A common polymorphism in the promoter of UCP2 is associated with decreased risk of obesity in middle-aged humans.

Obesity is the most common nutritional disorder in Western society. Uncoupling protein-2 (UCP2) is a recently identified member of the mitochondrial transporter superfamily that is expressed in many tissues, including adipose tissue. Like its close relatives UCP1 and UCP3, UCP2 uncouples proton entry in the mitochondrial matrix from ATP synthesis and is therefore a candidate gene for obesity. We show here that a common G/A polymorphism in the UCP2 promoter region is associated with enhanced adipose tissue mRNA expression in vivo and results in increased transcription of a reporter gene in the human adipocyte cell line PAZ-6. In analyzing 340 obese and 256 never-obese middle-aged subjects, we found a modest but significant reduction in obesity prevalence associated with the less-common allele. We confirmed this association in a population-based sample of 791 middle-aged subjects from the same geographic area. Despite its modest effect, but because of its high frequency (approximately 63%), the more-common risk allele conferred a relatively large population-attributable risk accounting for 15% of the obesity in the population studied.

Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet.

AIMS/HYPOTHESIS: Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. METHODS: Male and female littermate Tlr2(+/+) and Tlr2(-/-) mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. RESULTS: While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2(-/-) mice were protected from the adverse effects of HFD compared with Tlr2(+/+) littermate controls. Female Tlr2(-/-) mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2(-/-) mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2(-/-) mice did not increase IL-1beta expression in response to a NEFA mixture, whereas Tlr2(+/+) control tissues did. CONCLUSION/INTERPRETATION: These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.

Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.

BACKGROUND: Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML). PATIENTS AND METHODS: We treated six patients with imatinib-resistant CML in haematological relapse (leukocytes > 20,000 microL(-1)) with rapamycin at 2 mg per os daily for 14 consecutive days, with dose-adjustment allowed to reach a target rapamycin serum concentration of 10-20 pg mL(-1). RESULTS: A major leukocyte response with decrease to less than 10,000 microL(-1) was obtained in two patients, and a minor transient response was seen in two other patients. In responding patients, we also observed a decrease in vascular endothelial growth factor (VEGF) mRNA levels in circulating leukaemic cells. Side effects during rapamycin treatment were mild in most patients. In one patient, pneumonia developed. Rapamycin was also found to counteract growth of CML cells in vitro as determined by (3)H-thymidine incorporation. Moreover, rapamycin inhibited the growth of Ba/F3 cells exhibiting various imatinib-resistant mutants of BCR/ABL, including the T315I variant that exhibits resistance against most currently available BCR/ABL kinase inhibitors. CONCLUSIONS: Rapamycin shows antileukaemic effects in imatinib-resistant CML in vitro and in vivo. Larger trials with rapamycin or rapamycin-derivatives in combination with other targeted drugs are warranted to further determine clinical efficacy in CML.

Clinical and prognostic significance of histamine monitoring in patients with CML during treatment with imatinib (STI571).

BACKGROUND: Although imatinib is highly effective in chronic myeloid leukemia (CML), drug-resistance may occur. Therefore, monitoring of minimal residual disease (MRD) during treatment with imatinib is important. However, most MRD-parameters are expensive and require special technology. We determined the value of histamine as MRD-marker in CML. PATIENTS AND METHODS: Histamine levels were measured serially in whole blood samples before and during imatinib therapy in 80 CML patients by radioimmunoassay. RESULTS: Histamine levels were highly upregulated in CML at diagnosis compared to healthy controls, and correlated with the presence of basophils. During treatment with imatinib, histamine levels decreased and returned to normal levels in those achieving a complete cytogenetic response (CCR). Loss of CCR during therapy was invariably accompanied by an increase in histamine. Moreover, a histamine level of >100 ng/ml three or six months after start of imatinib was associated with a significantly reduced probability of survival (p<0.05). Whereas basophils were found to correlate well with histamine during imatinib, no correlations were found between histamine and Ph+ metaphases or histamine and BCR/ABL. CONCLUSION: Histamine-monitoring during treatment with imatinib is of prognostic significance.

Mesenchymal stem cells in patients with chronic myelogenous leukaemia or bi-phenotypic Ph+ acute leukaemia are not related to the leukaemic clone.

