Biosensor for lactate determination in biological fluids. 2. Interference studies

The selectivity of a yeast lactate biosensor with immobilized cells of aerobic yeast Hansenula anomala was studied. Reducing substances potentially present in blood plasma influenced both enzyme and yeast biosensors in the same way; the highest positive error was observed in the case of uric acid. With respect to the metabolic activity of the yeast cells the biosensor was absolutely specific for lactate during the first two weeks; later on the biosensor responded slightly to some other metabolites, especially some sugars and amino acids. Glucose could cause the highest degree of interference, its effect was however completely eliminated by adding sodium fluoride to the reaction solution. The concentration of other metabolites present in blood plasma is not great enough to call a significant positive error. The results thus support the general use of the yeast lactate biosensor for lactate determination in biological material.     

Despite structural similarities between gp91phox and FRE1, flavocytochrome b558 does not mediate iron uptake by myeloid cells.

Superoxide (O2-) generated by the phagocyte reduced nicotinamide adenine dinucleotide phosphate oxidase is dependent on electron transfer by flavocytochrome b558 (flavocytochrome b), a transmembrane heterodimer that forms the redox center of the oxidase at the plasma or phagosomal membrane. The larger of its two subunits, gp91phox, is homologous to the yeast iron reductase subunit FRE1, and these two proteins share many structural and functional characteristics. Because FRE1 is required for iron uptake in yeast, we hypothesized that flavocytochrome b might serve a similar function in human phagocytes and thus provide a mechanism for the transferrin-independent iron acquisition observed in myeloid cells. To determine whether flavocytochrome b was required for iron uptake, we compared iron acquisition by polymorphonuclear neutrophils (PMNs) or Epstein-Barr virus (EBV)-transformed B lymphocytes derived from individuals with X-linked chronic granulomatous disease (CGD) with iron acquisition by normal cells. Our results indicate that all cells acquired iron to the same extent and that uptake could be significantly enhanced in the presence of the trivalent metal gallium. The gallium enhancement of iron uptake observed in PMNs or in EBV-transformed B lymphocytes derived from healthy individuals was mirrored by those derived from individuals deficient in flavocytochrome b. Furthermore, both normal and CGD-derived EBV-transformed B lymphocytes had similar iron reductase activity, suggesting that flavocytochrome b is not a biologically significant iron reductase. In contrast to previously suggested hypotheses, these results show conclusively that flavocytochrome b is not necessary for cellular iron acquisition, despite structural and functional similarities between yeast iron reductases and flavocytochrome b.     

Silk/polyols/GOD microneedle based electrochemical biosensor for continuous glucose monitoring

This work illustrates the feasibility of a microneedle based electrochemical biosensor for continuous glucose monitoring. The device consists of three silk/d-sorbitol pyramidal microneedles integrated with platinum (Pt) and silver (Ag) wires and immobilized glucose selective enzyme (glucose oxidase, GOD) during fabrication. The silk/d-sorbitol composite can provide a biocompatible environment for the enzyme molecules. The break strength can be controlled by the ratio of silk to d-sorbitol, which guarantees microneedle penetrate into skin. The enzymatic-amperometric responses and glucose concentration were linearly correlated, and cover physiological conditions. The microneedle displays high stability both in long-term monitoring and storage, even at 37 °C. Our results reveal that this new microneedle biosensor is a promising tool for wearable minimally invasive continuous glucose monitoring in practical applications.

This work illustrates the feasibility of a microneedle based electrochemical biosensor for continuous glucose monitoring.     

Oxidation of unsaturated fatty acids by human fibroblasts with very-long-chain acyl-CoA dehydrogenase deficiency: aspects of substrate specificity and correlation with clinical phenotype

