Current Health Sciences Journal Vol. 35, No. 1, 2009 [602585]

Current Health Sciences Journal Vol. 35, No. 1, 2009
Actualities
Clinical Consequence of the Physicochemical
Properties of LDL Particles in Type 2 Diabetes
FLORIANA IONICĂ , FLORICA POPESCU , CĂTĂLINA PISOSCHI , ELIZA
GOFIȚĂ
Faculty of Pharmacy, University of Medicine and Farmacy, Craiova
ABSTRACT Atherosclerosis is the major cause of death in type 2 diabetes. LDL cholesterol and
atherosclerosis are correlated, both in nondiabetes people and those with diabetes, but people
with diabetes are more given to atheroma, even though their LDL cholesterol levels are si milar.
This review analysed the evidence that modification of physicochemical properties of LDL play a
role in the accelerated athero-sclerosis associated with type 2 diabetes.
KEY WORDS atherosclerosis, diabetes, LDL oxidation, LDL glycation, LDL size
Introduction
Diabetes and cardiovascular risk
Atherosclerosis is the leading cause of death in
type 2 diabetes. LDL cholesterol and
atherosclerosis are related, both in healthy people
and those with diabetes; people with diabetes are more prone to atheroma, even though their LDL
cholesterol levels are similar to those in their non –
diabetic persons. This is because LDL particles
are modified in the presence of diabetes to become
more atherogenic. These modifications include
glycation in response to high plasm a glucose
levels; oxidative reactions mediated by increased
oxidative stress; and transfer of cholesterol ester, which makes the particles smaller and denser. The
latter modification is strongly associated with
hypertriglyceridaemia. Oxidatively and non-
oxidatively modified LDL is involved in plaque
formation, and may thus contribute to the
accelerated atherosclerosis.
Atherosclerosis begins wi th endothelial
dysfunc tion, accumulation of lipids in
macrophages, and an inflammatory response, and results in pl aque formation and narrowing of the
lumen. The more vulnerable plaques are prone to rupture, which may lead to myocardial infarction
(MI) or stroke. Atherosclerotic cardiovascular
disease is the leading cause of morbidity and
mortality in patients with typ e 2 diabetes, and the
risk of developing the disease is two to four times higher than in non-diabetic subjects [1]. In
addition, the risk of cardiac morbidity and
mortality in individuals with type 2 diabetes without previous MI has been shown to be simila r
to that in non -diabetic subjects with a history of
MI. Although cardiovascular morbidity and
mortality are increased in patients with diabetes,
no more than 25% of the excess risk of cardiovascular disease can be explained by the co –
existence of traditio nal risk factors such as
hypertension, dyslipidaemia and central obesity
[2].
Lipid disorders in diabetes
LDL cholesterol is one of the strongest
predictors of CHD, both in individuals with and
without diabetes type 2 . It might be anticipated
that plasma l evels would be increased in diabetes;
however, the Heart Protection Study found that
plasma concentrations of LDL cholesterol are similar in control subjects and those with well –
controlled type 2 diabetes [ 3]. This study also
provided conclusive evidence t hat lowering
cholesterol is beneficial to people with diabetes.
Dyslipidaemia, characterised by hypertri –
glyceridaemia (fasting and postprandial) and a
reduced HDL cholesterol level, is common in
patients with the metabolic syndrome and type 2 diabetes, and is an independent predictor of
cardiovascular disease. In the Skaraborg Hypertension and Diabetes Project , HbA1c and
duration of diabetes were positively associated with plasma triglyceride concentrations [4], and
the subjects with poor glucose control have higher
concentrations of serum triglyceride —a
Assis t Floriana Ionică MD Student , Dept of Toxicology, Faculty of Pharmacy, U MF of Craiova 73

Floriana Elvira Ionică and colab: Clinical consequence of the physicochemical properties of LDL particles in type .
phenomenon that is often attributed to insulin
resistance (Fig . 1).
Insulinorezistența + hiperglicemia
apoCII apoCIII HSL

