Helen B. Burch

Enzymatic assay for glutathione

Abstract Methods for measuring total glutathione are described. These are based on the ability of glutathione and glutathione reductase (EC 1.6.4.2.) to catalyze the oxidation of NADPH by Ellmans reagent. Except for highest glutathione levels, NADP + rather than the reduced Ellman compound is measured. For intermediate sensitivity (2 × 10 −12 mol) NADP + is measured stoichiometrically by conversion to NADPH and determination of fluorescence. For smaller amounts (10 −14 mol) the NADP + generated is amplified by enzymatic cycling. These procedures have been tested extensively on kidney and are probably applicable to tissues in general.

Metabolic intermediates in liver of rats given large amounts of fructose or dihydroxyacetone.

Abstract The livers of rats given large injections of fructose or dihydroxyacetone were analyzed for glucose, glycogen, and 14 related metabolites. Dihydroxyacetone produced a large increase in glycogen and a modest increase in glucose and glucose-6-P. Fructose produced no increase in glycogen but a large increase in glucose and glucose-6-P. Changes in intermediates suggest in both cases diversion of part of the injected material into the Embden-Meyerhof pathway. Fructose caused a profound drop in ATP, UTP and UDPG. The fall in ATP is attributed to sequestration of phosphate into fructose-1-P, which increased to 18 mmole/kg. The decrease in ATP is believed to lead in turn to the fall in UTP and UDPG and the failure to deposit glycogen.

The location of glutamine synthetase within the rat and rabbit nephron

Abstract Glutamine synthetase (EC 6.3.1.2) was measured in seven different parts of the nephron in the rat and five in the rabbit, dissected from freeze-dried microtome slices. In the rat the enzyme is essentially confined to the proximal straight tubule. Acidosis did not change the activity; methionine sulfoximine abolished it. In the rabbit the enzyme is high in both proximal convoluted and straight tubules. Assays were made with a new method which measures glutamine formation per se . One of the products, NADH, is amplified by enzymatic cycling to provide sufficient sensitivity (2–10 pmol of glutamine).

Biochemical Changes in Liver Associated with Kwashiorkor

Kwashiorkor, the most prevalent form of severe protein malnutrition, is a serious disease, often fatal to young children and especially common from weaning to five years of age. There are insufficient data concerning the chemical changes which take place during development of the disease, although clinical information is extensive. Present knowledge about various aspects of the disease has been reviewed by Trowell, Davies, and Dean (1), by Brock (2, 3) and more recently by Scrimshaw and co-workers (4). Its biochemical characteristics include low levels of serum protein, amylase, alkaline phosphatase, pseudo-cholinesterase, cholesterol, riboflavin and vitamin A. Concomitantly the livers are usually found to be high in fat and low in protein. Waterlow (5, 6) did pioneering work on enzymes in liver biopsy specimens from malnourished children and reported that hepatic lactic dehydrogenase and cytochrome oxidase of four children in Gambia remained virtually unchanged after treatment whereas pseudo-cholinesterase was initially low but more than doubled on treatment. Later, Waterlow and Patrick (7, 8) studied levels of eight enzymes in liver biopsy samples from a large number of malnourished children in I This investigation was supported in part by the National Science Foundation, The Williams-Waterman Fund for the Combat of Dietary Diseases, the Nutrition Foundation, Inc., and National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health (A-981), Public Health Service. Reported in part at the annual meeting of the American Society of Biological Chemists, April, 1957. INCAP Scientific Publication I-86. 2Pan American Sanitary Bureau Consultant to INCAP from July 28 to September 9, 1956. S Staff members of the Institute of Nutrition of Central America and Panama (INCAP). 4Fellow of the World Health Organization of the United Nations. Jamaica on admission to the hospital and after treatment. For cytochrome oxidase, lactic, malic and glutamic dehydrogenases, succinoxidase, DPNH-cytochrome C reductase and transaminase they report unchanged activity following treatment. The only enzyme found to be reduced in activity in the disease was non-specific cholinesterase, which increased on treatment. Extensive dietary studies in various parts of the world where children suffer from kwashiorkor reveal inadequate intake of protein as well as low dietary levels of other essential nutrients (1, 3). The protein is often of poor quality and therefore unfavorable for the synthesis of tissue protein. If dietary situations exist such that protein and other nutrients necessary for the synthesis of hepatic enzymes are inadequate, the levels of some enzymes in the liver should reflect the lack. Certain hepatic flavin enzymes, particularly xanthine oxidase (9, 10), D-amino acid oxidase (11, 12), and glycolic acid oxidase (12), are greatly decreased either by riboflavin, protein or caloric deficiency in rats. Xanthine oxidase is also lowered by the lack of a single essential amino acid in the diet (13, 14). The levels of these flavoproteins and of riboflavin might conceivably be related to some of the changes in metabolism which occur in the livers of children with diseases of malnutrition, particularly where dietary protein and riboflavin are low. This report gives results of biochemical measurements on liver biopsy samples from Guatemalan children with kwashiorkor. Analyses were made for xanthine, D-amino, acid, and glycolic acid oxidases, DPNH-dehydrogenase, malic dehydrogenase, transaminase, riboflavin, total oxidized pyridine nucleotides, cholesterol, lipid and protein in the liver as well as the protein, cholinesterase and amylase in serum and riboflavin in red blood

Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.

