Presence Of Lactate Dehydrogenase Research Articles

Relevance of Plasmodium falciparum Biomarkers in the Treatment and Control of Malaria

We report here a dual-ELISA method to measure Malaria biomarkers concurrently in the same aliquot of blood sample. A correlation between the parasite numbers and ELISA values determined and the figures were used to establish a standard graph. Thick blood smears prepared from spiked blood samples were also Giemsa stained and parasite density determined by microscopy (It was thereby possible to undertake an objective comparison between lactate dehydrogenase and histidine- rich- proteins levels assessed by ELISA and parasite density determined by microscopy from the same spiked aliquot). The presence of lactate dehydrogenase and histidine- rich- proteins is localized and visualized by fluorescent antibody techniques. Twenty-two patient blood samples were retrospectively analyzed for the levels of pLDH and HRPs and their level was quantified in each blood sample. Fresh blood samples from malaria patients seeking care at a healthcare facility in an endemic area were then collected and separated into plasma and infected red blood cells and parasitemia levels determined. The biomarkers which were released into plasma from circulating and sequestered parasites provide a more accurate picture and indicate degree of disease severity. Since lactate dehydrogenase is known to have a short half-life compared to the histidine-rich proteins, its plasma level is believed to reflect on the number of metabolically active parasites more accurately and more reliable indicator of clinical outcome. In contrast, histidine -rich proteins accumulate in plasma and measurable levels persist long after patients have been treated and parasites cleared from their system. Best practice may now call for a pre-eminent role for microscopy and PCR in plasmodium species confirmation, a continued role for newer histidine-rich protein assays in epidemiological studies and control, and the preferential use of parasite lactate dehydrogenase in the clinical management of the disease. Data presented here from spiked blood samples as well as naturally infected patient samples provide evidence that by splitting whole blood samples into RBC-pellet and plasma, determination of parasite biomarkers in split sample fractions gives a true picture of malaria parasites present in humans and this may improve the accuracy of models that attempt to predict parasite burdens more accurately, and so clinical correlations.

Proteomic comparison of biofilm vs. planktonic Staphylococcus epidermidis cells suggests key metabolic differences between these conditions

Previous studies have shown that biofilm-forming bacteria are deficient in tricarboxylic acid (TCA) cycle metabolites, suggesting a relationship between these cellular processes. In this work, we compared the proteomes of planktonic vs biofilm cells from a clinical strain of Staphylococcus epidermidis using LC-MS/MS. A total of 168 proteins were identified from both growth conditions. The biofilm cells showed enrichment of proteins participating in glycolysis for the formation of pyruvate; however, the absence of TCA cycle proteins and the presence of lactate dehydrogenase, formate acetyltransferase, and acetoin reductase suggested that pyruvate was catabolized to their respective products: lactate, formate and acetoin. On the other hand, planktonic cells showed proteins participating in glycolysis and the TCA cycle, the pentose phosphate pathway, gluconeogenesis, ATP generation and the oxidative stress response. Functional networks with higher interconnection were predicted for planktonic proteins. We propose that in S. epidermidis, the relative absence of TCA cycle proteins is associated with the formation of biofilms and that lactate, formate and acetoin are the end products of partial glucose metabolism.

Development of an Automated Enzymatic Method to Quantify Pyruvate Kinase in Red Blood Cells.

