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Biomedical subjects

H L Schmidt

Publications and source records attributed to H L Schmidt.

At least 19 recordsLinked to original sources
PubMed2001

18O pattern and biosynthesis of natural plant products.

Oxygen atoms in plant products originate from CO(2), H(2)O and O(2), precursors with quite different delta18O values. Furthermore their incorporation by different reactions implies isotope effects. On this base the resulting non-statistical 18O distributions in natural compounds are discussed. The delta18O value of cellulose is correlated to that of the leaf water, and the observed 18O enrichment (approximately +27 per thousand) is generally attributed to an equilibrium isotope effect between carbonyl groups and water. However, as soluble and heterotrophically synthesised carbohydrates show other correlations, a non-statistical 18O distribution - originating from individual biosynthetic reactions - is postulated for carbohydrates. Similarly, the delta18O values of organic acids, carbonyl compounds, alcohols and esters indicate water-correlated, but individual 18O abundances (e.g. O from acyl groups approximately +19% above water), depending upon origin and biosyntheses. Alcoholic groups introduced by monooxygenase reactions, e.g. in sterols and phenols, show delta18O values near +5 per thousand, in agreement with an assumed isotope fractionation factor of approximately 1.02 on the reaction with atmospheric oxygen (delta18O=+23.5 per thousand). Correspondingly, a "thermodynamically ordered isotope distribution" is only observed for oxygen in some functional groups correlated to an origin from CO(2) and H(2)O, not from O(2). The individual isotopic increments of functional groups permit the prediction of global delta18O values of natural compounds on the basis of their biosynthesis.

Alcohols↗
PubMed2001

Electrocatalytic reduction of lipoic acid and electroenzymatic reduction of NAD(P)(+) for integrated dehydrogenase biosensors.

Biosensors for the oxidized substrates of NAD(P)(+)-specific dehydrogenases demand the reductive recycling of the coenzymes. So far, suitable catalysts for the corresponding two-electron transfer are not available. In the present paper, this transport has been realized by a combined electrocatalytical and electroenzymatic process. Lipoic acid has been reduced on graphite electrodes functionalized with Fe(II)-phthalocyanine in 95% yield at-1200 mV in phosphate buffer pH 7.0. With the electrocatalytically reduced product, dihydrolipoic acid, lipoamide dehydrogenase could reduce NAD(+) in 20% yield and thioredoxin reductase NADP(+) in 18.4% yield. So far, the combined electrocatalytic/electroenzymatic system has not yet been realized, mainly because at the potential needed for the lipoic acid reduction, a parallel one-electron reduction of NAD(P)(+) was observed, implying the dimerization of the coenzyme.

Biosensing Techniques↗
PubMed2000

Data model for the elimination of matrix effects in enzyme-based flow-injection systems.

This contribution presents a new conceptional enzyme-based flow injection analysis (FIA) system for the process and quality control of food processing and biotechnological systems. It provides the determination of different analytes in distinct process media on the base of a common experimental set-up. In contrast to known comparable systems, analysis is performed without the commonly used sample preparation and dilution steps. Instead, the adaptation to the necessary measurement range is realized by optimization of intrinsic system parameters. The central principle of the work presented is the elimination of occurring interferences by the heterogeneous matrix of the process sample. Based on a particular injection mode, the application of dehydrogenases as indicator enzymes and a specially developed data model using cognitive methods, cross sensitivities of the detector as well as disturbed reaction rates of the enzymes could be almost completely compensated. Two applications are presented, the analysis of ethanol in non-alcoholic beer and the online determination of D-/L-lactate during a lactic acid fermentation, which reveal the advantage of the developed system.

Alanine Transaminase↗
PubMed1998

Bioelectronic noses: a status report. Part II.

The present state of the art to record or to mimic electronically the human senses of olfaction and taste is characterized. In this part II, strategies are outlined to utilize chemical and biological structures with their different complexities which serve as sensor elements in (bio-) electronic noses. Finally a survey is given on the computer-science aspects of odor recognition based on these elements.

Animals↗
PubMed1997

Carbon isotope effects on the fructose-1,6-bisphosphate aldolase reaction, origin for non-statistical 13C distributions in carbohydrates.

