The results of this study, in which all participants had the presence or absence of CAD confirmed by coronary cineangiography, indicate that the plasma lipid atherogenic alterations classically associated with T2DM, i.e., low HDL-cholesterol and hypertriglyceridemia, did not discriminate between T2DM and T2DM + CAD patients. The triglycerides/HDL-cholesterol ratio which is also related with insulin resistance and atherogenesis [23, 24] was also not discriminative between the T2DM and T2DM + CAD groups. Rather, higher LDL-cholesterol, non-HDL-cholesterol and apo B were the lipid markers for T2DM + CAD. The ratio LDL-cholesterol/apo B, which is a surrogate for the more atherogenic small dense LDL sub-fraction, and is altered in T2DM was not different between the two groups.
Data in the literature comparing the plasma lipid profile of T2DM patients with and without CAD are scarce. In this respect, similar to our findings, Seviour et al.  reported that LDL-cholesterol and apo B concentrations were higher in T2DM with CAD than in T2DM patients without CAD, without differences in HDL-cholesterol or triglyceride values. In the study by Kahri et al. , all three plasma lipid parameters, LDL and HDL-cholesterol and triglycerides, were equal in T2DM with or without CAD. Thus, the two previous and the current study converge in the finding that low HDL-cholesterol and high triglycerides, were not differentiating factors between CAD and non-CAD T2DM patients. Likewise, NEFA, which is characteristically elevated in T2DM resulting from the mobilization of fatty acids in insulin action deficiency, were not different between the two groups. This is a first-time observation to our knowledge, NEFA was not examined as a CAD risk factor in T2DM patients in either transversal, as the current study, or in prospective studies from the literature.
The main novel finding in this study was the diminished transfer of unesterified cholesterol to the HDL fraction in T2DM + CAD, despite the fact that the HDL-cholesterol and apo A-I concentrations were equal in both groups. Diminished transfer of unesterified cholesterol was also found in patients with precocious (<50 years) CAD without T2DM in comparison with non-CAD control subjects. In that study, all analyzed traditional lipid risk factors such as LDL and HDL-cholesterol and triglycerides were equal in CAD and non-CAD subjects .
In T2DM + CAD unesterified cholesterol concentration in total and in non-HDL plasma fraction was higher than in T2DM. In contrast, in the HDL plasma fraction the unesterified cholesterol concentration was equal in the two groups. The association of higher unesterified cholesterol serum concentration with CAD without T2DM was reported by Frohlich and Dobiasova in women , but our current results in T2DM + CAD are a novel finding.
In the correlation study, we found that the unesterified cholesterol serum concentration correlates negatively with unesterified cholesterol transfer to HDL and unesterified cholesterol in HDL composition. The suggestion is then left that because less unesterified cholesterol enters in the HDL wherein it is esterified, more remains in the non-HDL fraction.
The activity of LCAT, the enzyme that catalyzes cholesterol esterification in the plasma, did not differ between T2DM + CAD and T2DM, but was negatively correlated with all lipid transfers. The status of LCAT activity is a controversial issue in atherosclerosis [28–30] and has not yet been investigated in T2DM with CAD. On the other hand, the fact that esterified cholesterol transfer was negatively correlated with LCAT activity suggests that higher rates of cholesterol esterification tend to stabilize the HDL fraction, so that the entry of all lipids into the lipoprotein particles is inhibited.
Esterification stabilizes the cholesterol pool because newly esterified cholesterol is sequestered into the lipoprotein core. Unesterified cholesterol at the lipoprotein surface can conceivably diffuse into the surrounding aqueous medium and eventually precipitate in the artery. In contrast, cholesteryl esters at the lipoprotein core are translocated from the lipoprotein only by a CETP-mediated process . In addition, cholesterol esterification is also a driving force for the reverse transport, since it creates the gradient for the surface of the HDL particles to receive additional unesterified cholesterol from the cells. Diminution of unesterified cholesterol transfer to HDL can then diminish the esterification process by diminishing the substrate offer, and impairing the cholesterol reverse transport.
