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PubMed1995

Osmotic responsiveness and cross talk involving oxytocin, but not vasopressin or amino acids, between the supraoptic nuclei in virgin and lactating rats.

Lactation is associated with complex changes of the hypothalamo-neurohypophysial system, and oxytocin released within the hypothalamic supraoptic (SON) and paraventricular nuclei may serve as a signal of communication between the magnocellular nuclei in lactating rats. In the first study, the intranuclear and peripheral release patterns of oxytocin and vasopressin in response to intraperitoneal hypertonic saline were studied in virgin and lactating rats to determine if the reduced osmoresponsiveness of the oxytocinergic and vasopressinergic systems during lactation is reflected by reduced release not only into blood, but also within the SON. Simultaneous microdialysis was performed within the SON and the jugular vein before and up to 6 hr after peripheral osmotic stimulation (3.0 M NaCl, 0.6 ml/100 gm body weight, i.p.). There was an immediate increase in secretion of both oxytocin and vasopressin into blood, whereas peptide release within the SON was delayed and peaked after 4-5 hr. Peripheral release of both peptides was significantly reduced in lactating animals, whereas within the SON release of oxytocin, but not vasopressin, was significantly reduced during lactation. In the second study, cross talk between the SONs--another phenomenon which seems to be characteristic for lactation--was studied. Microdialysis of one SON with hypertonic perfusion medium (with 1 M NaCl) significantly increased the release of oxytocin, vasopressin, and various amino acids (aspartate, glutamate, serine, glutamine, gamma amino butyric acid, and arginine) within the ipsilateral SON. In contrast to virgin female and male animals, this unilateral stimulation of the SON resulted in a transiently increased release of oxytocin in the contralateral SON of lactating rats. The release of vasopressin and amino acids within the contralateral SON of lactating rats remained unchanged, indicating specific activation of contralateral oxytocinergic neurons.

Amino Acids↗
PubMed1994

Demonstration of organic anion transport in T lymphocytes. L-lactate and fluo-3 are target molecules.

In this paper, we describe for the first time the existence of organic anion transport in T lymphocytes, exemplified by the transmembrane transport of the anions L-lactate and the Ca2+ indicator fluo-3. The transport of either anion was found to be inhibitable by probenecid, a common blocker of organic anion transport. Transport of L-lactate was observed in long-term cultured T cell lines, as well as in freshly ex vivo isolated T cells, and occurred via a saturable, pH-dependent, and stereospecific process. L-Lactate uptake was dependent on the activation state of the T cells, because activation of T cells by Con A strongly enhanced accumulation of L-lactate from the medium. Because L-lactate may be transported bidirectionally through the T cell membrane in vivo, different physiologic roles of L-lactate transport are discussed. L-Lactate uptake may serve as an alternative source of energy in an inflamed, glucose-deficient tissue or may represent a prerequisite for the earlier-published immunoregulatory function of this molecule on T cells. On the other hand, release of L-lactate emerging from glycolysis could be necessary to avoid acidification of the cell. The fact that the Ca2+ indicator fluo-3 is also transported through the cellular membranes of long-term cultured T cells via organic anion transport has important implications for the determination of Ca2+ influx into T cells. Even though the transport of both molecules, L-lactate and fluo-3, represents organic anion transport, evidence is presented that confirms that the respective transport systems are different.

Aniline Compounds↗
PubMed1996

Reproducibility of the blood lactate-running speed curve in horses under field conditions.

