Coupled reactions for the determination of analytes and enzymes based on the use of luminescence

Immobilized enzymes are widely used in the clinical laboratory in the assay of several analytes and enzymes. The use of immobilized enzymes makes these reagents recoverable and re-usable, and in most cases increases their stability and catalytic activity. In conjunction with bioluminescent enzymes (firefly and bacterial luciferases) and chemiluminescent catalyst (peroxidase) we set up high-sensitive flow methods based on the use of nylon tube coil or epoxy methacrylate column as solid support. All the NAD(P)/NAD(P)H-dependent dehydrogenases (bacterial luciferase), ATP-dependent enzymes (firefly luciferase) and oxidases producing H₂O₂ (peroxidase) can be immobilized and a large variety of analytes have been sensitively measured. As an alternative format we also reported a dry chemistry method in which all the enzymes, substrates and cofactors are ready to use, supported on dry cellulose disks. Methodological problems such as flow conditions, stability, pH, ionic strength and analytical performances are also reported.     

Effect of lactate and beta-hydroxybutyrate infusions on brain metabolism in the fetal sheep

Brain uptake of substrates other than glucose has been demonstrated in neonatal but not fetal animals in vivo. This study was undertaken to investigate the ability of the fetal sheep brain to use potential alternative substrates when they were provided in increased amounts. Brain substrate uptake was measured in chronically catheterised fetal sheep during 2-h infusions of neutralised lactate (n = 12) or beta-hydroxybutyrate (n = 12). Despite large increases in fetal arterial lactate and beta-hydroxybutyrate during the respective infusions, no significant uptake of either substrate was demonstrated. However during both types of infusion, the brain arterio-venous difference for glucose decreased 30% (P less than 0.05). Since the brain arterio-venous difference for oxygen was unchanged, and blood flow to the cerebral hemispheres (measured in 11 studies) was also unchanged, the infusions appeared to cause a true decrease in brain glucose uptake. This decrease paralleled the rise in lactate concentration during lactate infusions, and the rise in lactate and butyrate concentrations during the butyrate infusions. Both substrates have metabolic actions that may inhibit brain glucose uptake. We speculate that the deleterious effects of high lactate and ketone states in the perinatal period may in part be due to inhibition of brain glucose uptake.     

Lactography as an approach to monitor glucose metabolism on-line in brain and muscle

1. Thus far metabolic processes in the intact animal (or man) have been studied either by the analysis of body fluids, of biopsies, of tissue obtained post mortem or by techniques, requiring dedicated and expensive equipment (such as positron emission tomography or magnetic resonance spectroscopy). 2. Here we describe a relatively simple and inexpensive technique, that can be applied in vivo to study metabolism in brain regions and muscle in the freely moving rat and in human peripheral tissue. 3. The method is based on microdialysis allowing continuous sampling from the extracellular space, the enzymatic conversion of lactate and the on-line detection of fluorescent NADH. 4. Examples of the application of our technique include the monitoring of lactate efflux from various brain regions of behaving animals under a variety of stress exposures, during ischemia or hypoxia and drug treatments. 5. The results indicate that in brain lactate is not exclusively formed under hypoxia and that neuronal activation leads also to lactate formation, possibly due to the compartmentation of both the involved enzymes and the energy metabolism. 6. The increase of lactate formation in contracting or ischemic muscle or during exercise could also be followed on-line in the rat, suggesting that our approach allows the continuous monitoring of anaerobic metabolism in man e.g. during traumatic or arteriosclerotic limb ischemia or lactic acidosis in shock states. 7. The principle of our approach can easily be adapted to other metabolites, thus enabling to monitor other metabolic pathways in vivo as well.     

