Abstract
Pamaquine (6-methoxy-8-amino [N-diethylaminoisopentyl] quinoline) is recognized to be a potentially dangerous drug. However, a definitive appraisal of its hazard had not been achieved at a time when the further exploration of the antimalarial activity of the 8-aminoquinolines was considered advisable. Pamaquine toxicity can involve the gastro-intestinal tract, the central nervous system, and the circulating blood. Symptoms referable to the gastro-intestinal tract and the central nervous system may be annoying, but there is no evidence that they constitute a hazard to life or persist beyond the termination of therapy. Effects on the blood do constitute a serious hazard and are considered in this paper.
Abstract
Most of the patients utilized as subjects either were having malaria at the time of the observations or had had the disease from one to six weeks previously. None of the patients had malaria long enough to be considered likely subjects for the development of blackwater fever. Most subjects were suffering from asymptomatic neurosyphilis and many had received prior antiluetic therapy. (e) Fragility of erythrocytes to hypotonic saline. A 2 per cent suspension of erythrocytes in sterile physiological saline was prepared from blood containing one drop of saturated potassium oxalate per 5 ml. of blood. 0.5 ml. of the cell suspension was added as soon as possible to 2 ml. each of 0.9 per cent sodium chloride (control), to distilled water (total hemoglobin), and to a series of sodium chloride dilutions usually ranging from 0.48 per 121

However, whenever indicated, sufficient data were collected in normal subjects.

Plasma pamaquine levels were estimated by the method of Brodie, Taggart and Udenfriend (1) . A modification of the method of Horecker and Brackett (2) was utilized for the measurement of methemoglobin.
Special blood studies:
(a) Isoagglutinins. Fresh suspensions of group A, B,. and 0 erythrocytes were prepared each test day. The same donors were used throughout the study. The cells were washed once with sterile physiological saline and a 2 per cent solution in saline prepared. Test serum was obtained by allowing the whole blood to clot and centrifuging the sample. Half milliliter samples of the cell suspensions were added to 0.5 ml. of serial dilutions of the test serum in small glass test-tubes. The tubes were placed in a water-bath at 370 C. for three hours. The tubes were then removed, agitated gently, and observed for macroscopic agglutination in a well-lighted, white viewing box. Agglutination was recorded on a scale of 1 to 4 plus. The final titer was taken as the greatest serum dilution that caused agglutination.
Special blood studies:
(b) Serum hemolysins. The tubes containing the group O erythrocytes described in the isoagglutinin technique were examined for the presence of hemolysis.
Special blood studies:
(c) Cold hemagglutinins. Titers of cold hemagglutinins were measured by a technique based on that of Favour (3) . Indicator cells were derived from a group O donor whose serum contained no cold hemagglutinins. The titers were recorded as the maximum dilution of saline plus cell suspension at which visible agglutination occurred after 18 hours at 40 C.
Special blood studies:
(d) Autoagglutination was looked for by microscopic examination of a small drop of freshly drawn blood in a large drop of saline. If cold hemagglutinins were present, the slide was kept warm until the examination was completed.
Special blood studies:
(e) Fragility of erythrocytes to hypotonic saline. A 2 per cent suspension of erythrocytes in sterile physiological saline was prepared from blood containing one drop of saturated potassium oxalate per 5 ml. of blood. 0.5 ml. of the cell suspension was added as soon as possible to 2 ml. each of 0.9 per cent sodium chloride (control), to distilled water (total hemoglobin), and to a series of sodium chloride dilutions usually ranging from 0. 48 It is well known that methemoglobinemia is a common result of pamaquine therapy (5, 6) . The present study is concerned with the degree of methemoglobinemia achieved on various regimens of pamaquine, alone and in combination with quinacrine or quinine. More detailed studies of this phenomenon in relation to blood pigment metabolism will be reported elsewhere.
