Urinary Ascorbic Acid Research Articles

Toxicity of 2,4,4'-trichlorobiphenyl in rats following 90-day dietary exposure.

The toxicity of 2,4,4'-trichlorobiphenyl (PCB 28) was investigated in rats after a 90-d dietary exposure. Groups of 10 male and 10 female weanling Sprague-Dawley rats were administered PCB 28 in the diet at 0, 0.05, 0.50, 5.0, or 50.0 ppm for 13 wk. Growth rate and food consumption were not affected by treatment, and no clinical signs of toxicity were observed. Mottled liver was noted in both control and PCB-treated males, but was found with increased incidence in the highest treatment group. Increased urinary ascorbic acid and hepatic microsomal ethoxyresorufin O-deethylase activity were observed in the 50.0 ppm group of both sexes. The vitamin A content in liver, lung, and kidney was not significantly affected by treatment. Analysis of brain biogenic amines showed a decreased dopamine concentration in the substantia nigra region of female rats receiving 0.5 ppm PCB 28 and higher doses. Female rats appeared to be more sensitive than males to the neurochemical effects of PCB 28. Dose-dependent histologic changes were observed in the thyroid and liver, with biologically significant changes occurring at 5.0 ppm and above. Based on these data, the no observable-adverse-effect level (NOAEL) for this PCB congener was considered to be 0.5 ppm in diet or 36 micrograms/kg body weight/d.

Subchronic toxicity of a medium-chain chlorinated paraffin in the rat.

Groups of ten male and female weanling Sprague-Dawley rats were fed diet containing 0, 5, 50, 500 or 5000 ppm of a medium-chain chlorinated paraffin (C14-17, 52% chlorination) for a period of 13 weeks. Increased relative liver weight was observed at 500 and 5000 ppm in females and at 5000 ppm in males. Relative kidney weight was increased at 5000 ppm in both sexes. Serum cholesterol was increased in the females in a dose-related manner starting at 50 ppm. At 5000 ppm, animals of both sexes had elevated hepatic UDP-glucuronosyltransferase activity while only females showed increased aminopyrine N-demethylase activity. Increased urinary N-acetylglucosaminidase activity occurred at 5000 ppm in females. Increased urinary ascorbic acid excretion monitored at week 12 and a decreased hepatic vitamin A level were detected in females receiving the 500 ppm diet and male and female rats at 5000 ppm. Mild, adaptive histopathological changes were detected in the liver of rats of both sexes at 500 and 5000 ppm, and in the thyroid of males and females starting at 500 and 50 ppm respectively. Minimal changes were observed in the kidney proximal tubules of male rats fed the 5000 ppm diet and in the inner medulla tubules of female rats fed the 500 and 5000 ppm diets. These data indicate that the medium-chain chlorinated paraffin produces biochemical and histological changes at dietary levels of greater than or = 50 ppm in females and greater than or = 500 ppm in males.

Urinary ascorbic acid--HPLC determination and application as a noninvasive biomarker of hepatic response.

A high-performance liquid chromatograph (HPLC) procedure has been developed for the determination of rat urinary ascorbic acid, a major metabolite of the hepatic glucuronic acid pathway. The presence of EDTA and HCl effectively inhibited degradation of ascorbic acid during the collection of urine specimens. The reliability of the procedure was demonstrated by its high recovery (90%), specificity (characteristic absorption maximum and discrimination from isoascorbic acid), and reproducibility (2-3% coefficient of variation). The usefulness of this assay as an indicator of hepatic response was demonstrated in preliminary experiments where increases in urinary ascorbic acid excretion were detected in male rats treated with PCB 126 (3,3',4,4',5-pentachlorobiphenyl) or PCB 105 (2,3,3',4,4'-pentachlorobiphenyl). The HPLC measurement also showed that the two PCB congeners differed markedly in their potency in stimulating urinary ascorbic acid excretion. For example, 10 micrograms/kg bw/day of PCB 126 was sufficient to cause a fourfold increase in urinary ascorbic excretion while 5000 micrograms/kg bw/day of PCB 105 was required for a sevenfold increase. In response to the administration of PCB 105 or PCB 126, urinary ascorbic acid appeared to increase to the same extent as increases in hepatic ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyltransferase (UGT) activities, and to a much higher extent than changes in liver weight and hematological and serum clinical chemical parameters. The sensitivity and specificity, the ease in obtaining timed specimens, and the noninvasive nature make this assay a useful biomarker of hepatic response in dose-finding and various acute and chronic studies.

