Visual aptamer-based capillary assay for ethanolamine using magnetic particles and strand displacement.

This work describes an aptamer-based capillary assay for ethanolamine (EA). It is making use of strand displacement format and magnetic particles. The capillary tubes are coated with three layers, viz. (a) first with short oligonucleotides complementary to the aptamer (EA-comp.); (b) then with magnetic particles (Dynabeads) coated with EA-binding aptamer (EA-aptamer), and (c) with short oligonucleotide-coated magnetic particles (EA-comp.). On exposure to a sample containing ethanolamine, the DNA-coated magnetic particles are released and subsequently collected and spatially separated using a permanent magnet. This results in the formation of a characteristic black/brown spots. The assay has a visual limit of detection of 5 nM and only requires 5 min of incubation. Quantification is possible through capture and analysis of digital (RGB) photos in the 5 to 75 nM EA concentration range. Furthermore, results from tap water and serum spiked with EA samples showed that the platform performs well in complex samples and can be applied to real sample analysis. The combined use of plastic capillaries, visual detection and passive flow make the method suited for implementation into a point-of-care device. Graphical abstract Schematic representation of the capillary assay steps.

Bioactive 4-Oxoheptanedioic Monoamide Derivatives of Proteins and Ethanolaminephospholipids: Products of Docosahexaenoate Oxidation.

Oxidative stress causes lipid-derived oxidative modification of biomolecules that has been implicated in many pathological states. Phospholipids containing polyunsaturated fatty acids are major targets of free radical-initiated oxidation. Phospholipids that incorporate docosahexaenoate (DHA) are highly enriched in important neural structures including the brain and retina, where DHA comprises 40% and 60% of total fatty acids, respectively. Oxidative fragmentation of 2-docosahexaenoyl-1-palmityl-sn-glycerophosphocholine generates esters of 4-hydroxy-7-oxohept-5-enoic acid (HOHA) and 4-keto-7-oxohept-5-enoic acid (KOHA) with 2-lysophosphatidylcholine, HOHA-PC, and KOHA-PC. Covalent HOHA adducts that incorporate the primary amino groups of proteins and ethanolamine phospholipids in carboxyethylpyrrole (CEP) derivatives were detected immunologically with anti-CEP antibodies in human tumors, retina, and blood. Now, we generated an anti-OHdiA antibody to test the hypothesis that KOHA adducts, which incorporate the primary amino groups of proteins or ethanolamine phospholipids in 4-oxo-heptanedioic (OHdiA) monoamide derivatives, are present in vivo. However, whereas the anti-CEP antibody is highly specific and does not cross-react with the OHdiA monoamide epitope, the anti-OHdiA monoamide antibody cross-reacted with CEP epitopes making it of little value as an analytical tool for OHdiA monoamides but suggesting the possibility that OHdiA monoamides would exhibit receptor-mediated biological activity similar to that of CEP. An LC-MS/MS method was developed that allows quantification of OHdiA derivatives in biological samples. We now find that KOHA-PC forms OHdiA monoamide adducts of proteins and ethanolamine phospholipids and that OHdiA-protein levels are significantly higher than OHdiA-ethanloamine phospholipid levels in blood from healthy human subjects, 0.45 µM and 0.18 µM, respectively (n = 3, and p = 0.027). OHdiA monoamide epitopes are angiogenic, causing TLR2-dependent adhesion and tube formation by human umbilical vein endothelial cells. OHdiA monoamide epitopes are only slightly less potent than CEP epitopes that contribute to the pathological angiogenesis of age-related macular degeneration and tumor growth.

Metabolomic signatures in guinea pigs infected with epidemic-associated W-Beijing strains of Mycobacterium tuberculosis.

With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

Analysis of nitrogen mustard degradation products via post-pentafluorobenzoylation liquid chromatography-tandem mass spectrometry.

A pentafluorobenzoylation (PFBz)-liquid chromatography-tandem mass spectrometry method was developed for qualitative and quantitative analysis of ethanolamines (EAs, nitrogen mustard degradation products). With this method, highly hydrophilic EAs can be sufficiently analyzed with a commonly used reversed phase column (retention times: (PFBz)2-methyl diethanolamine, 9.1 min; (PFBz)2-ethyl diethanolamine, 9.8 min; and (PFBz)3-triethanolamine, 17.6 min). The applicability of the method for real samples was investigated via recovery tests. Methyl diethanolamine and ethyl diethanolamine were detected at concentrations as low as 1 ng/mL in serum and 10 ng/mL in urine, and quantified within the range of 1-1000 ng/mL and 10-1000 ng/mL, respectively.

