Orit Epstein

The heme biosynthetic pathway in lymphocytes of patients with malignant lymphoproliferative disorders

The metabolism of heme is impaired in lymphocytes of patients with malignant lymphoproliferative disorders (MLPO). Two of the enzymes of the heme biosynthetic pathway, delta-aminolevulinic acid dehydrase (ALAD) (EC 4.2.1.24) and ferrochelatase (FC) (EC 4.99.1.1) are markedly reduced. The activity of porphobilinogen deaminase (PBGD) (EC 4.3.1.8) is increased. The rate-limiting enzyme of heme biosynthesis in the liver, aminolevulinate synthase (ALAS) (EC 2.3.1.37) remains unchanged although the concentration of total heme in the lymphocytes is markedly reduced. This might reflect a lack of negative feedback inhibition by heme on ALAS activity in this system.

Erythrocyte uroporphyrinogen synthase activity as a possible diagnostic aid in the diagnosis of lymphoproliferative diseases

Patients with active lymphoproliferative diseases were shown to have high activity of erythrocyte uroporphyrinogen synthetase (URO‐S), the enzyme which converts porphobilinogen to uroporphyrinogen. In a few patients examined the lymphocyte URO‐S was markedly increased. No correlation was found between the high URO‐S activity and the degree of anemia, reticulocytosis, or the presence of hemolysis. Patients with epithelial malignancies and with some common viral diseases had normal erythrocyte URO‐S values. Three patients with nonalcoholic cirrhosis also had high erythrocyte URO‐S activities. The determination of erythrocyte and lymphocyte URO‐S activity may be of aid in the diagnosis of lymphoproliferative diseases. It may also indicate whether remission has been achieved and whether treatment should be continued or reinstituted. These preliminary observations justify the investigation of a larger patient and control material.

The effects of succinylacetone (4,6-dioxoheptanoic acid) on δ-aminolevulinate synthase activity and the content of heme in monolayers of chick embryo liver cells

Succinylacetone was shown to inhibit aminolevulinate dehydratase (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing), EC 4.2.1.24) to reduce cellular heme and porphyrins and to induce delta-aminolevulinate synthase (succinyl-CoA:glycine C-succinyltransferase (decarboxylating), EC 2.3.1.37) in monolayers of chick embryo liver cells. Marked synergistic effects on delta-aminolevulinate synthase activity were obtained by combining succinylacetone with levulinate and porphyrogenic drugs. The time course of delta-aminolevulinate synthase activity showed a delayed synergistic response.

The effects of metalloporphyrins, porphyrins and metals on the activity of delta-aminolevulinic acid synthase in monolayers of chick embryo liver cells

The effect of various metals, porphyrins and metalloporphyrins on the activity of delta-aminolevulinate synthase (ALAS) was measured in monolayers of chick embryo liver cells in order to determine whether the metal moiety of heme or heme itself is the regulator of ALAS activity. Iron, magnesium, zinc, copper, manganese and nickel did not decrease ALAS activity in non-induced and in cells induced by allyl-isopropylacetamide (AIA). Cobalt decreased both non-induced and induced activity. Porphyrins inhibited ALAS, apparently only after having been converted into metalloporphyrins. Almost all the metalloporphyrins examined decreased ALAS activity. None of the substances, at the concentrations used, was toxic to the cells. These observations indicate that probably heme and not iron is the regulator of ALAS activity in monolayers of chick embryo liver cells.

Inhibitory effect of membrane active compounds on induction of tyrosine aminotransferase in chick embryo liver cells in culture

In the course of investigations on the effect of beta-adrenergic receptor blocking agents in experimental porphyria we observed an inhibitory action of DL-propranolol and other membrane active compounds on the induced activity of delta-aminolevulinate synthetase Q-ALAS) (EC 3.2.1.37) both in vivo and in vitro [l-3] . Beta-adrenergic receptor blocking agents compete with catecholamines for activation of beta-adrenergic receptors. Some of these agents also possess non-specific membrane effects such as changes in depolarisation and/or repolarisation of the cellular membrane, local anesthetic and anti-arhythmic properties, etc. Other drugs, which have no betareceptor blocking effects, including quinidine and lydocain, have non-specific membrane activity and are regarded as membrane active compounds. The various membrane effects of these drugs and of betareceptor blockers are, at least partially, dissimilar. In order to determine whethertheinhibitory effect of DL-propanolol and other membrane active compounds is specific for induction of &-ALAS, we examined their effect on induction of another inducible enzyme, tyrosine aminotransferase (TAT) (EC 2.6.1 S). TAT is a soluble enzyme, the activity of which is increased by administration of steroid hormones with glucocorticoid activity, both in rat liver [4] and in hepatoma cells in culture [5,6]. Dexamethasone is known to be a potent inducer in cultured hepatoma cells. It raises TAT activity 4-8 fold in this system [7,8]. Our experiments were carried out in cultures of chick embryo liver cells.

