On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of α-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Convergent evolution of Trichomonas vaginalis lactate dehydrogenase from malate dehydrogenase

Lactate dehydrogenase (LDH) is present in the amitochondriate parasitic protist Trichomonas vaginalis and some but not all other trichomonad species. The derived amino acid sequence of T. vaginalis LDH (TvLDH) was found to be more closely related to the cytosolic malate dehydrogenase (MDH) of the same species than to any other LDH. A key difference between the two T. vaginalis sequences was that Arg91 of MDH, known to be important in coordinating the C-4 carboxyl of oxalacetate/malate, was replaced by Leu91 in LDH. The change Leu91Arg by site-directed mutagenesis converted TvLDH into an MDH. The reverse single amino acid change Arg91Leu in TvMDH, however, gave a product with no measurable LDH activity. Phylogenetic reconstructions indicate that TvLDH arose from an MDH relatively recently.

Interaction of two arginine residues in lactate oxidase with the enzyme flavin: Conversion of FMN to 8-formyl-FMN

Two arginine residues, Arg-181 and Arg-268, are conserved throughout the known family of FMN-containing enzymes that catalyze the oxidation of α-hydroxyacids. In the lactate oxidase from Aerococcus viridans, these residues have been changed to lysine in two single mutations and in a double mutant form. In addition, Arg-181 has been replaced by methionine to determine the effect of removing the positive charge on the residue. The effects of these replacements on the kinetic and thermodynamic properties are reported. With all mutant forms, there are only small effects on the reactivity of the reduced flavin with oxygen. On the other hand, the efficiency of reduction of the oxidized flavin by l-lactate is greatly reduced, particularly with the R268K mutant forms. The results demonstrate the importance of the two arginine residues in the binding of substrate and its interaction with the flavin, and are consistent with a previous hypothesis that they also play a role of charge neutralization in the transition state of substrate dehydrogenation. The replacement of Arg-268 by lysine also results in a slow conversion of the 8-CH3- substituent of FMN to yield 8-formyl-FMN, still tightly bound to the enzyme, and with significantly different physical and chemical properties from those of the FMN-enzyme.

Influence of intensive training on salivary flow, on salivary pH and on salivary lactate concentration: consequences for oral health

Abstract of the poster presented at the First international Congress of CiiEM “From Basic Sciences to Clinical Research”, 27-28 November 2015, Egas Moniz, Caparica, Portugal.

Determining the fetal scalp lactate level that indicates the need for intervention in labour

Background: Fetal scalp lactate testing has been shown to be as useful as pH with added benefits. One remaining question is What level of lactate should trigger intervention in the first stage of labour?' Aims: This study aimed to establish the lactate level in the first stage of labour that indicates the need for intervention to ensure satisfactory outcomes for both babies and mothers. Methods: A prospective study at Mater Mothers' Hospital, Brisbane, Australia, a tertiary referral centre. One hundred and forty women in labour, with non-reassuring fetal heart rate traces, were tested using fetal blood scalp sampling of 5 mu L of capillary blood tested on an Accusport (Boeringer, Mannheim, East Sussex, UK) lactate meter. Decision to intervene in labour was based on clinical assessment plus a predetermined cut off. Main outcome measures were APGAR scores, cord arterial pH, meconium stained liquor and Intensive Care Nursery admission. Results: Two-graph receiver operating characteristic (TG-ROC) analysis showed optimal specificity, and sensitivity for predicting adverse neonatal outcomes was a scalp lactate level above 4.2 mmol/L. Conclusions: Fetal blood sampling remains the standard for further investigating-non-reassuring cardiotocograph (CTG) traces. Even so, it is a poor predictor of fetal outcomes. Scalp lactate has been shown to be at least as good a predictor as scalp pH, with the advantages of being easier, cheaper and with a lower rate of technical failure. Our study, found that a cut off fetal scalp lactate level of 4.2 mmol/L, in combination with an assessment of the entire clinical picture, is a useful tool in identifying those women who need intervention.

Field evaluation of a novel colorimetric method - Double-site enzyme-linked lactate dehydrogenase immunodetection assay - To determine drug susceptibilities of Plasmodium falciparum clinical isolates from northwestern Thailand

A double-site enzyme-linked lactate dehydrogenase enzyme inummodetection assay was tested against field isolates of Plasmodium falciparum for assessing in vitro drug susceptibilities to a wide range of antimalarial drugs. Its sensitivity allowed the use of parasite densities as low as 200 parasites/mul of blood. Being a nonisotopic, colorimetric assay, it lies within the capabilities of a modest laboratory at the district level.