Pyruvate kinase (PK) deficiency is the most common cause of nonspherocytic hemolytic anemia owing to defective glycolysis. This study developed and validated an automated method to measure PK activity in red blood cells (RBCs). PK catalyzes the reaction of phosphoenolpyruvate with ADP to form pyruvate and ATP. The pyruvate is reduced in the presence of lactate dehydrogenase and NADH to produce lactate and NAD+. The rate of absorbance decrease at 340 nm is proportional to PK activity. PK and hemoglobin (Hb) measurements were performed on a Roche cobas c501 analyzer. After establishing a k-factor, accuracy, linearity, imprecision, sensitivity, and stability were validated and the reference interval was verified. The k-factor was -9477. Accuracy was evaluated by method comparison (n = 56). Linear regression yielded y = 1.0x - 0.57, and R2 of 0.93. Linearity was determined by combining a high sample with hemolyzing solution in 6 different ratios. Linear regression analysis yielded y = 1.02x - 2.68, and R2 of 1.0. The assay was linear to 87 U/dL. Precision was evaluated by testing hemolysates in 3 replicates/day for 10 days. Within-run imprecision was 1.9% and 2.5% and total imprecision was 4.0% and 5.6% at 14.0 and 8.1 U/g Hb, respectively. The limit of blank was 0.0, and the limit of detection was 1.0 U/dL. Stability was determined in 4 sample types at 3 different temperatures; the changes were all <10% when compared with t0. The current PK reference interval of 4.6 to 11.2 U/g Hb was verified. This automated assay for quantifying PK in RBCs has acceptable performance characteristics and is fit for intended use.

A Prospective Analysis of Breakthrough Hemolysis in 2 Phase 3 Randomized Studies of Ravulizumab (ALXN1210) Versus Eculizumab in Adults with Paroxysmal Nocturnal Hemoglobinuria

A Prospective Analysis of Breakthrough Hemolysis in 2 Phase 3 Randomized Studies of Ravulizumab (ALXN1210) Versus Eculizumab in Adults with Paroxysmal Nocturnal Hemoglobinuria

Synthesis of 1,4‐diaminoanthraquinones for the purification of lactate dehydrogenase

Cibacron blue 3G‐A is a dye commonly used for the purification of enzymes using affinity chromatography, often utilized in undergraduate biochemistry laboratory studies. Unfortunately, companies only have this product available intermittently, and when available it is sold at a fairly high price. Due to these issues, an alternative molecule that could be easily synthesized and would be similarly efficient at enzyme purification is desired. A distinct feature of Cibacron blue is its anthraquinone core. Anthraquinones are naturally‐occurring aromatic compounds commonly used in the manufacture of dyes. This synthesis utilizes a method of converting 1,4‐diaminoanthraquinones into diazonium salts to then allow the addition of various other functional groups, including anilines and phenols. After synthesis has been conducted, this small library of aminoanthraquinone derivatives will be tested for biological activity by examining spectrophotometric activity in the presence of lactate dehydrogenase (LDH). This will determine whether the products synthesized are efficient at binding to LDH and whether they can be successfully utilized for purification methods. This experiment details a simple, two‐step synthesis of various 1,4‐disubstituted aminoanthraquinone derivatives, which may be found to be useful alternatives to Cibacron blue. In addition, these products could potentially have other biological activity, due to the highly‐activated anthraquinone core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Factors contributing to adverse maternal outcomes in patients with HELLP syndrome

Objective: To determine the prognostic factors for adverse maternal outcomes in women with complete HELLP syndrome.Methods: A retrospective cohort study was carried out by searching the hospital data for the diagnosis of HELLP syndrome according to Tennessee classification from January 2007 to January 2014. Data included a total of 171 patients between January 2007 and January 2014. The data were collected from the hospital records including demographic data variables, presence of preeclampsia/chronic hypertension, gestational age at the time of diagnosis, and adverse maternal outcomes in terms of eclampsia, disseminated intravascular coagulopathy, acute renal failure (ARF), pulmonary edema, abruptio placentae placental abruption, pleural effusion, ascites, transfusion, and death were determined as adverse maternal outcomes. Independent prognostic factors for each complication with adverse maternal outcome were determined.Results: A total of 171 patients between January 2007 and January 2014 were included in the study. Clinical and laboratory parameters of 171 patients were analyzed. Risk factors for adverse maternal outcomes were as follows; aspartate amino transferase (AST) > 316 U/L, alanine aminotransferase (ALT) > 217 U/L, total bilirubin >2.0 mg/dL, lactate dehydrogenase (LDH) > 1290 U/L, blood urea nitrogen (BUN) > 44 mg/dL, and low platelets (<50,000/mm3). The risk of eclampsia increased 4.1 times and 3.4 times in the presence of LDH >1290 U/L and headache, respectively. Younger maternal age also increased the risk of eclampsia. Risk of ARF increased 15 times at the levels of bilirubin >2.0.Conclusions: Younger age, headache, bilirubin >2.0 mg/dL, LDH >1290 U/L, and low platelets (<50,000/mm3) were independent prognostic risk factors for predicting adverse maternal outcomes.