Special blood studies:
Observations. Serial methemoglobin determinations were made in patients given various doses of pamaquine, alone or in combination with quinacrine or quinine. These data are summarized in Table I No hemolytic anemias occurred among 20 patients (15 negroes, five whites) given 4 to 20 mgm. of pamaquine daily. Pamaquine was administered in serial dosage in amounts of 30 mgm. or more daily to 157 patients. The incidence of hemolytic anemia during the administration of pamaquine to these patients according to color, prior quinacrine therapy and plasma pamaquine levels is summarized in Table  IV . The most definite correlation achieved by these analyses was that between race and occurrence of hemolytic anemia.
Special blood studies:
It may be noted that all six acute hemolytic anemias occurred among 76 pigmented individuals, while only one subacute anemia was observed It was thought that malaria might cause changes in the serum or erythrocytes of certain patients that would predispose the cells to rupture to destruction. If such were the case, then pamaquine or some metabolic product could be considered the precipitating agent in the hemolytic reaction. Only one hemolytic anemia occurred among the patients subjected to these special studies. This precludes a definitive answer to the problem.
Special blood studies:
The following factors were examined:
Special blood studies:
(a) Isoagglutinins. It has been reported that malaria causes an increase in the titer of isoagglutinins, especially isoagglutinin a, and excessively high titers have been reported in two patients with blackwater fever (9) . Were this a cause or a predisposing factor in the precipitation of an acute hemolytic anemia, the patient would necessarily belong to Blood Group A or AB. The three patients in the present series who developed an hemolytic anemia during pamaquine administration and who were blood typed, belonged to groups A, A, and AB. However, no isoagglutinins were found in the serum of the group AB patient during the hemolytic episode. In addition, no evidence of increased isoagglutinin titers was found in 17 patients followed serially throughout the course of their malaria (five vivax, 12 falciparum).
Special blood studies:
(b) Isohemolysins. No isohemolysins were demonstrable among the patients described above in section (a).
Special blood studies:
(c) Cold hemagglutinins. Serial examination of the cold hemagglutinin titers was performed in 29 patients with malaria. There was an increase in the titer of even patients, the maximal titer in six of these without histories of recent respiratory infection being 1 to 40. The seventh patient had a maximum titer during malaria of 1 to 320. However, this patient had recently recovered from an undiagnosed pulmonary infection characterized by slight fever without leucocytosis or cough, and a patch of rales at the right lung base was found on several examinations at the time of the high titer.
Special blood studies:
Cold hemagglutinins increased to a maximal titer of 1 to 20 in the patient with falciparum malaria who developed hemolytic anemia due to pamaquine administration. However, this increase did not take place until two days after the onset of the anemia. In addition, pamaquine was given to six patients with cold hemagglutinin titers varying from 1 to 20 to 1 to 320 and in none of these was there evidence of an hemolytic anemia.
Special blood studies:
(d) Autoagglutinins. Autoagglutination, in the absence of cold hemagglutinins, was noted in wet drop preparations in three patients. Two patients were receiving pamaquine but no hemolytic anemia developed in either patient. Patient Fra exhibited autoagglutination only after the onset of his hemolytic anemia.
Special blood studies:
(e) Fragility of erythrocytes to hypotonic saline. The resistance of erythrocytes to hypotonic saline was examined serially in 25 attacks of P. vivax and P. falciparum malaria. The data show a moderate increase in hypotonic saline fragility during the initial days of the parasitemia and then a considerable decrease in fragility some seven to 12 days after the onset of parasitemia. Similar results were obtained in nine patients with active malaria during pamaquine administration. These data will be presented in more detail elsewhere.
Special blood studies:
(f) Resistance of erythrocytes to mechanical trauma. Each "mechanical fragility" (M. F.) was done in duplicate. The mean difference in the per cent hemolysis between the duplicates in 75 tests selected at random is 1.07 (a = 0.90). The mean difference between two tests done on different days in 27 subjects during control periods is 1.64 (va= 1.26 ). The mean M.F. in 38 patients (one to six tests per patient) during control periods is 8.0 (a = 1.95) per cent hemolysis.
Special blood studies:
The effect of malaria on the M.F. of erythrocytes was studied systematically in a series of patients during eight attacks of vivax malaria and 14 attacks of falciparum malaria. The mean M.F. during malarial attacks of varying duration and severity was 8.3 per cent as compared to 8.2 per cent for prior and post malaria control values. No increase in M.F. was noted in patients with malaria which was greater than 2.9 per cent (the mean difference between two consecutive tests in control subjects plus one a).
