The primary objective of this study was to investigate the sparing effects of dietary Se and AA on tissue vitamin C and AT concentrations. Guinea pigs were chosen for these experiments as they are similar to humans in their inability to make vitamin C and therefore likely provide a more relevant model system compared to previously used cell culture systems [17, 18, 20] or animal models that have the ability to make vitamin C . Further, we chose to investigate the effects of Se under conditions of marginal AA intake, given that Se may play a more biologically significant role in sparing vitamin C and AT when intake of AA is low.
Only guinea pigs fed the Se-D/MC diet developed paralysis of their limbs. In some cases, the paralysis was severe enough that the guinea pigs died or had to be euthanised. These results are consistent with previous studies demonstrating sensitivity of guinea pigs to disturbances in antioxidant status. Particularly, Se deficiency combined with vitamin E or C deficiency has been reported to cause skeletal muscle damage [24, 35]. Further, vitamin E combined with vitamin C deficiency has been shown to promote limb paralysis and death due to severe damage in the brainstem and spinal cord .
As part of the study design, guinea pigs were killed after 5 and 12 weeks on the experimental diets at 24 and 48 hrs following AA dosing. Although analyses of vitamin C data at each of the four separate time points revealed little difference between guinea pigs fed different levels of Se, a discernable decreasing trend for vitamin C concentrations in tissues with decreasing dietary Se was observed for week 5, 48 hrs and week 12, 24 hrs guinea pigs. In contrast, no trend was observed for week 5, 24 hrs and week 12, 48 hrs guinea pigs. The reason for the observed Se effect at different times post AA dosing for week 5 and 12 guinea pigs may be explained by differences in the metabolism of the dosed AA between younger (week 5) and older (week 12) guinea pigs. Notably, vitamin C concentrations were higher for guinea pigs killed at 24 compared to 48 hrs post dosing for both week 5 and 12 guinea pigs (data not shown) indicating that vitamin C concentrations rise in tissues following dosing and then fall over time as the vitamin is consumed. Increases in tissue vitamin C concentrations at early times post dosing and low concentrations after an extended time post dosing may mask any effects of Se on vitamin C concentrations. Therefore, if the younger and older guinea pigs metabolised the dosed AA differently (e.g. differences in AA absorption or rate of AA consumption by tissues), it would not be surprising that the Se effects on vitamin C are observed at different times post dosing for week 5 and 12 guinea pigs. However, additional studies are required to definitively show age related differences in AA metabolism in guinea pigs. Nonetheless, whatever the underlying mechanism for this difference, we clearly demonstrate here that dietary Se influences tissue vitamin C concentrations.
In vivo, AA is oxidised to DHAA. We show that Se or AA restriction decreases both the reduced (AA) and oxidised (DHAA) forms of vitamin C. Interestingly, liver was the only tissue that showed an increasing trend in DHAA with decreasing Se in the diet. Impaired regeneration of AA from DHAA with Se restriction may have resulted in accumulation of DHAA in liver, perhaps due to slower elimination of DHAA in liver compared to other tissues.
The observed sparing effects of Se on vitamin C may be explained by Se's role as a component of selenoproteins. It has been reported that the Se-dependent enzyme thioredoxin reductase (TR) can regenerate AA from DHAA  and the ascorbyl free radical . Although we were unsuccessful in developing an assay to measure TR activity in guinea pig tissues, it is possible that the low Se diets reduced TR activity which may have contributed to lower concentrations of vitamin C. Decreased antioxidant activity due to decreased activity of Se-dependent enzymes may also have contributed to the lower vitamin C and AT concentrations in tissues, since demand for their antioxidant activity may have been increased. The observed sparing effects of Se on AT may also be partly explained by a secondary effect of Se on AT given that vitamin C may play a role in the regeneration of vitamin E [37, 38]. In this regard, marginal AA intake reduced AT concentrations in liver, kidney and spleen.
A reduction in AT with decreased Se or AA intake was only observed in week 12 guinea pigs suggesting that longer-term Se or AA deficiency is more detrimental to tissue AT status than short-term deficiency. Previous studies with guinea pigs failed to observe reductions in AT in tissues with Se  or vitamin C  deficiency, including liver, which was depleted in AT in this study. However, in contrast to these previous studies, this study was of longer duration and Se-deficient guinea pigs were also fed a marginal AA diet.
AT concentrations were lower in tissues of Se-D/MC compared to Se-M/MC guinea pigs, but not Se-N/MC guinea pigs. Given the absence of significant differences between guinea pigs fed the Se-M/MC or Se-N/MC diets, these data are likely explained by the large variability in tissue AT concentrations between individual guinea pigs. However, these data suggest that marginal amounts of Se are sufficient to maintain tissue AT concentrations.
In most tissues, a large proportion of the total vitamin C was detected in the oxidised form. The large DHAA/AA ratios reported here are consistent with data from an earlier study by Hidiroglou et al  that reported comparably large DHAA/AA ratios in tissues of guinea pigs dosed with 1 mg AA/day. In addition, a study by Martensson et al  that used different methodology to measure vitamin C detected most of the total vitamin C in liver, lung, kidney and brain of control guinea pigs fed a standard guinea pig chow (Purina) diet as AA; however, when guinea pigs were fed an ascorbate-deficient diet for 21 days, 46 and 45% of the total vitamin C was detected as DHAA in liver and kidney, respectively. It should be noted that liver and kidney vitamin C concentrations reported in this study and that of Hidiroglou et al  are comparable to those of the ascorbate-deficient guinea pigs in the study by Martensson et al  showing large DHAA/AA ratios in tissues. The low tissue vitamin C concentrations reported in this study reflect the relatively low amounts of AA administered to the guinea pigs. Given these data, it is conceivable that reduced vitamin C intakes and consequently tissue vitamin C concentrations promote an increase in the DHAA/AA ratio in guinea pig tissues.
Se-GSHPx activity decreases with a reduction in Se status and is often used as a measure of Se nutriture in experimental animals, including guinea pigs [24, 42, 43]. Interestingly, guinea pigs dosed with marginal AA had lower Se-GSHPx activity compared to guinea pigs dosed with normal AA demonstrating a sparing effect of AA on Se-GSHPx activity. It remains to be determined whether the decrease in Se-GSHPx activity reflects a decrease in Se status or change in some other metabolic process that influences Se-GSHPx activity.
Lastly, since decreased antioxidant status can lead to oxidation of cellular components, we examined liver and plasma for oxidative modifications of proteins and lipids. We failed to detect any differences in protein carbonyl and lipid peroxide concentrations in liver cytosols or plasma between guinea pigs fed the different diets. Although these data indicate the absence of severe oxidative modifications to proteins and lipids in these tissues, we cannot rule out the presence of subtle changes that may be detected with more sensitive assays or differences in other markers of oxidative stress.