Intrapartum fetal scalp lactate sampling for fetal assessment in the presence of a non-reassuring fetal heart rate trace (Protocol)

This is the protocol for a review and there is no abstract. The objectives are as follows: To evaluate the effectiveness and risks of fetal scalp lactate sampling in the assessment of fetal wellbeing during labour, compared with no testing or alternative additional testing (pH, fetal pulse oximetry, etc) for women exhibiting a non-reassuring cardiotocograph trace. A secondary objective of the review is to determine whether effectiveness and risks of intrapartum fetal scalp lactate sampling is influenced by the following: stage of labour; gestation less than 37 completed weeks, greater than or equal to 37 completed weeks; additional tests performed to confirm the presence or absence of fetal acidemia during labour.

Functional astrocyte-neuron lactate shuttle in a human stem cell-derived neuronal network

The NT2.D1 cell line is one of the most well-documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of the neuronal cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time that human stem cell-derived astrocytes produce glycogen and that co-cultures of these cells demonstrate a functional astrocyte-neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake, which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2-derived neurons and astrocytes, we have shown that these cells modulate their glucose uptake in response to glutamate. Additionally, we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown after treatment with glutamate, potassium, isoproterenol, and dbcAMP. Together, these results demonstrate for the first time a functional ANLS in a human stem cell-derived co-culture. © 2013 ISCBFM.

Functional astrocyte-neuron lactate shuttle in a human stem cell derived neuronal network

The development of stem cell-derived neuronal networks will promote experimental system development for drug screening, toxicological testing and disease modelling, providing that they mirror closely the functional competencies of their in vivo counterparts. The NT2 cell line is one of the best documented embryocarcinoma cell lines, and can be differentiated into neurons and astrocytes. Great focus has also been placed on defining the electrophysiological properties of these cells, and more recently we have investigated the functional properties of their associated astrocytes. We now show for the first time in a human stem cell derived co-culture model that these cultures are also metabolically competent and demonstrate a functional astrocyte neuron lactate shuttle (ANLS). The ANLS hypothesis proposes that during neuronal activity, glutamate released into the synaptic cleft is taken up by astrocytes and triggers glucose uptake which is converted into lactate and released via monocarboxylate transporters for neuronal use. Using mixed cultures of NT2 derived neurons and astrocytes we have shown that these cells modulate their glucose uptake in response to glutamate, an effect that was blocked by cytochalasin B and ouabain. Additionally we demonstrate that in response to increased neuronal activity and under hypoglycaemic conditions, co-cultures modulate glycogen turnover and increase lactate production. Similar results were also shown following treatment with glutamate, potassium, Isoproterenol and dbcAMP. Together these results demonstrate for the first time a functional ANLS in a human stem cell derived co-culture.

Base-free, one-pot chemocatalytic conversion of glycerol to methyl lactate using supported gold catalysts

We report an efficient one-pot conversion of glycerol (GLY) to methyl lactate (MLACT) in methanol in good yields (73 % at 95 % GLY conversion) by using Au nanoparticles on commercially available ultra-stable zeolite-Y (USY) as the catalyst (160 °C, air, 47 bar pressure, 0.25 M GLY, GLY-to-Au mol ratio of 1407, 10 h). The best results were obtained with zeolite USY-600, a catalyst that has both Lewis and Brønsted sites. This methodology provides a direct chemo-catalytic route for the synthesis of MLACT from GLY. MLACT is stable under the reaction conditions, and the Au/USY catalyst was recycled without a decrease in the activity and selectivity. From glycerol to green building blocks and solvents! An efficient, base-free conversion of glycerol to methyl lactate in methanol is reported, achieving good yields (73 % at 95 % glycerol conversion) using Au/ultra-stable zeolite-Y (USY) as the catalyst and environmentally benign oxygen as the oxidant by combining two separate reaction steps efficiently in a one pot procedure. The Au/USY catalyst can be recycled without a decrease in the activity and selectivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Running economy and blood lactate accumulation in elite football players with high and low maximal aerobic power

