Iron-binding lipids of rabbit duodenal brush-border membrane

Rabbit duodenal brush-border membrane contains chloroform/methanol (2:1, v/v) extractable Fe-binding lipids (27.2 +/- 6.7 nmol/mg protein, mean +/- S.E. (n = 5)). Thin-layer chromatography in two solvent systems reveals that the major Fe-binding component(s) co-migrate with free fatty acids. Fe-binding by pure lipids reveals that phosphatidic acid, phosphatidylserine, oleic and stearic acids all show apparent Fe-binding in filtration assays, although oleic acid shows the highest apparent binding (5-10-fold) on a molar basis. The free fatty acid content of brush-border membrane vesicles is sufficient to account for the chloroform/methanol extractable Fe-binding observed in vesicle preparations. The pH dependence of Fe-binding by oleic acid is similar to that reported for the detergent extractable Fe-binding lipid which has been implicated in transport of Fe from Fe/ascorbate solutions by rabbit duodenal brush-border membrane vesicles (Simpson, R.J. and Peters, T.J. (1986) Biochim. Biophys. Acta 859, 227-236).     

Galactosyltransferase activities in mitochondrial outer membrane: biosynthesis of dolichylmonophosphate-galactose

Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.     

Chemical synthesis of all-trans-beta-retinoyl phosphat.

all-trans-beta-Retinoic acid is phosphorylated to retinoyl phosphate by bis(triethylamine) phosphate with yields of 10-15%. The product is soluble in methanol and is eluted from DEAE-cellulose acetate at a concentration of 0.1M-ammonium acetate in 99% (v/v) methanol. Its phosphate/retinoic acid molar ratio is 1. Retinoyl phosphate has an absorption maximum at 360nm in methanol, whereas retinoic acid has a maximum at 350 nm. The compound is hydrolysed at pH2 and pH13 for 20 min at 37 degrees C, but is relatively stable under the same conditions at pH4, 6, 8 and 10. Retinoyl phosphate (RF 0.1) can be separated from retinyl phosphate (RF 0.2) by chromatography on thin layers of silica gel in chloroform/methanol/water (60:25:4, by vol.).     

Biosynthesis of glycoproteins by membranes of Acer pseudoplatanus. Incorporation of mannose and N-acetylglucosamine.

Membrane preparations from Acer pseudoplatanus suspension cultures were demonstrated to incorporate radioactivity from GDP-[U-14C]mannose and UDP-N-acetyl-[6-(3)H]glucosamine into high-molecular-weight polymers characterized as glycoprotein. From 20 to 25% of the 14C was incorporated as fucose with the remainder as mannose, whereas 90% of the 3H was incorporated as N-acetylglucosamine with the remainder as N-acetylgalactosamine. Pronase digestion yielded radioactive glycopeptides that were separated into four fractions by gel-permeation chromatography and paper electrophoresis. The isolated glycopeptides differed in molecular weight and isotopes incorporated, as well as in amino-acid and monosaccharide composition. The membrane preparation also incorporated radioactivity from the added nucleotides into chloroform/methanol (2:1, v/v)- and chloroform/methanol/water (10:10:3, by vol.)-soluble lipids, and into an insoluble pellet.     

The use of non-aqueous chloroform/methanol extraction for the delipidation of brain with minimal loss of enzyme activities.

When freeze-dried brain was extracted at -4-0degrees C with dry chloroform/methanol (2:1 v/v), four of the five enzymes examined were recovered in the diethyl ether-washed residue without inactivation. By contrast, extraction with chloroform/methanol (2:1 v/v) in the presence of water destroyed activities of all the enzymes examined. The amounts of major lipids extracted were similar whether extraction was done in the absence or presence of water. The study was carried out with special interest in 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37), which is firmly bound to the membrane structures of brain white matter.     

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism

The two CHCl₃ activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and β-naphthoflavone). The present results provide direct evidence that CHCl₃ concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl₃ concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10–15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl₃ only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl₃ oxidative bioactivation do not participate in CHCl₃ reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl₃ activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity. © 1997 John Wiley & Sons, Inc. J Biochem Toxicol 11: 305–312, 1997.     

Virus safety of avital bone tissue transplants: evaluation of sterilization steps of spongiosa cuboids using a peracetic acid-methanol mixture.

