed in packed erythrocytes frozen at -20, -80°C and under liquid nitrogen (-196°C). Activities of EALT and EAST, with and without added PLP, were determined in fresh erythrocytes (d 0) and frozen samples on d 1, 7, 14, 28, 58 and 84. In -20°C samples, EALT basal activity decreased 17 and 22% (P 0.05 for both) by d 58 and 84, respectively, and EAST basal activity decreased 40% (P 0.05) by d 58. In -80 and -196°C samples, EALT and EAST basal activities did not change significantly. Activity coefficients did not differ significantly from d 0 at any storage temperature, but EAST-AC increased 9–19% (nonsignificant) in samples stored at -20 and -80°C for 7 to 84 d. Additionally, EAST-AC was significantly higher in -20 than -80 and -196°C samples on d 1 and 58, respectively. Erythrocytes may be frozen for 28 d at -20°C and 84 d at -80°C before analysis for EALT; for EAST, activity should be measured on fresh erythrocytes.

KEY WORDS: • erythrocyte alanine and aspartate aminotransferases • storage • stability • basal activity • activity coefficient

	INTRODUCTION

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The most widely used functional measure of vitamin B-6 status is the measurement of erythrocyte alanine [EALT,⁴ formerly erythrocyte glutamic pyruvic transaminase (EGPT); EC 2.6.1.2] and aspartate [EAST, formerly erythrocyte glutamic oxaloacetic transaminase (EGOT); EC 2.6.1.1] aminotransferase activity and/or stimulation by exogenous pyridoxal 5'-phosphate (PLP) (1 2 3 4) . These indicators are considered long-term measures of vitamin B-6 status because of the length of the erythrocyte’s lifespan. The specific tests employed are measurement of the basal activity of these aminotransferases and the ratio of activity in the presence of excess PLP to the basal activity (activity coefficient; AC).

According to recent studies in women, EALT and EAST activities (AC or percentage of stimulation) are sensitive measurements related to vitamin B-6 intake (1 ,2) , although EALT is considered to be more sensitive to changes in intake (1 ,5) . Huang et al. (2) , however, recently reported that EAST activity responded more readily to changes in vitamin B-6 intake than did EALT activity. Several researchers (2 ,6 ,7) have suggested that EALT and EAST analyses be conducted the same day that blood is drawn to prevent possible loss of activity due to freezing. However, no detailed studies pertaining to stability are available in the literature, despite the fact that such information is important when laboratory determinations must be delayed or a large number of samples analyzed. Thus, the stability of enzyme activities with frozen storage is important to consider.

In the present study, the stability of EALT and EAST activities was assessed in erythrocytes from three individuals frozen at different temperatures for varying periods of time ranging from 1 to 84 d. In particular, the usefulness of storage in ordinary and low temperature freezers (-20 and -80°C) was investigated.

	MATERIALS AND METHODS

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Subject selection.

Three healthy subjects (1 man, 2 women) between the ages of 24 and 52 y, who were nonsmokers and not using oral contraceptives, participated in this study. Subjects were screened for history of intestinal, renal or metabolic disorders that could affect absorption, excretion or metabolism of vitamin B-6 (8) . The study protocol was approved by the Human Subjects Institutional Review Board of Washington State University and informed consent was obtained from each subject.

Sample collection and storage.

Blood samples (60 mL) were taken from fasting subjects by venipuncture at the beginning of the study. Blood specimens were collected in heparinized Vacutainer tubes (Becton Dickinson, Rutherford, NJ). Samples were centrifuged at 1700 x g for 10 min at 4°C. Plasma was separated from erythrocytes and the buffy coat discarded. Erythrocytes were washed three times with normal saline. The erythrocytes from each subject were then separately pooled in 100-mL beakers in an ice slurry, gently mixed, divided into aliquots and stored frozen at -20 and -80°C, and under liquid nitrogen (-196°C). Fresh erythrocytes representing d 0 were assayed for EALT and EAST activities. Additional enzyme activities were measured from aliquots frozen at -20 and -80°C on d 1, 7, 14, 28, 58 and 84, and for aliquots frozen at -196°C on d 28, 58 and 84.

Sample analyses.

Erythrocyte alanine and aspartate aminotransferase activities with and without PLP stimulation in vitro were analyzed by the method of Woodring and Storvick (7) using 0.33 mol/L Tris buffer (pH 7.4). Hemolysates were prepared by diluting packed cells 1:5 and 1:10 with deionized water for EALT and EAST, respectively. Briefly, in both assays, 0.1 mL hemolysate was incubated with alanine or aspartate for 60 min at 37°C, in the presence and absence of excess PLP. After the reaction was stopped with trichloroacetic acid, 20 µL aniline citrate was added to the EAST reaction mixture to convert oxaloacetate to pyruvate. One milliliter of 1 g/L dinitrophenylhydrazine in 2.4 mol/L HCl, which reacts with pyruvate, was added and then extracted from the reaction mixture with 1 mL toluene. Pyruvate-dinitrophenylhydrazine formed a colored compound when 1 mL alcoholic potassium hydroxide (25 g NaOH/L ethanol) was added. The absorbance, read at 490 nm, is proportional to the concentration of product (pyruvate) formed. Hemoglobin (Hb) concentration of the packed erythrocytes was determined by a cyanmethemoglobin method (Sigma Chemical, St. Louis, MO). Enzyme activities were expressed as mmol pyruvate/(L packed erythrocytes · h) or mmol pyruvate/(g Hb · h) and converted to µkat/L RBC and µkat/g Hb. The activity coefficient was calculated by the ratio of stimulated (PLP added) to unstimulated activity. Erythrocyte aminotransferase activity measurements were carried out under yellow light to prevent photodestruction. All analyses were performed in triplicate.

