ascorbic-acid and pyridinoline

Vitamin C is an essential micronutrient. Absence from the diet will result in the deficiency disease scurvy, typically characterised by weakening of collagenous structures. High intakes of vitamin C have been associated with decreased incidence or severity of a number of diseases, including cancer and cardiovascular disease. These beneficial effects may be attributed to its antioxidant properties, although the exact mechanisms of action remain elusive. It is also unclear what intake levels are required for optimal health benefits. The task of defining optimal intakes is hindered by the lack of a reliable functional marker of tissue vitamin C status in man. Many different pathways have been investigated, but none of them have measurable outcome variables relating directly to scorbutic changes. The bone-collagen formation pathway has the potential to provide a functional index of tissue vitamin C adequacy. Vitamin C acts as a cofactor for the enzyme lysyl hydroxylase, which is required for the hydroxylation of lysine residues in procollagen chains. Pyridinoline is a mature collagen cross-link formed from three hydroxylysine residues, deoxypyridinoline is formed from two hydroxylysine and one lysine residue. Guinea-pig studies have shown an alteration in the pyridinium cross-link ratios in response to graded vitamin C intakes (Tsuchiya & Bates, 1998). In order to investigate whether these changes can be seen in a human population group, a study was carried out in rural Gambia, where there is a marked seasonal variation in dietary vitamin C. The present review discusses the rationale behind the study and presents some preliminary results.

Topics: Amino Acids; Antioxidants; Ascorbic Acid; Ascorbic Acid Deficiency; Bone and Bones; Collagen; Humans; Hydroxylation; Seasons

We have investigated the interactions between dietary vitamin C levels (at 33, 79, 135, and 424 mg kg-1 of wet mass feed) and growth rate on the collagen and cross-link contents of fast muscle in farmed juvenile Atlantic salmon (Salmo salar L.). The growth rate was measured over an 11 week period using the thermal growth coefficient (TGC). Alkaline-soluble (0.1 M NaOH) (a-s) hydroxyproline (HYP) and alkaline-insoluble (i-s) HYP were determined as a measure of collagen content and hydroxylysyl pyridinoline (PYD) as a measure of mature collagen cross-link concentration. There was a approximately 5-fold increase in muscle vitamin C concentration at similar feed conversion ratios ( approximately 0.82) as dietary vitamin C levels increased from 39 to 424 mg kg-1 of wet mass feed. However, even the lowest dietary vitamin C was sufficient for normal skeletal development and growth. The lowest dietary vitamin C level tested resulted in a approximately 27% decrease in the a-sHYP concentration relative to the other diets, whereas there was no significant effect of vitamin C on the i-sHYP and PYD concentrations. ANOVA revealed no significant interaction between vitamin C and growth rate, whereas the covariate TGC was significant for i-sHYP and PYD but not for a-sHYP. Pyridinoline cross-link and i-s HYP concentrations were 11.1 and 7.7% lower, respectively, in high (TGC > 3.9) mass than low (TGC < 3.9) growth rate fish. These small differences in collagen cross-linking were associated with a 15.6% decrease in fillet firmness measured with an instrumental texture analyzer. It was concluded that for healthy juvenile salmon reared under controlled growth conditions, the dietary vitamin C inclusion of 79 mg kg-1 of wet mass feed was sufficient to produce the required synthesis of soluble muscle collagen. Furthermore, post-translational modifications of the collagen leading to cross-linking showed a small decrease with increasing growth rate but was independent of vitamin C concentration in the diet at these levels.

Topics: Amino Acids; Animal Nutritional Physiological Phenomena; Animals; Ascorbic Acid; Collagen; Diet; Muscle, Skeletal; Salmon

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.

Topics: Adult; Amino Acids; Ascorbic Acid; Case-Control Studies; Collagen; Disease Susceptibility; Extraembryonic Membranes; Female; Fetal Membranes, Premature Rupture; Humans; Hydroxyproline; Infant, Newborn; Malondialdehyde; Oxidative Stress; Pregnancy; Proteins; Reference Values

Vitamin C (ascorbate) is essential for hydroxylation of prolyl and lysyl residues in nascent collagen, the failure of which leads to connective tissue lesions of scurvy. Of the pyridinium-type cross-links in mature collagen, pyridinoline requires more hydroxylysyl residues than does deoxypyridinoline. Our study tested the hypothesis that pyridinoline:deoxypyridinoline ratios in urinary degradation products may vary with ascorbate status in man. These ratios were compared between British and Gambian prepubertal boys, mean age 8.3 years, and in Gambian boys between two seasons with contrasting ascorbate availability. The mean cross-links ratio in 216 British boys was 4.36 (SD 0.71), significantly greater (P<0.0001) than in sixty-two Gambian boys: 3.83 (SD 0.52). In the Gambians the cross-links ratio was significantly higher in the dry season (with high ascorbate intake and status) than in the rains (with low intake and status). A 7-week controlled intervention was carried out in Gambian boys during the rainy season (the 'hungry' season, when vitamin C-containing foods are virtually unavailable): 100 mg ascorbate/d was given to one group of thirty-two Gambian boys and placebo to another group. The intervention did not, however, significantly alter the cross-link ratio, possibly because the response time and/or intervention-response delay is >7 weeks. If confirmed, the putative association between ascorbate and collagen cross-link ratios in man could become the basis for a functional test for adequacy of ascorbate status.