The aim of this study was to validate the virus-inactivating/eliminating capacity of the manufacturing process of spongiosa cuboids. Both the sterilization step with peracetic acid (PAA)/ethanol and the defatting step of bones with chloroform/methanol (2:1, v/v) were investigated. Relevant enveloped, non-enveloped, and model viruses belonging to different virus families were included in the investigation: human immunodeficiency virus type 2 (HIV-2), hepatitis A virus (HAV), poliovirus (PV-1), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine virus diarrhoea virus (BVDV). Treatment of virus-spiked spongiosa cuboids for 4 hours at room temperature (RT) with 1% PAA/24% ethanol (PES) efficiently inactivated most viruses. Titres were reduced by more than 4 log(10)with the exception of HAV. The defatting step with chloroform/methanol reduced HAV titres by a factor of >/=7.0 log(10). From these results it can be concluded that the treatment of spongiosa cuboids with (i) chloroform/methanol and (ii) 1% PAA/24% ethanol solution leads to a virus-safe medicinal product.     

Delipidization of membrane glycoproteins solubilized in acidified chloroform/methanol. Preparation of N-retinylidene opsin in a water soluble form without-detergent

Bovine retina membrane proteins and glycoproteins were insoluble in chloroform/methanol (2:1, v/v) unless the membrane suspension was precipitated with trichloroacetic acid and the organic solvent mixture added to the precipitated membranes. The presence of millimolar amount of trichloroacetic acid in the organic solvent led to the total solubilization of membranes. The glycoproteins precipitated at the interphase after partition of the acidified chloroform/methanol solution with water and were resolubilized from the interphase with chloroform/methanol/water (1:1:0.3, by vol). The solubility properties of the membrane glycoproteins in the acidified organic solvent mixtures allow to remove the bulk of membrane lipids and to recover from the chloroform/methanol/water solution the glycoprotein of rod outer segment membranes, rhodopsin, as protonated N-retinylidene opsin in a water soluble form.     

Isolation of a crustaceann-acetyl-d-glucosamine-1-phosphate transferase and its activation by phospholipids

Summary Then-acetyl-d-glucosamine-1-phosphate: dolichol phosphate transferase fromArtemia has been partially purified and characterized. The enzyme is solubilized from crude microsomes using Triton X-100, and after detergent removal appears to be associated with phospholipids. Using dolichol phosphate and UDP-n-acetyl-d-glucosamine as substrates, the enzyme catalyzes the formation of dolichol-pyrophosphate-n-acetyl-d-glucosamine. the product identity has been verified by TLC and paper chromatography following mild acid hydrolysis. Under the incubation conditions used only one product is made, i.e., Dol-p-p-GlcNAc. The formation of product is linear with increasing amounts of added protein and with time of incubation. The enzyme requires magnesium ions for activity. Activity of the enzyme is stimulated 6-fold by exogenous dolichol phosphate and is also stimulated by added phospholipids, with optimal activity being obtained in the presence of mixtures of phosphatidylcholine and phosphatidylglycerol. Enzymatic activity is not increased upon addition of GDP-mannose or dolichol phosphate mannose. The enzyme is rapidly inactivated by exposure to several detergents, including Triton X-100 and deoxycholate. The activity is inhibited by tunicamycin and by the purified B₂ homologue of this antibiotic. Other antibiotic inhibitors such as diumycin and polyoxin D have little effect on the enzyme. Both the microsomal and solubilized enzyme preparations are inactivated by 70% upon treatment with phospholipase A₂; activity may be restored by addition of phospholipids. Following hydrophobic interaction chromatography on Phenyl Sepharose, gel filtration chromatography on Sepharose CL-4B indicated that the enzyme, purified 81-fold, contained phophatidylcholine and phosphatidyl-ethanolamine.Abbreviations SDS sodium dodecyl sulfate - PMSF phenyl methanesulfonylfluoride - HEPES 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - GlcNAc N-acetyl-d-glucosamine - Dol-PP-GlcNAc dolichol pyrophosphate N-acetyl-d-glucosamine - Dol-P-man dolichol-phosphate-mannose - Dol-PP- (GlcNAc)₂ dolichol-pyrophosphate-di-N- acetylchitobiose - DMSO dimethylsulfoxide - C:M (2:1) chloroform:methanol (2:1) - C:M:W (10:10:3) chloroform:methanol:water (10:10:3) - GlcNAc-1-P N-acetyl-d-glucosamine-1-phosphate - Dol-P dolichol phosphate - EGTA ethylene glycol bis (b-aminoethyl ether)-NNNN tetraacetic acid     

Unusual fatty acid composition of cerebrosides from the filamentous soil fungus Mortierella alpina

The cerebrosides produced by the soil filamentous fungus Mortierella alpina strain KG-1/95 account for about 13% of the total polar lipids extractable from lyophilised cells with chloroform/methanol mixtures. By means of 1H NMR and (13)C NMR spectroscopy, matrix-assisted laser-desorption ionisation mass spectrometry, and chemical degradation experiment, they have been shown to be 1-O-beta-D-glucopyranosyl-2-N-(2'-D-hydroxyalkanoyl)-9-methylsphinga-4(E),8(E)-dienines, the fatty acid composition of which is unusual and consists of 2-hydroxytridecanoic (4%), 2-hydroxytetradecanoic (60%), 2-hydroxypentadecanoic (20%), and 2-hydroxyhexadecanoic (16%) acids.     

