Abstract

INTRODUCTION

Vitamin C (ascorbate) is an essential micronutrient with many important biological functions. Ascorbate is a cofactor for a variety of metalloenzymes that are necessary for the biosynthesis of collagen, carnitine, neurotransmitters, and peptide hormones as well as the regulation of transcription factors such as hypoxia-inducible factor-1 (1–3). Ascorbate also acts as a potent water-soluble antioxidant, with the ability to scavenge a wide variety of reactive oxygen and nitrogen species and regenerate other small molecule antioxidants from their respective radicals (4). Humans have lost the ability to synthesize ascorbate from glucose because of mutations in the gene encoding l-gulono-γ-lactone oxidase, which is the terminal enzyme in the ascorbate biosynthetic pathway (5). Therefore, an adequate and regular dietary intake is essential to prevent hypovitaminosis C and the potentially fatal deficiency disease scurvy (6). Clinical manifestations of scurvy include blood vessel fragility and bleeding, which result in petechial and other hemorrhages, skin changes that result in follicular hyperkeratosis, impaired wound healing, gum swelling and bleeding, joint pain and effusions, anemia, weakness, and fatigue (6–8). Many of these symptoms are attributed to the role of ascorbate in collagen and carnitine biosynthesis (9).

Plasma ascorbate status reflects recent dietary intake, whereas leukocyte concentrations are thought to more closely reflect tissue stores (10, 11). However, whether leukocytes are an accurate model for the ascorbate status of other tissues is uncertain. Our previous study with vitamin C–deficient Gulo knockout mice indicated that the maximal tissue uptake within different organs occurred at varying doses of ascorbate (12). Thus, the relative uptake of ascorbate by leukocytes may not be representative for all tissues or organs. Although ascorbate has previously been measured in skeletal muscle obtained at autopsy (13), to our knowledge, no studies have investigated the bioavailability of vitamin C to human skeletal muscle. Skeletal muscle contains relatively low concentrations of ascorbate compared with in other organs (13), but because of the large amount of skeletal muscle present in the body, it has been estimated to comprise ≤67% of the total body vitamin C (10) and, thus, represents the major pool of vitamin C in the body. The accumulation of ascorbate in muscle tissue is thought to protect these metabolically active cells against oxidative stress (14). Therefore, vitamin C is likely to be essential for both skeletal muscle structure and function because of its dual role as an antioxidant and as an enzyme cofactor for collagen and carnitine biosynthesis (15, 16).

Overall, it is likely that muscle tissue has a high requirement for and turnover of vitamin C. Therefore, the aims of the current study were to investigate the bioavailability of vitamin C to human skeletal muscle in relation to dietary intake and plasma concentrations and to compare this relation to the uptake of ascorbate by peripheral blood leukocytes. Zespri gold kiwifruit (Actinidia chinensis var. Hort 16A), which are an outstanding dietary source of vitamin C (17), were used as the intervention in this study. Our previous human study in which we investigated the bioavailability of vitamin C from gold kiwifruit indicated that the consumption of 0.5 kiwifruit/d resulted in a significant increase of plasma ascorbate concentrations in marginally deficient individuals, whereas the consumption of 2 kiwifruit/d was required to saturate the plasma, as shown by a significant increase in the urinary ascorbate excretion at this dose (18). In the current study, we supplemented participants with 0.5 or 2 kiwifruit/d for 6 wk and measured ascorbate concentrations in plasma, urine, leukocytes, and skeletal muscle (vastus lateralis) before and after intervention.

