OBJECTIVE: In this pilot study, we used untargeted metabolomics to identify biochemical mechanisms or biomarkers potentially underlying SLE-related fatigue.
METHODS: Metabolon conducted untargeted metabolomic plasma profiling using ultrahigh performance liquid chromatography/tandem mass spectrometry on plasma samples of 23 Black females with systemic lupus erythematosus (SLE) and 21 no SLE controls. Fatigue phenotypes of general fatigue, physical fatigue, mental fatigue, reduced activity, and reduced motivation were measured with the reliable and valid Multidimensional Fatigue Inventory (MFI).
RESULTS: A total of 290 metabolites were significantly different between the SLE and no SLE groups, encompassing metabolites related to glycolysis, TCA cycle activity, heme catabolism, branched chain amino acids, fatty acid metabolism, and steroids. Within the SLE group, controlling for age and co-morbidities, TCA cycle metabolites of alpha-ketoglutarate (AKG) and succinate were statistically significantly associated (p < .05) with physical and general fatigue.
CONCLUSIONS: While pervasive perturbations in the entire TCA cycle have been implicated as a potential mechanism for fatigue, our results suggest individual metabolites of AKG and succinate may be potential biomarkers or targets of intervention for fatigue symptom management in SLE. Additionally, perturbations in heme metabolism in the SLE group provide additional insights into mechanisms that promote systemic inflammation.

Alpha-ketoglutaric acid (α-KG), as an intermediate product of the tricarboxylic acid cycle, plays a crucial role in peptide and amino acid synthesis. In order to reduce costs and improve efficiency in the oxidative production of α-ketoglutaric acid, this study successfully synthesized and expressed L-glutamate oxidase (LGOXStr) from Streptomyces viridosporus R111 and catalase (KatGEsc) from Escherichia coli H736. Two immobilization methods and the conditions for one-step whole-cell catalysis of α-ketoglutaric acid were investigated. α-Ketoglutaric acid has broad applications in the pharmaceutical, food, and chemical industries. The specific research results are as follows: (1) By fusing the sfGFP tag, L-glutamate oxidase (LGOXStr r) and catalase (KatGEsc) were successfully anchored to the outer membrane of Escherichia coli cells, achieving one-step whole-cell catalysis of α-ketoglutaric acid with a conversion efficiency of up to 75%. (2) Through the co-immobilization of LGOXStr and KatGEsc, optimization of the preparation parameters of immobilized cells, and exploration of the immobilization method using E.coli@ZIF-8, immobilized cells with conversion rates of over 60% were obtained even after 10 cycles of reuse. Under the optimal conditions, the production rate of α-ketoglutaric acid reached 96.7% in a 12 h reaction, which is 1.1 times that of E. coli@SA and 1.29 times that of free cells.

Targeting molecular processes of aging will enable people to live healthier and longer lives by preventing age-related diseases. Geroprotectors are compounds with the potential to increase healthspan and lifespan. Even though many of them have been tested in animal models, the translation to humans is limited. Alpha-Ketoglutarate (AKG) has been studied widely in model animals, but there are few studies testing its geroprotective properties in humans. ABLE is a double blinded placebo-controlled randomized trial (RCT) of 1 g sustained release Ca-AKG versus placebo for 6 months of intervention and 3 months follow up including 120 40-60-year-old healthy individuals with a higher DNA methylation age compared to their chronological age. The primary outcome is the decrease in DNA methylation age from baseline to the end of the intervention. A total of 120 participants will be randomized to receive either sustained release Ca-AKG or placebo. Secondary outcomes include changes in the inflammatory and metabolic parameters in blood, handgrip strength and leg extension strength, arterial stiffness, skin autofluorescence, and aerobic capacity from baseline to 3 months, 6 months, and 9 months. This study will recruit middle-aged participants with an older DNA methylation age compared to their chronological age, and test whether supplementation with Ca-AKG can reduce DNA methylation age. This study is unique in its inclusion of biologically older participants.

