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The effectiveness of acetolysis as a tool in structural characterization of mono- and di-O-alkyl phosphoglycerides was investigated. Surprisingly, it was found that the di-O-alkyl phosphoglycerides were resistant to attack during acetolysis, whereas the mono-ether types, with a free hydroxyl function or an ester on carbon-2, were easily attacked at the glycerol-phosphate bond. On the other hand, Vitride reduction occurred readily with the mono-ether or di-ether phosphoglycerides. The implications of these findings as they relate to identification of ether phospholipids in tissues are discussed.

We have previously provided evidence for a mechanism by which acyl DHAP is converted enzymatically to O-alkyl DHAP. This mechanism involves, in part, the formation of an endiol of acyl DHAP, loss of the fatty acid by splitting of the DHAP carbon-1 to oxygen bond and the gain of a long chain fatty alcohol. It has been shown that acyl DHAP can exchange its fatty acid for one in the medium, presumably by the mediation of O-alkyl DHAP synthase. In the present ivestigation we have shown that the fatty acid which is gained by acyl DHAP in the exchange process retains both carboxyl oxygens, as predicted by our postulated mechanism. This reaction is exceptional because the usual action of acyl hydrolases is to cleave at the oxygen to acyl bond.

A facile route to the semi-synthesis of acetyl glycerylether phosphoethanolamine and, subsequently, its choline analogue (platelet-activating factor) has been developed. In essence, this technique takes advantage of the fact that the phosphatidylethanolamine fraction of bovine erythrocytes contains 75-80% of a 1-O-alkyl-2 fatty acyl derivative. Isolation of the latter by silicic acid chromatography followed by base-catalyzed methanolysis allowed good recovery (60-70%) of the 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine, which contained a mixture of long chain alkyl ethers. This compound was treated with acetic anhydride in the presence of trace amounts of perchloric acid for 45 sec to give, in excellent yield, 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoethanolamine (AGEPE). This procedure gave a 70% yield of purified AGEPE, based on the starting component, 1-O-alkyl-(lyso)-sn-glyceryl-3-phosphoethanolamine. Separation of AGEPE into fractions individually enriched in the 16:0, 18:0, and 18:1 alkylether substituents was accomplished by silica gel G combined with silver nitrate-impregnated silica gel H thin-layer chromatography. The AGEPE or its individual molecular species can be converted in high yields to the corresponding 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (AGEPC) analogues by reaction with methyl iodide in the presence of a crown ether. Characterization of the derivatives was achieved through thin-layer chromatography, infrared spectroscopy, gas-liquid chromatography, and combined gas-liquid chromatography-mass spectrometry. The ability of these analogues to induce irreversible aggregation and secretion of serotonin from washed rabbit platelets was evaluated.

Silica and C18 reverse phase high performance liquid chromatography (HPLC) were used to fractionate synthetic molecular species of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and semi-synthetic platelet-activating factor (PAF) synthesized from beef heart plasmalogens. A single coincident peak from silica HPLC was observed for either a mixture of synthetic AGEPC's with alkyl chain lengths from C12 to C18 or for beef heart-derived PAF. This peak was well separated from other classes of phospholipid standards including 2-lysophosphatidylcholine and 3H-labeled lyso-PAF. Subsequently, the synthetic AGEPC mixture or beef heart PAF was separated into individual species on a C18 reverse phase column. Beef heart-derived PAF was fractionated into at least four molecular species of PAF activity which had similar retention times as the radioactivity of 3H-labeled beef heart PAF. Approximately 56% of the radioactivity of 3H-labeled PAF was found in the fraction with a similar retention time as 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, 10% as 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, 11% as 1-O-pentadecyl-2-acetyl-sn-glycero-3-phosphocholine, and 13% in an unidentified fraction which eluted after C-16-AGEPC. The unidentified fraction did not correspond to any of the homologous series of synthetic AGEPCs with saturated alkyl chain lengths from C12 to C18. Recoveries of radioactive phospholipids from silica or reverse phase columns were greater than 95%.