Topics: Amino Acids; Anthropometry; Ascorbic Acid; Ascorbic Acid Deficiency; Biomarkers; Body Mass Index; Bone and Bones; Child; Child, Preschool; Collagen; Creatinine; Gambia; Humans; Male; Rain; Seasons; United Kingdom

Mild-to-moderate vitamin C depletion in weanling guinea-pigs affects pyridinoline:deoxypyridinoline (collagen cross-link) ratios in femur shaft and urine, attributed to impairment of hydroxylation of collagen lysine. We investigated: (1). whether the picture at two time points is compatible with progressive accumulation of abnormal collagen; (2). whether any changes are seen in skin, where little deoxypyridinoline occurs; (3). whether total food restriction has similar effects. Male weanling Dunkin-Hartley guinea-pigs were fed diets containing either 0.5 (vitamin C-restricted) or 160.0-320.0 (vitamin C-adequate) mg vitamin C/d. Two groups receiving the vitamin C-adequate diet received it ad libitum. Two other groups received the vitamin C-adequate diet in a restricted amount, limited to that which permitted nearly the same growth rate as in the vitamin C-restricted groups. Animals were fed for 4 or 8 weeks; urine was collected, and vitamin C and collagen indices were measured. In the femur shaft, the hydroxyproline content per unit weight was unaffected by vitamin C restriction or by total food restriction. Deoxypyridinoline was increased and the pyridinoline:deoxypyridinoline ratio was decreased in vitamin C-restricted groups, but not in food-restricted groups. Changes in the value of the ratio were greater after 8 than after 4 weeks. Urine indices mirrored bone indices. In skin, the main effect of vitamin C restriction was to reduce hydroxyproline. Here, the cross-link ratios changed less markedly than in bone, and there was less deoxypyridinoline. We conclude that the picture at two time points is compatible with a progressive accumulation of pyridinoline-enriched collagen in vitamin C-deprived animals, that the picture in skin differs from that of bone and urine, and that cross-link changes are not produced by total food restriction.

Topics: Amino Acids; Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Body Weight; Bone and Bones; Collagen; Eating; Food Deprivation; Guinea Pigs; Male; Skin

To clarify the role of L-ascorbic acid (AsA) in the formation of pyridinoline, we examined the effects of AsA in vitro using soluble collagen and partially purified lysyl oxidase from bovine aorta. The concentration of dehydrodihydroxylysinonorleucine decreased when AsA was added in the early stage of pyridinoline formation. However, when AsA was added in a later stage of pyridinoline formation, the concentration of pyridinoline was not affected. These findings indicated that AsA was involved in the initial enzymatic reaction in pyridinoline synthesis. We purified lysyl oxidase to confirm its association of AsA. AsA inhibited the enzyme activity. Erythorbic acid and 3,4-dihydroxybenzoate suppressed the enzyme activity as well as AsA did. The inhibition by AsA of the lysyl oxidase activity arose from characteristics of AsA structure. AsA might be important in the regulation of the oxidative reaction of lysine.

Topics: Amino Acids; Animals; Antioxidants; Aorta, Thoracic; Ascorbic Acid; Cartilage; Cattle; Collagen; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Oxidation-Reduction; Protein-Lysine 6-Oxidase

In order to elucidate the effect of L-ascorbic acid (AsA) on the formation of pyridinoline, a mature crosslink of collagen, its content in cartilage collagen of guinea pigs supplemented with and without AsA in the growing process (4-8 weeks of age) and in the period of maturity (10-14 weeks of age) was examined. The AsA-deficient animals, for four weeks during the growing process, had a significantly higher content of pyridinoline in their collagen than the AsA-supplemented group, indicating that the depletion of AsA induced increasing contents of pyridinoline. On the other hand, in the period of maturity, the pyridinoline content in the collagen decreased with age, whereas no difference between AsA-deficient and -supplemented groups was observed. Based on these results, it is assumed that AsA affects the formation of pyridinoline, especially in the growing period.

