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Modern lifestyle is often at odds with endogenously driven rhythmicity, which can lead to circadian disruption and metabolic syndrome. One signature for circadian disruption is a reduced or altered metabolite cycling in the circulating tissue reflecting the current metabolic status. Drosophila is a well-established model in chronobiology, but day-time dependent variations of transport metabolites in the fly circulation are poorly characterized. Here, we sampled fly hemolymph throughout the day and analyzed diacylglycerols (DGs), phosphoethanolamines (PEs) and phosphocholines (PCs) using LC-MS. In wild-type flies kept on sugar-only medium under a light-dark cycle, all transport lipid species showed a synchronized bimodal oscillation pattern with maxima at the beginning and end of the light phase which were impaired in period 01 clock mutants. In wild-type flies under constant dark conditions, the oscillation became monophasic with a maximum in the middle of the subjective day. In strong support of clock-driven oscillations, levels of the targeted lipids peaked once in the middle of the light phase under time-restricted feeding independent of the time of food intake. When wild-type flies were reared on full standard medium, the rhythmic alterations of hemolymph lipid levels were greatly attenuated. Our data suggest that the circadian clock aligns daily oscillations of DGs, PEs, and PCs in the hemolymph to the anabolic siesta phase, with a strong influence of light on phase and modality., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To assess alterations in the regional perfusion and oxygenation of the calf muscles in individuals with diabetes., Methods: Age-matched individuals with (n = 5) and without diabetes (n = 6) were investigated. Skeletal muscle perfusion, oxygen extraction fraction, and oxygen consumption rate were measured by newly developed noncontrast magnetic resonance imaging (MRI) techniques. The subjects lay supine on the MRI table with their foot firmly strapped to a custom-built isometric exercise device. The measurements were performed at rest and during an isometric plantar flexion muscle contraction., Results: Individuals without diabetes had up to a 10-fold increase in muscle perfusion, 25% elevation in muscle oxygen extraction fraction, and a 12-fold increase in oxygen consumption rate in the calf during the plantar flexion isometric contraction. In patients with diabetes, the increases in these parameters were only up to sixfold, 2%, and sixfold, respectively. Exercise oxygen consumption rate was inversely associated with blood HbA1c levels (r(2) = .91)., Conclusions: This is the first study to quantify regional skeletal muscle oxygenation in patients with diabetes using noncontrast MRI and warrants additional study. Attenuation of perfusion and oxygenation during exercise may have implications for understanding diabetic complications in the lower extremities., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Diaminofluorescein-dyes (DAFs) are widely used for visualizing NO· production in biological systems. Here it was examined whether DAF-fluorescence could be evoked by other means than nitrosation. Tobacco (Nicotiana tabacum) suspension cells treated with the fungal elicitor cryptogein released compound(s) which gave a fluorescence increase in the cell-free filtrate after addition of DAF-2 or DAF-FM or DAR-4M. DAF-reactive compounds were relatively stable and identified as reaction products of H(2)O(2) plus apoplastic peroxidase (PO). CPTIO prevented formation of these products. Horseradish-peroxidase (HR-PO) plus H(2)O(2) also generated DAF-fluorescence in vitro. Using RP-HPLC with fluorescence detection, DAF derivatives were further analyzed. In filtrates from cryptogein-treated cells, fluorescence originated from two novel DAF-derivatives also obtained in vitro with DAF-2+HR-PO+H(2)O(2). DAF-2T was only detected when an NO donor (DEA-NO) was present. Using high resolution mass spectrometry, the two above-described novel DAF-reaction products were tentatively identified as dimers. In cells preloaded with DAF-2 DA and incubated with or without cryptogein, DAF-fluorescence originated from a complex pattern of multiple products different from those obtained in vitro. One specific peak was responsive to exogenous H(2)O(2), and another, minor peak eluted at or close to DAF-2T. Thus, in contrast to the prevailing opinion, DAF-2 can be enzymatically converted into a variety of highly fluorescing derivatives, both inside and outside cells, of which none (outside) or only a minor part (inside) appeared NO· dependent. Accordingly, DAF-fluorescence and its prevention by cPTIO do not necessarily indicate NO· production., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The goal of rehabilitation following lower extremity amputation is to restore the highest level of independent function. As much as possible, this includes the functional use of a prosthetic device fitted to the residual limb. Early prosthetic fit depends, in turn, on rapid healing of the amputation site., Methods: We hypothesized that compliance with a novel custom-designed amputation protection and compression system (CAPCS) to the residual limb can accelerate and improve the likelihood of successful prosthesis use. We conducted a retrospective study of all patients who were offered CAPCS by certified prosthetists (Hanger Prosthetics and Orthotics, Bethesda, MD) during the period between April 2004 and November 2009. Variables included age, sex, indication for amputation, and compliance with CAPCS. Compliance was defined as consistent observed wearing of the CAPCS as directed. The primary end point was the fitting of a prosthetic device to the amputated limb, with time to prosthetic fit being the secondary outcome., Results: Out of 100 patients who were offered CAPCS (n = 100) during the study period, 76% were considered compliant (n = 76). Sixty five patients (65%) were ultimately fitted with prosthetic limbs. In multivariate analysis, we found that patients who had compliant use of CAPCS were significantly more likely to be successfully fit with prosthesis (72 vs. 42%, p = 0.005). At 100 days post amputation, the cumulative incidence of prosthesis fitting was significantly higher in CAPCS compliant patients (69.7 vs. 22.2%, p = 0.012)., Conclusions: Compliant use of a CAPCS following amputation is associated with earlier and more frequent use of a prosthetic. Based on this limited data set, a conclusion can be drawn that the potential exists to significantly improve functional outcomes after amputation, but well-designed prospective studies are needed to confirm this association., (Published by Elsevier Inc.)

