Primaquine Sensitivity (Glucose-6-Phosphate Dehydrogenase Deficiency)

PRIMAQUINE SENSITIVITY (GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY)

.An Inborn Error of Metabolism of Medical and Biolo Significance

Alvin R. Tarlov. M... D. .George J. Brewer, M. D.

From the University of Chicago-Army Medical Research Unit, Department of Medicine, Chicago, Illinois. "he previously unpublished studies gf the authors included in this review were supported by the Medical' Research and Development Command, Office of the Surgeon General, Department of the Army, under contracts DA-49-007-MD- 566 and DA 49-007-MDWwith the University of Chicago and were supplernent- ed by grants from Burroughs- Wellcome and Company, Winthrop Laboratories, , and the Douglas Smith Foundation, SUBMITTED TO THE

ARCHIVES OF INTERNAL MEDICINE

25 AUGUST I961 -

3. \, \, The potential danger of hemolysis from use of the 8-aminoquinoline

antim larial drug, pamaquine (plasmoquine@J , has been known since i.\\

1926 (l): Earle (2), in 1948, reported that pamaquine caused hemolysis . . '

in 5-10 percent of American Negroes, but rarely in Caucasians. Similar

observations were made in 1952 by Hockwald (3), during an evaluation of

the related, but less toxic drug, primaquine; it was further noted that the

severity of hemolysis was determined by the amount of drug administered.

In 1954, Dern (4) discovered that the susceptibility to hemolysis by

primaquine is due to an intrinsic abnormality of the erythrocyte. Dern,

Beutler and Alving (5) reported that the hemolysis is self-limited in that

clinical recovery occurs even if the daily dose of drug is continued. Many drugs can precipitate hemolysis (6 ) . This hypersusceptibility to hemolysis

by drugs is a genetically transmitted inborn error of metabolism (7 ) ,

- The first biochemical abnormality to characterize drug-sensitive cells, a low content of reduced glutathione (GSH), was discovered by Beutler (8).

This kind of susceptibility to drug-induced hemolysis is called

primaquine-sensitivity because the initial stbdies which led to its charact-

erization were made during investigations of the hemolytic property of

this antimalarial drug. It has recently also been designated glucose-6-

phosphate dehydrogenase (G-6-PD) deficiencx after the major known enzym-

atic defect in primaquine-sensitive individuals (9) but the phenotypic name, ‘,’ primaquine sensitivity, should not be discarded until the primary genetic 9 \;. defectkor basic biochemical abnormality responsible for the destruction of erythrocytes has bcen established with certainty. Important contrib- utions to an understanding of this inborn error of metabolism have been made by many scientific workers, Intensive investigations have been carried out in the United States, Israel, Italy, Germany and, more recently, in other countries.

Many drugs in common clinical use.__ may precieate hemolysis.___

‘Nu-iIaiig(*r of ialrogc11ii::iIQ ~iiidut.cdacxitc aticmia sliould always be - ~ ~._-___-_____-. weighed by physicians in their selection-. of therapeutic agents. The possib-

ility that _,some systemic discases -mznhancc the hemolytic effect of these drugs should alwTs be born in mind when an obscure hemolytic episode is encountered at the bedside.

MODE- OF. . IhYIERITANCE~. .. AND DISTRI- BUTION . - -- - __

Childs, Browne, and their co-workers (lo’ concluded from studies of American Negroes that primaquine-sensitivity is probably transmitted by a gene of partial dominance located on the X chromosome (sex- (12) linkage), Strong supporting evidence was soon presented by Gross ,

Szeinberg (I3), and Larizza (14). Definite proof for sex-linkage of the primaquine -sensitive gene in Sardinians was provided recently by chromosome; when colur blindness and primaquine-sensitivity coexist

in a single individual the two gent’s segregate identically through succeed-

ing generations in a given family (I5). Thcse conclusions are supported

by the investigations of Adam (I6) in Sephardic Jews.

