Derivatization of the mutagen MX (3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone) with butyl alcohols prior to GC-MS analysis.

An extremely potent mutagen, 3-chloro-4(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is commonly present in chlorinated drinking water. Due to its high mutagenic activity and according to WHO guidelines its concentration should be controlled in drinking waters. Determination of MX is difficult due to the low (ppt) levels at which the compound usually exists in drinking waters. Results obtained with butanols as MX derivatization agents are shown and derivatization with sec-butanol is presented as a method which significantly lowers GC/MS detection levels of MX.

Changes in the phospholipid composition of the arterial cell can result in severe atherosclerotic lesions.

The oxysterol concentration in the plasma and the phospholipid composition of vascular tissue obtained by coronary artery bypass grafting (CABG) were compared with plasma and vascular tissue from age and sex matched controls. The plasma from CABG patients had a higher concentration of oxysterols than was present in the controls. Human endothelial cells were cultured for 72 hours in a medium containing plasma obtained from CABG patients, from controls or from the same controls to which 5 oxysterols were added to make the total oxysterol level equivalent to that in the CABG plasma and then pulsed with calcium (45Ca(2+)) for one hr. A significantly higher influx of 45Ca(2+) was noted in the endothelial cells cultured in the plasma obtained from CABG patients and from the controls with 5 added oxysterols, but not in those cultured without added oxysterols indicating that oxysterols increased calcium influx into endothelial cells. A phospholipid analysis indicated that the arterial tissue from CABG patients had 48.2% sphingomyelin in its phospholipid fraction compared to 10% in arterial tissue from umbilical cords. The saphenous vein obtained during CABG surgery from the same patient had only 24% sphingomyelin in its phospholipid fraction and unlike the coronary arteries had no atherosclerotic lesions. The higher level of oxysterol in the plasma of patients suffering from severe atherosclerosis could increase the concentration of sphingomyelin in the arterial cell membrane and thereby increase calcium influx required for producing the calcific type VII lesions in the coronary arteries.

Headspace solid-phase microextraction use for the characterization of volatile compounds in vegetable oils of different sensory quality.

Headspace solid-phase microextraction (HS-SPME) was used to isolate the volatile compounds, which are formed during peroxidation of fatty acids in vegetable oils. Isolated compounds were characterized by GC-MS and quantified using GC with FID detection. Four fibers for HS-SPME method development were tested, and the divinylbenzene/carboxene/PDMS fiber was selected as providing the best detection of analyzed compounds. Extraction curves, limits of detection, repeatability, and linearity were investigated for 14 aldehydes, ketones, hydrocarbons, and alcohols being products of fatty acids autoxidation. Limits of detection for 11 of these were below 1 microg/L. For quantitative purposes, to minimize the influence of temperature on hydroperoxide formation and the changes in the volatiles profile of the extracts, sampling was performed at 20 degrees C. For compound characterization by GC-MS, sampling temperature of 50 degrees C was applied. The developed method was applied to the analysis of refined and cold-pressed rapeseed oil stored at 60 degrees C for 10 days, and for 10 different vegetable oils of various degree of peroxidation. All samples were subjected to sensory analysis. The results of PCA sensory analysis were related to the amount of volatile compounds isolated by SPME method. In cases where the amount of compounds was highest, the samples were perceived as the worst, whereas those with low levels of volatile compounds were the most desired ones according to sensory evaluation. The relation was observed for both total volatiles, quantified C5-C9 aldehydes, and 14 compounds selected in method development. SPME revealed to be a rapid and sensitive method for the extraction and quantitation of trace volatile compounds from plant oils even at ambient temperature.

Determination of cholesterol oxidation products in milk powder and infant formulas by gas chromatography and mass spectrometry.

In this paper a method for the cholesterol oxidation products (oxysterols) determination in milk powder and infant formulas has been presented. In the sample preparation step lipids transesterification has been performed. The recoveries of oxysterols have been determined and ranged from 94.2% to 99.9% for all but 20a-hydroxy cholesterol (74.2%). Detection limits were 0.018-0.034 ppm and the relative standard deviations (RSD) values were 4.6%-18.3%. The method has been utilized for the determination of oxysterols in milk-based infant formulas and milk powder available on the market. The concentration of oxysterols was between 0.04 and 4.20 ppm of a lipid extract fraction.