NEFA
LPL
VLDL
TG
RLP sdLDL ox-LDL LDL glicat
Ateroscleroza

Figura 1 – Mechanismfor the development of
atherosclerosis in type 2 diabetes.
Lipoprotein lipase (LPL), which hydrolyses
triglyceride s into monoglycerides and fatty acids,
is inhibited by apolipoprotein CIII and is activated
by apolipoprotein CII and insulin. Reduced insulin
action may thus increase plasma triglycerides by lowering their clearance rate. Increas ed hepatic
production of t riglyceride -rich VLDL also
contributes to the increase in triglyceride. In
central obesity, which is considered to play a n
important role in insulin resistance and the
metabolic syndrome, a process mediated by
hormone -sensitive lipase increases the
noneste rified fatty acid ( NEFA ) concentration.
This adipocyte -associated enzyme stimulates the
hydrolysis of stored triglyceride and is inhibited
by insulin. The NEFA flux from adipose tissue is
thus increased in insulin- resistant subjects and
will, in turn, contribute to increased synthesis and secretion of VLDL by the liver.
Since chylomicrons have to compete with
VLDL for entry to the lipolytic system, which delays their clearance, insulin resistance is al so
associated with postprandial hypertri –
glyceridaemia. High postprandial concentrations
of triglyceride -rich lipoproteins affect endothelial
function, promote atherogenesis, and are
associated with coronary artery disease (CAD) [5].
Remnants of these triglyceride-rich lipoproteins
are now considered to be ath erogenic since,
compared with LDL particles, they can deliver
more cholesterol to macrophages. High
concentrations of remnant -like particles (RLP)
have recently been found in type 2 diabetic patients [6]. LDL particle size
Each LDL particle (average 2.5 million)
contains one molecule of apolipoprotein B100
(apoB100) and approximately 3000 lipid
molecules. The core of the particle consists of
cholesterol ester and triglyceride. S urrounds this
core exists shell of protein, free unesterified
cholesterol and phospholipids, and some 3300
fatty acids are bound in different lipids,
approximately 50% of which are polyunsaturated
(specifically, lin oleic and arachidonic acid) (Table
1).
Table 1. Composition of LDL, expressed as the
number of lipid molecules per LDL particle and as a
percentage of the weight of the particle, determined
in 94 individuals with well-controlled,
normolipidaemic type 2 di abetes [8]
LDL is very heterogeneous with to lipid
composition, charge, density, and particle size and
shape and the strongest determinant of LDL
particle size in type 2 diabetes is the free
cholesterol content of the shell of the particle . A
number of studies have shown that LDL particle size is negatively correlated with plasma
triglycerid e concentrations and positively
correlated with HDL cholesterol levels. In contrast
to plasma triglyceride, the triglyceride content of LDL is not associated with LDL pa rticle size [7].
Triglyceride in the LDL particle is hydrolysed by
hepatic lipase, making it smaller and denser. In
response to elevated concentrations of plasma
triglyceride -rich lipoproteins, as is typically the
case in patients with type 2 diabetes, the rate of
transfer is increased.
An alternative, complementary theory for the
formation of small LDL is based on the existence
of multiple LDL precursors. There are two pools
of VLDL, large VLDL1 and small VLDL2, each
of which has a different metabolic fat e. Hepatic
synthesis of VLDL1 is increased at fasting
triglyceride concentrations exceeding 1.5 mmol/l. Paradoxically, large VLDL1 is preferentially
metabolised into small, dense LDL par ticles .
Multiple genes may contribute to LDL particle
size, and heritability investigations have suggested Molecule type Number of
molecules per LDL
particle Weight
(%)
ApoB100
Trigly cerides
Free cholesterol
Phospholipides
Cholesterol ester s 1
191±42
605±66
751±62
1534±143 20.5±1.1
6.7±1.6
9.4±0.8
23.5±0.9
39.9±2.2
Fatty acids
C16:0 (acid palmitic )
C18:0 (acid stearic )
C18:1 (acid oleic )
C18:2 (acid linoleic )
C20:4 (acid arahidonic ) 873±103
261±64
616±101
1328±213
207±5
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Current Health Sciences Journal Vol. 35, No. 1, 2009
that genetic influences account for one -third to
one-half of the variation in LDL peak particle
diameter in humans .
LDL particle size and association with
