Evidence for a recommended dietary allowance for vitamin C from pharmacokinetics: A comment and analysis

The current recommended dietary allowance (RDA) for vitamin C, as proposed by the Food and Nutrition Board/National Research Council in 1980 and reconfirmed in 1989, is 60 mg daily for nonsmoking adult males. Levine et al. [Levine, M., Conry-Cantilena, C., Wang, Y., Welch, R. W., Washko, P. W., et al. (1996) Proc. Natl. Acad. Sci. USA 93, 3704–3709], based on a study of vitamin C pharmacokinetics in seven healthy men, have now proposed that the RDA should be increased to 200 mg daily. I have examined, in brief, the experimental and conceptual bases for this new recommendation and its implications for public health and nutrition policy and programs. Using, for illustrative purposes only, data extracted from each of two recent dietary surveys of noninstitutionalized adult males living in households in the Netherlands and the United States, it is predicted that the prevalence of intakes inadequate to meet the individual’s own requirement would be about 96% or 84%, respectively, if the criteria of adequacy used for derivation of the 200 mg RDA are accepted. Depending upon the particular average requirement value for ascorbic acid that might be derived from their data, the proposal by Levine et al. would mean a desirable increase in mean intakes in these two populations by as much about 2- to 3-fold. Hence, before an action of this kind is to be recommended, an answer must be sought to the question whether current experimental data including the criteria selected (saturation kinetics) are adequate to establish a new set of requirements for vitamin C, which then carry such profound policy implications. This will require critical assessment of all of the available evidence emerging from laboratory, clinical, and epidemiological studies to determine whether it provides a sufficient rationale for accepting criteria of vitamin C adequacy such as those proposed by Levine et al. and the requirement estimates so derived.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.

Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance.

Determinants of the recommended dietary allowance (RDA) for vitamin C include the relationship between vitamin C dose and steady-state plasma concentration, bioavailability, urinary excretion, cell concentration, and potential adverse effects. Because current data are inadequate, an in-hospital depletion-repletion study was conducted. Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 mg of vitamin C daily. Steady-state plasma and tissue concentrations were determined at seven daily doses of vitamin C from 30 to 2500 mg. Vitamin C steady-state plasma concentrations as a function of dose displayed sigmoid kinetics. The steep portion of the curve occurred between the 30- and 100-mg daily dose, the current RDA of 60 mg daily was on the lower third of the curve, the first dose beyond the sigmoid portion of the curve was 200 mg daily, and complete plasma saturation occurred at 1000 mg daily. Neutrophils, monocytes, and lymphocytes saturated at 100 mg daily and contained concentrations at least 14-fold higher than plasma. Bioavailability was complete for 200 mg of vitamin C as a single dose. No vitamin C was excreted in urine of six of seven volunteers until the 100-mg dose. At single doses of 500 mg and higher, bioavailability declined and the absorbed amount was excreted. Oxalate and urate excretion were elevated at 1000 mg of vitamin C daily compared to lower doses. Based on these data and Institute of Medicine criteria, the current RDA of 60 mg daily should be increased to 200 mg daily, which can be obtained from fruits and vegetables. Safe doses of vitamin C are less than 1000 mg daily, and vitamin C daily doses above 400 mg have no evident value.

Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.

Homozygous deletions have been central to the discovery of several tumor-suppressor genes, but their finding has often been either serendipitous or the result of a directed search. A recently described technique [Lisitsyn, N., Lisitsyn, N. & Wigler, M. (1993) Science 259, 946-951] held out the potential to efficiently discover such events in an unbiased manner. Here we present the application of the representational difference analysis (RDA) to the study of cancer. We cloned two DNA fragments that identified a homozygous deletion in a human pancreatic adenocarcinoma, mapping to a 1-centimorgan region at chromosome 13q12.3 flanked by the markers D13S171 and D13S260. Interestingly, this lies within the 6-centimorgan region recently identified as the BRCA2 locus of heritable breast cancer susceptibility. This suggests that the same gene may be involved in multiple tumor types and that its function is that of a tumor suppressor rather than that of a dominant oncogene.