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C D Fitch

Publications and source records attributed to C D Fitch.

At least 19 recordsLinked to original sources
PubMed2000

A role for linoleic acid in erythrocytes infected with Plasmodium berghei.

Unesterified fatty acids were measured in mouse erythrocytes infected either with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) lines of Plasmodium berghei. This work was undertaken to identify candidates for the lipid involved in ferriprotoporphyrin IX (FP) polymerization. Linoleic, oleic, palmitic, and stearic acids were quantified by gas chromatography/mass spectrometry. In total, they increased 4-fold with CS infections and 6-fold with CR infections. Treating infected mice with chloroquine did not affect the amounts of unesterified fatty acids in erythrocytes. Of the four fatty acids, only linoleic acid increased disproportionately to the total. It increased 16-fold for the CS line and 35-fold for the CR line. The method could detect monoglycerides but they were below the limit of detection. It could not detect diglycerides, triglycerides or phospholipids. Triglycerides and phospholipids have been tested previously, however, and found to be ineffective at promoting FP polymerization. Therefore, other than linoleic acid, the lipids most likely to be involved in FP polymerization are diglycerides. We tested dilinoleolyglycerol in the present work and found it to be an effective promoter of FP polymerization. These results suggest that linoleic acid or a diglyceride containing it has the critical role of promoting FP polymerization in malaria parasites.

Animals↗
PubMed1999

Involvement of lipids in ferriprotoporphyrin IX polymerization in malaria.

Approximately 70% of the initial ferriprotoporphyrin IX polymerizing activity in cell-free preparations of erythrocytes infected with Plasmodium berghei was recovered in a chloroform extract. No polymerizing activity remained in the residue. In studies to identify substances that promote FP polymerization, arachidonic, linoleic, oleic, and palmitoleic acids, 1-mono- and di-oleoylglycerol, and the detergents, SDS, Tween 80, and n-octyl-glucopyranoside, were active. Tri-oleoylglycerol, cholesterol, di-oleoylphosphatidylethanolamine, and stearic and palmitic acids were inactive. The model lipid, mono-oleoylglycerol (250 nmol), co-precipitated with FP from a 0.09 M acetate medium at pH 5 and promoted the polymerization of 215 nmol (61%) of the ferriprotoporphyrin IX in the precipitate during a 24-h incubation at 37 degrees C. Polymerization was maximal at pH 5, it was approximately linear for 2 h, and it continued at a decreasing rate for 24 h. The polymer contained exclusively ferriprotoporphyrin IX (97+/-1.3%, mean+/-S.E., n=4) and exhibited the solubility and the electronic absorption and infrared spectral characteristics of the sequestered ferriprotoporphyrin IX of hemozoin. Detergents presumably promote polymerization in an acid medium by helping to dissolve monomeric FP. We suggest that unsaturated lipids co-precipitate with FP in the parasite's acidic food vacuole and also dissolve sufficient monomeric FP to allow polymerization.

Animals↗
PubMed1998

Involvement of heme in the antimalarial action of chloroquine.

When malaria parasites digest hemoglobin, they release FP intracellularly. FP is an oxidized form of heme which is toxic for biological membranes. The parasites are not poisoned when they digest hemoglobin, however, because they sequester FP in hemozoin. In fact, the refractile, dark brown substance in hemozoin is sequestered FP. Chloroquine binds tightly to nonhemozoin FP and, under certain circumstances, enhances its toxicity. In addition, chloroquine interferes with FP sequestration and causes toxic nonhemozoin FP to accumulate to lethal levels in erythrocytes parasitized with malaria parasites. Evidently, this is how chloroquine kills malaria parasites. It is desirable, therefore, to know more about FP sequestration and how it is affected by chloroquine. Malaria parasites possess a catalyst for FP sequestration which is modulated by treatment with quinoline antimalarial drugs such as chloroquine and quinine. Chloroquine treatment causes the activity of the catalyst to decrease by 80 to 90 percent. Quinine treatment has no obvious direct effect on the catalyst for FP sequestration. Nevertheless, quinine treatment antagonizes and reverses the chloroquine-induced loss of ability to sequester FP. The effect of chloroquine treatment also is antagonized by various metabolic inhibitors, including inhibitors of protein biosynthesis such as cycloheximide. These findings indicate that chloroquine, like quinine, does not interact directly with the catalyst for FP sequestration. Instead, they are evidence that chloroquine acts by increasing the amount, accessibility, or reactivity of a regulator of the catalyst for FP sequestration. I propose that chloroquine increases the amount of the regulator, which inactivates the catalyst for FP sequestration, which leads to accumulation of nonhemozoin FP, which binds with high-affinity to chloroquine and which ultimately kills the malaria parasite.

Animals↗
PubMed1997

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.

Animals↗
PubMed1994

Hemoglobin catabolism and the killing of intraerythrocytic Plasmodium falciparum by chloroquine.

