Elevated concentrations of Neu5Ac and Neu5,9Ac2 in human plasma: potential biomarkers of cardiovascular disease

Abstract

Cardiovascular disease (CVD) is a group of health conditions affecting the heart and vascular system with very high prevalence and mortality rates. The presence of CVD is characterised by high levels of inflammation which have previously been associated with increased plasma concentrations of N-acetyl neuraminic acid (Neu5Ac). While Neu5Ac has been studied in the context of CVD, Neu5,9Ac₂ has not, despite being the second most abundant sialic acid in human plasma. A small-scale pilot study of thirty plasma samples from patients with diagnosed CVD, and thirty age and sex-matched healthy controls, was designed to gain insight into sialic acids as biomarkers for CVD and potential future areas of study. Each sample was assayed for Neu5Ac and Neu5,9Ac₂ concentrations. Mean Neu5Ac and Neu5,9Ac₂ concentrations were significantly elevated in patients with CVD compared to healthy controls (Neu5Ac: P < 0.001; Neu5,9Ac₂: P < 0.04). Receiver operator curve (ROC) analysis indicated that both Neu5Ac and Neu5,9Ac₂ have reasonable predictive power for the presence of CVD (Neu5Ac AUC: 0.86; Neu5,9Ac₂ AUC: 0.71). However, while Neu5Ac had both good sensitivity (0.82) and specificity (0.81), Neu5,9Ac₂ had equivalent specificity (0.81) but very poor sensitivity (0.44). A combination marker of Neu5Ac + Neu5,9Ac₂ showed improvement over Neu5Ac alone in terms of predictive power (AUC: 0.93), sensitivity (0.87), and specificity (0.90). Comparison to a known inflammatory marker, high sensitivity c-reactive protein (hs-CRP: P-value: NS, ROC:0.50) was carried out, showing that both Neu5Ac and Neu5,9Ac₂ outperformed this marker. Further to this, hs-CRP values were combined with the three different sialic acid markers to determine any effect on the AUC values. A slight improvement in AUC was noted for each of the combinations, with Neu5Ac + Neu5,9Ac₂ + hs-CRP giving the best AUC of 0.97 overall. Thus, Neu5Ac would appear to offer good potential as a predictive marker for the presence of CVD, which the addition of Neu5,9Ac₂ predictive power improves, with further improvement seen by the addition of hs-CRP.

Keywords: Biomarker; Cardiovascular Disease; Inflammation; ROC analysis; Sialic acid.

Fig. 1

NeuAc (left) and Neu5,9Ac₂ (right) with additional acetyl group highlighted in red

Scheme 1

Sialic Acid release and DMB labelling reaction

Fig. 2

LC traces showing Neu5Ac and Neu5,9Ac₂. Left: Regular view; Right: Zoomed view to highlight Neu5,9Ac₂

Fig. 3

Boxplots showing the difference in plasma concentrations of Neu5Ac and Neu5,9Ac₂ between CVD cases and healthy controls

Fig. 4

ROC curves for Neu5Ac, Neu5,9Ac₂ and Neu5Ac + Neu5,9Ac₂

Fig. 5

Boxplots showing the difference in plasma concentrations of hs-CRP between CVD cases and healthy controls

Fig. 6

ROC curve for hs-CRP