K. Richard Bruckdorfer

Those confounded vitamins: what can we learn from the differences between observational versus randomised trial evidence?

Both these papers were published in The Lancet, both are thought to be methodologically sound, and both are widely cited, yet their conclusions are contradictory. In this example, in the randomised trial vitamin C was part of a multivitamin supplement, whereas in the observa- tional study plasma concentrations of vitamin C were assessed. However, it is difficult to see why a combination of vitamin C with other vitamins should reduce its protective effects, if they were real. Furthermore, in the case of other antioxidant vitamins, notably vitamin E, results of single-factor trials and observational studies show a similar discordance to those seen in our figure for vitamin C. 2-4 Why did observational studies and randomised controlled trials come up with different answers? Several reasons have been proposed for the disparity between the results of observational epidemiological studies and trials. First, antioxidants might be useful only for primary prevention of cardiovascular disease, and not protective once atherosclerosis is established. 6 However, trials 4,7 found that antioxidants did not reduce cardio- vascular disease risk in participants who had no evidence of this disorder at randomisation. Second, in many of the trials the choice of antioxidant regimen has been criticised. 6

Genetic variation at the SLC23A1 locus is associated with circulating concentrations of l-ascorbic acid (vitamin C): evidence from 5 independent studies with >15,000 participants

BACKGROUNDnL-ascorbic acid is an essential part of the human diet and has been associated with a wide range of chronic complex diseases, including cardiovascular outcomes. To date, there are no confirmed genetic correlates of circulating concentrations of L-ascorbic acid.nnnOBJECTIVEnWe aimed to confirm the existence of an association between common variation at the SLC23A1 gene locus and circulating concentrations of L-ascorbic acid.nnnDESIGNnWe used a 2-stage design, which included a discovery cohort (the British Womens Heart and Health Study), a series of follow-up cohorts, and meta-analysis (totaling 15,087 participants) to assess the relation between variation at SLC23A1 and circulating concentrations of L-ascorbic acid.nnnRESULTSnIn the discovery cohort, variation at rs33972313 was associated with a reduction in circulating concentrations of L-ascorbic acid (-4.15 micromol/L; 95% CI: -0.49, -7.81 micromol/L; P = 0.03 reduction per minor allele). Pooled analysis of the relation between rs33972313 and circulating L-ascorbic acid across all studies confirmed this and showed that each additional rare allele was associated with a reduction in circulating concentrations of L-ascorbic acid of -5.98 micromol/L (95% CI: -8.23, -3.73 micromol/L; P = 2.0 x 10(-7) per minor allele).nnnCONCLUSIONSnA genetic variant (rs33972313) in the SLC23A1 vitamin C active transporter locus was identified that is reliably associated with circulating concentrations of L-ascorbic acid in the general population. This finding has implications more generally for the epidemiologic investigation of relations between circulating L-ascorbic acid and health outcomes.

Antioxidants and CVD

The involvement of free radicals and reactive oxygen and nitrogen species in the pathology of inflammatory and degenerative disease has been widely accepted, although the centrality of these species to the outcome of these diseases is still a matter for debate. In the case of CVD, and particularly the development of the atherosclerotic plaque, the oxidation of LDL is of particular importance and appears to explain many of the events that occur during the life history of the plaque. The corollary of this situation is that antioxidants must be a benign force to protect the population from the modern scourge of heart disease. In fact, recent evidence from intervention studies with large doses of the antioxidant vitamins and other antioxidants in foods has been very disappointing. Here, the background for the belief that antioxidants ought to be beneficial is examined and an attempt made to explain why the results of these intervention studies have been unsuccessful. It is agreed that a diet rich in fruit and vegetables is protective for both CVD and cancer, but the explanation for this effect may not necessarily lie with the presence of antioxidants.

The nitration of proteins in platelets: significance in platelet function.

Exogenous peroxynitrite has been shown to inhibit or activate platelets according to the concentration added and, at the same time, nitrate platelet proteins. Here, recent evidence is discussed which indicates that nitration of proteins may also occur during normal platelet activation by collagen, by mechanical stimulation during isolation and by exposure to low levels of hydrogen peroxide. Furthermore, this nitration appears to be transient. The implications of these findings are discussed in terms of platelet biology and cell signaling processes.

The solubility of cholesterol and its exchange between membranes

It has been proposed that exchange between membrane cholesterol pools occurs by desorption of molecules into the aqueous environment rather than by formation of a transitory collision complex between the membranes. The rate of exchange is likely to be determined by the rate of dissociation of cholesterol from the membrane bilayer and by the concentration of cholesterol monomers or aggregates of cholesterol molecules in solution. The aim of this study was to measure the effects of agents known to increase cholesterol exchange rates on cholesterol solubility, critical micellar concentration and on the activation energy of exchange. A comparison was also made with regard to these parameters, of the exchange of cholesterol to that of 4-cholesten-3-one, another steroid which exchanges more rapidly than cholesterol. Acetone and dimethylsulphoxide increased cholesterol exchange between liposomes and erythrocytes, but only modestly increased the apparent solubility of cholesterol in saline and had no effect on the activation energy of the exchange process. However, acetone and dimethylsulphoxide increased the critical micellar concentration of the cholesterol 3-fold, although tetraethylammonium iodide, which had a smaller effect on exchange, did not. 4-Cholesten-3-one had a lower solubility and critical micellar concentration than that of cholesterol, but had the same activation energy for exchange. It is concluded that the apparent solubility of steroid aggregates are unlikely to determine the rate of exchange, but that agents which substantially increase exchange also increase the critical micellar concentration. The low critical micellar concentration of cholestenone suggests that the actual monomer concentration in an exchange system is low and that the rate of dissociation of the molecules from the liposomes must determine the exchange rate. This is not reflected in the activation energy measurements since these are a composite of all the elements of the exchange process.

