Effect of various levels of pyridoxine on erythrocyte aminotransferase activities in the rat

NUTRITION RESEARCH, Vol. 9, 195-204, 1989 0271-5317/89 $3.00 + .00 Printed in the USA. Pergamon Press plc. All r~ghts reserved.

EFFECT OF VARIOUS LEVELS OF PYEIDOXINE ON ERY~HEOCYTE AMINOTRANSFERASE ACTIVITIES IN THE RATJH Skala2,PhD, MC Schaeffer, PhD, DA Sampson, PhD, and D Gretz, MS Biochemistry Research Unit (JHS, DG), and Nutrient Intake and Performance Research Unit (MCS, DAS), Western Human Nutrition Research Center, US Department of Agriculture, P.O. Box 29997, Presidio of San Francisco, CA.

ABSTRACT

A s t u d y was d e s i g n e d t o e v a l u a t e t h e p e r f o r m a n c e o f e r y t h r o c y t e a l a n i n e (ALT) and a s p a r t a t e (AST) a m i n o t r a n s f e r a s e s i n t h e insufficient, marginal, adequate and elevated dietary vitamin B-6 intake ranges of the rat. Female Long-Evans rats, 12 weeks old, were 4-hour meal-fed an AIN 76A diet devoid of pyrldoxlne (PN) for 3 weeks. Rats were then blocked by weight and randomly assigned within block to one of six dietary treatments (n=12). Four diets were formulated to contain 0.25, 0.5, 1.0, or 7.0 mE PN.HCI/kE. The rats receiving these diets were palr-fed to the 0.25 group ; all were 4hour meal-fed. Two additional diet treatments of 7.0 and 1400 mg PN.HCI/kE were fed ad libltum. Blood samples were collected by cardiac puncture at the end of the devoid period and at 2, 4, and 6 weeks of repletion. ALT and AST endogenous and stimulated activities were determined by an automated procedure (recently published by the senior author). Aminotransferase activities differed significantly between devoid and control groups at the end of the devoid period. After repletion at various levels for six weeks a significant doseresponse was reached among the palr-fed groups. There was no difference in response between ad llhltum and palr-fed groups at the 7.0 mE PN.HCI/kg level. While there was a significant increase in the alanlne aminotransferase levels at nominal 1400 mg PN.HCI/kE, it was not suggestive of a dramatic change. KEY WORDS: V i t a m i n B-6, P y r i d o x i n e S t a t u s , P y r i d o x i n e - d e p e n d e n t Enzymes, Animal Study, Marginal Deficiency

INTRODUCTION

Erythrocyte aminotransferase activities have been used as biochemical markers of vitamin B-6 Status in experimental and clinical work. Investigators measure the activity of these vitamin B-6 dependent enzymes without (endogenous) and with (stimulated) in vitro addition of the active vitamin cofactor, 1

R e f e r e n c e to a company o r p r o d u c t name does n o t i m p l y a p p r o v a l o r r e c o m m e n d a t i o n of t h e p r o d u c t by t h e US D e p a r t m e n t o f A g r i c u l t u r e to t h e 2 e x c l u s i o n o f o t h e r s t h a t may be s u i t a b l e . To whom c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d .

195

196

J.H. SKALA et al.

pyrldoxal 5'-phosphate (PLP), to the assay medium, as well as the calculated activation coefficient (stimulated activlty/endogenous activity). While most agree that these measurements are useful in monitoring vitamin B-6 status (1,2,3), there is some debate as to their sensitivity in rats (2) and humans (3), particularly in the marginal ranges of dietary intake. Recent studies have measured erythrocyte aminotransferase activities in rats over a range of intakes close to or greater than the actual requirement (4p5). There is some evidence that hepatic amlnotransferase apoenzyme levels can be increased by administration of large doses of pyridoxine (PN) (6,7). Consumption of large doses of vitamin B-6 in the human population is apparently not uncommon 48). We wanted to determine: 1) if erythrocyte alanine (ALT) and aspartate (AST) aminotransferase activities were sensitive to vitamin B-6 status In the marginal range for the rat, and 2) if erythrocyte aminotransferase activities could be further increased above control levels by an excess of vitamin B-6. This collaborative study between the research units at our Center afforded the opportunity to evaluate the responsiveness of our simultaneous automated system for the aminotransferases (9) over dietary ranges estimated to be insufficient, marginal, adequate and excessive for the rat.

