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 A₂₅₀ 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 proteins by gel permeation chromatography

The ability of two high-performance liquid chromatography gel permeation columns to separate proteins was evaluated. These columns gave satisfactory molecular weight separations for some, but not all, proteins tested. These results indicate that there are limitations in confidence of molecular weight determinations made by this technique.     

High-performance liquid chromatography of apolipoproteins in serum high-density lipoproteins

A simple and rapid method for apolipoprotein analysis in serum high-density lipoproteins (HDL) has been developed using high-performance liquid chromatography (HPLC) with sodium phosphate buffer (pH 7.0) containing 0.1% sodium dodecyl sulphate (SDS) as eluent. In contrast to the use of urea solution as an eluent, apolipoproteins can be analysed by applying an incubation mixure of HDL and the eluent buffer. A TSK-GEL column of G3000SW was found to be more profitable than G2000SW or G4000SW for analysis of HDL apolipoproteins. Elution patterns monitored by absorbance at 280 nm using a G3000SW column can give precise quantitative as well as qualitative information about apolipoproteins of molecular weight between 10⁴ and 10⁵. HPLC patterns of HDL apolipoproteins were compared between individual human subjects with various diseases. Elution profiles for lipid components in an incubation mixture were also examined.     

Analysis of serum iron by gel permeation high-performance liquid chromatography

A high-performance liquid chromatographic procedure for the determination of serum iron is reported. Serum iron extracted with methyl isobutyl ketone was converted to dibenzoylmethane chelate (molecular weight 725), and it was separated from excess dibenzoylmethane (molecular weight 224) by gel permeation chromatography. The chelate was determined by measuring ultraviolet absorption at 280 nm. Good reproducibility, recovery, and correlation with the conventional colorimetric method were observed.     

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 μl) 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.     

High-performance liquid chromatography assay of rifapentine in human serum

A high-performance liquid chromatographic method for the determination of rifapentine in human serum was developed. The method utilized a Spherisorb C₁₈ column, ultraviolet detection (336 nm), rifampin as internal standard and a calibration curve (C = 7.010 A_s/A_in ± 0.156, R = 0.999) with reproducibility studies which yield a coefficient of variation (C.V.) of intra-day and inter-day assays lower than 10%. The average recovery of rifapentine from serum in the concentration range of 0.5 to 30 μg/ml was 92.93 ± 9.704%.     

Heterogeneity of human very low density lipoproteins by gel filtration chromatography

Very low density lipoproteins were separated by gel filtration on Sepharose 4B. A decrease in mean particle diameter and flotation rate was seen with increasing elution volumes. The smaller lipoproteins had relatively more protein and phospholipid and less triglyceride than the larger ones. No differences were noted in the relative contents of the various phospholipids or partial glycerides between small and large lipoproteins. Fatty acid patterns of triglycerides and cholesteryl esters were also similar for the various lipoproteins. Relatively more lecithin containing linoleoyl acyl groups was found in smaller lipoproteins of some subjects. More of the protein of smaller lipoproteins was apo-LDL protein. Apo-HDL peptide was lost from the very low density lipoprotein as a consequence of the gel filtration.     

Hight performance gel permeation chromatography of proteins

Proteins in the molecular weight range of 10 000–170 000 were separated by high performance gel permeation chromatography. Silica particles with 30 nm or 50 nm pores were derivatized with glycidoxy-propyltrimethoxysilane and used as support. The proteins were eluted with 50% formic acid. A protein fraction which induces endodermal and mesodermal tissues in amphibian gastrula ectoderm was purified by this method.     

Determination of neutral oligosaccharide fractions from human milk by gel permeation chromatography

A gel permeation chromatographic method for quantifying neutral oligosaccharide fractions from human milk has been developed. Oligosaccharides from monofucosyllactoses to trifucosyllacto-N-hexaoses were separated according to size on a Fractogel TSK HW 40 (S) column. Refractive index detection of monofucosyllactoses to difucosyllacto-N-tetraoses yielded a constant mass response factor of ca. 1 relative to glucose. After the addition of glucose as an internal standard, oligosaccharides were isolated from human milk by ethanol precipitation or two ultrafiltration procedures. The oligosaccharide concentrations found by the ultrafiltration procedures were significantly lower (significance level 0.05) than those determined by the ethanol precipitation procedure.     

Quantification of rotavirus-like particles by gel permeation chromatography

There is a lack of accurate and practical methods that require only small amounts of sample for quantifying virus-like particles (VLP). In this work, gel permeation (GP) HPLC was used to quantify double-layered rotavirus-like particles (dlRLP) produced in insect cells. The proposed methodology utilized two columns in series (pore sizes of 200 and 50 nm) and had a high precision (relative standard deviation below 5%). GP-HPLC not only allowed the routine quantification of dlRLP, but also of assembly intermediaries and other viral structures present in the samples. For the first time, kinetics of dlRLP accumulation could be followed. This methodology is valuable for designing new production processes and for optimizing dlRLP monitoring.     

