Handbook of Vitamin C Research

Human Specific Vitamin C Metabolism… 53follows: white blood cells, 6286 ± 1509/ L; red blood cells, 4.29 ± 0.40 million/ L;hemoglobin, 12.4 ± 1.1 g/dL; serum albumin, 4.6 ± 0.3 g/dL; and fasting blood glucose, 85 ±8 mg/dL.C. Mongolian group C for oxidative stress and VC levels: A total of 164 healthysubjects, ranging in age from 24 to 66 years (72 males and 92 females) were randomlyassembled by native researchers in July 2005 [9]. Subjects lived in Murun city, which is acentral town in Khuvsgul Prefecture, located in the northwest of Mongolia, 700 km awayfrom metropolitan Ulaanbaatar. For gene polymorphism analysis, Mongolians fromUlaanbaatar were selected. For the determination of gene polymorphism variant frequencies,Mongolians from Ulaanbaatar were selected from 7 areas in Asia Pacific region [58, 60].Experimental design was approved by the Human Genome Medical-Ethical Committeeof Kagawa Nutrition University (No 222-G, July 6, 2006, for groups A and B, and No. 185-G, May 18, 2005, for group C, and special approval by both Japanese and MongolianInstitutes for group C), and complied with the World Medical Association Declaration ofHelsinki (1964, 2000 version). Written consent was obtained from each subject afteradequate explanation of the purpose and contents of this study before the experiment. Noneof the subjects group B smoked during the experimental period, in order to eliminate thexenobiotic effects of smoking on VC metabolism [42], and there were no diabetics, asdiabetes inhibits DAsA transport through glucose transporters (GLUTs) [23, 40].(2) VC Loading Human Experiment (Figure 1)The study scheme of VC depleting-repleting experiment is shown in Figure 1 [4],following the classical human experiments on AsA and DAsA requirements [60]. In order tosaturate the in vivo VC pool of the subjects group B, 500 mg AsA (2.8 mmol) was orallyadministered 1 week before the loading experiment (Figure 1 left). Subjects group Bconsumed normal diet until 72 h (3 days) before AsA or DAsA loading (9:00), and thereafter,consumed low-VC diet (VC < 4.9 ± 0.1 mg/day; 1,700 kcal/day; protein 70 g/day) untildiscontinuation of the experiment [4]. Detailed composition of the diets was as previouslyreported [4]. Because a crossover study design was used, the same experiment was repeatedafter an interval of about 1 month for each subject [4].On the day of the loading experiment, 1 mmol AsA (176 mg) or DAsA (174 mg) crystalswas orally administered (Figure 1 middle). Urine was collected for 24 h before initiation ofthe low-VC diet (normal period, Figure 1 left) and two 12-h urine samples were collected onthe day before the loading experiment (deficiency period, Figure 1 middle). After oral VCloading, urine was collected after 3, 6, 9, 12 and 24 h (Figure right). To prevent thedeterioration of collected or stored urine, oxalic acid was added to collected urine (finalconcentration 5%). Blood was taken immediately before and at 1 and 3 h after VC loading.During the depletion-repletion experiment period, intake of anything other than the food thatwas cooked and offered (including drugs and supplements) was prohibited. For drinkingwater, bottled mineral water was provided. The crossover method was used; the sameexperiment was repeated after an interval of about 1 month for the subjects. During thisinterval detailed blood analyses, including VC [5], glutathione [9], vitamin E [9], malon