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Malaria parasites isolated from mouse erythrocytes are lysed by ferriprotoporphyrin IX chloride (hemin) or by a chloroquine-hemin complex in amounts that could be produced by release of less than 0.1 percent of the heme in erythrocytic hemoglobin. This effect of hemin may explain the protection against malaria provided by thalassemia and other conditions causing intracellular denaturation of hemoglobin. The toxicity of the chloroquine-hemin complex may explain the selective antimalarial action of chloroquine.

Ferriprotoporphyrin IX (FP) and a chloroquine-FP complex lysed isolated Plasmodium falciparum parasites as judged by decreases in the turbidity of parasite suspensions and by ultrastructural changes. Exposure of parasite suspensions to 50 ,uM FP or to a complex formed from 50 ,M FP and 20 ,uM chloroquine reduced the number of identifiable parasites and caused swelling and loss of internal detail in those that were identifiable. The amount of lysis was dosedependent over the range of 10 to 50 ,uM FP. Formation of a chloroquine-FP complex reduced, but did not eliminate, the toxicity of FP. Since there is evidence indicating that a chloroquine-FP complex forms when chloroquine-susceptible parasites are exposed to chloroquine, we suggest that accumulation of this complex may account for the chemotherapeutic effect of chloroquine against P. falciparum.

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A B S T R A C T Washed erythrocytes infected with chloroquine-susceptible (CS) or with chloroquine-resistant (CR) P. berghei were used in model systems in vitro to study the accumulation of chloroquine with high affinity. The CS model could achieve distribution ratios (chloroquine in cells: chloroquine in medium) of 100 in the absence of substrate, 200-300 in the presence of 10 mM pyruvate or lactate, and over 600 in the presence of 1 mM glucose or glycerol. In comparable studies of the CR model, the distribution ratios were 100 in the absence of substrate and 300 or less in the presence of glucose or glycerol. The presence of lactate stimulated chloroquine accumulation in the CR model, whereas the presence of pyruvate did not. Lactate production from glucose and glycerol was undiminished in the CR model, and ATP concentrations were higher than in the CS model. Cold, iodoacetate, 2,4-dinitrophenol, or decreasing pH inhibited chloroquine accumulation in both models. These findings demonstrate substrate involvement in the accumulation of chloroquine with high affinity.In studies of the CS model, certain compounds competitively inhibited chloroquine accumulation, while others did not. This finding is attributable to a specific receptor that imposes structural constraints on the process of accumulation. For chloroquine analogues, the position and length of the side chain, the terminal nitrogen atom of the side chain, and the nitrogen atom in the quinoline ring are important determinants of binding to this receptor.

The new antimalarial drug mefloquine bound with high affinity (Kd approximately 3 X 10-7 M) to membrane lipids of normal mouse erythrocytes and of erythrocytes infected either with chloroquine-susceptible or chloroquine-resistant Plasmodium berghei. Approximately 80 nmol of mefloquine was bound per mg of total lipid. Mefloquine also bound to purified phospholipids with high affinity (Kd approximately 3 X 10-7 M). Phosphatidylinositol and phosphatidylserine bound 300 to 400 nmol of mefloquine per mg. Phosphatidylcholine and phosphatidylethanolamine bound approximately 100 nmol of mefloquine per mg. Mefloquine did not bind to hemoglobin with high affinity, but it bound to free ferriprotoporphyrin IX with a Kd of approximately 3 X 10-7 M. In comparison with mefloquine, chloroquine did not bind effectively to purified phospholipids, although it is known to bind with high affinity to free ferriprotoporphyrin IX. Greater binding to phospholipids may account for the superiority of mefloquine in the treatment of chloroquine-resistant malaria.

To determine the diagnostic potential of a highly sensitive immunoradiometric assay (IRMA) for LH in children with normal puberty or altered tempo of sexual maturation, we compared serum LH levels by IRMA (LH IRMA) and standard RIA (LH RIA) in children with idiopathic precocious thelarche (IPT; n = 6), idiopathic premature adrenarche (IPA; n = 14), central precocious puberty (CPP; n = 15), and constitutional delay of puberty (DP; n = 15), and 160 control children (79 males and 81 females). Subjects in the latter group were staged, according to their genital or breast development, as early prepubertal (P1E; age, less than 8 yr), late prepubertal (P1L; 8-12 yr), or stage II-V (P2-P5; n = 22-34 for each subgroup). Serum LH IRMA levels in P1E, IPT, and IPA children were either undetectable (95% of subjects less than 0.25 IU/L) or barely detectable (5% of subjects, less than or equal to 0.5 IU/L). Serum LH IRMA levels were greater than 0.5 IU/L in 38% of P1L (mean +/- SD for the group, 1.0 +/- 1.3 IU/L) and 57% of P2 (1.4 +/- 1.3 IU/L); they were greater than 1.0 IU/L in 100% of P3 (2.6 +/- 1.3 IU/L), P4 (3.9 +/- 2 IU/L), and P5 (8.6 +/- 4 IU/L) children. Comparison of serum LH levels between contiguous pubertal stages showed significantly higher LH IRMA concentrations in P3 vs. P1E, P4 vs. P2, P5 vs. P4 (all P less than 0.001), and P3 vs. P1L (P less than 0.05). In contrast, LH RIA values were not significantly different in P1E (2.0 +/- 0.6 IU/L), P1L (2.3 +/- 0.6 IU/L), P2 (2.7 +/- 0.9 IU/L), P3 (3.2 +/- 1.3 IU/L), and P4 (3.7 +/- 2.2 IU/L), although they were higher in P5 (6.8 +/- 4 IU/L) than in P4 (P less than 0.001). From P1E to P5 LH IRMA levels increased 38-fold in females and 21-fold in males, while LH RIA increased 4- and 2.1-fold, respectively. Serum LH IRMA correlated significantly with serum testosterone levels in boys from P1L to P5 (r = 0.76; P less than 0.001), while LH RIA levels did not (r = 0.18). Serum LH IRMA concentrations were above the prepubertal range (greater than 0.5 IU/L) in 67% of children with CPP (group average, 1.8 +/- 1.4 IU/L) and 87% of children with DP (1.6 +/- 1.4 IU/L).(ABSTRACT TRUNCATED AT 400 WORDS)

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