The kinetic and equilibrium isotope effects on the fructose-1, 6-bisphosphate aldolase reaction have been determined using the rabbit muscle enzyme. The natural 13C abundance for both atoms participating in the bond splitting were measured in position C-1 of dihydroxyacetone phosphate and glyceraldehyde 3-P after irreversible conversion to glycerol-3-P and 3-phosphoglycerate, respectively, and chemical degradation. The carbon isotope effects were determined comparing the 13C content of the corresponding positions after partial and complete turnover, and after complete equilibration of the reactants. 13(Vmax/Km) on C-3 was 1.016 +/- 0.007 and 0.997 +/- 0.009 on position C-4, and the equilibrium isotope effects K12/K13 on these positions were 1.0036 +/- 0.0002 and 1.0049 +/- 0.0001. The observed kinetic isotope effect on C-3 is discussed to originate from the formation of the enamine, which comes to equilibrium before the rate determining release of glyceraldehyde 3-P from the ternary complex. The equilibrium isotope effect is seen as the reason for an earlier-found relative 13C enrichment in position C-3 and C-4 of glucose and for varying enrichments in 13C of carbohydrates from different compartments of cells. The kinetic isotope effect is suggested to cause 13C discriminations in the C-3 pool in context with the hexose formation in competition with other dihydroxyacetone phosphate turnover reactions.

Animals↗
PubMed1997

Application of fuzzy logic in multicomponent analysis by optodes.

Fuzzy logic can be a useful tool for the determination of substrate concentrations applying optode arrays in combination with flow injection analysis, UV-VIS spectroscopy and kinetics. The transient diffuse reflectance spectra in the visible wavelength region from four optodes were evaluated to carry out the simultaneous determination of artificial mixtures of ampicillin and penicillin. The discrimination of the samples was achieved by changing the composition of the receptor gel and working pH. Different algorithms of pre-processing were applied on the data to reduce the spectral information to a few analytic-specific variables. These variables were used to develop the fuzzy model. After calibration the model was validated by an independent test data set.

Ampicillin↗
PubMed1997

Determination of protein synthesis in human rectal cancer in situ by continuous [1-13C]leucine infusion.

Previous studies on human colorectal tumor protein synthesis in situ relied on techniques that required intra- or perioperative sampling to obtain a sufficient biopsy size. The purpose of the present study was to develop a new technique by use of new mass spectrometry equipment [capillary gas chromatography (GC)-combustion isotope ratio mass spectrometry (IRMS)], which allows reduction of the necessary sampling size. Thereby, tumor sampling could be done via conventional rectosigmoidoscopy, excluding the need for further disturbing invasive measures. Fifteen postabsorptive patients with localized rectal cancer received a primed-constant infusion of [1-13C]leucine (0.16 mumol.kg-1.min-1 constant, 9.6 mumol/kg prime). Forceps biopsies were taken after 3 and 6 h. In five subjects, tumor tissue and normal mucosa were studied simultaneously. Determination of protein-bound leucine enrichment was done by GC-IRMS, and GC-quadrupole MS was used to determine tracer-to-tracee ratios (tracer/tracee) for free intracellular leucine. GC-MS data demonstrated achievement of a steady state in the precursor pool enrichment after 3 h of isotope infusion (tracer/tracee at 3 h: 6.34 +/- 0.46%, at 6 h: 6.58 +/- 0.38%). Calculation of tumor protein synthesis yielded a fractional synthetic rate (FSR) of 1.06 +/- 0.11%/h or 25.5 +/- 2.6%/day (range 12.0-37.1%/day). At any time, protein-bound leucine enrichment was significantly higher in tumor tissue than in normal mucosa of the same subject. However, protein synthetic rates were comparable (tumor: 1.09 +/- 0.20%/h, mucosa: 1.29 +/- 0.28%/h). Thus combined GC-combustion IRMS and GC-/quadrupole MS provide a simple, reliable, and minimally invasive method to determine tumor FSR in situ, thereby excluding interferences common to previous methods. Tumor and mucosa tissues are similar with respect to protein synthesis, but they apparently differ with respect to leucine extraction from the arterial blood.

Aged↗
PubMed1995

D- and L-lactate determination in dairy products: presentation of a fast automated analysis system.

A flow-injection system is presented, which is versatile, automated and has an integrated enzyme system. Its efficiency at determining D- and L-lactate separately in dairy products is compared with the officially used reference method (DIN 10335). By means of a mathematical elimination of matrix effects, the pretreatment steps can be clearly reduced. As a result of the immobilization of the enzymes, an appropriate automation software and an adaptation of the system developed to the given medium, the analysis could be performed within 5 min and was distinctly cheaper than alternative methods. The values determined in 15 different dairy products deviated from those of the reference method by 3% at a maximum.