METHODS AND MATERIALS Diets. Diets were obtained from Dyeta, Inc. (Bethlehem, PA). The control (CON) diet was similar to AIN76A (I0,Ii) incorporating a vltamln-free casein and formulated to contain 7.0 mg pyrldoxlne (PN).HCI/kg. Appropriate amounts of PN.HCI were included in the AIN vitamin mix. Mean actual analyzed values (our laboratory) for the diets as expressed as mg PN.HCI/kg of diet are shown In parentheses after the nominal value: Devoid (0.04), 0.25 (0.25), 0.5 (0.51),

1.0 ( 0 . 9 7 ) ,

7.0 ( 6 . 8 9 ) ,

1400 (1379).

Animals and study design. After stabilization on rat chow for 5 days, 84 female Long-Evans rats (Charles River Laboratories, Bloomington MA), 12 weeks old, were oriented to a 4 hr meal-feeding routine on the CON diet. The mealfeeding (incorporated to equalize eating patterns) was scheduled for the beginning of the 12 hour dark phase of the lighting schedule daily at 1300 hr. After I week of meal-feeding orientation, 24 rats were randomly assigned to one or the other of two dietary treatment groups: the CON diet or the same dlet devoid of PN.HCI (DEF). The remaining sixty animals and the DEF group were all given access to the devoid diet during the 4 hour meal-feeding period. The CON group was palr-fed (calculated daily; group mean basis) to the DEF group as reference, and meal-fed. This protocol was followed for 3 weeks. The remaining 60 rats were then blocked by weight and randomly assigned within blocks to one of 6 dietary treatments. The DEF group was randomly distributed among these treatments, 2 animals per treatment, to compensate for any effect due to prior blood collections. This provided 12 rats per treatment group. The dietary treatments included 0.25, 0.5, 1.0, and 7.0 mg PN.HCI/kg palr-fed and meal-fed as before (0.25 PF, 0.5 PF, 1.0 PF, and 7.0 PF). In the repletion phase, the 0.25 PF was the pair-fed reference (calculated daily; group mean basis). A group fed the control diet, but on a 24-hr ad llbltum basis (7.0 AL), was included to control for possible effects of meal-feedlng on the response variables. The sixth treatment group was fed excess vitamin B-6 (about 200 times the NRC recommended level), also on a 24-hr ad libitum (1400 AL) basis. This protocol lasted 6 weeks.

DIET B-6 AND ALT/AST ACTIVITIES

197

Body weights were measured twice weekly and daily food intake was recorded using a computer assisted (San Diego Instruments, La Jolla, CA) automatic taring balance during both protocols. Sample collection, preparation, and analysis. Heparinlzed (sodium) blood specimens were obtained by cardiac puncture from the CON and DEF groups at the end of the 3-week devoid diet period, and from the 6 diet groups at 2, 4 and 6 weeks of repletion. Hematocrit determinations were made on duplicate aliquots of whole blood (12). The plasma was removed from measured aliquots of whole blood, and the red blood cells were washed 3 times with cold 0.9% (w/v) saline; 4.0 volumes (red cell volume assumed to be 50% of whole blood volume) of distilled water were mixed with the cells before they were frozen at -70 ~ C. Prior to analysis, the cell preparations were sonicated, frozen, then thawed, centrifuged and an aliquot diluted with 5.0 volumes of TRIS buffer. The sonication and freezing were necessary to minimize occlusion of the soluble enzymes by the dense cellular matrix/debris. This provided an hemolysate with an approximate (50% packed cell volume basis) 30-fold dilution of cells. Final calculated results took into consideration the actual cell volume (hematocrit) and the subsequent dilutions. ALT and AST endogenous and stimulated activities were determined by our automated simultaneous procedure (9). One U of an enzyme will catalyze the transformation of 1 micromole of substrate per minute at the specific assay temperature, which is 37~ C for the method used in this study. Statistical analyses. We determined (13) that I0 animals per group were required for confidence at the 95% level, based on ALT and AST determinations performed with prototype methodology (14). Descriptive statistics, analyses of variance and group comparisons (15), and regressions and curve fitting (16,17) were performed with standard statistical procedures using commercial microcomputer software (18,19). Group mean comparisons are indicated with an underscoring technique for clarity (20).