SUBJECTS AND METHODS

Study design

The study used a parallel-arm design (Figure 1), and participants were randomly assigned by the study coordinator into a low-dose group (0.5 kiwifruit/d) or a high-dose group (2 kiwifruit/d) by using a random-numbers chart. Blocking was used to account for the range in prestudy plasma ascorbate concentrations. The study was carried out in a phase I clinical trials unit and comprised a lead-in phase of 5 wk, an intervention phase of 6 wk, and a washout phase of 4 wk. Initially, participants were encouraged to reduce their dietary vitamin C intake by avoiding the consumption of juice and other vitamin C–fortified beverages and by substituting high–vitamin C–containing foods with low–vitamin C–containing foods. Fasting venous blood samples were drawn weekly throughout the study to monitor participant plasma ascorbate concentrations derived from normal daily diets and the intervention. Twenty-four–hour urine and extra blood for leukocyte isolations were collected at baseline (week 5), after intervention (week 11), and after washout (week 15). Muscle biopsies were carried out before and after intervention. Participants also completed 7-d food and beverage records on 4 occasions (at the beginning of the study, before intervention, after intervention, and after washout) to monitor their dietary vitamin C intakes.

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FIGURE 1.

Study design. Parallel arms comprised 0.5 or 2 kiwifruit/d for 6 wk.

RESULTS

Ascorbate status of plasma and urine

At baseline, the low-dose group had a mean plasma ascorbate concentration of 23 μmol/L and this increased to 46 μmol/L after 6 wk of intervention (Figure 2). The high-dose group had a baseline mean concentration of 25 μmol/L, and after intervention, this concentration increased to 63 μmol/L (Figure 2), which was close to saturating (ie, did not increase further after additional vitamin C intake) (18). Plasma ascorbate concentrations of the 2 groups were statistically different from each other within the first week of intervention, and this difference was maintained for the entire 6 wk of intervention (P < 0.001). At the end of the 4-wk washout period, plasma ascorbate concentrations for both high- and low-dose groups had decreased and were not different from baseline concentrations (Figure 2).

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FIGURE 2.

Mean ± SEM plasma ascorbate concentrations in the 0.5-kiwifruit/d (•; n = 18) and 2-kiwifruit/d (○; n = 17) groups during the lead-in, intervention, and washout phases of the study. Significant differences (P < 0.001) were observed between the 2 interventions from weeks 6 to 11; no differences were observed during the washout phase. *,**For comparison with baseline (week 5; 2-factor ANOVA with Fisher's pairwise multiple-comparison procedure): *P < 0.01, **P < 0.001.

After 6 wk of intervention, the urinary excretion of ascorbate had increased 2-fold in the low–kiwifruit-dose group and 15-fold in the high-dose group (Table 3), which suggested that the higher intake of kiwifruit resulted in plasma concentrations close to saturation (18).

TABLE 3

Increase in ascorbate concentrations of fluids, cells, and tissue after supplementation with 0.5 or 2 kiwifruit/d for 6 wk¹

	0.5 kiwifruit/d			2 kiwifruit/d
	Baseline	Intervention	Washout	Baseline	Intervention	Washout	Between-group intervention P
Plasma (μmol/L)	22.7 ± 2.5 (17)	45.5 ± 2.5*** (18)	37.9 ± 3.3. (15)	25.4 ± 3.3 (16)	62.6 ± 2.8*** (17)	31.8 ± 3.6 (17)	<0.0001
Urine (μmol/24 h)	32.3 ± 8.1 (18)	70.5 ± 17.0* (17)	64.7 ± 29.8 (16)	32.2 ± 11.2 (17)	485.0 ± 69.9*** (17)	64.3 ± 25.1 (17)	<0.0001
Mononuclear cells (nmol/108 cells)	60.5 ± 6.1 (18)	90.9 ± 6.1** (18)	75.2 ± 5.2 (17)	58.4 ± 5.6 (17)	91.0 ± 6.2** (16)	71.5 ± 5.0 (17)	0.998
Neutrophils (nmol/108 cells)	13.7 ± 2.4 (18)	30.4 ± 2.5*** (18)	24.8 ± 1.7*** (17)	16.2 ± 3.3 (17)	33.7 ± 2.9** (17)	24.7 ± 1.4* (17)	0.393
Skeletal muscle (nmol/g)2	15.1 ± 2.5 (17)	52.8 ± 5.0*** (18)	ND	17.1 ± 3.5 (17)	60.8 ± 5.5*** (17)	ND	0.284

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¹All values are means ± SEMs; n in parentheses. For 0.5- compared with 2-kiwifruit/d groups after intervention, P values were determined by using the unpaired t test. *,**,***For intervention compared with baseline (paired t tests): *P < 0.05, **P < 0.01, ***P < 0.001. ND, not determined.