To evaluate how chloroquine kills malaria parasites, hemoglobin catabolism was studied at the various stages of intraerythrocytic parasite development. We found that hemoglobin catabolism is switched off when Plasmodium falciparum parasites mature to the late trophozoite or early schizont stages and is switched on again during the ring stage. When hemoglobin catabolism is switched off, the parasites are resistant to the morphologic effects of chloroquine. Although the ring stage parasites failed to mature in the presence of chloroquine, some of them switched on hemoglobin ingestion and became stuffed with hemoglobin-filled vesicles, indicating a distal block in catabolism. In fact, we demonstrated a high-grade block in hemozoin production during a 22 h incubation of synchronized ring forms; ferriprotoporphyrin IX (FP) incorporation into the beta-hematin of hemozoin decreased from 900 to 50 pmol/10(6) parasitized erythrocytes. We propose that the primary effect of chloroquine on hemoglobin catabolism is to block FP polymerization to beta-hematin. Secondarily, toxic FP and FP-chloroquine complexes accumulate and are available to exert their several toxicities, which include inhibition of hemoglobin-degrading proteases and membrane damage. As a consequence, maturation is arrested and eventually the parasites die and lyse.

Animals↗
PubMed1993

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.

Animals↗
PubMed1993

Hemozoin production by Plasmodium falciparum: variation with strain and exposure to chloroquine.

Synchronized Plasmodium falciparum parasites were grown in erythrocytic culture for measurement of malaria pigment (hemozoin) production using a simple method based on the insolubility of beta-hematin, the principal pigment of hemozoin. In the last 44 h of the life cycle, one strain (chloroquine-susceptible) incorporated an average of 960 pmol of ferriprotoporphyrin IX (FP) from hemoglobin into beta-hematin per 10(6) parasitized erythrocytes. By comparison, another strain (chloroquine-resistant) incorporated 515 pmol of FP into beta-hematin. When exposed to 25 ng of chloroquine per ml of culture medium, chloroquine-susceptible P. falciparum incorporated 240 pmol of FP into beta-hematin per 10(6) parasitized erythrocytes in one intraerythrocytic life cycle. In contrast chloroquine-resistant P. falciparum exposed to 100 ng of chloroquine per ml incorporated 630 pmol of FP into beta-hematin. Thus, chloroquine inhibits hemozoin production in chloroquine-susceptible P. falciparum but not in chloroquine-resistant P. falciparum. On the contrary, sublethal concentrations of chloroquine partially reverse a deficiency of hemozoin production in chloroquine-resistant P. falciparum. These results indicate that the adaptation responsible for chloroquine resistance in P. falciparum prevents the accumulation of toxic FP by preventing chloroquine from uncoupling the processes of hemoglobin degradation and hemozoin production.

Animals↗
PubMed1992

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.

Animals↗
PubMed1989

Ferriprotoporphyrin IX: role in chloroquine susceptibility and resistance in malaria.

A review of all available evidence supports the hypothesis that FP is the receptor for chloroquine and mediator of its antimalarial activity. In accord with this hypothesis, previously unpublished experiments are described which demonstrate no effect of verapamil on chloroquine accumulation when chloroquine-resistant malaria parasites are nearly totally deficient in FP. Not all chloroquine-resistant parasites are so deficient in FP, however. It is important, therefore, to understand how the concentration of the toxic form of FP, which binds chloroquine, is regulated in erythrocytes infected with pigmented malaria parasites. This regulatory process involves the conversion of toxic FP to nontoxic aggregates (oligomers) of FP which have low oxygen contents. It is also possible that toxic FP is detoxified by decomposition, for example, by hydrogen peroxide.

Animals↗
PubMed1988

Ferriprotoporphyrin IX and cell lysis: a protective role for hydrogen peroxide.

Two potentially lytic substances, ferriprotoporphyrin IX (FP) and hydrogen peroxide, may coexist and partially detoxify each other in sickle cells and in erythrocytes infected with malaria parasites. Since hydrogen peroxide can decompose FP, its effect on hemolysis induced by FP and by the complex of FP with chloroquine was investigated. Human erythrocytes suspended at a concentration of 0.5% in a 50 microM solution of FP underwent approximately 42% hemolysis during the course of 2 hours. Twenty-five micromolar chloroquine potentiated hemolysis to 99%, and preincubation of 50 microM FP with 25 microM hydrogen peroxide for 5 minutes reduced hemolysis to 4%. Mixing either FP or hydrogen peroxide first with chloroquine abolished the effect of hydrogen peroxide. Detoxification of FP by hydrogen peroxide may be an important protective mechanism in certain hemolytic anemias, and inhibition of detoxification could account for the effectiveness of chloroquine in malaria.

Chloroquine↗
PubMed1987

The state of ferriprotoporphyrin IX in malaria pigment.