An excess concentration of oxysterols in the plasma is cytotoxic to cultured endothelial cells.

To test if there is an excess concentration of oxysterols in the plasma of the patients with cardiovascular disease, we analyzed the oxysterol content in the plasma from 105 cardiac catheterized patients with angina and 80+/-8% stenosis in their coronary arteries. The result showed that the plasma contained a significantly higher concentration of oxysterols than did plasma from 105 age- and sex-matched, non-catheterized and angina-free controls (P<0.05). We used endothelial cells (ECs) cultured in medium containing either [3H]thymidine, [3H]mevalonolactone or 45Ca(2+) to determine how the plasma from the patients influences cell growth and function. We found that less [3H]thymidine (P<0.05), less [3H]mevalonolactone (P<0.05) and more 45Ca(2+) (P<0.001) was incorporated into ECs cultured in the plasma from 36 patients with 83+/-4% stenosis than from the 36 controls. When synthetic 7beta-hydroxycholesterol, cholesterol 5beta,6beta-epoxide, cholesterol 5alpha,6alpha-epoxide and 7-ketocholesterol were added to the plasma from the controls, the influx of 45Ca(2+) into ECs then equaled that in the plasma of patients. The enhanced incorporation of 45Ca(2+) into the ECs cultured in the plasma both from the patients and from controls with added synthetic oxysterols substantiates in vitro the hypothesis that oxysterols increase the influx of calcium into cells. These data indicated that an excess of oxysterols in the plasma of the patients was cytotoxic to the cultured cells.

Bilirubin sensitized photooxidation of human plasma low density lipoprotein.

Previous investigations have shown that the bile pigment bilirubin can act as peroxyl radicals scavenger and transition metals trap, but also as a peroxidant, to erythrocyte ghost membranes through 1O2-driven photooxidation. In the present study we examined the changes occurring in the lipoprotein particle following bilirubin-sensitized photooxidation of isolated plasma LDL. The oxidative stress resulted in increased TBA reactivity, diene formation, free cholesterol oxidation, apo B fragmentation and enhanced uptake of the modified particle by the mouse macrophage scavenger receptors as well as the decrease binding to the native B, E-receptor on fibroblasts. The marked increase in TBARS production in D2O-enriched medium and the inhibition of lipid peroxidation of azide is consistent with singlet oxygen involvement in the oxidation process. The apo B-bound Cu2+ appears to become redox active during photooxidation since the presence of EDTA in the reaction mixture greatly reduced protein fragmentation. It was also found that BHT inhibited almost completely the lipid peroxidation, as determined by the TBA reaction but could not totally abolish the formation of 5 alpha-hydroxycholesterol, which is the main product formed by the direct attack of 1O2 on cholesterol. The results of this work strongly suggest that, through photooxidation by light-activated bilirubin, the lipoprotein particle may be modified in the blood stream as well, besides being modified in the well known oxidation site within the arterial wall. Our findings provide the rationale for extending these studies to clinical investigations, which aim at developing strategies for minimizing damage to arterial tissue following phototherapy of hyperbilirubinemic newborns or cancer patients after systemic administration of photosensitizers.

Production of volatile sesquiterpenes by Fusarium sambucinum strains with different abilities to synthesize trichothecenes.

