Estimated individual lifetime benefit from PCSK9 inhibition in statin-treated patients with coronary artery disease.

OBJECTIVE: In statin-treated patients with stable coronary artery disease (CAD), residual risk of cardiovascular events is partly explained by plasma levels of low-density lipoprotein cholesterol (LDL-C). This study aimed to estimate individual benefit of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition in CAD patients already treated with high-dose statin. METHODS: Individual lifetime benefit was estimated in months gain free of stroke or myocardial infarction (MI) until age 80 years. Predictions were based on two competing risk models developed in data from 4853 patients with CAD originating from the atorvastatin 80 mg arm of the Treating to New Targets (TNT) trial. The relative effect of PCSK9 inhibition was added to the models and was assumed based on average estimates from large clinical trials. We accounted for individual LDL-C levels, assuming 50% LDL-C reduction by PCSK9 inhibition and 21% cardiovascular risk reduction per mmol/L (39 mg/dL) LDL-C lowering. RESULTS: Estimated individual gain was <6 months in 61% of the patients, 6-12 months in 28% of the patients and ≥12 months in 10% of the patients (median 5, quartiles 2-8 months). Highest estimated benefit was observed in younger patients (aged 40-60 years) with high risk factor burden, particularly if LDL-C levels were >1.8 mmol/L (>70 mg/dL). Estimated benefit was lowest (≤5 months) in older patients (≥70 years), in particular if LDL-C and other risk factors levels were low. CONCLUSION: The individual estimated lifetime benefit from PCSK9 inhibition in patients with stable CAD on high-dose statin varied from <6 to ≥12 months free of stroke or MI. Highest benefit is expected in younger patients (age 40-60 years) with high risk factor burden and relatively high LDL-C levels. TRIAL REGISTRATION NUMBER: NCT00327691; Post-results.

Red wine polyphenols do not improve obesity-associated insulin resistance: A randomized controlled trial.

Preclinical studies have suggested that polyphenols extracted from red wine (RWPs) favourably affect insulin sensitivity, but there is controversy over whether RWPs exert similar effects in humans. The aim of the present study was to determine whether RWPs improve insulin sensitivity in obese volunteers. Obese (body mass index >30 kg/m2 ) volunteers were randomly allocated to RWPs 600 mg/d (n = 14) or matched placebo (n = 15) in a double-blind parallel-arm study for 8 weeks. The participants were investigated at baseline and at the end of the study. Insulin sensitivity was determined using a hyperinsulinaemic-euglycaemic clamp (M-value), a mixed-meal test (Matsuda index), and homeostatic model assessment of insulin resistance (HOMA-IR). RWPs elicited no significant changes in M-value (RWP group: median [interquartile range; IQR] baseline 3.0 [2.4; 3.6]; end of study 3.3 [2.4; 4.8] vs placebo group: median [IQR] baseline 3.4 [2.8; 4.4]; end of study 2.9 [2.8; 5.9] mg/kg/min; P = .65), in Matsuda index (RWP group: median [IQR] baseline 3.3 [2.2; 4.8]; end of study 3.6 [2.4; 4.8] vs placebo group: median [IQR] baseline 4.0 [3.0; 6.0]; end of study 4.0 [3.0; 5.2]; P = .88), or in HOMA-IR. This study showed that 8 weeks of RWP supplementation did not improve insulin sensitivity in 29 obese volunteers. Our findings were not consistent with the hypothesis that RWPs ameliorate insulin resistance in human obesity.

Folic Acid Impairs the Uptake of 5-Methyltetrahydrofolate in Human Umbilical Vascular Endothelial Cells.

BACKGROUND: Adequate folate status supports endothelial structure and function. Folic acid (FA), an oxidized synthetic folate, which is present in the plasma of patients consuming fortified food or FA supplements, may impair cellular uptake of physiological, reduced folates. We studied the effect of FA on uptake of the dominant circulatory folate, 5-methyltetrahydrofolate (5MTHF) in endothelial cells. METHODS AND RESULTS: For short-term effects of FA, primary human umbilical vein endothelial cells (HUVECs) were maintained in growth medium containing 200 nM 5MTHF and preincubated with 20 nM FA 10 minutes before the 5MTHF uptake assessment. For long-term effects, HUVECs were cultured for 3 passages in growth medium containing either 200 nM 5MTHF, or a combination of 100 nM 5MTHF and 100 nM FA. 5MTHF uptake was assessed after exposing cells to 200 nM [C5]-5MTHF, after which intracellular [C5]-5MTHF was quantified using liquid chromatography/tandem mass spectrometry. Acute FA exposure caused a 57% reduction in 5MTHF uptake compared with control conditions (51 ± 12 vs. 22 ± 7 fmol·min·mg protein; P = 0.01). Long-term exposure to FA reduced 5MTHF uptake by 41% (51 ± 12 vs. 30 ± 11 fmol·min·mg protein; P = 0.05) and reduced total cellular 5MTHF levels by 47 ± 21% in HUVEC (P = 0.02). CONCLUSION: Unmetabolized FA, which appears in the plasma after consumption of fortified food or FA supplements, may impair uptake of 5MTHF, the dominant bioactive form of folate, in HUVEC.

