Development of an anodic stripping voltammetric assay, using a disposable mercury-free screen-printed carbon electrode, for the determination of zinc in human sweat.

This paper reports on the development of a novel electrochemical assay for Zn(2+) in human sweat, which involves the use of disposable screen-printed carbon electrodes (SPCEs). Initially, SPCEs were used in conjunction with cyclic voltammetry to study the redox characteristics of Zn(2+) in a selection of supporting electrolytes. The best defined cathodic and anodic peaks were obtained with 0.1 M NaCl/0.1 M acetate buffer pH 6.0. The anodic peak was sharp and symmetrical which is typical for the oxidation of a thin metal film on the electrode surface. This behaviour was exploited in the development of a differential pulse anodic stripping voltammetric (DPASV) assay for zinc. It was shown that a deposition potential of -1.6 V versus Ag/AgCl and deposition time of 60 s with stirring (10 s equilibration) produced a well-defined stripping peak with E(pa) = -1.2 V versus Ag/AgCl. Using these conditions, the calibration plot was linear over the range 1x10(-8) to 5x10(-6) M Zn(2+). The precision was examined by carrying out six replicate measurements at a concentration of 2x10(-6) M; the coefficient of variation was calculated to be 5.6%. The method was applied to the determination of the analyte in sweat from 10 human volunteers. The concentrations were between 0.39 and 1.56 microg/mL, which agrees well with previously reported values. This simple, low-cost sensitive assay should have application in biomedical studies and for stress and fatigue in sports studies.

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays.

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 microM (270 microg l(-1)). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml(-1) LDH and 0.8 U ml(-1) LOD in reactions containing 246 microM pyruvate and 7.5 microM NADPH. PCP detection limits were an EC(10) of 800 nM (213 microg l(-1)) and a minimum inhibition detectable (MID) limit of 650 nM (173 microg l(-1)). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 microM. PCP detection limits were obtained for an assay containing 5 U ml(-1) LDH, 0.8 U ml(-1) LOD and 0.1 U ml(-1) GDH with 246 microM pyruvate, 400 mM glucose and 2 microM NADPH. The EC(10) limit was 150 nM (39.9 microg l(-1)) and the MID was 100 nM (26.6 microg l(-1)). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.

Ammonia vapour in the mouth as a diagnostic marker for Helicobacter pylori infection: preliminary 'proof of principle' pharmacological investigations.

Most current non-invasive tests for Helicobacter pylori depend on the conversion of labelled (13C or 14C) urea to labelled carbon dioxide (13CO2 or 14CO2) and ammonium (NH4+) by the enzyme urease, with the labelled CO2 detected in exhaled air. Despite suggestions going back over a number of years, the alternative possibility of using NH4+ (in the form of gaseous ammonia [NH3]) as the test parameter has received little or no attention. However, this approach is now being explored using a chemiresistive sensor detecting sub-parts per million concentrations of NH3. An in vitro 'glass stomach' (containing various volumes of hydrochloric acid [HCl] and ammonium chloride [NH4Cl]) was used to evaluate the means of increasing 'gastric' pH to that of the NH4+-->NH3 transition that occurs significantly at pH 9.24. This 'stomach' also was used to study mechanisms by which NH3 may be expelled in a pulse (as a surrogate belch), either by the in situ production of CO2 or through an exogenous source. On the basis of the protocols developed, H. pylori-negative subjects were tested before and after ingestion of 10 mg NH4Cl (as a surrogate for bacteria-produced NH4,), and H. pylori-positive subjects were tested without taking urea or NH4Cl. 'Intragastric' pH in the in vitro 'glass stomach' could be increased above pH 9.24 by adding a mixture of 15-30 mL magnesium hydroxide mixture (or the proprietary equivalent) and 50 mL water, and the resulting NH3 expelled by adding 100 mL CO2-saturated cold water (sparkling water). In vivo, NH3 levels in the oral cavity of H. pylori-negative subjects were increased after ingestion of 10 mg NH4Cl; however, levels in the oral cavity of a small number of H. pylori-positive subjects were two- to threefold higher after magnesium hydroxide and sparkling water. On the basis of in vitro studies, an in vivo protocol was developed to increase gastric pH above that required for the NH4+-->NH3 transition, and a mechanism established to release the NH3 into the oral cavity. Preliminary in vivo data confirm the chemiresistive sensor is sufficiently sensitive to NH3 to distinguish H. pylori-negative subjects who have taken 10 mg NH4Cl from those who have not, and clearly distinguish H. pylori-negative subjects from H. pylori-positive subjects. Ingestion of urea or other labelled tracers is not required, nor is belching; and the sensor takes less than two minutes to reach a maximum response. The data provide good evidence that the chemiresistive detection of NH3 has considerable potential as a rapid, point-of-care diagnostic test for H. pylori infection.

