Calcif Tissue Int 1996 Nov;59(5):352-6
Jie KG, Bots ML, Vermeer C, Witteman JC, Grobbee DE
Department of Biochemistry and Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

Gammacarboxyglutamate (Gla) is an uncommon amino acid formed by vitamin K action. Increasing evidence indicates that Gla-proteins are involved in the regulation of calcification processes in both bone tissue and atherosclerotic vessel wall. In a population-based study we have previously shown that in a group of 113< postmenopausal women the presence of abdominal aortic calcifications is associated with a reduced vitamin K status. In the present study we investigated whether this reduced vitamin K status was also associated with differences in bone mass or circulating calciotropic hormone levels. Serum immunoreactive osteocalcin with low affinity for hydroxyapatite (irOCfree) was used as a marker for vitamin K status. After correction for age it was found that women with atherosclerotic calcifications had a 7% lower bone mass as measured by metacarpal radiogrammetry (mean difference: 3.2 mm2, 95% CI: -0. 2-6.5, P = 0.06). No differences between both groups of women were observed for serum intact parathyroid hormone (PTH) and serum 25-hydroxyvitamin D levels. In the atherosclerotic women (n = 34), markers for vitamin K status were inversely associated with bone mass (r = -0.47, P = 0.013), whereas no such association was found in the nonatherosclerotic women (n = 79). It is concluded that the atherosclerotic women in this study may be at higher risk for osteoporotic fractures as evidenced by their lower bone mass and higher serum irOCfree levels. The finding that in atherosclerotic women vitamin K status is associated with bone mass supports our hypothesis that vitamin K status affects the mineralization processes in both bone and in atherosclerotic plaques.

High levels of plasma homocyst(e)ine (H[e]) are associated with increased vascular risk. Treatment is being contemplated, but the level at which patients should be treated is not known. We compared the response of carotid plaque to vitamin therapy in patients with H(e) above and below 14 micromol/L, a level commonly regarded as high enough to warrant treatment. Two-dimensional B-mode ultrasound measurement of carotid plaque was used to assess the response to vitamin therapy with folic acid 2.5 mg, pyridoxine 25 mg, and cyanocobalamin 250 microg daily, in 101 patients with vascular disease (51 with initial plasma levels above, and 50 below 14 micromol/L). Among patients with plasma H(e) >14 micromol/L, the rate of progression of plaque area was 0.21 +/- 0.41 cm2/year before vitamin therapy, and -0.049 +/- 0.24 cm2/year after vitamin therapy (P2 = .0001; paired t test). Among patients with levels <14 micromol/L, the rate of progression of plaque was 0.13 +/- 0.24 cm2/year before vitamin therapy and -0.024 +/- 0.29 cm2/year after vitamin therapy (P2 = .022, paired t test). The change in rate of progression was -0.15 +/- .44 cm2/year below 14 micromol/L, and -0.265 +/- 0.46 cm2/year above 14 micromol/L (P = 0.20). Vitamin therapy regresses carotid plaque in patients with H(e) levels both above and below 14 micromol/L. These observations support a causal relationship between homocyst(e)ine and atherosclerosis and, taken with epidemiologic evidence, suggest that in patients with vascular disease, the level to treat may be <9 micromol/L.

BMJ 1995 Jun 17;310(6994):1559-63
Khaw KT, Woodhouse P Clinical Gerontology Unit, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital.

OBJECTIVE--To examine the hypothesis that the increase in fibrinogen concentration and respiratory infections in winter is related to seasonal variations in VITAMIN C status (assessed with serum ascorbate concentration). DESIGN--Longitudinal study of individuals seen at intervals of two months over one year. SETTING--Cambridge. SUBJECTS--96 men and women aged 65-74 years living in their own homes. MAIN OUTCOME MEASURES--Haemostatic factors fibrinogen and factor VIIC; acute phase proteins; respiratory symptoms; respiratory function. RESULTS--Mean dietary intake of VITAMIN C varied from about 65 mg/24 h in winter to 90 mg/24 h in summer; mean serum ascorbate concentration ranged from 50 mumol/l in winter to 60 mumol/l in summer. Serum ascorbate concentration was strongly inversely related to haemostatic factors fibrinogen and factor VIIC as well as to acute phase proteins but not to self reported respiratory symptoms or neutrophil count. Serum ascorbate concentration was also related positively to forced expiratory volume in one second. An increase in dietary VITAMIN C of 60 mg daily (about one orange) was associated with a decrease in fibrinogen concentrations of 0.15 g/l, equivalent (according to prospective studies) to a decline of approximately 10% in risk of ischaemic heart disease. CONCLUSION--High intake of VITAMIN C has been suggested as being protective both for respiratory infection and for cardiovascular disease. These findings support the hypothesis that VITAMIN C may protect against cardiovascular disease through an effect on haemostatic factors at least partly through the response to infection; this may have implications both for our understanding of the pathogenetic mechanisms in respiratory and cardiovascular disease and for the prevention of such conditions. Comments: Comment in: BMJ 1995 Jun 17;310(6994):1548-9 PMID: 7787643, UI: 95307332

