Determination of non-volatile N-nitrosamines in baby bottle rubber nipples and pacifiers by high-performance liquid chromatography-thermal energy analysis.

A method is described for the determination of non-volatile N-nitrosamines in baby bottle rubber nipples and pacifiers. It consists of extraction of the sample with dichloromethane in the presence of ascorbyl palmitate (an inhibitor of artifactual formation of nitrosamines), clean-up on silica or basic alumina, and final analysis by high-performance liquid chromatography-thermal energy analysis, a technique which is highly specific for N-nitroso compounds. The method worked well for the determination of four rubber-related non-volatile nitrosamines, namely, N-nitrosomethylphenylamine, N-nitrosoethylphenylamine, N-nitrosodicyclohexylamine, and N-nitrosodibenzylamine (recoveries from spiked samples greater than 80%; detection limit, ca. 5 micrograms/kg for each). Eighteen out of twenty four samples analyzed were found to contain varying levels (mean, 41 micrograms/kg; range, 8-146 micrograms/kg) of N-nitrosodibenzylamine. The identity of the compound was confirmed by gas chromatography-thermal energy analysis as well as by gas chromatography-mass spectrometry analyses.

Improved method for determination of volatile nitrosamines in baby bottle rubber nipples and pacifiers.

A method is described for determining volatile nitrosamines in baby bottle rubber nipples and pacifiers. The method consists of overnight soaking of the sample with dichloromethane in the presence of propyl gallate, an inhibitor for artifactual formation of nitrosamines; extraction with dichloromethane using an ambient temperature column procedure; distillation from aqueous alkali; extraction of the aqueous distillate with dichloromethane and concentration of the extract using a Kuderna-Danish concentrator; and final analysis by gas chromatography with detection by thermal energy analyzer. Major improvements over other published methods include a simpler extraction, a more efficient distillation and concentration to minimize losses, and incorporation of propyl gallate inhibitor as well as of a test to detect unsuitable dichloromethane solvent that otherwise might lead to artifactual formation. The method is highly accurate (approximately 90% recovery) and precise (av. CV = 4.7% at greater than 9 ppb levels) and is applicable to determination of nitrosamines in both rubber- and plastic-based products.

Polar lipids in phototrophic bacteria of the Rhodospirillaceae and Chromatiaceae families.

The polar lipids of photosynthetic purple bacteria of the genera Chromatium, Thiocapsa, Thiocystis, Ectothiorhodospira, Rhodopseudomonas, Rhodospirillum, and Rhodomicrobium were analyzed. Characteristic compositions of the polar lipids were found for most of the Rhodospirillaceae and Chromatiaceae species. Phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin were the major phospholipids in most species. Phosphatidylcholine was present as a major component in all species of the genus Ectothiorhodospira, but was not detected in the remaining Chromatiaceae. It was also present in most of the Rhodospirillaceae species. No glycolipids were found in any of the Ectothiorhodospira species. In the Rhodospirillaceae, the glycolipids mono- and digalactosyl diglycerides were generally absent. Sulfoquinovosyl diglyceride was present in significant amounts in at least three species of the Rhodospirillaceae and may have been present in most of them, but only in traces. All of the Chromatiaceae species contained several glycolipids, one of which was similar to monogalactosyl diglyceride. Ornithine lipids were found in large amounts in most Rhodospirillaceae, but were absent in Ectothiorhodospira and in the other Chromatiaceae. The species examined could be divided into three groups on the basis of their lipid composition: (i) the genus Ectothiorhodospira; (ii) the remaining Chromatiaceae; and (iii) the Rhodospirillaceae. The data presented are compared with those available in the literature, and differences from other phototrophic organisms are discussed.

Novel polar lipids from the methanogen Methanospirillum hungatei GP1.