The development of an amperometric biosensor for the reduced glutathione determination in serum is described. The biosensor is based on glutathione peroxidase (GSH-Px, EC 1.11.1.9) immobilized onto a pyrolytic graphite-working electrode using carbodiimide as enzymatic condensing reagent. This resulted in an amperometric biosensor with good sensitivity and stability. The reduced glutathione (GSH) was enzymatically converted to glutathione disulfide (GSSG) in the presence of hydroperoxide, which was monitored amperometrically by its electrooxidation at +0.65 V vs. SCE (saturated calomel electrode). Glutathione measurement was carried out by maintaining the ratio between GSH and hydrogen peroxide at 2:1 (25 degrees C). The amperometric response of the biosensor was linearly proportional to the GSH concentration between 1.9x10(-5) and 1.4x10(-4) mol/l, in 0.1 mol/l phosphate buffer (pH=7.8), containing 0.1 mol/l KCl and 0.5 mmol/l Na(2)H(2)EDTA, as the supporting electrolyte. In presence of interfering compounds, the recoveries ranged between 97.2% and 110.7%. The biosensor useful lifetime was at least 2 months when it was evaluated after continuous use. Serum samples analyzed by this biosensor showed a good correlation with the results from the spectrophotometric method (Ellman's reagent) used as reference, presenting relative deviations lower than 7.0%. The low apparent Michaelis-Menten constant value, K(M)(app)=1.6 mmol/l, demonstrated that GSH-Px immobilized on pyrolytic graphite exhibited a high affinity to GSH, without loss of enzymatic activity.     

An integrated microchannel biosensor platform to analyse low density lactate metabolism in HepG2 cells in vitro

In this study, we developed an electrochemical microchannel biosensor platform to analyse lactate metabolism in cells. This biosensor platform was fabricated by photolithography, thin-film deposition and microfluidic technology. A kind of functional biomaterial was prepared by mixing lactate oxidase, single-walled carbon nanotubes and chitosan, and platinum as working and blank electrodes of the biosensor was modified by a thin Prussian blue layer. The lactate biosensor was obtained by dropping functional biomaterials on the electrode. The results demonstrated that the sensitivity of the electrochemical biosensor was up to 567 nA mM⁻¹ mm⁻² and the limit of detection was 4.5 μM (vs. Ag/AgCl as the counter/reference electrode). The biosensor used to quantitatively detect metabolic lactate concentrations in HepG2 cells cultured with cancer drugs showed high sensitivity, selectivity and stability, and has potential applications in organ-on-a-chip and tissue engineering technologies, which typically involve low concentrations of metabolites.

In this study, we developed an electrochemical microchannel biosensor platform to analyse lactate metabolism in cells.     

Clinical determination of glucose in human serum by a tomato skin biosensor

BACKGROUND: Glucose biosensors based on enzyme reaction of glucose oxidase were studied because the symptomatic therapy of diabetes mellitus requires reliable assessment of blood glucose level at frequent intervals. Tomato skin membranes have been successfully employed to entrap glucose oxidase for fabrication of glucose biosensor. METHODS: Glucose oxidase was immobilized onto the tomato skin and the enzyme membrane was then positioned on the surface of an oxygen electrode. The glucose concentration was quantified by the change of dissolved oxygen. All the serum samples were also simultaneously determined by a Hitachi 7060 chemistry analyzer. RESULTS: The response of the biosensor showed a linear relationship with a concentration range of 1.0-30.0 mmol/l glucose. The limit of detection was 0.20 mmol/l. Error Grid analysis demonstrated that 100% of the results fell within clinically acceptable zones A and B. The F- and t-tests showed no significant differences between the 2 methods. The recovery was 95.0-110.0% for 30 serum samples analysis. CONCLUSIONS: The tomato skin biosensor possesses the advantages of simple fabrication, fast response time, low cost and high sensitivity. The results of our method are more accurate than and match well with the current clinical instrument method.     

Influence of the redox state of glutathione upon pyruvate kinase in the intact erythrocyte

We have adapted the hexokinase glucose procedure to an immobilized enzyme stirrer for the determination of glucose concentrations in human blood plasma. The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity.     

Gel entrapped L-asparaginase: kinetic behavior and antitumor activity.