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

The content of nitric oxide in blood plasma of health persons depending on age.

The article considers results of study of total concentration of nitrites and nitrates (NOx) in blood plasma of healthy individuals residing in St. Petersburg. The samples from 58 healthy individuals were analyzed and out of them - 31 aged 18-25 years and 27 aged 48-63 years. The concentration of NOx in blood plasma was determined using Griess reagent after reduction of nitrates under effect of NADH-dependent recombinant nitrate reductase from Arabidopsis thaliana. The surplus of NADH preventing Griess reaction was removed at the expense of of enzyme reaction in presence of lactate dehydrogenase and sodium pyruvate. The analysis and its calibration was carried out using additions of solution of sodium nitrate to samples of blood plasma without deproteinization. The data was obtained concerning variation of content of NOx in healthy individuals of various age. In particular, it is demonstrated that the level of total content of nitrogen oxide in the group of elder age was reliably higher (p=0.0001) as compared with donors aged 18-25 years.

A Differential Effect of FGF‐2 on Resistance to Doxorubicin‐Induced Necrotic Versus Apoptotic‐Like Damage in Neonatal Rat Cardiomyocyte Cultures

Therapeutic agents like doxorubicin (DOX), an anti‐cancer anthracycline, can increase the risk of cardiac damage. Strategies are needed to protect the heart that still allows the benefits of drug treatment. “Basic” fibroblast growth factor‐2 (FGF‐2), and specifically the low (18 kDa) molecular weight isoform is cardioprotective, but this has not been reported for DOX‐induced injury. The clinical effects of DOX have been modeled in rat cardiomyocytes, and 0.1–1.0 μM DOX is sufficient to induce cell damage. Here, we assessed the effect of 10 nM FGF‐2 on 0.5 μM DOX‐induced necrotic versus apoptotic‐related damage over 24 hours, as detected by the presence of lactate dehydrogenase in the culture medium (LDH) versus DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). DOX induced a decrease in apparent (attached) cell number and a significant increase in LDH within 6 hours. The remaining cells continued to display muscle striations based on alpha‐actinin staining, but an increase in TUNEL intensity from 6 to 24 hours was also seen. Pre‐treatment (30 min) with FGF‐2 resulted in a significant decrease in LDH but not apparent TUNEL intensity. Additional assays to characterize these effects are being pursued. These data suggest that FGF‐2 can increase resistance to DOX‐induced plasma membrane damage but not DNA fragmentation under the conditions tested.

Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

Immobilization of lactate as an electroactive indicator on pencil graphite electrode for the development of a new electrochemical biosensor for the detection of lactate dehydrogenase

A biosensor was developed for the detection of lactate dehydrogenase (LDH) enzyme using a lactate modified pencil graphite electrode (PGE). The sensor relies on the immobilization of the lactate on PGE, and LDH detection is based on the decrease of lactate peak current following oxidation to pyruvate in the presence of LDH. Square wave voltammetric technique was used for the assay of signals in the range of −0.6 to 0.8 V and a frequency of 25 Hz for the determination of LDH. The dependence of the response was investigated in terms of reaction time, washing time and LDH and NAD+ amounts. Also, the electrochemical behavior of LDH treatment on the lactate modified PGE was studied. The electrode showed good selectivity, repeatability and an operational stability of about 90% of its original response for two weeks. Moreover, the sensor displayed a linear response range from 0.36–2.13 U μl−1 for LDH with a detection limit of 0.16 U μl−1. The response time of the LDH-treated lactate modified PGE was found to be 2 s. The relative standard deviation (RSD) obtained was 3.5% (for LDH 0.71 U μl−1 and n = 3).

