N. Grossowicz

Prevalence and causes of anemia in elderly hospitalized patients.

The prevalence and causes of anemia have been studied in 104 patients over 60 years of age admitted to a general medical ward in Jerusalem. In males and females, mean hemoglobin levels were about 1 g less than in the corresponding groups of healthy younger controls. A primary nutritional anemia could not be implicated in any of the 15 patients with hemoglobins below 11 g/dl. The most important causes of anemia were chronic renal failure, metastatic carcinoma, gastrointestinal bleeding, and infection. Conversely, in diseases with no adverse effect on erythropoiesis such as chronic ischemic heart disease, hypertension and diabetes, hemoglobin levels were equal to those of the younger controls. These findings indicate that although diminished serum iron and RBC folate levels may occasionally be found in elderly subjects, nutritional deficiency is seldom responsible for anemia in this age group in Israel- and anemia when present is often the manifestation of a chronic underlying disease.

Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.

Isotopic determination of vitamin B12 binding capacity and concentration.

Summary A method for measuring additional binding capacity (ABC) of Vit. B12 in serum with the aid of radio-B12 is described. Fixed amounts of untreated serum (fresh or frozen) and of Co60B12 are incubated for 30-60 minutes at 37°C and the excess of unbound vitamin adsorbed on charcoal; the radioactivity of the charcoal is counted directly in a well-type scintillator. ABC values in various physiological and pathological conditions are given. The ABC assay was also applied to measure Vit. B12 concentrations in biological material, by first releasing the vitamin from the carrier protein and incubating it together with a “standard serum” (of known ABC) with Co60B12. Vitamin content is determined by the increase in radioactivity in the charcoal over that found in the control containing “standard serum” only. Both methods are simple, rapid, and particularly suitable for serial determinations. The results obtained with these assays showed good agreement with microbiological and other radiometric methods. Various aspects of the assays are discussed.

A Microbiological Approach to Nutritional Evaluation of Proteins

Summary 1. A microbiological method to assess the biological value of proteins is described. It implies the digestion of proteins with pancreatin and subsequent testing of the growth-promoting activity of the hydrolysate for S. faecalis, in a medium devoid of the 10 essential amino acids. The nutritive value of the protein digest is determined in a single assay by the amino acid liberated in a limiting concentration. 2. The results obtained with casein, egg albumin, gelatin, gluten, and zein are presented and their agreement with the biological values obtained by the rat growth method is suggested. 3. The growth promoting activity (biological valued) of casein and egg albumin is high and about equal; gelatin and gluten, in comparison, are much less active, 29 and 15%, respectively, and zein still less (2%). 4. At the limited growth level, casein is deficient in: lysine, arginine, isoleucine, and threonine. Egg albumin under comparable conditions is limited in lysine, arginine, histidine, threonine, isoleucine, leucine, and valine. Gelatin is low in tryptophane, isoleucine, leucine, methionine, and threonine. Gluten and zein are short in lysine. 5. The suitability of the method described for studying imbalances of amino acids is proposed.

Fractionation of Serum Transcobalamins on Charged Cellulose Filters

Summary A simple and rapid fractionation procedure of the three transcobalamins, TCI, TCII, and TCIII, of human serum was achieved by filtration through a stack of charged cellulose filters composed of one cellulose-nitrate and three DEAE-cellulose (DE-81) disks. A reaction mixture containing microliter amounts of serum was incubated with excess of 57Co B12 of high specific activity, diluted with 0.1 M sodium borate buffer (pH 8.5), and passed through the filter stack by applying vacuum. Under these conditions TCII is selectively and quantitatively adsorbed to the cellulose-nitrate filter while both TCI and TCIII adsorb to the DE-81 filters. In the second step TCIII is selectively desorbed from the latter filters by a 0.05 M monopotassium phosphate solution of pH 4.6. Using sera of different distribution of transcobalamins the data obtained were comparable to those determined by the more laborious methods employing DE-52 column chromatography combined with procedures to remove TCII. This work was supported in part by Grant 015.0187 from the Joint Research Fund of The Hebrew University and Hadassah, and by a grant from The Ministry of Health of the Government of Israel.

Gamma-glutamyl transfer reactions in bacteria.