OBJECTIVE: To examine the reproducibility of blood lactate-running speed curve parameters derived by a curve-fit equation and by linear interpolation from the results of 4-speed tests of sport horses under field conditions. DESIGN: Thoroughbreds completed 10 test pairs with 3 to 11 days between tests and retest. ANIMALS: 7 Thoroughbreds. PROCEDURE: The 4-speed test consisted of 4 runs over a distance of 2,110 m. Exercise intensity was increased by 1 m/s for each run. Blood lactate concentration measured after each run was plotted against running speed to determine the blood lactate-running speed relation. By means of the blood lactate-running speed relation, velocity inducing 2, 2.5, 3, 3.5, or 4 mM blood lactate concentration was calculated by a curve-fit equation and by linear interpolation. RESULTS: The test-retest correlation coefficient of velocity inducing 2, 2.5, 3, 3.5, and 4 mM blood lactate concentration determined by curve-fit equation was 0.67, 0.78, 0.84, 0.86, and 0.86, respectively, and by linear interpolation was 0.42, 0.58, 0.77, 0.87, and 0.78, respectively. But the test-retest regression was equal to the line of identity only for velocity inducing 3.5 and 4 mM blood lactate concentration. CONCLUSIONS: The reproducibility of velocity inducing 3.5 and 4 mM blood lactate concentration was sufficient to be used to compare horses and evaluate conditioning effects. This holds both methods of calculation of the parameters derived from the blood lactate-running speed relation.

Animals↗
PubMed1997

The clinical value of lactate dehydrogenase in serum: a quantitative review.

The aim of this article is to describe guidelines for rational use of lactate dehydrogenase and its isoenzymes, in the diagnostic processes and during follow-up, based on a systematic review of relevant literature. Sources of data for this study were English-language scientific publications, obtained from the database of the National Library of Medicine (Medline), concerning the clinical application (diagnosis, monitoring or treatment of disease) of lactate dehydrogenase and lactate dehydrogenase isoenzyme measurements in serum in the following main clinical fields: cardiology, hepatology, haematology and oncology. For acceptance in the present review, studies had to include: a proper definition of the tested patient population, diagnostic criteria, sampling time, sampling frequency, and test characteristics. Estimation of the relation between lactate dehydrogenase or lactate dehydrogenase isoenzymes and specific diseases expressed as sensitivity, specificity, survival or remission rate were extracted. The application of serum lactate dehydrogenase is relevant in the diagnosis of myocardial infarction (late detection), haemolytic anaemia, ovarian dysgerminoma and testicular germ cell tumor. For monitoring the progress of a disease lactate dehydrogenase is relevant in establishing the survival duration and rate in Hodgkin's disease and non-Hodgkin's lymphoma, and in the follow-up of ovarian dysgerminoma. Rational use of lactate dehydrogenase can be achieved when requests for its determination are limited to the above mentioned conditions. No rationale could be found for measuring lactate dehydrogenase isoenzymes.

Animals↗
PubMed1997

The circumventricular organs form a potential neural pathway for lactate sensitivity: implications for panic disorder.

Patients with panic disorder experience panic attacks after intravenous sodium lactate infusions by an as yet unexplained mechanism. Lactate elicits a panic-like response in rats with chronic dysfunction of GABA neurotransmission in the dorsomedial hypothalamus (DMH). The circumventricular organs, organum vasculosum lamina terminalis (OVLT) and subfornical organ (SFO), are potential sites that could detect increases in plasma lactate levels and activate the DMH. To test this, we obtained baseline heart rate (HR) and blood pressure (BP) responses to lactate infusions in rats fit with femoral arterial and venous catheters. Next, unilateral chronic injection cannulae connected to an Alzet infusion pump filled with the GABA synthesis inhibitor L-allylglycine (L-AG) were implanted into the DMH. Another chronic injection cannula was implanted into the region of the OVLT, SFO, or an adjacent control site, the median preoptic area (MePOA). These rats were tested once again with lactate infusions after injection of either artificial cerebrospinal fluid (ACSF) or tetrodotoxin (TTX) into the CVO sites. Injecting TTX into the OVLT completely blocked the lactate-induced response, whereas TTX injections into the SFO or MePOA did not. Also, direct injections of lactate (100 or 500 nl) into the OVLT elicited robust anxiety-like responses in these rats. These results suggest that the OVLT may be the primary site that detects lactate infusions, activating an anxiety-like response in a compromised DMH, and provide the first neuroanatomical basis for lactate response in panic disorder.

Allylglycine↗
PubMed1997

Thyroxine, triiodothyronine and reverse-triiodothyronine concentrations in blood plasma in relation to lactational stage, milk yield, energy and dietary protein intake in Estonian dairy cows.