RESULTS

Groups of rats fed the recommended (7.0 AL) and elevated (1400 AL) levels of vitamin B-6 gained significantly more weight than the pair-fed meal-fed groups during the six week repletion phase and their final mean weight was greater (p<0.05) (Table i). There were no significant differences in body

OBSERVATION

TABLE 1 Summary of Body Weisht and Feed Intake Observations Palr-fed Meal-fed Groups (PF) Ad Libitum Groups (AL) Nominal mg PN.HCI/kg Nominal mg PN.HCI/kg 0.25 0.5 1.0 7.0 7.0 1400

Weight at End of 6 Weeks Repletion

Mean+SEM, g Final n

229+3.7 a 12

229+4.1 a 12

226+3.6 a 11--

229+3.3 a 11

247~6.0 b 11

243+3.9 b 12

Mean D a i l y PN I n t a k e Durin~ R e p l e t i o n P e r i o d s , ug/day+~EM 0-2 Weeks 2-4 Weeks 4-6 weeks

2.0+.06 2.3~.06 2.1~.05

4.0+.06 4.5~.08 4.2~.08

7.9+.07 8.4~.22 7.8~.22

56.0+.79 59.3+--1.4 56.4+-1.5

67.2+2.4 64.5+--2.0 64.2~2.6

14033+244 12979+-166 13262~272

Values not showing common superscripts are significantly different, p<0.05.

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J.H. SKALA et al.

weight between the AL groups, or among the PF groups. Mean body weights of the various repletion groups were notdlfferent at the beginning of the repletion phase. There were significant differences in PN intake (p<0.05) calculated after 6 wk of repletion among all treatment groups as intended by design and as might be expected from ad libitum consumption of diet by the two AL groups. The 7.0 AL group consumed approximately 14 % more diet and hence more PN per day than the 7.0 PF group for the entire repletion phase; the mean body weight of the 7.0 AL group was about 87. heavier. Actual g/day food intake did not differ among the 4 PF groups or between the 2 AL groups. The mean endogenous and PLP stimulated amlnotransferase activities, and activation coefficients (Act. Coef.; stimulated U/endogenous U) for DEF group rats differed significantly (p<0.05) from those of the CON group at the end of the depletion phase (Table 2). DEF group endogenous ALT activity was 56~ of the CON level, while DEF endogenous AST activity was 777. of the CON value. Stimulated DEF activities were 597. and 907. of CON values for ALT and AST, respectively.

ANALYSIS Endogenous Stimulated Act. Coef.

TABLE 2 Erythrocyte Enzyme Activities at End of Depletion Phase Alanine Amlnotransferase (ALT) Aspartate Aminotransferase (AST) ~VIL R B C ~ SEM)* (UIL R B C ~ SEH) DE CON DEF CON 113+6 125~6 1.10~.006

203+8 212+--8 1.05~.012

1753+85 2258~101 1.29~.014

2279+64 2513+--65 1.10~.006

Enzyme activity of red blood cells in International Units. One U of an enzyme will transform 1 micromole of substrate per minute. % group received PN devoid diet, and CON group received modified AIm control diet.

Some significant differences (p<0.05) in ALT activities became apparent in the palr-fed groups as early as the first blood collection (2 wk) during the repletion phase (Table 3). The 7.0 PF group endogenous and stimulated activities were already higher and the activation coefficient lower than the other restricted groups. The effect of the high ad libitum diet (1400 AL group) on endogenous ALT activity was also evident at this stage. At the 4 wk evaluation the ALT activities of the 0.25 PF group had not changed; endogenous and stimulated activities were significantly lower (p0.05) relationships were between all the measurements of the 7.0 PF and 7.0 AL groups, and the activation coefficients of the 7.0 AL and 1400 AL groups. The 6 wk ALT endogenous activities of the 0.25-7.0 PF groups were 52~, 78~, 125%, and 212~ of the DEF endogenous activity shown in Table 2. Differences in AST activities manifested themselves early (2 wk) in the repletion phase (Table 4). The endogenous and stimulated activities of the 0.25 and 7.0 PF groups were significantly (p<0.05) lower and higher, respectively, than the intermediate restricted groups. In contrast to the increase in ALT activity noted for the 1400 AL group (vs. the 7.0 AL group), there were no significant effects of the 1400 mg PN.HCI/kg diet on AST activities of the 1400 AL group at any observation point. There was slight improvement, compared to the 7.0 AL group, in the activation coefficient at weeks 4 and 6. The AST