The methanogenic bacterium Methanospirillum hungatei GP1 has been shown to contain two unusual phosphoglycolipids (phosphoglycolipid I and phosphoglycolipid II) that account for 64% (by wt.) of the total cellular lipids. These lipids are derivatives of the dibiphytanyldiglycerol tetraether. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid and the other is linked glycosidically to a disaccharide with structure alpha-Glcp-(1 leads to 2)-beta Gal phi in phosphoglycolipid I and beta-Gal phi-(1 leads to 6)-beta Gal phi in phosphoglycolipid II. Smaller amounts of the sn-2,3-diphytanylglycerol analog of phosphatidylglycerol and diglycosyldiphytanylglycerol ethers (DGD-I and DGD-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively, were identified, together with very small amounts of diglycosyldibiphytanyldiglycerol tetraethers (DGT-I and DGT-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively. A biosynthetic pathway involving head-to-head condensation of phosphatidylglycerol with DGD-I or DGD-II to form phosphoglycolipid I or phosphoglycolipid II, respectively, is proposed.

Novel complex polar lipids from the methanogenic archaebacterium Methanospirillum hungatei.

The methanogenic archaebacterium Methanospirillum hungatei contains two unusual phosphoglycolipids that account for 64 percent of the total cellular lipids. These lipids are derivatives of the dibiphytanyl diglycerol tetraether, previously identified in methanogens. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid, and the other is linked glycosidically to a disaccharide. The two phosphoglycolipids may function as covalently bonded lipid bilayers to impart stability and rigidity to methanogen membranes.

The lipids of Halobacterium marismortui, an extremely halophilic bacterium in the Dead Sea.

The lipids of an extremely halophilic bacterium, Halobacterium marismortui, isolated from the Dead Sea, were found to contain 86% polar lipids and 14% non-polar lipids. Four major polar lipids were detected, all derivatives of 2,3-di-O-phytanyl-sn-glycerol: (1) a novel glycolipid, 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl-(1'-6')-O-alpha-D-mannopyranosyl-( 1'-2')-O-alpha-D-glucopyranosyl]-sn-glycerol (11 mol%); (2) phosphatidylglycerol (11 mol%); (3) phosphatidylglycerophosphate (62 mol%); (4) phosphatidylglycerosulfate (17 mol%). In addition, a minor glycolipid (less than 1 mol%) was detected and partially characterized. Trace levels of two other unidentified glycolipids and of two unidentified phospholipids were also detected. In contrast to Halobacterium cutirubrum and H. halobium, H. marismortui did not contain any detectable sulfated glycolipid but appeared to compensate for this deficit in sulfate by having a high content of phosphatidylglycerosulfate compared to that in H. cutirubrum. The number of negative changes per mol ionic lipid appeared to be about the same for both halophiles. The non-polar lipids in H. marismortui consisted mostly of squalenes, vitamin MK-8 and bacterioruberins with traces of beta-carotene, lycopene and retinal, as in H. cutirubrum.

Exclusive formation of all-trans-phytoene by a colorless mutant of Halobacterium halobium.

A spontaneously occurring Halobacterium halobium mutant (strain W5002-1) was isolated which accumulated phytoene as the only C40 polyene. Its configuration was established as all-trans (7,8,11,12,7',8',11',12',-octahydro-psi,psi-carotene) by its chromatographic and spectral properties. Neither bacterioruberins (C50) nor retinal (C20) could be detected in this mutant strain. The biosynthetic pattern of isoprenoid compounds in this mutant strain confirms for the first time our conjecture that spontaneously arising H. halobium mutants can be isolated with defects in the carotenoid pathway and lends further support to our previous hypothesis suggesting all-trans-phytoene to be the precursor for more unsaturated carotenoids in halobacteria.

Effect of glycerol on carotenogenesis in the extreme halophile, Halobacterium cutirubrum.