Statistical analysis.

Data were analyzed using the JMP statistical analysis computer program and SAS version 6.12 (SAS Institute, Cary, NC). Means and standard deviations were calculated at each time point for all measurements. Repeated-measures ANOVA was used to test for statistical differences among means. Dunnett’s test was used to compare EALT and EAST mean basal activities and activity coefficients at each time point with d 0. The least significant difference (LSD) multiple comparison test was used to compare means of different storage temperatures at the same time point. Statistical comparisons were considered to be significant at P 0.05. Values are means ± SD.

	RESULTS

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On d 0, EALT and EAST activity coefficients (EALT-AC and EAST-AC) were measured. These vitamin B-6–dependent aminotransferase measurements reflect subjects’ vitamin B-6 status at the beginning of the study. EALT-AC and EAST-AC values (1.11 ± 0.09 and 1.28 ± 0.08, respectively) indicated vitamin B-6 nutritional adequacy because these measurements averaged <1.25 and 1.80, respectively (9) .

In erythrocytes frozen at -20°C, EALT basal activity was 16% lower (P 0.07) by d 14 compared with d 0 (Fig. 1 , upper panel). There were also 17 and 22% decreases in basal activity by d 58 and 84 (P 0.05 for both) in the samples frozen at -20°C. In addition, EALT basal activity in samples frozen at -20°C was 14% lower than in samples frozen at -80°C by d 14, 20% lower by d 58 and 24% lower by d 84 (P 0.05 for all comparisons). On d 28, 58 and 84, samples frozen at -20°C had 22, 21 and 29% lower basal activity, respectively, than samples frozen at -196°C (P 0.05 for all comparisons). Basal activity of EALT in samples frozen at -80°C and -196°C did not differ significantly from d 0 at any time points. Although the EALT-AC was not significantly different from d 0 at any time point in samples frozen at all three temperatures (Fig. 1 , lower panel), on d 7 and 28, the activity coefficients were 7% (P 0.05) and 5% higher (P 0.11) in samples frozen at -20°C than in those frozen at -80°C.

View larger version (20K): [in this window] [in a new window]   Figure 1. Human erythrocyte alanine aminotransferase basal activities (A) and activity coefficients (B) from hemolysates stored at -20, -80 and -196°C. Each value represents the mean ± SD, n = 3. The horizontal line represents the mean of d 0 measurements. Asterisks denote means that are significantly different from d 0, P 0.05.

View larger version (21K): [in this window] [in a new window]   Figure 2. Human erythrocyte aspartic acid aminotransferase basal activities (A) and activity coefficients (B) from hemolysates stored at -20, -80 and -196°C. Each value represents the mean ± SD, n = 3. The horizontal line represents the mean of d 0 measurements. Asterisk denotes mean that is significantly different from d 0, P 0.05.

	DISCUSSION

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The results of this study suggest a gradual loss of EALT basal activity when packed erythrocytes are stored frozen at -20°C, whereas enzyme activity of samples frozen at -80 and -196°C remains fairly stable. Similarly, Rose et al. (6) reported that after 10–15 d of erythrocyte hemolysate storage at -20°C, the activity of EALT decreased 45–49%. The greater decreases in EALT basal activity reported by Rose et al. (6) could be due to their use of a less concentrated frozen erythrocyte hemolysate preparation rather than packed cells. The reduced EALT basal activity that results from storage at -20°C may be due to increased labilization of the apoenzyme over time and/or degradation of dissociated PLP by phosphatases present in erythrocytes (10) . In support of our findings in samples frozen at lower temperatures, Skala et al. (11) reported that EALT basal activity was stable for 60 d at -70°C.

Rose et al. (6) also reported that EAST basal activity decreased 22–30% when hemolysates were stored for 10–15 d at -20°C. In the present study, EAST basal activity in packed cells frozen at -20°C decreased significantly (40%) by d 58 and then increased to greater than baseline on d 84. High intra- and interassay variability in the EAST assay is one possible explanation for this observation. Intra-assay variability between replicates in the EAST procedure was > 2 times higher than in the EALT procedure, whereas interassay variability in EAST basal activity measurements was 1–2 times higher than variability in EALT basal activity in samples frozen at -20 and -80°C throughout the course of the study. Skala et al. (11) reported that EAST basal activity was stable for up to 76 d at -70°C.