DIET B-6 AND ALT/AST ACTIVITIES

ANALYSIS

199

TABLE 3 Alanine Aminotransferase Results (ALT) (U/L RBC • SEM)* Ad Libitum Groups (AL) Pair-fed Meal-fed Groups (PF) Nominal mg PN.MCl/kg Nominal mg PN.HCI/kg 0.25 0.5 1.0 7.0 7.0 1400 REPLETED 2 WK

Endogenous

74+3

79+7

84+5

145+8

152+8

175+10

Stimulated

84+3

87+7

94+5

154+8

169+8

182+10

Act. Coef.

1.14+.007

1.12+.011

1.12+.006

1.06+.002

1.06+.002

1.04+.002

232+-1_13

220+-12

263+_15

245+14

232+13

277+13

1.06+.004

1.06+.003

1.05+.002

REPLETED 4 WK Endogenous

67~2

108+--8

Stimulated

79+2

120+8

Act. Coef.

1,18+.008

9A+_6 106+6

1.12+.009

1.13+.005

REPLETED 6 WK Endogenous

59+3

88+6

141+8

240+-15

232+_11

286+_16

Stimulated

68+3

99+6

153+8

255+16

246+11

301+16

Act. Coef.

1.16+.006

1.13+.004

1.08+.003

1.06+.002

1.06+.002

1.05+.007

Values not underscored by a continuous line differ significantly (p
response to lower dietary PN levels was manifested in the 6 wk observations. The apoenzyme levels of the 1.0 and 7.0 PF groups were equivalent by this time. Linear regression analyses were made on the endogenous amlnotransferase activities of diet groups 0.25-1.0 PF across time (2,4 and 6 wk) and are shown in Table 5. These analyses were made to confirm and interpret the apparent trends of data in Tables 3 and 4, possibly influenced by various elements of experimental error.

DISCUSSION

We f o u n d a s i g n i f i c a n t d o s e - r e s p o n s e r e l a t i o n b e t w e e n ALT a n d AST activities and activation coefficients, and dietary PN. This relationship manifested itself after six weeks of repletion at diet levels of 0.25, 0.5 and 1.0 mg PN.HCI/kg. At these apparently less than adequate dietary levels, enzyme response to diet was linear. Driskell et al. (4) fed both weanling rats and non-pregnant mature rats (250g), amounts of food having a dietary equivalence to 0.5-3.0 mg PN/kg and 0-800 mg PN/kg, respectively, for four weeks. Erythrocyte alanine amlnotransferase (ALT) was measured using a modified colorimetric procedure.

200

ANALYSIS

J.H. SKALA et al. TABLE 4 Aspartate Aminotransferase Results (AST) (U/L R B C • pair-fed Meal-fed Groups (PF) Ad Libitum Groups (AL) Nominal mg PN.MCl/kg Nominal mg PN.HCI/kg 0.25 0.5 1.0 7.0 7.0 1400 REPLETED 2 WK

Endogenous

1368+43

1605+108

1705+80

2223+119

2315+41

2442+67

Stimulated

1870+42

1974+118

1996+201

2427+118

2518+44

2621+72

Act. C o ef .

1.37+.014

1.24+.022

1.16+.106

1.10+.007

1.09+.007

1.07+.005

Endogenous

1354+36

1921+84

1979+82

2638+61

2677+75

2832+86

Stimulated

1999+46

2433+77

2457+94

2936+53

2956+88

3066+90

Act. Coef.