BACKGROUND: Human mesenchymal stem cells (MSCs) are thought to be multipotent cells which primarily reside in the bone marrow. Besides their well-known ability to replicate as undifferentiated cells and to differentiate into diverse lineages of mesenchymal tissues, they were recently suggested to also give rise to haematopoietic and leukaemic/cancer stem cells. In this study, the relationship between MSCs and leukemic stem cells in patients with either chronic myelogenous leukaemia (CML) or the more primitive variant, Ph+ bi-phenotypic leukaemia was investigated. PATIENTS AND METHODS: Cultured MSCs from 5 patients with CML and 3 patients with bi-phenotypic Ph+ leukaemia, all of them positive for BCP-ABL, were analysed with conventional cytogenetics, fluorescence in situ hybridisation (FISH) and polymerase chain reaction (PCR) for the presence of t(9;22) and BCR-ABL. MSCs were characterised phenotypically with surface markers (+CD73, +CD90, +CD105, -CD34, -CD45) and functionally through their potential to differentiate into both adipocytes and osteoblasts. RESULTS: MSCs could be cultivated from seven patients. These cells were BCR-ABL negative when analysed with conventional cytogenetics and FISH. Further cytogenetic analysis revealed a normal set of chromosomes without any aberrations. Two patients were BCR-ABL-positive when analysed with PCR, probably as a result of MSC contamination with macrophages. CONCLUSION: MSCs in patients with CML or Ph+ bi-phenotypic leukaemia are not related to the malignant cell clone.

Metabolism of 4-hydroxynonenal, a cytotoxic lipid peroxidation product, in Ehrlich mouse ascites cells at different proliferation stages.

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is cytotoxic at high concentrations (in the range of 100 microM); at low concentrations, it disturbs cell proliferation and exhibits genotoxic effects, and in the submicromolar range, HNE is chemotactic and stimulates phospholipase C. HNE is rapidly metabolized in eukaryotic cells. Here the metabolism of HNE was studied in suspensions of Ehrlich mouse ascites cells at different periods of the tumor age. The main products of HNE which were identified in the Ehrlich ascites cells, were glutathione-HNE-conjugate, hydroxynonenoic acid, and 1,4-dihydroxynonene. The formation of glutathione conjugates following the addition of HNE was higher in early phase cells when compared with cells in the late phase of tumor growth. That was in accordance with the increased consumption of the reduced form of glutathione. Ehrlich ascites tumor cells at the proliferation phase were able to reduce a higher amount of exogenous-added HNE, compared with cells at the stationary phase.

Role of oxidative stress in age dependent hepatocarcinogenesis by the peroxisome proliferator nafenopin in the rat.

Recently old rats were found to be much more susceptible than young rats to the hepatocarcinogenic effect of a 55-59-week treatment with the peroxisome proliferator nafenopin (NAF) (B. Kraupp-Grasl, W. Huber, H. Taper, and R. Schulte-Hermann, Cancer Res., 51: 666-671, 1991). In the present study indicators of oxidative stress were measured in the livers of the same animals (male Wistar). NAF enhanced peroxisomal beta-oxidation 10-12-fold and reduced glutathione peroxidase activity by 40-50%. Indicators of lipid peroxidation like thiobarbituric acid reactive substances and malondialdehyde were both decreased by one-third and two-thirds, respectively. Of the oxidation-sensitive polyunsaturated fatty acids linoleic acid and docosahexaenoic acid were decreased by 40% and two-thirds, respectively, but the particularly sensitive arachidonic acid remained unchanged. Taken together these data suggest that NAF did not significantly enhance lipid peroxidation in the present experiment. All NAF effects were of the same magnitude in the old and young animals. Therefore, the considerably stronger induction of hepatocarcinoma by NAF in the old animals was not associated with evidence of enhanced oxidative stress. These findings are consistent with the hypothesis that NAF acts hepatocarcinogenically by promotion of tumor development from preneoplastic lesions occurring spontaneously with age.

Early destruction of tryptophan residues of apolipoprotein B is a vitamin E-independent process during copper-mediated oxidation of LDL.