Dietary, Serum and Urine Ascorbic Acid Status in Male Athletes

The ascorbic acid (AA)-status of 14 marathon runners, 12 soccer players, 9 wrestlers, 9 basketball players and 16 controls was determined. A 7-day food weighed record was kept to quantify the AA-intake. In addition, the AA-serum concentrations and urinary ascorbate excretion were measured. The AA-intake of all 44 athletes (median, 26th-75th percentile) was 180.7 (188-239) mg/d, the serum concentration 70.6 (65.7-80.2 mumol/l) and the urine ascorbate excretion 1531 (391-2934) mumol/g creatine. No significant differences could be observed between the various sport groups, or between the sport groups and controls with respect to absolute (mg/d) and relative (mg/g body weight) AA-intake, serum and urine concentrations. Only a few of the athletes had AA-intake below the RDA or serum- or urine levels smaller than the decision limit. The absolute AA-intake (n = 44) from the 7-day record (r = 0.49, p < 0.0009) and the AA-intake on the last day (1-day) prior to urine collection (r = 0.90, p < 0.0000) correlate moderately/strongly with the urinary excretion. Between AA-intake (7-day) and serum concentration there is a correlation of r = 0.59, p < 0.0000. The AA-status of highly trained athletes does not differ significantly from the control group in spite of intensive daily training. Thus, AA-supplementation beyond the normal daily intake does not appear necessary.

Measurement of ascorbic acid in human plasma and urine by high-performance liquid chromatography results in healthy subjects and patients with idiopathic calcium urolithiasis

A simple, reliable high-performance liquid chromatographic method was developed to measure ascorbic acid (ASC), with ultraviolet detection (250 nm), in human plasma and urine. Immediately following blood withdrawal, the heparinized plasma samples were deproteinized with 10% m-phosphoric acid, while the freshly voided urine samples were diluted with m-phosphoric acid. ASC was separated on a reversed-phase column by elution with 0.1 M KH 2PO 4 adjusted to pH 2.35. In urine, after reduction of dehydroascorbic acid to ASC, total ASC was measured using the same mobile phase. The method was sensitive down to 0.1 and 0.4 mg ASC per litre of urine and plasma, respectively. In patients with idiopathic calcium urolithiasis, both plasma and urinary ASC were within the range observed in age-matched controls.

Effect of dietary addition of myo-inositol on the metabolic changes in rats exposed to 1,1,1-trichloro-2, 2-bis (P-chlorophenyl) ethane

Feeding of 0,1% 1,1,1-trichloro-2,2-bis (P-chlorophenyl) ethane (DDT)-containing diet for 13–14 days to rats caused a reduction in growth rate, which was significantly ameliorated by dietary addition of myo-inositol. Dietary DDT increased liver weight, liver lipids, serum cholesterol, serum phospholipid, serum copper or ceruloplasmin activity, liver and urinary ascorbic acid and activities of serum glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) as well as liver and serum thiobarbituric acid reactive substances (TBA-RS). The increases in liver weight, liver cholesterol, liver triglyceride, liver total lipids, serum cholesterol and serum GOT activity due to DDT were significantly suppressed by dietary addition of myo-inositol. In addition, dietary myo-inositol caused a decreasing trend in serum phospholipid and serum GPT activity in DDT-fed animals. On the other hand, the increase in urinary ascorbic acid due to DDT was potentiated with dietary myo-inositol. In the animals without receiving DDT, dietary myo-inositol caused no significant effect on these metabolic parameters except serum ceruloplasmin activity, which was decreased by myo-inositol intake. These results suggest that rats fed DDT containing diet may require an exogenous source of myo-inositol.

Vitamin C aspirin interactions in laboratory animals.

Ascorbic acid concentration has been determined in samples of plasma, leucocytes, urine, faeces and adrenal glands of guinea-pigs after administration of (i) 10, 25 or 100 mg ascorbic acid, (ii) 10 mg ascorbic acid plus 10, 25 or 50 mg aspirin and (iii) 25 mg aspirin and 25, 50 or 100 mg ascorbic acid. When the dose of aspirin was 25 mg or more, the transport of ascorbic acid into leucocytes was inhibited, the plasma concentration of vitamin C was elevated significantly and the excretion of ascorbic acid in the urine was increased in direct proportion to the aspirin dose. Ascorbic acid concentration in the adrenal glands was not significantly elevated after 3 h. When a constant dose of 25 mg of aspirin was given along with increasing doses of ascorbic acid both plasma and leucocyte ascorbic acid levels were elevated but not significantly after 2, 3 and 24 h. Urine ascorbic acid levels did not show any changes with the same doses.