Effect of vitamin B12 and folic acid supplementation on biomarkers of endothelial function and inflammation among elderly individuals with hyperhomocysteinemia.

B-vitamin trials failed to demonstrate beneficial effects on cardiovascular outcomes, but hyperhomocysteinemia still stands out as an independent cardiovascular risk factor, particularly in elderly individuals. B-vitamins may influence early vascular dysfunction, such as endothelial dysfunction, or may have adverse effects, for example on inflammation. We investigated the effect of B-vitamins on endothelial function and inflammation within an interventional study. This study was conducted within the framework of the B-PROOF trial, which included 2919 hyperhomocysteinemic elderly individuals, who received daily vitamin B12 (500 µg) and folic acid (400 µg) or placebo for 2 years. Using an electrochemiluminescence platform, we measured intercellular adhesion molecule 1 (ICAM-1), vascular adhesion molecule 1 (VCAM-1), serum amyloid A (SAA), vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) at baseline and follow-up in a subsample of 522 participants (271 intervention group; 251 placebo). Treatment effects were analyzed with ANCOVA. The participants had a mean age of 72 years, and 55% of them were male. At the 2-year follow-up, B-vitamins did not change the ICAM-1 (+36% change in the intervention group versus +32% change in the placebo group; p = 0.72), VCAM-1 (+27% vs +25%; p = 0.39), VEGF (-1% vs +4%; p = 0.40), SAA (+34% vs +38%; p = 0.85) or CRP levels (+26% vs +36%; p = 0.70) as compared to placebo. In conclusion, in elderly patients with hyperhomocysteinemia, vitamin B12 and folic acid are unlikely to influence either endothelial function or low-grade systemic inflammation. ClinicalTrials.gov Identifier: NCT00696514.

Effects of 2-year vitamin B12 and folic acid supplementation in hyperhomocysteinemic elderly on arterial stiffness and cardiovascular outcomes within the B-PROOF trial.

INTRODUCTION: Hyperhomocysteinemia is an important cardiovascular risk indicator in the oldest old, and is associated with elevated arterial stiffness in this age group. Since several intervention trials reported a lack of benefit of B-vitamin supplementation on cardiovascular outcomes, we aimed to investigate the effect of B-vitamin supplementation on arterial stiffness and atherosclerosis in hyperhomocysteinemic elderly patients. METHODS: The B-PROOF study is a double-blind, randomized controlled trial, including 2919 elderly aged at least 65 years, with hyperhomocysteinemia (12-50  µmol/l), treated with B-vitamins (500  µg vitamin B12 and 400  µg folic acid) or placebo for 2 years. In a subgroup (n = 569), the effect of B-vitamins on pulse wave velocity (PWV) was investigated as a measurement of arterial stiffness. To measure atherosclerosis, carotid intima-media thickness (IMT) measures had been used. Incidents of cardiovascular and cerebrovascular events were determined via structured questionnaires, and blood pressure was also measured. RESULTS: Compared to placebo, B-vitamin supplementation lowered serum homocysteine by 3.6  µmol/l (P < 0.001). Analysis of covariance showed no effect of supplementation on PWV levels, and this was not different for patients without increased arterial stiffness at baseline. Furthermore, no effect on carotid IMT was observed. DISCUSSION: Vitamin B12 and folic acid supplementation in hyperhomocysteinemic elderly patients have no effect on PWV or carotid IMT. Further research will still be necessary to unravel the effects and pathways of homocysteine-lowering treatment on cardiovascular outcomes.

Folic acid supplementation does not reduce intracellular homocysteine, and may disturb intracellular one-carbon metabolism.

