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Title: Immunological techniques applied to triacylglycerol rich lipoproteins in postprandial plasma and tissue slices
Author: Mohd-Esa, Norhaizan
ISNI:       0000 0001 3413 8380
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2001
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Apolipoprotein B-48 (apo B-48) is the specific transport protein of dietary fat, which is packaged as chylomicrons (CM) in enterocytes. Each CM or its remnant (CMR) contains one molecule of apo B-48 which remains within the particle until removal from plasma, making it an ideal marker for the metabolism of postprandial lipoproteins. By using our specific antiserum against apo B-48, which recognises the C-terminal end of the protein, an improved method for the immuno-detection and semi-quantitation of apo B-48 was developed which was 25-to 50-fold more sensitive than staining with Coomassie Brilliant Blue. This technique was relatively simple and the sample did not need a pre-delipidation step. In addition, samples with high protein concentrations but low levels of apo B could be used directly in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) method without an ultracentrifugation step. Furthermore, the separation of apo B-48 from apo B-100 was improved using a 3-16% (w/v) acrylamide gradient gel. In addition, this immunodetection method has the ability to quantify both apo B-48 and apo B-100 in the same blot, which reduces the amount of sample and time needed for apo B quantification. The apo B-48 specific antiserum and monoclonal antibody to the C-terminal end of apo B-100 were also used to examine the tissue localisation of these two apolipoproteins, of which the N-terminal 2152 amino acids are identical. Three types of human tissues were used: fatty streak, aorta and coronary artery. Immuno-cytochemistry was applied to human ileum and liver to check whether the system was working before it was applied to aorta and coronary artery. Apo B-48-containing lipoproteins were clearly visualised in ileum tissue whereas apo B-100-containing lipoproteins were detected by strong staining in the liver. Macrophages are known to be involved in the early stage of atherosclerosis (fatty streak). They are present in high concentrations in the foamy area in the intima layer of aorta and coronary artery. Apo B-48-containing lipoproteins and apo B-100-containing lipoproteins were present in the same area as macrophages, which suggests a role for both lipoproteins in causing the accumulation of lipid in these tissues. In addition, apo B-100 was also detected in the media area, which is rich in smooth muscle cells. Postprandial lipaemia is observed following the acute ingestion of ethanol. However, the effect of ethanol on apo B-48-containing lipoproteins when ethanol is consumed with a fatty meal has not been thoroughly investigated. To address this issue, we used an oral-fat load test in which CM metabolism was followed by analysis of triacylglycerol (TAG), apo B-48 and retinyl esters (RE). Ethanol consumption together with the test meal compared to control increased the area under the curve (AUC) for plasma-TAG (P < 0.01) and TAG rich lipoprotein (TAGRL)-TAG (P < 0.05). Even though the AUC for plasma-apo B-48 and RE were not significantly increased, the levels remained elevated after the test meal, which included ethanol, compared with control, mainly between 5-8 hours postprandially. The postprandial profile of plasma-apo B-48 induced by ethanol, as measured by SDS-PAGE was significantly higher than control (P < 0.05) after analysis by repeated measure ANOVA. The post-heparin lipoprotein lipase activities (PHLA) decreased about 13% (not significant) by ethanol. However, there was an inverse relationship between PHLA and plasma TAG (r=0.881, P < 0.005). This probably explains the delayed clearance of TAGRL after ethanol intake. The AUC of plasma non-esterified fatty acid (NEFA) was reduced after the ingestion of ethanol (P5 hours) of the study in contrast to insulin, which was higher than in the controls in the early postprandial phase (not significant). However, no significant differences were evident for plasma-cholesterol, total protein and apo B and the levels of high density lipoprotein (HDL)-cholesterol and low density lipoprotein (LDL)-cholesterol were also not significantly changed. This study demonstrates that the elevated TAG levels in the postprandial state after ethanol consumption is partly due to the accumulation of intestinally-derived lipoprotein particles.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Atherosclerosis