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Hyperpolarized pyruvate is a widely used marker to track metabolism in vivo and a benchmark molecule for hyperpolarization methods. Here, we show how a combination of improved bullet-DNP instrumentation, an optimized sample preparation and a further sensitivity increase via a 13 C- 1 H polarization transfer after dissolution enable the observation of pyruvate at a concentration of 250 nM immediately after dissolution. At this concentration, the experiment employs a total mass of pyruvate of only 20 ng or 180 pmol. If the concentration is increased to 45 μM, pyruvate may be detected 1 min after dissolution with a signal-to-noise ratio exceeding 50. The procedure can be extended to observe a mixture of amino acids at low micromolar concentrations.

Background: Cerebral perfusion pressure (CPP) management in the developing child with traumatic brain injury (TBI) is challenging. The pressure reactivity index (PRx) may serve as marker of cerebral pressure autoregulation (CPA) and optimal CPP (CPPopt) may be assessed by identifying the CPP level with best (lowest) PRx. To evaluate the potential of CPPopt guided management in children with severe TBI, cerebral microdialysis (CMD) monitoring levels of lactate and the lactate/pyruvate ratio (LPR) (indicators of ischemia) were related to actual CPP levels, autoregulatory state (PRx) and deviations from CPPopt (ΔCPPopt)., Methods: Retrospective study of 21 children ≤ 17 years with severe TBI who had both ICP and CMD monitoring were included. CPP, PRx, CPPopt and ΔCPPopt where calculated, dichotomized and compared with CMD lactate and lactate-pyruvate ratio., Results: Median age was 16 years (range 8-17) and median Glasgow coma scale motor score 5 (range 2-5). Both lactate (p = 0.010) and LPR (p =  < 0.001) were higher when CPP ≥ 70 mmHg than when CPP < 70. When PRx ≥ 0.1 both lactate and LPR were higher than when PRx < 0.1 (p =  < 0.001). LPR was lower (p = 0.012) when CPPopt ≥ 70 mmHg than when CPPopt < 70, but there were no differences in lactate levels. When ΔCPPopt > 10 both lactate (p = 0.026) and LPR (p = 0.002) were higher than when ΔCPPopt < -10., Conclusions: Increased levels of CMD lactate and LPR in children with severe TBI appears to be related to disturbed CPA (PRx). Increased lactate and LPR also seems to be associated with actual CPP levels ≥ 70 mmHg. However, higher lactate and LPR values were also seen when actual CPP was above CPPopt. Higher CPP appears harmful when CPP is above the upper limit of pressure autoregulation. The findings indicate that CPPopt guided CPP management may have potential in pediatric TBI., (© 2024. The Author(s).)

Objective: Hyperpolarized [1- 13 C]-pyruvate magnetic resonance imaging is an emerging metabolic imaging method that offers unprecedented spatiotemporal resolution for monitoring tumor metabolism in vivo. To establish robust imaging biomarkers of metabolism, we must characterize phenomena that may modulate the apparent pyruvate-to-lactate conversion rate (k PL ). Here, we investigate the potential effect of diffusion on pyruvate-to-lactate conversion, as failure to account for diffusion in pharmacokinetic analysis may obscure true intracellular chemical conversion rates., Methods: Changes in hyperpolarized pyruvate and lactate signal were calculated using a finite-difference time domain simulation of a two-dimensional tissue model. Signal evolution curves with intracellular k PL values from 0.02 to 1.00 s -1 were analyzed using spatially invariant one-compartment and two-compartment pharmacokinetic models. A second spatially variant simulation incorporating compartmental instantaneous mixing was fit with the same one-compartment model., Results: When fitting with the one-compartment model, apparent k PL underestimated intracellular k PL by approximately 50% at an intracellular k PL of 0.02 s -1 . This underestimation increased for larger k PL values. However, fitting the instantaneous mixing curves showed that diffusion accounted for only a small part of this underestimation. Fitting with the two-compartment model yielded more accurate intracellular k PL values., Significance: This work suggests diffusion is not a significant rate-limiting factor in pyruvate-to-lactate conversion given that our model assumptions hold true. In higher order models, diffusion effects may be accounted for by a term characterizing metabolite transport. Pharmacokinetic models used to analyze hyperpolarized pyruvate signal evolution should focus on carefully selecting the analytical model for fitting rather than accounting for diffusion effects.

Onion (Allium cepa L) is a major reservoir of important nutraceutical ingredients. Herein, nutraceutical profiling of elite germplasm was assessed and hybrids with improved nutraceutical quality were selected. The nutraceutical components were screened through Fourier Transform Infrared Spectroscopy (FTIR) analysis (scan range 4000-400cm-1) followed by spectrophotometric/colorimetric quantification in oven dried bulb samples. Line × Tester (L×T) analysis was used to identify potential hybrids with better nutraceutical quality. Based on common functional groups obtained from FTIR analysis, as well as bulb color, the onion genotypes were categorized into six groups viz., white, yellowish brown, light brown, dark brown, brown and purplish brown. Results indicated that the purplish brown, yellowish brown and dark brown genotypes had maximum concentration of pyruvic acid, total flavonoids and total phenolic content, while vitamin C content showed weak association with color pigmentation. The onion variety 'Onion Swat' contained the highest level of pyruvic acid (17.18 μM) and 'MKS8823GO' had the highest vitamin C content (13.83mg/100mL). The L×T analysis revealed that out of 35 crosses, 'MKS-77127 × Onion Swat' and 'MKS-77127 × MKS777' were the best hybrids with improved nutraceutical quality. Further, observations for specific combining ability, general combining ability, genetic versus environmental variance, heritability and heterosis indicated that the studied parameters were genetically inherited and could be improved significantly by adopting an appropriate breeding strategy., Competing Interests: The authors have declared that no competing interests exist.

Cancer cells meet their energy need by predominantly increased uptake of glucose, high rate of glycolysis, and increased production of lactate even in the presence of adequate oxygen.  This process was proposed by Otto Warburg and named after him as the Warburg effect. The development of drugs that target glucose intake and aerobic glycolysis or lactic acid secretion of cancer cells is a newer approach for drug discovery. We have tested five purified plants-derived compounds such as curcumin, quercetin, ellagic acid, resveratrol, and indole-3-carbinol in HeLa cells for cytotoxicity, inhibition of metastasis, and modulation of lactate-pyruvate metabolism. Standard biochemical methods were used for glucose, lactic acid, and pyruvic acid measurement. The cell viability was determined by MTT assay. Cell migration was checked by wound healing assay. A dose-dependent cytotoxic effect and inhibition of cell migration were observed in all the tested compounds. A decrease in the lactate and increase in pyruvate level was observed in all the tested compounds except ellagic acid. Our finding suggests that tested phytocompounds are associated with the metabolic reprogramming of cancer cells and execute the cytotoxic effect. These compounds could be used for cancer prevention and therapy., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Background: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden., Results: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate., Conclusions: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing., (© 2021. The Author(s).)

