plasma, neutrophil, and urinary ascorbate concentrations were determined during vitamin C depletion and repletion to steady-state. The rigorous criteria for achieving steady-state plasma concentrations (five daily samples that varied less than or equal to 10 percent) make the Levine et al. (1996a) data unique among depletion-repletion studies.

Seven apparently healthy male volunteers, aged 20 to 26 years, were studied as in-patients for 4 to 6 months. Subjects were depleted by being fed a diet containing less than 5 mg/day vitamin C. Depletion was defined as completed when plasma vitamin C concentrations ranged from 5 to 10 µmol/L (0.1 to 0.2 mg/dL) without signs or symptoms of scurvy. For repletion, seven consecutive doses of vitamin C (30, 60, 100, 200, 400, 1,000, and 2,500 mg/day) were given sequentially until steady-state plasma and leukocyte (neutrophils, monocytes, and lymphocytes) vitamin C concentrations were achieved at each dosage. The results for plasma and neutrophil concentrations can be seen in Figure 5-3 and Figure 5-4, and Table 5-10.

As seen in Figure 5-4 and Table 5-10, the ascorbate saturation concentration in neutrophils was approximately 1.3 mmol/L. This was attained by four of the seven subjects at a vitamin C intake of 100 mg/day. Monocytes and lymphocytes also reached maximum concentrations at 100 mg/day (Levine et al., 1996a). However, at neutrophil saturation, about 25 percent of the doses were excreted in the urine, whereas at 60 percent of maximum ascorbate (dose of 60 mg/day), essentially no ascorbate was excreted.

No data from the Levine at al. (1996a) study are available for vitamin C intakes between 60 and 100 mg/day. However, because 60 percent of maximal ascorbate concentration in neutrophils would provide less antioxidant protection than 80 or 100 percent (Figure 5-2) (Anderson and Lukey, 1987), and since 25 percent of the dose is excreted at 100 percent of maximum neutrophil ascorbate concention, the midpoint 80 percent of maximum (1.0 mmol/L) was chosen. This is assuming that antioxidant protection in this range is linear. This point should better estimate an approximate neutrophil target concentration that fulfills the criteria of adequate in vivo antioxidant protection with little or no urinary loss. From the equation of Figure 5-4, 80 percent of maximal neutrophil concentration (1.0 mmol/L) is equivalent to a vitamin C intake of about 75 mg/day. This represents an EAR, because 80 percent (1.0 mmol/L) neutrophil concentration is an average value, estimated by regression analysis, for the men consuming 75 mg/day of vitamin C as shown in Table 5-10.

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