BACKGROUND: Loss of endothelium-derived nitric oxide (EDNO) contributes to the clinical expression of coronary artery disease (CAD). Increased oxidative stress has been linked to impaired endothelial vasomotor function in atherosclerosis, and recent studies demonstrated that short-term ascorbic acid treatment improves endothelial function. METHODS AND RESULTS: In a randomized, double-blind, placebo-controlled study, we examined the effects of single-dose (2 g PO) and long-term (500 mg/d) ascorbic acid treatment on EDNO-dependent flow-mediated
dilation of the brachial artery in patients with angiographically established CAD. Flow-mediated dilation was examined by high-resolution vascular ultrasound at baseline, 2 hours after the single dose, and 30 days after long-term treatment in 46 patients with CAD. Flow-mediated dilation improved from 6.6+/-3.5% to 10.1+/-5.2% after single-dose treatment, and the effect was sustained after long-term treatment (9. 0+/-3.7%), whereas flow-mediated dilation was 8.6+/-4.7% at baseline and remained unchanged after single-dose (7.8+/-4.4%) and long-term (7.9+/-4.5%) treatment with placebo (P=0.005 by repeated-measures ANOVA). Plasma ascorbic acid concentrations increased from 41.4+/-12. 9 to 115.9+/-34.2 micromol/L after single-dose treatment and to 95. 0+/-36.1 micromol/L after long-term treatment (P<0.001). CONCLUSIONS: In
patients with CAD, long-term ascorbic acid treatment has a sustained beneficial effect on EDNO action. Because endothelial dysfunction may contribute to the pathogenesis of cardiovascular events, this study indicates that ascorbic acid treatment may benefit patients with CAD.

BACKGROUND: Hyperhomocysteinemia is a major and independent risk factor for vascular disease. The mechanisms by which homocysteine promotes atherosclerosis are not well understood. We hypothesized that elevated homocysteine concentrations are associated with rapid onset endothelial dysfunction, which is mediated through oxidant stress mechanisms and can be inhibited by the antioxidant vitamin c. Methods and RESULTS: We studied 17 healthy volunteers (10 male and 7 female) aged 33 (range 21 to 59) years. Brachial artery diameter responses to hyperemic flow (endothelium dependent), and glyceryltrinitrate (GTN, endothelium independent) were measured with high resolution ultrasound at 0 hours (fasting), 2 hours, and 4 hours after (1) oral methionine (L-methionine 100 mg/kg), (2) oral methionine preceded by vitamin c (1g/day, for 1 week), and (3) placebo, on separate days and in random order. Plasma homocysteine increased (0 hours, 12.8+/-1.4; 2 hours, 25.4+/-2.5; and 4 hours, 31. 2+/-3.1 micromol/l, P<0.001), and flow-mediated dilatation fell (0 hours, 4.3+/-0.7; 2 hours, 1.1+/-0.9; and 4 hours, -0.7+/-0.8%) after oral L-methionine. There was an inverse linear relationship between homocysteine concentration and flow-mediated dilatation (P<0. 001). Pretreatment with vitamin c did not affect the rise in homocysteine concentrations after methionine (0 hours, 13.6+/-1.6; 2 hours, 28.3+/-2.9; and 4 hours, 33.8+/-3.7 micromol/l, P=0.27), but did ameliorate the reduction in flow-mediated dilatation (0 hours, 4. 0+/-1.0; 2 hours, 3.5+/-1.2 and 4 hours, 2.8+/-0.7%, P=0.02). GTN-induced endothelium independent brachial artery dilatation was not affected after methionine or methionine preceded by vitamin c. CONCLUSIONS: We conclude that an elevation in homocysteine concentration is associated with an acute impairment of vascular endothelial function that can be prevented by pretreatment with vitamin c in healthy subjects. Our results support the hypothesis that the adverse effects of homocysteine on vascular endothelial cells are mediated through oxidative stress mechanisms.

Atherosclerosis is associated with stiffening of conduit arteries and increased platelet activation, partly as a result of reduced bioavailability of nitric oxide (NO), a mediator that normally has a variety of protective effects on blood vessels and platelets. Increased levels of oxygen free radicals are a feature of atherosclerosis that contributes to reduced NO bioavailability and might lead to increased arterial stiffness and platelet activation. Vitamin C is a dietary antioxidant that inactivates oxygen free radicals. This placebo-controlled, double-blind, randomized study was designed to establish whether acute oral administration of vitamin C (2 g), would reduce arterial stiffness and in vitro platelet aggregation in healthy male volunteers. Plasma vitamin C concentrations increased from 42+/-8 to 104+/-8 microM at 6 h after oral administration, and were associated with a significant reduction in augmentation index, a measure of arterial stiffness (by 9.6+/-3.0%; p = 0.016), and ADP-induced platelet aggregation (by 35+/-13%; p = 0.046). There was no change in these parameters after placebo. Vitamin C, therefore, appears to have beneficial effects, even in healthy subjects. The mechanism responsible is likely to involve protection of NO from inactivation by oxygen free radicals, but this requires confirmation. If similar effects are observed in patients with atherosclerosis or risk factors, vitamin C supplementation might prove an effective therapy in cardiovascular disease.