²To convert muscle ascorbate values from nanomoles per gram of tissue to micrograms per 100 mg of tissue, multiply by 0.0199.

Ascorbate status of leukocytes and muscle tissue

Baseline ascorbate concentrations of mononuclear cells and neutrophils are shown in Table 3. After 6 wk of intervention, mononuclear cell ascorbate concentrations increased ∼1.5-fold for both the low–kiwifruit-dose group (P = 0.004) and the high–kiwifruit-dose group (P = 0.004), whereas neutrophil ascorbate concentrations increased >2-fold (low-dose group: P = 0.0002; high-dose group: P = 0.003; Table 3). There were no significant differences in leukocyte ascorbate concentrations between the 2 intervention groups, which indicated that leukocytes were saturating with the low kiwifruit intake.

Baseline mean muscle tissue ascorbate concentrations were ∼16 nmol/g tissue (Table 3), with a range from 1.0 to 43.2 nmol/g tissue. After 6 wk of intervention, there was a ∼3.5-fold increase in mean muscle tissue ascorbate concentrations to 53 and 61 nmol/g tissue in the low- and high-dose groups, respectively (P < 0.0001; Table 3). Thus, muscle tissue appeared to have a significantly greater relative uptake of ascorbate than both mononuclear cells and neutrophils (Figure 3). There was no difference in muscle tissue ascorbate concentrations between the 2 intervention groups, which suggested that muscle tissue ascorbate concentrations were saturating with the low kiwifruit intake.

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FIGURE 3.

Mean ± SEM relative increases in ascorbate concentrations in peripheral blood mononuclear cells, neutrophils, and skeletal muscle tissue after supplementation with 0.5 kiwifruit/d (n = 18; black bars) or 2 kiwifruit/d (n = 18; gray bars). Baseline values are shown in Table 3. *,**For intervention compared with baseline (paired t test): *P < 0.01, **P < 0.001.

Two healthy male volunteers with saturating plasma ascorbate concentrations (74.6 ± 2.0 μmol/L) were recruited to compare with our participants. The muscle tissue ascorbate concentrations of the volunteers were 60.3 ± 13.6 nmol/g tissue, which compared well with those of study participants after intervention (Table 3).

Correlations of muscle ascorbate status with vitamin C intake and plasma and leukocyte concentrations were investigated by using linear regression analysis. Strong correlations were observed between muscle ascorbate status and vitamin C intake (R = 0.61, P < 0.001; Table 4) and plasma concentrations in the range of 5–80 μmol/L (R = 0.75, P < 0.001; Table 4, Figure 4A). These correlations were comparable with that of plasma ascorbate concentrations with dietary intake (R = 0.71, P < 0.001) and were greater than those exhibited for neutrophils and mononuclear cells (Table 4). Analysis of muscle ascorbate on the basis of quintiles of plasma ascorbate showed significantly lower muscle ascorbate for plasma concentrations <21 μmol/L, which provided a mean muscle ascorbate concentration of 11 nmol/g. Second and third quintiles had comparable mean muscle ascorbate of 25 and 29 nmol/g. Notably, a significant increase in muscle ascorbate was observed at plasma concentrations >47 μmol/L, with mean muscle ascorbate concentrations ∼60 nmol/g (Figure 4B).

TABLE 4

Correlation coefficients for dietary intake of vitamin C and concentrations of ascorbate in plasma, muscle tissue, neutrophils, and mononuclear cells¹

	Dietary intake			Plasma			Muscle
	R	P	n2	R	P	n	R	P	n
Plasma	0.707	<0.001	98	—	—	—	—	—	—
Muscle	0.613	<0.001	68	0.749	<0.001	67	—	—	—
Neutrophils	0.417	<0.001	100	0.432	<0.001	102	0.571	<0.001	69
Mononuclear cells	0.353	<0.001	99	0.460	<0.001	101	0.436	<0.001	68

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¹R and P values were determined by using linear regression analysis (Pearson's correlations).