Noninvasive monitoring of pneumococcal meningitis and evaluation of treatment efficacy in an experimental mouse model

Noninvasive real-time in vivo bioluminescent imaging was used to assess the spread of Streptococcus pneumoniae throughout the spinal cord and brain during the acute stages of bacterial meningitis. A mouse model was established by lumbar (LP) or intracisternal (IC) injection of bioluminescent S. pneumoniae into the subarachnoid space. Bacteria replicated initially at the site of inoculation and spread progressively from the spinal cord to the brain or from the brain down to the cervical part of the spinal column and to the lower vertebral levels. After 24 hr, animals showed strong bioluminescent signals throughout the spinal canal, indicating acute meningitis of the intracranial and intraspinal meninges. A decline in bacterial cell viability, as judged by a reduction in the bioluminescent signal, was observed over time in animals treated with ceftriaxone, but not in untreated groups. Mice treated with the antibiotic survived infection, whereas all mice in untreated groups became moribund, first in the IC group then in the LP group. No untreated animal survived beyond 48 hr after induction of infection. Colony counts of infected cerebrospinal fluid (CSF) correlated positively with bioluminescent signals. This methodology is especially appealing because it allows detecting infected mice as early as 3 hr after inoculation, provide temporal, sequential, and spatial distribution of bacteria within the brain and spinal cord throughout the entire disease process and the rapid monitoring of treatment efficacy in a nondestructive manner. Moreover, it avoids the need to sacrifice the animals for CSF sampling and the potential manipulative damage that can occur with other conventional methods.     

Lactate and pH in the Brain: Association and Dissociation in Different Pathophysiological States

Brain tissue pH and lactate content were measured in rats under three different experimental conditions, namely: during complete global cerebral ischemia; after reversible near-complete cerebral ischemia; and in experimental brain tumors. At the end of the experiments brains were frozen with liquid nitrogen. A series of 20-microns thick coronal sections was prepared in a cryostat and then used for the regional determination of tissue pH (umbelliferone technique) and tissue lactate (bioluminescent technique). In addition, tissue samples were taken for the quantitative measurement of brain lactate (enzymatic fluorometric technique). The relationship between lactate content and tissue pH was different for each of the three experimental models studied: only after short-term global cerebral ischemia did an increase in the lactate content correlate with a decrease in tissue pH (r = 0.94; p less than 0.001). A highly significant increase in the lactate content (p less than 0.001) was accompanied by physiological pH values (6.96 +/- 0.08 in comparison to 6.97 +/- 0.04 in controls) during recirculation after transient cerebral ischemia and in brain tumors even by an alkaline pH shift. In view of these observations the term "lactacidosis" should not be used without measuring both the lactate content and the pH. The observed dissociation between pH and lactate is due to the fact that both parameters are regulated independently. During anaerobiosis the main source of proton production is ATP hydrolysis rather than glycolysis. It is, therefore, suggested that the terms "acidosis" and "lactosis" should be used instead of "lactacidosis."     

Determinations of lactate and lactate dehydrogenase activity in serum with the flow injection analysis system involving immobilized enzyme column and chemiluminescence

The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods.     

Automated bioluminescent assays for NADH, glucose 6-phosphate, primary bile acids, and ATP

An automated flow system for the bioluminescent assay of various metabolites have been developed. The enzymes used in the assays have been coimmobilized onto Sepharose and packed into small flow cells. Assays for NADH, glucose 6-phosphate, and primary bile acids utilize the bacterial NADH:FMN oxidoreductase/luciferase and either glucose-6-phosphate dehydrogenase or 7 alpha-hydroxysteroid dehydrogenase. ATP assays were performed using immobilized firefly luciferase. In general, the lower limit of detection of the metabolites was at the picomole level, and light intensity was proportional to the substrate concentration from several picomoles to several hundred picomoles. The reproducibility was good with coefficient of variations in the range of 2-5%. The carryover was less than 5% and 30 samples per hour could be assayed. The flow cells were reusable for up to 700 consecutive assays. The major factor limiting their continued use was bacterial contamination of the Sepharose. The results obtained for serum primary bile acids using the bioluminescent assay wer in good agreement with independent measurements on the same samples using gas-liquid chromatography. The immobilized firefly luciferase system was successfully used to measure high levels of bacteria in urine specimens.     

