Associations between serum bilirubin and multiple CVD risk factors have been previously reported, but attribution of causality has been challenged by the potential for confounding and uncertainty regarding temporal ordering . Importantly, Lin et. al.  utilized genotype at the UGT1A1*28 locus to suggest a causal role for bilirubin in cardiovascular and coronary heart diseases. Most notably, using longitudinal data over 24 years of follow up, they demonstrated a striking unconfounded association with CVD events. While that study supports the hypothesis that increased bilirubin levels cause a decreased risk of CVD, it did not provide estimates of the causal relation of bilirubin with markers of cardiovascular disease burden and hence did not help identify the underlying pathway by which bilirubin might protect against CVD events. In this study, using Mendelian randomization, we attempt to extend previous studies by exploring the association between bilirubin and sub-clinical markers of vascular reactivity and CVD burden in "healthy" subjects, to help delineate potential early mechanistic pathways related to bilirubin. Of note, we find an association between bilirubin and brachial artery diameter and cold pressure reactivity, while utilizing genotype as an instrumental variable per Mendelian Randomization. This association between bilirubin and brachial artery diameter was not present in our data using the traditional regression approach, most likely due to the presence of confounding factors. In this case, it appears that confounding acts to hide or underestimate a potentially causal association. If one believes UGT1A1*28 locus is a reliable instrument of bilirubin levels, these data provide evidence that bilirubin may act on CVD via mechanism associated with brachial artery diameter.
While baseline brachial artery diameter had traditionally been measured to derive brachial flow mediated dilation measures as a surrogate of endothelial function, several studies have demonstrated an independent association between brachial artery diameter with coronary artery calcification , serum uric acid , blood pressure, serum lipids, smoking and glucose [27, 28], carotid IMT , pregnancy , severity of chronic heart failure , angiographic measures of coronary artery disease  and CVD events in a cohort of over 2500 subjects . Brachial artery size is also a determinant not only of both FMD and time to peak FMD, but also of non-endothelial related vasodilatory capacity . These studies clearly demonstrate repeatable and significant associations between brachial artery diameter and both CVD risk factors, outcomes and measures of vascular disease, and suggest that in certain populations, it may be more predictive of CVD than FMD measures. As such, our finding of smaller brachial artery diameter in hyperbilirubinemic subjects is consistent with the significant decrease in CVD disease noted in subjects with Gilbert's syndrome [1, 35] and raises a potential new mechanism associated with the CVD protective effects of bilirubin, separate from its described antioxidant and anti-inflammatory properties [24, 36]. The CPT, by measuring blood pressure change in response to cold stimulus, has been used as a measure of global sympathetic activity and/or response. Our study shows a modest association between bilirubin and increased CPT reactivity. While several studies have shown that increased reactivity in healthy populations may be associated with increased probability for hypertension, the results have not always been consistent, notably in healthy cohorts such as in this study [37, 38]. One reason for this could be that increased CPT reactivity may reflect either increased sympathetic activity, which is likely not protective of CVD or may be due to increased reactivity of blood vessels to sympathetic stimulus, which may be a reflection of healthy vasculature and less CVD. Indeed, ageing and obesity are associated with decreased vascular response to sympathetic activity and norepinephrine [39–41]. As such, it is plausible that higher bilirubin levels may be associated with increased vascular response capacity as opposed to an increase in sympathetic neural activity. The lack of association between the UGT1A1*28 genotype and heart rate also suggests no direct increase in baseline sympathetic activity. Mechanistic pathways for the well noted observation of smaller brachial artery diameter with decreased measures of CVD have not yet been elucidated. We did not find an association between the UGT1A1*28 genotype and markers of more advance vascular pathology such as PWV or IMT. This could be secondary to the absence of any such association or also because we studied a relatively young and healthy population with a low CVD disease burden.