L-Asparaginase from Escherichia coli was immobilized by entrapment in a gel based on poly(2-hydroxyethyl methacrylate) with an activity as high as 730 I.U./g of dry gel. The apparent Michaelis constant for these gels was similar to that of the free enzyme. At 37 degrees C the immobilized enzyme had a half-life of more than 40 days, in vitro. The gel was freeze-dried, crushed and sieved to pass a 38 mum screen, giving a median particle size of 12 mum. C3H mice were injected intraperitoneally with 40 I.U. of L-asparaginase; the peak plasma activity after 4 hours was only 0.9 I.U. for the gel entrapped enzyme compared to a peak activity of 5.0 I.U. after 2 hours for the native L-asparaginase. Ninety percent of the plasma enzyme activity for the gel entrapped case was sedimentable at 21,000 X g, indicating a small leakage of the enzyme from the gel; the clearance for the enzyme activity in plasma had an initial half-life of 13 hours in contrast to a half-life of 2 hours for the native preparation. After intraperitineal injection of 5.0 I.U. into C3H mice, plasma L-asparagine fell to undetectable levels for 4 days and reappeared by day 8 for both the native and immobilized enzymes. Subcutaneously transplanted 6C3HED murine lymphoma was inhibited by 35, 78 and 100% after single intraperitoneal injections of immobilized L-asparaginase of 2, 4 and 8 I.U., respectively, as compared to 36, 53 and 86% for the native enzyme by the 14th day. Body weight changes after receiving immobilized L-asparaginase were essentially similar to those of animals receiving a comparable dose of native enzyme. These results indicate that while most of the immobilized L-asparaginase remains at the injection site, it produces a significant plasma L-asparagine depression and antitumor acitivity comparable to that of the native preparation without major toxicity.     

Immunological methods for human placental alkaline phosphatase (Regan isoenzyme).

Four different immunological methods for the determination of the placental isoenzyme of alkaline phosphatase (Regan isoenzyme) were compared in 64 normal blood donors, 23 healthy laboratory and medical staff workers and 68 pregnant women: a. Inhibition by soluble antibodies to the placental enzyme. b. Precipitation with soluble antibodies. c. Precipitation with immobilized antibodies. d. Measurement of the activity of the placental alkaline phosphatase following binding to an immunoabsorbent that has been obtained by polymerization of anti-placental-alkaline phosphatase gamma-globulin using glutaraldehyde. The immunoabsorbent method yielded the best results. The optimal conditions were evaluated for the measurement of the activity of immunoabsorbent-fixed placental (tumoral) alkaline phosphatase activity. This method was applied to 209 normal blood donors and to 239 patients with different malignant tumors: 25.5 percent of the cancer patients exhibited an elevated Regan -isoenzyme activity in the serum.     

Lactic Acidosis as a Result of Iron Deficiency

Iron-deficient rats have an impaired work performance, even when their anemia is corrected by exchange transfusion. Muscle activity is associated with a higher blood lactate concentration than is observed in iron-replete animals. The accumulation of lactate is a result of excessive production as lactate clearance from the blood was shown to be unaffected. By adjusting the work load to a lower level, it was possible to divide iron-deficient animals into two groups, one capable of continued treadmill running and another in which animals stopped before 20 min. In the former, blood lactate concentration reached a plateau at moderate levels, whereas it continued to increase in the latter until the animal stopped running. Levels of alpha-glycerophosphate oxidase in skeletal muscle mitochondria were found to be much lower in the second group (P < 0.001). Lactate infusion into normal animals was shown to interfere with work performance, and maintenance of a normal pH in iron-deficient and iron-replete animals did not prevent the impairment in work associated with high blood lactate concentrations. Additional evidence was obtained that energy substrate (blood glucose and free fatty acids, muscle glycogen) was adequate in irondeficient animals. Oxygen tension in their vena caval blood was higher than in controls. Furthermore, the in situ behavior of electrically stimulated gastroenemius and soleus muscles appeared similar to that of control animals. Because the stimulation of the single muscle in the iron-deficient animal did not result in appreciable elevation of blood lactate and did not show impaired contractility further supported the hypothesis that the elevation of blood lactate caused the decreased work performance. It is concluded that iron deficiency by a depletion in the iron-containing mitochondrial enzyme, alpha-glycerophosphate oxidase, impairs glycolysis, resulting in excess lactate formation, which at high levels leads to cessation of physical activity.     