The decrease of the tryptophan fluorescence (Ex/Em = 282/331 nm) was used to monitor the kinetics of copper-mediated LDL oxidation. Cu2+ causes a concentration-dependent quenching of the LDL Trp-fluorescence, the maximum of about 22% suggests that 8-9 Trp residues (out of a total of 37) are accessible for Cu2+ ions. Decomposition of LDL tryptophan commences immediately after addition of Cu2+ and proceeds in two stages with quite different rates. At a molar ratio of Cu2+/LDL = 33:1 the LDL particle looses 1 Trp every 13.5 min in the initial slow phase and every 4.1 min in the subsequent rapid The second, stage temporarily coincides with the propagating lipid peroxidation. In the initial phase loss of Trp proceeds with a constant rate for up to 200 min depending on the copper concentration. Whereas lipid peroxidation accelerates after consumption of vitamin E, rate of Trp loss does not increase. Loading of LDL with vitamin E has also no effect on the initial rate of Trp loss. During the initial phase a loss of one Trp residue/LDL is accompanied by the loss of two alpha-tocopherols and the generation of two conjugated lipid hydroperoxides. The results suggest Trp residues play a role in initiating the lipid peroxidation process in the LDL particle. In such kinetic studies, precautions must be taken to avoid photodecomposition of LDL-Trp. The LDL vitamin E fluorescence (Ex/Em = 290/323 nm) does not interfere with the Trp fluorescence even at high concentrations.

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.

Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal.

The effects of the lipid peroxidation product 4-hydroxynonenal on freshly prepared human low-density lipoprotein (LDL) were studied. At a fixed LDL concentration (5.7 mg/ml) the amount of 4-hydroxynonenal incorporated into the LDL increased with increasing aldehyde concentration from 28-30 (0.2 mM) to 140 (1 mM) mol per mol LDL, whereas at a fixed aldehyde concentration (0.2 mM) its incorporation into LDL decreased with increasing LDL concentration from 48 (1 mg LDL/ml) to 26 (12 mg LDL/ml) mol 4-hydroxynonenal bound per mol LDL. Of the total hydroxynonenal taken up 78% was bound to the protein and 21% to the lipid moiety; the remaining 1% was dissolved as free aldehyde in the lipid fraction. Amino acid analysis of the apolipoprotein B revealed that 4-hydroxynonenal attacks mainly the lysine and tyrosine residues and to a lesser extent also serine, histidine and cysteine. Treatment of LDL with 4-hydroxynonenal results in a concentration-dependent increase of the negative charge of the LDL particle as evidenced by its increased electrophoretic mobility. Moreover, 4-hydroxynonenal treatment leads to a partial conversion of the apolipoprotein B-100 into higher molecular weight forms most probably apolipoproteins B-126 and B-151. Compared to malonaldehyde, 4-hydroxynonenal exhibits a much higher capacity to modify LDL and it is therefore believed that this aldehyde is a more likely candidate for being responsible for LDL modification under in vivo lipid peroxidation conditions.

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids.

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids. Identification of 4,5-dihydroxydecenal.

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.

Inhibition of copper- and peroxyl radical-induced LDL lipid oxidation by ebselen: antioxidant actions in addition to hydroperoxide-reducing activity.

The effects of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) on human LDL lipid oxidation induced by different fluxes of aqueous peroxyl radicals and cupric ion (at a Cu2+:LDL ratio of 17:1) were investigated. Addition of ebselen to LDL oxidised with Cu2+ prolonged the duration of the lag-phase typical for this oxidising condition, with the increase being proportional to the square of the ebselen concentration. Ebselen also prevented the formation of lipid hydroperoxides and inhibited the consumption of endogenous antioxidants during the early period of Cu(2+)-induced oxidation, during which time the drug was converted stoichiometrically into ebselen oxide (2-phenyl-1,2-benzisoselenazol-3(2H)-one-Se-oxide). Ebselen oxide itself was antioxidant inactive. Ebselen also inhibited formation of lipid-hydroperoxides and spared alpha-tocopherol during the initial stages of LDL oxidation mediated by low-flux of aqueous peroxyl radicals, where a lag-phase was not observed. When a higher flux of aqueous peroxyl radicals was used, ebselen increased the observed inhibited phase of peroxidation in a dose-dependent manner, though less pronounced than its prolongating effect on the lag-phase of Cu(2+)-induced LDL lipid oxidation. Ebselen was also able to directly interact with Cu1+, alkyl peroxyl radicals and alpha-tocopheroxyl radicals, demonstrating that the drug has a number of potential antioxidant activities in addition to its well-known hydroperoxide-reducing activity. We conclude that the antioxidant activities of ebselen are complex and that their relative importance likely vary depending on the experimental system used.

