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.

Adenylate kinase activity in various organs and tissues of mice with the obese-hyperglycemic syndrome (gene symbol Ob/Ob).

The activities of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in lyophilized cryostat sections of various organs from mice with the obese-hyperglycemic syndrome (gene symbol ob/ob) were measured fluorometrically. Skeletal and heart muscle tissues had the highest enzyme activities, i.e. 258 and 155 mmoles substrate converted per kg dry weight and per hr (MKH), respectively. Pancreatic islet specimens, which were composed mainly of B-cells, possessed the highest activitty of all parenchymatous cells studied, with 18 MKH as compared to 16 MKH for the cortical tubules of the kidney and 7 and 6 for the exocrine pancreas and liver cells, respectively. The granular layer of the cerebellum had enzyme activity of 31 MKH. The adenylate kinase activity in the B-cells was apparently not influenced by hyperglycemia since it was similar both in obese-hyperglycemic mice of different ages and in lean mice, although the blood sugar concentrations in such animals are much different.

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.

Effects of fructose on D-[6-3H]-glucose uptake and sorbitol metabolism of bovine retina in vitro.

In order to study the influence of fructose on sorbitol formation, bovine retinal tissue was incubated with different concentrations of glucose and fructose, and supplemented with tracer amounts of D-[6-3H]-glucose. Combining high-performance liquid chromatography (HPLC) with radioactivity determinations allowed detection of sorbitol and fructose derived from glucose in the incubation medium. In addition, the total amount of sorbitol was measured with a sensitive bioluminescence method. In this way, it was possible to distinguish between sorbitol formation from glucose and fructose. High concentrations of glucose in the medium increased the formation of sorbitol and fructose from glucose. Addition of fructose to the incubation medium diminished the sorbitol and fructose formation from glucose although the total amount of sorbitol increased significantly. Incubating retinal tissue with an aldose reductase inhibitor decreased sorbitol formation from glucose but did not influence the formation of sorbitol from fructose. Thus, the present findings clearly demonstrate the important influence exerted by fructose on sorbitol formation. The possible significance of the present finding is discussed with respect to diabetes retinopathy and retinal osmoregulation.

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.

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.

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.

Photokinetic microassay of adenylate kinase using the firefly luciferase reaction.

A new rapid photokinetic method is described for determining the activity of adenylate kinase (ATP:AMP phosphotranspherase, EC 2.7.4.3) in 0.1--5.0 micrograms of freeze-dried tissue. This represents a weight range far below that obtainable by fine-needle biopsy. The reaction 2 ADP in equilibrium with AMP + ATP was employed and the ATP formed assayed with firefly luciferase as light yielder. The light emission was recorded on a multi-channel scaler. The adenylate kinase activities found in tissues of mice were in the same range as previously described in a study using fluorometric microassay.

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.

Fluorometric microassays of adenylate kinase, an enzyme important in energy metabolism.

The adenylate kinase system offers a mechanism for the rapid provision of energy by catalysing the production of ATP from ADP. Fluormetric micromethods were developed for determination of the activity of this enzyme using either formation of ADP or ATP, in each case measured by coupling to suitable dehydrogenase reactions. Both procedures yielded results in good agreement, but when ADP formation was measured an interfering phosphatase splitting of ATP had to be corrected for. Therefore, ADP was preferred as the substrate and its conversion to ATP was determined in a coupled hexokinase-glucose-6-phosphate dehydrogenase reaction yielding stoichiometric amounts of NADPH which were measured by the native fluorescence of this form of the nucleotide. The sensitivity and reproducibility of our micro-method permitted assay of small samples (50-500 ng) such as a layer of cerebellar cortical nerve cells and of insulin producing cells from the islets of Langerhans. Although not reaching the high values in muscle, these cells showed significantly higher activities than parenchymatous cells from the liver and the exocrine pancreas. The sensitivity attained is more than required for assay of clinical fine needle biopsies and is quite satisfactory for detection and estimation of adenylate kinase contaminants in enzyme preparations.

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)

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.

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+.

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.