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Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • ascorbate • vitamin E • vitamin A • glutathione • cholesterol • glycation • ODS rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Byers and Perry 1992
), cataracts (Taylor 1992 and 1993
,
Taylor et al. 1997a
), and atherosclerosis (Simon 1992
). Many workers in this field also believe that other
biological antioxidants, including glutathione, vitamin E and
carotenoids, work in concert with ascorbate to scavenge reactive oxygen
species and thus protect organisms from oxidative damage. It has been
reported that ascorbate is coupled with the recycling of vitamin E and
glutathione in cell-free systems and in vivo (Packer et al. 1979
, Mukai et al. 1991
, Winkler et al. 1994
). For example, ascorbate deficiency for more than 2 wk in
osteogenic disorder Shionogi (ODS) rats has been shown to decrease
vitamin E levels in several tissues, whereas vitamin E deficiency
resulted in decreased plasma ascorbate levels (Tanaka et al. 1997
). Furthermore, glutathione deficiency decreased liver
ascorbate levels in Sprague-Dawley rats (Martensson and Meister 1991
), and supplementation of ascorbate elevated
glutathione levels in human blood cells (Johnston et al. 1993
). Because most of the results regarding the interactions
between ascorbate and vitamin E and glutathione were obtained under
ascorbate- or vitamin E–depleting conditions, it is not clear whether
the interactions of ascorbate with vitamin E and glutathione also occur
under nonscorbutic conditions.
In this study, we determined the effects of ascorbate on vitamin
E, glutathione and retinol in dietary ascorbate-requiring ODS rats
(Horio et al. 1985
, Kawai et al. 1992
)
under nonscorbutic conditions. We fed ODS rats diets that contained
different levels of ascorbate and determined the relationship between
dietary ascorbate intake and levels of ascorbate and glutathione in
liver and kidney. The relationships between dietary ascorbate intake
and levels of ascorbate, vitamin E, retinol, cholesterol and glycated
hemoglobin were also determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Forty 5-mo-old (equally distributed by sex) ODS rats (CLEA, Ringoes, NJ) were individually housed in 18 x 26 x 18 cm stainless steel, suspended rodent cages and were given free access to a nonpurified diet (Certified Guinea Pig Chow, PMI, Richmond, IN) and water for 1 mo. The animals were maintained in American Association for the Accreditation of Laboratory Animal Care accredited facilities in an environmentally controlled atmosphere (23°C, 45% relative humidity with 15 air changes of 100% fresh hepa-filtered air per hour and a 12-h light:dark cycle). This project was approved by the USDA HNRCA Animal Use and Care Committee. All animals were observed daily for clinical signs of disease.
Experimental procedures.
After 1 mo acclimation, all rats were weighed and were given free
access to an AIN 93M powdered diet (Reeves et al. 1993
)
supplemented with 1965 mg/kg ascorbate. The diet was replenished daily
and completely replaced every other day. Three weeks later, 1 mL blood
was collected from the lateral tail vein of 20 rats (10 male and 10
female), using a 22-gauge winged infusion set attached to a 3-mL
syringe. Rats were given free access to an AIN 93M powdered diet
supplemented with 1019 mg/kg ascorbate for 3 wk. Food intakes were
determined for each rat during wk 2, and blood was withdrawn by the end
of wk 3. Thereafter, rats were fed diets in which concentrations of
ascorbate were progressively reduced every 3 wk as follows: 768, 527,
380, 280 and 180 mg/kg. Food intake and body weight were determined,
and blood was withdrawn according to the procedure described above.
Control rats (n = 20) were fed a diet containing 1965 mg/kg ascorbate until the experimental rats finished the diet regimen. All of the rats were terminally exsanguinated under CO2 narcosis and organs were harvested.
Blood handling.
Blood samples were drawn into EDTA-coated Vacutainer glass tubes, shielded from light and kept on ice until processed. The whole blood was separated into plasma and erythrocytes by centrifugation at 1000 x g for 15 min. Plasma intended for total ascorbate analysis was deproteinized by mixing with equal volumes of 0.5 mol/L perchloric acid (PCA, Aldrich, Milwaukee, WI) followed by centrifugation; aliquots of the clear supernatant were stored at -70°C until analysis. Levels of cholesterol were determined immediately. All analyses were completed within 2 wk.