Bedside detection and early treatment of lasting cerebral ischemia may improve outcome after out-of-hospital cardiac arrest (OHCA). This feasibility study explores the possibilities to use microdialysis (MD) for continuous monitoring of cerebral energy metabolism by analyzing the draining cerebral venous blood. Eighteen comatose patients were continuously monitored with jugular bulb and radial artery (reference) MD following resuscitation. Median time from cardiac arrest to MD was 300 min (IQR 230-390) with median monitoring time 60 h (IQR 40-81). The lactate/pyruvate ratio in cerebral venous blood was increased during the first 20 h after OHCA, and significant differences in time-averaged mean MD metabolites between jugular venous and artery measurements, were documented (p < 0.02). In patients with unfavorable outcome (72%), cerebral venous lactate and pyruvate levels remained elevated during the study period. In conclusion, the study indicates that jugular bulb microdialysis (JBM) is feasible and safe. Biochemical signs of lasting ischemia and mitochondrial dysfunction are frequent and associated with unfavorable outcome. The technique may be used in comatose OHCA patients to monitor biochemical variables reflecting ongoing brain damage and support individualized treatment early after resuscitation., (© 2021. The Author(s).)

Background: Cerebral ischemia and neuroinflammation following aneurysmal subarachnoid hemorrhage (SAH) are major contributors to poor neurological outcome. Our study set out to investigate in an exploratory approach the interaction between NO and energy metabolism following SAH as both hypoxia and inflammation are known to affect nitric oxide (NO) metabolism and NO in turn affects mitochondria., Methods: In seven patients under continuous multimodality neuromonitoring suffering poor-grade aneurysmal SAH, cerebral metabolism and NO levels (determined as a sum of nitrite plus nitrate) were determined in cerebral microdialysate for 14 days following SAH. In additional ex vivo experiments, rat cortex homogenate was subjected to the NO concentrations determined in SAH patients to test whether these NO concentrations impair mitochondrial function (determined by means of high-resolution respirometry)., Results: NO levels showed biphasic kinetics with drastically increased levels during the first 7 days (74.5 ± 29.9 μM) and significantly lower levels thereafter (47.5 ± 18.7 μM; p = 0.02). Only during the first 7 days, NO levels showed a strong negative correlation with brain tissue oxygen tension (r = - 0.78; p < 0.001) and a positive correlation with cerebral lactate (r = 0.79; p < 0.001), pyruvate (r = 0.68; p < 0.001), glutamate (r = 0.65; p < 0.001), as well as the lactate-pyruvate ratio (r = 0.48; p = 0.01), suggesting mitochondrial dysfunction. Ex vivo experiments confirmed that the increase in NO levels determined in patients during the acute phase is sufficient to impair mitochondrial function (p < 0.001). Mitochondrial respiration was inhibited irrespectively of whether glutamate (substrate of complex I) or succinate (substrate of complex II) was used as mitochondrial substrate suggesting the inhibition of mitochondrial complex IV. The latter was confirmed by direct determination of complex IV activity., Conclusions: Exploratory analysis of our data suggests that during the acute phase of SAH, NO plays a key role in the neuronal damage impairing mitochondrial function and facilitating accumulation of mitochondrial substrate; further studies are required to understand mechanisms underlying this observation.

Objectives: The clinical and biochemical manifestations of acute rejection after liver transplantation are nonspecific, and a liver biopsy is often needed to verify the diagnosis. This may delay treatment. The aim of this study was to evaluate whether monitoring of intrahepatic glucose, lactate, pyruvate, and glycerol by microdialysis can be used to predict rejection early after liver transplantation., Methods: Seventy-one patients undergoing liver transplantation were included in the study. The patients were monitored using microdialysis for up to 6 days postoperatively. Patients who developed acute rejection within 1 month were identified according to standard protocol. Area under the curve (AUC) was calculated for 12-hour intervals for glucose, lactate, pyruvate, glycerol, and lactate/pyruvate ratio. Patients with and without rejection were compared with respect to these parameters, as well as standard liver blood investigations and time-zero biopsies., Results: The lactate/pyruvate ratio was higher at 0 to 12 hours in the group with rejection as compared to the group without rejection. Glucose was lower in the group with rejection at 24 to 48 hours. Also, the intrahepatic lactate levels at 48 to 72 hours and pyruvate levels at 60 to 72 hours after liver transplantation, were higher in the rejection group. The lactate/pyruvate ratio at 0 to 12 hours and lactate at 60 to 72 hours were two independent risk factors for rejection within the first month after liver transplantation. No significant differences in glycerol levels could be detected between the two patient groups., Conclusions: Microdialysis monitoring following liver transplantation may be useful in the detection of the metabolic events that precede rejection. The metabolic patterns detected by microdialysis early after transplantation indicate a possible relation between primary ischemia-reperfusion injury and the development of rejection. Identifying these patterns may help to identify patients at risk for the development of acute rejection and may help select those who may benefit from higher dose of immunosuppression early after liver transplantation., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

A buffer placed in brief contact in the skin was assayed by 1 H NMR spectroscopy. We found that this passive extraction of the skin surface yields abundant metabolites. Metabolites of the skin surface originate from a variety of sources, including the sweat gland, which produces lactate from the glucose received from its capillary bed. Little is known about how metabolites resident on and within the skin surface respond to a metabolic or hemodynamic perturbation. As a possible application of epidermal metabolite profiling, we asked whether metabolites extracted from the skin surface are indicative of heart failure. The levels of lactate and other molecules were significantly lower in patients in heart failure than in individuals who reported healthy heart function, possibly due to reduced blood flow to the sweat gland resulting in a lack of tissue perfusion. Most amino acids were unchanged in levels, except for glycine and serine that increased as a percentage of all amino acids. These results have the potential in the long term to help decide the extent to which a patient has heart failure for which objective measures are lacking. Moreover, the results suggest that epidermal metabolite profiling may be useful for other assessments of human health.

Hyperpolarized [1- 13 C]pyruvate magnetic resonance spectroscopic imaging (MRSI) is a noninvasive metabolic-imaging modality that probes carbon flux in tissues and infers the state of metabolic reprograming in tumors. Prevailing models attribute elevated hyperpolarized [1- 13 C]pyruvate-to-[1- 13 C]lactate conversion rates in aggressive tumors to enhanced glycolytic flux and lactate dehydrogenase A (LDHA) activity (Warburg effect). By contrast, we find by cross-sectional analysis using genetic and pharmacological tools in mechanistic studies applied to well-defined genetically engineered cell lines and tumors that initial hyperpolarized [1- 13 C]pyruvate-to-[1- 13 C]lactate conversion rates as well as global conversion were highly dependent on and critically rate-limited by the transmembrane influx of [1- 13 C]pyruvate mediated predominately by monocarboxylate transporter-1 (MCT1). Specifically, in a cell-encapsulated alginate bead model, induced short hairpin (shRNA) knockdown or overexpression of MCT1 quantitatively inhibited or enhanced, respectively, unidirectional pyruvate influxes and [1- 13 C]pyruvate-to-[1- 13 C]lactate conversion rates, independent of glycolysis or LDHA activity. Similarly, in tumor models in vivo, hyperpolarized [1- 13 C]pyruvate-to-[1- 13 C]lactate conversion was highly dependent on and critically rate-limited by the induced transmembrane influx of [1- 13 C]pyruvate mediated by MCT1. Thus, hyperpolarized [1- 13 C]pyruvate MRSI measures primarily MCT1-mediated [1- 13 C]pyruvate transmembrane influx in vivo, not glycolytic flux or LDHA activity, driving a reinterpretation of this maturing new technology during clinical translation. Indeed, Kaplan-Meier survival analysis for patients with pancreatic, renal, lung, and cervical cancers showed that high-level expression of MCT1 correlated with poor overall survival, and only in selected tumors, coincident with LDHA expression. Thus, hyperpolarized [1- 13 C]pyruvate MRSI provides a noninvasive functional assessment primarily of MCT1 as a clinical biomarker in relevant patient populations., Competing Interests: The authors declare no competing interest.

