Analysis of alpha-lipoprotein cholesterol in 50 microl of plasma

A micromethod for quantitation of alpha-lipoprotein cholesterol was devised for gas-liquid chromatography to minimize plasma sample size and facilitate lipid studies using capillary blood from children or small animals. alpha-Lipoprotein cholesterol was measured by gas-liquid chromatography in 20 micro l of the centrifuged supernate obtained after addition of 5 micro l of mixed heparin-manganese chloride solution 1:1 (v/v) to 50 micro l of plasma. Comparison of venous alpha-lipoprotein cholesterol measured by gas-liquid chromatography with venous alpha-lipoprotein cholesterol measured by conventional heparin-manganese precipitation and ferric chloride (colorimetric) cholesterol determination gave a correlation coefficient of 0.98 for 80 plasma samples. Capillary alpha-lipoprotein cholesterol and venous alpha-lipoprotein cholesterol were closely correlated in 31 patients (r = 0.97).     

Capillary gas-liquid chromatographic-mass spectrometric measurement of very long chain (C22 to C26) fatty acids in microliter samples of plasma

In order to quantify accurately the plasma content of very long chain fatty acids, we have developed a selected ion monitoring gas-liquid chromatographic-mass spectrometric micromethod which allows all of these acids (22:0, 24:1, 24:0, 26:1, and 26:0) to be determined simultaneously in the same 0.5-ml plasma sample; 17:0 and 27:0 fatty acids are used as assay internal standards. For plasma samples in the range equivalent to the various very long chain fatty acid physiological concentrations, assay precision was +/- 2%. The present method has been successfully applied to the biological recognition of patients with adrenoleukodystrophy, their heterozygote relatives, and of cerebro-hepato-renal syndrome and neonatal adrenoleukodystrophy.     

Determination of ketone body kinetics using a D-(-)-3-hydroxy[4,4,4-2H3]butyrate tracer

In studies where D-(-)-3-hydroxy[4,4,4-2H3]butyrate is employed as isotopic tracer in vivo, we have described a selected ion monitoring, gas-liquid chromatography-mass spectrometry micromethod which measures [2H3] tracer enrichment in 3-hydroxybutyrate and acetoacetate from 300-microliters blood samples. For plasma samples in the physiologic range, intra- and interassay precisions for each ketone averaged better than +/- 1% and +/- 2%, respectively. The use of the method was validated by comparing kinetic data obtained with the above tracer with simultaneous flux data obtained with conventional D-(-)-3-hydroxy[3-14C]butyrate tracer in five fasted rats.     

Ultracentrifugation micromethod for preparation of small experimental animal lipoproteins

Sequential flotation ultracentrifugation is commonly used in the preparation of plasma lipoproteins. However, protocols often require prolonged centrifugation time (48-72 h) and large plasma volumes (2-20 ml), which makes them unsuitable for studies on small laboratory animals. Although analytical techniques such as FPLC have often small sample requirements, further fraction analysis is often limited to the small fraction volume obtained. A sequential ultracentrifugation micromethod is described to obtain rat lipoprotein fractions from 400 microl of plasma in a cumulative centrifugation time of 7.5 h. Fraction volumes were determined and densities were adjusted to those of rat plasma lipoproteins. Polyacrylamide gel electrophoresis and enzymatic measurements of triglycerides, total cholesterol, and phospholipids were used to assess the purity of the lipoprotein fractions. The results were compared with those obtained from a classical sequential ultracentrifugation protocol. The micromethod presented here can be further adapted to other experimental animal species with little modifications.     

Micromethod System for Identification of Anaerobic Bacteria

A micromethod multitest system prepared by Analytab Products, Inc. and conventional tests employed at the Center for Disease Control for identification of anaerobes were compared. All procedures were conducted in an anaerobic glove box. A total of 104 cultures, including 18 reference strains and 86 diagnostic cultures, were examined. Ninety-one percent of the total tests performed with the two systems were in agreement. Greater than 90% agreement between the two systems was obtained with 12 of the 17 differential tests compared. The tests for nitrate reduction and H(2)S production gave the poorest agreement, 77.8 and 80.8%, respectively. Only 66% of the 86 diagnostic cultures could be presumptively identified with the micromethod system supplemented only with microscopy and colonial characteristics. However, when appropriate supplementary tests and gas-liquid chromatography were used with the micromethod system, 85% of the 86 strains could be identified. When Ehrlich reagent, instead of Kovac reagent, was used with the micromethod to test for indole, the agreement in identification was raised to 93%.     