Fecal neutral steroids were fractionated by high performance liquid chromatography (HPLC) into three major fractions: 5 beta-H, 3-keto steroids; 5 beta-H, 3 beta-hydroxy steroids; and 5 alpha-H and delta 5-3 beta-hydroxy steroids. This separation was achieved in about 10 minutes, with greater than 97% recovery of standards in each fraction. Gas-liquid chromatographic quantitation of fecal steroids fractionated by either HPLC or thin-layer chromatography gave nearly identical results. A method using both C18 reverse phase and silica HPLC to purify radiolabeled sterols is also described.

Methods are detailed for the preparation of [2-18O]glycolate from chloroacetic acid and for the direct conversion of these intermediates to regiospecifically labeled [2-18O]-2-O-acylglycolic acids containing approximately 90% 18O at the C-O-acyl bond. Methods are also detailed for optimization of reaction conditions and yields for each synthetic step in previously published methods for the preparation of 1-O-acyldihydroxyacetone-3-O-phosphate (DHAP) from acyloxyacetic acid (i.e., 2-O-acylglycolic acid), where acyl is tetradecanoyl, hexadecanoyl, or heptadecanoyl. The optimized reaction conditions generate 1-O-acyl DHAP in its acid form, both in high overall yield and in high purity, without requiring a final chromatographic purification of the product, 1-O-acyl DHAP. Combining these new methods, efficient and facile preparations of regiospecifically labeled [1-18O]-1-O-hexadecanoyl DHAP and [1-18O]-1-O-heptadecanoyl DHAP have now been demonstrated, in which approximately 90% 18O is specifically located only at the C-O-acyl position. Some mechanistic postulates are offered to account for the optimized yields, regioselectivities, and high 18O incorporation which are observed in the reactions we have employed to generate 1-O-acyl DHAP from glycolate intermediates.

The mechanism of fragmentation in the chemical ionization mass spectra of thermally demethylated choline esters has been investigated using various deuterated analogs of acetylcholine as models. The reagent gases employed included CH4, C2H4, NH3 and N2H3. Prominent [MH]+ or [M2H]+ ions were observed respectively in all cases. A 1,3-hydrogen rearrangement in the acetyl portion followed by loss of ketene was seen in CH4 chemical ionization spectra, as well as cleavage of the CH2N bond after protonation on the nitrogen. Alpha cleavage produced ions analogous to the m / z 58 ion which is found as the base peak in the electron impact spectra of demethylated choline esters. A major difference between the fragmentation after electron impact and chemical ionization was seen in the m/z 72 region. The McLafferty rearrangement observed after electron impact was not found after chemical ionization. Rather, a mechanism involving protonation of the carboxyl group followed by simple cleavage of the CH2O bond was consistent with the present results for the variously labelled analogs and reagent gases. Finally, in the chemical ionization mass spectra of demethylated acetylcholine little retention of the H+ or 2H+ was found in the observed fragment ions.

Deficiency of biotin at the tissue level can be assessed indirectly by measuring the urinary excretion of 3-hydroxyisovaleric acid. This paper describes the application of an improved method of quantifying urinary 3-hydroxyisovaleric acid using unlabeled and uniformly deuterated 3-hydroxyisovaleric acid. These compounds were synthesized by a modification of the lithioacetic acid method for generation of beta-hydroxy acids. Elemental analysis, nuclear magnetic resonance, gas chromatographic and gas chromatographic/mass spectrometric data demonstrated that the compounds are greater than 95% pure. Mass spectrometry confirmed the identity of the unlabeled compound, demonstrated that the deuterated compound is uniformly labeled, and offered insight into the pattern of mass fragmentation. The method for determination of the concentration of 3-hydroxyisovaleric acid in rat urine uses gas chromatographic/mass spectrometric quantification of the di-trimethylsilyl derivative with the deuterated compound as the internal standard. Results provide evidence that this method is more accurate than a previously published method that did not utilize the unlabeled and deuterated standards.

Norepinephrine, dopamine and serotonin concentrations were measured in mouse whole brain. Animals were killed either by decapitation or by exposure to 250 msec microwave irradiation which produces irreversible inactivation of brain enzymes. The biogenic amines were determined by mass fragmentometry, fluorometry and by a combination of high performance liquid chromatography and an electrochemical detector. No differences were found in the levels of these neurochemicals between the two methods of animal sacrifice. Therefore, rapid inactivation of brain enzymes is not necessary prior to analysis for catecholamines and serotonin in mouse whole brain.

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.