Characterization of lipoxygenase metabolites of arachidonic acid in cultured human skin fibroblasts.

Cultured human skin fibroblasts were incubated in the presence of [14C]arachidonic acid (50 microM; 1 m Ci/mmol) and the divalent cation ionophore A23187 at 37 degrees C for 60 min. The metabolites formed were extracted from the cell-free medium in diethyl ether, separated by thin layer chromatography and identified unequivocally by GC-MS. The distribution of the arachidonic acid metabolites as estimated from the recovered radioactivity showed as major product prostaglandin E2 (26%). Minor amounts of other prostaglandins, i.e., 6-oxo-prostaglandin F1 alpha (1%), prostaglandin F2 alpha (1%), prostaglandin D2 (0.5%) and prostaglandin A2 (1%) were also present. In addition to the prostaglandins, monohydroxy fatty acids (4.5%) were also detected. This fraction contained 33% 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), 22% 11-hydroxy-5,8,11,14-eicosatetraenoic acid (11-HETE) and 31% 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). Lipid extracts of the cells did not show any detectable amount of the monohydroxy fatty acids, indicating that they are not incorporated metabolically in the cellular lipids. The monohydroxy fatty acids originate mainly from the exogenously added arachidonic acid as evidenced by the 2H/H ratio (30:1) from experiments with octadeuterated arachidonic acid [( 2H8]arachidonic acid). Indomethacin inhibited the formation of all prostaglandins, HHT and 11-HETE; moreover, eicosatetraynoic acid (also blocked the formation of 15-HETE. From these results, it can be concluded that in human skin fibroblasts prostaglandin E2 is the major product of the cycloxygenase pathway, while 15-HETE is the main lipoxygenase product.

Detection of carbonyl functions in phospholipids of liver microsomes in CCl4- and BrCCl3-poisoned rats.

Since the peroxidative cleavage of unsaturated fatty acids can result in either the release of carbonyl compounds or the formation of carbonyl functions in the acyl residues, evidence for the presence of carbonyl groups in liver microsomal phospholipids was searched for in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been previously demonstrated. The spectrophotometric examination of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from the intoxicated animals showed absorption spectra similar to those observed for the dinitrophenylhydrazones of various carbonyls. Similar spectra, although magnified from a quantitative point of view, were also observed with 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation showed that the amount of 2,4-dinitrophenylhydrazine-reacting groups (carbonyl functions) in phospholipids of liver microsomes increases with the incubation time and is correlated to the amount of malonic dialdehyde formed in the incubation mixture. The kinetics of the production of 4-hydroxynonenal was somewhat similar to that of malonic dialdehyde formation. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of carbonyl functions in microsomal phospholipids, which was higher in the BrCCl3-intoxicated animals as compared to the CCl4-poisoned ones, was close to that found in the vitro condition in which lipid peroxidation is induced by 6 microM Fe2+. The possible pathological significance of formation of carbonyl functions in membrane phospholipids is discussed.

Evidence for aldehydes bound to liver microsomal protein following CCl4 or BrCCl3 poisoning.

Since it has been demonstrated in previous studies that peroxidation of liver microsomal lipids leads to the production of aldehydes provided with cytopathological activities--namely 4-hydroxyalkenals--evidence was searched for aldehydes bound to microsomal protein in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been demonstrated previously. The spectrophotometric analysis of 2,4-dinitrophenylhydrazine-treated non-lipoidal residues of liver microsomes from the intoxicated rats shows absorption spectra similar to those observed for the dinitrophenylhydrazones formed in the reaction of alkenals with -SH groups of proteins or low molecular weight thiols. Similar spectra, although magnified from a quantitative point of view, were obtained either with liver microsomes allowed to react with synthetic 4-hydroxynonenal or with liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation shows that the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases with the incubation time and is correlated to the amount of thiobarbituric acid-reacting products formed in the incubation mixture. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases from 15 min up to 2 h after poisoning and is higher, in every instance, in the BrCCl3-intoxicated animals compared to the CCl4-poisoned ones. Experiments carried out to ascertain the reliability of the spectrophotometric detection of protein-bound alkenals showed that in the in vitro system in which liver microsomes are allowed to react with 4-hydroxynonenal there is a good agreement between the binding value that can be calculated from the absorption spectrum and the binding value obtained by using labelled 4-hydroxynonenal.