Astrocytes are poised for lactate trafficking and release from activated brain and for supply of glucose to neurons

Brain is a highly-oxidative organ, but during activation, glycolytic flux is preferentially up-regulated even though oxygen supply is adequate. The biochemical and cellular basis of metabolic changes during brain activation and the fate of lactate produced within brain are important, unresolved issues central to understanding brain function, brain images, and spectroscopic data. Because in vivo brain imaging studies reveal rapid efflux of labeled glucose metabolites during activation, lactate trafficking among astrocytes and between astrocytes and neurons was examined after devising specific, real-time, sensitive enzymatic fluorescent assays to measure lactate and glucose levels in single cells in adult rat brain slices. Astrocytes have a 2- to 4-fold faster and higher capacity for lactate uptake from extracellular fluid and for lactate dispersal via the astrocytic syncytium compared to neuronal lactate uptake from extracellular fluid or shuttling of lactate to neurons from neighboring astrocytes. Astrocytes can also supply glucose to neurons as well as glucose can be taken up by neurons from extracellular fluid. Astrocytic networks can provide neuronal fuel and quickly remove lactate from activated glycolytic domains, and the lactate can be dispersed widely throughout the syncytium to endfeet along the vasculature for release to blood or other brain regions via perivascular fluid flow.     

Cellular mechanisms of brain energy metabolism and their relevance to functional brain imaging.

Despite striking advances in functional brain imaging, the cellular and molecular mechanisms that underlie the signals detected by these techniques are still largely unknown. The basic physiological principle of functional imaging is represented by the tight coupling existing between neuronal activity and the associated local increase in both blood flow and energy metabolism. Positron emission tomography (PET) signals detect blood flow, oxygen consumption and glucose use associated with neuronal activity; the degree of blood oxygenation is currently thought to contribute to the signal detected with functional magnetic resonance imaging, while magnetic resonance spectroscopy (MRS) identifies the spatio-temporal pattern of the activity-dependent appearance of certain metabolic intermediates such as glucose or lactate. Recent studies, including those of neurotransmitter-regulated metabolic fluxes in purified preparations and analyses of the cellular localization of enzymes and transporters involved in energy metabolism, as well as in vivo microdialysis and MRS approaches have identified the neurotransmitter glutamate and astrocytes, a specific type of glial cell, as pivotal elements in the coupling of synaptic activity with energy metabolism. Astrocytes are ideally positioned to sense increases in synaptic activity and to couple them with energy metabolism. Indeed they possess specialized processes that cover the surface of intraparenchymal capillaries, suggesting that astrocytes may be a likely site of prevalent glucose uptake. Other astrocyte processes are wrapped around synaptic contacts which possess receptors and reuptake sites for neurotransmitters. Glutamate stimulates glucose uptake into astrocytes. This effect is mediated by specific glutamate transporters present on these cells. The activity of these transporters, which is tightly coupled to the synaptic release of glutamate and operates the clearance of glutamate from the extracellular space, is driven by the electrochemical gradient of Na+. This Na(+)-dependent uptake of glutamate into astrocytes triggers a cascade of molecular events involving the Na+/K(+)-ATPase leading to the glycolytic processing of glucose and the release of lactate by astrocytes. The stoichiometry of this process is such that for one glutamate molecule taken up with three Na+ ions, one glucose molecule enters an astrocyte, two ATP molecules are produced through aerobic glycolysis and two lactate molecules are released. Within the astrocyte, one ATP molecule fuels one 'turn of the pump' while the other provides the energy needed to convert glutamate to glutamine by glutamine synthase. Evidence has been accumulated from structural as well as functional studies indicating that, under aerobic conditions, lactate may be the preferred energy substrate of activated neurons. Indeed, in the presence of oxygen, lactate is converted to pyruvate, which can be processed through the tricarboxylic acid cycle and the associated oxidative phosphorylation, to yield 17 ATP molecules per lactate molecule. These data suggest that during activation the brain may transiently resort to aerobic glycolysis occurring in astrocytes, followed by the oxidation of lactate by neurons. The proposed model provides a direct mechanism to couple synaptic activity with glucose use and is consistent with the notion that the signals detected during physiological activation with 18F-deoxyglucose (DG)-PET may reflect predominantly uptake of the tracer into astrocytes. This conclusion does not question the validity of the 2-DG-based techniques, rather it provides a cellular and molecular basis for these functional brain imaging techniques.     

Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.     

Acetylcholine esterase sensitivity to chronic administration of diphenylhydantoin and effects on cerebral enzymatic activities related to energy metabolism

The effect of chronic treatment (8 months) with diphenylhydantoin (DPH) on rat brain was studied. The activity of some enzymes related to energy transduction (lactate dehydrogenase, citrate synthase, and malate dehydrogenase; NADH-cytochromec reductase and cytochrome oxidase) and neurotransmission (acetylcholine esterase) was evaluated both in the whole brain homogenate and/or in the crude mitochondrial fraction. A clear-cut decrease of acetylcholine esterase activity was observed, the decrease continuing even after treatment was discontinued. Effects on energy metabolism and on lactate dehydrogenase, malate dehydrogenase, and cytochrome oxidase are discussed.     