The virion proteins of Pseudoalteromonas phage φRIO-1 were identified and quantitated by mass spectrometry and gel densitometry. Bioinformatic methods customized to deal with extreme divergence defined a φRIO-1 tail structure homology group of phages, which was further related to T7 tail and internal virion proteins (IVPs). Similarly, homologs of tubular tail components and internal virion proteins were identified in essentially all completely sequenced podoviruses other than those in the subfamily Picovirinae. The podoviruses were subdivided into several tail structure homology groups, in addition to the RIO-1 and T7 groups. Molecular phylogeny indicated that these groups all arose about the same ancient time as the φRIO-1/T7 split. Hence, the T7-like infection mechanism involving the IVPs was an ancestral property of most podoviruses. The IVPs were found to variably host both tail lysozyme domains and domains destined for the cytoplasm, including the N4 virion RNA polymerase embedded within an IVP-D homolog.

To investigate the apparent genomic complexity of long-genome bacteriophages, we have sequenced the 218,948-bp genome (6479-bp terminal repeat), and identified the virion proteins (55), of Bacillus thuringiensis bacteriophage 0305phi8-36. Phage 0305phi8-36 is an atypical myovirus with three large curly tail fibers. An accurate mode of DNA pyrosequencing was used to sequence the genome and mass spectrometry was used to accomplish the comprehensive virion protein survey. Advanced informatic techniques were used to identify classical morphogenesis genes. The 0305phi8-36 genes were highly diverged; 19% of 247 closely spaced genes have similarity to proteins with known functions. Genes for virion-associated, apparently fibrous proteins in a new class were found, in addition to strong candidates for the curly fiber genes. Phage 0305phi8-36 has twice the virion protein coding sequence of T4. Based on its genomic isolation, 0305phi8-36 is a resource for future studies of vertical gene transmission.

Direct derivatization of the acyl analogue of platelet-activating factor (acyl-PAF) with heptafluorobutyric anhydride results in replacement of the phosphocholine moiety with a heptafluorobutyryl (HFB) group. Electron capture (EC) mass spectrometric analysis of this compound that makes use of negative ion detection along with subsequent accurate mass measurement and tandem mass spectrometry studies revealed that in addition to expected fragmentation due to losses of elements of HF, ketene, and/or acetic acid, there is a rearrangement reaction between the HFB group and the substituent on carbon-2 of the glycerol backbone. For 2-acetyl isomers, this fragmentation yields a characteristic ion at m/z 237; for 1-acetyl isomers, the analogous ion is observed at [M-135]−, along with a corresponding carboxylate anion. The use of the HFB derivative is invaluable for analysis of PAF homologues and analogues because it provides detailed structural information in combination with the high sensitivity of a gas chromatography combined with EC-mass spectrometry assay.

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-alpha. Substantial increases of phosphorylation were observed for Thr(446) upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser(1078). This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C16:0 alkyl chain in the sn-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C15- and C18-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.

Fast atom bombardment mass spectrometry was used to identify molecular species of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes. Normal and reverse-phase high performance liquid chromatography were employed to separate the individual regions with PAF activity prior to mass spectrometric analysis. The following alkyl chain homologs of acetyl glyceryl ether phosphorylcholine (AGEPC) were found: C16:0, C17:0, C18:0 and C18:1. There was also evidence for the presence of the C15:0 homolog, as well as other species which have not yet been identified.

The development of sensitive analytical techniques for use in the identification and qualification of molecules of biological origin remains a constant challenge. Often the compounds of interest are present in a complex mixture and require extensive sample preparation prior to analysis. Thermospray liquid chromatography/mass spectrometry (LC/MS) has been shown to be a method which allows for both separation of target molecules from complex mixtures and sensitive specific detection using a mass spectrometer. In this work thermospray LC/MS is used for the direct detection of intact, underivatized choline and acetylcholine from mouse brain homogenate. The results of our analysis corroborate previous analyses using a variety of indirect measurement techniques and thereby show that this simple direct analysis has wide potential applicability.

