Sn-protoporphyrin suppresses chemically induced experimental hepatic porphyria. Potential clinical implications.

The ability of Sn(tin)-protoporphyrin to inhibit the induction of hepatic delta-aminolevulinate (ALA) synthase by allylisopropyl acetamide (AIA) was examined in the adult rat. Doses of Sn-protoporphyrin of 1, 10, and 50 mumol/kg body wt resulted in decreases in AIA-induced hepatic ALA-synthase activity of 32, 52, and 60%, respectively, compared with rats treated with AIA alone; inhibition of ALA-synthase was not a direct effect of Sn-protoporphyrin. This inhibition of the enzyme activity in liver was reflected in concurrent decreases in urinary excretion of ALA and porphobilinogen (PBG). The increased urinary excretion of ALA and PBG observed following AIA treatment was reduced by the lowest dose of Sn-protoporphyrin (1 mumol/kg body wt) and abolished completely by the higher doses of the metalloporphyrin (10 and 50 mumol/kg body wt). These findings in a rat model of hepatic porphyria suggest that Sn-protoporphyrin may be useful in the treatment of acute exacerbations of "inducible" hepatic porphyrias in man, especially since Sn-protoporphyrin, unlike hematin which is presently used for this purpose, is neither degraded by nor induces the activity of heme oxygenase.

Insulin binding and response in erythroblastic leukemic cells.

Mammalian erythrocytes have been shown to bind, but not to respond to, physiologic doses of insulin. Insulin binding was studied in normal rat erythrocytes and erythroblastic leukemic (EBL) cells by standard 125I-insulin competitive binding assays. EBL cells exhibited marked insulin degradation, which was time, temperature, and concentration dependent and was mediated by both cell-bound and soluble enzymes. Bacitracin or bovine serum albumin was used to inhibit such degradation in a dose-dependent fashion to allow meaningful data analysis. Insulin binding studies showed a greater than 10-fold increase of specific binding to EBL cells compared with erythrocytes. Scatchard analysis was consistent with increases predominantly in the number of receptors on EBL cells. Concordant with increased insulin binding, EBL cells demonstrated increased transport of alpha-aminolsobutyric acid and increased incorporation of uridine into ribonucleic acid in response to physiologic doses of insulin (100 microunits/ml). It can be concluded that EBL cells may serve as useful models of erythroblasts to explore the relationships between insulin binding, response, and cell maturation.

Molecular defects of uroporphyrinogen decarboxylase in a patient with mild hepatoerythropoietic porphyria.

The molecular defect of uroporphyrinogen decarboxylase (UROD) was examined in a patient with mild hepatoerythropoietic porphyria. To elucidate the UROD defect, we cloned UROD cDNAs from EBV-transformed lymphoblastoid cells of the proband using reverse transcriptase-polymerase chain reaction. Nucleotide sequence analysis of the cloned UROD cDNAs revealed two separate missense mutations, each occurring in a separate allele. One mutation was a Val134-->Gln transition, and was due to three sequential point mutations (T417G418T419-->CCA); the other mutation was a His220-->Pro transition (A677-->C). UROD phenotype studies demonstrated that the TGT-->CCA mutation was inherited from the father, and the A-->C mutation was inherited from the mother. In contrast to the null activity previously described for a mutant UROD from a patient with familial porphyria cutanea tarda, these mutant URODs had subnormal but substantial enzyme activities, when expressed in Chinese hamster ovary cells. This is the first demonstration of a mutation caused by three sequential base substitutions.

Thrombophlebitis and disturbed hemostasis following administration of intravenous hematin in normal volunteers.

PURPOSE: Acute porphyria episodes are routinely treated with hematin, but side effects, including disturbances of hemostasis and peripheral thrombophlebitis, are associated with the compound's use. Thrombophlebitis is particularly troublesome in patients who require repeated administration of hematin, and may eventually lead to the placement of central venous lines or implantation of indwelling venous access ports. We undertook this study to determine whether only patients with porphyria experienced peripheral thrombophlebitis and disturbed hemostasis following administration of hematin, or if this was a general phenomenon that could also be observed in normal volunteers. SUBJECTS AND METHODS: Hematin was administered intravenously in the doses customarily used in therapy of acute porphyria crises (4 mg/kg body weight) to nine normal male volunteers, who were screened by history, physical examination, routine blood cell counts, urinalysis, biochemical screening profile, and coagulation tests. Hemostasis tests were performed in each subject, and hematin concentrations were determined. RESULTS: Within the first 24 hours, the activated partial thromboplastin time was prolonged in all subjects (mean of 25 percent), the prothrombin time was increased in eight subjects (mean of 20 percent), and the thrombin time in five subjects rose (mean of 15 percent), whereas the concentration of circulating platelets decreased in three subjects (mean of 20 percent). In four subjects (45 percent), thrombophlebitis developed following hematin infusion. CONCLUSION: Although hematin is frequently effective in the treatment of acute porphyria crises, it is often associated with abnormalities in coagulation and these effects also occur in normal volunteers.