Full expression of the genetic defect occurs in affected hemizygous

males (E)because the mutant gene (2)is not opposed by a normal allele

(X). Full expression is rare in females because the homozygous female -_ (XX) must inherit a mutant gene from both parents. Usually, affected

females are heterozygous (e);they inherit a mutant gene from one

parent and a normal allele from the 0th r, and, therefore, have partial

expression of the trait.

In our study of Negro* volunteers at two penal institutions in

Illinois, 13 percent of the males and 3 percent of the females were fully (171 susceptible to hemolysis . Affected females manifested a wide

’ spectrum of expression of the clinical phenotype (hemolysis when 30 mg.

of primaquine base was administered daily); in some hemolysis was only detectable by isotopic labeling of the erythrooytes. In iit hers the hemolytic susceptibility and the biochemical abnormalities of the erythrocytes were

~~ ~~ +This paper concerns the inherited disorder of primaquine- sensitive American Negroes unless otherwise specified. Sensitive Caucasians (Sardinians, Sephardic Jews) are more severely affected and differ in some respects from sensitive Negroes.

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as severe, possibly even more severe, than in males with full expression.

Judged by the clinical phenotype 20 percent of the Negro women were heterzygous. ** Yet, deficient G-6-PD*** in the erythrocytes could be

demonstrated unequivocally only in approximately three-fourths of the

heterozygous females. This may mean that several enzymatic abnormal-

ities can influence the expression of the clinical phenotype or, alternatively,

that the --in vitro assay of G-6-PD in hemolysates does not accurately reflect a slight decrease in the --in vivo activity of this enzyme in the circulating

erythrocyte6.

Primaquine- sensitiyity has a broad geographic distribution. Among

Caucasians the defect is particularly concentrated in the Mediterranean

area. Racial and ethnic groups having a high incidence usually are more

darkly pigmented; the mutant gene rarely occurs in peoples of Northern

European origin. Primaquine-sensitivity occurs in 48 percent of some

Sardinian groups (") and in 2 to 36 percent of Mediterranean and Asiatic

(Sephardic) Jews (13' 22); it is rare in European (Ashkenazic) Jews. The incidence in American Negro males is approximately 13 percent (1 1,17). - **This figure is slightly lower than the expected incidence of heterozygosity calculated from 13 percent incidence of sensitivity in Negro men. This may ir$Licate that som heterozygous females are so weakly expressed that detection is impossible even when primaquine is administered. ***The correlation between hemolysis and enzyme defi ienc was not improved when the Clock and McLean assay of G-6-PDb, 1% was substituted for the Kornberg method (21,23,24,25) it is even higher in some African Negro tribes . Prima- quine-sensitivity has also been reported in Greeks (26), Iranian Moslems

(27), Asiatic Indians (28), Chinese (29), Filipinos(Z1) and many others

(‘l). The incidence is highest in tropical and semi-tropical zones. It tends to parallel the distribution of falciparum malaria (21).

GENETICAL AND ENVIRONMENTAL INFLUENCES

Analysis of gene frequencies often yield information concerning the adaptive values of the trait. Many mutant genes decrease fitness; therefore, they tend to disappear from the population. A mutant gene, if it increases fitness, may exist in a population in a greater frequency than would be expected if it did not produce this biological advantage.

I. Falciparum Malaria and Primaquine-Sensitivity:- The geographic distribution of primaquine-sensitivity (high frequency in tropical and semi-tropical malarious areas) suggests that the trait provides an evolutionary selective advantage against malaria*, as hypo- (31) thesized by Motulsky (‘”, and supported by the studies of Allison .

Malaria parasites require glutathione (GSH). (32) and an oxidative pathway of carbohydrate metabolism (32* a3) for optimum growth. The low GSH

*The incidence of the trait, however, is very low in Armenians who have lived for years in Iran where falciparum malaria is common and y&re the incidence of this trait in native Iranians varies from 5-1570. ( )

-. content and the diminished activity of the pentose phosphate pathway of primquine-sensitive erythrocytes mymake it difficult for the intracellular parasites to survive. The lower parasite density in the blood of young children whose erythrocytes are deficient in G-6-PD compared to children with normal enzyme activity has been interpreted as supporting this hypothesis. (31) Protection against malaria may similarly explain the geographic variation in frequency of sickle- (34, 35, 36) hemoglobin and thalassemia . Recently, however, caution (37, 65) has been advocated in interpreting data based on parasite densities I.