The effect of lipids on transepidermal water permeation in snakes

Transepidermal water permeation was measured for the shed skin of 8 species of snakes under conditions where the skin formed an interface between liquid water and dry air. Extraction with chloroform: methanol removed lipids amounting to 4.9-8.7% of the dry weight of skin and increased transepidermal water permeation 35- to 175-fold. Initial extraction of snake skin with hexane removed only 25-35% of the total extractable lipid but increased water permeation 3- to 10-fold. For totally delipidized skin, water-to-air permeation was much faster than air-to-air permeation, and was also faster than evaporation from a free water surface.     

Partial characterization of the embryo-derived platelet-activating factor in mice

The platelet-activating factor (PAF) produced by mouse embryos showed similar kinetics of action and dose-response curve, in a bioassay, as did 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether). The activity of the embryo-derived PAF was not affected by inhibitors of the ADP (pyruvate kinase with phosphoenol pyruvate) or cyclo-oxygenase (indomethacin) pathways of platelet activation. Chlorpromazine, an inhibitor of the PAF-acether pathway of platelet activation, caused a significant inhibition of the effects of embryo-derived PAF. Phospholipases A2, C and D significantly inhibited the activity while lipase had no effect, suggesting a phospholipid structure. All the embryo-derived PAF was found in the chloroform fraction after chloroform:methanol (2:1 v/v) extraction, as was PAF-acether. Both factors migrated at a similar rate (Rf 0.10-0.12) on silica thin-layer chromatography (chloroform:methanol:water; 65:35:4 by vol.). The embryo-derived PAF therefore displays chemical, biochemical and physiological properties similar to those of PAF-acether.     

Surface properties of membrane systems. Transport of staphylococcal delta-toxin from aqueous to membrane phase

Hemolytic delta-toxin from Staphylococcus aureus was soluble in either water, methanol or chloroform/methanol (2 : 1, v/v). The toxin spread readily from distilled water into films with pressures (pi) of 10 dynes/cm on water and 30 dynes/cm on 6 M urea; from chloroform/methanol it produced 40 dynes/cm pressure on distilled water. The toxin adsorbed barely from water (pi = 1 dyne/ cm) but it did rapidly from 6 M urea (pi = 35 dynes/cm). The protein films had unusually high surface potentials, which increased with the film pressure and decreased with increasing both pH and urea concentration in the aqueous phase. The fluorescence of 1-aniline 8-naphthalene sulfonate with delta-toxin was much greater than that with RNAase and dipalmitoyl phosphatidylcholine itself, indicating probably a marked lipid-binding character of the toxin. By circular dichroism the alpha-helix content of delta-toxin was 42% in water, 45% in methanol, 24% in 6 M urea. Infrared spectroscopy showed predominant alpha-helix in both 2H2O and deuterated chloroform/methanol as well as in films spread from either solvent on 2H2O. In spreading from 6 M [2H]urea, in which the major infrared absorption was that of [2H]urea with peaks at 1600 and 1480 cm(-1), the delta-toxin film showed prevalently non-alpha-helix structures with major peak intensities at 1633 cm(-1) > 1680 cm(-1), indicating the appearance of new beta-aggregated and beta-antiparallel pleated sheet structures in the film. The data prove that (1) high pressure protein films can consist of alpha-helix as well as non-alpha-helix structures and, differently from another cytolytic protein, melittin, delta-toxin does not resume the alpha-helix conformation in going into the film phase from the extended chain in 6 M urea; (2) conformational changes are important in the transport of proteins from aqueous to lipid or membrane phase; (3) delta-toxin is by far more versatile in structural dynamics and more surface active than alpha-toxin.     

一种简便的肌醇磷脂微量分析方法

分析磷脂酰肌醇循环(PI cycle)的磷脂组分常采用双向薄层层析法．建立了一个简单快速的单向薄层层析分离肌醇磷脂方法．首先采用不同的有机溶剂体系分别提取非多磷酸肌醇磷脂和多磷酸肌醇磷脂,然后用不同的层析展开体系,对两部分磷脂进行单向薄层层析分离．采用无载体 ³²P标记实验对该方法分离效果进行了观察．此法适用于同位素标记和非标记样品中肌醇磷脂组分的比较分析及多磷酸肌醇磷脂的提取、纯化和定量．     

Preparation of the proteolipid apoprotein from bovine heart, liver and kidney.