OXIDIZED AND REDUCED PYRIDINE NUCLEOTIDE LEVELS AND ENZYME ACTIVITIES IN BRAIN AND LIVER OF NIACIN DEFICIENT RATS

ADVANTAGE has been taken of the recent development of highly sensitive methods (LOWRY, PASSONEAU, SCHULZ and ROCK, 1961) to study the response of the individual pyridine nucleotides (NADf, NADH, NADP+, NADPHg) in the brain andliver of rats to a low tryptophan, niacin-free diet. The study of three pyridine nucleotide-dependent enzymes was included, prompted by the findings of BURCH et al. (1956,1960) that some flavin-dependent enzymes decrease in riboflavin deficiency. SINGAL, SYDENSTRICKER and LITTLEJOHN (1 948) measured the nicotinic acid levels in different tissues of rats on corn rations and found them to be subnormal in the brain, liver, and muscle of the deficient animals but normal in other tissues. BURCH et al. (1955) reported that rats fed low amounts of tryptophan and niacin had subnormal amounts of oxidized pyridine nucleotides in blood cells and liver. More recently, SPIRTES and ALPER (1 961) studied the diphosphopyridine nucleotide levels in the livers of niacin-deficient and protein-deficient mice. They found a decrease of both NAD+ and NADH in the liver of the niacin-deficient animals, with no change in the NAD+/NADH ratio.

METHODS FOR DETECTING AND EVALUATING ASCORBIC ACID DEFICIENCY IN MAN AND ANIMALS

The assessment of nutritional status with respect to ascorbic acid is more satisfactory than for any of the other nutrients. This is true despite the fact that the cellular biochemical action of ascorbic acid is still poorly understood. The first evaluations of nutritional status were based on clinical findings and the response of patients to dietary therapy. This method is still quite satisfactory for the determination of scurvy, the gross deficiency disease. However, the nutritional spectrum for ascorbic acid, or for any single nutrient, consists of a graded series of possible states with frank deficiency a t one end, optimal nutrition beginning at some region in the middle and, possibly, overnutrition at the far end. The transition from optimal to suboptimal nutrition is gradual with few signposts. The difference between optimal nutrition and less than optimal is of practical concern here. Biochemistry has been of decisive help in the measurement of the nutritional spectrum for ascorbic acid in both animals and human beings. Fortunately, a wealth of biochemical possibilities for the function and the mechanism of action of ascorbic acid has been explored. The extensive studies on the guinea pig have provided much information concerning ascorbic acid metabolism and tissue levels. The results of these studies parallel in many ways the less extensive data on human beings. Experimental results that define possible means for measuring tissue levels as an indication of the nutritional status for ascorbic acid follow.

Substrates of carbohydrate metabolism and their relation to enzyme levels in liver from rats of various ages

Abstract Biochemical differentiation in rat liver is being studied by measurement of enzyme activities in whole homogenates; by studies of the steady-state levels of substrates and products of these enzymes in quick frozen tissue; and by observing the changes in substrates in liver when it is cut off from its blood supply. Changes observed during development in the levels of various enzymes concerned with carbohydrate metabolism indicate a decrease in capacity for glucose utilization and glycolysis and an increase in capacity for glycogenolysis, glucose formation, and lactate utilization.

The distribution of six enzymes of oxidative metabolism along the rat nephron.

Using quantitative methods, citrate synthase (CS), fumarase, beta-hydroxyacyl-coenzyme A (CoA) dehydrogenase (beta OAC), 3-keto-acid CoA transferase (KCT), malic dehydrogenase (MDH), and malic enzyme were measured in seven defined parts of the nephron and in thin limb and papilla areas dissected from freeze-dried microtome sections of rat kidney. The results not only show a wide range of activity along the nephron for each of the enzymes, but that the proportions between the enzymes vary markedly among the different parts of the nephron. This suggests the existence of major regional differences in the capacity to oxidize specific metabolites. The ratio between two citrate cycle enzymes, fumarase and CS, was 4- or 5-fold higher in proximal segments than in the glomerulus or thin limb areas. The ratio between beta OAC (an enzyme of fatty acid oxidation) and CS was 3- to 5-fold higher in the middle proximal segments than in glomeruli or thin limb and papilla areas. The key enzyme for ketone body metabolism, KCT, was essentially confined to the thick tubule segments. Malic enzyme, in contrast to the other five enzymes, was highest in the proximal straight segments. New methods, sufficiently sensitive for this histochemical study, are described for malic enzyme and 3-keto-acid CoA transferase.

Effects of Prolonged High Dosage with Ascorbic Acid.

Results and Conclusion The data for the 4 subjects were so concordant, they have been averaged together and treated as replicates. There is clearly no progressive change in either the serum or white cell level, tolerance curve, or urinary excretion (Table I). The averages obscure the fact that the individual tolerance curves differed a little in shape. However, the individual character of the curves remained essentially unchanged throughout. There seems, therefore, to be no reason to believe that the prolonged high dosage of ascorbic acid had any qualitative or quantitative effect on the manner of disposal of excess acid by the body. Furthermore, in these fourpersons, at least, no harmful effects whatever were observed during the 3 months with 1000 mg daily intake.