Special blood studies:
The effect of pamaquine alone on the M.F. of 11 patients was also examined. The mean M.F.'s before and during drug administration were 8.4 and 8.8 per cent. There were no individuals with increases greater than 2.9 per cent.
Special blood studies:
M.F. was studied during the administration of pamaquine to ten patients with active malaria. The mean M.F. of the group during pamaquine administration was 11.4 per cent as compared to the mean control values of 8.0 per cent. In three instances the increases over control values were 6.6, 7.6 and 9.4 per cent, respectively, differences that are statistically significant. The greatest change occurred in a patient who subsequently developed an hemolytic anemia.
Special blood studies:
However, additional studies on eight patients with vivax malaria who were given large doses of quinine indicate that the M.F. was also significantly increased in this circumstance in four instances. Increased M.F. occurred in three instances when large doses of pamaquine were given to these patients after the malaria was terminated but while quinine therapy was still being given. None of these patients developed hemolytic anemia. These observations indicate that increased M.F. is not useful as a premonitory sign of hemolytic anemia due to pamaquine. Section III GRANULOCYTOPENIA DUE TO PAMAQUINE Hasselmann (10) noted one death due to agranulocytosis and "toxic hepatitis" among 103 subjects given 60 to 120 mgm. pamaquine hydroiodide daily. However, granulocytopenia had not been noted as a consistent manifestation of pamaquine therapy. During the course of studies on the curative action of pamaquine against vivax malaria, a striking effect on the polymorphonuclear neutrophilic leucocytes was noted. Independently, and at the same time that the present observations were made, Schmidt (11) observed granulocytopenias among monkeys given large doses of pamaquine.
Special blood studies:
Observations. Eight young adult white volunteers who had been infected by mosquitoes carrying Chesson strain vivax malaria were given 3.0 grams of quinine sulfate over 24 hours, followed by 0.65 gram every eight hours for 15 days, starting on the fourth day of fever. Ninety mgm. pamaquine base daily, in divided doses, were begun two days after the first quinine dose and continued for 14 days.
Special blood studies:
Daily leucocyte counts were done on each of these eight subjects. One patient, however, is not included in this analysis since pamaquine administration was stopped before the completion of 14 days of therapy. The total daily leucocyte counts for the seven subjects were averaged and presented in- Figure 1 , in relation to the days of pamaquine administration. This was possible as the curve in each patient was almost identical. A definite rise in leucocyte count occurred in each subject and reached its peak on the sixth or Further studies showed that neither concurrent quinine therapy nor a preceding malaria was necessary for the granulocytopenia observed during pamaquine administration, although quinacrine apparently potentiates the effect. Compare groups 3 and 4 in Table V .
GENERAL DISCUSSION
The adverse reactions due to pamaquine which are referable to the blood include the production of methemoglobin, the occurrence of hemolytic anemia, and of neutrophilic granulocytopenia. The occurrence of methemoglobin, per se, is rarely a deterrent to the continued administration of pamaquine since it is unusual, even with high doses, for the oxygen capacity of the blood to be reduced sufficiently to precipitate respiratory diseases.
GENERAL DISCUSSION
Granulocytopenia represents a potential danger to life, but this reaction has not been noted at doses ordinarily used in therapy. Furthermore, when it occurs, the process is not precipitous and rapidly disappears on cessation of dosage.
GENERAL DISCUSSION
It would appear that the hemolytic reaction is the most seriously toxic hazard of pamaquine. This judgment is based upon the information available in the literature as well as on the experience of this service with the administration of pamaquine under close clinical supervision to some 160 patients over a period of two years.
GENERAL DISCUSSION
The mechanisms whereby pamaquine exerts its effects on the formed elements of the blood have not been established by these studies. However, in the instances of the acute hemolytic anemias, it appears that pamaquine or, more likely, one of its metabolic products, acts as a precipitating factor capable of producing hemolysis when certain predisposing factors are present. This hypothesis is based on the observations that acute hemolytic anemia occurs in only a small proportion of individuals given pamaquine, that race has an effect on the incidence of the hemolytic episodes, and that there is probably no correlation between plasma pamaquine levels and the hemolytic reaction.