Glucose metabolism is accelerated by exposure to t-butylhydroperoxide during NADH consumption in human erythrocytes

Several mechanisms have been proposed to underlie the events that occur during oxidative damage in red blood cells (RBCs) exposed to reactive oxygen species. This work explores what happens when metabolites related to redox regulation in human RBCs are oxidized to form alkoxyl radical and peroxyl radical as a result of exposure to tert-buthylhydroperoxide (BHP). During exposure to BHP, the glutathione level and the ratio of NADPH to total nicotinamide adenine dinucleotide phosphate (NADPH plus NADP +) were significantly decreased. Although alteration in the concentration of monosaccharides metabolized in the pentose phosphate pathway (PPP) was not observed, exposing RBCs to BHP caused the formation of methemoglobin (metHb) and a significant decrease in NADH. Moreover, we detected a significant increase in one of the peaks during BHP exposure by using HPLC with dansyl hydrazine as a prelabel reagent. A complete enzymatic conversion procedure was used to identify the peak as pyruvate based on comparison with standards. These results suggest that the rapid recovery in the level of glutathione and the formation of metHb by BHP require NADPH and NADH consumption. Subsequently, glucose metabolism accelerates to reproduce NADPH and NADH, which results in pyruvate accumulation. Our findings indicate that the level of pyruvate markedly increases upon exposure to a radical-generating oxidant capable of forming metHb. Methemoglobin reductase requires NADH as a co-factor, and oxidized form (NHADP +) is reduced via the glycolytic reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase. Thus, the overall acceleration of glycolysis induced by BHP is strongly dependent on the NADH reproducing pathway. In addition, the decrease in NADH enhances the increase in pyruvate by inhibiting the conversion of pyruvate to lactate in the presence of lactate dehydrogenase.

Oestrogen changed cardiomyocyte contraction and β‐adrenoceptor expression in rat hearts subjected to ischaemia–reperfusion

Women with functional ovaries have a lower cardiovascular risk than men and postmenopausal women. However, oestrogen replacement therapy remains controversial. This study examined the effect of ovarian hormone deficiency and oestrogen replacement on ventricular myocyte contractile function and expression of beta-adrenoceptors (beta-ARs). Female Sprague-Dawley rats were subjected to bilateral ovariectomy (OVX) or sham operation (Sham). A subgroup of OVX rats received oestrogen (E2) replacement (40 microg kg(-1) day(-1)) for 4 weeks. Cardiomyocyte shortening was evaluated in basal conditions and in the presence of isoprenaline (ISO). The expression of beta-ARs was assessed by Western blotting. The presence of lactate dehydrogenase (LDH) activity in the coronary effluent was determined. Ovariectomy promoted body weight gain associated with reduced serum E2 and uterine weight, all of which were abolished by treatment with E2. Ovariectomy increased the amplitude of both basal and ISO-stimulated contractions, increased LDH release, upregulated beta1-AR expression and downregulated beta2-AR expression, all of which were restored by treatment with E2. A beta1-AR antagonist, CGP20712A, but not a beta2-AR antagonist, ICI118,551, significantly decreased the amplitude of ventricular myocyte shortening. Oestrogen decreased cardiomyocyte contraction and the expression of beta1-AR, and increased expression of beta2-AR, and all these effects were abolished by the E2 receptor antagonist, ICI182,780. These data suggest that oestrogen plays a cardioprotective role in female rat hearts subjected to ischaemia-reperfusion injury, and the effects of oestrogen are associated with decreased cardiomyocyte contraction and expression of beta1-AR, and increased expression of beta2-AR.

Mitochondrial Regulation of Caspase Activation by Cytochrome Oxidase and Tetramethylphenylenediamine via Cytosolic Cytochrome c Redox State

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.

Is there an intracellular lactate shuttle in skeletal muscle?