Evaluation of a biosensor for the measurement of lactate in whole blood

OBJECTIVES: Evaluation of the analytical performance of a biosensor for the measurement of lactate in whole blood samples and comparison of the results of patients samples with a routinely used spectrophotometric enzymatic method. DESIGN AND METHODS: Heparinized whole blood samples of patients and aqueous control samples were used to determine precision and carry-over. For comparison of the lactate biosensor and the enzymatic method human blood samples were split and measured. RESULTS: Satisfactory within-run (n = 7) and day-to-day (n = 15) precision was found, while carry-over was minimal (<0.2%). A statistically significant relation between the lactate levels in whole blood samples (n = 31) and the corresponding plasma samples was found: y = 0.98x - 0.05 and r = 1.00. No correlation was found between the hematocrit (range 0.23-0.51) and the difference in lactate concentration between plasma and whole blood. When comparing patient results (n = 722) obtained with the spectrophotometric method and the biosensor method, the biosensor measured 13% higher lactate levels. A correction on the basis of the hematocrit minimized this difference. CONCLUSION: The combination of analytical performance, easy handling, rapid analysis, and measurement in whole blood makes the biosensor suitable for lactate STAT-analyses. Care must be given to the interpretation of the results as well as to the preanalytical aspects.     

Determination of the lactate concentration in plasma using L-lactate:ferricytochrome c-oxidoreductase and tetraxolium salt

A method is described for the determination of lactate in plasma using cytochrome b2, with tetrazolium salt as an electron acceptor. The chief advantages over the usual methods of lactate determination are high sensitivity, satisfactory specificity, high absorbance, use of the visible region of the spectrum, and low cost.     

Determination of enzyme activity in biological fluids by means of electrochemical oxidation of NADH at a modified glassy carbon electrode

This amperometric technique for the determination of enzyme activity is based on detecting a decrease in the concentration of the NADH co-factor of the enzyme reaction. A glassy carbon electrode, modified by adsorption of Mg2+ and NADH, is used to measure the anodic peak current that corresponds to the oxidation of NADH. We found no significant difference between the enzyme activity of lactate dehydrogenase (E.C.1.1.1.27) preparations as measured by the above amperometric technique and by a spectrophotometric method.     

A microassay for Gaucher''s disease

We report a new assay for the detection of individuals heterozygous and homozygous for Gaucher's disease which requires relatively small samples of whole blood (0.3 ml), and which determines 4-methylumbelliferyl-β-D-glucopyranoside: β-glucosidase activity under conditions optimal for the determination of leukocyte glucocerebroside:β-glucocerebrosidase activity. The procedure involves the preparation of a leukocyte pellet from 50 μl of whole blood by hypotonic lysis of erythrocytes, followed by assay of β-glucosidase activity at pH 5.5 in the presence of sodium taurocholate (0.6 g/100 ml). The methods described may also prove to be useful for the diagnosis of other diseases of enzyme deficiency which use fluorogenic substrates and leukocytes as a source of enzyme, such as Fabry's disease, Tay-Sachs disease, and generalized gangliosidosis.     

Characterization of immobilized tyrosinase – an enzyme that is stable in organic solvent at 100 °C

Tyrosinase is a copper-containing enzyme present in plant and animal tissues, which catalyzes the production of melanin and other pigments. In organic solvent, tyrosinase can convert N-acetyl-l-tyrosine ethyl ester (insoluble in aqueous) to a derivative of l-dopamine (a drug used for the treatment of Parkinson''s disease). Thus, the performances of tyrosinase in organic solvent have attracted scientific attention since 1980. In this work, we investigated the stability of immobilized tyrosinase at high temperature in anhydrous organic solvent. Triethylaminoethyl cellulose (TEAE-Cellulose) performed the best out of six immobilization platforms. The dry immobilized tyrosinase became extremely thermostable in organic solvent, and the half-life of the dry immobilized tyrosinase in organic solvent is strongly related to the polarity of the organic solvent than their log P value. The immobilized tyrosinase loses its activity instantaneously in aqueous solution at 100 °C, but it keeps enzymatic activity within 10 min in hydrophilic methanol and over one month in hydrophobic hexane (log P: 4.66, non-polar) even incubating at 100 °C. This research provides valuable information for the design of new biocatalysts.

Immobilized tyrosinase in hexane can withstand 100 °C over one week, and the half-life of the dry immobilized tyrosinase in organic solvent is strongly related to the polarity of the organic solvent.     

Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated

We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.     