There are different studies that aim to enhance the production of nisin by Lactococcus lactis since its chemical synthesis is not possible. In this study, glutathione (GSH) and pyruvate, which are known to reduce the oxidative stress of cells, have been shown to trigger the production of nisin at both transcriptional and translational levels in L. lactis cells grown under aerobic condition. Presence of GSH and pyruvate caused more nisin yield than the heme-supplemented medium. Moreover, the expression of genes that encode stress-related enzymes were apparently upregulated in the presence of GSH and pyruvate. It can be concluded that GSH and pyruvate contribute to the defense system of L. lactis cells and so that higher biomass was obtained which in turn enhance nisin production. Antioxidant effect of GSH and pyruvate was known; however, their stimulating effect on nisin production was shown for the first time in this study.

Kinetic modeling of the in vivo pyruvate-to-lactate conversion is crucial to investigating aberrant cancer metabolism that demonstrates Warburg effect modifications. Non-invasive detection of alterations to metabolic flux might offer prognostic value and improve the monitoring of response to treatment. In this clinical research project, hyperpolarized [1- 13 C] pyruvate was intravenously injected in a total of 10 brain tumor patients to measure its rate of conversion to lactate ( k PL ) and bicarbonate ( k PB ) via echo-planar imaging. Our aim was to investigate new methods to provide k PL and k PB maps with whole-brain coverage. The approach was data-driven and addressed two main issues: selecting the optimal model for fitting our data and determining an appropriate goodness-of-fit metric. The statistical analysis suggested that an input-less model had the best agreement with the data. It was also found that selecting voxels based on post-fitting error criteria provided improved precision and wider spatial coverage compared to using signal-to-noise cutoffs alone.

Background: There is no uniform definition for cerebral microdialysis (CMD) probe location with respect to focal brain lesions, and the impact of CMD-probe location on measured molecule concentrations is unclear., Methods: We retrospectively analyzed data of 51 consecutive subarachnoid hemorrhage patients with CMD-monitoring between 2010 and 2016 included in a prospective observational cohort study. Microdialysis probe location was assessed on all brain computed tomography (CT) scans performed during CMD-monitoring and defined as perilesional in the presence of a focal hypodense or hyperdense lesion within a 1-cm radius of the gold tip of the CMD-probe, or otherwise as normal-appearing brain tissue., Results: Probe location was detected in normal-appearing brain tissue on 53/143 (37%) and in perilesional location on 90/143 (63%) CT scans. In the perilesional area, CMD-glucose levels were lower (p = 0.003), whereas CMD-lactate (p = 0.002), CMD-lactate-to-pyruvate-ratio (LPR; p < 0.001), CMD-glutamate (p = 0.002), and CMD-glycerol levels (p < 0.001) were higher. Neuroglucopenia (CMD-glucose < 0.7 mmol/l, p = 0.002), metabolic distress (p = 0.002), and mitochondrial dysfunction (p = 0.005) were more common in perilesional compared to normal-appearing brain tissue. Development of new lesions in the proximity of the CMD-probe (n = 13) was associated with a decrease in CMD-glucose levels, evidence of neuroglucopenia, metabolic distress, as well as increasing CMD-glutamate and CMD-glycerol levels. Neuroglucopenia was associated with poor outcome independent of probe location, whereas elevated CMD-lactate, CMD-LPR, CMD-glutamate, and CMD-glycerol levels were only predictive of poor outcome in normal-appearing brain tissue., Conclusions: Focal brain lesions significantly impact on concentrations of brain metabolites assessed by CMD. With the exception of CMD-glucose, the prognostic value of CMD-derived parameters seems to be higher when assessed in normal-appearing brain tissue. CMD was sensitive to detect the development of new focal lesions in vicinity to the neuromonitoring probe. Probe location should be described in the research reporting brain metabolic changes measured by CMD and integrated in statistical models.

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L-lactate dehydrogenase (L-LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K m values of pyruvate and NADH of the immobilized L-LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L-LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pyruvic acid (PA) has been demonstrated to be an important cancer biomarker. Herein, carbon/carbon nanotube paste electrode was modified with the newly synthesized PA-imprinted polymer (MIP) and used as an enzyme-free sensor for PA assay. Methacrylic acid and ethylene glycol dimethacrylate were copolymerized in the presence of PA to prepare PA-IP. The MIP was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. To analyze PA by the MIP/CNT-CP electrode, the electrode was incubated in the PA solution for a constant time and then, the anodic differential pulse voltammetry signal was recorded. Both extraction and electrochemical determination solutions were the same, making the procedure simple and fast. Presence of the CNT in the MIP electrode led to a great enhancement in the PA signal. The MIP material not only pre-concentrated PA at the electrode surface but also increased the electron-exchange rate. This was confirmed by electrochemical impedance spectroscopy. The effects of electrode composition, extraction condition, and voltammetry parameters on the sensing efficiency were optimized. Dynamic linear range, detection limit, and RSD of the sensor were estimated to be 0.1-200 μM, 0.048 μM (S/N), and 3.6% (n = 3), respectively. The utility of the method was confirmed by appropriate analysis results obtained for the determination of PA in the plasma and urine samples. Graphical Abstract.

Regulation of the energetic metabolism occurs fundamentally at the cellular level, so analytical strategies must aim to attain single cell resolution to fully embrace its inherent complexity. We have developed methods to utilize a toolset of metabolic FRET sensors for assessing lactate, pyruvate and 2-oxoglutarate levels of Drosophila tissues in vivo by imaging techniques. We show here how the energetic metabolism is altered by hypoxia: While some larval tissues respond to low oxygen levels by executing a metabolic switch towards lactic fermentation, the fat body and salivary glands do not alter their energetic metabolism. Analysis of tumor metabolism revealed that depending on the genetic background, some tumors undergo a lactogenic switch typical of the Warburg effect, while other tumors do not. This toolset allows for developmental and physiologic studies in genetically manipulated Drosophila individuals in vivo.

1. The purpose of this study was to investigate ATP levels and the activities of important enzymes involved in glycolysis and TCA cycle in livers of embryonated chicken eggs infected by infectious bursal disease virus (IBDV).2. Embryonated chicken eggs (9 days) were randomly divided into two groups (50 eggs per group). The first group was inoculated with a very virulent IBDV (vvIBDV) isolate into the chorioallantoic membrane. The second group was maintained as uninfected control eggs and inoculated with physiological saline. Embryo survival was assessed daily, and six embryos were sacrificed at 24, 48, 72, 96, and 120 hpi for examining livers. Viral loads in the livers were evaluated by qRT-PCR. A comparative analysis of markers associated with the regulation of energy metabolism across several functional classes (ATP, pyruvic and lactic acids, mitochondrial protein, NAD+/NADH ratios, and enolase, lactic acid dehydrogenase and the respiratory chain complex I activities) were examined in the context of IBDV infection.3. The results indicated that increases in the enzymatic activities associated with glycolytic metabolism in turn affected the synthesis and cytoplasmic concentrations of ATP at early timepoints in infected chicken embryos. Subsequently, energy metabolism was inhibited through the pathological perturbations of metabolic enzymes and mitochondrial damage, as inferred from reduced ATP generation.4. These results suggested impaired bioenergetics, which may lead to liver dysfunction consequent to IBDV infection, contributing to the disease pathogenesis.

