Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production.

The potential of accumulation of lipids by Lipomyces starkeyi when grown on sewage sludge was assessed. On a synthetic medium, accumulation of lipids strongly depended on the C/N ratio. The highest content of lipids was measured at a C/N-ratio of 150 with 68% lipids of the dry matter while at a C/N-ratio of 60 only 40% were accumulated. Within a pH range from 5.0 to 7.5 the highest lipid accumulation was found at pH 5.0 while the highest yield per litre was pH 6.5. Although sewage sludge had no inhibitory effects on growth or accumulation on L. starkeyi when added to synthetic medium, there was no significant growth on untreated sewage sludge. However, pretreatment of sludge by alkaline or acid hydrolysis, thermal or ultrasonic treatment lead to accumulation of lipids by L. starkeyi with highest values of 1 g L(-1) obtained with ultrasound pre-treatment. Based on the content of free fatty acids and phosphorus, lipids accumulated from sewage sludge could serve as a substrate for the production of biodiesel.

Macrophage-enhanced formation of cholesteryl ester-core aldehydes during oxidation of low density lipoprotein.

Oxidation of low density lipoproteins (LDL) results in changes to the lipoprotein particle that are potentially pro-atherogenic. To investigate mechanisms contributing to the formation of cholesteryl ester (CE)-core aldehydes (9-oxononanoyl- and 5-oxovaleroyl-cholesterol; 9-ONC and 5-OVC, respectively) LDL was incubated in the presence of mouse macrophages (J774 cells) under different culture conditions. Here we demonstrate that the formation of core aldehydes occurs only in transition metal-containing HAM's F10 medium but not in Dulbecco's modified Eagle's medium (DMEM), independent of supplementation with iron and copper at concentrations up to ten times higher than present in HAM's F10. The antioxidative properties of DMEM could be ascribed to the higher amino acid and vitamin content as compared to HAM's F10 medium. Supplementation with these components efficiently inhibited LDL oxidation in HAM's F10. Stimulation of J774 cells with phorbol ester (PMA) resulted in significantly enhanced 9-ONC and 5-OVC formation rates that were accompanied by increased consumption of LDL cholesteryl linoleate (Ch18:2) and cholesteryl arachidonate (Ch20:4) in the cellular supernatant. In PMA (10 ng/ml) activated cells, approximately 5% of Ch18:2 contained in LDL was converted to 9-ONC and 4% of Ch20:4 was converted to 5-OVC. With respect to core aldehyde formation, lipopolysaccharide (LPS, 10 microg/ml) was a less effective stimulant as compared to PMA. Part of the core aldehydes accumulated within the cells. Our study demonstrates that i) J774 macrophages are able to promote/accelerate core aldehyde formation in HAM's F10 medium, and ii) that core aldehyde formation rates can be increased by stimulation of the cells with PMA, and, although to a lesser extent, with LPS. Finally we could show that iii) a small amount of the core aldehydes is internalized by J774 macrophages.

Synthesis of 9-oxononanoyl cholesterol by ozonization.

A new route for the preparation of 9-oxononanoyl cholesterol (5) and its stable dimethylacetal (4) is described. The core aldehyde 5 is one of the major products formed during lipid peroxidation. The synthesis starts with the ozonization of oleic acid in methanol and further reduction with dimethyl sulfide to yield 9,9-dimethoxy nonanoic acid (2a). The condensation of 2a with cholesterol is achieved with N,N'-dicyclohexylcarbodiimide in dichloromethane to give 4. Further hydrolysis of 4 with the help of an acidic ion exchange resin yields 9-oxononanoyl cholesterol.

Femtomole analysis of 9-oxononanoyl cholesterol by high performance liquid chromatography.

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low (approximately 50) femtomole level, with an absolute limit of detection (signal: noise = 6) of 15 fmol. Intra-assay variation was < or = 5%, while inter-assay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol-12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization-mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28+/-14 micromol/mol cholesterol, n = 9) despite the presence of alpha-tocopherol (4+/-1.2 mmol/mol cholesterol, n = 9).

Synthesis of 4-methoxy-2,3,5-trimethylpyridine: a specific building block for compounds with gastric-acid inhibiting activity.

A new synthesis of 4-methoxy-2,3,5-trimethylpyridine (2), an important building block for the preparation of gastric-acid inhibiting compounds, is described. Condensation of ethyl 3-amino-2-methyl-2-butenoate (3) and diethyl 2-methylmalonate (4) gives 4-hydroxy-3,5,6-trimethyl-2(1H)-pyridone 5. Reaction of 5 with phosphoryl chloride affords 2,4-dichloro-3,5,6-trimethylpyridine (9a), which, upon hydrogenolysis with palladium on charcoal, gives 2,3,5-trimethylpyridine (10). However, selective hydrogenolysis in acidic solution yields 4-chloro-2-3-5-trimethylpyridine (11). Substitution of the chlorine in 11 with methoxide ion gives 4-methoxy-2,3,5-trimethylpyridine (2), which can be oxidized to the corresponding N-oxide (13). This constitutes a new and efficient route to compound 2 in an overall yield of 43%.