Average levels of thyroxine (T4), triiodothyronine (T3) and reverse-triiodothyronine (rT3) in blood plasma of 159 Estonian Red and Estonian Black and White cows were 55.2 nmol/L, 1.78 nmol/L and 0.25 nmol/L respectively. Animals were grouped according to stage of lactation. The T4 level was significantly lower during the early stage of lactation (45.1 nmol/L), compared with later stages, but increased as the stage of lactation progressed (late stage of lactation - 56.7, dry cows 64.3 nmol/L). The T3 level was significantly higher at the late stage of lactation (1.93 nmol/L) compared with the early stage of lactation (1.71 nmol/L) and level in dry cows (1.71 nmol/L). rT3 showed a trend similar to that found for T4 (lowest plasma concentration in early lactation, 0.19; highest in the dry period, 0.33; late lactation, 0.24 nmol/L). Levels of all thyroid hormones were negatively related to the daily milk yield (T4-r = -.51, rT3-r = -.47, calculated thyroid index as rT3 x T3 x T4-r = -.52, for all p < .0001; T3-r = -.32, p < .01). Plasma thyroid hormone concentrations were affected by energy and dietary protein intake. Differences were found in thyroid hormone levels between the 2 breeds and between summer and winter holding periods for Estonian Red cows, which could all be explained by differences in the feeding level and daily milk yield. It is suggested that maintaining low levels of thyroid hormones in early lactation may be one of the dairy cow's mechanisms of reducing metabolic demand.

Animals↗
PubMed1998

Leucine catabolism in mammary tissue, liver and skeletal muscle of dam rat during lactation and weaning.

BACKGROUND: This study was designed to determine the effect of lactation and weaning on the catabolism of branched-chain amino acids (BCAA). METHODS: Rates of transamination and oxidation of leucine and branched chain alpha-ketoacid dehydrogenase (BCKD) activity were measured in homogenates of mammary gland, skeletal muscle and liver on day 12 of lactation and 24 h after separation of dams from the litter (weaning). RESULTS: Lactating dams consumed 250% more protein than control rats, extra protein is required for protein synthesis by the mammary gland, the extent to which the excess of amino acids consumed during lactation is utilized or oxidized by different tissues is not known. The rate of transamination of [1-14C] leucine by mammary tissue of lactating dams was sixfold higher than in virgin rats. The rate of transamination remained elevated fourfold in postweaning dams. Rates of transamination were three times higher in mammary tissue than in muscle of lactating dams. Rate of oxidation [1-14C] leucine by lactating mammary tissue was tenfold higher than in control tissue. CONCLUSIONS: The capacity of mammary tissue for transamination and oxidation of leucine increased greatly during lactation, suggesting that the mammary gland may play an important role in the catabolism of BCAA during lactation.

Animals↗
PubMed2000

L-lactate inhibits L-cystine/L-glutamate exchange transport and decreases glutathione content in rat cultured astrocytes.

In several brain pathologies, the level of brain L-lactate increases. The stimulation of L-lactate production is a detrimental factor in promoting neuronal cell damage and astrocytic dysfunction. Astrocytic glutathione metabolism has an important role to protect brain cells against oxidative stress. In this study, effects of L-lactate on L-cystine uptake and glutathione level in rat-cultured astrocytes were examined. L-Lactate decreased the L-(35)S-cystine and Na(+)-independent L-(3)H-glutamate uptakes into astrocytes at the concentrations more than 2.5 mM. The L-lactate-induced decrease in L-(35)S-cystine uptake was neither affected by modification of extracellular pH nor mimicked by acetate, propionate and butyrate. The apparent Km value of the L-(35)S-cystine uptake was increased by L-lactate, while the Vmax was not changed. Astrocytic glutathione and nonprotein thiol content was decreased by incubation with 20 mM L-lactate for 48 hours (65% and 75% of control values, respectively). The decreases in astrocytic glutathione and nonprotein thiol content were restored to normal levels by withdrawal of L-lactate. These results suggest that L-lactate inhibits astrocytic L-cystine/L-glutamate exchangers and affects the glutathione contents.