²n, number of samples analyzed.

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FIGURE 4.

Correlation of muscle tissue ascorbate status with plasma ascorbate concentrations (A) and muscle tissue ascorbate status relative to quintiles of plasma ascorbate concentrations (B). Box plots show medians with 25th and 75th percentiles as boundaries, whiskers are for the 10th and 90th percentiles, and symbols indicate outliers. For trend across quintiles, P < 0.001 (1-factor ANOVA with Fisher's pairwise multiple-comparison procedure) (n = 67).

Because skeletal muscle ascorbate appears to be highly correlated with plasma ascorbate, it is likely that the variability observed in participant baseline muscle ascorbate (as previously indicated) was due to the variability observed in participant baseline plasma ascorbate (range: 3–61 μmol/L).

DISCUSSION

To our knowledge, our study provides novel data on the effects of vitamin C depletion and dietary supplementation on human skeletal muscle tissue ascorbate status. At baseline, mean plasma ascorbate concentrations of study participants were marginally deficient (24), and their skeletal muscle tissue ascorbate concentrations were similarly low, with a ∼3.5-fold uptake of ascorbate by muscle tissue observed after intervention. This uptake was significantly greater than the relative uptake of ascorbate by mononuclear cells and neutrophils, which suggestes that muscle is a more-labile compartment of whole-body vitamin C. Although kiwifruit was used as the source of vitamin C in this study, we (Carr et al, unpublished observations, June–August 2012) and other authors (25, 26) have shown no differences in the comparative bioavailability of synthetic and fruit-derived vitamin C.

We also investigated the previously unreported relation between human skeletal muscle ascorbate status and vitamin C intake and plasma concentrations. Plasma ascorbate is generally thought to reflect recent dietary intake, whereas tissue status and, in particular, leukocyte ascorbate concentrations are thought to be more indicative of the total body status (11). We showed skeletal muscle ascorbate concentrations were strongly correlated with dietary intake and plasma ascorbate status. The correlations between leukocytes and plasma ascorbate concentrations were weaker, which suggested that muscle is more responsive to changes in circulating ascorbate concentrations than are leukocytes. This effect may reflect either a higher turnover of ascorbate in metabolically active skeletal muscle or differences in uptake between muscle cells and leukocytes.

Ascorbate is transported into muscle cells primarily via sodium-dependent vitamin C transporter 2 (SVCT2), which is a high-affinity, low-capacity transporter (27–30). Although neutrophils express SVCT2 (31) when their respiratory burst is activated, they primarily transport dehydroascorbic acid (the oxidized form of ascorbate) via the glucose transporters, followed by the intracellular reduction to ascorbate (32). Thus, the difference in relative uptake of ascorbate between muscle cells and leukocytes could be due to differences in expression concentrations of SVCT2 in each cell type.

Because muscle tissue appears to comprise a significantly more labile vitamin C pool than leukocytes, it is likely to be prone to depletion when vitamin C intake is inadequate. This inadequacy could have a number of implications. Skeletal muscle fibers are greatly dependent on fatty acid oxidation for their energy requirements, and carnitine plays a vital role in the regulation of skeletal muscle fuel selection and physiologic function (33, 34). Indeed, skeletal muscle contains ∼98% of the body's total store of carnitine (16, 33), and ascorbate is required for its synthesis through its action as a cofactor for the metalloenzymes trimethyllysine hydroxylase and γ-butyrobetaine hydroxylase (35). Carnitine deficiency has been reported in human skeletal muscle, and deficient biosynthesis has been implicated in some cases (34, 36, 37). The effect of vitamin C deficiency on human skeletal muscle carnitine concentrations has not been explored but has been shown to result in a ≤50% reduction in skeletal muscle carnitine in guinea pigs (38–41). Hence, it is possible that muscle concentrations could be similarly decreased in humans. Reduced muscle carnitine is associated with muscle weakness (42) and may be associated with the symptom of fatigue observed in vitamin C–deficient individuals (43).