Dietary fatty acid composition impacts plasma fatty acid ethanolamide levels and body composition in golden Syrian hamsters.

Fatty acid ethanolamides (FAEs) are a class of lipid amides that regulate numerous pathophysiological functions. To date, pharmacological research in this area has focused on the endocannabinoid system, metabolic pathways, and biological significance of FAEs; however, limited nutritional studies have been conducted to understand the actions of FAEs on food intake and their role on overall body composition. Therefore, the present study was designed with the hypothesis that high C18:1n9 will attenuate food consumption in golden Syrian male hamsters (n = 105). Moreover, the long-term (two months) effects of feeding hamsters various dietary oil blends, namely, C+S, 25:75 corn oil:n9 safflower oil; F+S, 25:75 flaxseed oil:n6 safflower oil; H+DHA, 85:15 high oleic canola oil:docosahexaenoic acid; H+EPA, 85:15 high oleic canola oil:eicosapentaenoic acid; HOCO, high oleic canola oil; OO, olive oil; and RC, regular canola oil, on the plasma levels of seven different FAEs and fatty acids (FAs) composition were investigated. A further objective was to characterize the actions of these diets on energy expenditure and overall body composition to determine if dietary fatty acid (DFA) composition affects diet-induced obesity (DIO). The results show that DFA directly influenced plasma FA and FAE levels, with marked increases (p < 0.05) observed in plasma C18:1n9 levels after HOCO and OO treatments. Correspondingly, the most elevated plasma oleoylethanolamide (OEA) levels were observed with HOCO and OO treatments, which also decreased (p < 0.05) food intake by ∼8% when compared with H+EPA dietary treatment when measured at the endpoint. Diminished food intake subsequent to HOCO and OO feeding may have resulted from increased OEA concentrations, demonstrating the anorexic properties of the high C18:1n9 dietary components. No differences were observed across OO, HOCO, and HOCO diets with omega-3 FA blends in terms of body composition, energy expenditure, plasma C18:1n9 levels, or OEA concentrations. Based on these findings, we conclude that the addition of HOCO to diets aids in the reduction of food intake, which may contribute to the maintenance of healthy body weight.

Association of cholesterol efflux capacity with plasmalogen levels of high-density lipoprotein: A cross-sectional study in chronic kidney disease patients.

BACKGROUND AND AIM: Current research suggests that dysfunctional high-density lipoprotein (HDL) with low cholesterol efflux capacity may accelerate atherosclerosis, particularly in chronic kidney disease (CKD). We previously reported that serum levels of plasmalogens closely correlated with HDL concentration, and could serve as a novel biomarker for atherosclerosis. In the present study, we analyzed the association of cholesterol efflux capacity of HDL with clinical and biochemical parameters, including plasmalogens, in CKD patients. METHODS: We enrolled 24 mild-to-moderate CKD patients (CKD-3-4) and 33 end-stage renal disease (ESRD) patients nearing hemodialysis (CKD-5), and assessed physiological atherosclerotic scores, cholesterol efflux capacity, and plasmalogens levels in HDL. Furthermore, the effect of plasmalogen on cholesterol efflux capacity of HDL was examined by in vitro studies with re-constituted HDL (rHDL) and HDL prepared from CKD-5 patient (ESRD-HDL) with additional phospholipids. RESULTS: There were significant differences in many parameters between the two groups. In particular, plasmalogens levels and cholesterol efflux capacity of HDL were significantly reduced in the CKD-5 group compared to those in the CKD-3-4 group (-35.1%, p < 0.001, -36.8%, p < 0.001, respectively). Multivariate linear regression analyses revealed that ethanolamine plasmalogen levels of HDL were independently associated with cholesterol efflux capacity (p = 0.045) and plaque scores (p = 0.035). In vitro studies also indicated that additional plasmalogens augmented cholesterol efflux ability of HDL. CONCLUSIONS: High plasmalogens concentrations in HDL may correlate with acceleration of cholesterol efflux and their decreased levels may promote atherosclerosis in advanced CKD patients.

Serum ethanolamine and hepatocyte proliferation in perinatal and partially hepatectomized rats.