Heme binding to Leishmania mexicana amazonensis.

Leishmania mexicana amazonensis is a pathogenic parasite whose growth, due to a biosynthetic deficiency, is dependent on a supply of exogenous heme. Utilizing [55Fe]hemin, we have demonstrated that heme binding to non-dividing cultured promastigotes of L. m. amazonensis at 4 degrees C reaches equilibrium within 6 h, is 95% dissociable by 28 h and is elevated approximately 5-fold by decreasing the pH of the binding buffer to 5.4. Metalloporphyrins substituted either at the central metal atom or in the porphyrin ring all displaced [55Fe]hemin binding to varying extents. Scatchard analysis revealed the affinity of the interaction to be 0.03 nM-1 and the number of binding sites to be 400 per promastigote. These findings are remarkably similar to those demonstrated in murine erythroleukemia cells and are characteristic of a receptor-ligand interaction. During logarithmic growth, promastigote heme binding was increased approximately 10-fold compared to stationary phase organisms. The increase was caused by a 4-fold greater number of binding sites per promastigote with no significant change in affinity. These findings demonstrate not only that L. m. amazonensis promastigotes bind heme specifically, but that the binding may be regulated by the growth phase of the parasite.

Suppression of bilirubin production in the Crigler-Najjar type I syndrome: studies with the heme oxygenase inhibitor tin-mesoporphyrin.

The heme oxygenase inhibitor tin-mesoporphyrin was used to moderate hyperbilirubinemia in two 17-year-old boys with Crigler-Najjar type I syndrome. Both patients had histories of recent, progressive neurological deterioration and plasma bilirubin concentrations on admission to the hospital were 34.5 and 28.5 mg/dL. Throughout hospitalization lasting more than 400 days, both patients underwent 10 hours of phototherapy nightly and consumed constant weight-maintaining diets. They were treated with intermittent plasmapheresis and two periods of tin-mesoporphyrin therapy comprising, in the first study period, 40 doses of 0.5 mumol/kg body weight and in the second study period, 70 doses of 1.0 mumol/kg body weight. Plasma bilirubin concentrations were decreased in both patients to varying degrees as was the rebound hyperbilirubinemia which occurs after plasmapheresis. The prolonged treatments with the inhibitor were well-tolerated and no progression of the preexisting neurological impairments occurred during the clinical trials. The results of this study suggest that the clinical application of an effective heme oxygenase inhibitor can provide a potentially useful, pharmacological adjunct to presently available therapeutic modalities for controlling episodes of acute, severe jaundice in this but lethal disorder.

Prolonged clinical use of a heme oxygenase inhibitor: hematological evidence for an inducible but reversible iron-deficiency state.

The heme oxygenase inhibitor tin (Sn4+)-mesoporphyrin, administered to two 17-year-old Crigler-Najjar type I patients during a 400-day study to lower plasma bilirubin levels, also produced changes, beginning approximately 50 days after initiation of treatment, in hematological and iron metabolism indices consistent with the development of iron deficiency anemia. These indices were responsive to iron supplementation and reverted to normal after termination of inhibitor treatment. Tin-mesoporphyrin enhances biliary heme excretion and inhibits intestinal heme oxygenase when administered orally or parenterally; the changes in blood indices could thus reflect, in part, blockade of heme catabolism and therefore of uptake of heme-derived iron, by intestinal epithelium. This action of the inhibitor suggests that such agents may facilitate studies involving aberrant metabolism of heme-derived iron in humans and that they merit further investigation with respect to their potential value in enhancing iron disposal in certain disorders such as those related, for example, to transfusion-induced iron overload states.

Insulin binding to cultured B- and T-lymphocytes.

