Sven E. Brolin

Photokinetic micro assay based on dehydrogenase reactions and bacterial luciferase

Abstract Methods have been developed for photokinetic assay of metabolic intermediates at and below the picomole level. Our results indicate that it should be possible to measure all substances which can be converted in or coupled to a pyridine nucleotide dependent dehydrogenase reaction. Reduced pyridine nucleotides serve as determinable products by giving rise to a flash of light when injected into a solution of bacterial luciferase. Estimation of the oxidized forms requires a preceding reduction. In the photokinetic evaluations, the time profile of the flash was determined by employing a high-sensitivity photomultiplier with associated electronics and displaying the signal on an oscilloscope screen. The analyses included various reduced nucleotides, NAD, glucose and malate. The applicability was further tested in an assay of the NAD content of pancreatic islets and acini.

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.

Histochemical observations on the pancreatic islets in normal and obese-hyperglycemic mice

SummaryIn the islets of Langerhans in mice with the American variety of the obese-hyperglycemic syndrome (AO) and their thin litter mates (AN) the following observations were made:1.In cryostat sections of fresh pancreatic tissue from AN-mice, both more centrally situated islet cells with yellowish white granules, and peripherally situated cells with brighter silver white granules were found with dark field microscopy. While the central cells were considerably degranulated in the AO-mice, the peripheral cells were also well filled with granules in these animals. The technique described offers, especially in the AO-mice, better possibilities of differentiation of the islet cells than with the granule staining methods of fixed pancreatic material, which are now generally used.2.After intravenous injection of dithizone, abundant orange yellow zinc dithizonate granules were found in all islet cells with dark field microscopy of cryostat sections from fresh pancreatic tissue of AN-mice. By dissolving out these granules, the zinc content could be related to the different types of islet cells. It was possible in this way to show that the zinc in the islets of the AO-mice was mainly situated in the peripheral cells, containing the silver white granulation typical of untreated animals in dark field. The occurrence of a lower zinc content in the centrally situated islet cells in the AO-mice was also evident from the so-called “sulphide-silver method”.3.A positive islet reaction to both acid and alkaline phosphatases was obtained in the AN-mice. The alkaline phosphatase activity was high in the peripheral parts of the islets of AN-mice, but much lower in the AO-mice. While the majority of islet capillaries were strongly positive as regards alkaline phosphatases in the AN-mice, in the AO-mice they were for the most part completely negative.4.In spite of the existence of a prolonged hyperglycemia, no storage of glycogen could be demonstrated in the islets of the AO-mice. With fluorescence microscopy, the occurrence of masked lipids in the islets was observed in the AN-mice, and to an even higher degree in the AO-mice. On the other hand the sudanophilic reaction was negative throughout. The raised functional activity in the islet B-cells of the AO-mice was reflected in an enlargement and fragmentation of the Golgi apparatus.

Substrate analyses in single cells: I. Determination of ATP

Subfemtomole quantities of ATP were determined using luminescence analysis with firefly extracts. The methodological advances include development of microtechniques for sample handling, and recording of the time profile of the light emission by photon counting in connection with multichannel techniques. The amount of ATP in isolated single cells of the microorganisms Paramecium, Peridinium, and of macrophages from the abdominal cavity of mice were assayed. These cells represented a range from 0.07 to 2.5 ng dry mass as assessed with automatic interferometric scanning.

Effects of glucose on the content of ATP and glycogen and the rate of glucose phosphorylation of isolated pancreatic islets maintained in tissue culture

Summary Isolated mouse pancreatic islets cultured for 7 days in various glucose concentrations had higher ATP concentrations than freshly collagenase isolated ones. A pronounced increase of the glycogen content was observed in islets cultured in a high glucose medium. Such islets also revealed an enhanced rate of glucose phosphorylation, when assayed at a high glucose concentration, indicating an increased high Km hexokinase activity. The low Km hexokinase was unaffected by the glucose concentration of the culture medium. These findings further support the idea that the glucose metabolism of the pancreatic B-cell may show adaptive changes to a prolonged period of high extracellular glucose concentration.

Simplified luciferase assay of NAD+ applied to microsamples from liver, kidney and pancreatic islets

Abstract A new single-step procedure for the bioluminescence assay of NAD + , permitting measurements on the pmol level (10 −12 mol) is described. Acid extracts of NAD + were prepared in different tissues. The acidification destroys reduced pyridine nucleotides and most enzymes which are present in the tissue sample. After neutralization the extract is added to a light-yielding solution, and the luminescence is measured with a photomultiplier. The maximal height of the signal is measured by means of a digital voltmeter. The light yielder is bacterial luciferase with appropriate additives and supplemented with malate and malate dehydrogenase. The modified light-yielding solution provides for continuous formation of NADH resulting in a durable level of light emission. The cycle involved was shown not to operate with NADP + . The slow fading of the emission permits simplification of the measuring procedure. Rapid injection in front of the phototube can thus be omitted and replaced by ordinary mixing before the reaction cell is positioned for the measurement. Furthermore, the instrumentation required is less elaborate than in photokinetic assay, since it is not necessary to record and integrate the time course of the emission. To test the applicability of the method, analyses of pmol amounts were performed in the islets of Langerhans and in tissue samples of much smaller size than fine needle biopsies.

Photokinetic assay of NADH and NADPH in microdissected tissue samples

Abstract A high sensitivity is reached in photokinetic assay of reduced pyridine nucleotides with extracts of Achromobacter fischerii . When occurring together, NADH and NADPH are difficult to measure individually, since both initiate light emission. This problem has been solved by selective enzymic oxidation of either nucleotide before the measurement of the other. Assay of the reduced nucleotides in extracts of microdissected samples from pancreatic islets, exocrine pancreas, and liver demonstrates the applicability of the methods.

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

SummaryTo 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-hydroxubutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the fentomole 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. The single step performance is suitable for microassay of enzymes as in fine needle or punch biopsies, in search of contaminants for control of purification, for studies of small cell populations separated by cell sorters and whenever else a low level of detectability is essential to attain.

Attempts to simplify the analytical performance in micro assay of metabolites with bacterial luciferase

Abstract The formation of NADH in dehydrogenase reactions was utilized for simplified bioluminescence analyses of metabolites. The enzymatic reduction of NAD + was carried out in a reaction cell, containing bacterial luciferase and appropriate additives for light formation. In this way the emission faded very slowly which made measurements much easier than in the case of short light flashes. Pico-mole amounts of malate. L -glycerol-3-phosphate and 3-hydroxybutyrate were analysed in each case with the aid of the enzyme required for the reduction of NAD + . Thus a much simplified assay was found to be possible by the utilization of coupled reactions in a common vessel, thereby decreasing the demands on the equipment for fast mixing and measuring.

Photokinetic assay of pyruvate in the islets of Langerhans using bacterial luciferase.

A method is described determining the pyruvate content in 1–10 μg of lyophilized tissue. This represents a range of weight much below that of fine-needle biopsies. NAD+, generated through the enzymatic conversion of pyruvate to lactate, is reduced to NADH which is then assayed by its ability to induce light emission with luciferase extracts of Achromobacter fischerii. The sensitivity of the method is sufficient to determine 0.5 pmol of pyruvate. Values obtained by this method in lyophilized liver agree well with those obtained by spectrophotometric techniques. In applications of the method, it was found that the islet concentration of pyruvate was greater than in the exocrine pancreas but smaller than in the liver. The pyruvate content in tissues of the obese hyperglycemic mice was greater than in their lean litter mates, which may be explained by a more rapid rate of glucose utilization due to their hyperglycemia.