The aim of the study was to screen Yarrowia lipolytica strains for keto acid production and determine optimal conditions for pyruvic acid biosynthesis from glycerol by the best producer. The analyzed parameters were thiamine concentration, medium pH, stirring speed, and substrate concentration. The screening was performed in flask cultures, whereas pyruvic acid production was carried out in 5-L stirred-tank reactor with 2 L of working volume. In total, 24 Y. lipolytica strains were compared for their abilities to produce pyruvic and α-ketoglutaric acids. The total concentration of both acids ranged from 0.1 to 15.03 g/L. Ten strains were selected for keto acid biosynthesis in bioreactor. The Y. lipolytica SKO 6 strain was identified as the best producer of pyruvic acid. In the selected conditions (thiamine concentration 1.5 μg/L, pH 4.0, stirring speed 800 rpm, 150 g/L of glycerol), the strain Y. lipolytica SKO 6 produced 99.3 g/L of pyruvic acid, with process yield of 0.63 g/g and volumetric production rate of 1.18 g/L/h. Higher titer of pyruvic acid was obtained during fed-batch culture with 200 g/L of glycerol, reaching 125.8 g/L from pure glycerol (yield 0.68 g/g) and 124.4 g/L from crude glycerol (yield 0.62 g/g). Results obtained for the strain Y. lipolytica SKO 6 proved the suitability of microbial production of pyruvic acid at industrial scale.

Background: Acute Achilles tendon rupture (ATR) is a frequently disabling injury, which exhibits unclear variability in long-term functional and patient-reported outcomes. Biomarkers from early healing, which have been shown to be prognostic of long-term outcome would facilitate the development of improved treatment methods., Hypothesis/purpose: The aim of this study was to assess essential metabolites pyruvate and its product lactate, as early biomarkers in relation to long-term functional- and patient-reported outcome after ATR., Study Design: Prospective cohort study., Methods: A total of 124 patients (103 men, 21 women; mean age 40 ± 7 years) with ATR, treated with uniform anesthetic and surgical technique, were prospectively assessed. At two weeks post-injury pyruvate and lactate concentrations were assessed in both the injured and uninjured limbs using microdialysis followed by enzymatic quantification. The ratios of the concentration in the injured versus uninjured limb of pyruvate (pyruvate-r) and lactate (lactate-r) were calculated as well as the lactate/pyruvate ratios (L/P-r). At 12 months, patient-reported outcome was examined using self-reported questionnaires; Achilles tendon Total Rupture Score (ATRS), Foot and Ankle Outcome Score (FAOS), and physical activity score. At 12 months, functional outcome was studied using the validated heel-rise test., Results: Elevated pyruvate-r, at two weeks, was significantly associated with total ATRS (R = 0.254, P = 0.028), less loss in physical activity (R = 0.241, P = 0.039), less experience of pain in FAOS (R = 0.275, P = 0.032), and a higher number of heel-rise repetitions on injured side (R = 0.230, P = 0.040) at 12 months. Increased lactate-r was related with less strength limitations in the calf (R = 0.283, P = 0.011), while the elevated lactate-pyruvate ratio, notably, was related to more limitations in walking on uneven surface (R = -0,243, P = 0.027). The findings were verified by multiple linear regression taking confounding factors into consideration., Conclusion: This study established that the metabolite pyruvate is a good potential biomarker, prognostic of patient outcome at the one-year follow-up after ATR surgery. These novel findings suggest that local biomarkers could be developed at an early-stage screen for new ATR treatments., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The fundamental limit to in vivo imaging applications of hyperpolarized 13 C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13 C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1- 13 C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3°-5°). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

The work was aimed at the development of a biosensor array for the simultaneous determination of six solutes (glutamate, glucose, choline, acetylcholine, lactate, and pyruvate) in aqueous solutions. Enzymes selective for these substrates were immobilized on the surface of amperometric platinum disc electrodes and served as bioselective elements of a biosensor array. Direct enzymatic analysis by the developed biosensors provided high sensitivity to the tested substrates (limits of detection were 1-5 μM). The linear ranges of the biosensors were from 0.001-0.01 mM to 0.2-2.5 mM. The influence of solution pH, ionic strength and buffer capacity on the biosensor responses was investigated; the conditions for simultaneous operation of all the bioselective elements were optimized. The absence of any cross-influence of the substrates of enzymatic systems used was shown as well as a high selectivity of the biosensors and the absence of any impact of interfering substances (ascorbic acid, dopamine, cysteine, paracetamol). The developed biosensor array had good response reproducibility and storage stability. The array is suitable for rapid (0.5-1 min) and simple simultaneous determination of glutamate, glucose, choline, acetylcholine, lactate, and pyruvate in aqueous (biological) samples; furthermore, the creation of a single chip with six sensitive elements is possible as well as the addition of other biosensors., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Various excitation strategies have been proposed for dynamic imaging of hyperpolarized agents such as [1- 13 C]-pyruvate, but the impact of these strategies on quantitative evaluation of signal evolution remains unclear. To better understand their relative performance, we compared the accuracy and repeatability of measurements made using variable excitation angle strategies and conventional constant excitation angle strategies., Methods: Signal evolution for constant and variable excitation angle schedules was simulated using a pharmacokinetic model of hyperpolarized pyruvate with 2 chemical pools and 2 physical compartments. Noisy synthetic data were then fit using the same pharmacokinetic model with the apparent chemical exchange term as an unknown, and fit results were compared with simulation parameters to determine accuracy and reproducibility., Results: Constant excitations and a variable excitation strategy that maximizes the HP lactate signal yielded data that supported quantitative analyses with similar accuracy and repeatability. Variable excitation angle strategies that were designed to produce a constant signal level resulted in lower signal and worse quantitative accuracy and repeatability, particularly for longer acquisition times., Conclusions: These results suggest that either constant excitation angle or variable excitation angles that attempt to maximize total signal, as opposed to maintaining a constant signal level, are preferred for metabolic quantification using hyperpolarized pyruvate., (© 2019 International Society for Magnetic Resonance in Medicine.)