Oxidation of lipoprotein Lp(a). A comparison with low-density lipoproteins.

Aimed at identifying possible mechanisms of the suggested high atherogenicity of Lp(a), its susceptibility for Cu(II)-induced oxidation was studied and compared with that of LDL. Since the content of antioxidants as well as the fatty acid pattern of a lipoprotein greatly affects its oxidizability, Lp(a) and LDL were characterized first with respect to these substances. Paired samples of low-density lipoproteins (LDL) and Lp(a) were isolated from seven individual donors and compared with each other. This study showed that LDL and Lp(a) are very similar with respect to their fatty acid and antioxidant composition. LDL contains approx. 1132 nmol of total fatty acids/mg lipoprotein and LDL 1466 nmol total fatty acids/mg lipoprotein. Analysis of the fatty acid composition of individual lipid classes (cholesteryl esters, phospholipids and triacylglycerols) revealed also a high similarity in the composition of these lipid classes between the two lipoproteins. A comparison of the antioxidant composition showed that Lp(a) contains less alpha-tocopherol than LDL (1.6 +/- 0.35 nmol/mg vs. 2.1 +/- 0.25 nmol/mg LDL). In copper(II)-induced lipid peroxidation experiments we found a striking difference in the susceptibility of individual lipoprotein classes between all donors. In addition, Lp(a) exhibited a 1.2 to 2.4 longer lag-phase than the corresponding LDL preparation from the same blood donor. Treatment of Lp(a) with neuraminidase resulted in a drastic decrease of the lag-phase of Lp(a). Neuraminidase treatment of LDL on the other hand had no significant effects on its susceptibility to oxidation. Supplementation of neuraminidase-treated Lp(a) with N-acetylneuraminic acid (NANA) at concentrations comparable to the naturally occurring amounts of NANA in the Lp(a) protein moiety led to an increase of the lag-phase yielding values which were comparable to those observed with native Lp(a). These results demonstrate that the fatty acid composition as well as the antioxidant concentrations of Lp(a) and LDL are quite similar; despite this fact, Cu2(+)-mediated oxidation of Lp(a) is retarded in comparison to LDL which might be due to the higher content of NANA in Lp(a).

Studies on the mechanism of formation of 4-hydroxynonenal during microsomal lipid peroxidation.

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.

A sensitive chemiluminescence method to measure the lipoxygenase catalyzed oxygenation of complex substrates.

Oxidative modification of low-density lipoprotein (LDL) has been implicated as a patho-physiological process in early atherogenesis and 15-lipoxygenases (15-LOX) may be involved. While studying the in vitro kinetics of the 15-LOX/LDL interaction, we found that the conventional spectrophotometric assays failed in the range of substrate saturation owing to the high optical density of concentrated LDL solutions. Therefore, we developed a much more sensitive assay system which was based on peroxide induced isoluminol enhanced chemiluminescence. With this method reliable kinetic data were obtained at LDL concentrations of up to 1 mg/ml. To validate this luminometric method the kinetic parameters of 15-LOX catalyzed oxygenation of linoleic acid (Km=3.7 microM, kcat=17 s-1) were determined and we observed a good agreement with previously published data obtained with a spectrophotometric assay. Moreover, we found that the kinetic constants of 15-LOX catalyzed LDL oxidation (Km=0.64 microM, kcat=0.15 s-1) are quite different from those of free fatty acid oxygenation and that the cholesterol esters are preferentially oxidized during 15-LOX/LDL interaction. Vitamin E depletion does not reduce the rate of LDL oxidation and analysis of the structure of the oxygenation products suggests that the majority of the products were formed via direct LOX catalyzed oxidation of LDL ester lipids. The luminometric method described here is not restricted to the measurement of LOX catalyzed LDL oxidation, but may also be used to determine kinetic constants for the oxidation of other complex substrates such as biomembranes or liposomes.

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats.

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.