Animals↗
PubMed2001

Lactate reduces glutamate-induced neurotoxicity in rat cortex.

Experiments were carried out to test the hypothesis that lactate reduces the neurotoxicity of glutamate in vivo. MAP2 immunohistochemistry was used to measure lesion size, and microdialysis to measure the changes in glucose and lactate in the extracellular compartment. After implantation of a microdialysis probe 100 mM glutamate with or without 6 mM lactate was added to the perfusion medium and infused into the cortex of unanesthetized rats. Infusion of 100 mM glutamate for a period of 30 min produced a lesion of 6.05 +/- 0.64 mm(3), an increase in lactate of 124 +/- 19% above basal and a 21 +/- 9% reduction of glucose below basal level. When 6mM L-lactate was perfused together with 100 mM glutamate there was a significant reduction in the size of the lesion and there was no reduction in dialysate glucose. When L-lactate was replaced with D-lactate the lesion size and the increase in dialysate lactate were greater than after glutamate alone. The neuroprotective role of L-lactate is attributed to its ability to meet the increased energy demands of neurones exposed to high concentrations of glutamate.

Animals↗
PubMed1995

Blood lactate and pyruvate concentrations, and their ratio during exercise in healthy children: developmental perspective.

Blood concentrations of lactate normally increase during and after intense exercise as does the ratio of concentrations of lactate to pyruvate (L:P). Since there appear to be differences in blood lactate concentrations on exercise, in muscle metabolic enzyme activities, and in anaerobic capacity between children and adults, we speculated that there would be age related differences in lactate and pyruvate concentrations, and their ratio among children. Whole blood concentrations of lactate and pyruvate were measured in 28 healthy children aged 7-17 years, split into three age groups: less than 11, 11-14, and 15-17 years. Blood was drawn at rest, immediately after 6 min of exercise at one-third and two-thirds of maximum work capacity (Wmax), and 20 min after completion of work. Lactate and pyruvate concentrations increased significantly from rest to exercise at two-thirds Wmax [approximately 72% of peak oxygen consumption (VO2peak)]. Whereas greater increments in lactate concentration were seen with groups of increasing age, exercise-related increments in pyruvate concentrations were no different among age groups. There was a significant rise in L:P ratio on exercise, with greater increments found from the youngest to the oldest group. There were no sex differences. We concluded that in healthy children exercising at approximately 70% of VO2peak there is a rise in blood lactate concentration in excess of that of pyruvate, such that the L:P ratio rises to a degree determined by age. This suggests age dependent changes, perhaps coincident with puberty, in pathways involved in lactate production and/or elimination.

Adolescent↗
PubMed1996

Do cardiac output and serum lactate levels indicate blood transfusion requirements in anemia of prematurity?

BACKGROUND: Whether and when to transfuse in anemia of prematurity is highly controversial. Some authors suggest transfusions simply if the hemoglobin (Hb) level is below a defined normal range. Others propose the use of clinical or laboratory parameters in anemic patients to decide whether to transfuse or not. HYPOTHESIS: A decreasing amount of circulating Hb should cause a compensatory increase in cardiac output (CO) and an increase in arterial serum lactate. MATERIALS AND METHODS: In 56 anemic preterm infants (not in respiratory or hemodynamic failure) we analyzed CO after the first week of life using a Doppler sonographic method. At the same time serum lactate levels, Hb levels and oxygen saturation were registered. Nineteen of these patients were given transfusion when they demonstrated clinical signs of anemia by tachycardia > 180/min, tachypnea, retractions, apneas and centralization (group 2). The remaining 37 patients were not transfused (group 1). Serum lactate, CO, heart rate (HR), oxygen delivery, respiratory rate, capillary refill and Hb were analyzed in both groups and in group 2 before and 12-24 h after transfusion. Data between groups 1 and 2 and in group 2 before and after transfusion were compared. RESULTS: In the 56 patients studied no linear correlation between Hb and CO or between Hb and serum lactate was found. Nor could any correlation be demonstrated between the other variables studied. Examining the subgroups separately, a negative linear correlation was demonstrated between serum lactate and oxygen delivery in group 2. No other significant correlations were detected. However, when the pre- and post-transfusion data were compared in group 2 (increase of Hb from 9.45 (SD 3.44) to 12.5 (SD 3.8) g/100 ml), the CO decreased from 281.3 (SD 162.6) to 224 (SD 95.7) ml/kg per min (p < 0.01) and serum lactate decreased significantly from 3.23 mmol/l (SD 2.07) before to 1.71 (SD 0.83) after transfusion. Oxygen delivery was 35.8 (+/- 0.19) ml/kg per min group 1, 27.8 (+/- 0.05) pre- and 43.4 (+/- 0.07) post-transfusion in group 2 (p < 0.01). CONCLUSIONS: CO measurements and serum lactate levels add little information to the decision-making process for blood transfusions, as neither CO nor serum lactate levels correlate with HB levels in an otherwise asymptomatic population of preterm infants. In infants where the indication for blood transfusion is made based on traditionally accepted clinical criteria, serum lactate is an additional laboratory indicator of impaired oxygenation, as it correlates significantly with oxygen delivery. A significant lower oxygen delivery in patients in whom blood transfusion is indicated and an increase in oxygen induced by transfusion demonstrate the value of these criteria in identifying preterm infants who benefit from transfusion.