High Incidence of Significant Urinary Ascorbic Acid Concentrations in a West Coast Population—Implications for Routine Urinalysis

Examination of 4379 routine urinalysis specimens with dipsticks sensitive to ascorbic acid showed that 22.8% were positive specimens. The mean urinary vitamin C concentration in this population was 2120 mumol/L. There was a high rate of false-negative dipstick results for hemoglobin in patients with vitamin C in the urine. The highest false-negative rates were observed in urine samples containing less than 50 erythrocytes per high-power field. In further experiments when volunteers consumed supplemental oral USP vitamin C at doses of 100, 250, 500, and 1000 mg or vitamin C-containing fruit juices, even the lowest doses of oral vitamin C or juice resulted in sufficient urinary vitamin C to produce false-negative dipstick results in hemoglobin and glucose testing. To prevent potentially dangerous false-negative results, screening urinalysis protocols relying only on dipstick testing should include a check for urinary vitamin C or use a dipstick that is not subject to vitamin C interference.

Determination of ascorbic acid in pharmaceuticals and urine by reverse flow injection.

Two reverse flow injection (FI) methods, using spectrophotometric detection, are proposed for the determination of ascorbic acid. Both methods are based on its reaction with the ethylenediaminetetraacetic acid-CoIII complex in a medium of 5% diethylamine. In the first method, using the peak-height FI technique, ascorbic acid is determined over the range from 2 x 10(-4) to 5 x 10(-3) mol dm-3 and in the second, using the peak-width FI method, the working range is extended (2 x 10(-3)-5 x 10(-2) mol dm-3). Both FI methods were applied to the determination of ascorbic acid in pharmaceuticals while the peak-height FI technique was also used to determine ascorbic acid in urine.

Effect of supplementing methionine and threonine to a non-protein diet on the induction of enzymes by polychlorinated biphenyls in rats.

The N-sparing action of methionine and threonine in rats fed with a non-protein diet seems to indicate that methionine and threonine are the endogenously most-limiting amino acids. The effect of a dietary supplementation of methionine and threonine to a non-protein diet on enzyme induction by polychlorinated biphenyls (PCB) was investigated. Liver microsomal drug-metabolizing enzymes were further induced by the methionine and threonine supplement to the non-protein diet in PCB-receiving rats. Although feeding with PCB did not increase the urinary ascorbic acid excretion in rats of the non-protein diet group, the supplementation of methionine and threonine to the non-protein diet markedly increased this excretion. From these observations, dietary methionine and threonine might promote the synthesis of drug-metabolizing enzymes and the enzymes related to ascorbic acid biosynthesis in PCB-receiving rats.

Abnormalities of ascorbic acid metabolism and diabetic control: differences between diabetic patients and diabetic rats

Ascorbic acid is required in the synthesis of collagen and is also an important anti-oxidant. In a previous study, plasma ascorbic acid concentration was found to be decreased in diabetic patients but there was no relationship with blood glucose level. In the current study of diabetic patients, both plasma ascorbic acid and its urinary excretion correlated inversely with glycosylated hemoglobin level. Plasma ascorbic acid was also lower in diabetic rats but urinary ascorbic acid was elevated. The divergent trend in urinary ascorbic acid excretion observed in diabetic patients and diabetic rats may be due to difference in the ability of these two species to synthesize ascorbic acid. Difference in renal reabsorption of ascorbic acid may also be a relevant factor. The lower plasma and urinary ascorbic acid levels in diabetic patients with more severe hyperglycaemia indicates that this group of patients is particularly at risk of developing deficiency of this vitamin. As ascorbic acid has many important functions in the body, it may be necessary to supplement this vitamin in patients with chronically poorly controlled diabetes.

Ascorbic Acid Metabolism and Polyol Pathway in Diabetes

It has been reported previously that the plasma concentration of ascorbic acid (AA) is reduced in streptozocin-induced diabetic rats and can be normalized by treatment with the aldose reductase inhibitor tolrestat. This study was designed to investigate further the relationship between the polyol pathway and AA metabolism in diabetic rats. Disturbance of AA metabolism was demonstrable after 1 wk of 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 myo-inositol. 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. The interaction of the polyol and AA pathways suggests that this could be another site of action for aldose reductase inhibitors.

