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<i>Background and Objectives:</i> The key pathogenetic mechanism of glucose metabolism disorders, insulin resistance (IR), can be assessed using the Homeostasis Model Assessment of IR (HOMA-IR). However, its application in clinical practice is limited due to the absence of cut-offs. In this study, we aimed to define the cut-offs for the Czech population. <i>Methods:</i> After undergoing anthropometric and biochemical studies, the sample of 3539 individuals was divided into either nondiabetics, including both subjects with normal glucose tolerance (NGT, <i>n</i> = 1947) and prediabetics (<i>n</i> = 1459), or diabetics (<i>n</i> = 133). The optimal HOMA-IR cut-offs between subgroups were determined to maximize the sum of the sensitivity and specificity for diagnosing type 2 diabetes mellitus (T2DM) or prediabetes. The predictive accuracy was illustrated using receiver operating characteristic (ROC) curves. Logistic regression was performed to assess the association between a target variable (presence/absence of T2DM) depending on the HOMA-IR score as well as on the age and sex. <i>Results:</i> The HOMA-IR cut-off between nondiabetics and diabetics for both sexes together was 3.63, with a sensitivity of 0.56 and a specificity of 0.86. The area under the ROC curve was 0.73 for T2DM diagnosing in both sexes. The HOMA-IR cut-off between the NGT subjects and prediabetics was 1.82, with a sensitivity of 0.60 and a specificity of 0.66. Logistic regression showed that increased HOMA-IR is a risk factor for the presence of T2DM (odds ratio (OR) 1.2, 95% confidence interval (CI) 1.14&#8722;1.28, <i>p</i> &lt; 0.0001). The predictive ability of HOMA-IR in diagnosing T2DM is statistically significantly lower in females (OR 0.66, 95% CI 0.44&#8722;0.98). The results are valid for middle-aged European adults. <i>Conclusions:</i> The results suggest the existence of HOMA-IR cut-offs signaling established IR. Introduction of the instrument into common clinical practice, together with the known cut-offs, may contribute to preventing T2DM.