GENERAL DISCUSSION
In contrast, methemoglobinemia and granulocytic neutropenia regularly result when pamaquine is given in sufficient dosage, both phenomena are potentiated by quinacrine, which increases plasma pamaquine levels (12) , and both phenomena appear to be roughly correlated with plasma pamaquine levels. SUMMARY 1. The administration of pamaquine results in methemoglobinemia which is roughly proportional to dosage. The effect is potentiated by concurrent quinacrine treatment.
GENERAL DISCUSSION
2. Some 5 to 10 per cent of non-Caucasian patients given pamaquine develop acute hemolytic anemia on the third to fifth day of dosage. The incidence of hemolytic anemia among white patients given pamaquine is much lower.
GENERAL DISCUSSION
3. The administration of 90 mgm. of pamaquine for 14 days results in a significant reduction of mature neutrophilic granulocytes. Pamaquine in daily doses of 30 mgm. does not have this effect unless quinacrine is also administered. (13) indicate that this increase was not due to the anemia alone. There was a leucocytosis of 16,400 per cu. mm. on the second day of the reaction which promptly returned toward normal. There was a definite increase in the "indirect" bilirubin content of the plasma to 3.5 mgm. per cent while the "direct" bilirubin did not change. The resistance of the erythrocytes to hypotonic saline showed a slight increase during the reaction but this was probably due to the malaria and not to the pamaquine or anemia (see above, saline fragility). The (8) . Hemoglobin and an unidentified pigment with an absorption band between 475 and 500 A were found in the black urine voided after the reaction. The urine contained no methemoglobin or methemalbumin.
GENERAL DISCUSSION
The patient's blood group was AB (Rh positive, subgroup 0) 6 and, therefore, there were no isoagglutinins in his serum. There was a slight increase in the cold agglutinin titer after the hemolytic episode. No cold agglutinins were demonstrated before or on the first day of the reaction but the titer was 1: 10 on the second and 1:20 on the fifth day after the episode. Autoagglutination was not present before malaria, but the phenomenon was marked the day following the onset of the hemolytic anemia and before the appearance of cold agglutinins. The intensity of the autoagglutination reaction gradually diminished but still persisted in the fourth week of the follow-up period. The autoagglutination and the presence of cold agglutinins made cross-matching for transfusion difficult. However, good cross-matching was obtained using washed red blood cells and the tube technique at 370 C.
GENERAL DISCUSSION
Serum hemolysins against compatible group 0 erythrocytes were not demonstrable. However, incubation of group 0 erythrocytes for one hour with the patient's serum (obtained the day after the hemolytic reaction) increased the hypotonic saline fragility of the cells to a significantly greater degree than did serum from a normal subject. The erythrocytes showed no tendency to sickle.
GENERAL DISCUSSION
The treatment of the anemia in this patient consisted of the oral administration of sodium bicarbonate in large doses to maintain the urine at a high pH-and the giving of two blood transfusions. Quinacrine was given by mouth to control the malaria, even though it is known to decrease the rate of pamaquine degradation.
GENERAL DISCUSSION
2. Slowly developing hemolytic anemia. One of the 12 white subjects given 90 mgm. of pamaquine base and 2 grams of quinine daily during vivax malaria developed evidence of a mild, slowly developing hemolytic anemia. This represents the only hemolytic anemia observed on this service during administration of pamaquine to 81 white subjects. This patient did not develop signs of hemolytic anemia until the seventh day of pamaquine administration and then the hemoglobin fell gradually from 13.9 to 9.7 grams per cent over a period of six days. Prior to the onset of the signs of hemolytic anemia, this patient had a mean plasma pamaquine level of 198 gg. per liter, methemoglobin rose to 18.7 per cent of the total hemoglobin, and the resistance of the erythrocytes to hypotonic saline showed a slight but significant decrease. There were no significant changes in the erythrocyte mechanical fragility values.

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