Guilland and colleagues (1989) suggested that exercise may increase vitamin B-6 requirements in young athletes. They showed that more vitamin B-6 is required for maintenance of a normal erythrocyte aspartate aminotransferase activity coefficient (EAST-AC < 1.80)⁵ in exercising subjects than in sedentary subjects. This hypothesis is supported by research that shows increased urinary 4-pyridoxic acid (4-PA; the excretory form of vitamin B-6) excretion with exercise when athletes have vitamin B-6 intakes similar to the RDA and when supplemented with an additional 8-20 mg/d of vitamin B-6 (Dunton et al. 1992, Hatcher et al. 1982, Manore et al. 1987). However, Dreon and Butterfield (1986) observed that 4-PA excretion was decreased in their male subjects consuming 4-4.5 mg vitamin B-6/d and running either 8 or 16 km/d for 29 d. This would indicate that less of the vitamin was being converted to 4-PA. They also observed a decrease in urinary 4-PA and cystathionine excretion after a methionine load. They hypothesized that long-term exercise may stimulate the storage of vitamin B-6 in a labile pool that is capable of redistribution under circumstances of increased need (e.g., a methionine load test). In fact, to date, there is no clear documentation that long-term exercise increases the loss of vitamin B-6 from the system. Therefore, further research is required in the area of vitamin B-6 and exercise to determine if vitamin B-6 requirements are increased by endurance exercise.

Understanding how blood and tissue B-6 vitamers change is necessary to expand knowledge of vitamin B-6 and nutritional biochemistry during long-term exercise (i.e., training). Past studies on vitamin B-6 nutrition and metabolism have focused mainly on either pyridoxal 5-phosphate (PLP) or total vitamin B-6, although the vitamin occurs in several other forms (termed "vitamers") in mammalian tissues, including pyridoxal (PL), pyridoxamine 5-phosphate (PMP), pyridoxamine (PM), pyridoxine (PN) and 4-PA. HPLC now permits measurement of all forms of vitamin B-6 (Edwards et al. 1989, Sampson and O'Connor 1989a) and can be used to clarify how blood and tissue B-6 vitamer levels may vary.

The aim of the study reported here was to investigate exercise-related changes in B-6 vitamer distribution in blood and organ tissue in relation to three different dietary vitamin B-6 levels. Furthermore, urinary 4-PA excretion and erythrocyte aspartate aminotransferase activity were measured to assess vitamin B-6 status. For this purpose, a long-term feeding experiment with male Sprague-Dawley rats was started. The animals were kept under strictly controlled conditions and were either trained by swimming or rested.

Table 1. Composition of experimental diet [View Table]

MATERIALS AND METHODS

Table 2. Effect of training on growth performance and food intake of rats fed diets containing three different levels of pyridoxine-HCl (0, 2 or 7 mg PN-HCl/kg diet)1 [View Table]

Table 3. Weights of liver, gastrocnemius muscle, heart, kidneys, brain, adrenals, epididymal fat of sedentary and trained rats fed 0, 2 or 7 mg PN-HCl/kg diet1 [View Table]

Aliquots of plasma and resuspended erythrocytes (0.5 mL) were mixed with 1.0 volume of 0.8 mol/L cold perchloric acid containing an internal standard, deoxypyridoxine (100 µmol/L) and centrifuged at 13,000 × g for 3 min at 4°C to precipitate protein. Portions of tissues (1 g) were homogenized in 9 mL 0.4 mol/L cold perchloric acid containing deoxypyridoxine (100 µmol/L) and centrifuged as described above to precipitate protein. Perchloric acid has been used successfully to extract B-6 vitamers from plasma and tissues and gives acceptable extraction for other rat tissues described here, on the basis of recovery of external spikes (discussed below). Protein-free supernatants from plasma, erythrocytes and tissues were filtered (0.22-µm microcentrifuge tube filters, Waters, Saint-Quentin en Yvelines, France) before injection into the chromatographic system. Concentrations of B-6 vitamers in sample extracts were measured using a reverse-phase HPLC method described by Edwards et al. (1989). That method gives the following recoveries for PLP, PMP, PL, PN and PM, for rat liver: 91, 96, 96, 105, and 96%, respectively; for rat plasma: 96, 103, 98, 103 and 102%, respectively. These values are similar to or better than recoveries obtained with another reliable HPLC method (Sampson and O'Connor 1989a). B-6 vitamer concentrations measured with this method agree closely with values measured with the HPLC procedure of Sampson and O'Connor (1989a and 1989b). Precision of the reverse-phase HPLC method, measured as a percentage of the coefficient of variation for 10 injections of B-6 standards, ranged from 1.8 to 4.2% for different vitamers. Acceptable accuracy, precision and recoveries of the reverse-phase method justified its use in the current study.