SUMMARY: γ-Glutamyl transfer activity was found to be widely distributed in different bacterial species. The γ-glutamyl transfer from glutathione to water and acceptors other than water was studied with cell-free preparations of Proteus morganii. In the absence of added acceptor, the γ-glutamyl residue was predominantly transferred to water; however, some transfer to the substrate, resulting in the formation of γ-glutamylglutathione, was detected. In the presence of acceptors (amino acids or peptides) all the γ-glutamyl residue was transferred to the added acceptor. The different reactionproducts were isolated and identified. Kinetics and properties of the γ-glutamyl transfer reaction were studied.

Nystatin effects on cellular calcium in Saccharomyces cerevisiae

The primary effects of nystatin, a polyene antibiotic, on the yeast Saccharomyces cerevisiae were investigated. Though K+ leakage was observed shortly after the addition of nystatin, Ca2+ leakage was delayed 2-3 h after its application and it occurred only at an acidic pH and in the absence of K+, Na+ or Mg2+ from the medium. However, within 4 min after application nystatin induced a passive influx of Ca2+ into the cells even at a concentration of 1 microM in the medium. These results led to the conclusion that the primary membranal lesion induced by nystatin is not restricted to monovalent cations but is also manifested by increased permeability to Ca2+. The delayed leakage of Ca2+ is explained by the assumption that the bulk of cellular calcium is sequestered so that the concentration of free Ca2+ in the cytoplasm is very low. The sequestered calcium may be liberated 2-3 h after the addition of nystatin as a consequence of secondary damage to the cells such as intracellular acidification and loss of cations.

Determination of Vitamin B12 in Human Serum by a Mutant of Escherichia coli

Summary The vit. B12 assay using a mutant strain of E. coli was modified by addition of methionine-free casein hydrolysate to a mineral medium. This method gave better growth and allowed a more accurate estimation of the vit. B12 than previous methods using the same organism. The assay required only 40-48 hr as compared with 7-9 days when E. gracilis is used; the set-up, too, is very simple. The test proved satisfactory for the determination of vit. B12 in human serum. The vit. B12 content of sera of healthy subjects determined by this method ranged from 200 to 1000 μμg/ml, while sera of patients suffering from pernicious anemia contained only 50-130 μμg/ml.

The Absorption of Milk-Bound Pteroylglutamic Acid from Small Intestine Segments

Summary Binding tritiated pteroylglutam-ic acid to cow or to goat milk protein was associated with a substantial reduction in its absorption from rat jejunum, while the ileal absorption increased markedly. There was no difference in the amount of folate absorbed whether it was bound to cow milk or to goat milk. Despite the fourfold higher folate binding capacity of goat milk than of cow milk, there was no difference in the amount of folate absorbed after their administration. Folate depletion or folate overload had no effect on the amount of the milk-bound folate absorbed, while such treatments decreased the absorption of free folate.

Regulatory properties of an inducible aliphatic amidase in a thermophilic bacillus.

A thermophilic bacillus growing on acetamide as both carbon and nitrogen sources produces an inducible amidase. This amidase hydrolysed the following amides in decreasing order or activity, in comparison with acetamide (1.00): propionamide (0.97), fluoroacetamide (0.84), formamide (0.35) and glycinamide (0.12). Cyanoacetamide, dimethylacetamide, dimethylformamide and urea also induced the synthesis of the amidase, but were not substrates of the enzyme. Studies with protoplasts suggest that the amidase is located in the cytoplasm. Glucose strongly inhibited amidase synthesis; and limiting nitrogen did not release this inhibition. Urea strongly inhibited amidase activity in a competitive manner; but the inhibition caused by iodoacetamide and cyanoacetamide was non-competitive. Both thioacetamide and thiourea were effective inhibitors of enzyme induction. Bacteria grown on a succinate-minimal medium exhibited a lag in amidase synthesis, which could be eliminated by decreasing the concentration of succinate. Acetate- or pyruvate-grown cultures behaved similarly, while those grown on alanine or glutamate exhibited no lag in enzyme induction. In the mutant strain E21, repression of amidase synthesis by glucose was much less evident and no lag for induction was apparent with any of the other carbon sources mentioned.