Background & Aims: Breastfed infants may be at particular risk for iron deficiency because breast milk is low in iron. In a secondary analysis of data from a complementary feeding trial, indicators of iron status were examined, with particular focus on the development of iron status in those infants who were fully breastfed during the first 4 months of life., Methods: In this retrospective analysis of data from a randomized controlled trial infants were stratified according to their predominant milk diet during the first 4 months of life, a subgroup of breastfed infants (group BM, n=53) were compared with a subgroup of infants fed (iron-fortified) formula (group F, n=23). Dietary iron intake and indicators of iron status were analysed at 4 months of age (during the full milk feeding period), and during the complementary feeding period at 7 and 10 months of age., Results: Iron intake was low in the BM group, ranging below the Dietary Reference Intakes throughout the complementary feeding period, with the (estimated) bioavailable iron intake only just achieving the reference requirements. At 4 months, iron deficiency (ID, Ferritin <12.0 ng/mL) was observed in 3 infants in the BM group and in 1 infant in the F group; no infant developed iron deficiency anaemia (IDA, ID and Hb <10.5 g/dl). At 7 and at 10 months of age, iron status was adequate in all infants of the F group. In the BM group, at 7 (10) months of age, ID was diagnosed in 10 (11) infants and IDA was found in 2 (1) infants., Conclusions: Healthy infants, fully breastfed at 4 months of age, demonstrated ID in about 21% and IDA in up to 6% during the second half of infancy while fed according to the paediatric dietary guidelines. This finding supports the recommendation that supplementation with bioavailable iron via complementary foods should be started early (4-6 months of age) in order to prevent iron deficiency during infancy., (Copyright © 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

Supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) has been reported to reduce lipid peroxidation products formed from arachidonic acid (F(2)-isoprostanes) in healthy humans, as well as in those under oxidative stress. alpha-Linolenic acid (ALA) is a precursor to EPA and DHA; however, its conversion in humans is thought to be inefficient. ALA can also undergo free radical oxidation, forming compounds known as F(1)-phytoprostanes, which are found in all plants and are in high concentrations in plant pollens. In this study, we examined the effect of ALA supplementation on plasma and urine F(1)-phytoprostane and F(2)-isoprostane concentrations in men. Thirty-six nonsmoking men, aged 20-65 y, were recruited from the general population and randomly allocated to consume 9 g/d of either flaxseed oil (62% ALA, 5.4 g/d) or olive oil (placebo) for 4 wk in a parallel design. At baseline and after 4 wk of supplementation, blood samples and a 24-h urine sample were collected for measurement of plasma and urinary F(1)-phytoprostanes and F(2)-isoprostanes and plasma fatty acids. Compared with the olive oil group, plasma phospholipid ALA was greater (P < 0.0001), as were F(1)-phytoprostanes in plasma (P = 0.049) and urine (P = 0.06) in the flaxseed oil group after 4 wk supplementation. Flaxseed oil did not affect plasma or urinary F(2)-isoprostanes. The greater plasma F(1)-phytoprostane concentration in the flaxseed oil group most likely resulted from the increased plasma concentration of the ALA substrate and/or the F(1)-phytoprostane content of the flaxseed oil. Future studies are needed to determine the physiological importance of increased plasma and urine F(1)-phytoprostanes and their relevance to heart disease prevention.

Oxidized lipids in plants comprise a variety of reactive electrophiles that contain an alpha,beta-unsaturated carbonyl group. While some of these compounds are formed enzymatically, many of them are formed by non-enzymatic pathways. In addition to their chemical reactivity/toxicity low levels of these compounds are also biologically active. Despite their structural diversity and biosynthetic origin, common biological activities such as induction of defense genes, activation of detoxification responses and growth inhibition have been documented. However, reactive electrophilic oxylipins are poorly defined as a class of compounds but have at least two properties in common, i.e., lipophilicity and thiol-reactivity. Thiol-reactivity is a property of reactive oxylipins (RES) shared by reactive oxygen and nitrogen species (ROS and RNS) and enables these agents to modify proteins in vivo. Thiol-modification is assumed to represent a key mechanism involved in signal transduction. A metaanalysis of proteomic studies reveals that RES oxylipins, ROS and RNS apparently chemically modify a similar set of highly sensitive proteins, virtually all of which are targets for thioredoxins. Moreover, most of these proteins are redox-regulated, i.e., posttranslational thiol-modification alters the activity or function of these proteins. On the transcriptome level, effects of RES oxylipins and ROS on gene induction substantially overlap but are clearly different. Besides electrophilicity other structural properties such as target affinity apparently determine target selectivity and biological activity. In this context, different signalling mechanisms and signal transduction components identified in plants and non-plant organisms as well as putative functions of RES oxylipins are discussed.

Our aim was to investigate the effects of an oral supplementation of quercetin at 3 different doses on plasma concentrations of quercetin, parameters of oxidant/antioxidant status, inflammation, and metabolism. To this end, 35 healthy volunteers were randomly assigned to take 50, 100, or 150 mg/d (group Q50-Q150) quercetin for 2 wk. Fasting blood samples were collected at the beginning and end of the supplementation period. Compared with baseline, quercetin supplementation significantly increased plasma concentrations of quercetin by 178% (Q50), 359% (Q100), and 570% (Q150; P < 0.01 for all). High interindividual variation was found for plasma quercetin concentrations (36-57%). Quercetin did not affect concentrations of serum uric acid or plasma alpha- and gamma-tocopherols, oxidized LDL, and tumor necrosis factor-alpha, or plasma antioxidative capacity as assessed by the ferric-reducing antioxidant potential and oxygen radical absorbance capacity assays. In addition, serum lipids and lipoproteins, body composition, and resting energy expenditure did not significantly change during quercetin supplementation. Pharmacokinetics of quercetin were investigated in a subgroup of 15 volunteers. The areas under the plasma concentration-time curves ranged from 76.1 mumol.min.L(-1) to 305.8 mumol.min.L(-1) (50- and 150-mg dosages, respectively). Median maximum plasma concentrations of quercetin (431 nmol/L) were observed 360 min after intake of 150 mg quercetin. In conclusion, daily supplementation of healthy humans with graded concentrations of quercetin for 2 wk dose-dependently increased plasma quercetin concentrations but did not affect antioxidant status, oxidized LDL, inflammation, or metabolism.