BACKGROUND: In randomized trails, folic acid (FA) lowered plasma homocysteine, but failed to reduce cardiovascular risk. We hypothesize this is due to a discrepancy between plasma and intracellular effects of FA. METHODS: In a double-blind trial, 50 volunteers were randomized to received 500 µg FA daily for 8 weeks, or placebo. Plasma and peripheral blood mononuclear cell (PBMC) concentrations of homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine, methionine, cystathionine and 5-methyltetrahydrofolate (bioactive folate) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). PBMCs were used as a cellular model since they display the full spectrum of one-carbon (1C) enzymes and reactions. RESULTS: At baseline, plasma concentrations were a poor reflection of intracellular concentrations for most 1C metabolites, except 5-methyltetrahydrofolate (R=0.33, p=0.02), homocysteine (Hcy) (R=0.35, p=0.01), and cystathionine (R=0.45, p=0.001). FA significantly lowered plasma homocysteine (p=0.00), but failed to lower intracellular homocysteine or change the concentrations of any of the other PBMC 1C metabolites. At baseline, PBMC homocysteine concentrations correlated to PBMC SAM. After FA supplementation, PBMC homocysteine no longer correlated with PBMC SAM, suggesting a loss of SAM's regulatory function. In vitro experiments in lymphoblasts confirmed that at higher folate substrate concentrations, physiological concentrations of SAM no longer effectively inhibit the key regulatory enzyme methylenetetrahydrofolate reductase (MTHFR). CONCLUSIONS: FA supplementation does not reduce intracellular concentrations of Hcy or any of its closely related substances. Rather, FA may disturb physiological regulation of intracellular 1C metabolism by interfering with SAM's inhibitory effect on MTHFR activity.

Effect of a treatment strategy consisting of pravastatin, vitamin E, and homocysteine lowering on arterial compliance and distensibility in patients with mild-to-moderate chronic kidney disease.

BACKGROUND: Arterial stiffness is increased in chronic kidney disease (CKD). Intervention studies aimed at reduction of arterial stiffness in dialysis patients have been disappointing. We therefore investigated the effect of pravastatin, vitamin E, and homocysteine lowering on arterial compliance and distensibility coefficients in mild-to-moderate CKD. METHODS: This is a sub-study of the ATIC study, a randomized, double-blind trial in 93 CKD patients. The treatment group received pravastatin to which vitamin E supplementation was added after 6 months and homocysteine lowering therapy after another 6 months. Measurement of the distensibility coefficient (DC) and the compliance coefficient (CC) of the common carotid (CCA), femoral (FA) and brachial artery (BA) was performed at 0, 6, 12, 18 months. Young's elastic modulus (YEM) was measured in the common carotid artery. RESULTS: After 18 months, CCA-DC increased from mean (SD) 15.15 (6.67) to 16.52 (6.37) × 10-3kPa-1 in the treatment and decreased from 18.44 (8.19) to 16.26 (7.35) in the placebo group (p = 0.057). CCA-CC increased from 0.64 (0.24) to 0.71 (0.26) mm2kPa-1 in the treatment and decreased from 0.77 (0.28) to 0.69 (0.25) in the placebo group (p < 0.0001). FA-DC had increased from 6.64 (3.45) to 11.46 (6.83) in the treatment group, and from 6.46 (2.85) to 7.08 (2.73) in the placebo group (p = 0.0001). FA-CC had increased from 0.46 (0.24) to 0.74 (0.44) in the treatment group, and from 0.48 (0.27) to 0.53 (0.21) in the placebo group (p = 0.008). BA-DC and CC, and CCA YEM were not significantly different between the groups. CONCLUSION: In patients with mild-to-moderate CKD, 18 months of treatment consisting of pravastatin, vitamin E and homocysteine lowering resulted in significant improvement of compliance and distensibility in CCA and FA. Since pravastatin was used throughout the observation period, it remains unclear whether the beneficial effects are attributable solely to the ongoing effect of pravastatin treatment, or if the additional interventions further slowed the progression of vascular stiffness. Therefore, larger studies with a longer period of follow-up observing the separate effects are needed.

Capillary rarefaction in advanced chronic kidney disease is associated with high phosphorus and bicarbonate levels.

