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Technical Briefs |
1
Medicine and
2
Chemical Pathology, Univ. of Zimbabwe Medical School, Avondale Harare, Zimbabwe;
3
Div. of Hematology and Oncology, Dept. of Medicine, George Washington Univ. Medical Center, 2150 Pennsylvania Ave., Suite 3-428, Washington, DC 20037;
4
Dept. of Medicine, Univ. of the Witwatersrand, Parktown 2196 Johannesburg, South Africa;
5
National Institutes of Child Health and Human Development, Cell Biology and Metabolism Branch, Building 18T Room 101, Bethesda, MD 20892;
a author for
correspondence: Dept. of Chemical Pathology, Medical School, University of Zimbabwe, Box A178, Avondale Harare, Zimbabwe, fax 263-4-720640
Since Szent-Gyorgyi first purified ascorbic acid (vitamin C) in 1928 (1), a number of associations between ascorbic acid and iron metabolism have been described. At the molecular level, ascorbic acid mobilizes iron from the crystal core of ferritin in vitro by reducing Fe3 to Fe2 (2). Intracellularly, ascorbic acid enhances iron-induced translation of ferritin (3) by favoring the conversion of the iron regulatory protein (IRP) from the RNA binding form to aconitase (3). Ascorbic acid also retards degradation of ferritin by blocking lysosomal autophagy of ferritin and transformation to hemosiderin (4)(5). In humans, the oral administration of ascorbic acid enhances the absorption of non-heme iron from the diet (6) and leads to increases in serum iron in subjects with iron overload and ascorbic acid deficiency (7). Moreover, iron overload states are associated with reduced ascorbic acid concentrations, possibly because of increased catabolism of ascorbic acid (7)(8).
The amount of labile iron in the cytosol influences the stability of transferrin receptor mRNA and the translation of ferritin mRNA (9)(10)(11). Posttranscriptional regulation occurs by means of an interaction between IRPs, proteins that sense changes in the chelatable intracellular iron pool, and iron-responsive elements located on untranslated regions of transferrin receptor mRNA and ferritin mRNA (12)(13)(14). In states of iron abundance, an IRP has aconitase activity and does not bind to iron-responsive elements, producing increased transferrin receptor mRNA degradation and increased ferritin mRNA translation. Conversely, when iron is deficient, an IRP loses aconitase activity and binds to iron-responsive elements, causing increased transferrin receptor mRNA stability and a repression of ferritin mRNA translation (15)(16). Thus, intracellularly, the presence of iron leads to inhibition of transferrin receptor expression and promotion of higher ferritin expression, whereas the deprivation of iron leads to higher transferrin receptor expression and decreased ferritin expression.
The present study is based on the hypothesis that the administration of ascorbic acid to humans will affect the concentrations of transferrin receptor and ferritin in the plasma because of changes in the expression of these molecules at the cellular level. The inhibition of lysosomal conversion of ferritin to hemosiderin by ascorbic acid (4)(5) might be expected to increase bioavailable intracellular iron because iron is more readily mobilized from ferritin than from hemosiderin. The resulting increase in chelatable iron would be expected to lead to a down-regulation of transferrin receptor expression (16). The promotion of the conversion of IRPs to aconitase by ascorbic acid (3) would also be expected to down-regulate transferrin receptor expression. Finally, the effects of ascorbic acid in inhibiting ferritin degradation (5) and in facilitating iron-induced translation of ferritin mRNA (3) might lead to increased ferritin content in the cell. We therefore postulated that ascorbic acid would decrease serum transferrin receptor concentration and increase serum ferritin concentration as a consequence of these changes in intracellular iron.
The subjects were 178 volunteers from rural Swaziland and rural Zimbabwe, who were studied from 1993 to 1995 as part of an investigation of families with African iron overload and of community members with a history of traditional beer consumption. Traditional beer has high iron content and is associated with the development of iron overload in Africa (17). We estimated the years of traditional beer consumption and the lifetime consumption of the beverage in each subject through interviews conducted by medical personnel fluent in the local language and knowledge of local customs. To determine the drinking histories of the participants, each subject was asked to estimate his or her consumption of traditional beer: the amount ingested on a typical day of traditional beer-drinking, the number of days of traditional beer-drinking in a typical month, the year the subject began drinking traditional beer, and if no longer drinking, the year he or she stopped. These estimates provide only broad approximations rather than precise amounts because consumption probably was not uniform over time and because information is subject to recall bias.
Fasting, morning venous blood samples were drawn from each subject on three consecutive days for the determination of leukocyte ascorbic acid and serum concentrations of transferrin receptor, ferritin, and iron. A complete blood count, liver function tests, and erythrocyte sedimentation rate were measured on the first day. Each subject received 1–2 g of ascorbic acid orally 24 h before the second phlebotomy and 24 h before the third phlebotomy.