Anemia, Neonatal↗
PubMed2007

Improving the thermal stability of lactate oxidase by directed evolution.

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 degrees C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution.

Amino Acid Substitution↗
PubMed1997

Facilitated transport of lactate by rat jejunal enterocyte.

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K(m) of 39.2 +/- 4.8 mM and a Jmax of 8.9 +/- 0.7 nmoles mg protein-1 sec-1. A very small conductive pathway for L-lactate is present in basolateral membranes. In brush border membrane vesicles both Na+ and H+ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H(+)-lactate cotransporter, whereas in the apical membrane both H(+)-lactate and Na(+)-lactate cotransporters are present, even if they exhibit a low transport rate.

Animals↗
PubMed2006

Lactate production upon short-term non-ischemic forearm exercise in mitochondrial disorders and other myopathies.

BACKGROUND: The nonischemic forearm exercise test (NIFET) has been shown to be as effective as the classic ischemic forearm exercise test (IFET) in the diagnosis of patients with McArdle disease. Recently, the lactate increase normalized to the mechanical energy production in NIFET was suggested to have a intermediate sensitivity and satisfactory specifity for the screening of mitochondrial disorders. METHODS: NIFET at 80% maximal contraction force (MCF) was performed in normal controls (n = 41), patients with mitochondrial disorders (n = 15) and other myopathies (diseased controls, n = 20). 26 healthy volunteers also underwent IFET at 80% MCF. The ratio of lactate increase and workload was defined as specific lactate production (mmol x s/N x l). RESULTS: In normal controls there was no significant different lactate increase during NIFET and IFET. The workload performed showed only a weak significant positive correlation with the lactate increase in the NIFET in normal controls (r(2) = 0.20) but not in IFET and NIFET with patients. A moderate negative correlation of specific lactate production and the absolute workload was found in all groups and in both protocols (r(2) = 0.22-0.34). The specific lactate production was highest in patients with other myopathies, intermediate in patients with mitochondrial disorders and lowest in normal controls. NIFET showed a sensitivity of only 20 % and a specifity of 95% for normal controls, but only 75 % for diseased controls. CONCLUSION: The specific lactate production during NIFET is neither sufficiently specific nor sensitive for the diagnosis of mitochondrial disorders. Increased specific lactate production during rest-to-work transition period might be caused by increased acetyl group deficits.

Adolescent↗
PubMed2005

Lactate is a metabolic substrate that sustains extraocular muscle function.

