Resolution of adiponectin oligomers in human plasma using free flow electrophoresis.

Adiponectin is an important adipocytokine hormone which circulates in blood as homo-oligomers (trimer, hexamer and high molecular weight (HMW) forms) as well as a truncated form corresponding to the globular domain. Free flow electrophoresis (FFE) used in zone electrophoresis mode revealed the presence of isoforms within these oligomeric forms in plasma. HMW adiponectin oligomer showed two isoforms which carry different charge density at pH 4.7, only one of which is susceptible to dissociation by SDS. The adiponectin hexamer was shown to consist of a doublet and also shown to have at least two isoforms. A truncated form of adiponectin was identified as the main constituent of adiponectin in plasma and appeared to circulate bound to a basic protein, potentially one of the chemokines reported to bind to the globular domain. Analysis of the monomer composition of the oligomers revealed differences in monomeric isoforms used to build up the oligomers.

Determination of nitrotyrosine concentrations in plasma samples of diabetes mellitus patients by four different immunoassays leads to contradictive results and disqualifies the majority of the tests.

BACKGROUND: In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress. METHODS: Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33+/-11 years) following a standardized meal. RESULTS: The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples. CONCLUSIONS: All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma.

Comparison of the dose accuracy of prefilled insulin pens.

BACKGROUND: Prefilled insulin pens have become a convenient and accurate way for diabetes patients to inject insulin. Their ease of use has helped to reduce the resistance of patients with type 1 diabetes and type 2 diabetes in the United States and Europe toward initiation of insulin therapy. This study compared the dosing accuracy of two prefilled insulin pens (the SoloStar((R)) from Sanofi Aventis, Berlin, Germany, and the Next Generation [NG] FlexPen((R)) from Novo Nordisk, Mainz, Germany). METHODS: The dosing accuracy was tested for both pens with x 24 10 international units of insulin (IU) and 9 x 30 IU injection volumes to investigate whether the pens comply within the acceptable International Organization for Standardization (ISO) limits of 10% (±1 IU) for 10 IU and 5% (±1.5 IU) for 30 IU. The doses were applied each with a new needle strictly according to the instructions for use of the pen manufacturers. A sensitive pharmaceutical balance was used for the assessment of the applied volumes, and the results were corrected for the specific density of the insulin formulations. We used 18 insulin pens (from two different production lots each) for the two volumes, respectively, resulting in a total of 432 doses per pen with 10 IU and 162 doses per pen with 30 IU. RESULTS: Both pens showed a very good performance, which was better for the 10 IU dose than in comparative previous studies. The NG FlexPen (mean absolute percent deviation 10 IU/30 IU: 1.63 ± 0.84%/1.23 ± 0.76%) was even more accurate than the SoloStar (2.11 ± 0.92%/1.54 ± 0.84%, p < .001/p < .05 versus the NG FlexPen). Only 0.2% of the doses were outside the ISO limit at 10 IU, with the NG FlexPen (0.6% at 30 IU). The corresponding figures for the SoloStar were 0.4% and 1.8%, respectively. CONCLUSIONS: A direct head-to-head comparison of the two prefilled insulin pens with a standardized protocol resulted in a more stable dosing accuracy of both pens as compared to previous investigations. In this investigation, the NG FlexPen was more accurate than the SoloStar at both tested doses.

Effect of insulin glulisine on microvascular blood flow and endothelial function in the postprandial state.