Characterization and purification of the vitamin K1 2,3 epoxide reductases system from rat liver.

The enzyme vitamin K1 2,3 epoxide reductase is responsible for converting vitamin K1 2,3 epoxide to vitamin K1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagulant, R,S-warfarin. Purification of this protein would enable the interaction of the inhibitor with its target to be elucidated. To date a single protein possessing vitamin K1 2,3 epoxide reductase activity and binding R,S-warfarin has yet to be purified to homogeneity, but recent studies have indicated that the enzyme is in fact at least two interacting proteins. We report on the attempted purification of the vitamin K1 2,3 epoxide reductase complex from rat liver microsomes by ion exchange and size exclusion chromatography techniques. The intact system consisted of a warfarin-binding factor, which possessed no vitamin K1 2,3 epoxide reductase activity and a catalytic protein. This catalytic protein was purified 327-fold and was insensitive to R,S-warfarin inhibition at concentrations up to 5 mM. The addition of the S-200 size exclusion chromatography fraction containing the inhibitor-binding factor resulted in the return of R,S-warfarin inhibition. Thus, to function normally, the rat liver endoplasmic reticulum vitamin K1 2,3 epoxide reductase system requires the association of two components, one with catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thiol-disulphide redox centre that in the oxidized form binds R,S-warfarin.

The rapid potentiometric detection of catalase positive microorganisms.

The rate of fluoride ion release from the enzymatic cleavage of fluoride ion from 4-fluorophenol by horseradish peroxidase, in the presence of hydrogen peroxide, was measured using a fluoride ion selective electrode. Monitoring the utilisation of hydrogen peroxide by catalase (intracellularly present in almost all aerobic microorganisms) in the presence of 4-fluorophenol demonstrated the inhibition of the enzyme. Horseradish peroxidase appeared to impart a partial protective mechanism of this inhibition. The development of a sequential assay demonstrated the applicability of the proposed method in the assessment of aerobic microorganism numbers. The judicious variation of three parameters, the length of incubation, the concentration of the primary substrate (hydrogen peroxide) and the indicator enzyme activity (horseradish peroxidase), affected both the detection limit and the sensitivity of the assay. Typically with a 15 minute incubation, a detection limit for catalase activity of 1.5 x 10(-6) Uml-1 was obtained together with a sensitivity of 2.42 mumol l-1 s-1 per decade change in activity. Application of the developed catalase assay to the detection of Escherichia coli achieved a detection limit of 1 x 10(2) colony forming units (cfu) ml-1 with a sensitivity of 3.26 mumol l-1 s-1 per decade change in intact microorganisms. By lysis of the microorganisms the detection limit was further reduced to less than 10 cfu ml-1, indicating the future possibilities of the assay.

Interference in an electrochemical detection system for peroxidase-linked reactions based on a fluoride ion-selective electrode.

Use of the fluoride ion-selective electrode as a detector in a linked glucose oxidase (EC 1.1.3.4)/peroxidase (EC 1.11.1.7) method for glucose was investigated for possible interference from sugars, metabolites, drugs, and anticoagulants. The CV for the method was 1.5% (SD 0.37 mmol/L) at a glucose concentration of 25.0 mmol/L. Interference was studied with glucose at this concentration, interference being defined as any result differing by more than +/- 3 SD. When present in concentrations of clinical relevance, interference from urea, uric acid, or acetaminophen would preclude the use of this detection system for routine analysis.