Serum transferrin receptor concentrations were measured with the Quintikine enzyme immunoassay (R&D Systems). Serum ferritin was measured with the Ramco Spectroferritin enzyme immunoassay (Ramco Laboratories). Ascorbic acid in white blood cells was measured colorimetrically (18). A modification to the International Committee for Standardization in Hematology method was used to determine serum iron (19). Liver function tests were determined on an automated Cobas with reagents from Roche Diagnostic Systems. An automated analyzer (Coulter Electronics) was used for full blood counts. The erythrocyte sedimentation rate was determined by the Westergren method.
Because serum ferritin had a skewed distribution, this variable was log transformed before statistical analysis. Analysis of variance for repeated measures was used to examine the effect of ascorbic acid supplementation on the concentrations of leukocyte ascorbic acid and serum concentrations of transferrin receptors, ferritin, and iron.
Although most of the subjects gave a history of traditional beer consumption, they generally had liver function test results within reference values. In 52% of the subjects, serum ferritin was >300 µg/L, and in 36%, leukocyte ascorbic acid was <20 µg/10 leukocytes. In 8% of the subjects, serum ferritin was <20 µg/L. The clinical characteristics of the study population are available from the authors.
Table 1
shows the mean values for leukocyte ascorbic acid and serum
indicators of iron status on three consecutive days: day 1, before
ascorbic acid supplementation, and days 2 and 3, each after 1–2 g of
oral ascorbic acid. The mean leukocyte ascorbic acid progressively
increased from day 1 to day 3 (P <0.001), with the greatest
increase occurring between days 1 and 2. Mean transferrin receptor
concentrations in serum declined progressively from day 1 to day 3
(P <0.001). The increase in ascorbic acid and decrease in
serum transferrin receptor concentrations was observed in all subgroups
on the basis of iron status. Overall, serum concentrations of ferritin
did not change significantly, although there was a significant increase
in the subgroup of 15 subjects with iron deficiency. Serum iron
concentrations also did not change significantly overall, although
there was a significant decline in the subgroup of 70 subjects with
serum ferritin concentrations within reference values and a trend to an
increase in the subgroup of 93 subjects with increased serum ferritin
concentrations (Table 1
).
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In keeping with our hypothesis, serum transferrin receptor concentrations declined significantly after vitamin C administration. This finding appears to be in keeping with studies indicating that ascorbic acid may in some manner increase intracellular chelatable iron by facilitating mobilization of iron from the ferritin compartment (4). An increase in cytosolic nonstorage iron would then be expected to facilitate transferrin receptor down-regulation through the iron regulatory protein mechanism. Thus, the present findings are consistent with our hypothesis that ascorbic acid supplementation may down-regulate transferrin receptor synthesis and would seem to be in agreement with previous findings suggesting that ascorbic acid influences intracellular iron metabolism (5).
The hypothesized increase in serum ferritin concentrations with ascorbic acid supplementation did not occur when all 178 subjects were considered together, although it did occur in the subgroup of 15 individuals with iron deficiency. This result may suggest that serum ferritin is less a reflection of intracellular iron status than is serum transferrin receptor concentrations. Alternatively, perturbations of serum ferritin concentrations by effects of iron overload, of inflammation, and of hepatocellular dysfunction may have masked a small effect due to changes in chelatable intracellular iron (20)(21)(22). A high proportion of the subjects in this study gave a history of the consumption of traditional, iron-laden beer, and two-fifths of them had increased serum ferritin.
In contrast to our studies, Wapnick et al. (7) reported that serum iron concentrations increased dramatically after oral administration of vitamin C to iron-loaded, scorbutic subjects. The explanations for this difference probably lie in the timing of the vitamin C dose in relation to the serum iron measurement and the fact that the majority of the subjects in the present study did not have iron overload or ascorbic acid deficiency of the severity described by Wapnick et al. In addition, the vitamin C administered in this study was not sufficient to return leukocyte ascorbic acid to reference values in all of the iron-loaded subjects.
This study had limitations in that it was not possible to assess intracellular iron metabolism directly and to correlate these findings with serum concentrations of transferrin receptor, ferritin, and iron. In addition, we did not have a control group that did not receive ascorbic acid. Nevertheless, our results indicate that ascorbic acid status may influence serum transferrin receptor concentrations, probably through changes in intracellular iron metabolism.
Acknowledgments
This work was supported in part by a grant from the Office Of Minority Health to the Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development (Bethesda, MD); by National Institute of Child Health and Human Development Contract No. 1-HD 3–3196; and by grants from the JF Kapnek Charitable Trust (Philadelphia, PA), the South African Medical Research Council (Johannesburg, South Africa), the University of the Witwatersrand (Johannesburg, South Africa), and the Research Board of the University of Zimbabwe (Harare, Zimbabwe).
References
The following articles in journals at HighWire Press have cited this article:
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  N. Nabavi, Y. Urukova, M. Cardelli, J. E. Aubin, and R. E. Harrison Lysosome Dispersion in Osteoblasts Accommodates Enhanced Collagen Production during Differentiation J. Biol. Chem., July 11, 2008; 283(28): 19678 - 19690. [Abstract] [Full Text] [PDF]
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