II. Favism, Thalassemia and Primaquine-Sensitivity: Favism, hemolytic anemia caused by the fava bean, occurs only in primaquine- (38, 39.40.41 1 sensitive individuals . This condition is most common in

Sardinians. The mortality rate in children who experience acute hemolysis after ingestion of the beans is reported to be 8 percent (42) .

Clinical manifestations of acute icteric favism, however, are rare

among primaquine-sensitive individuals who have thalassemia minor

(41’43) and the observed frequency of individuals havinb both traits is

. The erythrocytes of individuals with thalassemia minor have high\Y’ (45,46, 41.33.40) a shortened survival and consequently, a young erythrocyte population. Young cells are relatively resistant to hemolysis deficiency is rare. Similarly, Rh negativity is rare (3.3 percent)

among Congolese Bantus in whom primaquine-sensitivity is common

(21 percent) (51) . More extensive statistical studies are needed for i

definitive evaluation of the relationship between primaquine-sensitivity

and the Rh blood groups. Although there is a tendency toward differences in the B- and -0 distribution in our study, the only significant difference (p=. 02) is

the relative rarity of E- in the sensitive group. This can not be inter-

preted at the present time because there are no known disorders caused * by the -E blood group. The incidence of primaquine -sensitivity in different population

groups appears to be balanced by some factors which increase fitness

(e, g., by resistance offered to falciparum malaria) and others which

lower fitness (e. g., susceptibility to hemolysis by vegetable foods and

infectious diseases, susceptibility to neonatal jaundice). In genetic

terms this represents a balanced polymorphism. Correlation of

primaquine-sensitivity and other diseases (e. g., atherosclerosis,

neoplasia) should be of great interest in defining the evolutionary sign-

ificance of the primaquine-sensitive mutation.

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HEMOLYTIC COMPOUNDS .*

The number of potentially hemolytic drugs continue8 to increase

(Table LI). Many of these are frequently used in clinical medicine, e. g. , sulfonamides, nitrofurans, and acetophenitidin (Phenacetin 8,. Most of the potentially hemolytic drugs may be administered to normal individuals in therapeutic doses without producing clinical hemolysis.

Some drugs which are only moderately hemolytic in normal people, i. e., phenylhydrazine, sulfones, and acetanilid, cause severe hemolysis in primaquine-sensitive individuals (61 . Finally, other drugs may cause no hemolysis even in sensitive persons unless aggravating or potent- iating factors are present.

CLINICAL COURSE OF HEMOLYSIS

Extensive studies of the hemolytic potential of primaquine have been performed by the University of Chicago-Army Medical Research

Unit at the Stateville Penitentiary. In these investigations the administration of primaquine, 30 mg. base daily, has been adopted as a standard procedure for inducing a reproducible, self-limited hemolysis.

The course of hemolysis (Figure 1) in primaquine-sensitive, but otherwise healthy, * adult Negro male volunteers under controlled hospital

*The hemolysis observed in an ill individual might be quite different due to complicating factors which myy mitigate or potentiate the hemolysis.

File: conditions taking this standard dose may arbitrarily be divided into

-~-. . ~ three phases as described by Dern (5):

1) Acute Hemolytic Phase: The acute hemolytic phase lasts

7- 12 days. The hematocrit usually begins to fall on the second day,

more rarely the third or fourth day, and drops to its lowest level

(approximately to 30 mm.) between the 8th and the 12th day. It has

been estimated that 30-50 percent of the red cell mass is destroyed by this dose of drug (5,621 . Symptoms are uncommon, but if present are related to the anemia. Intra-erythrocytic inclurfion bodies (Heinz

bodies) occur in large numbers during the first seve 1 days of drug (63) administration but disappear when rapid hemolysis takes11 place . The serum bilirubin, evenly divided between direct and indirect

reacting, rises to 3-5 mg. 70; the urine is dark, sometimes black,

and scleral icterus may occur. Erythrocyte destruction usually ceases

within 48-96 hours if primaquine is stopped during the acute phase. (62) Beutler demonstrated that erythrocytes 63-76 days old were

destroyed by the standard challenge dose of primaquine whereas cells

8-21 days old were not. Hemolytic susceptibility, therefore, is related

to the age of the erythrocytes. Kellermeyer (61) demonstrated that

there is a progressive increase in susceptibility as the cells age. Drug-

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I induced hemolysis is self-limited if the daily dose of 30 mg. primaquine is not exceeded and clinical recovery takes place even though drug is continued, because the surviving young erythrocyte population is relatively resistant to drug.