Purpose: To develop and translate a metabolite-specific imaging sequence using a symmetric echo planar readout for clinical hyperpolarized (HP) Carbon-13 ( 13 C) applications., Methods: Initial data were acquired from patients with prostate cancer (N = 3) and high-grade brain tumors (N = 3) on a 3T scanner. Samples of [1- 13 C]pyruvate were polarized for at least 2 h using a 5T SPINlab system operating at 0.8 K. Following injection of the HP substrate, pyruvate, lactate, and bicarbonate (for brain studies) were sequentially excited with a singleband spectral-spatial RF pulse and signal was rapidly encoded with a single-shot echo planar readout on a slice-by-slice basis. Data were acquired dynamically with a temporal resolution of 2 s for prostate studies and 3 s for brain studies., Results: High pyruvate signal was seen throughout the prostate and brain, with conversion to lactate being shown across studies, whereas bicarbonate production was also detected in the brain. No Nyquist ghost artifacts or obvious geometric distortion from the echo planar readout were observed. The average error in center frequency was 1.2 ± 17.0 and 4.5 ± 1.4 Hz for prostate and brain studies, respectively, below the threshold for spatial shift because of bulk off-resonance., Conclusion: This study demonstrated the feasibility of symmetric EPI to acquire HP 13 C metabolite maps in a clinical setting. As an advance over prior single-slice dynamic or single time point volumetric spectroscopic imaging approaches, this metabolite-specific EPI acquisition provided robust whole-organ coverage for brain and prostate studies while retaining high SNR, spatial resolution, and dynamic temporal resolution., (© 2018 International Society for Magnetic Resonance in Medicine.)

Purpose: Androgen receptor (AR) signaling affects prostate cancer (PCa) growth, metabolism, and progression. Often, PCa progresses from androgen-sensitive to castration-resistant prostate cancer (CRPC) following androgen-deprivation therapy. Clinicopathologic and genomic characterizations of CRPC tumors lead to subdividing CRPC into two subtypes: (1) AR-dependent CRPC containing dysregulation of AR signaling alterations in AR such as amplification, point mutations, and/or generation of splice variants in the AR gene; and (2) an aggressive variant PCa (AVPC) subtype that is phenotypically similar to small cell prostate cancer and is defined by chemotherapy sensitivity, gain of neuroendocrine or pro-neural marker expression, loss of AR expression, and combined alterations of PTEN, TP53, and RB1 tumor suppressors. Previously, we reported patient-derived xenograft (PDX) animal models that contain characteristics of these CRPC subtypes. In this study, we have employed the PDX models to test metabolic alterations in the CRPC subtypes., Procedures: Mass spectrometry and nuclear magnetic resonance analysis along with in vivo hyperpolarized 1-[ 13 C]pyruvate spectroscopy experiments were performed on prostate PDX animal models., Results: Using hyperpolarized 1-[ 13 C]pyruvate conversion to 1-[ 13 C]lactate in vivo as well as lactate measurements ex vivo, we have found increased lactate production in AR-dependent CRPC PDX models even under low-hormone levels (castrated mouse) compared to AR-negative AVPC PDX models., Conclusions: Our analysis underscores the potential of hyperpolarized metabolic imaging in determining the underlying biology and in vivo phenotyping of CRPC.

Background: Objective interpretation of laboratory test results used to diagnose and monitor diabetes mellitus in part requires the application of biological variation data (BVD). The quality of published BVD has been questioned. The aim of this study was to quality assess publications reporting BVD for diabetes-related analytes using the Biological Variation Data Critical Appraisal Checklist (BIVAC); to assess whether published BVD are fit for purpose and whether the study design and population attributes influence BVD estimates and to undertake a meta-analysis of the BVD from BIVAC-assessed publications., Methods: Publications reporting data for glucose, HbA 1c , adiponectin, C-peptide, fructosamine, insulin like growth factor 1 (IGF-1), insulin like growth factor binding protein 3 (IGFBP-3), insulin, lactate and pyruvate were identified using a systematic literature search. These publications were assessed using the BIVAC, receiving grades A, B, C or D, where A is of highest quality. A meta-analysis of the BVD from the assessed studies utilised weightings based upon BIVAC grades and the width of the data confidence intervals to generate global BVD estimates., Results: BIVAC assessment of 47 publications delivered 1 A, 3 B, 39C and 4 D gradings. Publications relating to adiponectin, C-peptide, IGF-1, IGFBP-3, lactate and pyruvate were all assessed as grade C. Meta-analysis enabled global BV estimates for all analytes except pyruvate, lactate and fructosamine., Conclusions: This study delivers updated and evidence-based BV estimates for diabetes-related analytes. There remains a need for delivery of new high-quality BV studies for several clinically important analytes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 μM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.

Background: To facilitate faster phenotyping of onions (Allium cepa L.), Fourier-transform mid infrared (FT-MIR) spectroscopy with partial least squares (PLS) regression modelling was evaluated for the determination of pungency (pyruvate), sweetness (free sugars) and fructan in juice samples (n = 605) expressed from bulbs from breeding populations., Results: Fourier-transform infrared (FTIR) spectra (range 1700-900 cm -1 ) were obtained from droplets (30 μL) of unprocessed juice. Goodness-of-fit (r 2 ) and prediction errors (standard error of cross validation) for optimal PLS models were: soluble solids (0.997, 0.1 °Brix), pyruvate [0.825, 0.8 μmol g -1 fresh weight (FW)], fructan (0.98, 1.9 mg g -1 FW), glucose (0.941, 1.1 mg g -1 FW), fructose (0.967, 1.0 mg g -1 FW) and sucrose (0.919, 1.7 mg g -1 FW). FTIR models for industry sweetness indices based on glucose or sucrose equivalents were also developed. Because of its very low concentration (0.8-12 μmol g -1 FW) relative to other compounds, pyruvate was the weakest model developed. Fructan could be determined spectroscopically without the need for enzymatic digestion., Conclusions: All of the chemometric models developed are acceptable for screening purposes. Those for soluble solids, fructan and fructose are also suitable for routine analysis. FT-MIR can therefore be utilised for the simultaneous determination of pungency, sweetness and fructan in this crop. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

The novel [Pt( O , O' -acac)(γ-acac)(DMS)], Ptac2S, Pt(II) complex has recently gained increasing attention as a potential anticancer agent for its pharmacological activity shown in different tumor cell lines, studied both in vitro and in vivo. The mechanism of action of Ptac2S, operating on non-genomic targets, is known to be very different from that of cis -[PtCl₂(NH₃)₂], cisplatin, targeting nucleic acids. In this work, we evaluated the cytotoxicity of Ptac2S on the cisplatin resistant Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells, by the MTT assay. A ¹H-NMR metabolomic approach coupled with multivariate statistical analysis was used for the first time for Ptac2S to figure out the biological mechanisms of action of the complex. The metabolic variations of intracellular metabolites and the composition of the corresponding extracellular culture media were compared to those of cisplatin (cells were treated at the IC 50 doses of both drugs). The reported comparative metabolomic analysis revealed a very different metabolic profile between Ptac2S and cisplatin treated samples, thus confirming the different mechanism of action of Ptac2S also in the Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells line. In particular, higher levels of pyruvate were observed in Ptac2S treated, with respect to cisplatin treated, cells (in both aqueous and culture media). In addition, a very different lipid expression resulted after the exposure to the two drugs (Ptac2S and cisplatin). These results suggest a possible explanation for the Ptac2S ability to circumvent cisplatin resistance in SKOV-3 cells.

