Mechanism of hemolysis induced by ferriprotoporphyrin IX.

Incubation of a 0.5% suspension of washed, normal mouse erythrocytes with ferriprotoporphyrin IX (FP) at 37 degrees C and pH 7.4 caused potassium loss, swelling, increased susceptibility to hypotonic lysis, and finally hemolysis. Hemolysis was not inhibited by incubation in the dark, malonyldialdehyde was not produced, and various free radical scavengers had no effect on the hemolysis. Only the sulfhydryl compounds, cysteine, dithiothreitol, and mercaptoethanol protected erythrocytes from FP. Potassium loss reached 90% within 30 min of exposure to 5 microM FP. This amount of FP caused greater than 50% hemolysis within 2.5 h. Sucrose (0.1 M) completely prevented hemolysis but had no effect on potassium loss. Likewise, reducing the temperature from 37 to 25 degrees C greatly retarded hemolysis but had no effect on potassium loss. These observations indicate that FP impairs the erythrocyte's ability to maintain cation gradients and induces hemolysis by a colloid-osmotic mechanism.

Hemolysis of mouse erythrocytes by ferriprotoporphyrin IX and chloroquine. Chemotherapeutic implications.

Incubation of a 0.5% suspension of washed normal mouse erythrocytes with ferriprotoporphyrin IX (FP) for 2.5 h at 37 degrees C and pH 7.4 results in sufficient membrane damage to produce hemolysis. A sigmoidal dose-response curve is followed with 50% hemolysis being produced by 4 microM FP. Complete hemolysis is produced by 6 microM FP. The hemolytic process has at least two phases: a lag phase of approximately 45 min, during which little hemolysis occurs, and a phase characterized by rapid hemolysis. Chloroquine, which binds tightly to FP, enhances the effect of FP by eliminating the lag phase. Under the conditions of these experiments, maximum enhancement is observed with chloroquine concentrations in the range of 5-25 microM. Since FP is produced when malaria parasites digest hemoglobin, it may mediate a chemotherapeutic effect of chloroquine by forming a complex with the drug that could enhance the toxicity of FP for biological membranes, including those of the parasite.

Acute effects of N-methyl-N'-nitro-N-nitrosoguanidine on canine gastric mucosa.

The electrophysiological effects of the chemical gastric carcinogen N-methyl-N'-nitro-N-nitrosoquanidine (MNNG) were determined in an in vivo chambered canine stomach and in an in vitro canine gastric mucosal preparation. In the in vivo stomach, the topical application of 2.5 mg MNNG/ml decreased the transmural electrical potential difference, and the systemic blood pressure was essentially unchanged. In the in vitro preparation, exposure of the mucosal side of the isolated canine gastric mucosa to 0.25 and 2.5 mg MNNG/ml for 1 hour sequentially or exposure of the serosal side to 2.5 mg MNNG/ml for 2 hours inhibited net Na+ and Cl- fluxes. With longer duration, the undirectional fluxes of Na+ and Cl- increased, indicating an increase in permeability. These findings suggested that inhibition of active transport in the gastric mucosa may have an important function in the gastric carcinogenicity of MNNG.

The in vitro metabolism of 13-cis-retinoic acid in a model sebaceous structure, the rat preputial gland.

In order to study the metabolism of 13-cis-retinoic acid (13-cis-RA) in animal sebaceous glands and analogues, preputial glands from normal and vitamin A-deficient male rats were incubated with [3H]13-cis-RA for up to 24 h; vitamin A-normal hamster costovertebral glands (flank organs) were incubated for 24 h as well. High-performance liquid chromatography was used to identify the metabolites. [3H]13-cis-RA was rapidly converted to a less polar compound, [3H]all-trans-retinoic acid, by the preputial glands from both normal and deficient rats. In normal preputial glands, the level of [3H]all-trans-RA decreases and two more polar compounds, metabolite I and [3H]4-keto-13-cis-RA appear. In contrast, [3H]all-trans-RA is not metabolized further by the preputial glands from deficient rats, while [3H]13-cis-RA in the hamster costovertebral glands remains intact for up to 24 h. The major metabolite of [3H]13-cis-RA in rat preputial glands is [3H]4-keto-13-cis-RA. Initially, [3H]13-cis-RA is converted to [3H]all-trans-RA. In vitamin A-deficient rats the preputial glands fail to further metabolize [3H]13-cis-RA to the more polar [3H]13-cis-RA derivatives. This may be due to the reduced level of P-450 enzyme in vitamin A-deficient rat preputial glands.