Effects of dietary ethanol on ascorbic acid and lipid metabolism, and liver drug-metabolizing enzymes in rats.

Effects of dietary ethanol on ascorbic acid and lipid metabolism, and liver drug-metabolizing enzymes in rats fed a semi-purified diet containing a powdered ethanol preparation (30 cal% in the diet) were studied. Administration of ethanol increased urinary ascorbic acid excretion (p less than 0.001) and ascorbic acid level in the liver (p less than 0.001) and the spleen (p less than 0.01). The activity of hepatic aniline hydroxylase was increased (p less than 0.05) by ethanol feeding but that of aminopyrine N-demethylase was not. Increases of serum total and high-density-lipoprotein (HDL) cholesterol level, commonly observed by the administration of xenobiotics, were not observed. These results showed ethanol possessed rather similar properties to xenobiotics such as polychlorinated biphenyls (PCB) or 1,1,1-trichloro-2,2-bis(p-chlorophenyl)-ethane (DDT) in some metabolic changes. In this study, no accumulation of lipid in the liver was observed.

Urinary Ascorbic Acid Levels Following the Withdrawal of Large Doses of Ascorbic Acid in Guinea Pigs

Male guinea pigs received sodium ascorbate solution [equivalent to 1 g ascorbic acid/(kg body weight.d)] by intraperitoneal injection for 4 wk. During the ascorbic acid treatment period, plasma and urinary ascorbic acid levels rose markedly. Three weeks after the ascorbic acid treatment was withdrawn, mean urinary ascorbic acid levels were significantly lower than their corresponding basal levels. At both 2 and 5 wk after withdrawal of ascorbic acid treatment, mean plasma ascorbic acid levels were below normal. The results indicate that these animals had experienced a transient withdrawal effect after administration of large doses of ascorbic acid that lasted about 1 wk. This, in turn, indicates that the rate of ascorbic acid turnover was probably increased during treatment, and this effect persisted even after the ascorbic acid was withdrawn. Examination of data from each individual experimental animal revealed that the pattern of urinary ascorbic acid excretion after the withdrawal of large doses of ascorbic acid varied from animal to animal. Among the twelve experimental guinea pigs, seven had abnormally low urinary ascorbic acid levels 2-4 wk after the withdrawal of the large doses of ascorbic acid.

Effect of polychlorinated biphenyls on lipids and ascorbic acid metabolism in streptozotocin-induced diabetic rats.

Metabolic changes in lipids, ascorbic acid, and hepatic microsomal cytochrome P-450 by feeding polychlorinated biphenyls (PCB) were investigated in streptozotocin-induced diabetic rats. Streptozotocin (STZ, 60 mg/kg body weight) was injected in Wistar male rats intraperitoneally. Diabetic and non-diabetic rats were fed ad libitum a 20% casein-based control diet or a PCB-containing diet (200 mg/kg diet) for 9 days. Body weight decreased significantly in STZ-induced diabetic rats with or without PCB (groups PD and D, respectively). In rats of group D, urinary ascorbic acid excretion was 15 times higher than that in non-diabetic rats fed a control diet (group C). Dietary PCB caused 30-fold higher urinary ascorbic acid excretion in non-diabetic rats (group P) than that in group C. In group PD, urinary ascorbic acid was nearly 60 times higher than that in group C. Ascorbic acid in liver and kidney was significantly lower in group D than in group C, and it was significantly lower in group PD than in group P. Liver microsomal cytochrome P-450 and b5 were both increased by dietary PCB in group P. Addition increase in these enzymes was observed in diabetic rats by PCB. Serum total cholesterol was 1.8 times higher in group P than in group C. Further increase in serum total cholesterol was observed in group PD. These data suggest that metabolic changes in lipids and ascorbic acid induced by the dietary xenobiotic were magnified in STZ-induced diabetic rats.

Biochemical indices of human vitamin C status

Biochemical indicators of ascorbic acid (AA) status were studied in eleven young adult males fed the same AA deficient diet for 14 wk in a live-in metabolic unit. Supplements of AA were added to the diet to give AA-intake periods of 65 mg/d (2 wk), 5 mg/d (4 wk), 605 mg/d (3 wk), 5 mg/d (4 wk), 605 mg/d (4 d), and 65 mg/d (3 d). Blood plasma, erythrocyte, and leukocyte AA levels all reflected AA intake, however, plasma AA showed less variability than red cell AA levels and was considerably easier to determine than leukocyte AA. Plasma AA values less than 0.40 mg/dL (23 mumol/L) reflected marginal AA status. The daily AA intake calculated to maintain plasma AA levels of at least 0.4 mg/dL (23 mumol/L) in healthy young men was 41 mg. The average AA intake estimated to maximize the total body pool was 138 mg/d. Urine and salivary AA levels were not useful indicators of AA status because urinary AA levels did not discriminate well between adequate and deficient AA intakes and salivary AA levels did not consistently reflect AA intake.

