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S E Brolin

Publications and source records attributed to S E Brolin.

At least 19 recordsLinked to original sources
PubMed1992

Bioluminescence analysis of NAD(P)H and NAD(P)+ preventing mutual interference by selective nucleotide destruction and enhanced specific light emission.

Improved bioluminescence analysis of pyridine nucleotides has been designed based on the fact that the luminescence intensity expresses the velocity of the light formation. The bacterial luciferase system is, in principle, composed of two reactions with two different velocities, one for energy supply by the oxidation of NAD(P)H and the other for the subsequent light generation. The rate setting can be arranged such that an emission maximum is produced 30 to 40 s after mixing the sample with the light-yielding solution, hence providing for a convenient analytical performance. The maximal intensity which is easily recorded, e.g., by a tracking volt-meter, is proportional to the concentration of the reduced nucleotide. Discriminative analysis of the various pyridine nucleotides is facilitated by selective destruction of the oxidized forms with alkali and the reduced forms with acid. Erroneous conversion of NAD(P)H to NAD(P)+ may be induced by haemoglobin in a tissue sample but this is prevented by the presence of 2 mM ascorbic acid at the instant of the acidification. Simultaneous coupling of the ongoing reduction of a pyridine nucleotide to the oxidation in the bacterial luciferase system generates a light-yielding cycle which offers important advantages. With NAD(P)+ as the analytic target compound, direct measurement replaces a preceding separate conversion to NAD(P)H. The four nucleotide forms become determinable in a sample by combining selective destruction of either the reduced or oxidized species with a nucleotide-specific reduction in the cycle. Discriminative analyses are furthermore facilitated by the enhanced emission which is due to the energy derived from the continuous specific reduction, whereas initial light signals from side reactions fade out. It is often possible to suppress disturbing analytical errors by the design of the light-yielding cycle. If the rate of the dehydrogenase reactions is kept low compared with the overall rate of the luciferase system, moderately impaired function of some of its components may only give rise to a slight and tolerable decrease in emission intensity. Kinetic evaluations and model experiments are presented and supplemented with applications to tissue samples.

Algorithms↗
PubMed1991

Analytical applications of bioluminescence--a matter of proper kinetic design and recording.

The way bioluminescence analysis employs photometric technique is illustrated in relation to the resulting demands on signal processing and detectability. The analytical reaction may be regarded as composed of a supply reaction providing the excited species followed by a decay reaction in which light is emitted. Bioluminescence analysis implies the recording of a velocity, hence rate regulation forms the basis of the development of an analytical set-up. In principle two means of design are used, either the application of a pulse technique or the monitoring of a durable emission. Both methods have their respective pros and cons but operating in the intermediate time range is sometimes favorable. The pulse technique is an arrangement where the entire emission develops out during a limited amount of time usually as a flash of light in the subminute range. It is accompanied by demands for rapid mixing and initiation of the analytical process as well as fast recording techniques. Durable emission measurements are based on a slowing down of the process, e.g., by reducing the concentration of enzyme or by the addition of inhibitors, so that the light intensity may be regarded as constant during the measurements. This facilitates the measuring procedure and provides for simplified handling, but occurs at a cost of emission intensity and sensitivity. Bioluminescence analysis mimics metabolic routes yielding great possibilities for coupling with other metabolic pathways. Such coupled systems are suited for analysis of a wide variety of metabolites and enzymes. By proper kinetic design it is possible to make the analyses largely insensitive to variations in activity of the reagents.

Animals↗
PubMed1991

Influence of glucose, fructose and aldose reductase inhibition on retinal sorbitol metabolism.

The sorbitol shunt has been studied in bovine retinal tissue at incubation times from 1 to 24 h. It was shown that an elevated glucose concentration (22 mM) of the medium was accompanied by a slight increase in sorbitol content already after 3 h. At longer incubation times, but lower glucose concentration (11.1 mM) there was a similar increase. Addition of an aldose reductase (AR) inhibitor prevented the sorbitol increase. Addition of fructose to the medium significantly increased the sorbitol accumulation above the effect seen with glucose alone and this effect was not influenced by the AR inhibitor. Thus the sorbitol concentration in the retina may be increased after a short incubation time and further enhanced by the presence of fructose.