RESULTS

Food intake differed significantly among the six groups studied (Table 2). Rats fed the deficient diet consumed less food than rats fed either the suboptimal or control diets, and some of them had scaley dermatitis on their paws and around the mouth, nose and ears. Daily food intake averaged 19.2 g for the deficient rats (n = 24), 22.0 g for the rats fed the suboptimal diet (n = 24), and 22.2 g for the rats fed the control diet (n = 24), corresponding to a daily pyridoxine intake of ~13 nmol (3 µg), 170 nmol (35 µg), and 725 nmol (150 µg), respectively.

Basal erythrocyte aspartate aminotransferase activities were higher in rats consuming the 2 or 7 mg PN-HCl/kg diet compared with rats consuming the 0 mg PN-HCl/kg diet (Table 4). Similar results were obtained when additional cofactor was added to the same hemolysate to measure the degree of coenzyme deficit in the erythrocytes (stimulated EAST). In each dietary group, the activities observed in erythrocytes from sedentary rats were not significantly different than those of trained rats.

Table 4. Basal and stimulated erythrocyte aspartate aminotransferase (EAST) activities and erythrocyte aspartate aminotransferase activity coefficients (EAST-AC) of sedentary or trained rats fed three different levels of pyridoxine (PN-HCl)1 [View Table]

B-6 vitamer data for plasma, erythrocytes and different tissues are presented in Tables 5 and 6 and Figure 4. The distribution of principal B-6 vitamers varied in blood and tissues, with PLP and PL predominating in plasma and erythrocytes, and PLP and PMP predominating in tissues. Minor vitamers (i.e., those comprising <10% of total B-6 vitamers) that were affected by diet or exercise are reported, whereas those unaffected by diet or exercise are reported in table and figure footnotes as pooled means ± SEM. In erythrocytes of control groups, PL concentration exceeded PLP concentration three-fold, whereas in plasma, PL concentration was similar to PLP concentration (Table 5). After 9 wk, erythrocytes and plasma PLP concentration were about 95% lower in deficient groups than in control groups. Training had no effect on PLP, PL and total vitamin B-6 concentrations in plasma or erythrocytes. The concentrations of PMP, PLP and total vitamin B-6 in muscle were affected by both training status and dietary treatment (Table 6). They were significantly higher in rats fed the suboptimal and control diets than in rats fed the deficient diet. The concentration of vitamin B-6 in muscle of the deficient group was about 58% that of the control group. The muscle PLP level was slightly but significantly higher in trained rats (5-16%) than in sedentary rats. Concentration of PMP, PLP, PL, 4-PA and total vitamin B-6 in liver of deficient rats was significantly lower than that of rats of the two other dietary groups (Table 6). The concentrations of total vitamin B-6 in liver of the deficient group was about 44% that of the control group. Liver PLP, but not PMP, was slightly lower in the trained rats than in the sedentary rats. The concentration of PL in liver was significantly higher in the trained than in the sedentary rats. The liver total vitamin B-6 levels were not significantly different between the trained and the sedentary rats. Heart, brain, adrenal glands and kidney vitamin B-6 concentrations were not affected by exercise (Fig. 4).

Table 5. Concentration of B-6 vitamers in plasma and erythrocytes of sedentary or trained rats fed three different levels of pyridoxine-HCl (0, 2 or 7 mg PN-HCl/kg diet) [View Table]

Table 6. Concentration of B-6 vitamers in muscle and liver of sedentary or trained rats fed three different levels of pyridoxine-HCl (0, 2 or 7 mg PN-HCl/kg diet) [View Table]

DISCUSSION

The use of deficient and suboptimal dietary levels of vitamin B-6 during training allowed a more complete study of long-term effects of exercise on vitamin B-6 status, even though the use of suboptimal levels of dietary vitamin B-6 can be considered a more realistic animal model with which to study the effects of vitamin B-6 inadequacy than complete deprivation because a complete deprivation in unlikely to occur in athletes (Guilland et al. 1989). In fact, by inducing states of deficient and suboptimal vitamin B-6 nutriture in rats, our objective was to evaluate more easily whether chronic exercise (training) would affect vitamin B-6 status.