The AAD-1 enzyme belongs to the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent nonheme aryloxyalkanoate dioxygenase family (AADs), which catalyzes the breakdown of 2,4-dichlorophenoxyacetic acid (2,4-D, an active ingredient of thousands of commercial herbicides) by using the highly active Fe(IV)═O complex. Multiple species of bacteria degrade 2,4-D via a pathway initiated by AADs; however, the detail of how they promote the cleavage of the ether C-O bond to generate 2,4-dichlorophenol (2,4-DCP) and glyoxylate is still unclear, which is the prerequisite for the further degradation of these halogenated aromatics. In this work, based on the crystal structure of AAD-1, the computational models were constructed, and a series of QM/MM and QM-only calculations were performed to explore the cleavage of the ether bond in 2,4-D with the catalysis of AAD-1. Our calculations reveal that AAD-1 may be only responsible for the hydroxylation of the substrate to generate the intermediate hemiacetal, which corresponds to an overall energy barrier of 14.2 kcal/mol on the quintet state surface, and the decomposition of the hemiacetal in the active site center of AAD-1 was calculated to be rather slow, corresponding to an energy barrier of 24.5 kcal/mol. In contrast, the decomposition of the free hemiacetal molecule in a solvent was calculated to be quite easy. Whether the decomposition of the hemiacetal occurs inside or outside the activation site is still worthy of experimental verification.

Invited for the cover of this issue are Christo Z. Christov and co-workers at Michigan Technological University, University of Oxford, and Michigan State University. The image depicts the oxygen diffusion channel in class 7 histone demethylase (PHF8) and ethylene-forming enzyme (EFE) and changes in the enzymes\' conformations upon binding. Read the full text of the article at 10.1002/chem.202300138.

This study investigates dioxygen binding and 2-oxoglutarate (2OG) coordination by two model non-heme FeII /2OG enzymes: a class 7 histone demethylase (PHF8) that catalyzes the hydroxylation of its H3K9me2 histone substrate leading to demethylation reactivity and the ethylene-forming enzyme (EFE), which catalyzes two competing reactions of ethylene generation and substrate l-Arg hydroxylation. Although both enzymes initially bind 2OG by using an off-line 2OG coordination mode, in PHF8, the substrate oxidation requires a transition to an in-line mode, whereas EFE is catalytically productive for ethylene production from 2OG in the off-line mode. We used classical molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM metadynamics (QM/MM-MetD) simulations to reveal that it is the dioxygen binding process and, ultimately, the protein environment that control the formation of the in-line FeIII -OO⋅- intermediate in PHF8 and the off-line FeIII -OO⋅- intermediate in EFE.

2-oxoglutarate (2OG), an intermediate metabolite in the tricarboxylic acid cycle, has been found to associate with chronic heart failure (HF), but its effect on short-term adverse outcomes in patients with acute HF (AHF) is uncertain.
This prospective cohort study included 411 consecutive hospitalized patients with AHF. During hospitalization, fasting plasma samples were collected within the first 24 h of admission. Plasma 2OG levels were measured by hydrophilic interaction liquid chromatography-liquid chromatography tandem mass spectrometry (HILIC-LC/MS/MS). All participants were followed up for six months. Multiple logistic regression was used to determine the odds ratio (OR) and 95% confidence interval (CI) for primary outcomes.
The AHF cohort consisted of HF with preserved ejection fraction (EF) (64.7%), mid-range EF (16.1%), and reduced EF (19.2%), the mean age was 65 (±13) years, and 65.2% were male. Participants were divided into two groups based on median 2OG levels (μg/ml): low group (< 6.0, n = 205) and high group (≥6.0, n = 206). There was a relatively modest correlation between 2OG and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels (r = 0.25; p < 0.001). After adjusting for age, sex, and body mass index, we found that the progression of the NYHA classification was associated with a gradual increase in plasma 2OG levels (p for trend< 0.001). After six months of follow-up, 76 (18.5%) events were identified. A high baseline 2OG level was positively associated with a short-term rehospitalization and all-cause mortality (OR: 2.2, 95% CI 1.3-3.7, p = 0.003), even after adjusting for NT-proBNP and estimated glomerular filtration rate (eGFR) (OR: 1.9, 95% CI 1.1-3.4, p = 0.032). After a similar multivariable adjustment, the OR was 1.4 (95% CI 1.1-1.7, p = 0.018) for a per-SD increase in 2OG level.
High baseline 2OG levels are associated with adverse short-term outcomes in patients with AHF independent of NT-proBNP and eGFR. Hence plasma 2OG measurements may be helpful for risk stratification and treatment monitoring in AHF.
ChiCTR-ROC-17011240 . Registered 25 April 2017.