AIMS: There is evidence that formation of free radicals increases in patients with hypertension or hypercholesterolaemia, which may contribute to endothelial dysfunction of epicardial coronary arteries due to inactivation of the vasodilator NO. The present study was designed to test whether the abnormal constriction of epicardial coronary arteries due to sympathetic stimulation by the cold pressor test in patients with essential hypertension or hypercholesterolaemia could be reversed by administration of the antioxidant vitamin C. METHODS and RESULTS: In 28 patients without relevant coronary artery stenosis the cold pressor test was performed before and after a 3 g infusion of vitamin C. In five normal controls the cold pressor test led to a similar increase in luminal area before and after vitamin C (3.7+/-1.3% and 1.9+/-0.8%, ns vs before vitamin C). In nine hypercholesterolaemic patients the cold pressor test led to a -14.1+/-2.8% reduction in cross-sectional area before vitamin C. This constriction was significantly improved after vitamin C to -7.6%+/-2.0, P=0.027 vs before vitamin C. In nine hypertensive patients, the cold pressor test led to a -17.1+/-3.2% decrease in cross-sectional area before vitamin C, which was improved to -7.1+/-3.1 after vitamin C, P=0.004 vs before vitamin C. This increase in luminal area was significant in each group in comparison with normal controls (each P<0.05). Administration of saline (placebo group, five patients) had no significant effect on cold pressor test-induced constriction (-6.9+/-3.9% before and -6. 8+/-3.7% after saline). CONCLUSION: The antioxidant vitamin C reverses cold pressor test-induced vasoconstriction of epicardial coronary arteries in patients with hypertension or hypercholesterolaemia. Our data suggest that enhanced oxidative stress contributes to impaired endothelial function in this patient population.

CONTEXT: Increased levels of homocysteine are associated with risk of cardiovascular disease. Homocysteine may cause this risk by impairing endothelial cell function. OBJECTIVE: To evaluate the effect of acute hyperhomocysteinemia with and without antioxidant vitamin pretreatment on cardiovascular risk factors and endothelial functions. DESIGN AND SETTING: Observer-blinded, randomized crossover study conducted at a university hospital in Italy. SUBJECTS: Twenty healthy hospital staff volunteers (10 men, 10 women) aged 25 to 45 years. INTERVENTIONS: Subjects were given each of 3 loads in random order at 1-week intervals: oral methionine, 100 mg/kg in fruit juice; the same methionine load immediately following ingestion of antioxidant vitamin E, 800 IU, and ascorbic acid, 1000 mg; and methionine-free fruit juice (placebo). Ten of the 20 subjects also ingested a placebo load with vitamins. MAIN OUTCOME MEASURES: Lipid, coagulation, glucose, and circulating adhesion molecule parameters, blood pressure, and endothelial functions as assessed by hemodynamic and rheologic responses to L-arginine, evaluated at baseline and 4 hours following ingestion of the loads. RESULTS: The oral methionine load increased mean (SD) plasma homocysteine level from 10.5 (3.8) micromol/L at baseline to 27.1 (6.7) micromol/L at 4 hours (P<.001). A similar increase was observed with the same load plus vitamins (10.0 [4.0] to 22.7 [7.8] micromol/L; P<.001) but no significant increase was observed with placebo (10.1 [3.7] to 10.4 [3.2] micromol/L; P=.75). Coagulation and circulating adhesion molecule levels significantly increased after methionine ingestion alone (P<.05) but not after placebo or methionine ingestion with vitamins. While the mean (SD) blood pressure (-7.0% [2.7%]; P<.001), platelet aggregation response to adenosine diphosphate (-11.4% [4.5%]; P=.009) and blood viscosity (-3.0% [1.2%]; P=.04) declined in these parameters 10 minutes after an L-arginine load (3 g) following placebo, the increase after methionine alone (-2.3% [1.5%], 4.0% [3.0%], and 1.5% [1.0%], respectively; P<.05), did not occur following methionine load with vitamin pretreatment (-6.3% [2.5%], -7.9% [3.5%], and -1.5% [1.0%], respectively; P=.24). CONCLUSION: Our data suggest that mild to moderate elevations of plasma homocysteine levels in healthy subjects activate coagulation, modify the adhesive properties of endothelium, and impair the vascular responses to L-arginine. Pretreatment with antioxidant vitamin E and ascorbic acid blocks the effects of hyperhomocysteinemia, suggesting an oxidative mechanism.