BACKGROUND: In patients with chronic kidney disease (CKD), disorders of mineral metabolism are associated with vascular calcifications and mortality. Microvascular dysfunction, by affecting flow resistance and tissue perfusion, may explain the cardiovascular sequelae of CKD-associated disorders of mineral metabolism. We investigated whether advanced CKD is associated with a decrease in the functional and structural number of capillaries in skin and subsequently whether capillary rarefaction is related to mineral metabolism. METHODS: Capillary density was measured by nailfold microscopy in 19 predialysis and 35 CKD Stage 5 (CKD5) patients and 19 controls. In CKD patients, calcium, phosphorus, parathyroid hormone, 25-hydroxyvitaminD3 (25vitD3) and 1,25-dihydroxyvitaminD3 (1,25vitD3) were analysed as well. RESULTS: Capillary density at baseline was 42 ± 15/mm(2) in predialysis patients, 45 ± 17/mm(2) in CKD5 patients and 56 ± 20/mm(2) in controls (patients versus controls, respectively, P < 0.05 and P = 0.05). Absolute capillary recruitment during post-occlusive reactive hyperaemia was 17 ± 7/mm(2), 14 ± 6/mm(2) and 23 ± 8/mm(2), respectively (P < 0.05 for both patients and controls). Capillary density during venous occlusion was 59 ± 20/mm(2), 59 ± 21/mm(2) and 77 ± 21/mm(2), respectively (P < 0.05 for both patients and controls). In multiple regression analysis, both serum phosphorus and bicarbonate values were independently and inversely associated with capillary density at baseline (r(2) of model = 19%) as well as during venous occlusion (r(2) of model = 28%). Furthermore, both serum phosphorus and bicarbonate were inversely and female gender positively correlated with capillary density during recruitment (r(2) of model = 37%). CONCLUSION: Advanced CKD is characterized by an impaired functional and structural capillary density in skin, which is related to both high phosphorus and bicarbonate values.

The homocysteine controversy.

Mild to moderate hyperhomocysteinemia has been identified as a strong predictor of cardiovascular disease, independent from classical atherothrombotic risk factors. In the last decade, a number of large intervention trials using B vitamins have been performed and have shown no benefit of homocysteine-lowering therapy in high-risk patients. In addition, Mendelian randomization studies failed to convincingly demonstrate that a genetic polymorphism commonly associated with higher homocysteine levels (methylenetetrahydrofolate reductase 677 C>T) is a risk factor for cardiovascular disease. Together, these findings have cast doubt on the role of homocysteine in cardiovascular disease pathogenesis, and the homocysteine hypothesis has turned into a homocysteine controversy. In this review, we attempt to find solutions to this controversy. First, we explain that the Mendelian randomization analyses have limitations that preclude final conclusions. Second, several characteristics of intervention trials limit interpretation and generalizability of their results. Finally, the possibility that homocysteine lowering is in itself beneficial but is offset by adverse side effects of B vitamins on atherosclerosis deserves serious attention. As we explain, such side effects may relate to direct adverse effects of the B-vitamin regimen (in particular, the use of high-dose folic acid) or to proinflammatory and proproliferative effects of B vitamins on advanced atherosclerotic lesions.

Effect of B vitamin supplementation on plasma homocysteine levels in celiac disease.

AIM: To investigate the effect of vitamin supplements on homocysteine levels in patients with celiac disease. METHODS: Vitamin B6, folate, vitamin B12, and fasting plasma homocysteine levels were measured in 51 consecutive adults with celiac disease [median (range) age 56 (18-63) years; 40% men, 26 (51%) had villous atrophy, and 25 (49%) used B-vitamin supplements] and 50 healthy control individuals matched for age and sex. Finally, the C677T polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) was evaluated in 46 patients with celiac disease and all control individuals. RESULTS: Patients with celiac disease and using vitamin supplements had higher serum vitamin B6 (P = 0.003), folate (P < 0.001), and vitamin B12 (P = 0.012) levels than patients who did not or healthy controls (P = 0.035, P < 0.001, P = 0.007, for vitamin B6, folate, and vitamin B12, respectively). Lower plasma homocysteine levels were found in patients using vitamin supplements than in patients who did not (P = 0.001) or healthy controls (P = 0.003). However, vitamin B6 and folate, not vitamin B12, were significantly and independently associated with homocysteine levels. Twenty-four (48%) of 50 controls and 23 (50%) of 46 patients with celiac disease carried the MTHFR thermolabile variant T-allele (P = 0.89). CONCLUSION: Homocysteine levels are dependent on Marsh classification and the regular use of B-vitamin supplements is effective in reduction of homocysteine levels in patients with celiac disease and should be considered in disease management.