Twenty-five strains of Fusarium sambucinum grown on wheat kernels were examined for trichothecene production and the synthesis of volatile sesquiterpenes. The volatiles were purged with air and collected on Tenax traps. Adsorbed compounds were eluted from the traps and injected into a gas chromatograph coupled with a mass spectrometer. Ten strains isolated from potato tubers produced high amounts of diacetoxyscirpenol and its derivatives. These strains were characterized by the production of high amounts of diverse sesquiterpenes. In 10 cultures, 19 compounds were detected, of which 6 were predominant and composed as much as 82% of the volatile sesquiterpene fraction (e.g., beta-farnesene, beta-chamigrene, beta-bisabolene, alpha-farnesene, trichodiene, and an unidentified compound). Fifteen strains isolated from various sources that did not produce trichothecenes produced much less volatile sesquiterpenes, with less chemical diversity. No more than six compounds were present in cultures. Two of these compounds were present in the toxigenic strains isolated from potatoes (beta-farnesene and acoradiene), but four were unique to the strains not producing trichothecenes (longifolene, isocaryophyllene, delta-elemene, and an unidentified one). The pattern of volatile sesquiterpenes was characteristic and distinctive for both toxic and nontoxic strains.

Inhibition of metal-catalyzed oxidation of low-density lipoprotein by free and albumin-bound bilirubin.

Both free and albumin-bound bilirubin are known to scavenge peroxyl radicals in vitro. In the present work we showed that free and albumin-bound bilirubin at the physiological concentration of the bile pigment in blood plasma could greatly inhibit the metal-catalyzed oxidation of low density lipoprotein (LDL) as shown by the reduced thiobarbituric acid reactivity, smaller or no shifts in electrophoretic mobility, less apo B fragmentation and a decreased amount of cholesterol oxidation products as detected by gas chromatography. Free bilirubin (BR) was more effective in inhibiting the production of thiobarbituric acid reactive substances in iron-catalyzed LDL peroxidation as compared to the copper-catalyzed reaction up to a BR to metal molar ratio of 4:1. Above this ratio the same degree of inhibition was observed for both metal ions. It was found that serum albumin provided full protection against Cu(2+)-dependent oxidative stress only at very high protein to metal molar ratio, i.e., 30:1, that is similar to that in human plasma. Complexation of BR to albumin brought about a marked increase in the capacity of the complex to bind metal ions, particularly iron, as opposed to albumin alone. At a molar ratio of metal ion to albumin-BR of 1:1 the inhibition of lipid peroxidation was about 96% and it was almost complete at a molar ratio of 1:2. The ability of albumin-BR complex to inhibit effectively the transition metals-dependent oxidative stress could be important in the extravascular space where local concentrations of metal ions may exceed the protein binding capacity. In addition, the strong binding of iron to the albumin-BR complex may be clinically important, especially in iron loaded sera of hemochromatosis patients, where the transferrin is fully saturated with this ion and the free iron could catalyze lipid peroxidation unless bound by a metal trapping device such as the albumin-BR complex.

Esterification of oxysterols by human plasma lecithin-cholesterol acyltransferase.

In the present study, lecithin-cholesterol acyltransferase (LCAT) catalyzed esterification of oxysterols was investigated by using discoidal bilayer particles (DBP) containing various oxysterols, phosphatidylcholines, and apolipoprotein A-I. The esterified oxysterols were analyzed by high pressure liquid chromatography, gas chromatography, and mass spectrometry. LCAT esterified all oxysterols tested that are known to be present in human plasma. The esterification yields in almost all cases were relatively high, often as high as the yield of cholesterol esterification. When DBP preparations containing 27-hydroxycholesterol and various phosphatidylcholines were used for the LCAT reaction, both monoesters and diesters were produced. The mass spectrometry analysis showed that the monoester was produced by the esterification of the 3 beta-hydroxyl group and not the 27-hydroxyl group. The diesters were apparently produced by the esterification of the 27-hydroxyl group only after the esterification of the 3 beta-hydroxyl group. Phosphatidylcholine containing a saturated acyl group at sn-1 position and an unsaturated acyl group at sn-2 position gave generally high esterification yield. The esterification of various oxysterols was compared by using DBP containing dioleoyl-phosphatidylcholine and individual oxysterols. All oxysterols produced 3 beta-oleoyl monoesters. Unlike 27-hydroxycholesterol, 25-hydroxycholesterol, 7 alpha-hydroxycholesterol, 7 beta-hydroxycholesterol, or cholestanetriol did not produce diesters. Various factors influencing the formation of the monoesters and diesters from 27-hydroxycholesterol were investigated. When dioleoyl-phosphatidylcholine was used as the acyl donor, prolonged dialysis of DBP preparations and increase in the ratio of the enzyme concentration to substrate particle concentration increased the diester formation. Significant amounts of diesters were also produced by using 1-palmitoyl-2-oleoyl-phosphatidylcholine and other phosphatidylcholines as the acyl donors. By analyzing the conditions of monoester and diester formation, a scheme for the LCAT reaction pathway was proposed.