Animals↗
PubMed1993

An automatic dehydrogenase-based flow-injection system: application for the continuous determination of glucose and lactate in mammalian cell-cultures.

A concept for the development of an automatic flow-injection analyzer with integrated dehydrogenase columns and its application in the control of industrial processes is presented. The system is based upon a kernel consisting of a nested-loop injection unit, pumps for the filling of the injection loops and the transport of buffer and values for switching on the one hand between sample and standard solutions and on the other hand between different enzyme columns. A Microsoft Windows 3.x application 'WIN-FIA' controls interactively the whole system and can be easily adapted to a specific solution of an analytical problem. As an example, the flow-injection system was used for the continuous determination of glucose and lactate, using glucose dehydrogenase (GDH) and lactate dehydrogenase (LDH) as indicator enzymes, in a mammalian cell-culture fermentation process. The resulting concentration values are in good agreement with those obtained by discontinuously taken standard spectrophotometric enzyme assays.

Animals↗
PubMed1992

Flow injection analysis with immobilized enzymes for process control of pullulan production by fermentation.

A flow injection system is described for the parallel determination of pullulan and glucose during a fermentation of the fungus Aureobasidium pullulans. The polysaccharide was hydrolyzed by pullulanase and amyloglucosidase, immobilized to controlled-pore glass (CPG). The glucose produced was oxidized by glucose dehydrogenase and the NADH formed determined photometrically. The pullulan concentration was calculated from the difference to the response obtained for free glucose. The calibration curves for monomer and polymer were both linear between 2 mg dm-3 and 20 mg dm-3. Analysis of one sample for the determination of glucose and pullulan took about 10 min.

Enzymes, Immobilized↗
PubMed1992

Stabilization of NAD(+)-dependent dehydrogenases and diaphorase by bilayer encagement.

A feasibility study aimed at stabilization of L-lactate-dehydrogenase, L-malate-dehydrogenase, alcohol-dehydrogenase and diaphorase by the recently described method of enzyme 'encagement' was conducted. This method involves derivatizing the enzymes with polyglutaraldehyde, followed by secondary crosslinking with amino derivatives of polyacrylamide. Encagement conditions were optimized for each of the four enzymes, so as to achieve the highest thermal stability combined with highest catalytic activity. Depending on the encagement conditions, residual activities were in the range of 18% to 96% with higher values in the presence of cofactors. Increases in thermal stabilization of up to 26-fold were obtained. For high retention of enzymic activity and stability, the most significant factor was the concentration of polyglutaraldehyde; the crosslinking polymers had only a negligible effect. Furthermore, the significant enhancement in thermal stability could be attained without perturbing the kinetic parameters: Km values for NADH and pH optima remained unaltered for the stabilized enzymes.

Acrylic Resins↗
PubMed1992

Use of naturally enriched mixed food in 13C breath tests applied in young suckling calves.

Utilization of three milk diets including cream, casein or whey, each naturally labelled with 13C (1 mmol 13C excess) from C4 sources, by six young male calves of the Deutsche Fleckvieh breed was investigated in a Latin-square split-plot design. Each milk diet was examined under resting conditions and during a short period of physical exercise on a treadmill. Delta 13C values (/1000) in carbon dioxide in expired air were measured at intervals of about 1 h during 6.5 h after food intake. Expired air samples for CO2 isolation, subsequent isotopic analysis, measurement of CO2 production and respiratory quotient were taken at about hourly intervals and 13C recovery rates over 6.5 h were calculated. Feeding milk containing enriched milk casein, cream, or whey resulted in maximal significant 13C enrichments over background (delta 13C) in CO2 of +1, +2.4 and +2.2 /1000, and recovery rates of 3.6, 9.9 and 12.2% respectively. This comparison shows the different kinetic behaviour of the main nutrients during the oxidation in tissues. The short exercise period (5 min at 1 J/s per kg body-weight +5 min at 2 J/s per kg body-weight) did not influence the recovery rates significantly. However, after 10 min of muscular exercise there was a brief decrease in delta 13C value of expired air which disappeared within the first 5 min of rest. These experiments demonstrate for the first time the applicability of the 13C breath test with naturally enriched diets in animal nutrition research and that quantitative results may be obtained.