A novel group of cyclopentenone prostaglandin-like compounds, deoxy phytoprostanes J(1), together with their precursors, phytoprostanes D(1), were identified in tobacco, tomato and Arabidopsis. Previously, it was thought that 14,15-dehydro-12-oxo-phytodienoic acid, a member of the deoxy phytoprostanes J(1) family, is derived from either 12-oxo-phytodienoic acid or diketols via the allene oxide synthase pathway. Results suggest that 14,15-dehydro-12-oxo-phytodienoic acid as well as structurally related cyclopentenones of the chromomoric acid family are synthesized via the phytoprostane D(1) pathway in planta. Notably, 14,15-dehydro-12-oxo-phytodienoic acid is more abundant than 12-oxo-phytodienoic acid in all three species so far analyzed.

Screening of 18 suspension plant cell cultures of taxonomically distant species revealed that a methyl jasmonate hydrolysing enzyme activity (0.21-5.67 pkat/mg) occurs in all species so far analysed. The methyl jasmonate hydrolysing esterase was purified from cell cultures of Lycopersicon esculentum using a five-step procedure including anion-exchange chromatography, gel-filtration and chromatography on hydroxylapatite. The esterase was purified 767-fold to give an almost homogenous protein in a yield of 2.2%. The native enzyme exhibited a M(r) of 26 kDa (gel-filtration chromatography), which was similar to the M(r) determined by SDS-PAGE and MALDI-TOF analysis (M(r) of 28547 kDa). Enzyme kinetics revealed a K(m) value of 15 microM and a V(max) value of 7.97 nkat/mg, an pH optimum of 9.0 and a temperature optimum of 40 degrees C. The enzyme also efficiently hydrolyzed methyl esters of abscisic acid, indole-3-acetic acid, and fatty acids. In contrast, methyl esters of salicylic acid, benzoic acid and cinnamic acid were only poor substrates for the enzyme. N-Methylmaleimide, iodacetamide, bestatin and pepstatin (inhibitors of thiol-, metal- and carboxyproteases, respectively) did not inactivate the enzyme while a serine protease inhibitor, phenylmethylsulfonyl fluoride, at a concentration of 5 mM led to irreversible and complete inhibition of enzyme activity. Proteolysis of the pure enzyme with endoproteinase LysC revealed three peptide fragments with 11-14 amino acids. N-Terminal sequencing yielded an additional peptide fragment with 10 amino acids. Sequence alignment of these fragments showed high homologies to certain plant esterases and hydroxynitrile lyases that belong to the alpha/beta hydrolase fold protein superfamily.

Animal prostaglandins and plant jasmonates are well-known enzymatically formed cyclopentanoic lipids that have regulatory functions and serve as inducible mediators of host defense reactions. A novel group of prostaglandin-like compounds, the isoprostanes, generated in animals and plants by a nonenzymatic, free radical-catalyzed process, are now suspected to be mediators of oxidant injury in vivo.

Jasmonic acid can be assayed by the highly sensitive and reproducible gas chromatography-negative ion chemical ionization-mass spectrometry (GC-NICI-MS) technique. We describe an optimized sample preparation method for routine analysis of jasmonic acids which allows the analysis of more than 10 samples/day. The basis of the method resides in a one-step extraction and phase partition, selective adsorption/elution of acids with an aminopropyl column, conversion of acids to pentafluorobenzyl esters, final purification using a silica column, and GC-NICI-MS. Extracted standard curves were linear over an assay range of 1 to 1000 ng; correlation coefficients were typically greater than 0.9999. The limit of detection was 500 fg (2.4 fmol) jasmonic acid/injection at a signal to noise ratio of 10:1. Furthermore, the method was modified for steric analysis of jasmonic acid stereoisomers at the nanogram level. (3R,7S)-(+)-7-Iso-jasmonic acid was found to be biosynthesized from plant tissue cell cultures upon elicitation. This stereoisomer epimerizes rapidly under alkaline and acidic conditions and in the presence of albumin. Epimerization rate constants were determined at different pH values.