Publisher Summary This chapter discusses preparation of isolated rat liver cells The early mechanical and chemical methods for liver-cell preparation were relatively successful in converting liver tissue to a suspension of isolated cells The successful preparation of intact liver cells by perfusion with collagenase is technically quite difficult The major method for preparation of nonparenchymal liver cells is based on the selective sensitivity of parenchymal cells toward proteases By incubation of rat liver minces with pronase, most of the liver parenchyma is digested, while nonparenchymal cells remain intact and can be recovered from the incubate Similar results have been reported with trypsin digestion of collagenase-dispersed liver minces, but pronase appears to be more effective The most common procedure is to perfuse the liver briefly with pronase before it is minced and incubated with the enzyme Such direct pronase methods have been used by several investigators with yields of nonparenchymal liver cells reported to be in the range 2–15 × 10 6 cells/gm liver

Preparation of isolated rat liver cells.

I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

Increasing evidence in both experimental and clinical studies suggests that oxidative stress plays a major role in the pathogenesis of both types of diabetes mellitus. Free radicals are formed disproportionately in diabetes by glucose oxidation, nonenzymatic glycation of proteins, and the subsequent oxidative degradation of glycated proteins. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defense mechanisms can lead to damage of cellular organelles and enzymes, increased lipid peroxidation, and development of insulin resistance. These consequences of oxidative stress can promote the development of complications of diabetes mellitus. Changes in oxidative stress biomarkers, including superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione levels, vitamins, lipid peroxidation, nitrite concentration, nonenzymatic glycosylated proteins, and hyperglycemia in diabetes, and their consequences, are discussed in this review. In vivo studies of the effects of various conventional and alternative drugs on these biomarkers are surveyed. There is a need to continue to explore the relationship between free radicals, diabetes, and its complications, and to elucidate the mechanisms by which increased oxidative stress accelerates the development of diabetic complications, in an effort to expand treatment options.

Diabetes, oxidative stress, and antioxidants: a review

Publisher Summary
This chapter discusses glucose-6-phosphate dehydrogenase (G6P-DH), which was first isolated from erythrocytes and from fermenting yeast by Warburg et al., who carried out an extensive purification and characterization of the enzyme. Blood cells, adipose tissue, and lactating mammary gland are especially rich sources of the enzyme. Some human and animal tumors contain high activity of the enzyme. G6P-DH is applied in biochemistry and clinical chemistry. Triethanolamine buffer (50 mM, pH 7.5) containing 5 mM EDTA has proved best. Measurements are made on tissue samples with 0.67 mM G-6-P and 0.5 mM NADP, which are optimum concentrations for the enzyme from erythrocytes. G6P-DH is inhibited by primaquine and other 8-aminoquinolines (antimalarial drugs) in millimolar concentration, as well as by phenylhydrazine. Nevertheless, the therapeutic concentration of these substances is more than tenfold lower and therefore, they have no significant effect on the measurements.

Glucose-6-phosphate Dehydrogenase

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.

Hyperglycemic Pseudohypoxia and Diabetic Complications

Renewed interest in the direct oxidative pathway of glucose 6-phosphate metabolism during the last few years has revealed that this pathway is by no means restricted to erythrocytes, yeast and microorganisms. The triphosphopyridine-nucleotide(TPN)-specific glucose 6-phosphate and 6-phosphogluconate dehydrogenases are also widely distributed in mammalian tissues (Dickens & Glock, 1950, 1951; Horecker & Smyrniotis, 1951), in a variety of lower plants and animals (Cohen, 1950) and also in higher plants (Conn & Vennesland, 1951; Gibbs, 1952). The significance of this pathway in animal tissues, its physiological control and the relative importance of the direct oxidative and glycolytic routes of carbohydrate metabolism are still, however, chiefly matters of conjecture. An essential preliminary step to such an investigation is to devise a satisfactory procedure for the assay of glucose 6-phosphate and 6-phosphogluconate dehydrogenases in animal tissues and it was with this object in view that the present work was undertaken.

Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

In a previous publication from this laboratory (Dickens & Glock, 1951), data ofa semi-quantitative nature were given on the distribution of enzymes of the direct oxidative pathway of carbohydrate metabolism in several mammalian tissues. Apart from this work, however, and the quantitative data of Horecker & Smyrniotis (1951) on the 6-phosphogluconate dehydrogenase activities ofsome acetonedried tissues, the distribution of the enzymes of this pathway has not been systematically investigated in animal tissues. Following the development of quantitative methods of assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (Glock & McLean, 1953) and of methods for quantitatively estimating the breakdown of ribose 5-phosphate, the enzyme activity levels in these consecutive stages of the direct oxidative pathway have been determined in a variety of normal mammalian tissues and tumours. The results are reported in the present paper.

Levels of enzymes of the direct oxidative pathway of carbohydrate metabolism in mammalian tissues and tumours.

1. The DPN+, DPNH, TPN+ and TPNH contents of a variety of animal tissues have been determined. 2. In all the tissues investigated DPN is present mainly in the oxidized form, whereas TPN is present chiefly and sometimes exclusively in the reduced form. 3. High total concentrations of both DPN and TPN are found in liver, adrenals, kidney and lactating mammary glands. The high level of TPN in ovary and the low levels in brain and voluntary muscle are also of interest.

A preliminary investigation of the hormonal control of the hexose monophosphate oxidative pathway

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Levels of oxidized and reduced diphosphopyridine nucleotide and triphosphopyridine nucleotide in animal tissues.

Small, L. D., Bailey, J. H. & Cavallito, C. J. (1947). J. Amer. chem. Soc. 69, 1710. Stoll, A. & Seebeck, E. (1948). Helv. chim. acta, 31, 189. Stoll, A. & Seebeck, E. (1949a). Helv. chim. acta, 32, 197. Stoll, A. & Seebeck, E. (1949b). Helv. chim. acta, 32, 866. Stoll, A. & Seebeck, E. (1951). Helv. chim. acta, 34, 481. Szymona, M. (1952). Acta Microbiol. Polon., 1, 5. (Quoted from Chem. Abstr. 47, 2412.) Twiss, D. F. (1914). J. chem. Soc. 105, 36. Vinokurow, S. I., Bronz, L. M. & Korsak, S. E. (1947). Bull. Biol. Med. exp. U.R.S.S. 23, 296. (Quoted from Chem. Ab8tr. 42, 6864.) Wills, E. D. (1954). Biochem. J. 57, 109. Wills, E. D. & Wormall, A. (1950). Biochem. J. 47, 158.

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Patricia McLean

Papers

Further studies on the properties and assay of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Levels of enzymes of the direct oxidative pathway of carbohydrate metabolism in mammalian tissues and tumours.

A preliminary investigation of the hormonal control of the hexose monophosphate oxidative pathway

Levels of oxidized and reduced diphosphopyridine nucleotide and triphosphopyridine nucleotide in animal tissues.

Pathways of glucose catabolism in rat liver in alloxan diabetes and hyperthyroidism