Aldehyde Reductase↗
PubMed1989

Design of coupled reactions for simplification of bioluminescence analysis.

Luminescence analysis may be applied to many substances by arranging a prior reaction producing a species entering the light-emitting reaction. Under favourable conditions the two consecutive reactions are carried out simultaneously as a one-step procedure. In a bioluminescence assay, luciferase stability is frequently a problem, making it desirable to develop analytical schemes where the analytical response becomes largely independent of any impaired luciferase activity. The value of maximal emission or an approached steady-state level is a convenient and usually well-defined analytical parameter. When recording this level it is important to design the participating reactions in a way that compensates for changes in luciferase activity.

Chemical Phenomena↗
PubMed1989

Sorbitol metabolism in retina studied in vitro.

A new method is described which allows study of the retinal sorbitol metabolism in vitro. Bovine retinal tissue was incubated in tissue culture medium for 24 h and after this time showed only small morphological changes. The sorbitol content doubled at a glucose level of 5.5 mM compared with freshly prepared noncultured retina. Increasing glucose concentrations led to a gradual increase of the sorbitol content. Supplementation of the culture medium with fructose likewise enhanced the retinal sorbitol accumulation. An aldose reductase inhibitor (ICI 128436; Statil) significantly decreased the sorbitol content.

Animals↗
PubMed1989

Influence of sorbitol accumulation on growth and development of embryos cultured in elevated levels of glucose and fructose.

Day-9 rat embryos, cultured for 48 hours in 67 mmol/l D-glucose, showed impaired growth, increased malformation rate and elevated concentration of sorbitol compared to embryos cultured in medium without additional glucose supplement. Supplementing the high-glucose cultures with an aldose reductase inhibitor markedly decreased the sorbitol levels without affecting the malformations or the retarded growth of the embryos. Since embryos cultured in 6.6 mmol/l D-fructose showed normal growth and morphology despite increased accumulation of sorbitol, this study suggests a dissociation between raised sorbitol levels and glucose-induced maldevelopment in rat embryos.

Abnormalities, Drug-Induced↗
PubMed1988

Sorbitol metabolism in the retina, optic nerve, and sural nerve of diabetic rats treated with an aldose reductase inhibitor.

Sorbitol concentration has been measured in retina, optic, and sural nerve of normal, diabetic, and aldose reductase inhibitor-treated diabetic rats. The sural nerve displayed significantly higher sorbitol content than the retina and the optic nerve both in control animals and in diabetic animals. In the sural nerve the response to treatment with an aldose reductase inhibitor was more marked than in the two other tissues. The activities of aldose reductase and sorbitol dehydrogenase were not influenced by diabetes. It is suggested that aldose reductase inhibition may be of greater use for alleviating peripheral nervous system accumulation of sorbitol than for hindering CNS accumulation of the polyol.

Aldehyde Reductase↗
PubMed1988

Sorbitol in aortic endothelium of diabetic rats.

Sorbitol metabolism of the endothelial cells of the aorta has been studied in normal and alloxan diabetic rats by the aid of a new time- and gradient governed elution method. In the normal rats the amount of sorbitol was small whereas in the diabetic animals the content was significantly increased (p less than 0.001). The activity of sorbitol-dehydrogenase was not changed. It is possible that a metabolic injury of endothelial cells induced by a glucose overload may be mediated by formation of sorbitol.

Animals↗
PubMed1987

A new approach to the biochemical pathology of the vascular system, using time governed laminar elution and bioluminescence analyses.

Endothelial cells which cover the inner wall of blood vessels were extracted for bioluminescence analyses of nucleotides and enzymes. The contaminating blood was removed by heparinization and rinsing with ammonium chloride. The content of the endothelial cells of the rat aorta was reached by time governed laminar elution, using a saponin solution to disrupt the cell membranes. Uniformity of extraction was achieved with a Hamilton programmable pump. All the analyses of the eluted fractions showed a characteristic and reproducible peak. The activities of glucose-6-phosphate dehydrogenase and adenylate kinase were significantly higher in the diabetic animals whereas the amount of nucleotides did not differ between diabetic and control rats. The laminar elution technique combined with bioluminescence assay represents a new approach to studies of biochemical alterations in the endothelial cells. The method is also useful for extraction and analyses of other surface layers.

