Ichiro Hara

High-performance liquid chromatography of serum lipoproteins

Publisher Summary High-performance liquid chromatography (HPLC) has been widely used for the separation and analysis of many substances due to the short experimental time, small sample for analysis, high resolution, and excellent reproducibility. A simple and rapid method for lipoprotein analysis has been developed using HPLC with the gel permeation column. A direct quantitation method for cholesterol in each lipoprotein class using a very small amount of intact serum has been developed through a combination of the methods of (1) the separation by HPLC with gel permeation column, and (2) the selective detection of cholesterol in a post column effluent using an enzymatic reaction reagent. The HPLC technique of monitoring lipids in a post column effluent holds promise as a means of serum lipoprotein analysis from the following standpoints: simplicity of procedure, an analysis time of less than one hour, no need for sample pretreatment, small sample amount required (less than 20/μl of intact serum), high resolution, and good reproducibility.

High-performance aqueous gel permeation chromatography of human serum lipoproteins

A new application of high-performance aqueous gel permeation chromatography was developed for the analysis of human serum lipoproteins. A good combination of columns (TSK GEL, type PW and type SW) was found for the separation of serum lipoproteins: very low-density lipoprotein, low-density lipoprotein and high-density lipoproteins. Analyses of serum lipoproteins from individual normal subjects and pathological subjects were performed by this combination of columns. The effects of pH and salt concentration fo the eluent on the separation of lipoproteins were also investigated.

High-performance aqueous gel permeation chromatography of serum lipoproteins: Selective detection of cholesterol by enzymatic reaction

A rapid method for the quantitation of cholesterol in each lipoprotein fraction has been developed which utilizes high-performance aqueous gel permeation chromatography followed by enzymatic reaction using reaction-type high-performance chromatography. Cholesterol in serum lipoproteins eluted from the column could be sensitively and selectively detected by the absorbance at 550 nm following the enzymatic reaction. The sensitivity of the detection for cholesterol measured by A550 was compared with that for protein measured by A280 using the standard lipoprotein fractions: low-density lipoprotein (LDL) and high-density lipoproteins (HDL2 and HDL3). The effects of changing the flow-rate and lengthening the column on the resolution of LDL and HDL were examined. Analyses of serum protein and cholesterol were performed with this method for human and animal subjects.

High-performance liquid chromatography of human serum lipoproteins : Selective detection of triglycerides by enzymatic reaction

A simple and convenient method for detection and quantitation of triglycerides in each lipoprotein fraction (chylomicron, VLDL, LDL and HDL) has been developed by high-performance liquid chromatography followed by enzymatic reaction using a high-speed reaction type chromatograph. Triglycerides in serum lipoproteins eluted from the gel permeation column (TSK GEL) could be sensitively and selectively detected by the absorbance at 550 nm using a commercial reagent kit. The distribution of triglycerides in each lipoprotein fraction could be examined with a small amount of serum (10-50 microliters) in less than 50 min by this method. Moreover, free glycerol could be detected as a sharp peak at the elution volume of total permeation of the column. This technique was found to be suitable for the study of triglyceride-rich lipoproteins such as chylomicron and VLDL.

Phase transitions of phospholipids in monolayers and surface viscosity

Abstract The surface pressures and the surface viscosities of lecithin, cephalin and its analogs were measured at the air—water and the oil—water interfaces. It was found that the surface viscosity of the phospholipids used in this study was very high, and was comparable to those of some polymer films at the oil—water interface as well as at the air—water interface under the conditions where the monolayers were condensed. The plateaus indicating the phase transitions in monolayers were clearly observed on the pressure-area curves at the oil—water interface in all of the specimens studied. It was found that the phase transitions exactly corresponded to the abrupt increases in surface viscosity. From the results thus obtained, an intermolecular ionic linkage between neighboring molecules in the monolayers is discussed.

Critical structure formation in ampholytic phospholipid monolayers as analyzed by calcium binding characteristics

Abstract The binding of calcium ions was studied by radiotracer method for the monolayer of three ampholytic phospholipids differing in structural separation between phosphate and ammonium groups. The phospholipids used were DL-α-dipalmitoylphosphatidylethanolamine (PE), -n-propanolamine (PNP), and -iso-propanolamine (PIP), all synthesized and purified by thin layer chromatography. The reactivity of these lipids to calcium ions decreased in the following order PE

High-performance liquid chromatography of human serum lipoproteins. Selective detection of choline-containing phospholipids by enzymatic reaction.

A convenient method for the quantitation of choline-containing phospholipids in each lipoprotein fraction has been developed by combining separation by high-performance liquid chromatography with gel permeation columns and selective detection by enzymatic reaction in the post-column effluent. The elution patterns monitored by choline-containing phospholipids were compared with those monitored by cholesterol. The elution patterns of choline-containing phospholipids were found to give much more information about the distribution of lipoproteins according to their particle-size differentiation than analyses done by cholesterol. This choline-containing phospholipid monitoring method not only resolves lipoprotein peaks of the major classes (chylomicron + VLDL, LDL, HDL2 and HDL3) quantitatively, but also detects the presence of abnormal lipoproteins containing a large amount of choline-containing phospholipids. We could detect these abnormal lipoproteins using a small amount of whole serum (10-20 microliter) from patients with various liver diseases. Our examination of HDL subclasses using this technique showed that the HDL fraction was composed of several subfractions due to their particle-size differentiation.

Synthesis of phospholipids. III. Synthesis of 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene)

Abstract The chemical synthesis of two new glycerophosphatide analogues containing steroid groups, i.e., 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-3′β-cholesterol and 1,2-dipalmitoyl-rac-glyceryl-3-phosphoryl-20′-(3β-hydroxy norpregn-5-ene) is described.

Temperature dependent ionic structure of phospholipid monolayers.

Radiotracer studies of calcium adsorption to dipalmitoylphosphatidyl-alkanolamine monolayers measured at various temperatures showed that the binding constant of calcium increased with temperature up to around 30 degrees C but the decreased on exceeding this critical temperature. The temperature dependent ionic structure of ampholytic phospholipid monolayers are discussed.

Ionic properties of phospholipids at the oil/water interface

Abstract Surface pressures and surface viscosities of dipalmitoylphosphatidylethanolamine and its polar group analogues were measured at the hexane/water interface as a function of bulk pH. The monolayers expanded as the bulk pH was shifted from neutral to alkaline and gave rise to an increase in surface pressure at a constant area, and the surface viscosity was simultaneously reduced at high pH. For the increase in surface pressure, theoretical values were calculated using the Gouy-Chapman equation for the electrical double layers produced by shifting pH, and good agreements were obtained with the measured ones from which a simple mechanism was deduced for the increase in pressure. The ionic dissociation characteristics of amino groups of the lipids were discussed taking p K a values given in the above calculations into account. The reduction of the surface viscosity was thought to be attributable to disintegration of zwitter ionic structure in the condensed monolayers.