It has been shown that the administration of ethanolamine (Etn) to partially hepatectomized rats enhances stimulation of DNA synthesis in regenerating hepatocytes. The present study aimed to test the hypothesis that the level of serum Etn in vivo may be regulated to control the growth of hepatocytes. Concentrations of serum Etn were determined in rats 1) of varying ages (from embryonic-19 (E-19) to 7-week-old), and 2) during regeneration following two-thirds hepatectomy (PH), to investigate whether serum Etn concentration correlates with the rate of proliferation of hepatocytes in growing animals or during regeneration. Serum Etn levels were 3 fold higher in E-19 fetuses and newborns than in adults, and were increased 2 fold 4 h after PH and remained high for at least 24 h. Results in both systems indicated a significant positive correlation between the rate of hepatocyte proliferation and serum Etn levels. Furthermore, Etn supplementation of 0.1 to 1 mmol immediately after PH promoted a significant weight gain and stimulated phosphatidylethanolamine (PE) and phosphatidylcholine (PC) synthesis in the regenerating liver. We also observed that whenever serum Etn levels were elevated, the metabolism of PE and PC in the liver changed dynamically, first by elevating the net synthesis of PE. Taken together, these results suggested that the levels of serum Etn might be regulated based on the physiological state of an animal, which consequently regulates the proliferation of hepatocytes.

Body composition in patients with classical homocystinuria: body mass relates to homocysteine and choline metabolism.

INTRODUCTION: Classical homocystinuria is a rare genetic disease caused by cystathionine ß-synthase deficiency, resulting in homocysteine accumulation. Growing evidence suggests that reduced fat mass in patients with classical homocystinuria may be associated with alterations in choline and homocysteine pathways. This study aimed to evaluate the body composition of patients with classical homocystinuria, identifying changes in body fat percentage and correlating findings with biochemical markers of homocysteine and choline pathways, lipoprotein levels and bone mineral density (BMD) T-scores. METHODS: Nine patients with classical homocystinuria were included in the study. Levels of homocysteine, methionine, cysteine, choline, betaine, dimethylglycine and ethanolamine were determined. Body composition was assessed by bioelectrical impedance analysis (BIA) in patients and in 18 controls. Data on the last BMD measurement and lipoprotein profile were obtained from medical records. RESULTS: Of 9 patients, 4 (44%) had a low body fat percentage, but no statistically significant differences were found between patients and controls. Homocysteine and methionine levels were negatively correlated with body mass index (BMI), while cysteine showed a positive correlation with BMI (p<0.05). There was a trend between total choline levels and body fat percentage (r=0.439, p=0.07). HDL cholesterol correlated with choline and ethanolamine levels (r=0.757, p=0.049; r=0.847, p=0.016, respectively), and total cholesterol also correlated with choline levels (r=0.775, p=0.041). There was no association between BMD T-scores and body composition. CONCLUSIONS: These results suggest that reduced fat mass is common in patients with classical homocystinuria, and that alterations in homocysteine and choline pathways affect body mass and lipid metabolism.

Adverse effect of obesity on red cell membrane arachidonic and docosahexaenoic acids in gestational diabetes.

AIMS/HYPOTHESIS: Gestational diabetes is a metabolic disorder affecting 2-5% of women and is a predictor of obesity, Type 2 diabetes mellitus and cardiovascular disease. Insulin resistance, a characteristic of gestational diabetes and obesity, is correlated with the fatty acids profile of the red cell and skeletal muscle membranes. We investigated the plasma and red cell fatty acid status of gestational diabetes. The effect of obesity on membrane fatty acids was also examined. METHODS: Fasting blood obtained at diagnosis was analysed for the fatty acids in plasma choline phosphoglycerides and red cell choline and ethanolamine phosphoglycerides. RESULTS: There were reductions in arachidonic acid (controls 10.74+/-2.35 vs gestational diabetes 8.35+/-3.49, p<0.01) and docosahexaenoic acid (controls 6.31+/-2.67 vs gestational diabetes 3.25+/-2.00, p<0.0001) in the red cell choline phosphoglycerides in gestational diabetes. A similar pattern was found in the ethanolamine phosphoglycerides. Moreover, the arachidonic and docosahexaenoic acids depletion in the red cell choline phosphoglycerides was much greater in overweight/obese gestational diabetes (arachidonic acid=7.49+/-3.37, docosahexaenoic acid=2.98+/-2.18, p<0.01) compared with lean gestational diabetes (arachidonic acid=10.03+/-2.74, docosahexaenoic acid=4.18+/-1.42). CONCLUSION/INTERPRETATION: Apparently normal plasma choline phosphoglycerides fatty acids profile in the gestational diabetic women suggested that membrane lipid abnormality is associated specifically with perturbation in the membrane. The fact that the lipid abnormality is more pronounced in the outer leaflet of the membrane where most of receptor binding and enzyme activities take place might provide an explanation for the increased insulin resistance in gestational diabetes and obesity.