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients

BACKGROUND/AIM: Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN: Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS: Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS: Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.     

Improved quantitation of plasma lipids by direct gas-liquid chromatography

A preliminary digestion of total plasma lipid extracts with phospholipase C, which converts the lysolecithins, lecithins, and sphingomyelins into monoglycerides, diglycerides, and ceramides, respectively, has been shown to facilitate subsequent determination of the plasma lipids by gas-liquid chromatography. A further improvement in the chromatographic elution pattern results from acetylation or trimethylsilylation of the liberated alcohol moieties prior to injection into the chromatograph. If tridecanoin is used as internal standard, quantitative estimates can be rapidly obtained for plasma lysolecithins, free cholesterol, lecithins, sphingomyelins, cholesteryl esters, and triglycerides, as well as for free fatty acids. Other plasma lipids do not occur in sufficiently high concentrations to interfere with the analysis. The determination requires 0.1-0.5 ml of plasma and about 6 hr of processing, but many samples can be processed at a time.     

Sensitive detection of specific repair endonucleases: radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31.0% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.     

Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Presence and synthesis of cholesterol in stable staphylococcal L-forms.

The sterol which was present in two strains of a stable staphylococcal L-form was analyzed by gas-liquid chromatography and combined gas-liquid chromatography-mass spectrometry. The retention time of the sterol on gas-liquid chromatography was the same as that of authentic cholesterol. Analysis of the sterol by mass spectrometry showed a molecular ion at an m/e of 386 and the same patterns of major ions above an m/e of 145 as those of authentic cholesterol. As a result, the sterol in staphylococcal L-form was identified as cholesterol. A parent strain and its L-forms were cultured in medium containing [14C]acetate, and the synthesis of cholesterol was examined. In the L-forms, 0.52% of the total lipid radioactivity was found in cholesterol fraction, whereas no significant radioactivity was detected in the cholesterol fraction of the parent strain, indicating that staphylococcal L-forms have acquired the capacity to synthesize cholesterol.     

Polyunsaturated fatty acids are enriched in the plasma membranes of mitogen-stimulated T-lymphocytes

Plasma membranes were purified from T-lymphocytes from rabbit thymus stimulated with concanavalin A. Lipids were extracted and the fatty acid composition of the individual phospholipid species was determined by gas-liquid chromatography. Compared to the plasma membranes derived from control cells, the plasma membranes from mitogen-stimulated cells were enriched in polyunsaturated fatty acids. This increase in unsaturation was found in phosphatidylcholine, phosphatidylinositol and phosphatidylserine, while the fatty acid composition of phosphatidylethanolamine was not significantly altered. The phospholipid composition remained almost unchanged during the period of stimulation. The molar ratio cholesterol to phospholipid was decreased. These changes in the lipid composition of plasma membranes from mitogen-stimulated T-lymphocytes are discussed with regard to functional implications.     

Pentacyclic triterpenoids and sterols from seven species of mangrove

The isolation of pentacyclic triterpenoids from seven species of fresh mangrove leaves using a simple and rapid method is described. The leaves were homogenized using chloroform—methanol and the extract was diluted with water to precipitate out triterpenoids which were separated into neutral and acidic fractions. These were analysed by gas-liquid chromatography as acetyl and trimethylsilyl ether derivatives on a 3% OV-17 column. Sterols were isolated from the chloroform layer by preparative thin layer chromatography and were analysed by gas-liquid chromatography as their trimethylsilyl ether derivatives on a 3% OV-17 column. The triterpenoids found were α-amyrin, β-amyrin, lupeol, oleanolic acid and ursolic acid in most of the samples. Sterols found in all the samples were cholesterol, campesterol, stigmasterol, sitosterol and stigmast-7-en-3β-ol. Retention indices of the triterpenoids and sterols have been determined.     