Insulin receptors have been demonstrated on activated but not resting T-lymphocytes. We compared insulin binding to several T- and B-cell tissue culture lines transformed by either radiation or lymphotrophic Herpes virus. Two T-cell lines derived from mouse lymphomas, one T-cell derived from the cotton top marmoset and one mouse B-cell line bound, at most, 3% of a tracer dose of [125I]insulin. In contrast, positive B-cell line (1605 L) derived from a leukemic cotton top marmoset, bound 40% of tracer [125I]insulin. The binding was of high affinity (1.9 nM-1), displayed analogue specificity characteristic of the insulin receptor and receptor number per cell was 16,600. The data show that a transformed B-lymphocyte binds substantial levels of insulin. The magnitude of such expression might be related to the fact that this B-cell line also expresses Epstein-Barr virus.

Decreased estrogen hydroxylation in male rat liver following cimetidine treatment.

Administration of cimetidine (600 mumol/kg x 5) to adult male rats resulted in 55 and 25% decreases, respectively, in estradiol 2- and 16 alpha-hydroxylation. The same treatment also decreased the activities of ethylmorphine demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase and heme oxygenase but did not inhibit the activities of 7-ethoxycoumarin de-ethylase and delta-aminolevulinic acid synthase or decrease cytochrome P-450 content. In vitro addition of cimetidine (10-300 microM) also inhibited estradiol hydroxylations, and the effect was additive in rats pretreated with cimetidine in vivo; the other enzymic activities studied were completely unaffected by in vitro addition of cimetidine. In contrast, there was no effect of cimetidine either in vivo or in vitro on any of these activities in female rats. The results point to a wide variation in the susceptibilities of different isozymes of cytochrome P-450 to inhibition by cimetidine and suggest that such differential susceptibilities are also highly dependent on the sex of the animal.

Hypothalamic mechanism for cobalt protoporphyrin-induced hypophagia and weight loss: inhibition of the feeding response to NPY.

The mechanism whereby neurally or peripherally administered cobalt-protoporphyrin (CoPP) leads to transient hypophagia and prolonged weight reduction in normal and genetically obese animals is unknown. Neuropeptide Y (NPY) is a known endogenous stimulator of feeding behavior and is elevated in the hypothalamus of food-deprived rats. Accordingly, we examined the interaction between CoPP and NPY in the central nervous system. Concentrations of NPY mRNA in the hypothalami of rats treated intracerebroventricularly with vehicle or CoPP responded to decreased food intake with comparable increases. However, intracerebroventricular infusions of NPY elicited increased intake of food in vehicle-treated rats but were without effect in CoPP-treated animals. The results suggest that CoPP acts, at least in part, by blocking the feeding response to NPY.

Suppression of 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activity in rat pituitary by cobalt protoporphyrin: implications for testosterone homeostasis.

Cobalt protoporphyrin (CoPP) administered subcutaneously to adult male rats caused a marked reduction in the conversion of 5 alpha-androstane-3 beta-17 beta-diol (3 beta-adiol) to its main triol derivative (6 alpha-atriol) by homogenates of the pituitary but not of the prostate or brain (ventromedial hypothalamus and cortex). No effect in the brain was observed when this heme analogue was infused intracerebroventricularly. 3 beta-adiol hydroxylase, the enzyme responsible for the reaction and whose main function is thought to be the elimination of dihydrotestosterone and its metabolites from target tissues, was also inhibited by CoPP and SKF-525A added in vitro. The reaction was microsomal and dependent on NADPH. It is proposed that the lack of reciprocal elevation of luteinizing hormone in the face of the low testosterone levels observed following treatment with CoPP may be due, in part, to increased levels of androstanediols. These metabolites accumulate because of increased production from testosterone and decreased conversion to their triol derivatives in the pituitary.

Cimetidine inhibits catechol estrogen metabolism in women.

Chronic cimetidine use in men is associated with hyperestrogenic side effects such as gynecomastia, which may be linked to inhibition of estradiol 2-hydroxylation. As this property of the drug might be helpful in hypoestrogenic states such as osteoporosis, we investigated the effect of cimetidine on estradiol metabolism in premenopausal and postmenopausal women. Using an in vivo radiometric assay, we found that the extent of estradiol 2-hydroxylation in premenopausal women (n = 9) was decreased by a 1-month course of cimetidine, 800 mg twice daily (44.0% +/- 3.5% v 31.2% +/- 4.1%, P less than .001). Among premenopausal smokers (n = 3), the response to cimetidine was approximately the same as nonsmokers. Serum estradiol levels (follicular phase) in these women were unaltered by cimetidine after 1 month, while concentrations of sex hormone-binding globulin (SHBG) were decreased by 30% (P = .018). Postmenopausal women (n = 5) initially received a lower dose of cimetidine (600 mg twice daily) for 2 weeks, followed by a higher dose (1200 mg twice daily) for another 2 weeks. The extent of estradiol 2-hydroxylation was significantly reduced by the low dose (44.4% +/- 4.5% v 24.3% +/- 3.0%, P less than .005), with minimal further reduction after the high dose (21.7% +/- 1.6%). After 4 weeks of cimetidine treatment, serum estradiol levels increased significantly from 30.0 +/- 6.4 to 59.8 +/- 13.1 pg/mL (P = .033), while SHBG was unaffected. Cimetidine was found to have little effect on selected biochemical indices of bone and calcium metabolism in both groups of women.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exogenous thyroxine on C-2 and C-16 alpha hydroxylations of estradiol in humans.

