Apolipoprotein-defined and NMR lipoprotein subclasses in the Veterans Affairs Diabetes Trial☆,☆☆
Introduction
The Veterans Affairs Diabetes Trial (VADT) was a randomized clinical trial designed to determine whether intensive glucose management would improve cardiovascular (CV) outcomes. The study cohort comprised US veterans (mean ± SD; age 60.4 ± 9.0 years) with long-standing Type 2 diabetes (T2DM) (time since diagnosis: 11.5 ± 7.0 years). At baseline, 40% had established cardiovascular disease (CVD) and all others were at high risk (Duckworth et al., 2009). The primary endpoint was time to first occurrence of a composite of major CV events. A total of 1791 participants (97% men) were randomized to intensive (target HbA1c < 6%) vs. standard control, aiming for an absolute separation of 1.5% in HbA1c levels between randomization groups (Duckworth et al., 2009). The study concluded that intensive glucose control yielded only a non-significant 11.9% reduction in the primary endpoint (Duckworth et al., 2009). In accordance with the study protocol, blood pressure and conventional lipid profiles were maintained at similar levels between randomization groups throughout (Duckworth et al., 2009, Meyers et al., 2006).
Several hypotheses have been advanced to explain the absence of CVD benefit with intensive glucose management, a finding consistent with those of other large, recent cohort studies (Gerstein et al., 2008, Gerstein et al., 2011, Patel et al., 2008, Zoungas et al., 2010). Intensive management may have been initiated too late: there was a significant burden of disease at study entry, and an earlier, sustained intervention might have been more effective, as suggested by UKPDS (Holman, Paul, Bethel, Matthews, & Neil, 2008). Additionally, in the VADT, rosiglitazone was the first-line oral hypoglycemic agent, and was taken by a majority of participants in both randomization groups (Abraira, Duckworth, & Moritz, 2009). During the VADT, concerns developed that rosiglitazone may be implicated in CVD (Ajjan and Grant, 2008, Nissen, 2007a, Nissen, 2007b, Nissen and Wolski, 2007, Nissen and Wolski, 2010). While the randomization groups were comparable in terms of conventional lipid profiles, detailed studies of lipoprotein subclasses have not yet been reported, and have the potential to yield useful insights.
In this study, we employed two complementary methods to measure lipoprotein subclass profiles. First, apolipoprotein-defined lipoprotein subclasses (ADLS) define particles according to their qualitative apolipoprotein complement (Alaupovic, 1991, Alaupovic, 1996, Alaupovic, 2003). Lipoproteins belong to one of two ‘families’, containing either ApoAI or ApoB. The ApoAI family is broadly equivalent to HDL, and includes two subclasses (LpAI and LpAI:AII). The ApoB family is equivalent to VLDL, IDL, and LDL, and includes five subclasses (LpB, LpB:E, LpB:C, LpB:C:E and LpAII:B:C:D:E). In each case, the particle name reflects its qualitative apolipoprotein complement. Since apolipoproteins are critically important determinants of lipoprotein interactions with cells and enzymes, ADLS measures are believed to reflect particle metabolism (Alaupovic, McConathy, Fesmire, Tavella, & Bard, 1988). We also measured total apolipoprotein C-III (ApoCIII), which is involved in the transport and catabolism of triacylglycerols (Alaupovic, 1981, Ginsberg et al., 1986), as well as ApoCIII bound to ApoAI-containing lipoproteins (ApoCIII-HS, heparin soluble) and ApoCIII bound to ApoB-containing lipoproteins (Apo CIII-HP, heparin precipitate).
Second, Nuclear Magnetic Resonance (NMR) subclass analyses categorize lipoproteins according to particle diameter. The method does not involve physical separation of particles, and, unlike ADLS, is rapid and clinically applicable. It yields measures of three lipoprotein subclasses in the VLDL range, two in LDL, and three in HDL. It also provides estimates of molar particle concentrations and mean particle diameter for VLDL, LDL, and HDL. In contrast to ADLS, NMR provides structural, not functional measures of lipoprotein subclasses.
We report cross-sectional, post-randomization measures of conventional, ADLS, and NMR lipoprotein analyses in a subgroup of the VADT cohort, analyzing the data according to randomization group (intensive vs. standard glucose management).
Section snippets
Subjects
As previously described (Duckworth et al., 2009), the VADT was conducted at twenty centers in the United States. A total of 1791 participants (1739 men), recruited from December 2000 through May 2003, were randomized to standard vs. intensive glucose control, with a target HbA1c separation of 1.5% (Duckworth et al., 2009). The study was approved by the Institutional Review Board at each site. The pre-defined, initial choice of oral hypoglycemic agents was rosiglitazone, followed by metformin
Conventional lipids
Conventional plasma lipid profiles (TC, LDL-C, TG, and HDL-C) were analyzed at clinical laboratories at each VADT site and centrally at Tufts University (Boston, MA). The central readings were utilized for analysis.
ADLS and ApoCIII analyses
ADLS were measured by immunoprecipitation of plasma samples as previously described (Alaupovic, 1991, Alaupovic, 1996). ApoCIII was measured by electroimmunoassay (Alaupovic, 1981, Ginsberg et al., 1986). We calculated the ratio of ApoCIII-HS to ApoCIII-HP, which is considered a
Demographic and clinical characteristics
The clinical characteristics of the 740 men from whom samples were obtained are summarized in Table 1. Of these, 368 had been randomized to intensive, and 372 to standard management. The subset was generally representative of the entire VADT cohort as shown in Supplemental Table 1, with the exception of TC, HDL-C, and LDL-C, which, for reasons that are unclear, were slightly lower in our subset than in non-participants (p < 0.03). Based on a Kaplan–Meier estimate of time-to-death distribution,
Discussion
In a large subset of male VADT participants studied approximately two years after randomization to intensive vs. standard glucose control, we found no significant differences in conventional lipid profiles between the intensive and standard glycemic management groups. This finding was not unexpected: the two groups were treated to defined targets of HbA1C and with the goal of achieving similar lipid levels. Detailed lipoprotein subclass analyses using ADLS also revealed no differences between
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Supported by National Heart Lung and Blood Institute Research Grant P01 HL55782; National Institute for Diabetes, Digestive, and Kidney Diseases Grants R01DK080043 and R21 HL80921; American Diabetes Association Research Grant (1-09-CR-38); the Medical University of South Carolina General Clinical Research Center (Grant M01-RR-1070); the University of Oklahoma General Clinical Research Center (Grant MO1-RR-14467, 1-09-CR-38 and 7-12-CT-46). The VA Diabetes Trial was supported by the Veterans Affairs Cooperative Studies Program, Department of Veterans Affairs Office of Research and Development; the American Diabetes Association; and the National Eye Institute. Pharmaceutical and other supplies and financial assistance for VADT were provided by GlaxoSmithKline, Novo Nordisk, Roche Diagnostics, Sanofi-Aventis, Amylin, and Kos Pharmaceuticals.
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Disclosure Statement: The authors have nothing to disclose.
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These authors contributed equally to the manuscript.