OBJECTIVE: To investigate the effect of insulin glulisine on postprandial microvascular blood flow in type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 15 patients with type 2 diabetes received insulin glulisine or human insulin before a liquid meal test. Thereafter, skin microvascular blood flow was measured by laser Doppler fluxmetry and blood samples were taken for measurement of plasma levels of glucose, insulin, intact proinsulin, asymmetric dimethylarginine, nitrotyrosine, interleukin-18, matrix metalloproteinase-9, oxidized LDL, and free fatty acids. RESULTS: Insulin glulisine injections resulted in higher postprandial insulin levels (means +/- SEM area under the curve [AUC](0-120) 51.0 +/- 6.8 vs. 38.2 +/- 5.4 mU/l; P = 0.004), while plasma glucose (AUC(0-240) 158 +/- 9 vs. 180 +/- 9 mg/dl; P < 0.05) and intact proinsulin (AUC(0-240) 26.2 +/- 3.5 vs. 31.2 +/- 4.3 pmol/l; P = 0.002) were lower. Microvascular blood flow increased after insulin glulisine injection (27.9 +/- 3.1 to 51.7 +/- 9.9 arbitrary units [AU]; P < 0.05), while only a minor increase was found during human insulin (27.9 +/- 3.1 to 34.4 +/- 7.8 AU; not significant). Asymmetric dimethylarginine and nitrotyrosine levels were reduced after insulin glulisine (P < 0.05). CONCLUSIONS: Insulin glulisine is superior to human insulin in restoring postprandial metabolic and microvascular physiology.

Anaerobic cometabolic transformation of polycyclic and heterocyclic aromatic hydrocarbons: evidence from laboratory and field studies.

The sulfate-reducing enrichment culture N47 can grow on naphthalene or 2-methylnaphthalene as the sole carbon and energy source. Here we show that the culture can furthermore cometabolicallytransform a variety of polycyclic and heteroaromatic compounds with naphthalene or methylnaphthalene as the auxiliary substrate. Most of the cosubstrates were converted to the corresponding carboxylic acids, frequently to several isomers. The mass spectra of specific metabolites that were extracted from supernatants of cultures containing the cosubstrates benzothiophene, benzofuran, and 1-methylnaphthalene resembled known intermediates of the anaerobic naphthalene and 2-methylnaphthalene degradation pathways (i.e., naphthyl-2-methylsuccinic acid and naphthyl-2-methylenesuccinic acid). This indicates that some of the tested compounds were first methylated and then transformed to the corresponding methylsuccinic acids by a fumarate addition to the methyl group. For some of the cosubstrates, a partial or total inhibition of growth on the auxiliary substrate was observed. This was not caused by the toxicity of the individual cosubstrate itself, but by a specific combination of auxiliary substrate and cosubstrate. None of the cosubstrates tested could be utilized as the sole carbon source and electron donor by the enrichment culture N47. Field investigations at the tar-oil-contaminated aquifer, where strain N47 originated, revealed the presence of a number of metabolites similar to the ones identified in batch culture supernatants. Our findings suggest that aromatic hydrocarbons and heterocyclic compounds can be converted by aquifer organisms and produce a variety of polar compounds that become mobile in groundwater.

Methylation is the initial reaction in anaerobic naphthalene degradation by a sulfate-reducing enrichment culture.

The sulfate-reducing culture N47 can utilize naphthalene or 2-methylnaphthalene as the sole carbon source and electron donor. Here we show that the initial reaction in the naphthalene degradation pathway is a methylation to 2-methylnaphthalene which then undergoes the subsequent oxidation to the central metabolite 2-naphthoic acid, ring reduction and cleavage. Specific metabolites occurring exclusively during anaerobic degradation of 2-methylnaphthalene were detected during growth on naphthalene, i.e. naphthyl-2-methyl-succinate and naphthyl-2-methylene-succinate. Additionally, all three enzymes involved in anaerobic degradation of 2-methylnaphthalene to 2-naphthoic acid that could be measured in vitro so far, i.e. naphthyl-2-methyl-succinate synthase, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase and naphthyl-2-methyl-succinyl-CoA dehydrogenase were also detected in naphthalene-grown cells with similar activities. Induction experiments were performed to study the growth behaviour of the cell when transferred from naphthalene to 2-methylnaphthalene or vice versa. When the cells were transferred from naphthalene to 2-methylnaphthalene they grew immediately, indicating that no new enzymes had to be induced. On the contrary, the transfer of cells from 2-methylnaphthalene to naphthalene caused a lag-phase of almost 100 days demonstrating that an additional catabolic enzyme has to be activated in this case. We propose the methylation as a novel general mechanism of activation reactions in anaerobic degradation of unsubstituted aromatic hydrocarbons.