Progressive neuropathologic lesions in vitamin E-deficient rhesus monkeys.

A consistent group of progressive central and peripheral nervous system lesions developed in seven rhesus monkeys maintained on a vitamin E-deficient diet for 30 to 33 months. These lesions were absent from vitamin E-supplemented monkeys. The principal neuropathologic alteration was loss of sensory axons in the posterior columns, sensory roots, and peripheral nerves. Morphologic and morphometric studies indicated that the distal segments of the axons were affected most severely and large-caliber myelinated fibers are selectively involved. Swollen, dystrophic axons (spheroids) occurred infrequently. Degeneration and phagocytosis of small numbers of neuronal perikarya were observed in the dorsal root ganglia and the anterior horns. The number of affected neurons was not proportional to the number of affected axons. Accumulation of lipopigment was evident in neuronal perikarya and CNS endothelial cells. The nervous system lesion were usually accompanied by a chronic necrotizing myopathy. The neuropathologic lesions in vitamin E-deficient monkeys are compared with those in vitamin E-deficient rats and in humans with low serum vitamin E concentrations. A similar type of sensory axonopathy is associated with chronic deficiency of vitamin E in these three species.

Heme polymerase: modulation by chloroquine treatment of a rodent malaria.

The biosynthesis of the beta-hematin of malarial pigment (hemozoin) is catalyzed by a newly discovered enzyme, heme polymerase, which is described for Plasmodium berghei in this report. This novel enzyme is present in the insoluble fraction of hemolysates of infected erythrocytes but is not present in normal erythrocytes. The substrate is ferriprotoporphyrin IX (FP) released from hemoglobin. At pH 5 and 37 degrees C the enzyme is saturated by 100 microM FP. The pH optimum is between 5 and 6 and the reaction is linear for 6 hours. All heme polymerase activity is destroyed by heating at 100 degrees C for 3 minutes. Chloroquine treatment of malarious mice reduces by 80 percent the activity of this enzyme, without inhibiting release of FP from hemoglobin, and thereby causes excess nonpolymerized, nonhemozoin FP to accumulate. Since the accumulated FP is accessible to bind chloroquine, we propose that it is the mediator of the antimalarial activity of chloroquine.

Fitting contact lenses after myopic keratomileusis.

PURPOSE: To evaluate the long-term efficacy and results of contact lens fitting following myopic keratomileusis (MKM). SETTING: Department of Ophthalmology, Manhattan Eye, Ear, and Throat Hospital, and Swinger Vision Center, New York, New York, USA. METHODS: Postoperative fitting of contact lenses was studied as part of a prospective evaluation of myopic keratomileusis. In this series, 27 eyes of 20 patients had residual postoperative refractive errors that were corrected with contact lenses. Patients were fit at a mean of 9.4 months after surgery by the trial-lens method. Preoperative keratometry readings and refractions, as well as postoperative keratometry readings, refractions, and contact lens specifications, were recorded and used for fitting. RESULTS: Twenty-six eyes (96%) were fit successfully: 24 (89%) with rigid gas-permeable lenses and 2 (7%) with daily-wear soft lenses. The mean diameter of the rigid lenses was 10.00 mm (range 9.4 to 11.0 mm) and the mean base curve, 8.52 mm (range 7.9 to 9.2 mm). The 2 soft lenses had base curves of 8.6 and 8.9 mm. The mean lens power was -5.24 diopters (D) (range -0.37 to -14.75 D), which was, on average, 4.06 D more myopic than the postoperative spectacle refraction. Postoperative keratometry provided a good starting point for the trial lens. Lenses were tolerated for up to 16 years. One eye, fit with a soft lens, developed significant myopia during the fifth year. CONCLUSION: After lamellar refractive surgery, the topography of the cornea is significantly altered. Although the postoperative keratometry readings are steeper than the actual curvature, they are reasonably reliable for determining the base curve of the initial trial lens, validating the use of conventional methods of fitting rigid contact lenses in patients who have had MKM.