To evaluate the state of ferriprotoporphyrin IX (FP) in malaria pigment, mouse erythrocytes infected with Plasmodium berghei NYU-2 parasites were lysed by hypotonic shock, and hemoglobin and other soluble material were removed by extensive washing. The amount of FP recovered in the insoluble pellet was 2.1 mumol/ml of packed infected erythrocytes, of which approximately 1% was attributable to hemoglobin contamination. This crude preparation then was digested with a nonspecific protease from Streptomyces griseus and extracted with chloroform/methanol. The residue of insoluble dark brown material had the spectral and solubility properties characteristic of the FP of malaria pigment, and various different preparations contained from 82 to 99% of FP by weight. By elemental analysis, highly purified preparations contained no chlorine and had an oxygen content consistent with 1 mol of hydroxyl/mol of FP (oxygen content: calculated, 12.6%; found, 12.5%). In comparison to hematin purchased from Sigma, which had a measured oxygen content of 14.7%, the low oxygen form of hematin purified from malaria pigment was remarkably less soluble in ethanol, 3% sodium bicarbonate, and chloroform.

Animals↗
PubMed1986

Thyrotoxic myopathy in mice: accentuation by a creatine transport inhibitor.

To demonstrate the importance of creatine and phosphocreatine in skeletal muscle during periods of metabolic stress, thyrotoxicosis was induced in mice fed the creatine transport inhibitor, beta-guanidinopropionic acid (beta-GPA). Adding 2% of beta-GPA to the diet of normal mice inhibited weight gain and caused a 75% reduction of creatine and phosphocreatine concentrations in skeletal muscle. Addition of 0.25% or 2% of thyroid powder to the diet of normal mice was associated with hyperactivity, cardiomegaly, and a high mortality rate. Superimposing thyrotoxicosis on mice already depleted of creatine and phosphocreatine resulted in degeneration of muscle fibers. These results indicate that high concentrations of creatine and phosphocreatine are essential for the maintenance of muscle integrity during periods of metabolic stress.

Animals↗
PubMed1986

Excess release of ferriheme in G6PD-deficient erythrocytes: possible cause of hemolysis and resistance to malaria.

Hemoglobin in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes is abnormally vulnerable to oxidative denaturation, which may release ferriheme, a known cytolytic agent. We found 13.3 nmol of ferriheme in G6PD-deficient erythrocyte membranes (per gram of total erythrocyte hemoglobin) using a spectrophotometric assay, as compared to 9.8 in normal membranes (P less than .05). After incubation of erythrocytes with 250 mumol/L menadione, an oxidant drug, the values increased by 37.4 nmol in G6PD-deficient membranes and by 26 in normal membranes (P less than .005), indicating increased hemoglobin denaturation. To verify that hemoglobin denaturation in G6PD-deficient erythrocytes releases ferriheme in a form available to interact with other ligands, [14C]-chloroquine binding to intact erythrocytes was measured. With an initial concentration of 5 mumol/L chloroquine in a medium containing no menadione, an excess of 14.8 nmol of chloroquine was bound in G6PD-deficient erythrocytes (per gram of hemoglobin) as compared to normal erythrocytes (P less than .005). In the presence of 250 mumol/L menadione, chloroquine binding increased by 17.9 nmol in G6PD-deficient and by 7.2 in normal erythrocytes (P less than .005). These results indicate that ferriheme becomes available to interact with endogenous ligands and, thus, to mediate menadione-induced hemolysis in patients with G6PD deficiency. Furthermore, the increase in ferriheme may mediate the selective toxicity of menadione for Plasmodium falciparum parasites growing in G6PD-deficient erythrocytes. Ferriheme release in response to the intraerythrocytic oxidant stress introduced by malaria parasites also may account for the resistance to malaria afforded by G6PD deficiency. This is a US government work. There are no restrictions on its use.

Chloroquine↗
PubMed1985

Toxic heme in sickle cells: an explanation for death of malaria parasites.

In an effort to elucidate a mechanism of genetic resistance to malaria, we asked whether a toxic form of heme is included in the excess of ferriprotoporphyrin IX (FP) which has been reported to accumulate as hemichromes in sickle cells. When FP is bound to certain erythrocytic elements, such as native hemoglobin, it is inaccessible to bind chloroquine with high affinity and is nontoxic. However, when FP is accessible to bind chloroquine with high affinity, it has been demonstrated to be sufficiently free to have membrane toxicity and, under certain conditions, to lyse Plasmodium falciparum parasites. [14C]-chloroquine was used, therefore, as a reporter molecule to evaluate the quantity, accessibility, and potential toxicity of FP released from hemoglobin. Intact erythrocytes from subjects with sickle cell anemia bound approximately 71 mumoles of chloroquine per kg with an apparent Kd of 10(-6) M. Erythrocytes from normal subjects or subjects with sickle trait bound little or no chloroquine with high affinity. Since the oxidant stress introduced by malaria parasites would increase the tendency for denaturation of hemoglobin S with additional release of FP, we suggest that FP toxicity accounts for the death of malaria parasites in sickle cells.

Adolescent↗