Direct-measurement ion-selective electrodes: analytical error in hyponatremia.

Using two commercial direct ion-selective-electrode sodium and potassium analyzers, we found lower sodium values in cases of hyponatremia as compared with those by flame emission spectrophotometry or indirect ion-selective-electrode analyzers. It is shown that these errors can be eliminated by modifying the calibrant, and that there is a requirement for internationally agreed-upon reference standards for use by manufacturers and laboratory personnel to assess analytical performance of these analyzers.

Effects of somatostatin and a long-acting somatostatin analogue on the prevention and treatment of experimentally induced acute pancreatitis in the rat.

The effects of somatostatin (SRIF) and its long-acting analogue, SMS 201-995 on the prevention and treatment of acute pancreatitis were studied in rats. Acute pancreatitis was established by ligating the bile duct at the point of entry into the duodenum, thereby allowing reflux of bile into the pancreas. Administration of SRIF (4 micrograms kg-1 body wt IV followed by a 12 h infusion of 4 micrograms kg-1 body wt h-1) or SMS 201-995 (2 micrograms kg-1 body wt SC) at the time of bile duct ligation prevented the increase in the serum concentrations of amylase and lipase observed in control rats 12 h after bile duct ligation. Moreover, SRIF and SMS 201-995 administration prevented development of the histological changes consistent with acute pancreatitis observed in control animals. These results suggest that SRIF or SMS 201-995 may be of value in preventing acute pancreatitis following ERCP or after surgery on the pancreas. In rats with established pancreatitis, SRIF (IV bolus of 4 micrograms kg-1 body wt followed by a 24 h continuous infusion of 4 micrograms kg-1 body wt h-1) or SMS 201-995 (2 micrograms kg-1 body wt SC followed by a similar dose 12 h later): (1) significantly improved survival; (2) produced histological changes in the pancreas consistent with organization and healing; (3) prevented the accumulation of ascitic fluid; (4) reduced the serum levels of amylase and lipase. These results suggest that SRIF and SMS 201-995 may prove valuable in the treatment of established acute pancreatitis in man.

Ionic fluoride: a study of its physiological variation in man.

The serum ionic fluoride concentrations in 497 normal individuals, from areas with non-fluoridated water supplies, ranged from 0.25 to 2.20 micromol F-/1 and were positively correlated with age (r = 0.31, p less than 0.001). Distribution of the data with age, coupled with the distribution of serum calcium with age, suggests a possible change in bone metabolism between 26 and 35 years of age. Serum fluoride levels vary with the time of day with a mean minimum value at 0800 and a mean peak value at 2200. Renal clearance studies gave fluoride ion clearances ranging from 19.5 to 44.0 ml/min and tubular reabsorption of fluoride ion ranging from 61.5 to 86.5%. After oral ingestion of 0.48 mmol sodium fluoride, peak serum levels occurred at 60 to 90 minutes; the peak levels were significantly higher in women than in men.

Automated fluoride ion determination. Determination of urine fluoride ion levels.

An automated method is described, using standard continuous flow techniques, for the determination of urine fluoride ion concentration using a fluoride ion selective electrode. It is shown that the kinetics of the electrode response to changes in fluoride ion can be used for the accurate measurement of fluoride ion concentration in urine, and that equilibration of the electrode response is not a prerequisite for the measurement of fluoride ion. Recovery experiments are in the range 83 to 90%; in-batch precision is between 0.9 and 1.6% and carryover 2.5% or less.

Automated fluoride ion determination. Determination of serum fluoride ion levels.

A method is described, using standard continuous flow techniques, for the automated determination of serum fluoride ion concentration using a fluoride ion selective electrode. It is shown that the kinetics of the electrode response to changes in fluoride ion can be used for the accurate measurement of fluoride ion concentration in serum, and that equilibration of the electrode response is not a prerequisite for the measurement of fluoride ion. Recovery experiments are in the range 86.6-98.5%, in-batch precision is 1.0-6.1%, between-batch precision 5.5%, with carryover of -2.99%.