Id6 rca 3/18

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http://diabetes.diabetesjournals.org/content/37/3/359.full-text.pdf

Diabetes 1988 Mar; 37(3): 359-361. https://doi.org/10.2337/diab.37.3.359

Deficiency of Ascorbic Acid in Experimental Diabetes: Relationship With Collagen and Polyol Pathway Abnormalities

ABSTRACT

The plasma and tissue concentration of ascorbic acid (AA) is reduced in diabetes. This study was designed to investigate the mechanism and significance of this phenomenon. The low plasma AA concentration of diabetic rats can be normalized by dietary AA supplement (20-40 mg/day), a dosage approximately equal to the maximal synthetic rate of this substance in the rats. Treatment of diabetic rats with this regime prevented the decrease in activity of granulation tissue prolyl hydroxylase (PRLase), an AA-dependent enzyme required for maintaining the normal properties of collagen. The decreased plasma AA concentration and granulation tissue PRLase activity in diabetes can also be normalized by the aldose reductase inhibitor tolrestat. We conclude that in diabetic animals there is a true deficiency of AA that may be responsible for some of the changes of collagen observed in diabetes. Treatment with AA or an aldose reductase inhibitor may prevent some of the diabetic complications with underlying collagen abnormalities. Diabetes 37:359-61, 1988.

The plasma and tissue concentration of ascorbic acid (AA) has been reported to be decreased in diabetic animals and humans (1-3). The mechanism of this reduction in AA level in diabetes is not well understood, and its functional significance is equally uncertain. AA regulates the biosynthesis and posttranslational modifications of collagen, actions substantially mediated by its ability to maintain the activity of the enzyme prolyl hydroxylase (PRLase; EC 1.14.11.2) (4-6). Because collagen is quantitatively the most important structural protein, any disturbance of AA metabolism in diabetes can lead From the Department of Medicine, The University of Sydney, and the Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, New South Wales, Australia. Address correspondence and reprint requests to Dr. D.K. Yue, Department of Medicine, The University of Sydney, Sydney, NSW, Australia 2006. Received for publication 28 September 1987 and accepted in revised form to tissue damage. Investigations into the mechanism and functional effects of the AA abnormalities in diabetes may provide valuable information on the pathogenesis of some diabetic complications. In this study, the effects of AA supplementation on plasma AA concentration and granulation tissue PRLase activity of diabetic animals were investigated. Because the polyol pathway is implicated in many biochemical changes in diabetes, we also examined the effects of aldose reductase inhibition by tolrestat (Ay-27,773, Ayerst, New York) on AA level and PRLase activity in diabetes.

MATERIALS AND METHODS

Animals. Female Wistar rats weighing 180-200 g were used for this study. Each experiment was started with ~30 rats divided into approximately equal numbers for each of the four subgroups (normal and diabetes ± treatment with AA or normal and diabetes ± tolrestat). Diabetes was induced by the injection of streptozocin (65 mg/kg i.v.; Calbiochem, San Diego, CA), and only animals with tail blood glucose levels >20 mM were used. After 4 wk of treatment by the appropriate agent (AA or tolrestat), the animals were killed by an overdose of ketamine (Parke Davis, Sydney, Australia), and blood and tissue were obtained for study. All experiments were approved by the Animal Experiments Ethics Review Committee of The University of Sydney.

Ascorbic acid and tolrestat treatment

AA was measured by HRLC with uBondapak C-18 (Waters, Mollord, MA.  Blood was obtained from rats by cardiac puncture after death by an overdose of ketamine (200 mg/kg). … problem copying PDF….

DISCUSSION The metabolism of AA is abnormal in diabetes, with AA concentration reported to be decreased in both plasma and tissues (1-3). Despite these observations, it has not been established whether the decreased AA in diabetes has any functional significance or whether diabetic patients should receive AA treatment. The best-known function of AA is the prevention of scurvy, an effect mediated by its action on the synthesis and posttranslational modifications of collagen (4). The interaction between AA and collagen biosynthesis is complex, but a major factor is the ability of AA to maintain in vivo the enzyme PRLase in its active state (5,6). This enzyme catalyzes the hydroxylation of proline to form hydroxyproline, an amino acid required for the stability of the collagen molecule (9).

We and others have previously observed a reduction of PRLase activity in the tissues of diabetic animals (10,11). Results herein showed that this phenomenon is a manifestation of AA depletion, which suggests a true deficiency of AA in diabetes and that its supplementation should be considered in the treatment of diabetic patients. This has the potential of preventing the abnormal synthesis and biomechanical properties of collagen in diabetes.

Because AA has many other important biochemical actions, including the scavenging of free radicals (4), its therapeutic usage in diabetes has even wider implications. Further studies are required to determine if these findings are also applicable to humans. The pattern of changes in AA metabolism is very similar in diabetic rats and humans, although rats can synthesize AA, whereas humans must rely on exogenous sources.

The aldose reductase inhibitor tolrestat, which normalized the plasma AA concentration in diabetes, also prevented the fall in PRLase activity, further supporting a causal relationship between these two phenomena. How aldose reductase inhibition leads to normalization of plasma AA is uncertain but may be related to the ability of this class of substance to raise the tissue concentration of reduced glutathione, which is required for the recycling of dehydroascorbate to AA by both enzymatic and nonenzymatic mechanisms (12,13). The urinary excretion of AA and myo-inositol is increased in experimental diabetes, and aldose reductase inhibition could also affect the metabolism of AA at this level (14).

This study confirms the low AA level in diabetes and reveals its relationship with the abnormalities of collagen and the polyol pathway. The depletion of AA in diabetes may not be a harmless phenomenon, and further studies are required to evaluate the need of treating diabetic patients with this vitamin.