Proteolipid apoproteins have been prepared from heart, kidney, and liver by dialysis in chloroform/methanol against chloroform/methanol, acidified chloroform/methanol, and chloroform/methanol in succession. They are free of lipids (less than 0.05% P; less than 0.1% carbohydrate). They show a high content of non-polar amino acids, methionine, and tryptophan and contain little or no half-cystine. The differ from neural proteolipid apoproteins by absence of half-cystine, and of covalently bound fatty acids. As recovered from chloroform/methanol solutions, they are soluble in chloroform/methanol and insoluble in water, but a water-soluble form can be prepared by changing the solvent from chloroform/methanol to water in a stream of nitrogen. The chloroform-methanol-soluble form and the water-soluble form are interconvertible. ORD and CD spectra of all proteolipid apoproteins indicate 60-70% alpha-helix content in chloroform/methanol solution and 20-30% alpha-helix in water solution. Sodium dodecyl sulfate gel electrophoresis resolves proteolipid apoprotein into two major components corresponding to ca. 12 000 and 34 000 daltons. With sodium dodecyl sulfate/urea numerous bands appear, with a major one at 30 000 daltons and 8 to 10, ranging downward to 2500. For comparison, neural proteolipid apoproteins also show numerous bands with a major one at 25 000. The marked chemical and physical similarities among all proteolipid apoproteins studied suggest a common role in membrane structures.     

Structure in solution of biscyclo(dipeptide) restricted by CSSC gauche effect

Biscyclo(Cys-Sar) [I] and biscyclo(Cys-Pro) [II] were prepared by the extension of amino acid moieties from cystine. Compound I equilibrates between two rotamers around the CSSC bond of the cystine residue in methanol, showing dual negative CD transitions (CS-SC) at 270 and 255 nm, and dual S-S vibrations at 507 and 539 cm^?1 in Raman spectra. In contrast, a conformation of P-helix type is suggested in CH₃CN, which shows a distinct negative CD at 270 nm and only one Raman band at 507 cm^?1 for the S-S bond. Compound II rotates freely in dimethylsulfoxide (Me₂SO) but takes a relatively stable conformer of P-helix type in methanol, chloroform/Me₂SO (9:1), and chloroform/trifluoroacetic acid (9:1). The conformation in chloroform is retained even on addition of trifluoroacetic acid. A more complete conformation of compound II in water was determined from the negative CD of S-S transition and ¹H-nmr spectra of the Cys-β-CH₂ protons.     

The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino-acid sequence of the second light-harvestng polypeptide B 880-beta (B 870-beta) of Rhodospirillum rubrum S 1 and the carotenoidless mutant G-9+. carotenoidless mutant G-9+

The light-harvesting complex B 880 from Rhodospirillum rubrum S 1 (wild type) and B 870 from the carotenoidless mutant G-9+ was shown to consist mainly of an organic solvent-(chloroform/methanol-) soluble and an organic solvent-insoluble polypeptide. The isolation and separation of these two low-molecular-mass polypeptides (Mr 6101 and Mr 6079) were achieved by a two-step extraction procedure of chromatophores using in the first step chloroform/methanol containing 0.1M ammonium acetate. Following Sephadex LH-60 chromatography of this first extract a light-harvesting polypeptide (B 870-alpha) was isolated and its complete amino acid sequence was determined (R. Brunisholz et al. (1981) FEBS Lett. 129/1, 150-154, B 880-alpha: G. Gogel et al. (1983) Biochim. Biophys. Acta 746, 32-39). Upon reextraction of the chromatophore pellet with chloroform/methanol/ammonium acetate containing in addition acetic acid a second low-molecular-mass polypeptide (B 880-beta of B 870-beta) was generated. The complete amino acid sequences of the chloroform/methanol-insoluble light-harvesting polypeptide of Rs. rubrum S 1 (B 880-beta) and of Rs. rubrum G-9+ (B 870-beta) were determined. They are identical and consist of 54 amino acid residues. The conserved histidine residue within the hydrophobic stretch raises more evidence for ligand complexation of bacteriochlorophyll to this specific histidine residue which therefore possibly plays the key role in pigment-protein interactions. Both polypeptides (B 880-alpha and B 880-beta) are part of the light-harvesting complex B 880 in an apparent ratio of 1:1. Based on the primary structure data a possible arrangement of both light-harvesting polypeptides within the membrane will be discussed.     