Relative levels of hexokinase in isolated neuronal, astrocytic, and oligodendroglial fractions from rat brain

The levels of hexokinase, as well as those of the cytoplasmic glycolytic enzyme lactate dehydrogenase and the mitochondrial tricarboxylic acid cycle enzymes fumarase and citrate synthase, have been determined in whole rat brain and in neuronal, astrocytic, and oligodendroglial fractions isolated from rat brain. Compared with either whole brain or with isolated neurons or astrocytes, oligodendroglia are low in hexokinase content. This provides direct confirmation for the conclusion, based on an electron microscopic immunohistochemical method, that oligodendroglia, compared with other neural structures, contain relatively low levels of this key enzyme of glucose metabolism. Based on this confirmation, it is concluded that the electron-microscopic immunohistochemical procedure provides a valid indication of hexokinase content, and thus that other structures shown to stain weakly by the latter technique (e.g., dendritic terminals of cerebellar granule and Purkinje cells) are, indeed, low in hexokinase activity.     

U-14C]glucose metabolism of the perfused cat brain with blood flow disturbances

—In order to study changes of the glycolytic-respiratory system and amino acid metabolism associated with blood flow disturbance, the cat brain perfusion was conducted with artificial blood containing [U-¹⁴C]glucose and the results were compared with those of standard perfusion keeping the cerebral blood flow at constant rate. The findings of the present study are briefly summarized: (1) In blood flow disturbance there was observed an accumulation of lactate just as seen in the low functional state observable in the standard perfusion. However the increase in relative specific activity of lactate was not so marked as the rise in cerebral lactate content, and this indicates that there is an increase of lactate production from substrates other than glucose as well as an increase of net flow of glucose carbon to lactate. (2) In blood flow disturbance relative specific activities of glutamate, aspartate, glutamine and respiratory CO₂ were decreased as compared with those in the brain of high functional state. The relative specific activity of GABA in the reduced blood flow brain was at the same level as that of the brain at high functional state and it was higher than the relative specific activity of glutamate. (3) The relative specific activity and content of alanine were increased in the low function brain with standard perfusion.     

Disposable luciferase‐based microfluidic chip for rapid assay of water pollution

In the present study, we demonstrate the use of a disposable luciferase‐based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3‐dihydroxybenzene and 1,4‐benzoquinone for the proposed microfluidic biosensor measured 3 μM, 15 mM, and 2 μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.     

Immobilized enzymes in analysis

The sensitivity and specificity of enzyme reactions has led to the widespread use of enzymes as reagents in analysis. This article surveys the applications of immobilized enzymes in clinical analysis with particular emphasis on bioluminescent and chemiluminescent assays and test devices intended for use outside the main clinical laboratory.     

Localized magnetic resonance spectroscopy measurement of brain lactate during intravenous lactate infusion in healthy volunteers.

Proton magnetic resonance spectroscopy (1H MRS) localized to the left temporal-parietal region in 8 healthy volunteers detected a 2.1-fold +/- 0.7-fold increase (all values +/-SD) in brain lactate during intravenous infusion of 0.5 molar (M) sodium lactate (5 meq/kg over 20 minutes). Significant increases in brain lactate occurred within 5-10 minutes after starting lactate infusion, progressively rose during the infusion, then decreased towards baseline levels during 30 minutes post-infusion. Venous lactate concentration increased from 0.8 +/- 0.2 mM to 10.9 +/- 4.1 mM or 13.6-fold during the infusion. Flow phantom findings in vitro suggest attenuation of 1H MRS blood lactate signal from arteries and veins as a result of flow velocity effects. Correlations between paired blood and brain lactate measurements at each sampling time indicate a non-linear relationship between compartments during lactate infusion.     