The human T cell lymphotrophic virus type I (HTLV-I) has recently been identified in a T cell lymphoma associated with hypercalcemia and increased bone turnover. Since increased serum concentrations of 1,25-dihydroxyvitamin D have been reported in this disease, we have examined the capacity of HTLV-I-infected cord blood lymphocytes to metabolize 25-hydroxyvitamin D3. Our results demonstrate that HTLV-I-infected cells have the capacity to metabolize 25-hydroxyvitamin D3 to a substance that co-migrates with 1,25-dihydroxyvitamin D3 by high performance liquid chromatography over a silica column using either 12% isopropanol in hexane or 5% isopropanol in dichloromethane. The metabolite binds to the 1,25-dihydroxyvitamin D3 receptor in rat osteosarcoma cells and stimulates bone resorption in cultures of fetal rat long bones. Mass spectrometric analysis of the metabolite confirmed the presence of 1,25-dihydroxyvitamin D3. Production of 1,25-dihydroxyvitamin D by lymphoma cells may contribute to the pathogenesis of the hypercalcemia seen in patients with HTLV-I-associated T cell lymphomas.

We have compared the competitive inhibitory effects of 2-deoxyglucose, glucosamine, N-acetylglucosamine, N-benzoylglucosamine, and the commonly used radiographic and density gradient agent metrizamide (2-[3 - acetamido - 2,4,6 - triiodo -5-(N- methylacetamido) benzamido]-2-deoxyglucose) on the mitochondrial and soluble forms of human brain hexokinase. Metrizamide produces a classical competitive inhibition with glucose for human brain hexokinase, with Kis of 2.8 and 2.5 mM, respectively, for the mitochondrial and soluble forms. Glucosamine exhibited Kis of 0.58 and 0.29 mM, while 2-deoxyglucose exhibited Kis of 0.074 and 0.15 mM and N-acetylglucosamine 0.098 and 0.092 mM for these two forms, respectively. N-Benzoylglucosamine was by far the most effective inhibitor tested, with Ki values of 0.0086 and 0.022 mM, respectively. In order of increasing potency as a competitive inhibitor for mitochondrial hexokinase are metrizamide, glucosamine, N-acetylglucosamine, 2-deoxyglucose, and N-benzoylglucosamine. For the soluble form of the enzyme in increasing potency are metrizamide, glucosamine, 2-deoxyglucose, N-acetylglucosamine, and N-benzoylglucosamine. Since N-benzoylglucosamine was over 100 times more potent than metrizamide, some of the effects of metrizamide could be due to contamination by N-benzoylglucosamine. However, gas chromatography-mass spectrometry analysis of metrizamide did not indicate the presence of N-benzoylglucosamine. In addition, column chromatographic separation of commercially available metrizamide and reconstitution of freeze-dried eluate fractions localized the inhibitory effect to the metrizamide peak.

It was first noted by Schweitzer and Wright that acetylcholine (ACh) might be involved in carotid body chemoreception, but it was Eyzaguirre and co-workers (4) who later studied this problem systematically and concluded that ACh might be a sensory transmitter released from the glomus (type I) cells to excite neighboring afferent nerve terminals. However, ACh has never been chemically identified in carotid body tissue, although bioassays have suggested the presence of an ACh-like substance in carotid body extracts (5,8). Furthermore, Osborne and Butler (9), proceeding from Biscoe’s (1) suggestion that the synapses on the glomus cells are efferent, not afferent, have recently proposed that ACh is the transmitter at this junction and that consequently this substance is contained in the nerve terminals, not in the glomus cells.

The chemistry and selected biological activity of lipopolysaccharide (LPS) from Bacteroides gingivalis strains W50, W83, and ATCC 33277 were compared, as well as the role of this molecule as a mediator of selected inflammatory responses. Chemically, the LPSs consisted of 47-58% Lipid A, 5-10% carbohydrate, 0.05% 3-deoxy 2-octulosonic acid, 0.3% heptose, 3.8-5.2% hexosamine, and 2% phosphate. Rhamnose represented the dominant sugar (26-36%), with lesser amounts of glucose (18-34%), galactose (18-25%), mannose (9-12%), glucosamine (7-11%), and galactosamine (2-5%). The major fatty acids were: 13-methyl-tetradecanoate (42-45%), 3-OH-heptadecanoate (21-23%), hexadecanoate (16-19%), and 12-methyl-tetradecanoate (6-8%). SDS-PAGE and sodium deoxycholate-PAGE revealed the LPS to be a smooth chemotype. Differences in migration patterns between the virulent and avirulent strain LPSs also occurred. C3H/HeN macrophages (Mo) exposed to 1 microgram/ml of LPS released 3.2-4.2 ng of prostaglandin E (PGE)/ml of supernatant, representing 236-278% of control. Interleukin-1 (IL-1) activity in C3H/HeN and C3H/HeJ Mo exposed to 50 micrograms of LPS/ml was 382-724% and 270-300% of control, respectively; similar Mo exposed to 10 micrograms of LPS/ml released 1.6-2.0 ng and 0.3-0.5 ng of tumor necrosis factor (TNF)/ml of supernatant, respectively. Maximum TNF release in C3H/HeN Mo occurred in response to 50 micrograms of LPS/ml, and was sustained for up to 96 hours. These results suggest that LPS from the B. gingivalis strains stimulate cytokine production from Mo which, in turn, may play a role in orchestrating the inflammatory response for the development of periodontal diseases.

