الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
The involvement of glucose in atherosclerosis both in diabetic and in non-diabetic people is currently uncertain. One mechanism by which microvascuhir complications of diabetes arise is through protein glycation. The serum concentrations of glycated apoB arc higher in diabetic and hypcrlipidacmic patients as compared to healthy individuals. The levels of glycated apoB are higher both in them and also in healthy people than those of oxidised low density lipoprotein (ox-LDL).
Oxidative modification of LDL remains the most frequently stated hypothesis for its participation in athcrogencsis. However, clinical trials using anti-oxidant and vitamins as therapeutic tools failed to prove any protection against CHD. Glycated apoB could potentially participate in atherosclerosis through many mechanisms of atherosclerosis suggested for oxidised LDL. Therefore, we focused on glycation of apoB, as an important index for increased CVD risk. The current study therefore, aimed mainly to:
1. Investigate the distribution of glycated apoB in lipoproteins from diabetics, hyperlipidaemic and healthy individuals.
2. Characterize some physical and chemical modifications that might occur during in vitro glycation of LDL and its sub-tract ions.
3. Clarify the relationship between in W/ro-induced glycation and the lipid peroxides generated in LDL and its different density subtractions.
4. Establish the protective role of ITDL during LDL glycation in vitro and ’whether the paraoxonase located in HDL is important in retarding LDL glycation.
5. Characterize and quantify the extent of foam cell formation by glycated LDL.
Two hundred and ninety one subjects from those attending Lipid Clinic, Peripheral Vascular Disease Clinic and Manchester Diabetes Centre at Manchester Royal Infirmary and healthy volunteers from those working in Manchester University and Manchester Royal Infirmary were recruited to participate in this study. We looked at glycated apoB distribution among different density apoB-containing lipoproteins in 48 subjects including diabetic, hyperlipidaemic patients and healthy
uwu* giuuj^a aiumvu wcit ciuici ii;uciving or noi receiving hypolipidaemic drugs.
We also isolated LDL and its subfractions from 173 subjects (healthy volunteers, diabetic and hyperlipidaemic patients). Non-enzymatic glycation was induced in vitro using 0-80 mmol/I glucose under N2. Glycation induced was assessed as a percentage glycaiion after separating glycatcd from non-glycatcd LDL by column chromatography and analysing it by in-house apoB ELISA. Moreover, glycation was characterised both physically and chemically by the assessment of REM and loss of free ami no group. Lipid peroxides were also measured in the glycated Lipoprotein.
To study the protective effect of HDL, mainly its PON-1 activity, on the in vitro glycation of LDL, 30 subjects were grouped according to their serum PON-1 activity into low and high PON-1 activity groups. Total LDL and HDL were isolated from scrum rather than plasma in order to maintain calcium which is required for PON-1 activity. In vitro glycation of LDL was carried out in the presence and absence of HDL. Glycation was assessed as mentioned earlier.
Glycation was also induced in LDL isolated from another 4 healthy individuals by glucose to be loaded on to cultured macrophages to examine its uptake and the formation of foam cell. The uptake and foam cell formation was assessed by quantifying the amount of free cholesterol and cholesterol esters in cell lysates and also by oil red O staining.
Results showed that
I- Glycated apoB distribution
1- LDL is the richest lipoprotein in apoB among apoB-containing lipoproteins (VLDL, IDL and LDL) in all groups studied.
2- More apoB is located in LDL1, 2 as compared to LDL3 in all groups studied except in diabetic patients who were not receiving hypolipidaemic medication.
patients receiving no hypolipidaemic treatment followed by the hyperlipidaemic patients.
4- A positive correlation exists between plasma glycated apoB and
that located in total LDL and LDL3. Plasma glycated apoB in diabetic patients as expected correlates positively with fasting glucose.