Agarose--starch gel electrophoresis of rat serum lipoproteins

Rat serum lipoproteins were separated into at least four fractions by agarose-starch gel electrophoresis. The system used was discontinuous in that glycine and sodium barbitone buffer was used in the reservoirs and Tris buffer was used for the gels. The four major bands could be related to the pattern obtained by ultracentrifugation. The high density lipoproteins consisted of at least two poorly resolved bands and were not separated from albumin. The vertical gel apparatus was further modified to accept 0.4 ml of rat plasma, which was prestained with Sudan black. After electrophoresis the different lipoprotein bands could conveniently be cut out and the lipid phosphorus determined. The addition of Sudan black B decreased the recovery of the low and high density lipoproteins by 5-9%. However, the recovery of phospholipids was reproducible (80 +/- 2%) and the high density lipoproteins contained over two-thirds of the plasma lipid phosphorus.     

Urinary protein profiling by high-performance gel permeation chromatography

We describe a new application of high-performance aqueous gel permeation chromatography for the analysis of human proteinuria. Separations of urinary proteins from normal subjects and patients with renal impairment were performed with TSK G 3000 SW columns. The effects of pH and icnic strength of the eluent on the separation of urinary proteins were investigated. Albumins were selectively separated from urine by affinity chromatography on Blue Sepharose CL-6B. According to the results of clinical investigations, urinary protein pattern derived from gel permeation chromatography revealed a good prediction of the site of renal involvement. Predominant excretion of proteins with lower molecular weight than albumin correlated with tubular damage. Albumin and higher molecular weight protein patterns were associated with glomerular disease. Absorbance measurements of the eluent at 280 nm were used for quantitative determination of total urinary protein. Gel permeation chromatography was compared to sodium dodecyl sulfate—polyacrylamide gel electrophoresis and the resulting protein patterns are in good agreement.     

Separation and size determination of human serum lipoproteins by agarose gel filtration

A method is described for the separation of the three major classes of human serum lipoproteins by gel filtration on columns of 4 and 6% agarose gel. After calibration of the columns, the elution volumes of the lipoproteins were used to calculate the molecular sizes and molecular weights of these macromolecules. The technique was employed to demonstrate aggregation of low density lipoprotein following partial delipidation, partial proteolysis, or mild heat denaturation. Agarose gel filtration shows promise as a useful method for the isolation, purification, and characterization of lipoproteins.     

Derivatization-free gel permeation chromatography elucidates enzymatic cellulose hydrolysis

Background

The analysis of cellulose molecular weight distributions by gel permeation chromatography (GPC) is a powerful tool to obtain detailed information on enzymatic cellulose hydrolysis, supporting the development of economically viable biorefinery processes. Unfortunately, due to work and time consuming sample preparation, the measurement of cellulose molecular weight distributions has a limited applicability until now.

Results

In this work we present a new method to analyze cellulose molecular weight distributions that does not require any prior cellulose swelling, activation, or derivatization. The cellulose samples were directly dissolved in dimethylformamide (DMF) containing 10-20% (v/v) 1-ethyl-3-methylimidazolium acetate (EMIM Ac) for 60?minutes, thereby reducing the sample preparation time from several days to a few hours. The samples were filtrated 0.2?μm to avoid column blocking, separated at 0.5?mL/min using hydrophilic separation media and were detected using differential refractive index/multi angle laser light scattering (dRI/MALLS). The applicability of this method was evaluated for the three cellulose types Avicel, α-cellulose and Sigmacell. Afterwards, this method was used to measure the changes in molecular weight distributions during the enzymatic hydrolysis of the different untreated and ionic liquid pretreated cellulose substrates. The molecular weight distributions showed a stronger shift to smaller molecular weights during enzymatic hydrolysis using a commercial cellulase preparation for cellulose with lower crystallinity. This was even more pronounced for ionic liquid-pretreated cellulose.

Conclusions

In conclusion, this strongly simplified GPC method for cellulose molecular weight distribution allowed for the first time to demonstrate the influence of cellulose properties and pretreatment on the mode of enzymatic hydrolysis.

     

Studies on human serum high-density lipoproteins. Self-association of human serum apolipoprotein A-II in aqueous solutions.

Some of the solution properties of pure preparations of human serum high-density apolipoprotein A-II were studied by sedimentation equilibrium ultracentrifugation, conducted at different apoprotein concentrations and at several speeds. The concentration dependence of the apparent weight average molecular weight indicated that apolipoprotein A-II, when dissolved in 0.02 MEDTA (pH 8.6), undergoes self-association. Over a protein concentration range between 0.8 and 1.5 mg/ml, the self-association could best be described by a monomer-dimer-trimer step association, although indefinite self-association could not be ruled out. The equilibrium constants obtained were sufficient to describe the system over the concentration range investigated.     

Purification of plant hormone extracts by gel permeation chromatography

Gel permeation chromatography on porous polystyrene beads has been adapted for the purification of plant extracts prior to analysis for plant hormones. The retention characteristics of gibberellins, indoleacetic acid, cytokinins and abscisic acid are presented along with chromatograms of some typical plant extracts.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.