1.48+.016

1.27+.018

1.24+.008

1.12+.008

1.10+.006

1.08+.004

REPLETED 4 WK

REPLETED 6 WK Endogenous

1356+49

1904+62

2475+79

2631+58

2759+53

2901+65

Stimulated

1956+42

2440+58

2871+84

2873+64

3021+54

3110+68

Act. Coef.

1.45+.026

1.29+.016

1.16+.008

1.09+.002

1.10+.006

1.07+.005

Values not underscored by a continuous llne differ significantly (p<0.05) from others in the comparison groupings as determined by ANOVA and the least significant difference. Comparisons were made among PF groups, between the 7.0 PF and 7.0 AL groups, and between the 7.0 AL and 1400 AL groups.

TABLE 5 Linear Regression Analysis of Aminotransferase Endogenous Activity Response Over Time to Repletion With Lower Vitamin B-6 Levels Aminotransferase Diet Regression Correlation Significance Group Equation Coefficient (p
0.25 PF 0.5 PF 1.0 PF

y=81.2+(-3.7)x yffi82.2+2.4x y=45.7+14.9x

-0.55 0.15 0.76

+ +

Aspartate (AST)

0.25 PF 0.5 PF 1.0 PF

y=1371+(-3)x y=1511+75x y=1296+190x

-0.03 0.38 0.76

+ +

ALT activity response was significantly different for weanling groups fed 0.5 or 1.0 mg PN/kg compared to 1.5-3.0 mg PN/kg of diet. The difference in enzyme response between 0.5 and 1.0 mg Pn/ kg diet levels were not significant. This is in contrast to our limits of detection at these low dietary intake levels. No significant differences in ALT activities were noted in mature rats fed 3, 6, 9, 12, 18, 24, 80 and 800 mg PN/kg.

DIET B-6 AND ALT/AST ACTIVITIES

201

Lumeng and coworkers (1) maintained weanling rats for 9 weeks on liquid diets supplying 0, 4, 12, 24 and i00 ug of PN daily, and measured erythrocyte ALT and AST by a colorimetric procedure, as well as other plasma and tissue parameters. They concluded that the enzyme activities were useful markers for vitamin B-6 status but that the activation coefficients (alpha factors) were insensitive in the rat. Ink and Henderson (2) reviewed their paper and concluded that the erythrocyte aminotransferase activities "were found to be useful but insensitive indicators". We found that both activity and activation coefficient were sensitive in the range of dietary inadequacy for the rat. A review by Shane (3) noted that the utility of AST measurements had been demonstrated in controlled severe deficiencies but its applicability in detection of marginal deficiency had not been documented. He also noted that publications dealing with responsiveness of the activation coefficient of ALT were equivocal. More recently Lequeu et al. (5) measured ALT and AST activities using a spectrophotometric method in weanling rats fed four levels of PN for 13 days. They found significant differences in enzyme activities between groups fed 0.01 or 0.5 mg PN.HCI/kg and those fed 2 or 8 mg PN.HCI/kg. They found no dietary effects on AST activation coefficients; differences in ALT coefficients were noted only between the lowest group and the groups fed 2 and 8 mg PN.HCI/kg. By contrast we observed distinct enzyme activity responses to 0.25, 0.5 and 1.0 mg PN.HCI/kg diet. While we found activation coefficients for both enzymes were responsive in this low range of intake, more marked changes were noted in AST coefficients than in those for ALT. Categorization of nutritional status (i.e., deficient, marginal, adequate) using vltamin-dependent enzyme activities requires consideration of several limiting factors for accurate deductions to be reached. These include allowance of time for equilibration of erythrocyte cofactor concentration, subsequent formation of holoenzyme, and erythrocyte turnover (18), as well as various analytical details. We analyzed the data in Tables 3 and 4 in two general schemes: a) by diet group across time of repletion (Table 5); and b) at a single time-polnt across diets. The regression analysis (Table 5) and values for endogenous ALT and AST activities in Tables 3 and 4 indicate that the PN status of the 0.25 PF group declined during repletion. ALT activities of the 0.5 PF group did not change significantly with time; AST activities increased significantly at week 4 of repletion and leveled off. Activities of both enzymes in the 0.25 and 0.5 PF groups were markedly lower (p<0.05) than the DEF values (Table 2). The slope of the regression lines (Table 5) for activities of both enzymes in the 1.0 PF group indicates continued improvement in status during repletion (as sampled in thls study). It is possible that endogenous activities (1.0 PF) might have peaked with longer repletion time. ALT responded slower (59% of 7.0 PF group activity) than AST (947~ to 6 weeks of repletion at 1.0 mg PN.HCI/kg diet. Moreover, there was no difference between the stimulated AST activities of the 1.0 and 7.0 PF groups. Brln (22) has described a 5 step sequence for the development of vitamin deficiency; enzyme cofactor insufficiency occurs in the marginal phase. Our observation that AST activities of the 1.0 PF group were virtually equivalent to those of the 7.0 PF group at 6 weeks, whereas the ALT activities were not, supports the conclusion that this diet (1.0 mg PN.HCI/kg) is close to a marginal dietary level for these rats as it was consumed in this study (actual intake about 8 ug/d). In a generic sense, marginal intake might be defined as the point just below the lowest limit of adequacy. The d a t a f o r e a c h o f t h e 7 . 0 PF, 7 . 0 AL and 1400 AL g r o u p s ( T a b l e s 3 a n d