In a previous study, we injected lipidic nanoemulsions with labeled unesterified cholesterol and cholesteryl ester in CAD and control non-CAD subjects, all with presence or absence of CAD documented by coronary cineangiography: the removal of unesterified cholesterol from the plasma was faster than that of cholesteryl ester in CAD, indicating that in CAD patients unesterified cholesterol was being dissociated from the nanoparticles . The possibility was raised that unesterified cholesterol dissociated from the particles could be deposited in the arterial wall, as suggested by analyzing vessel fragments of patients who underwent revascularization previously injected with the labeled nanoemulsion .
Thus, in CAD patients, regardless of their having T2DM or not, the suggestion is left from the lipid transfer assay results and unesterified cholesterol serum concentration that the presence of CAD is associated with dynamic aspects of unesterified cholesterol intravascular metabolism. It would be tempting to examine whether the transfer of unesterified cholesterol from cultivated murine macrophages to HDL would also be decreased in T2DM + CAD, as Khera et al. showed in non-diabetic CAD patients . Similarly to our previous study in non-diabetic CAD patients, our results of diminished transfer of unesterified cholesterol to HDL in T2DM + CAD also point to a second defective step in unesterified cholesterol metabolism, now involving the entry of unesterified cholesterol from lipoproteins in the HDL fraction. Indeed, the ability of HDL to accept unesterified cholesterol is essential for cholesterol esterification and drives reverse cholesterol transport.
Another outcome of the lipid transfer study was the decrease in the transfer of cholesteryl ester to HDL in T2DM + CAD. Since CETP facilitates cholesteryl ester transfers, this was probably related to the diminution of the concentration of CETP observed in T2DM + CAD. In T2DM, CETP concentration was reportedly high in two studies [34, 35] and low in one . The current study is the first to compare T2DM patients without and with CAD. In this context, it could be postulated that high CETP concentration, together with high cholesteryl ester transfer to HDL is protective against CAD in T2DM patients. Phospholipid and triglyceride transfers were not altered in T2DM + CAD relative to T2DM. In respect to triglycerides, since CETP concentration was decreased in T2DM + CAD it would be expected that the triglyceride entry would also be decreased, since triglyceride transfers are also mediated by CETP and, as expected, in our correlation study, decreased CETP concentration corresponded to decreased triglyceride transfer to HDL.
Cholesteryl ester transfer protein also positively correlated with the triglyceride content in HDL composition. This is quite expected because the net result of CETP action is enrichment of the HDL fraction with triglycerides.
It is worthwhile to point out that many factors present in the plasma that are difficult to account for can interfere in the lipid transfers to HDL. In this respect, Morton et al.  reported that the unesterified cholesterol content of the donor lipoproteins could modulate cholesteryl ester transfer without changing triglyceride transfer. Since the unesterified cholesterol pool in the non-HDL is increased in T2DM + CAD, it is possible that the donor nanoemulsion becomes unesterified cholesterol-enriched in the plasma of those patients, which would contribute for the results of T2DM + CAD vs T2DM.
The lipid composition of the HDL fraction did not differ between T2DM + CAD and T2DM, which is somewhat unexpected in view of the decreased transfers to HDL of both unesterified cholesterol and cholesteryl ester. However, lipid transfers are bidirectional and the transfer assay used here measures only the entry of lipids into the HDL fraction and not the lipid exit. Thus, the fact that the unesterified cholesterol or the cholesteryl ester transfers were diminished does not necessarily imply a reduction of those lipids in HDL composition.
As limitations of the current study, some of the great number of variables that are present in T2DM, such as time from diabetes diagnosis, anti-glycemic and anti-hypertensive medications, as well as lifestyle interventions, were not equal in the two groups and may have eventually influenced the results.
An important finding of the correlation analysis was that the transfer of all four lipids positively correlated with the HDL-cholesterol and with the apo A-I concentration. This reflects the mass effect observed in the methodological study on lipid transfers , such that the greater the HDL concentration, expressed by the HDL-cholesterol and apo A-I concentrations, the higher the transfer of lipids to the HDL fraction.