Background: Individuals with inherited deficiencies of the pyruvate dehydrogenase complex or the respiratory chain complex can have increased concentrations of cerebrospinal fluid (CSF) lactate. Such measurements are clinical useful when measured in conjunction with pyruvate in order to calculate the lactate:pyruvate (L:P) ratio, a useful surrogate of cytosolic redox status. CSF pyruvate is measured in a protein-free supernatant prepared by the addition of CSF to perchloric acid while lactate is measured in untreated CSF. Utilizing the same sample for both lactate and pyruvate measurements is desirable., Objective: To develop a method to measure lactate in perchloric-acid precipitated CSF and validate the L:P ratio as calculated from the analysis of both analytes in the same sample., Methods: Samples were prepared by the addition of 1 mL CSF to 2 mL 8% (w/v) cold perchloric acid, incubated on ice for 10 min, then centrifuged to obtain a protein-free supernatant. Lactate was measured by its oxidation to pyruvate and hydrogen peroxide using lactate oxidase and the absorbance of the resulting chromogen determined at 540 nm on a Roche cobas c501 chemistry analyzer. Method accuracy, linearity, imprecision and sensitivity were determined and a reference interval was verified., Results: To assess accuracy, this method was compared to lactate determined in unaltered CSF at another laboratory using 41 specimens with lactate concentrations from 0.6-11.9 mmol/L. Linear regression produced a slope of 1.09 and y-intercept of 0.26 (R 2  = 1.00). Recovery was performed by ad-mixes of a high lactate standard and a CSF pool in different ratios to create a set of 19 samples prior to preparing protein-free supernatants. Recovery was 94.6-100% (mean ± SD was 97.4 ± 1.4%) at lactate concentrations of 2.68 to 12.63 mmol/L. Linearity was determined by combining two supernatants with low and high lactate concentrations in different ratios to create a set of six samples (0.15-12.70 mmol/L) that were tested in duplicate. Linear regression generated a slope of 1.01, y-intercept of -0.04 (R 2  = 1.00). Precision was verified by analyzing quality control materials (acid-treated lactate standard) in 3 replicates each day for 5 days. Within-laboratory imprecision was 2.3% at 1.5 mmol/L and 1.5% at 10.5 mmol/L. The limit of blank was 0.05 mmol/L as determined by the mean added to three standard deviations determined from 10 replicates of perchloric-acid treated saline pool. The limit of detection was determined to be 0.12 mmol/L calculated from 10 replicates of a patient sample treated with perchloric-acid. The manufacturer's reference interval of 1.1-2.4 mmol/L was verified using 20 residual patient CSF samples., Conclusions: CSF lactate can be measured with accuracy and precision using the same perchloric-acid treated sample that is used for pyruvate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the interface effect between five types of natural precipitated dust and two normal floras. Five kinds of natural dust (FC-1 # , FC-2 # , FC-15 # , FC-18 # , and FC-21 # ) were collected, and particle size and chemical components were detected by laser particle size analyzer and X-ray fluorescence (XRF). The elements, bacterial count, glucose (GLU) consumption, pH, and three biochemical indicators were measured after being co-cultured with Escherichia coli and Staphylococcus epidermidis in vitro. In addition, the changes of bacterial morphology were observed by scanning electron microscopy (SEM). Results showed that most particles contained a high level of SiO 2 , which diameter ranged from 0.3 to 1.0 μm. The concentration of Ca showed s significant increase upon interaction with E. coli and S. epidermidis in all dusts (p < 0.01). Moreover, FC-1 # and FC-21 # induced obvious growth in bacterial count, glucose consumption, and pH after they reacted with two normal floras (p < 0.05). Besides, the results also showed an apparent increase in the concentration of pyruvate, β-galactosidase, and alkaline phosphatase (AKP) after being co-cultured with E. coli and S. epidermidis, in which FC-1 # is enhanced in the most obvious. The E. coli interacted with dust made more indentations in surface, and the configuration became thin and long. Some broken bacteria were present, and bacterial wreckage was visible. Plenty of S. epidermidis interacted with dust gathered in the indentations of dust, particularly in pleated surfaces. Further, these findings demonstrated that the alkaline dust with higher Ca content stimulated the growth of bacteria, and irregularly shaped or thin dust would be easier to combine with bacteria and conduct interface effect.

Purpose: To explore the effects of noise and error on kinetic analyses of tumor metabolism using hyperpolarized [1- 13 C] pyruvate., Methods: Numerical simulations were performed to systematically investigate the effects of noise, the number of unknowns, and error in kinetic parameter estimates on kinetic analysis of the apparent rate of chemical conversion from hyperpolarized pyruvate to lactate (k PL ). A pharmacokinetic model with two physical and two chemical pools of hyperpolarized spins was used to generate and analyze the synthetic data., Results: The reproducibility of k PL estimates worsened quickly when peak signal-to-noise ratio for hyperpolarized pyruvate was below approximately 20. The accuracy of k PL estimates was most sensitive to errors in high excitation angles, the vascular blood volume fraction (v b ), and the rate of pyruvate extravasation (k ve ), and was least sensitive to errors in the T 1 of pyruvate. When v b and/or k ve were fit as additional unknowns, the accuracy of k PL estimates suffered, and when the vascular input function of pyruvate was also fit, the reproducibility of k PL estimates worsened., Conclusions: The accuracy and precision of k PL estimates improve substantially for peak signal-to-noise ratio above approximately 20. Accurate estimates of perfusion parameters (combinations of v b , k ve , and the pyruvate vascular input function) and transmit calibration at high excitation angles have the greatest effect on the accuracy of kinetic analyses. Magn Reson Med 79:3239-3248, 2018. © 2017 International Society for Magnetic Resonance in Medicine., (© 2017 International Society for Magnetic Resonance in Medicine.)

Rationale: Current cardiovascular clinical imaging techniques offer only limited assessment of innate immune cell-driven inflammation, which is a potential therapeutic target in myocardial infarction (MI) and other diseases. Hyperpolarized magnetic resonance (MR) is an emerging imaging technology that generates contrast agents with 10- to 20 000-fold improvements in MR signal, enabling cardiac metabolite mapping., Objective: To determine whether hyperpolarized MR using [1- 13 C]pyruvate can assess the local cardiac inflammatory response after MI., Methods and Results: We performed hyperpolarized [1- 13 C]pyruvate MR studies in small and large animal models of MI and in macrophage-like cell lines and measured the resulting [1- 13 C]lactate signals. MI caused intense [1- 13 C]lactate signal in healing myocardial segments at both day 3 and 7 after rodent MI, which was normalized at both time points after monocyte/macrophage depletion. A near-identical [1- 13 C]lactate signature was also seen at day 7 after experimental MI in pigs. Hyperpolarized [1- 13 C]pyruvate MR spectroscopy in macrophage-like cell suspensions demonstrated that macrophage activation and polarization with lipopolysaccharide almost doubled hyperpolarized lactate label flux rates in vitro; blockade of glycolysis with 2-deoxyglucose in activated cells normalized lactate label flux rates and markedly inhibited the production of key proinflammatory cytokines. Systemic administration of 2-deoxyglucose after rodent MI normalized the hyperpolarized [1- 13 C]lactate signal in healing myocardial segments at day 3 and also caused dose-dependent improvement in IL (interleukin)-1β expression in infarct tissue without impairing the production of key reparative cytokines. Cine MRI demonstrated improvements in systolic function in 2-DG (2-deoxyglucose)-treated rats at 3 months., Conclusions: Hyperpolarized MR using [1- 13 C]pyruvate provides a novel method for the assessment of innate immune cell-driven inflammation in the heart after MI, with broad potential applicability across other cardiovascular disease states and suitability for early clinical translation., (© 2018 The Authors.)