Growth rate determines activity of porphobilinogen deaminase both in nonmalignant and malignant cell lines

PBGD activity and growth rate were determined in cultures of rat embryo fibroblasts, nontransformed and MLV/MS transformed fibroblastic cell lines; NIH-3T3 cells, and in a mouse lymphosarcoma cell line [L-929]. The two parameters examined correlate positively (P less than 0.001). The results of this investigation would seem to indicate clearly that porphobilinogen deaminase activity is related to growth. However, these experiments do not rule out the possibility that malignant transformation per se also causes changes in porphobilinogen deaminase activity.

A method for obtaining high recovery of purified subcellular fractions of rat liver homogenate

Abstract A method for obtaining highly purified subcellular fractions of rat liver is described. The recovery of each fraction approaches 100%. The method is based on differential centrifugation and the use of appropriate concentrations of Triton X-100 and MgCl 2 at certain specific steps in the fractionation procedure.

Inhibitory effect of membrane active compounds on the uptake of 14C-∝-aminoisobutyric acid (AIB) in cultured chick embryo liver cells

Abstract High concentrations of beta-adrenoceptor blocking drugs with membrane active properties and of the membrane active compounds quinidine and lidocaine inhibit the uptake of ∝-aminoisobutyric acid by chick embryo liver cells in culture. Beta-adrenoceptor blockers without membrane active properties were without effect. These results are in accordance with previous findings which showed partial inhibition of incorporation of amino acids into proteins caused by membrane active drugs in this system.

Inhibitory effect of some membrane active drugs on RNA and DNA synthesis in cultured chick embryo liver cells

DL-Propranolol and oxprenolol, both beta-adrenergic receptor blocking agents with membrane active properties, were found to inhibit the incorporation of uridine and thymidine into RNA and DNA respectively in cultures of chick embryo liver cells. The membrane active compounds quinidine and lidocaine had similar effects. D-Propranolol, which has a very slight beta-blocking action only but has a membrane activity similar to DL-propranolol, showed inhibition like that of DL-propranolol. Pindolol and practolol, beta-adrenergic blocking agents nearly devoid of membrane activity, had no effect on incorporation. The inhibition is reversible and non-competitive. It seems to be related to the membrane active properties of the drugs examined.

The effect of beta-adrenergic blocking agents on experimental porphyria induced by 3,5-diethoxycarbonyl-1,4-dihydrocollindine (DDC) in vivo and in vitro

Abstract The effect of DL-propranolol on 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced experimental porphyria was studied. dl -Propranolol, a beta-adrenergic blocking agent with non-specific membrane effects, partially inhibited 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine-induced delta-aminolevulinate synthetase activity both in rats and in chick embryo liver cells in culture. In rats, DL-propranolol decreased urinary delta-aminolevulinate and porphobilinogen but no change occurred in the 24-h urinary excretion of total porphyrins and in the concentration of porphyrins in the liver. In cultured chick embryo liver cells treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine, DL-propranolol decreased accumulation of porphyrins in the medium. d -Propranolol, oxprenolol and quinidine acted like dl -propranolol in chick embryo liver cells in culture treated with 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine. Pindolol, practolol and lidocaine had no effect. Phenobarbitone had a synergistic effect on the induction of delta-aminolevulinate synthetase by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine in cultures of chick embryo liver cells. This induction was partially inhibited by propranolol. However, the increased accumulation of porphyrins in the medium caused by 3′,5′-diethoxycarbonyl-1,4-dihydrocollidine was inhibited by the addition of phenobarbitone. This inhibited induction was further decreased by propranolol. Most of our results indicate that the drugs tested act mainly by their effects on membranes.