Lactic acid is considered the end product of glycolysis and is a major cause of muscle fatigue. However, the lactate dehydrogenase (LDH) reaction is bidirectional: Lactate can be oxidized to pyruvate and used as a substrate for the Krebs cycle. Therefore, our hypothesis was that lactate sustains the contractile function of rat extraocular muscles during periods of increased activity. The study used extraocular and extensor digitorum longus (EDL) muscles from adult Sprague-Dawley rats to determine LDH isoform expression, total LDH activity, and contractile function in vitro. To evaluate the role of lactate on fatigue, we tested the effect of cinnamate, a blocker of lactate transport, and exogenous lactate on fatigue resistance. Cinnamate accelerated fatigue in the extraocular muscles: Endurance and residual force decreased significantly. Conversely, cinnamate did not affect the endurance or residual force of EDL muscles. Replacing glucose with exogenous lactate increased EDL fatigability but had no effect on the extraocular muscles. However, the extraocular muscles fatigued faster when exposed to exogenous lactate combined with cinnamate. The LDH-A and LDH-C isoforms were expressed at lower levels in extraocular muscle; LDH-B was equally abundant in the EDL and extraocular muscles. Total LDH activity in the extraocular muscles was only approximately 32% of the level in EDL. These results support the hypothesis that lactate sustains the contractile performance of the extraocular muscles.

Animals↗
PubMed2006

Impact of lactate in the perfusate on function and metabolic parameters of isolated working rat heart.

The goal of this study was to investigate the effect of 1 mM exogenous lactate on cardiac function, and some metabolic parameters, such as glycolysis, glucose oxidation, lactate oxidation, and fatty acid oxidation, in isolated working rat hearts. Hearts from male Sprague-Dawley rats were isolated and perfused with 5 mM glucose, 1.2 mM palmitate, and 100 microU/ml insulin with or without 1 mM lactate. The rates of glycolysis, glucose, lactate, and fatty acid oxidation were determined by supplementing the buffer with radiolabeled substrates. Cardiac function was similar between lactate+ and lactate- hearts. Glycolysis was not affected by 1 mM lactate. The addition of lactate did not alter glucose oxidation rates. Interestingly, palmitate oxidation rates almost doubled when 1 mM lactate was present in the perfusate. This study suggests that subst rate supply to the heart is crucially important when evaluating the data from metabolic studies.

Acetyl Coenzyme A↗
PubMed2006

Complex glutamate labeling from [U-13C]glucose or [U-13C]lactate in co-cultures of cerebellar neurons and astrocytes.

Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-(13)C]glucose (2.5 mM) and lactate (1 mM) or [U-(13)C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-(13)C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. (13)C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-(13)C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-(13)C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-(13)C]glucose and [U-(13)C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-(13)C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.

Alanine↗
PubMed2004

Amperometric determination of lactate with novel trienzyme/poly(carbamoyl) sulfonate hydrogel-based sensor.

A novel trienzyme sensor for the amperometric determination of lactate was constructed by immobilizing salicylate hydroxylase (SHL, E.C. 1.14.13.1), l-lactate dehydrogenase (LDH, E.C. 1.1.1.27), and pyruvate oxidase (PyOD, E.C. 1.2.3.3) on a Clark-type oxygen electrode. The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. LDH catalyzes the specific dehydrogenation of lactate consuming NAD(+). SHL catalyzes the irreversible decarboxylation and the hydroxylation of salicylate in the presence of oxygen and NADH produced by LDH. PyOD decarboxylates pyruvate using oxygen and phosphate. SHL and PyOD force the equilibrium of dehydrogenation of lactate by LDH to the product side by consuming NADH and pyruvate, respectively. Dissolved oxygen acts as an essential material for both PyOD and SHL during their respective enzymatic reactions. Therefore, an amplified signal, caused by the consumptions of dissolved oxygen by the two enzymes, was observed in the measurement of lactate. Regeneration of cofactor was found in the trienzyme system. A Teflon membrane was used to fabricate the sensor in order to avoid interferences. The sensor has a fast response (2s) and short recovery times (2 min). The total test time for a measurement by using this lactate sensor (4 min) was faster than using a commercial lactate testing kit (up to 10 min). The sensor has a linear range between 10 and 400 microM lactate, with a detection limit of 4.3 microM. A good agreement (R2 = 0.9984) with a commercial lactate testing kit was obtained in beverage sample measurements.

Biosensing Techniques↗