Analysis of total ascorbate.
The total ascorbic acid assay is a modification of a method described
by Behrens and Madere (1987)
. Complete reduction of the
dehydroascorbic acid in a 100-µL aliquot of
deproteinized supernatant was achieved by adding 30 µL
of 0.22 mol/L homocysteine in a 2.58 mol/L
K2HPO4 buffer to the supernatant and mixing
with a vortex for 15 min. Following this, 170 µL of
0.50 mol/L perchloric acid was added. The sample was mixed for an
additional 15 s and centrifuged at 14,000 x g
for 10 min. The supernatant was then injected onto a Biosil ODS 5S 150
x 4 mm column (Bio-Rad, Richmond, CA). The mobile phase consisted
of 40 mmol/L sodium acetate, 0.25 mL/L n-octaylamine
(Sigma Chemical, St. Louis, MO) and 0.2 g/L EDTA, pH 4.0. The HPLC
instrumentation consisted of a Waters Model 510 pump, a Waters 710B
autosampler (Waters, Milford, MA), and an LC4B Bioanalytical Systems
(West Lafayette, IN) electrochemical detector with amperometric
detection. The intra-assay CV for this method was 4.2%, whereas
the interassay CV was 5.1%.
Analysis of glutathione.
Levels of reduced and oxidized glutathione in liver were determined
using the HPLC procedure described by Fariss and Reed (1987)
. Livers and kidneys were homogenized with 1.2 mol/L
perchloric acid containing 1 mmol/L bathophenanthrolinedislfonic acid
(10 mL/g liver or 5 mL/g kidney). After centrifugation at 1000 x g for 10 min, the supernatant was first reacted with
iodoacetic acid to convert reduced glutathione and other thiols to
stable S-carboxymethyl derivatives. Then
1-fluroro-2,4-dinitrobenzene was added to convert the primary amine to
dinitrophenyl derivatives. After derivatization, samples were separated
with the HP 1100 HPLC system (Wilmington, DE) using an amine column and
monitored at 365 nm. 
-Glu-glu was used as an internal standard.
Retinol and 
-tocopherol analysis.
In an adaptation of the method described by Bieri et al. (1979)
, retinol and -tocopherol levels in plasma were
determined simultaneously by reversed-phase HPLC. Retinyl acetate
(Sigma) was used as an internal standard. After the addition of the
internal standard solution, the samples were extracted with
HPLC-grade hexane (VWR, Piscataway, NJ), centrifuged and the
solvent layer evaporated under a stream of nitrogen. The dried samples
were then dissolved in a mixture of methanol and ethyl ether and were
injected onto a reversed-phase column, Bio-Sil ODS-5S 150
x 4 mm (Bio-Rad). The mobile phase was 100% HPLC degassed
methanol. Intra-assay CV for the vitamin A and vitamin E assays
were 0.9 and 1.5%, whereas the interassay CV were 4.9 and 5.0%,
respectively.
Determination of total cholesterol.
Total cholesterol levels in plasma were determined by a colorimetric
method initially described by Allain et al. (1974)
and
modified by Roche Diagnostics to be run on a Cobas Mira Analyzer (Roche
Diagnostics, Nutley, NJ). Assayed, lyophilized control materials
purchased from Ciba Corning Diagnostics (Irvine, CA) were run to track
precision and accuracy. Interassay CV for three levels of controls were
2.2, 1.5 and 1.3%, whereas intra-assay CVwere 1.8, 1.8 and 1.1%.
Determination of glycosylated hemoglobin.
Glycosylated hemoglobin levels were determined as described by
Klenk et al. (1982)
using GlycoTest II affinity
chromatography columns (Pierce Chemical, Rockford, IL). These columns
were initially loaded with an aliquot of hemolysate and then washed
with an equilibration buffer to elute the nonglycosylated proteins. An
elution buffer containing sorbitol was subsequently used to displace
the bound glycosylated hemoglobin from the column. Absorbance readings
of the nonglycosylated and glycosylated effluents were measured at 414
nm on a Cobas Fara II Centrifugal Analyzer (Roche Diagnostics). Assayed
control materials (normal control, mean 4.2%, and abnormal control,
mean 14.6%) from Pierce were analyzed during each run to test the
reliability of this assay. Intra-assay CV for the normal and
elevated controls were 3.4 and 0.9%, respectively, whereas the
interassay CV for the normal and abnormal controls was 2.6%.