The data of the present study showed that the very low level of pyridoxine (0.68 µmol/kg) inherent in the deficient diet produced a severe deficiency as shown by the dryness and scurfiness of the fur on the hind paws, chest and neck occurring during the seventh week of feeding. The second major clinical effect was poor weight gain. The reported effects of vitamin B-6 deficiency on body weights and food intakes have been observed by numerous investigators (Driskell and Kirskey 1971, Felice and Kirskey 1981). It is interesting to note that the rats consuming 2 mg PN-HCl/kg diet (corresponding to 35 µg PN-HCl/d) grew, but their body weight gain was lower than the body weight gain of the rats consuming the control diet (corresponding to 150 µg PN-HCl/d) (Fig. 1 and Table 2). In fact, the National Research Council recommended in 1962 that 8-25 µg pyridoxine/d was needed for adequate growth (NRC 1962); in 1972, this recommendation was increased to 5 mg of the vitamin per kilogram diet for growth and reproduction and to 7 mg of the vitamin per kilogram diet for maintenance of normal erythrocyte transaminase activity (NRC 1972). Our data confirmed that growing rats require a daily pyridoxine intake higher than 45 µg for growth. In the three dietary groups, body weight gain and food intake were significantly higher in sedentary animals than in rats trained by swimming (Table 2). The reported effects of chronic exercise on body weight gain and food intake are in agreement with others (Crews et al. 1969, Guilland et al. 1988), showing both decreased food intake and body weight in trained male rats subjected to programs of regularly performed treadmill running or swimming. In addition to reduced food intake, the slower rate of body weight gain could be due to an increased energy expenditure associated with exercise. The significant decrease in growth resulting from either pyridoxine deprivation or chronic exercise was associated with lower epididymal fat weight and higher heart weight in relation to body weight (Table 3). It is generally held that exercise and/or training programs can augment lean body mass as well as cause a decrease in body fat (Forbes 1991).

The training-dependent changes in vitamin B-6 status indices (plasma PLP, EAST-AC) observed in our cohort of growing rats (Tables 4 and 5) were not significant. Furthermore, they did not compare well with those observed in other experiments conducted in humans. The functional index of vitamin B-6 status, i.e., the EAST-activity coefficient test, did not indicate a poorer vitamin B-6 status in trained than in sedentary rats. On the contrary, research reporting the vitamin B-6 status of athletes suggests that some athletes may have poor status (Fogelholm et al. 1993, Guilland et al. 1989, Telford et al. 1992). These three studies reported that 40-60% of the athletes tested had poor vitamin B-6 status on the basis of enzyme stimulation indices. In the study done by Guilland et al. (1989), supplementation for 1 mo improved the vitamin B-6 status of the control subjects but did not entirely improve that of the young athletes studied. In fact, some studies on humans and animals have shown that EAST was relatively unresponsive (Bosse and Donald 1979, Brown et al. 1975). This lack of response may be due to a strong affinity in vivo by the enzyme for the coenzyme. From an in vitro stimulation test on human erythrocyte aminotransferases, Cavill and Jacobs (1967) concluded that the apoenzyme-coenzyme binding was greater for aspartate aminotransferase (AST) than for alanine aminotransferase (ALT). Also, several investigators have reported that ALT was more severely affected than AST in vitamin B-6 deficiency (Bosse and Donald 1979, Brin et al. 1960). Unfortunately, in the current study, we did not measure erythrocyte ALT activity. The data presented here indicate that erythrocyte AST basal and stimulated activities of rats receiving 35 µg pyridoxine daily was as high as that of those receiving 150 µg daily. The aspartate aminotransferase activities of rats receiving 35 and 150 µg of the vitamin daily were significantly higher than those of the rats consuming the deficient diet (3 µg/d), even when additional cofactor was added. Lumeng et al. (1978) found that erythrocyte alanine aminotransferase activity increased steadily with increasing vitamin B-6 intake, whereas aspartate aminotransferase started to plateau at an intake of 142 nmol/d (29 µg/d).

Urinary 4-pyridoxic acid (4-PA) is a direct measure of vitamin B-6 turnover. Produced in the liver, 4-PA is the major metabolic product of vitamin B-6 (Leklem 1990). The contribution of other tissues to urinary 4-PA is not known. Because 4-PA is produced by oxidation of PL, PN and PM must first be converted to PLP and then to PL before they can contribute to urinary 4-PA. Thus, the turnover of PLP via conversion to PL is the main control point determining the subsequent amount of 4-PA. The importance of phosphatase activity in the regulation of cellular PLP content is shown in studies by others (Li et al. 1974), which provide evidence that the regulation of PLP metabolism principally resides with PLP degradation.