As detailed in my review in this journal (Gladden, 2004), a fundamental change in the overall view of the role of La− in metabolism occurred with the introduction of the lactate shuttle hypothesis (now called the cell-to-cell lactate shuttle) by George Brooks in the mid-1980s (Brooks, 1985). Since then, evidence for the cell-to-cell lactate shuttle has been essentially unanimous. It is now clear that O2 limitation is not a requirement for net La− formation, and that La− is an important intermediary in numerous metabolic processes, a mobile fuel for aerobic metabolism, and perhaps a mediator of redox state among various compartments both within and between cells. Skeletal muscle, once considered to be simply the major site of La− production, is now known to be a ready consumer of La− in many circumstances, including steady state exercise. Since 1998, Brooks and colleagues (e.g. Brooks et al. 1999) have also promulgated the idea of an intracellular lactate shuttle in skeletal muscle, cardiac muscle and liver. If verified, this shuttle would require a revision of biochemistry textbooks. The basic idea is that cytosolic activity of the enzyme lactate dehydrogenase (LDH) is so high that pyruvate-to La−-conversion is prevalent, making La− the primary endproduct of glycolysis even under aerobic conditions. La− would then diffuse to mitochondria and into the mitochondrial matrix via facilitated diffusion across the inner membrane with the assistance of a monocarboxylate transporter (MCT). In the matrix, La− would be converted back to pyruvate in a reaction catalysed by intramitochondrial LDH. Such a shuttle would bring reducing equivalents into the mitochondria, and at least to some extent, replicate or replace the role of the malate–aspartate and glycerol phosphate shuttles. This shuttle would also be a major pathway for pyruvate transfer from the cytosol to the mitochondrial matrix. Although there is some disagreement about the exact isoform of the MCT, there is accord on the premise that MCTs are present in mitochondria, apparently on the inner membrane. The catch is that LDH must be present in the mitochondrial matrix, and there is strong disagreement on this point. In this issue of The Journal of Physiology, Bonen and coworkers (Yoshida et al. 2007) offer strong evidence against the presence of LDH in mitochondria and against the ability of mitochondria to directly oxidize La− without prior extramitochondrial conversion of La− to pyruvate. They (Yoshida et al. 2007) isolated both subsarcolemmal and intermyofibrillar mitochondria from both oxidative and glycolytic rat skeletal muscle and report these key findings: (1) in agreement with previous work, MCTs are present on both types of mitochondria; (2) [La−] has to be 57–139 times higher than [pyruvate] to achieve the same mitochondrial oxidation rate; (3) addition of LDH increases the rate of La− oxidation, but not pyruvate oxidation; (4) excess pyruvate inhibits the oxidation of palmitate and La− whereas excess La− does not inhibit either palmitate or pyruvate oxidation; (5) AICAR (a cell-permeant adenosine analogue) increases pyruvate but not La− oxidation; and (6) LDH activity in either type of mitochondria is only about 0.4–0.5% of that in whole muscle. In toto, these results along with previous congruous reports by other investigators may lay to rest the idea of intramitochondrial oxidation of La−. However, correctly in my opinion, Bonen and colleagues (Yoshida et al. 2007) retain the idea of an intracellular lactate shuttle that does not involve the mitochondrial matrix. This is a concept originally proposed by Stainsby & Brooks (1990) and it is compatible with recent observations of Brooks and colleagues (Hashimoto et al. 2006) suggesting the presence of a lactate oxidation complex (an association of MCT, CD147, LDH and cytochrome oxidase) within the inner mitochondrial membrane. In this model, La− would still be the primary endproduct of glycolysis in the cytosol. Uptake of pyruvate and subsequent intramitochondrial oxidation would create a sink for pyruvate in areas adjacent to mitochondria, perhaps in the mitochondrial intermembrane space. A low [pyruvate] near the mitochondria would in turn create a sink for La− via reversal of the LDH reaction, creating a [La−] gradient from cytosolic sites of La− production to mitochondrial sites of La− removal. Due to the higher concentration of La− relative to pyruvate, this [La−] gradient would be much larger than the [pyruvate] gradient making La− the primary diffusive species. Such a model is compatible with the compartmentation of metabolism. Is it possible that La− derived from glycolysis compartmentalized with sarcolemmal Na+–K+-ATPase activity is shuttled towards subsarcolemmal mitochondria whereas La− derived from glycolysis associated with sarcoplasmic Ca2+ pumping is shuttled towards intermyofibrillar mitochondria? Next comes the arguably more difficult task of detecting, quantifying and explaining an intracellular lactate shuttle that does not require a mitochondrial matrix component.

