The results of these rat and human studies indicate that inflammation directly affects vitamin B6 metabolism differently in different tissues. Furthermore, the low vitamin B6 level is unlikely to be due to a decrease in food intake or the excessive excretion of vitamin B6. Adjuvant arthritis in Lewis rats is a useful animal model for studying vitamin B6 status during inflammation. Adjuvant arthritis decreased the pyridoxal 5'-phosphate pools in the circulation and liver, whereas it did not alter the pyridoxal 5'-phosphate pool in the skeletal muscle. Liver was studied because of its significant metabolic relevance and muscle was studied because it is the major store for vitamin B6. The lower plasma pyridoxal 5'-phosphate concentration in arthritic animals during inflammation was found to be in the physiological range seen in humans with rheumatoid arthritis. Despite the limited number of human subjects, the difference between patients and controls in plasma pyridoxal 5'-phosphate concentration was significant. The average plasma pyridoxal 5'-phosphate concentration in patients with rheumatoid arthritis was about 55% of the level seen in the healthy controls. The mean pyridoxal 5'-phosphate concentration in rats with adjuvant arthritis was about 53% of the controls at peak inflammation on day 21. Rat adjuvant arthritis reflected the altered plasma pyridoxal 5'-phosphate and it is a potential model for studying vitamin B6 status during inflammation.
Lumeng and colleagues suggested that plasma pyridoxal 5'-phosphate concentration reflects vitamin B6
status in the liver in healthy humans [21
]. Kinetic studies in rats also indicate that changes in plasma pyridoxal 5'-phosphate content primarily reflect changes in the relatively small, but metabolically relevant and more rapidly exchanging, liver pool (as compared with muscle) [22
]. However it was not clear whether this is true during inflammation. The reduced plasma pyridoxal 5'-phosphate level in our study implies that vitamin B6
status in the liver in these patients was altered, because we previously found good correlations between circulating pyridoxal 5'-phosphate level and vitamin B6
functional status measured by methionine load and tryptophan load in these patients [14
]. Results from adjuvant arthritis were in agreement with this postulation. In the rat arthritis model, both plasma and hepatic pyridoxal 5'-phosphate concentrations were lower (Table ). Furthermore, plasma pyridoxal 5'-phosphate concentration was correlated with hepatic pyridoxal 5'-phosphate content. These results suggest that the lower circulating pyridoxal 5'-phosphate levels observed in rheumatoid arthritis could reflect a decrease in hepatic pyridoxal 5'-phosphate pools, and plasma pyridoxal 5'-phosphate is a good indicator of liver B6
status during inflammation.
Effects of adjuvant arthritis on B vitamin status during inflammation in rats
Our data imply that there are distinct metabolic roles for plasma and erythrocytes in vitamin B6
metabolism during inflammation, and that the impact of inflammation on vitamin B6
is tissue specific. In human subjects, despite the significantly lower pyridoxal 5'-phosphate in plasma (and possibly in liver), erythrocyte pyridoxal 5'-phosphate seemed to be adequate in patients with rheumatoid arthritis, because no difference was found between patients and healthy controls in the erythrocyte pyridoxal 5'-phosphate level or the activity coefficient of erythrocyte aspartate aminotransferase (Table ). These observations are in agreement with the findings by Talwar and colleagues, which showed that pyridoxal 5'-phosphate decreases in plasma but not erythrocytes during systemic inflammatory response [23
Indices of vitamin B status in patients with rheumatoid arthritis and control subjects
Data from our animal model imply localized vitamin B6 depletion during inflammation. Before the present study it was not clear how the pyridoxal 5'-phosphate pool in muscle might react to an inflammatory process. In rats with adjuvant arthritis, hepatic pyridoxal 5'-phosphate content was decreased, whereas muscle pyridoxal 5'-phosphate content remained unaltered, suggesting localized vitamin B6 deficiency during inflammation.
Skeletal muscle seems to be less sensitive to vitamin B6
deficiency in humans. In young healthy males receiving a defined diet restricted in vitamin B6
, the muscle content of vitamin B6
is relatively resistant to vitamin B6
deficiency, whereas plasma pyridoxal 5'-phosphate is more sensitive to dietary vitamin B6
]. We conclude that liver and muscle have distinctive roles as the body undergoes metabolic changes; skeletal muscle, the body's major storage site of vitamin B6
, may turn over very slowly during inflammation.
