"Investigation of the Lipid Composition of Blood HDL Lipoproteins in Prediabetic and Diabetic Patients"

Abstract

Prediabetes is a pathological condition in which patients have blood glucose levels higher than normal (fasting blood glucose level > 100 mg/dL) but lower than patients with Type 2 Diabetes Mellitus, T2DM (again, fasting blood glucose level ≤ 125 mg/dL). Prediabetes is a complex multifactorial metabolic disorder with many serious complications. Patients who suffer from prediabetes show impaired fasting glucose, IFG, with increased glucose production, or impaired glucose tolerance, IGT. Individuals with IGT develop decreased glucose disposal mainly in skeletal muscles. These two different pathological conditions refer as prediabetes, with their main difference to be the tissue which shows the main insulin resistance. Insulin resistance in the liver appears mostly in patients with IFG, while in patients with IGT the skeletal muscles are the tissue which is not able to receive insulin’s signal in order to take up glucose and reduce its levels in the bloodstream. In addition to this main difference, a characteristic of prediabetes is the dysfunction of beta-pancreatic cells, resulting in an increased amount of insulin which is not able to reduce glucose levels, a condition that ultimately leads to the depletion of these cells. Unless the condition of prediabetes is not reversed, patients with prediabetes are more likely to develop type 2 diabetes mellitus. Type 2 diabetes mellitus is a chronic disease that shows many serious complications, with cardiovascular diseases being the most serious and dangerous complication. The condition of a diabetic patient is essentially a deteriorating image of a prediabetic patient, as the pancreatic cells, due to their continuous insulin production, have reached the point of being dysfunctional and unable to compensate the reduced tissue sensitivity to insulin. At this point skeletal muscles appears 85-95% of the insulin resistance. HDLs are lipoproteins which in their mature form appear as spherical particles consisting of a hydrophobic core, which contains triglycerides, 5-10% (of total lipids of the particle) and cholesterol esters at 30-40%, and from a lipid monolayer as the surface of the particle, containing phospholipids, 40-60%, with their main representative, phosphatidylcholine (33-45%), free cholesterol at 3-12%, (part of the cholesterol molecule penetrates the core) and apolipoproteins, mainly the apo-AI apolipoprotein. HDL are particles with shown a number of properties, such as atheroprotective, antiinflammatory, antioxidant, antithrombotic and antidiabetic. Lipidomics is a methodology that aims at the analysis of the overall lipid profile of a biological matrix. In the last decade progress has been made on the qualitative analysis of HDL particles, as it has become apparent that the quality of these particles is a better indicator of their functionality than the amount of cholesterol in the particle, or their quantity, in general, in the bloodstream.

In this in particular thesis, we used proton nuclear magnetic resonance spectroscopy, NMR, as analytical technique to study the HDL particles’ lipid profile of three groups of patients: patients with normal glucose tolerance, patients with prediabetes and patients with type II diabetes mellitus, aiming to compare their profiles and examine both the course of glycaemic disorder and its impact on the HDL lipoprotein particle composition. In particular for this study 120 subjects were selected and divided into three groups of 40 subjects each, where: a) patients with fasting glucose levels 90,45 ± 7,48 mg/dL and glycated hemoglobin, HbA1c, 4,99 ± 0,68 % formed the healthy population, control group, b) patients with fasting glucose levels 105,58 ± 7,91 mg/dL and HbA1c 5,95 ± 0,35% formed the prediabetic population and c) patients with fasting glucose levels 156,73 ± 24,40 mg/dL and HbA1c 7,28 ± 0,69 % formed the population with type 2 diabetes mellitus. During the experimental part of the research, the HDL lipoproteins were isolated from non-HDL lipoproteins with precipitation and their lipids were extracted using a chloroformmethanol mixture. The final sample was re-dissolved in a solution of deuterated solvents and a ΝΜR spectrum was taken. The study and analysis of the received spectrums showed a gradual change in lipid composition as glycemic control worsened. Specifically, we showed a general elevation in total cholesterol and in the triglycerides of the core, while the phospholipids of the surface showed a significant decrease. In more detail, there was a significant decrease in core’s cholesterol in HDL particles of prediabetic individuals compared to those with T2DM, while the increase in free cholesterol on the particle’s surface was significant among all three groups, as prediabetic individuals showed richer HDL in free cholesterol than in the control group, but poorer than the T2DM group. Regarding phospholipids, both glycerophospholipids and etheric glycerolipids decreased between the three groups, while sphingolipids were the only phospholipids that showed increase. In particular for glycerophospholipids, was observed a significant reduction in phosphatidylethanolamine and remaining lipids (corresponding to phosphatidylserine and phosphatidylglycerol) in prediabetic’s HDL compared to the HDL of the control group. The only glycerophospholipid that increased was lysophosphatidylcholine, with this elevation being significant for prediabetic compared, again, to the control group. For etheric glycerolipids there was a decrease in HDL’s plasmalogens of prediabetic individuals compared to HDL of patients with T2DM, while in terms of the remaining etheric lipids (PAF) the reduction was significant between all three groups. Lastly, the increase that observed in sphingolipids of prediabetic’s HDL compared to HDL of control group was mainly due to the increased levels of the remaining sphingolipids, as sphingomyelin, the main sphingolipid in HDL, was decreased between these groups. In addition to the lipid type, in this study also analyzed the content of fatty acids in these lipids, which showed an increase in saturated fatty acids and a decrease in unsaturated, both monounsaturated and polyunsaturated fatty acids.

For polyunsaturated fatty acids, we further analyzed acids such as linoleic, which showed a significant reduction in HDL from prediabetics compared to HDL from patients with T2DM, arachidonic and eicosapentaenoic acid, with the reduction being significant in prediabetic’s HDL compared to the HDL of control group and docosahexaenoic acid which showed a decrease in HDL of prediabetic compared to both control group and the group of patients with T2DM. Similar alterations have been observed in previous studies, however, the results of most studies focused on comparing HDL lipoproteins of healthy patients with HDL from patients with T2DM. Also, in most of the studies analyzed the rate of cholesterol in the HDL particles, as well as the percentage of triglycerides and phospholipids in the particle, without considered the overall lipid profile of HDL. Thus, this study contributes to the knowledge of the lipid profile of HDL lipoproteins not only for the T2DM patients, but also for the patients in an intermediate condition, i.e., the state of prediabetes, showing us that the prediabetic’ HDL show significant alterations compared to healthy patients, whereas the corresponding alterations compared to patients with T2DM are not all statistically significant. The above results highlight the already known risk of prediabetes as a disease and the need for better monitoring and control of this condition. The existence of additional studies with the same content but with larger population could help validate these results, so that in future the lipid composition of HDL will take into consideration both as a diagnostic tool of prediabetes and as an indication of metabolic disorder in a patient.

KARAKITSOU Kiriaki