2) Recovery Phase: Clinical recovery takeg place between the

10th and the 40th day. The reticulocyte count increases to a peak of

8-12 percent. The hemoglobin and hematocrit slowly rise and reach noymal values by the 4th or 5th week. \ 3) Equilibrium or Resistant Phase: The equilibrium phase begins when the anemia disappears and continues as long as the same dose of primaquine is administered. Mild hemolysis, detectable only by erythrocytic survival studies, persists during the equilibrium phase

(61). The hemolysis is not clinically apparent because it is compensated by a slightly increased rate of erythropoiesis. When circulating erythrocytes reach a critical age they are no longer able to resist this dose of primaquine. The resistance of the younger cells to hemolysis is not absolute; Like the severity of hemolysis, it is related to the dosage of drug. With stepwise increases of drug dosage destruction of progressively younger erythrocytes occurs.

After the drug has been discontinued hemolysis of equal severity to the original episode'cannot be produced by the same dose until a -11-

ri period of 4 to b months has elapsed, the time necessary for the circulating erythrocyte population to return to mean pre-drug age.

FACTORS WHICH INFLUENCE THE SEVERITY OF HEMOLYSIS

I. Concentration of the Drug in the Blood:- The severity of hemolysis is determined by the concentration of the drug (or its active degradation product) in the blood, and the length of time that critical concentrations are maintained.

Drug Dosage: - Aspirin, which is not hemolytic in small dosages is mildly hemolytic at the high dosages (10 grams daily) sometimes employed in the treatm nt of severe rheumatic disease; nitrofurantoin

(Furadantin@ ), primaquine (Fig. Z), and salicylazo-sulfapyridine

(Azulfidine @I, are only moderately hemolytic when administered in therapeutic doses to healthy sensitive volunteers but produce severe hemolyses when these dosages are doubled (64, 58)

Concurrent Renal or Liver Disease: - Kidney and liver' diseases may affect the concentration of drugs in the blood by altering their metabolism or excretion. As an example, therapeutic doses of nitrofurantoin, ordinarily only mildly hemolytic, may cause severe hemolysis in sensitive individuals with severe renal impairment (65).

II. Viral and Bacterial Infections: - Acute hemolytic anemia caused by infections (upper respiratory infection, viral hepatitis, " infectious mononucleosis. pneumonia, and influenza) have been (67) reported in both sensitive Caucasians (14' 66) and Negroes . (68,691) may Furthermore, the hemolysis due to drugs (e. g., aspirin be markedly accentuated in sensitive individuals during some bacterial, viral, or other febrile illnesses. It is not known whether or not this enhancement of susceptibility to hemolysis is caused by a toxic

product of the infecting organism, by a toxic metabolite produced by

the host, or by the concurrent development of autoimmunity.

Ill. Diabetic Acidosis: - Diabetic acidosis increases the (691, susceptibility of the erythrocytes to hemolysis by drugs

Furthermore, Gant (70)has reported the occurrence of acute self-

limited hemolytic anemia during diabetic acidosis in two sensitive

$/ Negro males. The hemolysis could not be attributed to infection or

to tdov ingestion of drugs.

\$ \ IV. Hypoglycemia of the Newborn: Acute hemolytic anemia

of the newborn following the administration of high doees of soluble

vitamin K derivatives is probably more common than is recognized.