Low vitamin B6 status is unlikely to be due to lower intake or excessive excretion
Dietary intake is known to be a major determinant of vitamin B6 status. The arthritic and control rats showed decreases in plasma pyridoxal 5'-phosphate from the baseline levels. This was partly due to a decrease in overall food intake in both groups. However, the different vitamin B6 status observed between animals with adjuvant arthritis and control animals in the present study was not caused by different food intake between the two groups. Food intake of individual rats in the arthritis group was recorded daily, then a one-to-one match (each rat with adjuvant arthritis had its own weight-matched, saline-injected control) in food intake was arranged. This pair-feeding regimen in our animal experiments minimized the confounding effects of anorexia on the measures of vitamin B6.
Despite the significantly lower plasma pyridoxal 5'-phosphate in patients, 24-hour urinary 4-pyridoxic acid excretion in patients with rheumatoid arthritis did not differ from that of the healthy control subjects (Table ). The low circulating pyridoxal 5'-phosphate level seen in these patients therefore did not result from excessive catabolism of vitamin B6. This is in agreement with the observation in our animal model. The 24-hour urinary excretion of 4-pyridoxic acid did not differ between control and rats with adjuvant arthritis, despite lower pyridoxal 5'-phosphate levels in plasma and liver in the adjuvant arthritic rats. To summarize these observations, the abnormal vitamin B6 status in rheumatoid arthritis results from the inflammatory process, and it is unlikely that it resulted from insufficient intake or excessive excretion of vitamin B6.
Potential factors involved in the compartmentalization of pyridoxal 5'-phosphate during inflammation
In healthy populations, the variance in plasma pyridoxal 5'-phosphate can be explained to a great extent by vitamin intake, serum albumin, and alkaline phosphatase. The later two are physiological variables directly related to pyridoxal 5'-phosphate metabolism. In an elderly Dutch population it was reported that a combination of vitamin B6
intake, alkaline phosphatase, alcohol consumption, and albumin accounted for 30 to 40% variance in plasma pyridoxal 5'-phosphate [25
]. Serum albumin is an acute-phase reactant that decreases during the flaring of active arthritis [26
]. As the major protein for pyridoxal 5'-phosphate transport in the circulation, albumin might protect pyridoxal 5'-phosphate from hydrolysis [27
]. In the present study, serum albumin was found to be correlated with plasma pyridoxal 5'-phosphate in patients whereas no such correlation was detected in the control subjects. Lower albumin levels in patients with more active arthritis may partly contribute to the lower pyridoxal 5'-phosphate level in these patients, although further study is needed for this postulation.
Many patients with arthritis have been reported to have elevated alkaline phosphatase [28
], including those in the present study. Although still in the normal range, mean serum alkaline phosphatase levels in our patients were 26% elevated compared with healthy control subjects. Alkaline phosphatase hydrolyzes the phosphorylated form of vitamin B6
]; we therefore speculate that serum alkaline phosphatase could be another key determinant of the concentration of circulating vitamin B6
coenzyme during inflammation. Alkaline phosphatase has been shown to regulate extracellular levels of pyridoxal 5'-phosphate in humans [30
], and abnormal vitamin B6
metabolism was found in alkaline phosphatase knock-out mice [32
]. We found that the serum alkaline phosphatase level was inversely correlated with the plasma pyridoxal 5'-phosphate level in our subjects, which indirectly supports the above hypothesis. Compartmentalization of pyridoxal 5'-phosphate has been reported in the acute-phase response, such as the acute phase of myocardial infarction [33
]. Because erythrocyte pyridoxal 5'-phosphate level seem to be normal whereas plasma and hepatic pyridoxal 5'-phosphate levels are significantly lower during inflammation, pyridoxal 5'-phosphate might be compartmentalized between tissues. The elevated alkaline phosphatase during inflammation may facilitate the mobilization and uptake of B6
vitamers, because vitamin B6
is taken up by tissues primarily in the form of pyridoxal.
In contrast, elevated serum alkaline phosphatase or reduced albumin did not provide a satisfactory explanation for the lower plasma pyridoxal 5'-phosphate level in rheumatoid arthritis, because the presence of disease remained a significant determinant of plasma pyridoxal 5'-phosphate concentrations after adjustment for serum alkaline phosphatase and albumin concentrations [34
]. The low plasma pyridoxal 5'-phosphate level in patients with rheumatoid arthritis may also be attributed to elevated pyridoxal phosphatase activity during inflammation. It has been reported that the decrease in plasma pyridoxal 5'-phosphate characteristically seen in cirrhosis may be related to a substantial elevation of hepatic pyridoxal 5'-phosphate phosphatase activity [35
]. McCarty hypothesized that the pro-inflammatory cytokine interleukin-6 might stimulate the activity of pyridoxal phosphatase in hepatocytes, in these patients, and the elevated enzyme may result in reduced plasma pyridoxal 5'-phosphate concentrations [36
It remains uncertain whether the activity of pyridoxal 5'-phosphate phosphatase is altered in patients with arthritis, and this should be considered for future studies.