II- Susceptibility of LDL and its subclasses lo non-enzymatic glyeation
1- In vitro non-enzymatic tzlycation
i. Total LDL
Glucose concentration- and lime-dependent increase in LDL glyeation was observed in all studied groups. LDL isolated from diabetic patients who were not receiving hypolipidaemic drugs showed the highest susceptibility lo glyeation in contrast to those receiving hypolipidaemic treatment. ii. LDL sub-classes
LDL3 was more susceptible to glyeation than LDL1, 2 in all groups.
2- Lipid pcroxidation during lipoprotcin glycation i. Total LDL
Lipid peroxidation increased with increased glucose concentration during non-enzymatic glyeation of LDL isolated from all patients and control groups. These were correlated to the increase achieved in glyeation percentage, ii. LDL sub-classes
j Lipid peroxides increased rapidly in response to increased glucose
concentrations till 50mmol/l and then the change become steady or even decreased.
3- Electrophoretic mobility
Glycaled lippproteins showed higher mobility than native fractions due to loss . of the positive charge of lysine amino acid as a.result of glyeation. The increased RF.M was parallel to the increase in both glyeation and lipid pcroxidations.
4- Free amino groups
Free amino groups in apoB molecules in LDL and/or its subtractions were consumed during glyeation as assessed by TNBS assay. This confirms the loss of free lysine amino acids (positive charge) during the process.
III- HDL protective effect during the non-enzymatic glycation of LDL
HDL with high PON-1 activity acts as protector against non-enzymatic glycation of LDL in vitro. This protection was confirmed by the decreased levels of lipid peroxides, REM and loss of free amino groups indeed decreased glycation.
IV- Glycated LPT, uptake by cultured macrophages
Uptake of glycated LDL by cultured macrophage was higher than that of native LDL as confirmed by increased cholesterol and its esters in cells incubated with glycLDL.
Conclusions:
We can conclude the following:
1- ApoB is expressed to be important than cholesterol indices in evaluation of cardiovascular risk factors especially when chemically modified (glycated).
2- Glycated apoB occurs most abundantly in small, dense LDL.
3- Even in non-diabetic people, in addition to its susceptibility to oxidation, small, dense LDL may be rendered more atherogenic, because it is more abundantly glycated than more buoyant LDL.
4- Not only was circulating small, dense LDL demonstrating heavily glycation, but also it was preferentially glycated when different density LDL subfractions were rendered glycated in vitro.
5- Glycation itself resulted in moderate oxidation of LDL when incubated with glucose under N? (away from oxidation). In other words, glycation plays an important and perhaps primary role in initiating lipid oxidation in vivo.
6- HDL with increased PON-1 activity might nonetheless have protective role against Glycation (increase resistance).
7- Glycation of LDL stimulates substantial cholesterol accumulation in cultured macrophages.
III- HDL protective effect during the non-enzymatic glycation of LDL
HDL with high PON-1 activity acts as protector against non-enzymatic glycation of LDL in vitro. This protection was confirmed by the decreased levels of lipid peroxides, REM and loss of free amino groups indeed decreased glycation.
IV- Glycated LPT, uptake by cultured macrophages
Uptake of glycated LDL by cultured macrophage was higher than that of native LDL as confirmed by increased cholesterol and its esters in cells incubated with glycLDL.
Conclusions:
We can conclude the following:
1- ApoB is expressed to be important than cholesterol indices in evaluation of cardiovascular risk factors especially when chemically modified (glycated).
2- Glycated apoB occurs most abundantly in small, dense LDL.
3- Even in non-diabetic people, in addition to its susceptibility to oxidation, small, dense LDL may be rendered more atherogenic, because it is more abundantly glycated than more buoyant LDL.
4- Not only was circulating small, dense LDL demonstrating heavily glycation, but also it was preferentially glycated when different density LDL subfractions were rendered glycated in vitro.
5- Glycation itself resulted in moderate oxidation of LDL when incubated with glucose under N? (away from oxidation). In other words, glycation plays an important and perhaps primary role in initiating lipid oxidation in vivo.
6- HDL with increased PON-1 activity might nonetheless have protective role against Glycation (increase resistance).
7- Glycation of LDL stimulates substantial cholesterol accumulation in cultured macrophages.