In some cases the infants have the genetically determined enzymatic

defect (71); the erythrocytes of others, however, have normal glucose-

6-phosphate dehydrogenase activity but a marked instability of

reduced glutathione (GSH) --in vitro which may be corrected by addition

*Clinical observations of the authors...... ,. I’ -13-

of glucose to the blood (72). Physiological hypoglycemia of the newborn may accentuate the vulnerability of sensitive erythrocytes to destruction and may even render normal erythrocytes susceptible to hemolysis when drugs are administered. Zinkham (57) has recommended using not more than 2.5 mg. of the water soluble vitamin K compounds (menadione sodium bisulfite, Hykinone 9 sodium menadiol diphosphate, Synkayvite @I or 1.0 mg. of vitamin K1 (Konakion@), in newborns. These doses are adequate to correct hypoprothrombinemia of the newborn but probably not large enough to cause clinically significant red cell destruction.

V. Age of the Erythrocytes: - Conditions w ch lead to a pre- ponderance of young cells (e. g., concurrent acute o1 chronic blood loss, chronic hemolytic anemia, or recent recovery from acute hemolysis) may prevent the development or mitigate the severity of an acute drug-induced hemolysis.

VI. Schedule of Drug Administration: It is possible to prevent clinical hemolysis, without loss of therapeutic effectiveness in the radical cure of vivax malaria, by giving large doses of primaquine

(45 mg. base) once a week rather than smaller doses daily (52)

(Fig. 3). It is not known whether intermittent dosages of some other

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drugs would be similarly useful; this possibility should be investigated,

H EMATO LOG1 C C HARACT E FUSTI CS OF PRJ MAQUIN E - SENS1 TI VE ERYTHROCYTES (FULLY EXPRESSED NEGRO MALES)

In their early studies butler, Dern, and Alving (63) showed that primaquine-sensitivity could not be predicted prior to drug

administration by the Light-microscope morphology of the erythrocytes, tht; hemoglobin type, the alkali resistant hemoglobin, nor by the

dir'i! ?ct Coombs test. The reticulocyte count, hemoglobin, and hemato- crita were reported to be within normal limits except during drug- induced hemolysis. Subsequent studies have revealed the following hematologic abnormalities:

I. Shortened Erythrocyte Survival Time: Brewer has recently demonstrated that the life span of erythrocytes of primaquine- sensitive Negro males is about 25 percent shorter than normal: even before drug is administered. This is not sufficient to cause chronic anemia. The abnormally young m an age of the circulating erythrocytes must be taken into considgation whenever sensitive and normal cells are compared because many characteristics of the erythrocyte, including the activity of several enzymes, change with age (73,74) (55) LI. Decreased Osmotic Fragility: - Tarlov has shown that the erythrocytes of most drug sensitive Negro males are more -15-

resistant to osmotic lysis than normal, i. e. they have decreased osmotic fragility (Fig. 4). The entire osmotic fragility curve is symmetrically shifted to tb less fragile side indicating that all fractions of the erythrocyte population are more resistant than normal. The hyper-resistance may be a manifestation of their younger mean age (76). Other factors must be studied before this * explanation can be accepted as final.

The difference in resistance of primaquine-sensitive and normal erythrocytes to osmotic lysis is small and can only be detected by precise research techniques not used in earlier studies (63). The determination of osmotic fragility is unsuitable as a screening test for primaquine-sensitivity because of the small difference, and the overlap, between normal and sensitive values,

III. Falsely Elevated Hematocrit Values: - In recent studies

Tarlov (55) showed that the erythrocytes of drug-sensitive Negro males trap more plasma intercellularly during centrifugation than do normal cells (Table III). Consequently, falsely elevated hematocrit values are produced when determined by the Wintrobe method. This is compatible with the younger mean age of sensitive red cells because more plasma is trapped by young than by old erythrocytes ('7'cv, 78). The capillary micro-hematocrit, which employs higher

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centrifugal forces, yields values which are closer to the true

hematocrit of both normal and sensitive blood when the duration

of centrifugation is carefully controlled (Table UI).