ACKNOWLEDGMENTS:

This study was supported by the National Health and Medical Research Council of Australia, The Kellion Foundation, and The Hoechst Foundation of Australia. Tolrestat was a gift of Ayerst Company.

REFERENCES

1. Som S, Basu D, Mukherjee S, Deb S, Choudhary PR, Mukherjee SN, Chatterjee SN, Chatterjee IB: Ascorbic acid metabolism in diabetes mellitus. Metabolism 30:572-77, 1981 2. Yew MS: Effect of streptozotocin diabetes on tissue ascorbic acid and dehydroascorbic acid. Horm Metab Res 15:158, 1983 3. Chen MS, Hutchinson ML, Pecoraro RE, Lee WYL, Labbe RF: Hyperglycemia-induced intracellular depletion of ascorbic acid in human mononuclear leukocytes. Diabetes 32:1078-81, 1983 4. Levine M: New concepts in the biology and biochemistry of ascorbic acid. N Engl J Med 314:892-902, 1986 5. Barnes MJ: Function of ascorbic acid in collagen metabolism. Ann NY Acad Sci 258:264-75, 1976 6. Myllyla R, Kuutti-Savolainen ER, Kivirikko Kl: The role of ascorbate in the prolyl hydroxylase reaction. Biochem Biophys Res Commun 83:441-48, 1978 7. Yue DK, Swanson S, McLennan S, Marsh M, Spaliviero J, Delbridge L, Reeve T, Turtle JR: Abnormalities of granulation tissue and collagen formation in experimental diabetes, uraemia and malnutrition. Diabetic Med 3:221-25, 1986 8. Peterkofsky B, DiBlasio R: Modifications of the tritium-release assays for prolyl and lysyl hydroxylase using Dowex-50 columns. Anal Biochem 66:279-86, 1975 9. Berg RA, Prockop DJ: The thermal transition of a non-hydroxylated form of collagen: evidence for a role for hydroxyproline in stabilizing the triplehelix of collagen. Biochem Biophys Res Commun 52:115-20, 1973 10. Ramamurthy NS, Greenwald RA, Schneir M, Golub LM: The effect of alloxan diabetes on prolyl and lysyl hydroxylase activity in uninflamed and inflamed rat gingiva. Arch Oral Biol 30:679-83, 1985 11. Yue DK, McLennan SV, Dunwoodie SL, Turtle JR: Prolyl hydroxylase deficiency in diabetic collagen (Abstract). Diabetes 36 (Suppl. 1):101A, 1987 12. Gonzales AM, Sochor M, McLean P: The effect of an aldose reductase inhibitor (Sorbinil) on the level of metabolites in lenses of diabetic rats. Biochem J 210:775-81, 1983 13. Rigley R, Riddle M, Layman D, Stankova L: Human cell dehydroascorbate reductase kinetic and functional properties. Biochim Biophys Acta 659:15-22, 1981 14. Straumfjord JV Jr, West ES: In vivo synthesis of ascorbic acid by the alloxan diabetic rat. Proc Soc Exp Biol Med 94:566-68, 1957

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From http://diabetes.diabetesjournals.org/content/38/2/257.short   1989

Ascorbic Acid Metabolism and Polyol Pathway in Diabetes

Dietary myo-inositol supplementation was effective in normalizing plasma AA levels, as was treatment with tolrestat. In untreated diabetes, despite low plasma AA concentration, there was increased urinary excretion of AA that was reversed by treatment with either tolrestat or myoinositol. In contrast, AA supplementation normalized plasma AA concentrations while further increasing urinary AA excretion. The abnormality of AA metabolism was less severe in galactose-fed rats, which had normal plasma AA levels and only minor increases in urinary AA excretion. These studies demonstrated a disturbance in the regulation of plasma and urinary AA concentration in experimental diabetes and confirmed the relationship of AA with the polyol pathway. Because AA has many important biological functions, abnormalities of AA metabolism could be important in the pathogenesis of some diabetic complications.

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http://diabetes.diabetesjournals.org/content/38/8/1036.short           Diabetes 1989 Aug; 38(8): 1036-1041

In Vitro and In Vivo Reduction of Erythrocyte Sorbitol by Ascorbic Acid

Abstract

The in vitro accumulation of sorbitol by human erythrocytes incubated in a physiological glucose medium was found to be strongly reduced by the addition of ascorbic acid (AA). A maximal inhibition of sorbitol in the erythrocytes of 98.3% occurred when the concentration of AA was at its peak in the cells. After incubation, the erythrocyte sorbitol was found to be inversely correlated with the concentration of AA in the erythrocytes. A human supplementation study was conducted with 10 normo-glycemic subjects. Each was given 500 mg/day AA alone or in a citrus fruit medium. Each supplementation lasted 2 wk and was followed by a 10-day washout. The citrus fruit medium produced a significantly greater increase in erythrocyte AA compared with AA alone. AA alone and in citrus fruit medium decreased erythrocyte sorbitol 12.6 and 27.2%, respectively, with the latter being significantly more effective. In a study with 4 subjects, 2000 mg/day AA resulted in a reduction in erythrocyte sorbitol of 56.1%. As in the in vitro study, there was an inverse relationship between erythrocyte AA and sorbitol. Two thousand milligrams of AA per day (AA or citrus fruit medium) was given to 8 diabetic subjects in a preliminary 3-wk supplementation trial in which erythrocyte sorbitol levels were decreased by 44.5%. These results suggest that AA supplementation for diabetic subjects may provide a simple means of preventing and ameliorating the complications of diabetes without the use of drugs.

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