The effectiveness of 0.5% atropine in controlling high myopia in children.

Twenty highly myopic children (> or = -6.0 D) were treated with 0.5% atropine eyedrops once per night. Twelve subjects were initially treated with a short-acting cycloplegic agent, tropicamide (0.5%) (Group A), and the other eight subjects did not receive any myopic therapy before atropine (Group B). These cases were followed for up to five years. In Group A, the mean myopic progression rate after 0.5% atropine treatment was -0.01 +/- 0.04 D/M (Diopter/Month), which was significantly lower than that of the period during tropicamide treatment (-0.12 +/- 0.09 D/M) (p < 0.05). In Group B, the mean myopic progression rate after atropine therapy was begun was -0.04 +/- 0.06 D/M, which was also significantly slower than that of non-medication, -0.14 +/- 0.07 D/M (p < 0.05). The results suggested that 0.5% atropine is effective for slowing down myopic progression, even in highly myopic children.

Effects of different concentrations of atropine on controlling myopia in myopic children.

Although 1% atropine effectively slows myopia progression, it is associated with adverse effects, including photophobia, blurred near vision, and poor compliance. We investigated whether lower doses of atropine would control myopia progression. One hundred and eighty-six children, from 6 to 13 years of age, were treated each night with different concentrations of atropine eye drops or a control treatment for up to 2 years. The mean myopic progression in each of the groups was 0.04 +/-0.63 diopter per year (D/Y) in the 0.5% atropine group, 0.45+/-0.55 D/Y in the 0.25% atropine group, and 0.47+/-0.91 D/Y in the 0.1% atropine group. All atropine groups showed significantly less myopic progression than the control group (1.06+/-0.61 D/Y) (p<0.01). Our study also showed that 61% of students in the 0.5% atropine group, 49% in the 0.25% atropine group and 42% in the 0.1% atropine group had no myopic progression. However, 4% of children in the 0.5% atropine group, 17% in the 0.25% atropine group, and 33% in the 0.1% atropine group still had fast myopic progression (>-1.0 D/Y). In contrast, only 8% of the control group showed no myopic progression and 44% had fast myopic progression. These results suggest that all three concentrations of atropine had significant effects on controlling myopia; however, treatment with 0.5% atropine was the most effective.

Control of heme polymerase by chloroquine and other quinoline derivatives.

To evaluate the response of heme polymerase to treatment of malaria with chloroquine, we used mice infected with Plasmodium berghei. Six hours after treatment with 3 mumoles of chloroquine intraperitoneally per mouse, heme polymerase activity in parasitized erythrocytes decreased from 238 to 37 nanomoles of ferriprotoporphyrin IX polymerized per hour per mumole of ferriprotoporphyrin IX in preformed hemozoin, and nonhemozoin ferriprotoporphyrin IX increased in vivo from 40 to 123 nanomoles per ml of packed, parasitized erythrocytes. Other 4-aminoquinoline derivatives were similar in effect to chloroquine. Treatment with quinine, mefloquine, primaquine, or naphthalene derivatives caused no reduction in heme polymerase activity. In contrast to 4-aminoquinoline derivatives, quinine and mefloquine, which are quinolinemethanol derivatives, antagonized the effect of chloroquine.

Regulation of heme polymerizing activity and the antimalarial action of chloroquine.

Mice infected with Plasmodium berghei served as donors of erythrocytes with a high level of parasitemia for the study of ferriprotoporphyrin IX (FP) polymerization. Six hours after treatment of these mice with 3 micromol of chloroquine per 25 g of body weight, there were significant losses of heme polymerase I (HPA I). For chloroquine-susceptible (CS) P. berghei, the rate of FP polymerization decreased from 541 +/- 42 (mean +/- standard deviation; n = 12) to 51 +/- 19 (n = 8) nmol of FP polymerized per h per ml of packed erythrocytes (normalized to represent 1,000 parasites per 1,000 erythrocytes). For chloroquine-resistant (CR) P. berghei, the rate decreased from 284 +/- 19 (n = 16) to 124 +/- 11 (n = 6) nmol per h per ml. The chloroquine-induced loss of HPA I was accompanied by the accumulation of unpolymerized FP in CS P. berghei but not in CR P. berghei, which is consistent with the hypothesis that FP mediates the antimalarial action of chloroquine. Quinine treatment partially reversed the effects of chloroquine in CS P. berghei but not in CR P. berghei. Cycloheximide treatment antagonized the effects of chloroquine in both lines of parasites. To explain these findings, we propose that chloroquine, quinine, and cycloheximide perturb a regulatory process for HPA I. Furthermore, we propose that when chloroquine engages its target in the regulatory process, it initiates a chain of events which culminates in increased production, accessibility, or reactivity of a regulator (inactivator) of HPA I.