Adenine Nucleotides↗
PubMed1987

Quantification and gel electrophoretic analysis of proteins extracted from vascular endothelial cells.

The inner walls of tubular organs and body cavities are lined with a sheet composed of cells which control the passage through the walls and thus are of considerable physiological and biochemical interest. They are difficult to prepare but their content can be extracted and analysed, using laminar extraction and sensitive bioluminescence methods. This has been shown for the endothelial cells which line the interior of blood vessels. Although it is dealing with small amounts of material, only a minor part of it is consumed in the analyses. The bioluminescence measurements can thus be combined with other analytical procedures such as protein assay and electrophoresis. The possibilities of extending the biochemical information is of interest in the efforts to clarify the pathogenesis of vascular diseases. Laminar elution of protein from endothelial cells of the rat aorta was traced by the simultaneous efflux of adenine nucleotides. These were determined in a bioluminescence assay after conversion to ATP, while the fluorescamine reaction was used for the quantification of protein. The elution patterns obtained by these two methods showed an initial peak with a common maximum. The new possibilities of detecting relevant biochemical changes were evidenced by the finding of a marked protein loss in the fractions containing the outflux from the endothelial cells of diabetic rats. Electrophoresis of proteins eluted from endothelial cells resulted in separation into a large number of bands, but no differences were detected in the electropherograms at comparisons between normal and diabetic rats. It remains to be clarified, whether the protein depletion of the endothelial sheet is due to a concomitant loss of cells or to a cytoplasmatic loss without profound changes of the cytoplasmatic composition.

Adenine Nucleotides↗
PubMed1987

The sorbitol shunt in the retina and the optic nerve of mice with inherited and STZ-induced diabetes.

Micromethods designed for studying the sorbitol shunt permitted studies of the retina and the optic nerve from mice. A significant accumulation of sorbitol was found in the retina of 5-months-old obese-hyper-glycaemic and severely STZ diabetic mice. The latter mice also showed increased sorbitol concentrations in the optic nerve. The activities of aldose reductase were about 3 times higher in the optic nerve than in the retina. Our findings show that not only the retina but also the optic nerve accumulates sorbitol. It is suggested that this hypothetically may lead to degeneration of the optic nerve in severe diabetes as in peripheral nerves.

Animals↗
PubMed1986

Laminar elution of tubular organs for bioluminescence analysis of the interior cell layer.

Cells lining the interior of tubular organs are of considerable interest from physiological and pathological aspects but are very difficult to prepare for biochemical analyses. The contents of such cells can be extracted by infusion of a suitable detergent serving as a membrane destroyer. The tiny ureter of the rat has been used as experimental model. Time governed elution with saponin, using a Hamilton programmable microlab for the infusion results in an effluent pattern which can be determined by sensitive bioluminescence assays. The time course of the outflux of nucleotides and enzymes showed two maxima in agreement with the presence of two epithelial layers in the ureter.

Adenine Nucleotides↗
PubMed1986

Increased accumulation of sorbitol in offspring of manifest diabetic rats.

The effects of maternal diabetes on somatic development and activity of the polyol pathway were investigated during early and late gestation in a rat model for diabetic pregnancy. We studied embryo-fetal growth, mortality, and malformation rate in the offspring of nondiabetic rats and in the offspring of diabetic rats either treated with an aldose reductase inhibitor during gestation or left untreated. The numbers of embryo-fetal resorptions and malformations were significantly increased in the diabetic groups compared with the controls despite maternal treatment with the aldose reductase inhibitor. The sorbitol content of embryos and membranes from the diabetic rats in early gestation was increased 3-5 times over the control values. Similarly, elevated sorbitol levels were observed in the fetal livers and placentas of the diabetic rats in late gestation. Administration of the aldose reductase inhibitor to the pregnant diabetic rats normalized the sorbitol levels in the embryos and their membranes, whereas the sorbitol contents of the fetal livers and placentas were significantly lowered but not completely corrected. Furthermore, in the diabetic groups, no differences in sorbitol levels could be demonstrated between malformed and nonmalformed offspring. The results of this study suggest that enhanced polyol metabolism leading to increased sorbitol accumulation is present in the embryos of diabetic mothers as early as organogenesis. This accumulation is apparently not a major factor in the early developmental disturbances (e.g., growth perturbations and congenital malformations) of diabetic pregnancy.