Failure of vitamin E to protect cultured human arterial smooth muscle cells against oxysterol-induced cytotoxicity.

OBJECTIVE: The cytotoxicity of oxysterols including 7 alpha-hydroxycholesterol (7 alpha OHC), 7 beta-hydroxycholesterol (7 beta OHC), cholesterol 5 alpha,6 alpha-epoxide (alpha epoxyC), cholesterol 5 beta,6 beta-epoxide (beta epoxyC), 7-ketocholesterol (7ketoC), 26-hydroxycholesterol (26OHC), cholesterol-3 beta,5 alpha,6 beta-triol (TriolC) and the possible protecting effect of vitamin E on 26OHC-induced cytotoxicity were investigated in smooth muscle cells isolated from the arteries of human umbilical cords. METHODS: To study the cytotoxicity of oxysterols, the cells were incubated with each oxysterol at a level of 10 micrograms/ml from 24 to 120 hours, then 45Ca++ uptake, cytosolic free Ca++ level, [3H]thymidine incorporation, total DNA content and viable cell number were measured. Cholesterol was used as a control. For tracing the possible origin of cytotoxicity of 26OHC, cholesterol, phospholipid and 26OHC content in the membrane were investigated from 24 to 72 hours. For determining whether antioxidants had a protective effect against the cytotoxicity of 26OHC, vitamin E and butylated hydroxytoluene (BHT) were used. RESULTS: The results indicated that the oxysterols elevated 45Ca++ uptake and cytosolic free Ca++ level, but diminished [3H]thymidine incorporation, total DNA content and viable cell number. 26OHC lowered the cholesterol content of the membrane and incorporated into the membrane after 24 hours of the incubation, but did not alter the total phospholipid content of the membrane until 72 hours. Neither vitamin E or BHT significantly protected the cells from the 26OHC-induced alterations. CONCLUSION: We suggest that the cytotoxicity of oxysterols, which might result in an alteration in Ca++ ion flow into the cell by decreasing cholesterol content and incorporating oxysterol itself into the membranes, could not be protected by vitamin E.

Changes in arterial membrane lipid composition may precede growth factor influence in the pathogenesis of atherosclerosis.

A recent review article on the pathogenesis of atherosclerosis stated that the lesions result from an excessive inflammatory-fibroproliferative response to various forms of insult of the endothelium and smooth muscle cells of the arterial wall, that a large number of growth factors participate in this process and that the injury is most apparent at branching points of the arterial tree. We found a significant increase in sphingomyelin and a decrease in other phospholipid components in the arterial wall at the branching points as compared to the non branching points of human and swine arteries. Because of the higher transition temperature of sphingomyelin, its replacement of other phospholipid components may increase the rigidity of the cell membrane and may alter negatively charged bilayers in such a way that they interact more strongly with cholesterol and calcium. Duplication of such conditions with arterial cells in tissue culture caused calcium infiltration into the cell. Furthermore, platelet derived growth factor (PDGF) synthesis was increased in smooth muscle cells grown in magnesium deficient media. It is possible that a change in phospholipid and cholesterol composition of arterial cells at the branching points of arteries and that factors other than PDGF may precede inflammatory-fibroproliferative response to various forms of insult.

Plasma lipid physical properties in swine fed margarine or butter in relation to dietary magnesium intake.