Accumulation of cholesterol precursors and plant sterols in human stenotic aortic valves

The pathogenesis of aortic valve stenosis (AS) is characterized by the accumulation of LDL-derived cholesterol in the diseased valves. Since LDL particles also contain plant sterols, we investigated whether plant sterols accumulate in aortic valve lesions. Serum samples were collected from 82 patients with severe AS and from 12 control subjects. Aortic valves were obtained from a subpopulation of 21 AS patients undergoing valve surgery and from 10 controls. Serum and valvular total cholesterol and noncholesterol sterols were measured by gas-liquid chromatography. Noncholesterol sterols, including both cholesterol precursors and sterols reflecting cholesterol absorption, were detected in serum samples and aortic valves. The higher the ratios to cholesterol of the cholesterol precursors and absorption markers in serum, the higher their ratios in the stenotic aortic valves (r=0.74, P<0.001 for lathosterol and r=0.88, P<0.001 for campesterol). The valvular ratio to cholesterol of lathosterol correlated negatively with the aortic valve area (r= -0.47, P=0.045), suggesting attenuation of cholesterol synthesis with increasing severity of AS. The higher the absorption of cholesterol, the higher the plant sterol contents in stenotic aortic valves. These findings suggest that local accumulation of plant sterols and cholesterol precursors may participate in the pathobiology of aortic valve disease.     

Simultaneous determination of nicotine and cotinine in plasma using capillary column gas chromatography with nitrogen-sensitive detection

A rapid and sensitive method is described for the simultaneous determination of nicotine and its principal metabolite, cotinine, in plasma. A one-step extraction procedure is employed and the quantitative analyses are performed by capillary column gas chromatography using a thermionic specific detector. Other special measures to avoid contamination from external sources such as atmosphere, solvents and laboratory equipment, which constitutes the major limiting factor of nicotine assay, were also undertaken. The structural analogues of nicotine and cotinine, N-methylanabasine and N-ethylnorcotinine, are used as internal standards. Moreover, a micromethod, which requires only 0.1 ml of plasma and found to be suitable for analysis of cotinine in finger-tip samples of blood, is described. Linearity over the concentration ranges 5–100 ng of nicotine per ml of plasma and 5–500 ng of cotinine per ml of plasma is demonstrated. The precision of the method has been investigated by determining the reproducibility at different levels of nicotine and cotinine within the working ranges, for both 1-ml and 0.1-ml samples of plasma.     

Gas chromatographic determination of novel valproyl taurinamide derivatives in mouse and dog plasma

Valproyl taurinamides are a novel group of compounds that possess anticonvulsant activity. In this study a gas chromatographic micromethod was developed for the quantification of selected valproyl taurinamides and some of their metabolites in biological samples. Valproyl taurinamide (VTD), N-methyl valproyl taurinamide (M-VTD), N,N-dimethyl valproyl taurinamide (DM-VTD) and N-isopropyl valproyl taurinamide (I-VTD) were analyzed in mouse and dog plasma and in dog urine using gas chromatography. Flame ionization detection and mass spectrometric detection were compared. The plasma samples were prepared by solid-phase extraction using C(18) cartridges. The urine samples were prepared by liquid-liquid extraction. The sample volume used was 100 microl of dog plasma, 50 microl of mouse plasma and 20 microl of dog or mouse urine. The quantification range of the method was 1.5-50 mg/l in dog plasma (VTD only), 2.5-250 mg/l in mouse plasma (0.7-90 pmol injected) and 0.04-2 mg/ml in dog urine (VTD only). The inter-day precision in plasma and urine samples was around 10% for all quantified concentrations except LOQ (15-20%). The accuracy for all four compounds was between 90 and 110% within the entire concentration range. The developed method was suitable for quantification of a series of CNS-active valproyl taurineamide derivatives in biological samples at relevant in vivo concentrations.     

A microassay for tetrazolium-reductase activity of polymorphonuclear leukocytes--comparison with a test-tube technique

A micromethod for quantitative INT test is described to assay the activity of human peripheral blood PMNL. PMNL are obtained from a pellet after the separation of mononuclear cells. The test is performed in microtiter plates with 96 wells. The estimation of the activity is based on the colour change following the INT reduction to formazane. As a stimulating agent starch (rice species--Amylum oryzae) or zymosan is used. Absorbance alternations are measured using a microplate reader (e.g. MR 580, Dynatech). Conditions necessary for standard results (i.e. PMNL concentration, type and concentration of stimulating agent, incubation time, sedimentation time for PMNL isolation) were verified experimentally. Blood samples of 60 blood donors were simultaneously tested by the micromethod and the test-tube method. The statistical analysis (F-test of homogeneity of variances, t-test with paired observations, correlation coefficient of tightness of dependence) proved that the results of the micromethod were comparable to the test-tube assay. The advantages of the micromethod are as follows: low blood consumption, possibility to perform more parallel examinations of the same sample, and to test more samples at a time, standard and highly reproducible results, saving of reagents, reduction of labour time, possibility of some further automation.     