Summary Both NO and N2O were identified by GC/MS as gaseous products of nitrite reduction catalyzed by the cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa . Gas production was inhibited by cyanide. The enzyme also catalyzed the reduction of NO to N2O. No N2 was identified as a consequence of either nitrite or NO reduction.

– The time course for accumulation of acetylcholine was measured in rat brain regions after treatment with 15 mg/kg, i.v., dichlorvos. With this dose of dichlorvos 84-96% of the brain cholinester-ase is inhibited within 1 min. After killing and concomitant enzyme inactivation through microwave irradiation, the acetylcholine levels were measured by pyrolysis-gas chromatography. In the brain regions studied, the striatum had the highest rate of accumulation of acetylcholine and the cerebellum had the lowest. The calculated turnover time in minutes for the regions of the brain were cerebral cortex 0.9; hippocampus 1; striatum 1.4; cerebellum 1.7; medulla-pons 2.2; midbrain 4.5; thalamus 5.6.

The effect of chronic lead ingestion on the central cholinergic system of immature rats was investigated. Rats were given 1% lead acetate solution in the drinking water before and after weaning. Tap water and sodium acetate controls were used. The growth of rats treated with lead was significantly inhibited as compared to controls. After chronic lead ingestion, a significant decrease in cholinesterase activity was seen in the medulla-pons, midbrain, and diencephalon, and a significant increase in choline acetyltransferase activity was found in the medulla-pons, hippocampus, and cerebral cortex. The only brain region exhibiting a change in acetylcholine content after chronic lead administration was the diencephalon, where a significant increase was observed. Thus, significant changes in the central cholinergic system have been found after chronic lead ingestion.

— Heating with 2450 MHz microwave radiation has been investigated as a means for animal sacrifice concurrent with enzyme inactivation. Uniform inactivation of cholinesterase (EC 3.1.1.8) in the entire brain can be effected in the rat within 4 s and in the mouse within 2 s without destruction of acetylcholine. The acetylcholine content in the whole brain of a rat was found to be 25.4 ± 1.5 nmol/g after irradiation, in comparison to 13.8 ± 1.7 nmol/g after standard methods of sacrifice. In the mouse whole brain, the comparable acetylcholine contents were 25.5 ± 2.6 and 13.7 ± 1.7 nmol/g, respectively. The value of this procedure for rapid inactivation of enzymes in the study of acetylcholine turnover is discussed.

Previously we found that Ho:YAG laser (2120 nm) lithotripsy of uric acid stones produced cyanide, a known thermal breakdown product of uric acid. We now report that alloxan, another thermal breakdown product, is also likely produced. Uric acid stones (approximately 98% pure) of human origin were placed in distilled water and subjected to one of the following experimental treatments: unexposed control, exposed to Ho:YAG laser, Nd:YAG laser, or mechanically crushed. Samples were then processed for HPLC analysis with UV detection. Peaks were identified by comparison to authentic standards. All samples contained uric acid, with retention time (RT) about 6 min. All of the laser-exposed samples contained a peak that eluted at 2.5 min, identical to the RT of authentic alloxan. Ho:YAG laser irradiation, however, produced a larger presumed alloxan peak than did the Nd:YAG laser. The peak at 2.5 min, as well as unidentified later-eluting peaks, were present in the laser-exposed, but not the unexposed or mechanically crushed, samples. These results confirm the thermal nature of lithotripsy performed with long-pulse IR lasers.