Predictive Properties of Plasma Amino Acid Profile for Cardiovascular Disease in Patients with Type 2 Diabetes

<div><p>Prevention of cardiovascular disease (CVD) is an important therapeutic object of diabetes care. This study assessed whether an index based on plasma free amino acid (PFAA) profiles could predict the onset of CVD in diabetic patients. The baseline concentrations of 31 PFAAs were measured with high-performance liquid chromatography-electrospray ionization-mass spectrometry in 385 Japanese patients with type 2 diabetes registered in 2001 for our prospective observational follow-up study. During 10 years of follow-up, 63 patients developed cardiovascular composite endpoints (myocardial infarction, angina pectoris, worsening of heart failure and stroke). Using the PFAA profiles and clinical information, an index (CVD-AI) consisting of six amino acids to predict the onset of any endpoints was retrospectively constructed. CVD-AI levels were significantly higher in patients who did than did not develop CVD. The area under the receiver-operator characteristic curve of CVD-AI (0.72 [95% confidence interval (CI): 0.64–0.79]) showed equal or slightly better discriminatory capacity than urinary albumin excretion rate (0.69 [95% CI: 0.62–0.77]) on predicting endpoints. A multivariate Cox proportional hazards regression analysis showed that the high level of CVD-AI was identified as an independent risk factor for CVD (adjusted hazard ratio: 2.86 [95% CI: 1.57–5.19]). This predictive effect of CVD-AI was observed even in patients with normoalbuminuria, as well as those with albuminuria. In conclusion, these results suggest that CVD-AI based on PFAA profiles is useful for identifying diabetic patients at risk for CVD regardless of the degree of albuminuria, or for improving the discriminative capability by combining it with albuminuria.</p></div

Baseline characteristics of patients who did (cases) and did not (controls) experience cardiovascular events during follow-up.

<p>Data are expressed as mean ± SD for normally distributed continuous variables or median (interquartile range) for skewed continuous variables.</p><p><i>Abbreviations:</i> GFR, glomerular filtration rate; HDL, high density lipoprotein; baPWV, brachial-ankle pulse wave velocity.</p

Crude and multivariate-adjusted hazard ratios for the cardiovascular composite endpoint in patient subgroups stratified according to urinary albumin excretion rate and the CVD-AI.

<p>Subjects were categorized as being above or below a UAER of 20 µg/min and above or below the CVD-AI cut-off value of −1.662. Crude (unadjusted) and adjusted hazard ratios were calculated using Cox proportional hazards regression models.</p>a<p>Estimates were adjusted for the conventional risk factors of cardiovascular disease, including age, sex, HbA1c, total cholesterol, triglyceride, high density lipoprotein cholesterol, estimated glomerular filtration rate, body mass index and hypertension.</p><p><i>Abbreviations:</i> CVD-AI, cardiovascular disease-amino acid based index; UAER, urinary albumin excretion rate.</p

Areas under the receiver-operating characteristic curves distinguishing patients who did (cases) and did not (controls) experience cardiovascular events during follow-up.

<p><i>Abbreviations:</i> AUC, area under the receiver-operator characteristic curve; β-AIBA, β-amino-iso-butyric acid; 3MeHis, 3-methylhistidine; Cit, citrulline; Trp, tryptophan; Cys, cystine; CVD-AI, cardiovascular disease-amino acid based index; UAER, urinary albumin excretion rate.</p

Hazard ratios for the cardiovascular composite endpoint.

<p>The variables listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101219#pone-0101219-t001" target="_blank">Table 1</a> and CVD-AI were firstly assessed in the univariate analysis of the Cox proportional hazards regression model. Only variables shown to be statistically significant in the univariate model are shown in this table.</p>a<p>Each estimate was adjusted for all variables shown in this table.</p><p><i>Abbreviations:</i> BP, blood pressure; CI, confidence interval, CVD-AI, cardiovascular disease-amino acid based index; HDL, high density lipoprotein; UAER, urinary albumin excretion rate; eGFR, estimated glomerular filtration rate; baPWV, brachial-ankle pulse wave velocity.</p

Absolute levels of 31 plasma amino acids in patients who did (cases) and did not (controls) experience cardiovascular events during follow-up.

<p><i>Abbreviations:</i> HMDB ID: Human Metabolome Database ID.</p

Results of area under the curve of receiver-operator characteristics curve analysis for both CVD-AI and urinary album excretion rate to distinguish cases from controls in all subjects and those with/without albuminuria.

<p>Results of area under the curve of receiver-operator characteristics curve analysis for both CVD-AI and urinary album excretion rate to distinguish cases from controls in all subjects and those with/without albuminuria.</p

Interaction Analysis of FABP4 Inhibitors by X‑ray Crystallography and Fragment Molecular Orbital Analysis

X-ray crystal structural determination of FABP4 in complex with four inhibitors revealed the complex binding modes, and the resulting observations led to improvement of the inhibitory potency of FABP4 inhibitors. However, the detailed structure–activity relationship (SAR) could not be explained from these structural observations. For a more detailed understanding of the interactions between FABP4 and inhibitors, fragment molecular orbital analyses were performed. These analyses revealed that the total interfragment interaction energies of FABP4 and each inhibitor correlated with the ranking of the <i>K</i>_i value for the four inhibitors. Furthermore, interactions between each inhibitor and amino acid residues in FABP4 were identified. The oxygen atom of Lys58 in FABP4 was found to be very important for strong interactions with FABP4. These results might provide useful information for the development of novel potent FABP4 inhibitors