A sensitive and convenient method for lipoprotein profile analysis of individual mouse plasma samples

A simple and convenient method to determine plasma cholesterol profiles in individual mouse plasma samples is not presently available. With commonly used methods, plasma samples from several animals in a study group must often be pooled and analyzed, usually by the fast phase liquid chromatography (FPLC) method. The Column Lipoprotein Profile (or CLiP) method described here is a modification of the FPLC method that provides a simple and convenient procedure for determining plasma lipoprotein cholesterol profiles in small sample volumes, allowing determination of profiles from individual animals rather than from pooled plasma. The CLiP method is reproducible; a human sample measured five times over several days produced coefficients of variation as follows: VLDL, 10.0%; LDL, 0.93%; and HDL, 2.51%. CLiP-derived total cholesterol values of five different human samples (with total cholesterol levels ranging from 198 to 263 mg/dL) differed from VAP-II by -1.88% +/- 2.57%. Linearity of differing concentrations for each of the lipoprotein classes was determined by measuring the same sample with different aliquot sizes. The linear regression from VLDL had an r value of 0.996, while LDL, HDL, and total cholesterol all had r values of greater than 0.999. We present a direct comparison of plasma cholesterol profiles from several mouse models with gene modification or expression of transgenic proteins.In conclusion, the CLiP method provides a simple, reliable, and reproducible procedure for determination of plasma cholesterol profiles from individual plasma samples with very low sample volumes, using readily available equipment and reagents.     

An improved gas-liquid chromatographic method for the determination of fecal neutral sterols

The analysis of fecal neutral sterols has been improved by use of a highly selective gas-liquid chromatography column packed with SP-2401. This chromatographic column allows separation of cholesterol and cholestanol and delta 5-5 alpha plant sterol homologs without employing silver nitrate thin-layer chromatography. Furthermore, there is no need to derivatize neutral sterols before injection. The main fecal neutral sterols are well resolved; retention times are reproducible; detector response is reproducible, linear, and sensitive to 0.2 micrograms. This method, successfully used for fecal samples, may be suggested as a routine method for the clinical study of cholesterol metabolism.     

A reliable analysis of tissue free fatty acids by gas-liquid chromatography

A convenient and reliable gas-liquid chromatographic method for determining the free fatty acids in biological specimens is described. The free fatty acids were extracted with hexane in the presence of H3PO4 and then back-extracted from the hexane phase into a very small volume of trimethyl (alpha, alpha, alpha-trifluoro-m-tolyl)ammonium hydroxide solution. Direct injection of the resultant quaternary ammonium salts of the fatty acids into a gas-liquid chromatograph unit gave their methyl esters, with a high recovery. The presence of triglycerides, phospholipids, or cholesterol esters did not interfere with the determination of free fatty acids. This method was applied to determination of free fatty acids in the samples of serum or brain. The results were more precise and reliable than those reported with the conventional methods with TLC separation. This method should be a useful aid for providing precise information about the physiological or pathological roles of free fatty acids.     

Composition of neutral lipids from erythrocytes of common mammals

The neutral lipids of the erythrocytes were investigated in several common mammals: cow, dog, goat, horse, pig, rabbit, rat, and sheep. Cholesterol content was determined by gas-liquid, thin-layer, and column chromatography, the last in conjunction with the IR spectrophotometry. The three methods yielded similar results. In every species investigated, cholesterol was the major neutral lipid; cholesteryl esters, triglycerides, and free fatty acids were detected only in trace amounts. It is concluded that these substances may have been contaminants from plasma lipoproteins or leukocytes rather than true constituents of the erythrocyte. In the erythrocytes of all species, cholesterol content was close to 30% of the total lipids extracted from the cells, and the molar ratio of cholesterol to phospholipid was approximately one. The significance of the data is discussed in relation to current concepts of the structure of the cell membrane involving cholesterol-polar lipid complexes.