Alcoholic beverages are enjoyed together with meals worldwide, but their excessive intake is associated with an increased risk of various diseases. We investigated whether S-allyl-L-cysteine sulfoxide (ACSO), a sulfuric odor precursor of garlic, suppresses elevation in plasma ethanol concentration by accelerating ethanol metabolism and preventing ethanol absorption from the gut in rats. ACSO and garlic extract with a high ACSO content (Garlic-H) suppressed elevation in concentrations of ethanol and acetaldehyde in plasma and promoted the activities of alcohol dehydrogenase and aldehyde dehydrogenase. However, ACSO and Garlic-H did not affect plasma acetate so much. Furthermore, we examined the change in plasma ethanol concentration by injecting ACSO or Garlic-H into the ligated stomach or jejunum together with ethanol solution. ACSO and Garlic-H suppressed the absorption of ethanol from the stomach and jejunum, but suppression in the jejunum was less than in the stomach. In conclusion, ACSO inhibits ethanol absorption and accelerates ethanol metabolism.

The present study describes the rapid microplate method to determine pyruvic acid content in different varieties of onions. Onion juice was treated with 2,4-dinitrophenylhydrazine to obtain hydrazone, which was further treated with potassium hydroxide to get stable colored complex. The stability of potassium complex was enhanced up to two hours and the structures of hydrazones were confirmed by LC-MS for the first time. The developed method was optimized by testing different bases, acids with varying concentrations of dinitrophenyl hydrazine to get stable color and results were comparable to developed method. Repeatability and precision showed <9% relative standard deviation. Moreover, sweet onion juice was stored for four weeks at different temperatures for the stability; the pyruvate remained stable at all temperatures except at 25°C. Thus, the developed method has good potential to determine of pungency in large number of onions in a short time using minimal amount of reagents., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Alterations in muscle milieu are suggested as important activity of peripheral drive in patients with chronic musculoskeletal pain (CMP). Microdialysis (MD) has been used in monitoring altered metabolic response pattern in muscles. However, the insertion of MD probe causes a local tissue trauma. Whether and how metabolites in trapezius muscle are affected by acute tissue trauma is unknown. Hence, this study investigated the metabolic response and nociceptive reaction of the tissue following MD probe insertion in patients with CMP and healthy individuals. Fifty-nine patients and forty pain-free volunteers were recruited. Pressure pain thresholds (PPTs) were obtained at the trapezius and tibialis muscles. Pain questionnaires determined the levels of pain related aspects. MD (20 kDa cut-off) was performed in the trapezius and samples were collected within 40 min. Interstitial concentration of the metabolites was analyzed by a two-way-mixed-ANOVA. The metabolic response pattern changed over time and alterations in the level of metabolites could be seen in both CMP and healthy controls. Pain questionnaires and pain intensities manifested clinical aspects of pain closely to what CMP patients describe. Analyzing metabolites due to acute tissue trauma by aid of MD may be a useful model to investigate altered metabolic response effect in CMP.

Herein, a fluorescent capillary biosensor was developed for quantifying micro-volume intracellular pyruvate (PA), in which AuNPs and lactate dehydrogenase (LDH) were modified on the inner surface of an amination capillary (20 μL) via a self-assembly technique. The PA concentration was quantified by the change in the value of the fluorescence of NADH after sucking a mixed solution of the sample and NADH into the biosensor. This study investigated factors including the degree of protonation of the amino groups on the surface of the capillary, the AuNP concentration and time for self-assembly, the activity concentration and time for the LDH self-assembly, the flow rate and acidity for LDH immobilization, pH, temperature, and reaction time for the NADH/PA/LDH reaction system. Under the optimized conditions, the linear response range of the biosensor towards PA was 2.5-120 μmol L -1 , in which the determination limit and detection limit were 2.5 and 0.75 μmol L -1 , respectively. The biosensor could be reused more than 41 times when its relative standard deviation (RSD) was controlled at less than 1.5%. At room temperature (approximately 25 °C), the intracellular PA in the erythrocyte of a healthy person was measured using the biosensor, and the PA content was observed to be 241.76 ± 68.05 μmol L -1 (n = 8). The standard addition recovery was 95-106%. Employment of the AuNPs in the PA biosensor not only improved the affinity of the immobilized LDH towards PA and its stability, but also significantly enhanced the service life of the PA biosensor.

Drug-induced off-target cardiotoxicity, particularly following anti-cancer therapy, is a major concern in new drug discovery and development. To ensure patient safety and efficient pharmaceutical drug development, there is an urgent need to develop more predictive cell model systems and distinct toxicity signatures. In this study, we applied our previously proposed repeated exposure toxicity methodology and performed 1 H NMR spectroscopy-based extracellular metabolic profiling in culture medium of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to doxorubicin (DOX), an anti-cancer agent. Single exposure to DOX did not show alteration in the basal level of extracellular metabolites while repeated exposure to DOX caused reduction in the utilization of pyruvate and acetate, and accumulation of formate compared to control culture medium. During drug washout, only pyruvate showed reversible effect and restored its utilization by hiPSC-CMs. On the other hand, formate and acetate showed irreversible effect in response to DOX exposure. DOX repeated exposure increased release of lactate dehydrogenase (LDH) in culture medium suggesting cytotoxicity events, while declined ATP levels in hiPSC-CMs. Our data suggests DOX perturbed mitochondrial metabolism in hiPSC-CMs. Pyruvate, acetate and formate can be used as metabolite signatures of DOX induced cardiotoxicity. Moreover, the hiPSC-CMs model system coupled with metabolomics technology offers a novel and powerful approach to strengthen cardiac safety assessment during new drug discovery and development.

A consumer study was conducted to evaluate preferences in Vidalia onions, and define consumer acceptability thresholds for commonly analyzed flavor compounds associated with pungency. Two varieties of Vidalia onions (Plethora and Sapelo Sweet) were grown at 3 fertilizer application rates (37.5 and 0; 134.5 and 59.4; and 190 and 118.8 kg/ha of nitrogen and sulfur, respectively), creating 6 treatments with various flavor attributes to use in the study. Bulb soluble solids, sugars, pyruvic acid, lachrymatory factor (LF; propanethial S-oxide), and methyl thiosulfinate (MT) content were determined and compared to sensory responses for overall liking, intensity of the sharp/pungent/burning sensation (SPB), and intent to buy provided by 142 consumers. Onion pyruvate, LF, MT, and sugar content increased as fertilization rate increased, regardless of onion variety. Consumer responses showed participants preferred onions with low SPB, which correlated positively to lower pyruvate, LF and MT concentrations, but showed no relationship to total sugars in the onion bulb. Regression analyses revealed that the majority of consumers (≥55%) found the flavor of Vidalia onions acceptable when the concentrations of LF, pyruvic acid, and MT within the bulbs were below 2.21, 4.83, and 0.43 nmol/mL, respectively. These values will support future studies aimed at identifying the optimal cultivation practices for production of sweet Vidalia onions, and can serve as an industry benchmark for quality control, thus ensuring the flavor of Vidalia onions will be acceptable to the majority of consumers., Practical Application: This study identified the relationship between consumer preferences and commonly analyzed flavor compounds in Vidalia onions, and established thresholds for these compounds at concentrations which the majority of consumers will find desirable. These relationships and thresholds will support future research investigating how cultural practices impact onion quality, and can be used to assist growers in variety selection decisions. In addition, this information will provide a benchmark to Vidalia onion producers for quality control of the sweet onions produced, ensuring that the onions are consistently of a desired quality, thereby increasing consumer's reliability in the Vidalia onion brand., (© 2017 Institute of Food Technologists®.)