Plasma lipids obtained from swine which had been fed butter or margarine at two dietary magnesium (Mg) levels indicated that the level of dietary Mg was more significant to plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels than was the presence of butter or margarine. At 270 mg Mg/kg, which is considered adequate for swine, there was a significant difference in the plasma TC between swine fed margarine and those fed butterfat (105 and 126 mg %, respectively). Plasma LDL-C was higher in swine fed butter than in those fed margarine (88 and 71 mg %, respectively). In swine fed an additional 247 mg Mg/kg, however, there was no significant difference in plasma TC between those fed margarine or butter. Although at 247 mg Mg/kg, however, there was no significant difference in plasma TC between those fed margarine or butter. Although at 247 mg Mg/kg plasma LDL-C was higher in swine fed margarine and HDL-C was higher in those fed butter, there were no significant differences in the order parameters of LDL and HDL. Studies in which the influences of dietary fats on plasma cholesterol were first noted were carried out on liquid diets deficient in Mg. Mg, a cofactor in the enzymes involved in desaturation of saturated fatty acids, is also necessary in desaturation of linoleic to arachidonic acid.

Structure of the rigid-layer of rhizobium cell wall. II. Evidence for a covalent bond between peptidoglycan and cellodextrins.

Covalent linkages between peptidoglycan and cellodextrins in the cell walls of Rhizobium were defined by the analysis of lysozyme split products. Digestion of peptidoglycan with lysozyme resulted in the liberation, beside disaccharide tetrapeptide fragments composed of glucosamine, muramic acid, alanine, glutamic acid and diaminopimelic acid in a molar ratio 1:1:2:1:1, also significant amounts of glucose and its polymers. The neutral carbohydrates composed of glucose, were further purified and determined as cellobiose, cellotriose and cellotetrose. Peptidoglycans pretreated with cellulase, which librated glucose and cellobiose, still contains glucose linked by lysozyme sensitive but cellulase insensitive bond.

Volatile Flavor Compounds Produced by Molds of Aspergillus, Penicillium, and Fungi imperfecti.

Strains of molds Aspergillus niger, A. ochraceus, A. oryzae, A. parasiticus, Penicillium chrysogenum, P. citrinum, P. funiculosum, P. raistrickii, P. viridicatum, Alternaria, Cephalosporium, and Fusarium sp. were grown on sterile coarse wheat meal at 26 to 28 C for 120 h. The volatiles from mature cultures were distilled at low temperature under reduced pressure. The distillates from traps -40 and -78 C were extracted with methylene chloride and subsequently concentrated. All the concentrates thus obtained were analyzed by gas-liquid chromatography, mass spectrometry, chemical reactions of functional groups, and olfactory evaluation. Six components detected in the culture distillates were identified positively: 3-methylbutanol, 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, and 2-octen-1-ol. They represented 67 to 97% of all the volatiles occurring in the concentrated distillate. The following 14 components were identified tentatively: octane, isobutyl alcohol, butyl alcohol, butyl acetate, amyl acetate, octyl acetate, pyridine, hexanol, nonanone, dimethylpyrazine, tetramethylpyrazine, benzaldehyde, propylbenzene, and phenethyl alcohol. Among the volatiles produced by molds, 1-octen-3-ol yielding a characteristic fungal odor was found predominant.

Identification of the predominant volatile compounds produced by Aspergillus flavus.

A culture of Aspergillus flavus grown on moistened wheat meal was homogenized with a blendor, and the resulting slurry was vacuum-distilled at 5 mm of Hg and 35 C. The aqueous distillate was collected in traps cooled to -10 to -80 C. The culture volatiles were extracted from the distillate with CH(2)Cl(2), and, after removal of the bulk of the solvent, the concentrated volatiles were examined by packed-column gas chromatography. Nineteen peaks were observed, and coupled gas chromatography-mass spectrometry was employed to identify the larger components. The compounds identified were: 3-methyl-butanol, 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, and cis-2-octen-1-ol. The two octenols were the predominant compounds, and sufficient sample was trapped from the gas chromatograph for infrared analyses; this confirmed the mass spectral identifications and permitted the assignment of the cis designation to 2-octen-1-ol. Both oct-1-en-3-ol and cis-2-octen-1-ol are thought to be responsible for the characteristic musty-fungal odor of certain fungi; the latter compound may be a useful chemical index of fungal growth.