The purpose was to determine running economy and lactate threshold among a selection of male elite football players with high and low aerobic power. Forty male elite football players from the highest Swedish division (“Allsvenskan”) participated in the study. In a test of running economy (RE) and blood lactate accumulation the participants ran four minutes each at 10, 12, 14, and 16 km•h-1 at horizontal level with one minute rest in between each four minutes interval. After the last sub-maximal speed level the participants got two minutes of rest before test of maximal oxygen uptake (VO2max). Players that had a maximal oxygen uptake lower than the average for the total population of 57.0 mL O2•kg-1•minute-1 were assigned to the low aerobic power group (LAP) (n=17). The players that had a VO2max equal to or higher than 57.0 mL O2•kg-1•minute-1 were selected for the high aerobic power group (HAP) (n=23). The VO2max was significantly different between the HAP and LAP group. The average RE, measured as oxygen uptake at 12, 14 and 16km•h-1 was significantly lower but the blood lactate concentration was significantly higher at 14 and 16 km•h-1 for theLAP group compared with the HAP group.

Ruthenibacterium lactatiformans gen. nov., sp.nov., an anaerobic, lactate-producing member of the family Ruminococcaceae isolated from human faeces

Two novel strains of Gram-stain-negative, rod-shaped, obligately anaerobic, non-spore-forming, non-motile bacteria were isolated from the faeces of healthy human subjects. The strains, designated as 585-1T and 668, were characterized by mesophilic fermentative metabolism, production of d-lactic acid, succinic acid and acetic acid as end products of d-glucose fermentation, prevalence of C18 : 1 ω9, C18 : 1 ω9 aldehyde, C16 : 0 and C16 : 1 ω7c fatty acids, presence of glycine, glutamic acid, lysine, alanine and aspartic acid in the petidoglycan peptide moiety and lack of respiratory quinones. Whole genome sequencing revealed the DNA G+C content was 56.4–56.6 mol%. The complete 16S rRNA gene sequences of the two strains shared 91.7/91.6 % similarity with Anaerofilum pentosovorans FaeT, 91.3/91.2 % with Gemmiger formicilis ATCC 27749T and 88.9/88.8 % with Faecalibacterium prausnitzii ATCC 27768T. On the basis of chemotaxonomic and genomic properties it was concluded that the strains represent a novel species in a new genus within the family Ruminococcaceae , for which the name Ruthenibacterium lactatiformans gen. nov., sp. nov. is proposed. The type strain of Ruthenibacterium lactatiformans is 585-1T (=DSM 100348T=VKM B-2901T).

Point-of-care lactate and creatinine analysis for sick obstetric patients at Queen Elizabeth Central Hospital in Blantyre, Malawi: A feasibility study

Background: To achieve good outcomes in critically ill obstetric patients, it is necessary to identify organ dysfunction rapidly so that life-saving interventions can be appropriately commenced. However, timely access to clinical chemistry results is problematic, even in referral institutions, in the sub-Saharan African region. Reliable point-of-care tests licensed for clinical use are now available for lactate and creatinine. Aim: We aimed to assess whether implementation of point-of-care testing for lactate and creatinine is feasible in the obstetric unit at the Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, by obtaining the opinions of clinical staff on the use of these tests in practice. Methods: During a two-month evaluation period nurse-midwives, medical interns, clinical officers, registrars, and consultants were given the opportunity to use StatStrip® and StatSensor® (Nova Biomedical, Waltham, USA) devices, for lactate and creatinine estimation, as part of their routine clinical practice in the obstetric unit. They were subsequently asked to complete a short questionnaire. Results: Thirty-seven questionnaires were returned by participants: 22 from nurse-midwives and the remainder from clinicians. The mean satisfaction score for the devices was 7.6/10 amongst clinicians and 8.0/10 amongst nurse-midwives. The majority of participants stated that the obstetric high dependency unit (HDU) was the most suitable location for the devices. For lactate, 31 participants strongly agreed that testing should be continued and 24 strongly agreed that it would influence patient management. For creatinine, 29 strongly agreed that testing should be continued and 28 strongly agreed that it would influence their patient management. Twenty participants strongly agreed that they trust point-of-care devices. Conclusions: Point-of-care clinical chemistry testing was feasible, practical, and well received by staff, and was considered to have a useful role to play in the clinical care of sick obstetric patients at this referral centre.