The role of ascorbic acid in the biosynthesis of collagen. II. Site and nature of ascorbic acid participation.
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Leucocyte ascorbic acid was measured in 79 pregnant women in the 34th to 38th week of gestation and on the first, and third or fifth days post partum. No differences were found for time of year or age of the subjects. There was a significantly lower vitamin C status in social classes IV and V. The leucocyte ascorbic acid concentration fell significantly on the first day post partum and rose again to initial values by the fifth day. In late pregnancy 40 per cent of all subjects had leucocyte ascorbic acid concentrations less than the lower limit of normal (100 nmol/10(8) leucocytes). This rose to 75 per cent on the first day post partum.
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An ascorbic acid tolerance test is described for assessing vitamin C status. The test is simple to administer and suitable for elderly patients. It involves giving an oral load of 1 g ascorbic acid in water and then measuring urinary excretion of vitamin C over the next 6 h. The excretion pattern at dosing has been studied in ten young subjects. The result of the ascorbic acid tolerance test in these young subjects was significantly different after supplementation with 1 g ascorbic acid daily for 1 month. Two series of elderly patients were also studied with the ascorbic acid tolerance test. They had low initial plasma ascorbic acid levels and much less vitamin C was excreted in the urine after dosing. Seven of these elderly patients were then supplemented with 1 g ascorbic acid for 1 month. After supplementation the initial plasma levels and their response to the ascorbic acid tolerance test became similar to that seen in younger subjects.
Fluorescence and non-enzymatic browning were observed in reactions between ascorbic acid (AH2) and amino acids (AA) as well as in reactions involving AH2 autoxidation and/or polymerization in the presence of trace amounts of adventitious iron (less than or equal to 10 microM). These reaction products exhibited fluorescent spectra (400-490 nm) akin to those of extracts from lipofuscin-rich tissues. Lengthy incubation of AH2 at 37 degrees C in phosphate buffer (pH 7.0) caused a concentration- and time-dependent increase in absorbance at 412 nm, paralleling increase in 377/440 nm fluorescence, due to formation of autoxidation/polymerization products. The fluorescences of these substances were increased by acidity and quenched at alkaline conditions but restored by neutralization. The reactions between AH2 (0.1-2.0 mM) and a number of AA (1.0-4.0 mM) were also found to result in products with blue fluorescence. Following TBA test, the AH2 autoxidation/polymerization products and AH2/AA reaction products showed only moderate and slight absorbance at 535 nm, respectively, indicating a little or minute formation of aldehydes with MDA-like reactivity. The findings in this study, nevertheless, suggest possible misinterpretations of results in previous studies dealing with AH2-dependent, oxygen free radical induced, 'lipofuscin-related fluorescence'. Thus, similar to nonenzymatic glycosylation (Maillard) reactions, AH2 autoxidation as well as reactions between AH2 and AA may result in 'lipofuscin-like material', as judged from their fluorescence spectral patterns.
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The effect of ascorbic acid deficiency on the urinary excretion of nitrate was investigated using a mutant strain of rats (osteogenic disorder syndrome rats; ODS rats) unable to synthesize ascorbic acid. The amount of urinary nitrate excreted by ODS rats with or without ascorbic acid supplementation were measured before and after the intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS). Urinary nitrate excretion increased markedly after LPS injection. Urinary nitrate excretion by ODS rats not supplied with ascorbic acid was significantly less than that of those supplied with ascorbic acid both before and after LPS injection. These results show that ascorbic acid enhances both LPS-stimulated and constitutive nitrate production in vivo.
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1. Glucuronic acid was excreted in the urine of the brushtail possum, Trichosurus vulpecula, in response to dietary terpenes (essential oils found in Eucalyptus foliage). 2. The relationship between urinary glucuronic acid and the estimated terpene content of the diet was not equimolar, but varied from 1-17%, with levels of 5-39% found in animals maintained on terpenes or eucalypt leaves for several weeks. 3. Blood levels of ascorbate and the turnover rate of ascorbate were increased in the brushtail possum in response to dietary terpenes. 4. The presence of an active glucuronic acid pathway and associated glucose-ascorbate-glucose cycle was postulated for the brushtail possum and the other arboreal marsupials, Pseudocheirus peregrinus (common ringtail possum) and Petauroides volans (greater glider). 5. By means of these pathways these animals may utilise the high ascorbate content of Eucalyptus leaves to conserve glucose required for synthesis of glucuronic acid used for conjugation of dietary terpenes.
Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbic acid and dehydroascorbic acid (DHAA) were measured by HPLC in the striatum of rats whose fronto-parietal cortex had been unilaterally ablated after a single injection of apomorphine (1 mg/kg s.c.), scopolamine (0.6 mg/kg s.c.) or L-glutamate (500 mg/kg i.p.). Unilateral cortical ablation decreased striatal levels of glutamate in both striata ipsilateral (35%) and contralateral (17-25%) to the lesion. Apomorphine and scopolamine significantly increased (+94 and +122%, respectively) the DHAA/ascorbic acid ratio in the striata ipsilateral to the lesion in unoperated and sham-operated rats (+72 and +34%, respectively), but both drugs failed to increase it in ablated rats. L-Glutamate significantly increased the DHAA/ascorbic acid ratio in unoperated (+53%) and ablated rats (+37%). The increase in sham-operated rats (+34%) did not reach statistical significance. Apomorphine and scopolamine significantly decreased the DOPAC/DA ratio in the striata ipsilateral to the lesion of unoperated, sham-operated and ablated rats. The decrease in the DOPAC/DA ratio induced by apomorphine and scopolamine was greater in ablated rats than in sham-operated rats. L-Glutamate induced only minor changes in striatal DA and DOPAC levels. We conclude that the apomorphine- and scopolamine-induced increase in ascorbic acid oxidation in the striatum requires intact cortico-striatal glutamatergic pathways. Cortical ablation potentiates the apomorphine- and scopolamine-induced inhibition of striatal DA turnover.
The concentrations of total ascorbic acid, reduced and oxidized forms of ascorbic acid, the ESR intensity of ascorbate radical, and the ratio of oxidized form of ascorbic acid to total ascorbic acid (DAsA/AsA) were estimated on 217 healthy controls, whose ages ranged from 12 to 96 years, in order to examine influences of sex and age. The concentration of total ascorbic acid was higher in females than in males throughout all age classes, but the oxidized form did not show a sex difference. Then it was found that the reduced form was higher in females than in males throughout all age classes. The concentrations of total ascorbic acid and reduced and oxidized forms of ascorbic acid, and the ESR intensity declined with age, but the DAsA/AsA ratio increased with age.
The stromal cell line ST2, derived from mouse bone marrow, differentiated into osteoblast-like cells in response to ascorbic acid. Ascorbic acid induced alkaline phosphatase (ALPase) activity, the expression of mRNAs for proteins that are markers of osteoblastic differentiation, the deposition of calcium, and the formation of mineralized nodules by ST2 cells. We investigated the mechanism whereby ascorbic acid induced the differentiation of ST2 cells. Inhibitors of the formation of collagen triple helices completely blocked the effects of ascorbic acid on ST2 cells, an indication that matrix formation by type I collagen is essential for the induction of osteoblastic differentiation of ST2 cells by ascorbic acid. We furthermore examined the effects of bone morphogenetic proteins (BMPs) on the differentiation of ST2 cells induced by ascorbic acid. Ascorbic acid had no effect on the expression of mRNAs for BMP-4 and the BMP receptors. However, a soluble form of BMP receptor IA inhibited the induction of ALPase activity by ascorbic acid. These results suggest that ascorbic acid might promote the differentiation of ST2 cells into osteoblast-like cells by inducing the formation of a matrix of type I collagen, with subsequent activation of the signaling pathways that involve BMPs.
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Ascorbate oxidase from zucchini squash was immobilized onto CH-Sepharose via carbodiimide. The properties of the immobilized enzyme were found to be similar to those of the free ascorbate oxidase. The immobilized enzyme was utilized in a flow-through system equipped with a polarographic detector which monitors the oxygen depletion due to the reaction ascorbic acid + 1/2 O2----dehydroascorbic acid + H2O. This method, the response of which is linear between 3 X 10(-7) and 5 X 10(-4) M ascorbate, was utilized to measure the ascorbic acid in biological samples such as human plasma and fruit juices at a rate of about 60 determinations every hour with a standard deviation lower than 5%.
Marmosets can tolerate an ascorbic acid (AA) deficiency for several weeks without clinical symptoms. After being fed an AA-free diet for 3 months, nonspecific deficiency symptoms became obvious. Different dietary levels of AA resulted in corresponding serum ascorbate levels. The kidney threshold of AA in marmosets is comparable to that in humans. When the minimal AA requirement is defined as the amount that is necessary to maintain a serum AA level above the kidney threshold, then about 20 mg AA/kg body weight is needed. This intake was achieved in our trial with a diet containing 500 ppm AA. Thus, the AA requirement of marmosets is severalfold higher than the AA requirement of humans.
Ascorbic acid uptake in AtT-20 tumor cells and primary cultures of rat anterior and intermediate pituitary was sodium-dependent and showed half-maximal saturation between 9 and 18 microM ascorbate. When incubated in [14C]ascorbic acid at concentrations similar to those in serum (50 microM), all of the cells concentrated ascorbate 20- to 40-fold, producing intracellular ascorbate concentrations of 1-2 mM. HPLC analyses showed that over 90% of the intracellular label comigrated with authentic ascorbic acid. Although ascorbate was rapidly oxidized in culture medium in the absence of cells, incubation of ascorbate in culture medium in the presence of cells stabilized the ascorbate substantially. Unlike systems that transport dehydroascorbic acid, the ascorbate transport systems in all three preparations were not inhibited by glucose. Thus all three systems possess similar saturable, high-affinity, sodium-dependent active transport systems for ascorbic acid.