Long-Term Effects of Dietary Polychlorinated Biphenyl and High Level of Vitamin E on Ascorbic Acid and Lipid Metabolism in Rats

Long-term feeding of purified diets containing (per kg diet) 100 mg of polychlorinated biphenyl (PCB) and 1000 mg of vitamin E (RRR-α-tocopheryl acetate) to male Wistar rats was carried out. Rats fed a diet containing PCB rapidly became hypercholesterolemic and maintained high cholesterol levels throughout the 240 d of the experiment. Rats fed a high dietary level of vitamin E plus PCB had higher serum cholesterol and lower liver cholesterol than rats fed a lower level of vitamin E plus PCB. In rats fed PCB, urinary excretion of ascorbic acid was higher than in rats not fed PCB. Urinary ascorbic acid was lower in rats fed high levels of vitamin E plus PCB than in those fed the normal levels of vitamin E plus PCB. Rats fed PCB had lower liver vitamin A storage and higher vitamin A in kidney than rats not fed PCB. This implies that a redistribution of vitamin A occurred in rats fed PCB. Histological observations revealed that central halves of the hepatic lobules of rats fed PCB showed distinct changes consisting of hypertrophy of hepatocytes in the perivenous region and accumulation of vacuoles (lipid droplets) in the cells in the remaining affected portion. Administration of a high dose of vitamin E could not ameliorate this lesion while the treatment depressed effectively the lipid peroxidation. This suggests that the lipid peroxidation was not responsible for the hepatic damage induced by PCB.

Oxidation of Urinary Ascorbic Acid by Ascorbate Oxidase Immobilized onto a Test Tube Wall

Oxidation of Urinary Ascorbic Acid by Ascorbate Oxidase Immobilized onto a Test Tube Wall

Determination of ascorbic acid in human urine by high-performance liquid chromatography coupled with fluorimetry after post-column derivatization with benzamidine

High-performance liquid chromatography on two Asahipak GS-320 hydrophilic gel columns (50 × 0.76 cm I.D.), connected in series, with 0.015 M tartrate buffer (pH 3.0), containing 2 m M ethylenediaminetetraacetate and 0.05% β-thiodiglycol as eluent allowed the separation of glucose, diketogulonic acid + diketogluconic acid, dehydroisoascorbic acid, dehydroascorbic acid, ascorbic acid, and isoascorbic acid within 55 min. Ascorbic acid in a urine sample was stabilized by the addition of an equal volume of 5% metaphosphoric acid solution, containing 0.5% of β-thiodiglycol. Filtration of the mixture through a column of Dowex 50W-X8 (H +) facilatated the determination of ascorbic acid and isoascorbic acid in human urine. Samples could be analyzed every 20 min.

Effects of Dietary Methionine, Cystine and Potassium Sulfate on Serum Cholesterol and Urinary Ascorbic Acid in Rats Fed PCB

Liver weight, liver and urinary ascorbic acid levels and serum cholesterol concentration were higher in rats fed polychlorinated biphenyls (PCB) than in controls. The influences of methionine, cystine and potassium sulfate on these metabolic responses were studied. Methionine or equivalent moles of cystine or potassium sulfate were added to a basal diet containing 10% soy protein isolate. The basal diet contained 0.3% of total sulfur-containing amino acids (S-AAs). When methionine was added to the basal diet, maximum gain in body weight was obtained with 0.5% of dietary S-AAs, while the highest values in serum cholesterol and urinary ascorbic acid were obtained with 0.8% of dietary S-AAs in rats fed PCB. Dietary addition of cystine had little effect on body weight gain. Nevertheless, in rat fed PCB, urinary ascorbic acid and serum cholesterol were significantly higher in rats fed the cystine-supplemented diet than in those fed the unsupplementd diet. Addition of potassium sulfate had no effect on body weight gain, urinary ascorbic acid or serum cholesterol. These results suggest that more S-AAs are required for the highest metabolic response to PCB than for maximum growth, and the higher requirement for S-AAs cannot be replaced by inorganic sulfate.