Statistical analysis.
The data were analyzed by using repeated-measures ANOVA with diet as a within-subjects factor and sex as a between-subjects factor. Because a considerable amount of data were missing (assuming to be missing at random), the mixed procedure from SAS, Version 6.12 (SAS Institute, Cary, NC) was used to perform the analysis. Unstructured correlations between the repeated measurements were specified. When the diet-by-sex interaction did not reach significance, overall diet and sex differences were examined. Otherwise, diet-specific sex differences or sex-specific diet differences were examined. Tukey's honestly significant difference test was used to compare individual diets. Student's t test for independent samples was used to compare males and females fed the same diet and to compare the ascorbate and glutathione in liver and kidney.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine the dietary ascorbate requirements and associated
interactions with other antioxidants in ODS rats, we progressively
reduced the ascorbate concentration in the diet from 1965 to 180 mg/kg.
Although an increase in food intake was associated with decreasing
dietary ascorbate levels in males (P < 0.001),
reducing dietary ascorbate did not affect food intake in females
(Table 1). Both male and female rats gained body weight over the course of
the study (P < 0.05). Even when the ascorbate
level in the diet was reduced to 180 mg/kg for 3 wk, no scorbutic signs
were observed, and rats appeared healthy for the duration of the
experiment.
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),
plasma ascorbate levels were related to dietary intake. The maximal
plasma ascorbate level was reached in female rats when the ascorbate
concentration in the diet was at least 527 mg/kg (Table 1)
, which
provided ~7 mg ascorbate/d or 25 mg/kg body weight. However, in the
males, saturating levels of plasma ascorbate were observed only when
the concentration of dietary ascorbate was at least 768 mg/kg,
providing 34 mg ascorbate/kg body weight. However, there were no
significant differences between male and females in the plasma
ascorbate levels at any given dietary ascorbate level. When the
concentration of ascorbate in the diet was <527 mg/kg (for female
rats) or 768 mg/kg (for male rats), plasma ascorbate declined
significantly with decreasing dietary ascorbate (P
< 0. 001). Plasma concentrations of ascorbate decreased 72 and
78% in female and male rats, respectively, when ascorbate levels in
the diet decreased from 1965 to 180 mg/kg. These data confirm that ODS
rats are dependent on dietary ascorbate and that levels of ascorbate in
plasma of ODS rats can be manipulated by altering ascorbate intake. Relationship between levels of ascorbate and glutathione in liver and kidney.
Total ascorbate levels in liver and kidney (Table 2) were also related to dietary ascorbate intake. The ascorbate
level in liver was higher than in kidney, regardless of the dietary
ascorbate level. Ascorbate levels in liver and kidney of rats fed the
diet containing 1965 mg/kg ascorbate were 134 and 228% higher,
respectively, than ascorbate levels in liver and kidney of rats fed the
diet containing 180 mg/kg ascorbate (progressively reduced from 1965 to
180 mg/kg). As previously described in mouse tissues (Mune et al. 1995
), glutathione in liver was 100- to 200-fold higher in
liver than in kidney. Levels of glutathione in liver and kidney
remained constant despite the significant changes in the level of
ascorbate (Table 2)
. The ratio of reduced glutathione and oxidized
glutathione also did not differ between rats fed high and low ascorbate
diets, in contrast to reports that ascorbate can elevate glutathione
levels (Johnston et al. 1993
, Martensson and Meister 1991
). This apparent inconsistency may arise because
the studies that showed the relationship between levels of ascorbate
and glutathione were performed in glutathione-depleted rats
(Martensson and Meister 1991
). Therefore, these data do
not support the suggestion of a direct correlation between ascorbate
and glutathione under nonscorbutic conditions.