The role of protein nitration in the inhibition of platelet activation by peroxynitrite

Peroxynitrite at low concentrations (3–10 μM) inhibited agonist‐induced platelet aggregation by a mechanism not dependent on the formation of cyclic guanosine monophosphate. Platelets recovered completely from peroxynitrite‐induced inhibition within 30 min. Peroxynitrite induced nitration of cytosolic proteins, but this diminished to near basal levels within 60 min of exposure to the oxidant. During this period there was a reduction in tyrosine phosphorylation of specific proteins such as syk, but this was not due to direct nitration of these same proteins. The inhibition of phosphorylation was reversible with platelet proteins recovering the ability to be phosphorylated within 15 min of exposure to peroxynitrite. Conversely, peroxynitrite increased phosphorylation of other proteins, but again these events were not directly linked to nitration. Nitration may affect the phosphorylation of tyrosine residues in a number of proteins, but by an indirect route, possibly by acting on proteins upstream in the signalling cascades. We suggest that low concentrations of peroxynitrite reversibly inhibit platelet aggregation by preventing the phosphorylation of key signalling proteins.

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes.

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.

The nitration of proteins in platelets.

Nitric oxide has many important physiological functions, but it may also form an important oxidant, peroxynitrite, as a consequence of its reaction with superoxide anions. Peroxynitrite is capable of nitrating the aromatic amino acids in proteins, particularly tyrosine. Nitrated proteins are found in tissues of a variety of diseases where inflammation occurs. However, our recent work suggests that more selective nitration of specific proteins may occur during normal physiological processes, such as platelet activation by collagen. It is not yet clear what role this may play in the normal cell biology, but there is potential to be a role in signal transduction mechanisms, possibly by influencing tyrosine phosphorylation or dephosphorylation.

The nitration of platelet vasodilator stimulated phosphoprotein following exposure to low concentrations of hydrogen peroxide.

Hydrogen peroxide (H2O2) at biologically relevant concentrations acts as a signaling molecule. We have shown previously that H2O2 acts synergistically with nitric oxide (NO) to inhibit platelet aggregation. We found that this synergism may be associated with the increased serine phosphorylation of vasodilator-sensitive phosphoprotein (VASP) by H2O2. In this study we demonstrate that H2O2 in the absence of NO or exogenous haem- containing proteins induces nitration of platelet VASP and other unidentified proteins by a mechanism that may involve the formation of peroxynitrite. The nitration was NO-dependent, but independent of oxidative stress and guanylyl cyclcase. The flavanoid epigallocatechin gallate (ECGC) completely suppressed nitration and was also shown to inhibit partially platelet activation by other agonists. Importantly, protein nitration was reversible, or at least the nitrated tyrosine residues are converted to a form not recognized by anti-nitrotyrosine antibodies. The loss of nitrated VASP was still evident in the presence of membrane permeable protease inhibitors. In conclusion, as H2O2 can inhibit platelet function, the nitration of VASP, a protein critical for actin cytoskeletal rearrangement, may represent a novel mechanism important in the regulation of platelets shape change leading to inhibition of platelets aggregation and the formation of blood clot.

Variation in the SLC23A1 gene does not influence cardiometabolic outcomes to the extent expected given its association with l-ascorbic acid

Background: Observational studies showed that circulating l-ascorbic acid (vitamin C) is inversely associated with cardiometabolic traits. However, these studies were susceptible to confounding and reverse causation. Objectives: We assessed the relation between l-ascorbic acid and 10 cardiometabolic traits by using a single nucleotide polymorphism in the solute carrier family 23 member 1 (SLC23A1) gene (rs33972313) associated with circulating l-ascorbic acid concentrations. The observed association between rs33972313 and cardiometabolic outcomes was compared with that expected given the rs33972313-l-ascorbic acid and l-ascorbic acid–outcome associations. Design: A meta-analysis was performed in the following 5 independent studies: the British Womens Heart and Health Study (n = 1833), the MIDSPAN study (n = 1138), the Ten Towns study (n = 1324), the British Regional Heart Study (n = 2521), and the European Prospective Investigation into Cancer (n = 3737). Results: With the use of a meta-analysis of observational estimates, inverse associations were shown between l-ascorbic acid and systolic blood pressure, triglycerides, and the waist-hip ratio [the strongest of which was the waist-hip ratio (−0.13-SD change; 95% CI: −0.20-, −0.07-SD change; P = 0.0001) per SD increase in l-ascorbic acid], and a positive association was shown with high-density lipoprotein (HDL) cholesterol. The variation at rs33972313 was associated with a 0.18-SD (95% CI: 0.10-, 0.25-SD; P = 3.34 × 10−6) increase in l-ascorbic acid per effect allele. There was no evidence of a relation between the variation at rs33972313 and any cardiometabolic outcome. Although observed estimates were not statistically different from expected associations between rs33972313 and cardiometabolic outcomes, estimates for low-density lipoprotein cholesterol, HDL cholesterol, triglycerides, glucose, and body mass index were in the opposite direction to those expected. Conclusions: The nature of the genetic association exploited in this study led to limited statistical application, but despite this, when all cardiometabolic traits were assessed, there was no evidence of any trend supporting a protective role of l-ascorbic acid. In the context of existing work, these results add to the suggestion that observational relations between l-ascorbic acid and cardiometabolic health may be attributable to confounding and reverse causation.