Purpose: Balanced steady-state free precession (bSSFP) sequences can provide superior signal-to-noise ratio efficiency for hyperpolarized (HP) carbon-13 ( 13 C) magnetic resonance imaging by efficiently utilizing the nonrecoverable magnetization, but managing their spectral response is challenging in the context of metabolic imaging. A new spectrally selective bSSFP sequence was developed for fast imaging of multiple HP 13 C metabolites with high spatiotemporal resolution., Theory and Methods: This novel approach for bSSFP spectral selectivity incorporates optimized short-duration spectrally selective radiofrequency pulses within a bSSFP pulse train and a carefully chosen repetition time to avoid banding artifacts., Results: The sequence enabled subsecond 3D dynamic spectrally selective imaging of 13 C metabolites of copolarized [1- 13 C]pyruvate and [ 13 C]urea at 2-mm isotropic resolution, with excellent spectral selectivity (∼100:1). The sequence was successfully tested in phantom studies and in vivo studies with normal mice., Conclusion: This sequence is expected to benefit applications requiring dynamic volumetric imaging of metabolically active 13 C compounds at high spatiotemporal resolution, including preclinical studies at high field and, potentially, clinical studies. Magn Reson Med 78:963-975, 2017. © 2016 International Society for Magnetic Resonance in Medicine., (© 2016 International Society for Magnetic Resonance in Medicine.)

This study investigated the possible biological, biochemical and histological changes in Bradybaena similaris(Gastropoda: Pulmonata) infected by Heterorhabditis indica (Rhabditida: Heterorhabditidae), strain LPP1. Two groups of 16 snails were formed: the control group (unexposed) and the treated group, which was exposed for three weeks to infective juveniles (J 3 ) of H. indica LPP1. The experiment was conducted in duplicate, using a total of 64 snails. After the exposure period, the snails were dissected to collect the hemolymph and tissues, for evaluation of the physiological changes caused by the infection. The number of eggs laid/snail and the viability of these eggs were also assessed as indicators of the reproductive activity of B. similaris. Intense glycogenolysis was accompanied by a significant reduction (p < 0.05) in the glucose content of the hemolymph of the exposed snails, indicating that infection by H. indica induces breakdown of the host's glycemic homeostasis. Significant variations (p < 0.05) in the lactate dehydrogenase activity occurred together with changes in the concentration of pyruvic and lactic acid in the hemolymph of the infected B. similaris snails, corroborating the transition from aerobic to anaerobic metabolism in the hosts. These metabolic alterations reflect the parasitic castration process in this interface. The results suggest that the use of H. indica LPP1 is a potential alternative for biological control of B. similaris., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Secondary peritonitis is a condition associated with high morbidity and mortality. Continuous postoperative monitoring of patients to ensure timely intervention to treat complications without delay is important for survival and outcome. We aimed to (1) investigate potential differences in postoperative intraperitoneal biomarker levels between patients with upper and lower gastrointestinal tract lesion, and (2) compare postoperative biomarker levels between complicated and uncomplicated patients., Methods: We included a total of 15 consecutive patients operated for upper (n = 7) and lower (n = 8) gastrointestinal tract perforation. We registered postoperative complications during a 30 days follow up-period. Complications were defined as intraabdominal complications, septic shock, and mortality. 5 patients were complicated. A microdialysis catheter was placed intraperitoneally in each patient. Samples were collected every 4th hour for up to 7 postoperative days. Samples were analysed for concentrations of glucose, lactate, pyruvate and glycerol., Results: Microdialysis results showed that patients with upper gastrointestinal tract lesions had significantly higher levels of postoperative intraperitoneal glucose and glycerol concentrations, as well as lower lactate/pyruvate ratios and lactate/glucose ratios. In the group with perforation of the lower gastrointestinal tract, those patients with a complicated course showed lower levels of postoperative intraperitoneal glucose concentration and glycerol concentration and higher lactate/pyruvate ratios and lactate/glucose ratios than those patients with an uncomplicated course., Conclusion: Patients with upper and lower gastrointestinal tract lesions showed differences in postoperative biomarker levels. A difference was also seen between patients with complicated and uncomplicated postoperative courses.

Onion pyruvate concentration is used as a predictor of flavor intensity and nutraceutical value. The protocol of Schwimmer and Weston (SW) (1961) is the most widespread methodology for estimating onion pyruvate. Anthon and Barret (AB) (2003) proposed modifications to this procedure. Here, we compared these spectrophotometry-based procedures for pyruvate analysis using a diverse collection of onion cultivars. The SW method always led to over-estimation of pyruvate levels in colored, but not in white onions, by up to 65%. Identification of light-absorbance interfering compounds was performed by spectrophotometry and HPLC analysis. Interference by quercetin and anthocyanins, jointly, accounted for more than 90% of the over-estimation of pyruvate. Pyruvate determinations according to AB significantly reduced absorbance interference from compounds other than pyruvate. This study provides evidence about the mechanistic basis underlying differences between the SW and AB methods for indirect assessment of onion flavor and nutraceutical value., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L -1 , 0.30 g g -1 , and 0.32 g L -1  h -1 in the resulting strain W4209 with CaCO 3 as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L -1 , 0.31 g g -1 , and 0.32 g L -1  h -1 , respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.

Purpose: To determine the effects of a blockade of nitric oxide (NO) synthesis on hyperbaric oxygen (HBO₂) therapy during cyanide (CN) intoxication., Methods: 39 anesthetized female Sprague-Dawley rats were exposed to CN intoxication (5.4 mg/kg intra-arterially) with or without previous nitric oxide synthase (NOS) inhibition by L-NG-nitroarginine methyl ester (L-NAME) injection (40 mg/kg intraperitoneally). Subsequently, either HBO₂ therapy (284 kPa/90 minutes), normobaric oxygen therapy (100% oxygen/90 minutes) or nothing was administered. Intracerebral microdialysis was used to measure the interstitial brain concentration of lactate, glucose, glycerol and lactate/pyruvate ratios., Results: L-NAME potentiated CN intoxication by higher maximum and prolonged lactate (in mM: 0. 5 ± 0.3 vs. 0.7 ± 0.4, P ⟨ 0.005) concentrations compared with solely CN-intoxicated rats. The same trend was found for mean glucose, glycerol and lactate/pyruvate ratio levels. During HBO₂ treatment a sustained reduction occurred in mean lactate levels (in mM: 0.5 ± 0.5 vs. 0.7 ± 0.4, P ⟨ 0.01) regardless of NOS blockade by L-NAME. The same trend was found for mean glucose and glycerol levels., Conclusion: The results suggest that blocking NOS using L-NAME can worsen acute CN intoxication. HBO₂ treatment can partially overcome this block and continue to ameliorate CN intoxication., Competing Interests: The authors of this paper declare no conflicts of interest exist with this submission.

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.