Growth of cells of Halobacterium cutirubrum in complex medium in the presence of 0.1 to 0.5% glycerol was slightly stimulated (about 10%), but the content of the major red pigment, bacterioruberin, was reduced fourfold and that of the minor red pigments, mono- and bisanhydrobacterioruberins, was also reduced but to a lesser extent. The content of the C40 carotenes, beta-carotene and lycopene, however, was greatly increased in 0.1 to 0.5% glycerol but was then reduced to negligible amounts at higher glycerol concentrations (1 to 5%). Similar effects of glycerol on carotenogenesis were observed when cells were grown in a chemically defined medium (BSMK), but growth of cells was considerably slower.

Studies of the biosynthesis of C50 carotenoids in Halobacterium cutirubrum.

Cells of Halobacterium cutirubrum were grown in the presence of [14C]mevalonate with and without 3 mM nicotine. Growth of cells was inhibited to a maximum extent of 25% but overall incorporation of 14C into total, neutral, or polar lipids was only inhibited about 16% during active growth and to a much lesser extent or not at all in stationary phase. Little effect of nicotine on labelling of squalenes, vitamin MK-8, geranylgeraniol, and phytoene was observed. However, labelling of bacterioruberin and monoanhydrobacterioruberin was extensively inhibited while that of lycopene and bisanhydrobacterioruberin was increased reciprocally by growth in the presence of 3 mM nicotine. Reincubation of the labelled nicotine-grown cells in fresh cold medium without nicotine restored the 14C-labelling of bacterioruberin and monoanhydrobacterioruberin at the expense of lycopene and bisanhydrobacterioruberin. These results confirm our previous findings suggesting that the C50 bacterioruberin is made by addition of a C5-isoprene unit to each end of the C40-lycopene chain, followed by introduction of four hydroxyl groups.

Occurrence of indole in cells of extremely halophilic bacteria.

The neutral lipids of several halophilic bacteria contain a non-isoprenoid compound which was identified by its chromatographic and spectroscopic properties as indole.

Lipid composition of Neurospora crassa.

The lipids of Neurospora crassa, isolated in pure form from freeze-dried mycelium, were found to contain squalene, sterol esters, triglycerides, free fatty acids, geranylgeraniol, free sterols, carotenoids, cardiolipin, phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, and phosphatidic acid. The above compounds were isolated in pure form by column and thin layer chromatography and were characterized by infrared spectroscopy and chromatographic mobilities. Fatty acid moieties were characterized by gas liquid chromatographic retention times of their methyl esters relative to those of authentic standards. The fatty acid composition of the triglycerides was found to be similar to that of phosphatidic acid, cardiolipin, and lecithin.

Enzymatic synthesis of C40 carotenes by cell-free preparation from Halobacterium cutirubrum.

[14C]Mevalonate or (14C)isopentenyl pyrophosphate was found to be converted to transphytoene, trans-phytofluene, lycopene, and beta-carotene by a cell-free 270 000 X g supernatant fraction prepared from Halobacterium cutirubrum cells that were broken by manual grinding with glass beads. Incubations were done under N2 in the dark at 37 degrees C in 4 M NaCl in presence of FAD, NADP, and MgCl2; ATP was also added when mevalonate was the substrate. This system was also capable of converting trans-(14C)phytoene to beta-carotene via the intermediates trans-phytofluene, zeta-carotene, neurosporene, lycopene, and gamma-carotene. Each of these labelled intermediates on incubation separately with the same enzyme system was shown to be converted to the intermediates farther down the pathway. The results of this study show that the biosynthetic pathway for the formation of C40 carotenes in H. cutirubrum proceeds as follows: isopentenyl pyrophosphate leads to trans-phytoene leads to trans-phytofluene leads to zeta-carotene leads to neurosporene leads to lycopene leads to gamma-carotene leads to beta-carotene. This pathway differs from that in higher plants in that the cis isomers of phytoene and phytofluene are not on the main pathway of carotene biosynthesis, as they are in higher plants. Furthermore, trans-phytoene, which has not been reported to have any role in higher plants, appears to be the main intermediate in carotene biosynthesis in H. cutirubrum.