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Although the plasma ascorbate level decreased significantly with
decreasing ascorbate intake, there were no detectable changes in plasma
vitamin E or retinol (Table 1)
. It is of interest to note that vitamin
E levels in plasma of female ODS rats were significantly higher than
the levels of male ODS rats (P < 0.001). Similar data
were obtained in Emory mice (Scrofano et al. 1998
).
However, when expressed relative to plasma cholesterol, the difference
between male and female was much less (P = 0.12; data
not shown). This indicates that the sex-related difference in
plasma vitamin E is due mainly to differences in plasma lipids or
lipoproteins because levels of cholesterol in females were also higher
than in males (see below).
Effect of dietary ascorbate intake on plasma cholesterol levels.
In ODS rats, ascorbate deficiency results in a significant
elevation in plasma cholesterol and triglyceride concentrations
(Horio et al. 1987 and 1991
, Uchida et al. 1990
). In this study, we determined the relationship between
ascorbate intake and total cholesterol levels under nonscorbutic
conditions. Consistent with previous studies, decreasing dietary
ascorbate was associated with an increase in total plasma cholesterol
in females (Table 1)
. There was no significant increase in plasma
cholesterol with decreasing ascorbate intake in male ODS rats.
Effect of dietary ascorbate intake and levels of glycated hemoglobin.
Glycated hemoglobin levels correlated with the dietary ascorbate intake
in both males and females. When the concentration of ascorbate
decreased from 1695 to 527 mg/kg, although changes in plasma ascorbate
were marginal, the level of glycated hemoglobin decreased significantly
(Table 1)
. When the ascorbate concentrations in the diet were further
decreased from 527 to 180 mg/kg, plasma ascorbate decreased
dramatically, but levels of glycated hemoglobin decreased only slightly
(Table 1)
. Considering the turnover rate of glycated proteins, levels
of glycated hemoglobin may be related to the residual effect from
previous dietary ascorbate levels. Therefore, these data indicate that
the ascorbate-associated elevation in levels of glycated hemoglobin
occurs mainly when the plasma ascorbate is maximal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Due to its size and
tolerance, the ODS rat is an ideal animal to augment the use of the
guinea pig for research regarding physiologic roles of ascorbate. In
contrast to data obtained from scorbutic animals, this study shows that
there is no significant effect of ascorbate on levels of glutathione in
liver or kidney or on plasma, vitamin E and retinol, suggesting that
the interaction of ascorbate with other antioxidants is negligible
under nonscorbutic conditions. However, taken together with earlier
research, these data imply that having reserves of ascorbate may
diminish stress induced by insufficient levels of other antioxidants.
This study also confirms that decreasing ascorbate intake is associated
with an increase of total plasma cholesterol levels, especially in
females (Meyers and Maloley 1993
). The effect of
ascorbate on plasma cholesterol may be the mechanism underlying the
beneficial effects of supplementation of ascorbate in reducing the risk
of cardiovascular diseases (Simon 1992
).
Protein glycation and oxidative stress are major contributing
factors for aging and age-related diseases (Kristal and Yu 1992
, Wolff et al. 1991
). Ascorbate is one of
the major biological antioxidants and plays an important role in
ameliorating oxidative stress. However, ascorbate is also a glycating
agent. Excessive ascorbate may result in protein glycation
(Bensch et al. 1985
, Ortwerth et al. 1988
, Ortwerth and Olesen 1988
, Saxena et al. 1996
). This study confirms that excessive intake of
ascorbate elevates the level of glycated hemoglobin. Because protein
glycation is a contributing factor for aging and age-related
diseases, including cataract development, excessive intake of ascorbate
should be avoided. To achieve maximal benefits of ascorbate
supplementation, the optimal doses of ascorbate intake should be
established. Because the elevation in levels of glycated hemoglobin was
significant when the dietary ascorbate levels were beyond the level
required to maintain saturating levels of plasma ascorbate, these
results indicate that the optimal dose should be equal to or less than
the doses required to achieve the saturating level of plasma ascorbate.
Optimal levels of ascorbate intake will provide maximal protection
against oxidative stress and avoid the adverse effects of glycation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received November 2, 1998. Revision accepted February 23, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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