On 2015 Jun 10, Paul Golding commented:

Baggott and Tamura imply that my experiment was flawed because: “The study design by Golding (2014) should have included a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.” and advise that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”.

My full response is available here.

Was the 500 mg/day vitamin C supplement excessive, and could it have abnormally extended the time taken to deplete the liver folate store?

Baggott and Tamura claim that my 500 mg/day vitamin C supplement was excessive, and that my saturated vitamin C status was abnormal. They propose that by abnormally protecting the reduced (unstable) form of folate from oxidation, which they claim would not have been preserved by recommended vitamin C intakes, the “excessive” vitamin C preserved the liver folate store. I strongly disagree with these assertions.

Carr et al (2012), Levine et al. (1996) and Lykkesfeldt and Poulsen (2010) recommend that vitamin C intake should be sufficient to ensure plasma saturation. If the plasma vitamin C concentration is insufficient to prevent oxidation of fully reduced folate, although sufficient to prevent scurvy, there would be sub-optimal vitamin C status.

There is no evidence that, once there is sufficient vitamin C present to ensure plasma saturation, and thereby protect the fully reduced folates, additional amounts will affect the time taken to develop folate deficiency.

Levine et al. (1996) recommend 200 mg vitamin C daily, very significantly higher than the 70 mg taken by Herbert (Herbert 1962), and a level that ensures plasma saturation. They plotted plasma vitamin C concentration against the daily vitamin C dose (Levine et al. 1996 Figure 1C); this produced a sigmoid curve with 200 mg the lowest dose to reach a plateau.

I used data from Levine et al. (1996 Table 1) to produce a chart to illustrate the relationship between vitamin C dose and the plasma concentration, at relevant levels. At Herbert’s dose of 70 mg/day, plasma concentration is 32 µmol/L; at 200 mg/day recommended by Levine et al. (1996), plasma concentration is 66 µmol/L; at my dose of 500 mg/day, plasma concentration is 71 µmol/L. A 500 mg dose of vitamin C will not produce a significantly higher plasma concentration than the 200 mg dose recommended by Levine et al. (1996), but a 70 mg dose will produce a very significantly lower plasma concentration. The Excel file, high-resolution PDF and Microsoft PowerPoint slide for my chart are available at the link above.

The predicted plasma vitamin C concentration of 71 µmol/L, produced by my intake of 500 mg/day, is very close to the value considered by Carr et al. (2012) to be necessary for good health.

The vitamin C supplement used by me was integrated with the iron tablets taken to prevent the iron deficiency reported by Herbert. As stated in Golding (2014), the iron tablets (Abbott Australia Ferro-Grad C) comprised 325 mg ferrous sulphate (equivalent to 105 mg elemental iron), with 562 mg sodium ascorbate (equivalent to 500 mg vitamin C). Sodium ascorbate is necessary to ensure adequate absorption of the iron (Hurrell 2002, McCurdy 1968).

Was the 1000 µg/day vitamin B12 supplement excessive, and could it have abnormally extended the time taken for me to deplete my liver folate store?

Although Baggott and Tamura raise the possibility of such a confounding effect, they have not suggested any mechanism for it, or cited any published reports of such findings.

I found no published reports of vitamin B12 used to overcome a dietary folate deficiency, or any findings of interference by vitamin B12 supplements with folate metabolism where there was no initial vitamin B12 deficiency. An initially untreated vitamin B12 deficiency can cause a secondary functional folate deficiency; the “methylfolate trap” (Tisman and Herbert 1973). Such a secondary folate deficiency might be corrected by vitamin B12 supplements, depending on the cause of the vitamin B12 deficiency, but there is no published evidence that increasing the vitamin B12 intake beyond that will have any effect on folate metabolism.

As explained in Golding (2014), the 1000 µg/day vitamin B12 supplement was necessary to prevent vitamin B12 deficiency. Extensive testing during my previous vitamin B12 investigation showed that my vitamin B12 is absorbed and transported to cells normally but is not utilised adequately within the cells, presumably because of a deficiency of one of the B12 cofactors. At least 250 µg was needed to avoid deficiency but 1000 µg is the most readily available, allows a reasonable margin for error, and is within the recommended range for the oral vitamin B12 dose (Kuzminski et al. 1998).

Did the experiment include appropriate supplementation of micronutrients and was the title adequate?

Baggott and Tamura have not provided any evidence that taking either of these supplements could have interfered with the development of folate deficiency in my experiment, and have not offered any plausible mechanism for such confounding effects. In addition, my supplementation with vitamin C and vitamin B12 was reasonable, in the doses used, to prevent deficiencies.

