Agarose Electrophoresis Assay for the Quantitative Determination of Lipoprotein-X Using Novel Enzymatic Staining

for Total and Free Cholesterol Authors: B. Askew1, S. J. Pattman2, M. Hudson3, R. D. G. Neely2

1HB Innovations, Newcastle University, Newcastle upon Tyne, United Kingdom 2Department of Clinical Biochemistry, Newcastle upon Tyne NHS Hospitals, Newcastle upon Tyne, United Kingdom

3Department of Hepatology, Freeman Hospital, Newcastle upon Tyne, United Kingdom

Gel showing paired samples: Lanes 1-6 are samples treated with Total Cholesterol Reagent. Lanes 7-12 are the same samples treated with Free Cholesterol Reagent. First sample (lanes 1,7) is that of a patient with biliary obstruction. Note the different migration of the Non-HDL Cholesterol and also the higher Total:Free ratio, as shown in scans below.

Conclusions: Agarose electrophoresis with simultaneous enzymatic staining for total and free cholesterol permits accurate identification and quantification of Lp-X. Application of this method may prove useful in diagnosis and monitoring of patients with cholestatic liver disease.

Template: Enables 2 different reagents to be added to the same gel.

Simul taneous enzymat ic s ta in ing for cholesterol was performed on 2 halves of the gel, using a unique reagent template to separate the original ‘Total Cholesterol’ and new ‘Free Cholesterol’ reagents. In this way the ratio could then be measured between the 2 reagents. By using densitometry, the area under the curve for the total and free cholesterol was quantified in non-HDL-C fractions. This allowed a ratio between total and free cholesterol to be calculated and Lp-X in the non-HDL-C fraction to be quantitated.

Methods: A cholesterol reagent which contains both cholesterol esterase and cholesterol dehydrogenase (Helena Biosciences Europe) was modified in order to measure both total cholesterol and free cholesterol on the same gel. Agarose Electrophoresis of duplicate plasma samples using the Helena Biosciences Europe SAS-MX HDL Agarose gel with a Tris buffer produced separation of lipoproteins in plasma samples.

Results: To establish a normal range, samples from 54 patients were analysed after exclusion of secondary causes of dyslipidaemia and any values outlying the three standard deviation range from the mean. The normal range for total:free ratio of Non-HDL Cholesterol was calculated to be 2.6 - 5.3.

The ratio of total:free cholesterol in combination with the electrophoretic mobility was used to detect the presence of Lp-X in samples from a patient with biliary obstruction pre- and post-treatment and the quantities of total and free cholesterol, cholesterol ester and Lp-X were calculated.

Between gel precision was calculated using 18 repeated samples giving a coefficient of variation (CV) of the total:free cholesterol ratio of 14%. Within gel precision of six samples gave a ratio CV of 7%. Linearity for the method has been demonstrated up to 6.5mmol/L of non-HDL-Cholesterol.

Introduction: Lipoprotein X (Lp-X) is an abnormal lipoprotein found in lecithin cholesterol acyltransferase (LCAT) deficiency and frequently associated with severe hypercholesterolaemia in cholestatic liver disease (normal lipoproteins being reduced or absent). Lp-X displays a unique cathodic mobility on agarose gel electrophoresis, however it overlaps significantly with low density lipoprotein (LDL-C).

It is important to distinguish Lp-X from LDL-C in view of its diagnostic significance, however currently there are no commercial methods for detection and quantification of Lp-X. Utilising the fact that the cholesterol component of Lp-X is almost entirely unesterified cholesterol, we describe a novel adaptation of an established commercial electrophoretic method for simultaneous estimation of total and free cholesterol enabling quantification of Lp-X in dyslipidaemic samples.

Sample A: Patient with biliary obstruction Sample B: Normal patient sample

Patient with biliary obstruction (Sample A) shows a Total:Free Cholesterol ratio (black:green) of 1.31.

Normal patient (Sample B) shows a much higher Total:Free Cholesterol ratio of 3.51. The Non-HDL-C migration shape and position is also markedly different.

With thanks to Helena Biosciences Europe for the provision of gels, reagents and electrophoresis equipment.