Effects of vitamin A status on cellular retinoic acid-binding protein in rat skin and testes.

Cellular retinoic acid-binding protein levels were determined in the skin and testes of normal and retinol-deficient rats. All-trans [3H]retinoic acid (1.1 TBq/mmol) was used to titrate the specific binding sites in tissue cytosol preparations. Scatchard plot analyses were used to determine the concentration of cellular retinoic acid-binding protein and its binding affinity (Kd) for all-trans-retinoic acid. In normal rat skin the concentration of cellular retinoic acid-binding protein was 3317 +/- 924 (SD) fmol/mg protein and the Kd was 1.98 +/- 1.0 x 10(-9) mol/l. In retinol-deficient rat skin the concentration of cellular retinoic acid-binding protein was 2584 +/- 1205 fmol/mg protein and the Kd was 3.30 +/- 3.4 x 10(-9) mol/l. In the normal rat testes the concentration of cellular retinoic acid-binding protein was 2965 +/- 1187 fmol/mg protein and the Kd was 2.30 +/- 2.1 x 10(-9) mol/l. In retinol-deficient rat testes the concentration of cellular retinoic acid-binding protein was 2439 +/- 383 fmol/mg protein and the Kd was 0.3 +/- 0.2 x 10(-9) mol/l. These findings indicate that there are no significant differences in the levels of cellular retinoic acid-binding protein between normal and deficient rat skin and testes (p greater than 0.1, by Wilcoxon rank sum test). We therefore conclude that the level of cellular retinoic acid-binding protein in skin and testes may not be controlled by the availability of vitamin A.

Abnormalities of iron metabolism and erythropoiesis in vitamin E-deficient rabbits.

Rabbits fed a vitamin E-deficient diet developed severe muscular dystrophy in 3-4 wk, but they did not become anemic. Nevertheless, reticulocyte counts increased in deficient rabbits (3.2%) compared to control rabbits (0.9%), and erythroid hyperplasia was evident in the bone marrow. Comparing deficient rabbits to controls, the plasma iron concentration was lower (134.4 versus 206.6 microgram/dl); the TIBC was higher (335.9 versus 228.3 microgram/dl); the whole blood protoporphyrin concentration was higher (131.6 versus 81.7 microgram/dl); and the total iron content was lower in spleen (71 versus 153 microgram), higher in skeletal muscle (4956 versus 3054 microgram), and unchanged in bone marrow, liver, and heart. Studies of iron absorption and excretion using 59Fe showed no abnormalities in deficient rabbits. There were abnormalities of ferrokinetics, however. The half-time of disappearance of 59Fe was shorter (100.6 versus 169.4 min), the plasma iron turnover was greater (1.25 versus 0.95 mg/dl blood/day), and the reappearance of 59Fe in circulating erythrocytes at day 9 was greater (77.2% versus 57.2%) in deficient rabbits. Anemia induced by phlebotomy accentuated the abnormal iron metabolism of deficient rabbits, and the animals were unable to correct the anemia. These findings show that vitamin E deficiency in rabbits causes abnormal erythropoiesis associated with abnormal iron metabolism and sequestration of iron in skeletal muscle.

Effects of dopamine on endotoxin and hemorrhagic shock in the canine stomach.

