Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of <1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Catalyst Deactivation on a Two-Stroke Engine

With the legislative demands increasing on recreational vehicles and utility engined applications, the two-stroke engine is facing increasing pressure to meet these requirements. One method of achieving the required reduction is via the introduction of a catalytic converter. The catalytic converter not only has to deal with the characteristically higher CO and HC concentration, but also any oil which is added to lubricate the engine. In a conventional two-stroke engine with a total loss lubrication system, the oil is either scavenged straight out the exhaust port or is entrained, involved in combustion and is later exhausted. This oil can have a significant effect on the performance of the catalyst.
To investigate the oiling effect, three catalytic converters were aged using a 400cm₃ DI two-stroke engine. A finite level of oil was added to the inlet air of the engine to lubricate the internal workings. The oil flow rate is independent of the engine speed and load.

Three catalysts were aged for 50 hours, experiencing a constant space velocity and set engine conditions. The engine was fueled on petrol and later on propane to eliminate the effects, if any, of petrol additives on catalyst deactivation. The oiling rate was varied to investigated deactivation from oil contamination. Post-mortem analysis was performed on the three catalysts. This consisted of a controlled light-off test performed on a catalyst rig, during which period, temperatures were measured and recorded towere aged for 50 hours, experiencing a constant space velocity and set engine conditions. The engine was fueled on petrol and later on propane to eliminate the effects, if any, of petrol additives on catalyst deactivation. The oiling rate was varied to investigated deactivation from oil contamination. Post-mortem analysis was performed on the three catalysts. This consisted of a controlled light-off test performed on a catalyst rig, during which period, temperatures were measured and recorded to find out where deactivation of each catalyst was occurring. The recorded results were all analyzed and these showed that from the measured light-off temperatures the aged catalysts behaved similarly. However the pattern in the light-off was significantly different when the engine was fueled by propane as opposed to gasoline.