Ex vivo analysis of lactate and glucose metabolism in the rat brain under different states of depressed activity

Brain metabolism of glucose and lactate was analyzed by ex vivo NMR spectroscopy in rats presenting different cerebral activities induced after the administration of pentobarbital, alpha-chloralose, or morphine. The animals were infused with a solution of either [1-(13)C]glucose plus lactate or glucose plus [3-(13)C]lactate for 20 min. Brain metabolite contents and enrichments were determined from analyses of brain tissue perchloric acid extracts according to their post-mortem evolution kinetics. When amino acid enrichments were compared, both the brain metabolic activity and the contribution of blood glucose relative to that of blood lactate to brain metabolism were linked with cerebral activity. The data also indicated the production in the brain of lactate from glycolysis in a compartment other than the neurons, presumably the astrocytes, and its subsequent oxidative metabolism in neurons. Therefore, a brain electrical activity-dependent increase in the relative contribution of blood glucose to brain metabolism occurred via the increase in the metabolism of lactate generated from brain glycolysis at the expense of that of blood lactate. This result strengthens the hypothesis that brain lactate is involved in the coupling between neuronal activation and metabolism.     

Bioluminescent immunoassay for alpha-fetoprotein

A bioluminescent immunoassay of alpha-fetoprotein is described. It uses monoclonal antibodies labeled with glucose-6-phosphate dehydrogenase and polyclonal antibodies coimmobilized on Sepharose with bioluminescent enzymes from marine bacteria. The bioluminescent reaction which occurs in the immunosorbent is proportional to the amount of alpha-fetoprotein in the assay. The protocol is simple and rapid, and no separation step is required to remove the excess labeled antibodies. The assay can be performed directly on 25 microliters serum and it is as sensitive as other immunometric assays.     

Comparative study of free and bound glycolytic enzymes from sea scorpion brain.

Free and bound forms of hexokinase, pyruvate kinase, and lactate dehydrogenase were prepared from the brain of the sea scorpion (Scorpaena porcus) in a low ionic strength medium. Properties of the free and bound forms were compared to determine whether binding to particulate matter could influence enzyme function or stability in vivo. Changes in pH differently affected the activity of the free and bound forms of all three enzymes. Furthermore, bound forms of hexokinase and pyruvate kinase were more stable than the free enzymes to heating at 45 degrees C. Bound hexokinase showed higher affinity for substrates (ATP, glucose) than the free form and bound lactate dehydrogenase had greater affinity for pyruvate and NADH. Although the affinities of the two forms of pyruvate kinase for substrates were similar, Hill coefficients for phosphoenolpyruvate as well as inhibition by ATP differed between the two enzyme forms. Free and bound lactate dehydrogenase also showed differences in Hill coefficients and bound lactate dehydrogenase was less sensitive to substrate inhibition by high pyruvate concentrations. The possible physiological role of the binding of these glycolytic enzymes to subcellular structures is discussed.     

Brain fatty acids in perinatal asphyxia

In hypoxic or ischemic states the release of fatty acids is proposed to have several harmful effects on brain structure and function. We therefore decided to study brain FFA in a simple, clinically related animal model resembling intrauterine perinatal asphyxia (PA). Cerebral blood flow (CBF), brain fatty acids (C14:0, C16:1, C16:0, C18:1, C1 8:0, sigma C), plasma glucose, lactate, beta-hydroxybutyrate (beta-OHB), non-esterified fatty acids (NEFA) and insulin were determined in PA and compared to the normoxic state. Brain C 14:0 FFA were not significantly different from normoxic rats. Brain FFA C 16:0 were comparable between groups but significantly decreased at 20 min of PA. C 18:0 FFA showed a trend to increase with the length of PA reaching significance at 10 min of asphyxia only and were declining at 20 min, however, not significantly. Brain C 16:1 and C 18:1 FFA concentrations were comparable between groups. The parameters cerebral blood flow, glucose and lactate showed a stepwise and significant increase with the length of PA, whereas beta-HOB, NEFA and insulin showed no changes. CBF, glucose and lactate showed a strong association whereas other parameters failed to correlate with each other. Only inconsistent trends of increased brain FFA were found and the association between brain glucose and brain FFA could be ruled out. Although CBF was manifold and significantly elevated in PA, brain FFA pattern suggests that the increase of CBF is obviously not mediated by brain FFA. We conclude that FFA may not be involved in the early phase-pathogenesis of PA.