Thyroid dysfunction in humans is known to alter the excretory pattern of estrogen metabolites, suggesting that thyroid hormone directly influences the oxidative metabolism of estradiol. We examined the extent to which a brief period of hyperthyroidism specifically affected estradiol hydroxylation at C-2 and C-16 alpha, the two primary and competing sites of estrogen oxidation, using an in vivo radiometric assay in healthy male volunteers. Hydroxylation at C-2 was increased by a 2-week course of thyroxine (4.3 micrograms/kg/d) from 29.9% +/- 2.6% to 35.9% +/- 3.1% (P = 0.04), while 16 alpha-hydroxylation was unchanged (10.3% +/- 0.8% versus 9.3% +/- 0.5%). The greater extent of oxidation at C-2 was evidenced by a twofold increase in the urinary excretion of 2-hydroxyestrone (2.88 +/- 0.32 versus 5.30 +/- 0.85 micrograms/g creatinine), while the excreted products of 16 alpha-hydroxylation were unchanged. At the same time, significant reductions in total cholesterol (173.8 +/- 7.9 versus 139.4 +/- 8.9 mg/dl), low-density lipoprotein cholesterol (110.0 +/- 5.3 versus 83.8 +/- 7.7 mg/dl), and apolipoprotein B (68.2 +/- 3.3 versus 53.1 +/- 3.6 mg/dl) were observed. Serum levels of estrone, estradiol, sex hormone-binding globulin, high-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, triglycerides, and apolipoprotein A-I were not significantly affected. This study adds to the growing evidence that catechol estrogen production in humans is more readily regulated than 16 alpha-hydroxylation, which is relatively refractory to treatment.

Lack of NPY-induced feeding in cobalt protoporphyrin-treated rats is a postreceptor defect.

The administration of cobalt protoporphyrin results in transient decreases in food intake and prolonged weight loss in rats. After IVC injection of cobalt protoporphyrin, the food intake of treated rats falls to 10% of vehicle-treated control rats within 48 h. At the same time, the concentrations of mRNA for neuropeptide Y increase approximately twofold in the hypothalamus. The failure of these animals to display a feedings response to elevation of endogenous NPY concentration is mimicked by their failure to respond to exogenous. ICV injections of neuropeptide Y. Because NPY binding studies are confounded by high nonspecific binding, radiolabeled PYY was used to measure binding to hypothalamic membranes and for autoradiography with hypothalamic sections. No abnormalities in the number of receptors or the affinity of the binding interaction were noted. In addition, hypothalamic concentrations of cyclic AMP were unchanged following treatment with either cobalt protoporphyrin or NPY. These results indicate that the locus of the failure of CoPP-treated animals to feed after administration of NPY must be either distal to, or unrelated to, the NPY receptor in the hypothalamus.

Heme binding to Hep G2 human hepatoma cells.

Utilizing high specific activity [55Fe]hemin, the interaction of heme with monolayer cultures of human Hep G2 hepatoblastoma cells was examined. Initial characterization was performed at 4 degrees C to minimize the possibility of heme internalization. Specific binding of [55Fe]hemin at 4 degrees C reached equilibrium within 6 h and was 66% dissociable after 18 h in the presence of fresh binding buffer. Stereospecificity of the binding interaction was evidenced by variable degrees of competition with different metalloporphyrins. Scatchard analysis revealed a Ka of 0.01 nM-1 and the number of binding sites per cell was approx. 200,000. Elevation of the temperature during binding to 22 degrees C resulted in a small decrease in the affinity of the interaction and the apparent number of sites per cell was doubled, possibly due to artefactual elevation of the cell-associated radioactivity secondary to intracellular accumulation. These data are interpreted to indicate the presence of a specific heme receptor on the plasmalemma of Hep G2 cells.