METABOLIC CHARACTERISTICS OF PRIMAQUINE-SENSITIVE ERYTHROCYTES (FULLY EXPRESSED NEGRO MALES)

The energy available to human erythrocytes is derived from \ the1 catabolism of glucose via at least two metabolic pathways (Fig. 5). The Embden-Meyerhof Pathway, (non-oxidative) metabol- (79) izes approximately 90 percent of glucose and produces potential

energy by the formation of high energy phosphate bonds in ATP.

Reduced diphosphopyridine- nucleotide (DPNH), apparently the preferred coenzyme for methemoglobin reduction under physiological

circumstances, is also generated in this pathway, The Pentose

Phosphate Pathway (oxidative) normally metabolizes approximately

10 percent of the glucose, according to Murphy (79), and provides

the sole mechanism for regeneration of reduced triphosphopyridine

nucleotide (TPNH) in mature human erythrocytee. TPNH is an

essential co-factor in many reductive processes, (e. g., methemo-

globin rectuction in the presence of oxidant or redox drugs and dyes,

glutathione reduction, the degradation of drugs, and lipid synthesis).

Glucose-6-phosphate dehydrogenase (G-6-PD) can control the rate -17-

of TPNH regeneration by catalyzing the initial step of the pentose phosphate pathway. Oxygen consumption and carbon dioxide production (respiration) are also controlled entirely by this pathway in the non-nucleated erythrocyte,

The most severe, possibly the basic, enzymatic Befect of drug sensitive erythrocytes is a deficiency of G-6-PD aa first shown by Carson (9). The activity of G-6-PD in the erythrocyte6 of affected

(hemizygous) Negro males is approximately 10 to 15 percent of normal, Most of the other abnormalities of primaquine-sensitive v erythrocytes (Table IV) are related to the deficiency of G-6-PD.

In erythrocytes having normal G-6-PD activity the availability of its obligatory coenzyme, TPN', is probably the rate limiting" factor in the pentose phosphate pathway. In normal erythrocytes this pathway can be greatly stimulated by artificial electron carriers

(e. g. , redox compounds such as methylene blue, hemolytic drugs, ascorbic acid) (99' 'O0) which oxidize TPkto TPe. In sensitive erythrocytes the deficient G-b-PD, rather than the TPN', limits the activity of the pentose pathway; their ability to respond to redox compounds, therefore, is limited. Studies of the rates of oxygen consumption (82,831, methemoglobin reduction (84,851 , pentose accumulation (83), dye reduction ("), and glucose utilization (55) -18-

(Fig. 6) by sensitive erythrocytes in the presence of redox dyes

all reveal a diminished responsiveness of the pentose pathway to

I stimulation. The inability of this pathway to respond may be the I , Achilles Heel of the drug-sensitive erythrocytes. (The role of I decreased catalase activity in drug-induced hemolysis has yet to

be defined). I BIOCHEMICAL CHANGES DURING HEMOLYSIS

FULLY EXPRESSED NEGRO MALES Fig. 7

I. Fall in Catalase Acltrivity: - The catalase activity in

hemolysates of sensitive men and fully expressed women, and some,

but not all, intermediate (heterozygous) women is diminished 60-80 percent prior to drug administration (98). Some evidence suggests

that low catalase activity may increase the susceptibility of the erythrocytes of intermediate females to hemolysis (98).

When the catalase activity is initially low, it falls sharply during hemolysis (97)., the fall is the first detectable change in the

erythrocytes. It may fall as early as 24 hours after the initial dose

'$\ of dru? and may precede the fall in glutathione by several hours.

Its low t value, representing a fall of 35 peecent, is rea&hed in

about a,se *XI days. The catalase activity rises slowly after hemolysis. \ -1

...... - 19-

The catalase activity, unlike the GSH and G-6-PR0 does not rise above the baseline during reticulocytosis, but remains low during the recovery and equilibrium phases and commonly does not return to pre-drug values for 2-4 months. The catalase of normal Negroes, and of intermediate females who have normal catalase activity, is not affected by primaquine administration. ” Catalase, like hemoglobin, is an iron-porphyrin protein,

Redox drugs may ofidize Feu- catalase to Fe++-catalase. Unlike

Fe++- hemoglobin, however, the oxidation of Fe++- catalase may be irreversible. Thus, the catalase activity of the circulating erythrocytes does not return to pre-drug values until affected cells have been completely replaced by a new erythrocyte population produced in a marrow which is free of drug and free of tlE stress of acute anemia (101)