I therefore disagree with their advice that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”, and with what they imply in their conclusion; that my experiment was flawed because the study design failed to include “a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.”

I also disagree with their assertion that “The title … should have contained words clearly indicating that the subject had a high initial folate status and was saturated with ascorbate.” My replete folate status was clearly stated in Objective, Experiment Design and The Subject. According to Levine et al. (1996), Carr et al. (2012) and Lykkesfeldt and Poulsen (2010), plasma saturation is the normal optimal vitamin C status.

References

Baggott JE, Tamura T (2014) The second study of experimental human folate deficiency. SpringerPlus 3:719

Carr AC, Pullar JM, Moran S, Vissers MC (2012) Bioavailability of vitamin C from kiwifruit in non-smoking males: determination of 'healthy' and 'optimal' intakes. J Nutr Sci 1:e14

Golding PH (2014) Severe experimental folate deficiency in a human subject – a longitudinal study of biochemical and haematological responses as megaloblastic anaemia develops. SpringerPlus 3:442

Herbert V (1962) Experimental nutritional folate deficiency in man. Trans Assoc Am Physicians 75:307–320

Herbert V (1987) Recommended dietary intakes (RDI) of folate in humans. Am J Clin Nutr 45(4):661–670

Hurrell RF (2002) Fortification: overcoming technical and practical barriers. J Nutr 132(4 Suppl):806S–812S

Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J (1998) Effective treatment of cobalamin deficiency with oral cobalamin. Blood 92(4):1191-1198

Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR (1996) Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci 93(8):3704–3709

Lykkesfeldt J, Poulsen HE (2010) Is vitamin C supplementation beneficial? Lessons learned from randomised controlled trials. Br J Nutr 103(9):1251-1259

McCurdy PR, Dern RJ (1968) Some therapeutic implications of ferrous sulfate-ascorbic acid mixtures. Am J Clin Nutr 21(4):284–288

Tisman G, Herbert V (1973) B12 dependence of cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B 12 deficiency. Blood 41(3):465–469

On 2015 Jun 10, Paul Golding commented:

Baggott and Tamura imply that my experiment was flawed because: “The study design by Golding (2014) should have included a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.” and advise that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”.

My full response is available here.

Was the 500 mg/day vitamin C supplement excessive, and could it have abnormally extended the time taken to deplete the liver folate store?

Baggott and Tamura claim that my 500 mg/day vitamin C supplement was excessive, and that my saturated vitamin C status was abnormal. They propose that by abnormally protecting the reduced (unstable) form of folate from oxidation, which they claim would not have been preserved by recommended vitamin C intakes, the “excessive” vitamin C preserved the liver folate store. I strongly disagree with these assertions.

Carr et al (2012), Levine et al. (1996) and Lykkesfeldt and Poulsen (2010) recommend that vitamin C intake should be sufficient to ensure plasma saturation. If the plasma vitamin C concentration is insufficient to prevent oxidation of fully reduced folate, although sufficient to prevent scurvy, there would be sub-optimal vitamin C status.

There is no evidence that, once there is sufficient vitamin C present to ensure plasma saturation, and thereby protect the fully reduced folates, additional amounts will affect the time taken to develop folate deficiency.

Levine et al. (1996) recommend 200 mg vitamin C daily, very significantly higher than the 70 mg taken by Herbert (Herbert 1962), and a level that ensures plasma saturation. They plotted plasma vitamin C concentration against the daily vitamin C dose (Levine et al. 1996 Figure 1C); this produced a sigmoid curve with 200 mg the lowest dose to reach a plateau.

I used data from Levine et al. (1996 Table 1) to produce a chart to illustrate the relationship between vitamin C dose and the plasma concentration, at relevant levels. At Herbert’s dose of 70 mg/day, plasma concentration is 32 µmol/L; at 200 mg/day recommended by Levine et al. (1996), plasma concentration is 66 µmol/L; at my dose of 500 mg/day, plasma concentration is 71 µmol/L. A 500 mg dose of vitamin C will not produce a significantly higher plasma concentration than the 200 mg dose recommended by Levine et al. (1996), but a 70 mg dose will produce a very significantly lower plasma concentration. The Excel file, high-resolution PDF and Microsoft PowerPoint slide for my chart are available at the link above.

The predicted plasma vitamin C concentration of 71 µmol/L, produced by my intake of 500 mg/day, is very close to the value considered by Carr et al. (2012) to be necessary for good health.