Animal Feed↗
PubMed1991

Evidence for a nonstatistical carbon isotope distribution in natural glucose.

The relative carbon isotope content (delta(13)C value) in each position of glucose from a C(4) plant (maize starch) and a C(3) plant (sugar beet sucrose) has been determined by stepwise chemical and biochemical degradation of the molecule and stable isotope ratio measurement of the fragments. The suitability of the degradation methods has been tested through their chemical yield and isotope balance. The results from both methods agreed perfectly, revealing a defined and reproducible (13)C distribution in glucose from both origins. Most prominent was a relative (13)C enrichment by 5 to 6 delta-units in position 4 and a depletion by about 5 delta-units in carbon 6. As possible reasons for these nonstatistical isotope distributions, isotope effects of the aldolase, the triose phosphate isomerase, and the transketolase reactions during carbohydrate biosynthesis are discussed. The practical importance of the results in regard to isotope distributions in secondary plant products as a means for food authenticity control is outlined.

Journal Article↗
PubMed1990

Prerequisites for the on-line control of microbial processes by flow injection analysis.

Problems associated with the use of biosensors in process control, e.g. difficulties of sterilization and sensor fouling, are shortly displayed, and possibilities to overcome them are outlined. The advantages of flow injection analysis (FIA) are demonstrated and examples for efficient sampling systems connected with this method are reviewed. Special emphasis is given to problem-orientated sample pretreatments, preventing fast inactivation of immobilized enzymes in the analysis system. Examples of problem-orientated sample pretreatment units are given. A proposal for a computer-controlled self-calibrating FIA system is given.

Bacteria↗
PubMed1990

Dependence of the carbon-isotope contents of breath carbon dioxide, milk, serum and rumen fermentation products on the delta 13C value of food in dairy cows.

Six dairy cows of two breeds were fed during three alternating periods with products from C3- and C4- plants to yield different natural 13C enrichments of the diet (delta 13C range: -28.0 to -13.7%). The resulting changes in the 13C enrichment of breath carbon dioxide, serum and milk of the animals followed the 13C:12C of the food, in agreement with the individual biological half-lives of those products, and established isotope discriminations. Breath CO2 was more enriched in 13C than expected. This could be related to isotope discriminations during rumen fermentation. From these results an isotopic balance model for the breath CO2 could be established.

Animal Feed↗
PubMed1989

Oxygen isotope effects on the ribulosebisphosphate oxygenase reaction.

The oxygen isotope effect at the substrate O2 on the oxygenase reaction of ribulose bisphosphate carboxylase/oxygenase from spinach is pH and metal dependent. The pH dependence between pH 7.4 and 8.9 is different with Mg2+ (steady decrease in this isotope effect from 1.036 to 1.030) and Mn2+ (minimum isotope effect of 1.028 at pH 8.0). Deuteration of the substrate ([3-2H]ribulose bisphosphate) has no influence on the isotope effect. The results are interpreted as a direct participation of the metal ion in the oxygen-sensitive step, i.e. carbon-oxygen bond formation and the stabilization of the intermediates. In the overall reaction oxygen addition is a major rate-limiting step, and the observed isotope effect is probably close to the intrinsic oxygen isotope effect of the reaction. The basic mechanisms for carboxylation and oxygenation of ribulose bisphosphate appear to be the same.

Carbon Isotopes↗
PubMed1988

Carbon isotope effects on the decarboxylation of carboxylic acids. Comparison of the lactate oxidase reaction and the degradation of pyruvate by H2O2.

The isotope effect at C-1 on the H2O2-catalysed decarboxylation of pyruvate (used as a model reaction for the enzymic reaction) increases between pH 3 and 10 from 1.0007 +/- 0.0004 to 1.0283 +/- 0.0014 (25 degrees C). This result indicates a change in the rate-determining step from formation of the tetrahedral intermediate to decarboxylation of this intermediate. Practically no isotope fractionation at C-1 (1.0011 +/- 0.0002, pH 6.0, 25 degrees C) is found in the lactate oxidase-catalysed decarboxylation of lactate, which is indicative for the existence of an irreversible O2-dependent step prior to the enzyme-catalysed decarboxylation. In addition, the result provides further evidence that dissociation of pyruvate and H2O2 from the enzyme can be excluded. The isotope effect at C-2 of lactate in the enzymic reaction (1.0048 +/- 0.0004) is attributed to the hydrogen transfer step from lactate to the coenzyme.

Carbon Isotopes↗