Animals↗
PubMed1983

Single-step bioluminescence analyses of enzymes, using Cibacrone Blue chromatography for removal of interfering dehydrogenases.

To provide for bioluminescence measurements of the enzymatic activities of dehydrogenases, disturbing contaminants were removed from a bacterial luciferase extract by chromatography, using Blue Sepharose CL-6B, a cross-linked agarose to which Cibacrone Blue F3G-A is covalently attached. This compound has a strong affinity to the dinucleotide fold, which is a region in enzymes binding NAD(H) or NADP(H). In contrast to the absorbed dehydrogenases, both luciferase and oxidoreductase were easily eluted and appeared close to the main bulk of UV-absorbing but analytically less important material. A rapid recording of the elution of luciferase was accomplished with a new electrochemical bioluminescence assay. Due to this and the early elution of the desired material, it could be chromatographed, recognized and collected in less than two hours. Thereby the light-yielding capacity of the sensitive material was well preserved. For bioluminescence assay solutions composed of pooled oxidoreductase-luciferase fractions, FMN and a long chain aldehyde were prepared and supplemented with NAD+ and either lactate, malate or 3-hydroxybutyrate. The analyses were carried out in a single step performance by adding the enzyme sample to the luciferase solution. Minute amounts of lactate dehydrogenase, malate dehydrogenase and 3-hydroxybutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the femtomole region. In case a dehydrogenase does not occur as a contaminant of a commercial luciferase preparation, purification with Cibacrone Blue can be omitted as demonstrated for glucose-6-phosphate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Dinitrobenzenes↗
PubMed1981

Potentialities of bioluminescence analyses in research on the pancreatic islets.

Progress in bioluminescence assay permits not only determinations of nucleotide and substrate concentrations, but also estimation of concentration shifts. The analyses can be extended to comprise Ca2+ since the Aequorea system is sensitive enough for applications in islet research. By connecting the bioluminometer to a microprocessor with a suitable readout device, it is possible to collect and evaluate large amounts of data which may be required in studies of concentration shifts. Thus, blanks, samples and standards can be processed completely within short time periods so that the light-yielding solutions remain stable.

Animals↗
PubMed1980

Photokinetic microanalysis of NADP+, using bacterial luciferase.

Bioluminescence photokinetic assay of NADP+ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. the analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP+ as measurable product and for nucleotide assay in cell samples of 0.5--10 microgram dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP+. Previously, FMN, NADH, NAD+ and NADH have been analysed with the bacterial luciferase system. Its applicability has not been extended by the assay of NADP+.

Enzymes↗
PubMed1980

Adenine nucleotide concentrations in A2-cell rich and normal pancreatic islets of the guinea pig.

Adenine nucleotides were measured in normal and A2-cell rich pancreatic islets of guinea pigs in order to evaluate the bioenergetic properties of the islet cells. The islets were either freshly isolated or cultured for one week. Immediately after isolation the ATP concentration of the normal islets was not significantly different from that of the A2-cell rich islets. Tissue culture raised the ATP concentrations in both experimental groups, leading to a significantly higher ATP content in the normal islets compared to the A2-cell rich islets. The total adenosine phosphate pool remained unchanged. Hypoxia for one minute markedly lowered the ATP concentrations in both normal and A2-cell rich islets. The ATP values could be normalized by renewed culture. The high interconversion rate of adenine nucleotides observed could provide the energy supply which may be required for fast changes in the hormonal secretions of both B- and A2-cells.

Adenine Nucleotides↗