Nevertheless, studies evaluating vitamin B-6 status usually employ activity coefficients rather than basal activities. Activity coefficients of EALT did not differ from d 0 at any of the time points or storage temperatures, although there was a downward trend after 28 d of storage. There were, however, nonsignificant increases (17 and 18%, P = 0.60 and 0.25, respectively) in EAST activity coefficients in samples frozen at -20 and -80°C by d 7, and EAST-AC was significantly higher (38%) in samples frozen at -20°C compared with samples frozen at -80°C after only 1 d of storage. The tendency for EAST basal activity to decrease while the activity coefficient increases again implies that with frozen storage, the PLP may be dissociating from the apoenzyme, which exposes it to degradation by phosphatases present in the erythrocytes (10) . These data suggest that the EALT activity coefficient is a more reliable measure than the EAST activity coefficient when samples must be frozen before analysis.

Another consideration is that the subjects in this study all had adequate vitamin B-6 status. In vitamin B-6–deficient subjects, proportionally more of the apoenzyme form would be present. The apoenzyme form may be more susceptible to degradation during storage than the holoenzyme form. Thus, aminotransferase activities may be less stable in frozen erythrocytes from vitamin B-6–deficient individuals.

In summary, the present study demonstrates that both EALT and EAST enzymes are prone to loss of basal activity when frozen at -20°C, whereas the EALT-AC is a more stable and reliable measure of vitamin B-6 status than EAST-AC. Packed erythrocyte preparations may provide greater EALT and EAST enzyme stability than frozen diluted hemolysate preparations. If EALT activity cannot be measured the same day blood is drawn, samples may be frozen at -20°C for 28 d or at -80°C for no > 84 d; EAST activity should be measured on fresh erythrocytes.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the statistical advice of Marc Evans, Program in Statistics, Washington State University.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 2000, April 2000, San Diego, CA [Hansen, C. M. & Shultz, T. D. ,s>(2000) Stability of vitamin B-6 dependent aminotransferase activity in frozen packed erythrocytes. FASEB J. 14: A241 (abs.)].

² Supported in part by U.S. Department of Agriculture NRICGP grant #97–35200–4238 and the Agricultural Research Station, College of Agriculture and Home Economics, Washington State University, Pullman, WA.

⁴ Abbreviations used: AC, activity coefficient; EALT, erythrocyte alanine aminotransferase; EAST, erythrocyte aspartate aminotransferase; EGOT, erythrocyte glutamic oxaloacetic transaminase; EGPT, erythrocyte glutamic pyruvic transaminase; Hb, hemoglobin; PLP, pyridoxal 5'-phosphate.

Manuscript received September 25, 2000. Revision accepted February 8, 2001.

	REFERENCES

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1. Hansen C. M., Leklem J. E., Miller L.T. Changes in vitamin B-6 status indicators of women fed a constant protein diet with varying levels of vitamin B-6. Am. J. Clin. Nutr. 1997;66:1379-1387[Abstract/Free Full Text]

2. Huang Y. C., Chen W., Evans M. A., Mitchell M. E., Shultz T. D. Vitamin B-6 requirement and status assessment of young women fed a high-protein diet with various levels of vitamin B-6. Am. J. Clin. Nutr. 1998;67:208-220[Abstract]

3. Leklem J. E. Vitamin B-6. Shils M. E. Olson J. A. Shike M. Ross A. C. eds. Modern Nutrition in Health and Disease 1999:413-421 Williams and Wilkins Baltimore, MD.

4. Sauberlich H. E. Vitamin B-6 (pyridoxine). Sauberlich H. E. eds. Laboratory Tests for the Assessment of Nutritional Status 1999:71-102 CRC Press New York, NY.

5. Brown R. R., Rose D. P., Leklem J. E., Linkswiler H., Anand R. Urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B-6. Am. J. Clin. Nutr. 1975;28:10-19[Abstract/Free Full Text]

6. Rose D. P., Strong R., Folkard J., Adams P. W. Erythrocyte aminotransferase activities in women using oral contraceptives and the effect of vitamin B-6 supplementation. Am. J. Clin. Nutr. 1973;26:48-52

7. Woodring M. J., Storvick C. A. Effect of pyridoxine supplementation on glutamic-pyruvic transaminase and in vitro stimulation in erythrocytes of normal women. Am. J. Clin. Nutr. 1970;23:1385-1395[Medline]

8. Merrill A. H., Henderson J. M. Diseases associated with defects in vitamin B-6 metabolism or utilization. Annu. Rev. Nutr. 1987;7:137-156[Medline]

9. Leklem J. E. Vitamin B-6: a status report. J. Nutr. 1990;120:1503-1507

10. Anderson B. B., Fulford-Jones C. E., Child J. A., Beard M.E.J., Bateman C.J.T. Conversion of vitamin B₆ compounds to active forms in the red blood cell. J. Clin. Invest. 1971;50:1901-1909

11. Skala J. H., Waring P. P., Lyons M. F., Rusnak M. G., Alletto J. S. Methodology for determination of blood aminotransferases. Leklem J. E. Reynolds R. D. eds. Methods in Vitamin B-6 Nutrition 1981:171-202 Plenum Press New York, NY.

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