Determination of choline-containing phospholipids in human bile and serum by a new enzyme sensor

A simple method for direct determination both of choline and lecithin (phosphatidylcholine) in human bile and blood sera was developed. An enzyme electrode, based on immobilized choline oxidase on nylon net and an oxygen Clark electrode was assembled. Phosphatidylcholine can be determined by use of phospholipase D as hydrolyzing agent. Reliable results were obtained in the case of determination of lecithin and choline in bile samples.     

The study of the characteristics and hydrolysis properties of naringinase immobilized by porous silica material

Silica material has high specific surface area and excellent chemical stability, which make it useful for enzyme immobilization. In this work, naringinase was immobilized from fermentation broth of Aspergillus niger FFCC uv-11 by silica materials with different pore diameters of 2 nm (MCM-41), 7.7 nm (SBA-15) and 80 nm (silica gel). It was shown that SBA-15 had the highest naringinase activity, and this was chosen as a suitable carrier material for naringinase immobilization. First, SBA-15 was modified by glutaraldehyde at a concentration of 7% at 25 °C for 2 h, and it was then used for the immobilization of naringinase. At pH 3.5, the immobilized naringinase activity reached 467.62 U g⁻¹ at 40 °C for 4 h when the initial naringinase activity was 89.04 U mL⁻¹. Furthermore, at the optimal reaction temperature of 45 °C and pH of 4.5, the binding efficiency, activity recovery rate and specific activity of the immobilized naringinase were 63.66%, 87.64% and 517.43 U g⁻¹, respectively. Compared with free naringinase, in naringin hydrolysis, the immobilized naringinase performed over a wide pH application range and had good thermal stability. Even more important, the immobilized naringinase retained 61.81% of the residual naringinase activity after eight consecutive cycles, and kept 80.95% of the residual naringinase activity after one month of storage. This study provides an ideal carrier material and some basic data for naringinase immobilization technology, which will greatly promote the application of naringinase in industrial fruit juice processing.

SBA-15 is an innovative silica material for naringinase immobilization, which achieved improved naringinase activity and had excellent hydrolysis properties.     

New system of continuous monitoring of enzyme activities and determination of some substrates

The proposed system of continuous monitoring of enzyme activities is based primarily on the electrochemical behaviour of thiol compounds, and the experimental equipment is extremely simple. The determination of cholinesterase (EC 3.1.1.8) activity is described. The normal values obtained for men (73.9, s +/- 10.3 microkat/l) and for women (71.1, s +/- 10.2 microkat/l), lie within the usual range of analogous photometric methods. Systems for determination of the activities of alkaline phosphatase (EC 3.1.3.1) and adenosylhomocysteinase (EC 3.3.1.1) are described. The activity of aspartate aminotransferase (EC 2.6.1.1) is determined by a combination of enzyme reactions, in which CoA is released from acetyl-CoA. Analogous procedures are discussed for determinations of alanine aminotransferase (EC 2.6.1.2), lactate dehydrogenase (EC 1.1.1.27), lipase (EC 3.1.1.2), and phospholipase A2 (EC 3.1.1.4) activities, and for determination of substrates, e.g., acetate and carnitine. Possible determinations of an additional 199 enzyme activities and of some of substrates are suggested. By determining electrochemically active groups other than thiols this method becomes almost universally applicable.     

Methods for studying the hepatic metabolism of drugs in man

Various methods can be used to investigate human hepatic drug metabolism in vivo. a) Indirect methods based on the assessment of atoxic substances metabolism in order to investigate: --hepatic oxidative activity by antipyrine or caffeine clearance and aminopyrine breath test determination. Such an activity can be modified by drug-induced enzyme induction or inhibition and in case of hepatic disease; --genetic polymorphism of hepatic drug metabolism assessed by the determination of debrisoquine or dextrometorphane metabolic ratio and of N-acetylation phénotype. Unusual therapeutic or adverse effects could be explained by such a polymorphism; --hepatic blood flow, which variations could modify hepatic clearance of drugs with a high hepatic extraction ratio, by the assessment of indocyanine green clearance. b) Direct methods based on pharmacokinetics data and their alterations under various circumstances: simultaneous administration of other drugs, liver disease.