Protein Glycation: Ascorbate Antagonism.

Bulletin Amer Physical Society, 33(3):296, 1988 (abst).

C9 1 Protein Glycation: Ascorbate Antagonism. J.T.A. Ely, U Washington, G.A. Warner, Northwest Oncology Clinic, D.H. Read & G.A. Santisteban, Seattle U -- Loss of elasticity and solubility, and degradation of other physical properties of proteins and nucleic acids have been reported to result from non enzymatic glycation as well as the associated cross-linking. (1). We present evidence that glycation of hemoglobin is significantly antagonized in animals and humans by pharmacologic daily supplementation of ascorbate at 50mg/kg and above. We explain that : (1) the antagonism is expected to be even stronger for other proteins; and (2) continuous elevation of ascorbate may be necessary for effective antagonism. We discuss the possible relevance to aging , birth defects, cancer, cataracts and other lesions.

*Supported by Northwest Oncology Foundation.
1. A. Cerami, H. Vlassara & M. Brownlee, Scientific American 256(5), 82 (1987)

Ascorbic Acid and Glycohemoglobin in Humans and Mice

Data published in Clinical Chemistry and the New Zealand Medical Journal [N Z Med J 2002 Aug 23; 115(1160): http://www.nzma.org.nz/journal/115-1160/156], describe the ascorbic acid-associated decrease in glycation of hemoglobin in mice and in humans, respectively.

Blood glucose (BG) concentrations averaged over a 2-3 month period are represented by blood glycohemoglobin (GHb) as a percentage of total hemoglobin. Strict glycemic control is important to the successful outcome of diabetic pregnancy and in avoiding development of many diabetic complications (1). Hyperglycemia in non-diabetics is a major risk factor in cardiovascular disease and cancer (2). Thus, knowledge of an individual's true GHb value is vital. This need has prompted continuing investigations of potential interferences in GHb assays (3,4), including conflicting reports on ascorbic acid's effect on GHb (5-7).

As a part of our research on effects of dietary variables on tumor growth, two groups of 18 mice each were provided either ascorbic acid (AA, vitamin C) in drinking water (2.5 mg/ml) or unsupplemented water. Blood glucose was measured six times during the 2 month study (Glucometer, Bayer Diagnostics, NY). We observed no differences in BG between the groups. At the beginning of the study, baseline BG (mean ± standard deviation) for the non-AA and AA groups were 82 ± 16 and 79 ± 15 mg/100 ml, respectively. The BGs at the end of the study were, respectively, 90±16 and 87±18 mg/100 ml. Also at the end of the study, GHb by affinity chromatography (Glycotest; Pierce, Rockford, IL) was determined. For this parameter, the AA group exhibited a significantly lower value (i.e., GHb 4.39% ± 0.78 for non-AA mice and 3.39 ± 0.60 for the AA supplemented mice; t = 4.324, p = 0.0001); a 23% reduction in GHb in the mice consuming approximately 7.5 mg AA/day.

In 1988, Ely et al (5) reported antagonism of hemoglobin glycation by AA in animals and humans. Since then, two contradictory reports of the effect of AA supplementation (750 - 1500 mg/day) on GHb in humans have appeared (6,7). When using affinity chromatography, Davies et al (6) found an 18% decrease in GHb, whereas Weykamp et al (7) found no significant change.

Recently, we observed a significant "false" lowering of GHb in humans supplementing AA at multigram levels. We studied 139 consecutive consenting non-diabetic patients in an oncology clinic. The patients had been encouraged as part of their treatment to supplement AA. Self-reported daily intake varied from 0 to 20 g/day. The plasma AA levels ranged from 11.4 to 517 µmol/L and correlated well with the reported intake. Regression analysis of their GHb and plasma AA values showed a statistically significant inverse association (e.g., each 30 µmole/L increase in plasma AA concentration resulted in a decrease of 0.1 in GHb). A 1 g oral dose of AA can raise plasma AA to 130 µmole/L within an hour and such doses at intervals of about 2 hours throughout the day can maintain ~230 µmole AA/L. Similar levels could also be achieved by use of sustained-release AA tablets. This AA concentration would induce an approximate 0.7 depression in GHb.

Ascorbic acid can lead to interferences in GHb assays, particularly in some based on charge separation (e.g., electrophoresis and ion exchange) where a positive interference has been observed (6). The affinity chromatography method we used, however, would not be affected. This suggests that the AA-associated lowering of GHb reflected a genuine in vivo decrease in glycation. If this is true, the effect has implications not only for interpretation of GHb, but also for human aging where glycation of proteins plays a prominent role in age-related degenerative changes.

A misleading GHb decrease of the magnitude we observed can be clinically significant. Current recommendations for diabetics suggest that GHb be maintained at 7, a level which is associated with acceptable control and decreased risk of complications; when GHb exceeds 8, re-evaluation of treatment is necessary. Moreover, relatively small increases in average blood sugar (i.e., GHb) can accompany adverse reproductive effects. A difference in mean maternal GHb of 0.8 was found for women giving birth to infants without or with congenital malformations. In either of these circumstances, an underestimation of GHb could obscure the need for more aggressive intervention.

Ascorbic acid is the most commonly consumed nutritional supplement after multivitamins (8) and in the western United States, over 11% of adults take an AA supplement daily. Laboratory, epidemiological and intervention studies suggest that antioxidant vitamins, especially AA, have long-term benefits in attenuating the progression of diabetic complications, and diabetics are encouraged to take AA. In light of these facts and the importance of BG in other aspects of human health including immunity, aging, etc., it seems prudent for primary care health providers to inquire regarding the AA intake of patients, especially diabetics, when using GHb for diagnosis or treatment monitoring.

References
  1. The Diabetes Control and Complications Trial Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med 1993;329:977-86.
  2. Ely JTA. Unrecognized pandemic subclinical diabetes of the affluent nations: causes, cost and prevention. J Orthomolec Med 1996;11:95-9.
  3. Weykamp CW, Penders TJ, Sieber CWM, Muskiet FAJ, van der Slik W. Interference of carbamylated and acetylated hemoglobins in assays of glycohemoglobin by HPLC, electrophoresis, affinity chromatography and enzyme immunoassay. Clin Chem 1993;39:138-42.
  4. Weykamp CW, Miedema K, de Haan T, Doelman CJA. Carbamylated hemoglobin interference in glycohemoglobin assays. Clin Chem 1999;45:438-40.
  5. Ely JTA, Warner GA, Read DH, Santisteban GA. Protein glycation: ascorbate antagonism. (Abstract) Bull Amer Phys Soc (Div Biol Phys) 1988;33:296.
  6. Davie SJ, Gould BJ, Yudkin JS. Effect of vitamin C on glycosylation of proteins. Diabetes 1992;41:167-73.
  7. Weykamp CW, Penders TJ, Baadenhuijsen H, Muskiet AJ, Martina W, van der Slik W. Vitamin C and glycohemoglobin. Clin Chem 1995;41:713-6.
  8. Selsinski MJ, Subar AF, Kahle LL. Dietary intake of fat, fiber and other nutrients is related to the use of vitamin and mineral supplements in the United States: the 1992 National Health Interview Study. J Nutr 1996;126:3001-8.


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