In the control group, absolute 4-PA excretion tended to become significantly lower in trained rats than in sedentary rats over the 9 wk of chronic swimming (Fig. 2). Prolonged swimming was also found to cause a reduction in urinary 4-PA excretion (Efremov and Zaburkin 1972). Similarly, basal 4-PA excretion was significantly lower in trained men after they ran 8 or 16 km/d than in free-living inactive men (Dreon and Butterfield 1986). Our results and those of others may indicate that long-term physical activity is associated with lower 4-PA excretion, reflecting less conversion of vitamin B-6 to 4-PA. The possible mechanisms for such a difference in conversion rates between sedentary and trained animals could include changes in the amount and the proportion of metabolites excreted, or actual increases in the need for the vitamin. The possibility that training could alter the form in which the vitamin was excreted, thus giving the appearance of decreased excretion in the form measured, was not examined in this experiment. A reduction in activity of plasma alkaline phosphatase would also explain this observation. Such changes in activity of this enzyme have been reported in various physiological conditions (Furth-Walker et al. 1989) and disease states (Leklem 1992, Reynolds et al. 1991) to explain the observed alterations in plasma PLP. On the other hand, because the proportion of dietary vitamin excreted as 4-PA by sedentary and trained rats generally did not differ (Fig. 3), we cannot assert that there was a genuine increase in need for vitamin B-6 in the exercising rats compared with the sedentary rats.

Several general patterns emerge from our data concerning B-6 vitamer concentrations in plasma, erythrocytes and organs. First, individual and total B-6 vitamers in blood and organs responded well to dietary vitamin B-6 depletion. These results extend previous reports showing that PLP in plasma, liver and brain declined rapidly in response to vitamin B-6-deficient diets (Sampson and O'Connor 1989b, Takami et al. 1968).

Second, individual and total B-6 vitamers in organs were less affected by exercise than by dietary PN-HCl level. Only muscle and liver vitamin B-6 levels were affected by chronic exercise. PMP, PLP and total vitamin B-6 concentrations (nmol/g) in gastrocnemius muscle were significantly higher in trained rats than in sedentary rats. Because it accounts for 45% of adult body mass in rats, humans and other mammals (Munro 1969), skeletal muscle constitutes a large pool of vitamin B-6. For example, muscle contains 80% of total body vitamin B-6 in rats (Coburn et al. 1989). In the present study, muscle mass was lower in trained rats at the end of the experimental period than in sedentary rats, because body weight gain was lower in the former group. One can assume that muscle accounted for 55% of body weight in the trained rats because exercise and/or training programs can augment lean body mass as well as cause a decrease in body fat. Many athletes of both sexes tend to have a larger lean body mass than their sedentary peers (Forbes 1991). Concentrations of muscle vitamin B-6 vitamers tended to be higher in the trained rats than in the sedentary rats. Content of muscle B-6 vitamers [estimated as (concentration × final mean body weight × 0.45 or 0.55)] was also higher in trained rats. For muscle PLP, estimates of content were 1.8, 6.2 and 7.2 µmol for the sedentary rats and 2.3, 7.1 and 8.1 µmol for the trained rats fed 0, 2 and 7 mg PN-HCl/kg diet, respectively. These estimates suggest that training caused accumulation of PLP in muscles. Estimates of muscle content of PMP and total vitamin B-6 showed similar patterns. These observations are consistent with previous work in rats (Hadj-Saad et al. 1995), showing that acute exercise induces changes in vitamin B-6 metabolism including a retentive capacity and temporary deposition of vitamin B-6 stores in skeletal muscle.

The response of B-6 vitamers differed in two ways in other organs compared with muscle. First, vitamer concentrations and content (concentration × tissue wet weight) in heart, kidney, brain and adrenal glands were not significantly higher in exercising rats as occurred for muscle vitamin B-6 content. Second, the concentration and content of PLP in liver was significantly lower in the trained rats. These observations showed that training led to PLP depletion in liver but not in heart, kidney, brain and adrenal glands. More work will be necessary to clarify inter- and intraorgan response to chronic exercise. It is worth noting that previous work indicating an interorgan transfer of the vitamin has been based on indirect measure of vitamin B-6 metabolism as plasma PLP level (Crozier et al. 1994, Hoffmann et al. 1991, Leklem and Shultz 1983) or urinary 4-PA excretion (Leklem 1985, Manore et al. 1987). Measurement of all B-6 vitamers directly with HPLC, coupled with the measurement of activity of the enzymes regulating vitamin B-6 metabolism, should provide a useful analytical tool for clarifying these relationships in future work.

In conclusion, training seems to alter vitamin B-6 metabolism, as indicated by higher muscle PLP concentration and reduced liver PLP concentration, without an alteration in erythrocyte AST activity, plasma PLP concentration or urinary excretion of 4-PA. Thus, our hypothesis that chronic exercise would depress vitamin B-6 status was not supported by our data.

ACKNOWLEDGMENT