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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
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,
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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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