II. Fall in Glutathione (GSH): - The reduced glutathione

(GSH) content of sensitive erythrocytes is usually, but not invariably, below normal in dmg sensitive Negro males (’). The GSH falls further within 36 hours after the initial dose of drug and before the i!, \ (102) major hemolysis takes place (Fig. 7). By the fifth to seventh day avdecrease\\ in GSH content of approximately 20 percent \ -20-

has occurred after which the GSH rises to, and may exceed, the pre-drug levels by the 14-18 day. The secondary rise in GSH is a reflection of 1) the destruction of the older erythrocytes by drug

and 2) the reticulocytosis. A rise in oxidized glutathione (GSSG), if it occurs at all, is slight and transient.

Barron first recognized that GSH is necessary to the stability of sulfhydryl containing enzymes (103). Reduced g tathione not only (1 04,'105)It protects many proteins, including hemoglobin , enzymes (106, 107) (96) and coenzymes against oxidation but also is bound to

some enzymes ('08) and is a co-factor for others (109)

The fall in GSH without an equivalent rise in its oxidized form

(GSSG) during drug-induced hemolysis may be explained by the formation of mixed disulfides of GSH with the sulfhydryl groups of the globin molecule followed by the denaturative precipitation of hemoglobin into Heinz bodies. (lo5) The susceptibility of GSH to destruction in G-6-PD deficient erythrocytes may not only contribute to the hyper-vulnerability of hemoglobin to oxidative denaturation, but may similarly contribute to the destruction of some enzymes

(7 catalase) and, as suggested by Jacob (110), may eventually lead to the lysis of the erythrocytic membrane. .

. .. III. Rise in Methemoglobin and Heinz Bodies: - Brewer

(89.90) demonstrated that sensitive men develop higher levels of methemoglobinemia than do normal individuals when sodium nitrite is administered. Yet, when primaquine is administered, the methemoglobinemia is slight and transient in sensitive men, whereas the methemoglobinemia increases three to five fold in normal men.

Similarly, the number of erythrocytes containing Heinz bodies increases during the first few days of drug administration to sensitive men but the Heinz bodies disappear as hemolysis takes place (63 1.

The explanation of these paradoxes is that the oldes cells, which

i)il form the most methemoglobin and presumably the most Heinz bodies, are the first to be destroyed by drug (90).

IV. Fall in Total Red Cell Lipids: - The total lipids of sensitive erythrocytes (expressed per gram of hemoglobin) are decreased (Table V) prior to drug administration (55). This finding points to an abnormality in the lipoprotein membrane of sensitive erythrocytes because 90 percent of the lipids are in the membrane.

Definitive interpretation is difficult but decreased lipids may reflect a diminished TPNH availability (and a decreased rate of lipid synthesis) in the marrow.

A slow decrease in red cell Lipids, sometimes preceded by -22-

/ a transient rise, during drug-inddced hemolysis was observed I

by Tarlov (55) (Fig. 7). The lowest values, 15 percent below the '

; ,-drug baseline, are reached in two to three weeks, The lipids

remain depressed after drug is discontinued and usually do not

return to pre-drug values for two to three months when the erythro-

cyte population has been completely replaced by cells produced in

a drug-free environment. In contrast, a rise in total red cell

lipids occurs when a young cell population is produced in sensitive (55) men by repeated phlebotomy . These observations indicate that the erythrocytes produced by the marrow in sensitive men

during primaquine administration differ biochemically from the erythrocytes produced prior to drug. V. Glucose-6-Phosphate Dehydrogenase: I/ - The G-6-PD activity, which is initially low, rises during hemolysis and then returns to the baseline (Fig. 7) in parallel to the rise and fall in the number of circulating reticulocytes, a reflection of the higher activity of this enzyme in the youngest cells, as demonstrated by