Effect of nicotine on biosynthesis of C50 carotenoids in Halobacterium cutirubrum.

The major carotenoids in pigmented extreme halophiles are the hydroxylated C50 red pigments bacterioruberin (major, 84%) and monoanhydrobacterioruberin (minor, 14%). When cells of Halobacterium cutirubrum were grown in the presence of nicotine they accumulated lycopene and bisanhydrobacterioruberin; maximal accumulation (80% lycopene, 20% bisanhydrobacterioruberin) occurred at 3mM nicotine. Removal of nicotine resulted in the reformation of monoanhydrobacterioruberin and bacterioruberin at the expense of lycopene and bisanhydrobacterioruberin. Thus nicotine does not inhibit the addition of the two extra isoprene units plus two hydroxyl groups (at C1 and C1') to a C40 carotenoid skeleton. It does, however, inhibit the hydration steps at C3'' and C3''' that convert bisanhydrobacterioruberin to monoanhydrobacterioruberin and finally bacterioruberin. These results support the view that the C50 carotenoids can be formed from a C40 carotene, lycopene.

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.

Nonpolar lipids of a halotolerant species of Staphylococcus epidermidis.

The nonpolar lipids of a halotolerant Staphylococcus epidermidis, isolated in pure culture from a growth medium for extreme halophiles containing 25% sodium chloride, were found to contain squalene, menaquinone-7, free fatty acids (mainly anteiso-15:0 and anteiso-17:0), undecaprenol, nonaprenol with predominately cis-isoprene residues, heptaprenol, with predominately trans-isoprene residues, and 1,2- and 1,3-diglycerides containing anteiso- 15:0 and anteiso-17:0 branched chain fatty acid residues. The above compounds were isolated in pure form by column and thin-layer chromatography and were characterized by ultraviolet, proton magnetic resonance, and mass spectra. Fatty acid moieities were characterized by gas-liquid chromatographic retention times of their methyl esters.

Isolation and characterization of C50-carotenoid pigments and other polar isoprenoids from Halobacterium cutirubrum.

The polar acetone-soluble lipids of Halobacterium cutirubrum were found to contain (in addition to the previously reported vitamin MK-8 and retinal) neo-bacterioruberin U, bacterioruberin, monoanhydrobacterioruberin, bis-anhydrobacterioruberin, an isomer of geranylgeraniol (with one internal cis-isoprene residue), 2,3,-di-O-phytanyl-sn-glycerol and two unidentified polar isoprenoids. All compounds were isolated in pure form by column and thin-layer chromatography, quantitated and characterized by their visible, ultraviolet, infrared, proton magnetic resonance and mass spectra and the spectra of their acetyl or silyl derivatives and/or dehydrated products.

Characterization and composition of the purple and red membrane from Halobacterium cutirubrum;.

The purple membrance (bacteriorhodopsin) isolated from cells of Halobacterium cutirubrum grown anaerobically in the light was shown to contain 77% protein and 20% lipils by weight. The protein component consisted of a single protein moeity, having a molecular weight of (19.6 plus or minus 0.8) times 10-3, complexed with retinal in mole ratio of 2:1, respectively. The protein moeity is not glycosylated but may be phosphorylated (ca. 2 mol of phosphate per mole of protein). The red membrance contains 56%protein and 38% lipids, including bacterioruberins. Several polypeptide components are present including some which may be glycosylated and/or phosphorylated. The lipids of both membranes contained phosphatidyl glycerophosphate (52%) and phosphatidyl glycerol (3-4%) but the sulfated lipid components, glycolipid sulfate and phosphatidyl glycerosulfate, were present exclusively in the purple membrane, the red membrane containing instead two unidentified glycolipids. Neutral lipids (squalenes, vitamin 75-8, etc.) were present in both membranes to the extent of 7-9%.