Insulin-Stimulated NADH/NAD + Redox State Increases NAD(P)H Oxidase Activity in Cultured Rat Vascular Smooth Muscle Cells

We reported that insulin modestly stimulates NAD(P)H oxidase activity in cultured rat vascular smooth muscle cells (VSMC) and synergistically stimulates enzyme activity with angiotensin II (Ang II), leading to synergistic stimulation of VSMC migration. The aim of this study was to determine the mechanism of insulin-stimulated NAD(P)H oxidase activity. Cultured rat VSMC O(2)(-) and H(2)O(2) production was measured by lucigenin and luminol luminescence, respectively, and lactate and pyruvate content of cell lysates determined by measuring increase or loss, respectively, of NADH in the presence of lactate dehydrogenase. Migration of VSMC was determined by a wound closure method. Administration of 1 nmol/L insulin increased the lactate/pyruvate ratio (LPR), and hence the NADH/NAD(+) ratio by 122 +/- 16% (P < .05). Exogenous lactate (5 mmol/L), which increased the LPR similarly to insulin, increased O(2)(-) production by 123% +/- 32% (P < .05); Ang II (50 nmol/L) increased it by 60% +/- 9% (P < .05); but together these agents synergistically increased O(2)(-) production to 343% +/- 72% above the control value (P < .05 v the sum of the lactate-and Ang II-stimulated values), all in a diphenyleneiodonium-sensitive or apocynin-sensitive manner. Blocking the increase of insulin in the LPR with exogenous pyruvate or oxaloacetate blocked insulin's stimulation and insulin's plus Ang II's synergistic stimulation of O(2)(-)production as well as insulin's stimulation of migration of Ang II-treated VSMC. Insulin plus Ang II also stimulated H(2)O(2) production. Neither wortmannin nor pertussis toxin, which inhibit insulin-stimulated NAD(P)H oxidase activity in other cell types, affected insulin or insulin plus Ang II-stimulated O(2)(-)production. Insulin stimulates NAD(P)H oxidase activity and with Ang II synergistically stimulates it in cultured rat VSMC by increasing the NADH/NAD(+) redox potential, but not by phosphatidylinositol 3-kinase or heterotrimeric G(iota) protein-dependent pathways.

Viability and volume of in situ bovine articular chondrocytes—changes following a single impact and effects of medium osmolarity

Mechanical stress above the physiological range can profoundly influence articular cartilage causing matrix damage, changes to chondrocyte metabolism and cell injury/death. It has also been implicated as a risk factor in the development of osteoarthritis (OA). The mechanism of cell damage is not understood, but chondrocyte volume could be a determinant of the sensitivity and subsequent response to load. For example, in OA, it is possible that the chondrocyte swelling that occurs renders the cells more sensitive to the damaging effects of mechanical stress. This study had two aims: (1) to investigate the changes to the volume and viability of in situ chondrocytes near an injury to cartilage resulting from a single blunt impact, and (2) to determine if alterations to chondrocyte volume at the time of impact influenced cell viability. Explants of bovine articular cartilage were incubated with the fluorescent indicators calcein-AM and propidium iodide permitting the measurement of cell volume and viability, respectively, using confocal laser scanning microscopy (CLSM). Cartilage was then subjected to a single impact (optimally 100g from 10 cm) delivered from a drop tower which caused areas of chondrocyte injury/death within the superficial zone (SZ). The presence of lactate dehydrogenase (LDH; an enzyme released following cell injury) was used to determine the effects of medium osmolarity on the response of chondrocytes to a single impact. A single impact caused discrete areas of chondrocyte injury/death which were almost exclusively within the SZ of cartilage. There appeared to be two phases of cell death, a rapid phase lasting approximately 3 min, followed by a slower progressive 'wave of cell death' away from the initial area lasting for approximately 20 min. The volume of the majority (88.1+/-5.99% (n=7) of the viable chondrocytes in this region decreased significantly (P<0.006). By monitoring LDH release, a single impact 5 min after changing the culture medium to hyper-, or hypo-osmolarity, reduced or stimulated chondrocyte injury, respectively. A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.