The vitamin C supplement used by me was integrated with the iron tablets taken to prevent the iron deficiency reported by Herbert. As stated in Golding (2014), the iron tablets (Abbott Australia Ferro-Grad C) comprised 325 mg ferrous sulphate (equivalent to 105 mg elemental iron), with 562 mg sodium ascorbate (equivalent to 500 mg vitamin C). Sodium ascorbate is necessary to ensure adequate absorption of the iron (Hurrell 2002, McCurdy 1968).

Was the 1000 µg/day vitamin B12 supplement excessive, and could it have abnormally extended the time taken for me to deplete my liver folate store?

Although Baggott and Tamura raise the possibility of such a confounding effect, they have not suggested any mechanism for it, or cited any published reports of such findings.

I found no published reports of vitamin B12 used to overcome a dietary folate deficiency, or any findings of interference by vitamin B12 supplements with folate metabolism where there was no initial vitamin B12 deficiency. An initially untreated vitamin B12 deficiency can cause a secondary functional folate deficiency; the “methylfolate trap” (Tisman and Herbert 1973). Such a secondary folate deficiency might be corrected by vitamin B12 supplements, depending on the cause of the vitamin B12 deficiency, but there is no published evidence that increasing the vitamin B12 intake beyond that will have any effect on folate metabolism.

As explained in Golding (2014), the 1000 µg/day vitamin B12 supplement was necessary to prevent vitamin B12 deficiency. Extensive testing during my previous vitamin B12 investigation showed that my vitamin B12 is absorbed and transported to cells normally but is not utilised adequately within the cells, presumably because of a deficiency of one of the B12 cofactors. At least 250 µg was needed to avoid deficiency but 1000 µg is the most readily available, allows a reasonable margin for error, and is within the recommended range for the oral vitamin B12 dose (Kuzminski et al. 1998).

Did the experiment include appropriate supplementation of micronutrients and was the title adequate?

Baggott and Tamura have not provided any evidence that taking either of these supplements could have interfered with the development of folate deficiency in my experiment, and have not offered any plausible mechanism for such confounding effects. In addition, my supplementation with vitamin C and vitamin B12 was reasonable, in the doses used, to prevent deficiencies.

I therefore disagree with their advice that “Therefore, the data presented by Golding (2014) must be cautiously interpreted”, and with what they imply in their conclusion; that my experiment was flawed because the study design failed to include “a balanced supplementation of micronutrients at the level of generally recommended dietary intakes.”

I also disagree with their assertion that “The title … should have contained words clearly indicating that the subject had a high initial folate status and was saturated with ascorbate.” My replete folate status was clearly stated in Objective, Experiment Design and The Subject. According to Levine et al. (1996), Carr et al. (2012) and Lykkesfeldt and Poulsen (2010), plasma saturation is the normal optimal vitamin C status.

References

Baggott JE, Tamura T (2014) The second study of experimental human folate deficiency. SpringerPlus 3:719

Carr AC, Pullar JM, Moran S, Vissers MC (2012) Bioavailability of vitamin C from kiwifruit in non-smoking males: determination of 'healthy' and 'optimal' intakes. J Nutr Sci 1:e14

Golding PH (2014) Severe experimental folate deficiency in a human subject – a longitudinal study of biochemical and haematological responses as megaloblastic anaemia develops. SpringerPlus 3:442

Herbert V (1962) Experimental nutritional folate deficiency in man. Trans Assoc Am Physicians 75:307–320

Herbert V (1987) Recommended dietary intakes (RDI) of folate in humans. Am J Clin Nutr 45(4):661–670

Hurrell RF (2002) Fortification: overcoming technical and practical barriers. J Nutr 132(4 Suppl):806S–812S

Kuzminski AM, Del Giacco EJ, Allen RH, Stabler SP, Lindenbaum J (1998) Effective treatment of cobalamin deficiency with oral cobalamin. Blood 92(4):1191-1198

Levine M, Conry-Cantilena C, Wang Y, Welch RW, Washko PW, Dhariwal KR, Park JB, Lazarev A, Graumlich JF, King J, Cantilena LR (1996) Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance. Proc Natl Acad Sci 93(8):3704–3709

Lykkesfeldt J, Poulsen HE (2010) Is vitamin C supplementation beneficial? Lessons learned from randomised controlled trials. Br J Nutr 103(9):1251-1259

McCurdy PR, Dern RJ (1968) Some therapeutic implications of ferrous sulfate-ascorbic acid mixtures. Am J Clin Nutr 21(4):284–288

Tisman G, Herbert V (1973) B12 dependence of cell uptake of serum folate: an explanation for high serum folate and cell folate depletion in B 12 deficiency. Blood 41(3):465–469