Intensified training may lead to fatigue or even a state of overreaching with temporary reductions in performance. Any aid helping to prevent these consequences and to better tolerate such a training regime would be of great importance. 5-hydroxymethylfurfural (5-HMF) and α-ketoglutaric acid (α-KG) supplementation has been suggested to support favorable training outcomes but its effectiveness to facilitate adaptations during an intensified training period has never been investigated. During an in-season competition break (2 weeks), seventeen young outfield soccer players (age:14.7 ± 0.4 yr) performed a 9-day lasting shock microcyle including 5-7 repeated sprint exercise sessions in addition to the regular training (∼6 sessions/wk) and match (1-2 matches/wk) schedule. Before the training period a treadmill test to exhaustion, a YOYO intermittent recovery level 2 (YYIR2) and a repeated sprint ability (RSA) test were performed. The treadmill test was repeated 3 days after the shock microcycle whereas the YYIR2 and the RSA test on day 10 after the training. Magnitude based inference analysis showed likely positive effects of the 5-HMF/α-KG compared to the control group for changes in the maximal running velocity (+0.3 ± 0.7 vs. -0.3 ± 0.8 km/h) and running velocity at lactate turn-point 1 (+0.2 ± 0.4 vs. -0.2 ± 0.6) and lactate turn-point 2 (+0.4 ± 0.4 vs. -0.2 ± 0.6 km/h, for the 5-HMF/α-KG and placebo group, respectively). Training improved YYIR2 performance (+180 ± 67 vs. +200 ± 168m) and RSA (mean time: -0.1 ± 0.1 vs. -0.1 ± 0.1s, for the 5-HMF/α-KG and placebo group, respectively) in both groups and to the same extent. In conclusion, an in-season shock microcyle including repeated sprint training improves YYIR2 performance and RSA in youth soccer players. Supplementation with 5-HMF/α-KG did not modify training adaptations but led to likely positive exercise performance responses shortly after the intensified training regime.

Considering the pharmacological effects of chiral drugs, enantiopure drugs may differ from their racemic mixture formulation in efficacy, potency, or adverse effects. Levomethorphan (LVM) and Dextromethorphan (DXM) act on the central nervous system and exhibit different pharmacological features. LVM, the l-stereoisomer of methorphan, shows many similarities to opiates such as heroin, morphine and codeine, including the potential for addiction, while the d-stereoisomer, DXM, does not have the same opioid effect. In the present study, NMR-based metabolomics were performed on the urine of rats treated with these stereoisomers, and showed significant differences in metabolic profiles. In urine within 24 h after treatment of these samples, levels of citrate, 2-oxoglutarate, creatine, and dimethylglycine were higher in LVM-treated rats than in DXM-treated rats. While urinary levels of hippurate and creatinine gradually increased over 72 h in DXM-treated rats, these metabolites were decreased in the urine by 48-72 h after treatment with LVM. The levels of these changed metabolites may provide the first evidence for different cellular responses to the metabolism of stereoisomers.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fiehn et al., 2016), that described how we intended to replicate selected experiments from the paper \"The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate\" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.