Urinary L-lactate excretion is increased in renal Fanconi syndrome.

Measurement of l-lactate in body fluids is an established clinical tool to identify disorders of cellular respiration. However, there is very little known about the clinical value of urinary lactate measurements. We investigated urinary lactate excretion in children with renal Fanconi syndrome. Freshly voided urine samples were obtained from children with Fanconi syndrome and controls both with and without renal disease. Urine lactate was estimated by conversion to pyruvate in the presence of lactate dehydrogenase and NAD. The NADH produced was measured photometrically. Urine lactate was factored for urine creatinine. Children with Fanconi syndrome had a significantly higher urine lactate/creatinine ratio [mean: 84 x 10(-2) mmol/mmol; 95% confidence interval (CI): 40.8-127.1 x 10(-2) mmol/mmol] than healthy controls (mean: 1.3 x 10(-2) mmol/mmol; CI: 1.1-1.5 x 10(-2) mmol/ mmol) and those with a variety of renal diseases (mean: 3.1 x 10(-2) mmol/mmol; CI: 1.8-4.5 x 10(-2) mmol/mmol). Urinary lactate is increased in Fanconi syndrome. The increase is likely to be due to reduced lactate co-transport in the proximal tubule. Urinary lactate/creatinine has clinical utility as a sensitive test of disordered proximal renal tubular function.

Analysis of NAD(P)+/NAD(P)H cofactors by imprinted polymer membranes associated with ion-sensitive field-effect transistor devices and Au-quartz crystals.

Specific recognition sites for the NAD(P)+ and NAD(P)H cofactors are imprinted in a cross-linked acrylamide-acrylamidophenylboronic acid copolymer membrane. The imprinted membranes, associated with pH-sensitive field-effect transistors (ISFETs) or Au-quartz piezoelectric crystals, enable the potentiometric or microgravimetric analysis of the oxidized NAD(P)+ cofactors and the reduced NAD(P)H cofactors, respectively. The NAD+- and NADP+-imprinted membranes associated with the ISFET allow the analysis of NAD+ and NADP+ with sensitivities that correspond to 15.0 and 18.0 mVdecade(-1) and detection limits of 4 x 10(-7) and 2 x 10(-7) M, respectively. The NADH- and NADPH-imprinted membranes associated with the ISFET device enable the analysis of NADH and NADPH with sensitivities that correspond to 24.2 and 21.8 mV x decade(-1) and lower detection limits that are 1 x 10(-7) and 2 x 10(-7) M, respectively. The ISFET devices functionalized with the NADH and NADPH membranes are employed in the analysis of the biocatalyzed oxidation of lactic acid and ethanol in the presence of lactate dehydrogenase and alcohol dehydrogenase, respectively.

Magneto-switchable electrocatalytic and bioelectrocatalytic transformations.

Magnetic switching of redox reactions and bioelectrocatalytic transformations is accomplished in the presence of relay-functionalized magnetite particles (Fe(3)O(4)). The electrochemistry of a naphthoquinone (1), pyrroloquinoline quinone (2; PQQ), microperoxidase-11 (3), a ferrocene derivative (4) and a bipyridinium derivative (5), functionalized magnetic particles, is switched "ON" and "OFF" by an external magnet upon the attraction of the magnetic particles to an electrode or their retraction from the electrode, respectively. The magneto-switchable activation and deactivation of the electrochemical oxidation of the ferrocene-functionalized magnetic particles and the electrochemical reduction of the bipyridinium-functionalized magnetic particles are used for the triggering of mediated bioelectrocatalytic oxidation of glucose, in the presence of glucose oxidase (GOx), and bioelectrocatalytic reduction of nitrate (NO(3) (-)), in the presence of nitrate reductase (NR), respectively. Magnetic particles functionalized with a PQQ-NAD(+) dyad are used for the magnetic switching of the bioelectrocatalytic oxidation of lactate in the presence of lactate dehydrogenase (LDH). The coupling of these particles with a ferrocene-monolayer-functionalized electrode allows the dual and selective sensing of lactate and glucose in the presence of LDH and GOx, respectively, by using an external magnet to switch the detection mode.