atherosclerosis and diabetes
Small LDL particles differ from no rmal-sized
LDL particles in terms of metabolism and
atherogenicity. Smaller LDL particles penetrate more easily into the arterial intima exhibit
increased binding to arterial wall proteoglycans
[9] and are more prone to oxidative stress. These
small and dense LDL particles also have a
prolonged plasma half -life because of their lower
bindin g affinity for the LDL receptor . The
presence of small, dense LDL particles in plasma
is therefore considered pro -atherogenic [10-12].
LDL particle size may be used in conjunction
with plasma LDL cholesterol measurements to
provide an indication of the number of circulating
LDL particles. Two individuals may have the
same LDL cholesterol concentration, but the one
with predominantly smaller particles will require
more LDL particles to carry the same load of
cholesterol. The observation that those with diabetes have smaller LDL particles but normal
levels of plasma LDL cholesterol suggests that the number of particles is increased in diabetes. A
rough estimate of the number of LDL (and VLDL)
particles may be obtained by assessing the
concentration of apoB100, because these
lipoproteins contain one molecule of the protein.
The Insulin Resistance Atherosclerosis Study
found that apoB100 outperformed LDL
cholesterol in the asse ssment of cardiovascular
risk [13], and Health Professionals’ Follow -up
Study showed that, compared with LDL
cholesterol, apoB100 was a stronger predictor of cardiovascular disease among diabetic men .
Assessment of LDL particle number by
measurement of LDL size or apoB100
concentration will thus enhance the precision of
the risk estimates based on LDL cholesterol.
LDL oxidation and oxidative stress
Oxidation of LDL initiates a series of events
that ultimately lead to the enhanced uptake of LDL by macrophag es, foam cell formati on and
plaque development .
Oxidised LDL and antibodies against the
modified form of the lipoprotein have been found
in human atherosclerotic lesions [14], but not in normal arteries or veins. Several characteristics of
oxidised LDL pla y a role in the development of
atherosclerotic plaques: oxidised LDL is toxic to
endothelial cells, recruits leucocytes to atherosclerotic lesions, and promotes the
proliferatio n of macrophages within plaques .
The rate of LDL oxidation in the intima
depends on multiple factors, including LDL
concentration, endothelial barrier function, the
intrinsic resistance of LDL to oxidation, and the
local concentration of free radicals; the latter will depend upon the balance between the production of free radicals an d the scaveng ing capacity of the
antioxidant defence system. Although the
initiating stimulus for LDL oxidation remains
unknown, several potential mechanisms have been
identified, including reactions with reactive
oxygen species (ROS), haem proteins, and
enzymes such as lipoxygenase and
myeloperoxidase (Fig. 2 ).
There is good evidence for the involvement of
enhanced oxidative stress in the pathogenesis of
cardiovascular disease in diabetes. Free radicals
can damage the double bonds of polyunsaturated
fatty acids in the cell membrane, leading to a
chain of chemical reactions called lipid
peroxidation, during which aldehydes are formed.
The measurement of malondialdehyde (MDA) by the thiobarbituric acid test is an indirect way of
quantifying oxidative stress. Although this method
lacks specificity and selectivity, it is widely used
to estimate the level of lipid peroxidation.
Plasma MDA concentrations are increased in
subjects with type 2 diabetes compared with those in type 1 diabetic subjects and healthy con trol
subjects [15]. F2 -isoprostanes, prostaglandin F2-
like compounds formed by the nonenzymatic oxidation of arachidonic acid, are currently
considered the most reliable biomarkers of in vivo
oxidative stress, and several studies have reported
that the lev el of isoprostanes is increased in type 2
diabetic patients .
The antioxidant capacity of diabetic patients,
both enzymatic and non-enzymatic, has also been examined in detail. Plasma vitamin E
concentrations are lower in diabetic patients than
in control s ubjects; leucocytes from type 2
diabetic patients have reduced levels of vitamin C;
and erythrocyte superoxide dismutase activity is
reduced in diabetes.