202

J.H. SKALA et al.

4) peaked after 4 weeks of repletion and leveled off, reflecting a faster equilibrium than occurred at lower PN levels. For this reason, data for these groups do not fit a linear regression line over the entire repletion period. A series of curve fitting procedures were performed to find the best description of the dose-response relationship between dietary PN and endogenous enzyme activities after repletion for 6 weeks. These analyses were not performed for predictive purposes, but to confirm and formally describe the nonlinear response observed in the isolated data points (Tables 3 and 4). Data analysis across diet groups indicated a significant (p<0.05) linear doseresponse for endogenous enzyme activities (ALT and AST) at 6 weeks on PN intake by rats in diet groups 0.25 PF, 0.5 PF and 1.0 PF. The linear regression equations for the data (Tables 3 and 4) are y=30.1+11.5x (r=0.853) for ALT and y=1017+157x (r=.896) for AST. However a lack of fit test indicated that when the 7.0 PF groups was included, a linear model did not fit the dose-response relationship. A quadratic model was statistically adequate to describe the effect of diet on endogenous ALT and AST activities over the range from 0.25-7.0 mg PN.HCI/kg. A more optimal fit was obtained using the equation for an asymptotic curve (19). This was performed on log transformed data (In) for ALT and original data for AST. Predicted mean y values for both enzymes at the four PF diet treatment levels agreed extremely well with observed means. The practical consequence of this is that these enzyme activities are more sensitive to incremental increase in PN intake in the lower dietary range than in the higher range. The methodology and study design employed for this study allowed distinction between narrower dietary vitamin B-6 levels (and intakes) and associated enzyme activities than previously reported (1,4,5). More points in the lower intake range were examined here. ApoAST appeared to be more stable (biologically) than apoALT, accounting for more marked changes in AST activation coefficient in this study. Other studies have noted lower ALT activation coefficient responses (1,5,23,24). Significant differences in this calculated factor were noted here at the lower intake levels contrasted with limited (5,23) or no response (i) in previous reports. We conclude that the aminotransferases are quite responsive to marginal or insufficient PN intake in the rat, in fact, more so at lower than at adequate levels of PN intake. There are notable differences in response of the two enzymes. The changes in ALT activities were more marked than in AST at lower intakes. A modest, significant increase in ALT activity was noted in response to excess dietary PN (1400 AL), while none was noted in AST activity. In controlled animal studies, changes in the endogenous aminotransferase activity appear to be an adequate marker for changes in PN status below the actual rat requirement. ACKNOWLEDGMENTS

The authors thank Laurie Campbell and Dennis O'Connor for technical assistance, and Bruce Mackey and Linda Whitehand of the USDA Pacific West Area office for advice on statistical procedures.

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Hamfelt A. Pyridoxal phosphate concentration and aminotransferase activity in human blood cells. Clin Chim Acta 1967; 16:19-28.

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Accepted for publication November 5, 1988.