Incorporation of N-acetyl-D-glucosamine from UDP-N-acetyl-D-glucosamine by isolated membranes of Bacillus subtilis. Identification of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate).

Membrane isolated from Bacillus subtilis strain 168 incorporated GlcNAc from UDP-GlcNAc directly onto undecaprenyl phosphate via transphosphorylation and subsequent transglucosylations. Chain lengths of 6, 4, and 1 units of GlcNAc were found. Approximately 80% of the isotope incorporated was extracted into chloroform:methanol (2:1 v/v), and could be distinguished from the undecaprenyl disaccharide cell wall intermediate by a different elution pattern on DEAE-cellulose (acetate form). The GlcNAc-lipid(s) were eluted from a similar column in chloroform:methanol:water (10:10:3, v/v) with 6 mM NH4COOH indicating a pyrophosphate linkage between the lipid and the GlcNAc. The GlcNAc-lipid(s) were not degraded by conditions which completely deacylated [32P]glyceryl phospholipids, but were rapidly hydrolyzed by mild acid treatment (0.005 N HCl, 90 degrees) with the release of oligosaccharide phosphate (typical of sugars linked to undecaprenyl pyrophosphate). Catalytic hydrogenation of the GlcNAc-lipid(s) resulted in the release of water-soluble sugar phosphate. Under these same conditions, undecaprenyl pyrophosphate and undecaprenyl disaccharide cell wall intermediate were similarly effected while [32P]glyceryl phospholipids remained intact. The formation of GlcNAc-lipid(s) in vitro was inhibited if membranes were prepared from cells previously treated with bacitracin. Thus, the GlcNAc-lipid(s) has the properties of undecaprenyl poly(N-acetylglucosaminyl pyrophosphate) and may represent a new synthetic role of the polyisoprenyl lipid in B. subtilis.     

Effects of heat treatment on oil-binding ability of rice flour

Heat-treated (120 °C for 120 min) rice flour showed high affinity to oil (oil-binding ability). This oil-binding ability could be observed by shaking the heat-treated rice flour (2.0 g), oil (4.0 mL), and water (20 mL) vigorously in a test tube, and the oil bound to the rice flour sank into the water. To examine the time-dependent levels of the oil-binding ability, rice flour was heat-treated at 120 °C for 10, 20, 40, 60, and 120 min, and the precipitated volume of oil/rice flour complex increased with an increase of the heating time. The oil-binding ability of the rice flour was not affected by the treatments with diethyl ether or boiled chloroform/methanol (2:1) solutions, which suggested no relationship to the oil in the rice flour, but was lost upon alkali (0.2% NaOH solution) or pepsin treatment, which suggested its relationship to the rice proteins.     

Measurement of choline and choline metabolite concentrations using high-pressure liquid chromatography and gas chromatography-mass spectrometry

We have developed a reproducible and sensitive procedure for the isolation and measurement of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, lysophosphatidylcholine and acetylcholine in a single 100-mg sample of biological tissue. Tissues were spiked with 14C-methyl- and 2H-methyl- or 15N-choline labeled internal standards for each compound. They were extracted with chloroform/methanol/water and the aqueous and organic phases were dried. The organic phase was resuspended in chloroform/methanol (1/1, v/v) and an aliquot was applied to a silica-gel thin-layer chromatography plate. The plate was developed in chloroform/methanol/water (65/30/4, v/v). Segments which cochromatographed with external standards of phosphatidylcholine and lysophosphatidylcholine were stained, scraped, and hydrolyzed in 6 M methanolic-HCl at 80 degrees C for 60 min, liberating free choline. The aqueous phase was resuspended in methanol/water and injected onto a silica HPLC column. Choline and its metabolites were eluted using a binary nonlinear gradient of acetonitrile/ethanol/acetic acid/1 M ammonium acetate/water/0.1 M sodium phosphate (800/68/2/3/127/10, v/v changing to 400/68/44/88/400/10, v/v). Peaks were detected with an on-line radiometric detector, collected, and dried under vacuum. Each choline ester was digested in 6 M HCl at 80 degrees C to form choline. Choline was then converted to the propionyl ester and demethylated with sodium benzenethiolate. This volatile derivative was then isolated using gas chromatography and measured with a mass selective detector. Deuterated internal standards were used to correct for variations in recovery. Choline, glycerophosphocholine, phosphocholine, phosphatidylcholine, lysophosphatidylcholine, and acetylcholine were measured in rat liver, heart, muscle, kidney, plasma, red blood cells, and brain and in human plasma.(ABSTRACT TRUNCATED AT 250 WORDS)