75

Floriana Elvira Ionică and colab: Clinical consequence of the physicochemical properties of LDL particles in type .
LDL nativ

Colesterol liber Fosfolipide Trigliceride ApoB100
Colesterol esterificat
Lyso -PC
Diene conjugate MDA -apoB100 Oxisteroli
LDL oxidat

Pro-oxidanți
Anti-oxidanți

Fig.2. Schematic diagram of the oxidation products formed during free radical -mediated LDL oxidat ion.
Several oxysterols are formed during oxidation of cholesterol; phosphatidylcholine is converted into lyso –
phosphatidylcholine; and lipid hydroperoxides are cleaved to form shorter aldehydes (e.g. MDA) which, in
turn, may react with apoB100. Pro -oxidan ts are m yeloperoxidase, lipoxygenase, haem proteins, p eroxynitrite,
metal ions, ROS, NO; endogenous and plasma antioxidants are Co enzyme Q10, v itamin E vitamin C, β –
carotene, flavonoids, albumin.
LDL glycation
The initial products generated by glycatio n,
otherwise known as non-enzymatic glycosylation,
undergo intramolecular rearrangements over time
and transform into AGE (Advanced Glycation End
products) . Since AGE form predominantly on
long-lived proteins such as collagen, their clinical
significance i s beyond the scope of this review.
However, the initial glycation products that arise
from the reaction of glucose with the lysine
residues of apoB100 represent an important modification of LDL. The LDL of diabetic
patients is more glycated than that of no n-diabetic
individuals.
This is especially true in those with poor
glycaemic control. Glycation of LDL affects its biological function. For example, compared with
normal LDL, glycated LDL is catabolised more
slowly. Oxidation may also damage glucose direct ly, with concomitant generation of free
radicals; consequently, hyperglycaemia may lead
to oxidative stress. The combination of glycation
and oxidation is termed ‘glycoxidation’, and Wolff and Dean have demonstrated that, under
diabetic conditions, ROS are produced via glucose
auto-oxidation. Consistent with this, it has been
shown that hyperglycaemia results in an increased oxidative load and that glucose intake stimulates
ROS generation [16]. Some studies have shown
that glycation accelerates the oxidisab ility of LDL
in vitro. For example, when native LDL was glycated with different concentrations of glucose (0, 5, 10 and 20 mmol/l) and then oxidised by copper ions, the amount of thiobarbituric acid
reactive substances (TBARS) in LDL —a
parameter of lipid o xidation—increased with
increasing glucose concentrations in a dose-
dependent mode . Consistent with this, the
glycation level of native LDL is positively correlated with LDL oxidation, as assessed by
measuring TBAR S during a 4-h oxidation period.
In summa ry, apoB100 glycation is
approximately twice as high in the LDL of
diabetic patients than in the LDL of non -diabetic
subjects. Hyperglycaemia is associated with
enhanced glycation of LDL and an increase in free radical production. It therefore seems reason able
to assume that this contributes to the accelerated atherosclerosis associated with diabetes, and this
assumption should be explored in longitudinal
studies.
Conclusions and perspectives
There is growing evidence that modifications
of LDL enhance its atherogenicity. Since modified
LDL is not a single homogeneous entity, there is
no single diagnostic or prognostic marker that
adequately reflects the risk of cardiovascular
disease associated with modified LDL. However,
there is no conclusive evidence of enhanced in
vitro oxidisability of LDL in type 2 diabetic
subjects. This may be because in vitro
oxidisability of LDL merely reflects its
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Current Health Sciences Journal Vol. 35, No. 1, 2009
susceptibility to oxidation, which is only one of
many factors that determine the rate of LDL
oxidation in the vascular wall. Other factors
include the permeability of the endothelium,
retention of LDL by the components of the
extracellular matrix, and local oxidant stress. Following oxidation in the vascular wall, some
oxidised LDL particles may escape scavenging by
resid ent macrophages and return to the circulation.
Unlike in vitro measures of LDL oxidisability,
circulating oxidised LDL therefore probably reflects the overall process of LDL oxidation. Circulating levels of in vivo oxidised LDL are
higher in patients with type 2 diabetes than in
control subjects, and further studies are needed to
determine whether assessment of oxidised LDL is
of value for the identification of patients at high risk of cardiovascular disease.
The presence of small, dense LDL particles in
plasma is associated with hypertriglyceridaemia
and an increased risk of CAD. Small LDL
particles predominate in individuals with the
insulin resistance syndrome, and an inverse relationship has been observed between LDL size
and circulating in vivo oxidised LDL in type 2
diabetic patients. LDL particle size can be
favourably affected by establishing good blood
glucose control. This supports the importance of tight glycaemic control in diabetes, above and
beyond its established benefits in microvascular
disease. LDL particle size is increased by
fenofibrate, whereas statin therapy, which strongly reduces the total amount of LDL cholesterol, does
not affect LDL particle size [18] . Glycation of
LDL is enhanced in individuals with type 2
diabetes, and this may co ntribute to accelerated
atherosclerosis via an increase in free radical production and possibly by rendering LDL more
susceptible to oxidation.
In conclusion, LDL cholesterol is a poor
predictor of the CAD risk associated with type 2
diabetes. Several oxid ative and non -oxidative
LDL modifications, such as oxidation and
glycation, contribute to the accelerated
atherosclerosis associated with this condition.
Measurement of these LDL modifications is
technically demanding, and therefore unsuitable
for routine practice. Plasma triglyceride
concentrations are closely linked to a
preponderance of small LDL particles and
increased concentrations of oxidized LDL. In addition, the number of LDL particles is positively
related to the risk of CAD. This can be assessed
by measuring LDL size or estimated by measuring
apoB100, but cannot be measured by
determination of the LDL chole sterol concentration. Prediction of future CAD in type 2 diabetic patients could thus be improved by the
routine measurement of triglyceride an d apoB100
in addition to LDL cholesterol.
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Corresponding Adress: Assistant. Floriana Ionic MD student , Dept of Toxicology Faculty of Ph armacy,
University of Medicine and Pharmacy. Craiova , E-mail: floriana_umf@yahoo.com
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