1. High-density lipoprotein subclasses in subjects with impaired fasting glucose Filippatos TD 1, Barkas F 1, Klouras E 1, Liontos A 1, Rizos EC 1, Gazi I 1, Tselepis A 2, Elisaf M 1. 1Department of Internal Medicine, University of Ioannina, Ioannina, Greece 2 Laboratory of Biochemistry, Department of Chemistry, University of Ioannina, Ioannina, Greece

2. Introduction  The term “prediabetes” describes the intermediate condition in which fasting plasma glucose level is found above the highest normal value and below the cut-off level used to determine the diagnosis of T2DM.  Impaired fasting glucose (IFG) is defined as fasting plasma glucose concentration of 100–125 mg/dL according to the definition of American Diabetes Association (ADA).  Prediabetic subjects are at high risk for the development of T2DM and may exhibit increased cardiovascular disease (CVD) risk. Diabetes Care 2008;31:596-615 Diabetes Care 2012;35 Suppl 1:S11-63 Moutzouri et al. Eur J Pharmacol 2011;672:9-19.

3. Introduction (II)  High-density lipoprotein (HDL) includes discrete subfractions that differ with respect to size, density, composition, and other physicochemical properties.  HDL particles carry enzymes, such as the HDL-associated lipoprotein-associated phospholipase A2 (HDL-LpPLA2) and paraoxonase-1 (PON1), which may play an essential role in their anti-atherogenic functions.  The anti-atherogenic properties of HDL are compromised in T2DM patients, an effect that may play a role in their increased CVD risk. Kontush et al. Nat Clin Pract Cardiovasc Med 2006;3:144-153 Mueller et al. Clin Chem Lab Med 2008;46:490-498 Tselepis et al. Atheroscler Suppl 2002;3:57-68 James R. Clin Chem Lab Med 2006;44:1052-1059

4. Aims of the study The aim of the present study was to assess any differences in HDL subclass distribution and in activities of HDL-associated enzymes between subjects with IFG and non-prediabetic subjects.

5. Methods Greek subjects (n = 185) with IFG attending the Outpatient Lipid and Obesity Clinic of the University Hospital of Ioannina (Greece) participated in the present study. Exclusion criteria  Coronary heart disease (CHD) or any other clinically evident vascular disease.  Abnormal hepatic function (aminotransferase activity > 3 times ULN, and/or history of chronic liver disease  Serum creatinine levels > 1.8 mg/dL (159 μmol/L)  T2DM [fasting blood glucose > 126 mg/dL (7.0 mmol/L)]  Thyroid-stimulating hormone levels > 5.0 μU/L  Administration of any drug during recruitment

6. Results Variable IFG (glucose ≥100 mg/dL) Glucose <100 mg/dLp-value N (females/males)80 (40/40)105 (56/49)NS Age, years51 ± 11 NS BMI, kg/m 2 33.4 ± 6.130.4 ± 5.90.01 Waist circumference, cm107 ± 14102 ± 120.008 Glucose, mg/dL109 ± 890 ± 10<0.001 Insulin, μU/mL12.5 (7.4-18.0)9.1 (6.8–12.3)<0.01 HOMA index3.6 (2.0-4.7)2.0 (1.5-2.9)<0.001 Systolic BP, mmHg138 ± 16133 ± 150.026 Diastolic BP, mmHg89 ± 984 ± 100.003 Values are given as mean ± standard deviation or median (interquartile range) for parametric and non-parametric variables, respectively. IFG = impaired fasting glucose; BMI = body mass index; BP = blood pressure; HOMA = homeostasis model assessment; NS = not significant.

7. Results (II) Variable Prediabetes (glucose ≥100 mg/dL) Glucose <100 mg/dLp-value Total cholesterol, mg/dL231 ± 32228 ± 39NS Triglycerides, mg/dL160 (109-230)127 (92-179)0.02 HDL-C, mg/dL50 ± 1153 ± 10NS LDL-C, mg/dL149 ± 25147 ± 32NS apoA-I, mg/dL137 ± 25141 ± 22NS apoB, mg/dL108 ± 23109 ± 25NS apoE, mg/dL43 ± 2241 ± 14NS Lp(a), mg/dL10.3 (5.4-14.8)10.5 (5.5-18.5)NS apoC-II (mg/dL)5.3 (4.4-6.3)5.6 (4-6.5)NS apoC-III (mg/dL)12.5 (10.6-15.7)13.9 (10.6-17)NS

8. Results (III) Variable IFG (glucose ≥100 mg/dL) Glucose <100 mg/dLp-value sdLDL-C, mg/dL10 (4-22)8 (3-18)NS (0.08) sdLDL, %7.6 (2.7-18.6)6.0 (1.8-15.1)NS (0.09) LDL size, Å267 (260-270)267 (261-271)NS Large HDL2, mg/dL14 (9-19)15 (12-20)NS Small HDL3, mg/dL37 (31-42)36 (31-43)NS Small HDL3, %73 (65-80)69 (60-77)0.03 LpPLA 2 activity, nmol/mL/min 55 (44-66)52 (46-62)NS HDL-LpPLA 2 activity, nmol/mL/min 2.35 (1.7-3.3)2.9 (2.0-3.7)0.016 PON1 (paraoxon), U/L57 (40-106)65 (41-136)NS PON1 (arylesterase), U/mL46 (33-62)43 (35-59)NS

9. Multivariate regression analysis for the prediction of small HDL3 concentration and its proportion over HDL-C levels Small HDL3 concentrationSmall HDL3 proportion Variablebetap p TG0.10NS0.24<0.01 HDL-C0.56<0.01-0.53<0.01 TC0.45<0.010.43<0.01 Waist circumference0.140.010.09NS Prediabetes presence0.05NS0.130.04 R 2 = 0.71R 2 = 0.52

10. Conclusions  The proportion of small HDL3 over HDL-C was significantly increased in prediabetic subjects compared with their controls (p<0.05).  The activity of the anti-atherogenic HDL-associated lipoprotein-associated phospholipase A2 (HDL-LpPLA2) was significantly lower in subjects with prediabetes (p<0.05), whereas the activity of PON1 did not significantly differ between subjects with or without prediabetes.  In a stepwise linear regression analysis, the proportion of small HDL3 over HDL-C concentration was independently associated with the presence of prediabetes and with total cholesterol and TG concentration (positively), as well as with HDL-C levels (negatively).