Enzymatic reactions in anaerobic 2-methylnaphthalene degradation by the sulphate-reducing enrichment culture N 47.

The upper pathway of anaerobic degradation of 2-methylnaphthalene was studied with a sulphate-reducing enrichment culture, which is able to grow with naphthalene or 2-methylnaphthalene as sole carbon source and electron donor. Anaerobic degradation of 2-methylnaphthalene is initiated by an addition of fumarate to the methyl-group producing the first intermediate, naphthyl-2-methyl-succinate. In a subsequent beta-oxidation of the original methyl atom, the central metabolite 2-naphthoic acid is generated. In the following pathway, the aromatic ring system is reduced, cleaved, and finally oxidised to CO(2). Here, we present two new enzymatic reactions of the 2-methylnaphthalene degradation pathway that were measured in crude cell extracts. All metabolites were identified with HPLC by co-elution with synthesised reference substances. The first enzyme, succinyl-CoA:naphthyl-2-methyl-succinate CoA-transferase, catalyses the activation of naphthyl-2-methyl-succinic acid to the corresponding CoA ester. The average specific activity of this enzyme was 19.6 nmol x min(-1) x mg of protein(-1). The CoA-transfer was not inhibited by sodium borohydride and only partially by hydroxylamine, indicating that this enzyme belongs to the family III of CoA-transferases like the corresponding enzyme in the anaerobic toluene degradation pathway. The product of this CoA-transfer reaction, naphthyl-2-methyl-succinyl-CoA is then oxidised in a reaction to naphthyl-2-methylene-succinyl-CoA by the enzyme naphthyl-2-methyl-succinyl-CoA dehydrogenase. The specific activity of this enzyme was 0.115 nmol x min(-1) x mg of protein(-1). The enzymatic activity could only be detected using phenazine methosulphate as electron acceptor. No activity was observed with natural electron acceptors such as nicotinamide adenine dinucleotide or flavin adenine dinucleotide. The two novel reactions presented here demonstrate that the original methyl-group of 2-methylnaphthalene is oxidised to the carboxyl group of 2-naphthoic acid in the upper part of the anaerobic degradation pathway.

Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer.

To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.

Anaerobic degradation of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are among the most important contaminants of groundwater. The 2- and 3-ring PAHs are of particular concern because they are water soluble in the 1-200 mug/l range and are transported with the groundwater over significant distances. Anaerobic degradation of PAH has been demonstrated in several microcosm studies with nitrate, ferric iron, or sulfate as electron acceptors and under methanogenic conditions. The biochemical degradation pathways were studied with naphthalene-degrading pure and enrichment cultures and revealed that 2-naphthoic acid is a central metabolite. Naphthalene is activated by addition of a C(1)-unit to generate 2-naphthoic acid, whereas methylnaphthalene is activated by addition of fumarate to the methyl group and further degraded to 2-naphthoic acid. In the central 2-naphthoic acid degradation pathway the ring system is reduced prior to ring cleavage generating e.g. 5,6,7,8-tetrahydro-2-naphthoic acid. The ring cleavage produces metabolites such as 2-carboxycyclohexylacetic acid indicating that further degradation goes via cyclohexane derivatives and not via aromatic compounds. Anaerobic degradation of PAH has also been demonstrated in situ in contaminated aquifers by identification of compound specific metabolites and using stable isotope fraction studies. Detection of specific metabolites of anaerobic PAH degradation such as naphthyl-2-methylsuccinate indicated anaerobic degradation of 2-methylnaphthalene in situ whereas 2-naphthoic acid was indicative of naphthalene and 2-methylnaphthalene degradation. Other carboxylic acids that were detected in groundwater indicated anaerobic degradation of a wide range of PAH and heterocyclic compounds. Degradation of naphthalenes in contaminated aquifers could also be confirmed by carbon stable isotope shifts in the residual substrate fraction.