Studies were conducted to evaluate the effects of dopamine on gastric electrophysiopathology in endotoxin and hemorrhagic shock. Intraarterial infusion of dopamine (15.5 or 31 microgram/kg/min) in the in vivo stomach preparation produced an immediate decrease in electrical potential difference (PD), which then returned and exceeded control values. No changes in resistance (R) and blood pressure were observed. These electrophysiological responses of the gastric mucosa to dopamine are very similar to the actions of epinephrine. The in vitro studies demonstrated that active transport of Na+ was stimulated with an addition of dopamine or epinephrine (2 X 10(-4) M) to the serosal solutions of the isolated gastric mucosa. In addition, the in vivo studies demonstrated that both 40% hemorrhage and 1 mg/kg of endotoxin given as an intravenous bolus decreased PD and blood pressure and increased R although dopamine was continuously infused intraarterially for 60 minutes prior to and following hemorrhage or endotoxin. Administration of endotoxin at the onset of dopamine infusion decreased both blood pressure and PD initially. While PD showed a complete recovery at a later stage, blood pressure never returned to control levels. These results, combined with previous observations, suggest that: 1) dopamine has no beneficial action on the gastric mucosa during hemorrhagic or endotoxin shock; 2) dopamine acts on the electrophysiology in vivo and Na+ fluxes in vitro in the gastric mucosa in a manner similar to epinephrine; and 3) decrease in blood flow may be responsible for the observed decrease in transmural PD after dopamine and epinephrine in vivo.

Intracellular ferriprotoporphyrin IX is a lytic agent.

Human erythrocytes were treated with menadione to oxidatively denature hemoglobin and release ferriprotoporphyrin IX (ferriheme, FP) intracellularly. The high affinity of FP for chloroquine was used to detect its release. After incubation for 1 hr at 37 degrees C and pH 7.4 with 0.5 mM menadione, erythrocytes bound 14C-chloroquine with an apparent dissociation constant of 10(-6)M. Untreated erythrocytes did not bind chloroquine with high affinity. At a chloroquine concentration in the medium of 2 microM, for example, menadione-treated erythrocytes bound 70 mumole chloroquine/kg and untreated erythrocytes bound 13.4 mumole/kg. The intracellular location of FP released by menadione was verified by finding that Tween 80 did not prevent chloroquine binding. By contrast, Tween 80 inhibited the binding of chloroquine to erythrocytes treated with extracellular FP. The hemolytic response to menadione was characteristic of the hemolytic response to FP. Thus, 5 microM chloroquine caused hemolysis to increase to 60% from baseline values of 5% in experiments using erythrocytes treated either with 0.5 mM menadione or with 5 microM FP; and, in both cases, the potentiating effect of chloroquine was inhibited by 1 microM mefloquine or 10 microM quinine. Higher concentrations of menadione caused hemolysis in the absence of chloroquine. We conclude that FP released by menadione exists intracellularly in a form that is accessible to bind chloroquine and to express its lytic activity.

Iron is sequestered as ferritin in macrophages in skeletal muscle of vitamin E-deficient rabbits.

Weanling rabbits were fed a purified diet with or without vitamin E supplementation to evaluate the abnormal sequestration of iron in skeletal muscle associated with vitamin E deficiency. A severe myopathy developed in unsupplemented rabbits within 3 to 4 weeks. At this time, the concentration of soluble nonheme iron in biceps femoris muscles had increased from 2.1 +/- 0.4 microgram/g wet weight (mean +/- SD) for six control rabbits to 4.3 +/- 1.4 for 10 vitamin E-deficient rabbits, and total nonheme iron had increased from 5.0 +/- 1.2 to 8.4 +/- 3.3. Soleus muscles had even greater increases in total and soluble nonheme iron concentrations. Intramuscular injection of iron-dextran caused large increases in total and soluble nonheme iron in noninjected muscle of vitamin E-deficient rabbits, which further exaggerated the difference between the two groups. By radioimmunoassay using an antibody to rabbit liver ferritin, the concentration of ferritin in biceps femoris muscles increased from 0.47 +/- 0.18 microgram/g wet weight for seven control rabbits to 6.34 +/- 1.70 for 14 vitamin E-deficient rabbits. Uptake of intravenously injected transferrin-bound iron into muscle of vitamin E-deficient rabbits was not increased in a short term experiment (6 h), but radioiron did accumulate in muscle in a long term experiment (6 days). There was no trapping of heat-damaged erythrocytes, no phagocytosis of intravenously injected carbon particles, and no erythrophagocytosis in muscle. An immunohistological staining method designed to detect ferritin in tissue sections stained muscle from normal rabbits very scantily but intensely stained macrophages in the muscle of vitamin E-deficient rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)