Topics: Adrenal Glands; Aging; Amino Acids; Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Cartilage; Collagen; Guinea Pigs; Liver; Male; Weight Gain

Ehlers-Danlos syndrome type VI (EDS VI) is an autosomal recessive disorder of connective tissue characterized by hyperextensible, friable skin and joint hypermobility. Severe scoliosis and ocular fragility are present in some patients. This disease is caused by defective collagen lsyl hydroxylase, a vitamin C-dependent enzyme that converts lysyl residues to hydroxylysine on procollagen peptides. Hydroxylysine is essential for the formation of the covalent pyridinium cross-links pyridinoline (Pyr) and deoxypyridinoline (Dpyr), among mature collagen molecules. Pyr derives from three hydroxylysyl residues, whereas Dpyr derives from one lysyl and two hydroxylysyl residues. Patients with EDS VI have high urinary excretion of Dpyr, resulting in a high ratio of Dpyr-Pyr. In this study, we evaluate content and production of pyridinium cross-links in the skin and cultured fibroblasts from patients with EDS VI. The skin of normal controls contained both Pyr and Dpyr, with a marked predominance of Pyr as observed in normal urine. The skin of patients with EDS VI had reduced total content of pyridinium cross-links, with the presence of Dpyr but not Pyr. Long-term cultures of control fibroblasts produced both Pyr and Dpyr, with a pattern resembling that of normal skin. By contrast, cross-links were not detected in dermal fibroblasts cultured from patients with EDS VI. Vitamin C, which improves the clinical manifestations of some patients with EDS VI, decreased Dpyr accumulation though only minimally affecting Pyr content in control cells. By contrast, addition of vitamin C to fibroblasts from patients with EDS VI stimulated the formation of Dpyr more than that of Pyr and greatly increased total pyridinium cross-link formation. These results indicate that qualitative and quantitative alterations of pyridinium cross-links occur in skin and in cultured dermal fibroblasts of patients with EDS VI and may be responsible for their abnormal skin findings. The vitamin C-stimulated production of Dpyr and Pyr in fibroblasts from patients with EDS VI may explain at least in part the therapeutic effects of this vitamin in EDS VI.

Topics: Adolescent; Amino Acids; Ascorbic Acid; Cells, Cultured; Child; Collagen; Cross-Linking Reagents; Ehlers-Danlos Syndrome; Female; Fibroblasts; Humans; Hydroxylysine; Lysine; Pyridinium Compounds; Skin

The purpose of this study was, first to explore metabolic interactions between Cu and ascorbic acid in guinea-pigs, particularly with respect to any possible disadvantages of high ascorbate in the presence of low Cu intakes, and second, to test the hypothesis that variations in ascorbate and/or Cu status might influence collagen cross-linking, either by inducing a change in the crosslinks: hydroxyproline ratio, or by inducing a change in the pyridinoline: deoxypyridinoline crosslinks ratio. Four matched groups, each of eight male weanling Dunkin-Hartley guinea-pigs, were maintained on purified diets containing either no added Cu, or 150 mg Cu/kg diet, and either 0.1 g or 30 g ascorbic acid/kg diet. They were then killed 8 weeks later, and the following indices were measured: body and organ weights; blood haemoglobin; adrenal ascorbate concentrations; Cu concentrations in plasma, liver and femur; superoxide dismutase (EC 1.15.1.1) activity in whole blood and liver; hydroxyproline, pyridinoline and deoxypridinoline in femur and in urine. The principal observations were: Cu intake significantly affected blood and tissue Cu concentrations and superoxide dismutase activity; and ascorbic acid intake significantly affected adrenal ascorbate levels and the deoxypyridinoline: pyridinoline cross-links ratio, especially in bone (femur). There was evidence of a significant interaction between ascorbate and Cu with respect to adrenal and plasma Cu concentrations, blood superoxide dismutase activity and body weights. We conclude that interactions between ascorbate and Cu at the functional level were present but modest, and that a new and potentially powerful functional index of ascorbate status may exist within the deoxypyridinoline:pyridinoline collagen cross-link ratio.

Topics: Adrenal Glands; Amino Acids; Analysis of Variance; Animals; Ascorbic Acid; Bone and Bones; Copper; Diet; Guinea Pigs; Hemoglobins; Liver; Male; Superoxide Dismutase

Age-related changes of pyridinoline, a mature cross-link of collagen fibers, in tissues of guinea pigs which cannot synthesize L-ascorbic acid (AsA) were investigated. Male guinea pigs, 2 weeks old, were fed a commercial diet until they were 40 weeks old. Based on the data of body weight gain, it is assumed that guinea pigs require 10 weeks to reach maturity. The content of AsA in serum, adrenals, and liver of the animals increased with growth, then decreased after 8-10 weeks. The pyridinoline content in cartilage and tendon collagen was very low in 2-week-old animals, whereas it increased markedly with growth. After 10 weeks, the pyridinoline tended to decrease in cartilage collagen; however, it was not changed in tendon collagen. The age-related changes of pyridinoline content in guinea pigs cartilage were similar to those of humans--increasing with growth and decreasing after adolescence.

Topics: Achilles Tendon; Adrenal Glands; Aging; Amino Acids; Animals; Ascorbic Acid; Cartilage; Collagen; Guinea Pigs; Heart; Liver; Lung; Male; Myocardium; Organ Size