It is well established that reactive oxygen species are generated in skeletal muscle during exercise, and this generation has been associated with muscle damage and inflammation (44, 45). Although a number of studies have shown that antioxidants such as ascorbate can attenuate the oxidative stress associated with exercise, whether this effect is desirable remains to be elucidated (46). Instead, there may be beneficial adaptive responses to elevated concentrations of reactive oxygen species, such as the increased activity of antioxidant enzymes, upregulation of redox-sensitive gene expression, and increased mitochondrial biogenesis (46). As such, high-dose vitamin C supplementation is not recommended for athletes who are undergoing endurance exercise training; however, an adequate dietary intake of the vitamin is likely to be required for normal muscle function (46, 47).

On the basis of the dietary analysis of our participants, it appears that a mean intake of ∼70 mg vitamin C/d is sufficient to saturate muscle tissue, whereas an intake of ∼30 mg/d is clearly inadequate. High nondietary intakes of vitamin C do not appear necessary to replenish normal muscle tissue, and the dietary intakes recommended in the United States and Canada (75 mg vitamin C/d for women and 90 mg vitamin C/d for men) should be sufficient. In contrast, the British and Australasian recommended dietary vitamin C intakes for adults of 40 and 45 mg/d, respectively, may not be sufficient to saturate muscle tissue, and additional studies need to be carried out to establish this.

Because of potential inaccuracies of dietary intake questionnaires and the variability between vitamin C intake and plasma concentrations in different individuals, due to genetic polymorphisms, smoking status and illness, plasma ascorbate concentrations are generally considered a more accurate marker of vitamin C status (48). This relation has been confirmed by our study, whereby we observed a higher correlation between plasma ascorbate concentrations and muscle status than that between vitamin C intake and muscle status. When plasma ascorbate was separated into quintiles, we observed significantly lower muscle ascorbate at plasma concentrations <21 μmol/L and significantly higher muscle ascorbate at plasma concentrations >47 μmol/L. Lykkesfeldt and Poulsen (24) suggested that plasma ascorbate concentrations between 23 and 50 μmol/L should be considered suboptimal, and concentrations <23 μmol/L should be considered deficient, and our muscle data supported this premise. Thus, plasma ascorbate concentrations ≥50 μmol/L should be aimed for to optimize muscle tissue status. However, even at plasma ascorbate concentrations ≥60 μmol and greater, some individuals presented with relatively low muscle ascorbate concentrations. The reason for this variability is not certain and did not appear to be related to BMI (data not shown).

An early study by Schaus (13) determined that pectoral muscle obtained from 63 individuals at autopsy contained 3.3 μg vitamin C/100 mg wet weight. This concentration is higher than that observed in our postintervention male participants and 2 healthy male volunteers (ie, ∼1.2 μg ascorbate/100 mg wet weight), but likely reflected differences in methodology (49). Schaus (13) observed higher vitamin C concentrations in the myocardium and pituitary of women; however, Schaus (13) reported no significant differences between men and women for pectoral muscle or the cerebral cortex. Therefore, because of sex differences that are known to influence vitamin C status, more studies are needed to confirm if skeletal muscle ascorbate concentrations differ between men and women.

Overall, we have shown that skeletal muscle ascorbate exhibits a strong association with vitamin C intake and plasma concentrations, which indicated that muscle tissue is very responsive to recent vitamin C intake and, thus, represents a relatively labile pool. This premise was supported by an animal study that showed a mean ascorbate transit time <18 h in skeletal muscle (50). Because of the accessibility and ease of sampling, leukocytes are often used as a model for tissue ascorbate status. However, it is apparent from our study that the uptake of ascorbate into mononuclear cells and neutrophils significantly underestimates the relative uptake of ascorbate into muscle tissue. Because of the multiple potential functions of ascorbate within muscle tissue, a daily vitamin C intake that provides plasma ascorbate concentrations ≥50 μmol/L should be consumed to maintain an optimal skeletal muscle ascorbate status.

Acknowledgments

Footnotes

REFERENCES