Marks (73) (93) The activity of glutathione reductase (") and of aldolase were reported by Schrier to be higher in drug-sensitivity than in -23-

normal erythrocytes. The increased activity of these enzymes suggests that a compensatory mechanism is operating to regulate the metabolism of these cells, but the increased activity may, in part at least, also reflect the younger mean age of sensitive erythrocytes. The activity of glutathione and of aldolase remain unchanged during drug-induced hemolysis (88,93) METABOLISM OF PFUMAQUINE I/ During the administration of primaquine an "anti-catalase" factor can be demonstrated in the plasma of both normal and sensitive individuals. Tarlov ('01) observed a profound fall in catalase activity of primaquine-sensitive erythrocytes* --in vitro during their incubation with plasma from a normal individual who ingested a single dose of 120 mg. primaquine base. The following observations were made (Fig. 6):- (1) The anti-catalase activity could not be demonstrated in the plasma until 4-6 hours after drug administration; (2) The maximum anti-catalase activity was attained in the plasma in 8 hours and was maintaineci for at least another 16 hours; (3) A gradual alteration in the metabolism of primaquine

*To demonstrate this effect intact sensitive cells must be used; the catalase activity of lysed sensitive cells or of intact normal cells are affected to only a minor extent.

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appeared to develop in one normal man to whom 120 mg. of primaquine base was administered daily for seventy-five days (Fig. 8).

After the second day of drug the anti-catalase activity in his plasma progressively decreased; after two and one half months of drug administration the anti-catalase activity was only 55 percent of the maximum observed on the first day. Thus, the equilibrium phase of hemolysis may not only result from the younger man age and the higher enzyme activity of the surviving erythrocytes but may, in ?, part kt least, be attributed to an alteration in drug metabolism if its administration is continued, although in earlier experiments Dern (5) reported ItThe capacity of drug-sensitive volunteers to hemolyse known primaquine -sensitive cells was not altered during long-term drug administration". This point should be re-evaluated.

The 6-methoxy- 8-aminoquinolines are rapidly metabolized; the concentration of %degraded"** drug in the plasma bears little or no relation to their therapeutic effect. Zubrod (' and Arnold (112) observed that the highest plasma concentration of the "undegraded" drug is reached in 2-4 hours, and thereafter falls rapidly and is barely detectable in 24 hours. In contrast, the anti-catalase activity does not

**The "undegraded" drug has the 6-methoxy group intact and still gives the diazo reaction with sulfanilic acid.

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.. . -25-

appear in the plasma until several hours after the peak concentration of the ”undegraded“ drug has passed and remains high when little or no “undegraded” drug can be demonstrated. It may be assumed that the anti-catalase component is similar or identical to the hemolytic component because a fall in catalase activity of the erythrocytes occurs -~in vivo during drug-induced hemolysis. The hemolytic effect of primaquine, therefore, is probably due to a degradation product, an oxidant quinone .(Fig. 9). This hypothesis is in accordance with the observations of Carson (’ 15) that the degradation products of primaquine produced by ultraviolet irradiation cause oxidative changes in sensitive cells --in vitro similar to those produced during drug- induced hemolysis, i. e., oxidation of hemoglobin to methemoglobin, and oxidation of GSH.

MECHAZlSI11- Ob’ HEhlOLYSlS -. (116) . Emerson, Ham and Castlc, in 1949, pointed out that most hemolytic compounds are oxidants. These compounds (or their degradation products) have a tautomeric structure and are, or can be transformed into, resonating compounds; thus, like methylene blue, they constitute reversible oxidation- reduction mediators (1 16)

Under physiological circumstances oxidation ordinarily proceeds slowly

.. . .. I -..

Washington National Record Center Ofice of the Army Surgeon General Record Group 112 Accession #: 6 7 A - y813 Box #: -26-

in erythrocytes, despite their high concentration of oxygen, because an electron hiatus exists between molecular oxygen and intracellular hydrogen (electron) donors. Jandl (117) has summarized the evidence which indicates that the hemolytic drugs can bridge this hiatus by mediating reversible hydrogen transfers and thus enabling oxygen to transmit its high oxidation potential to hemoglobin and other cellular components. The transfer of hydrogen fr TPNH, GSH, +,I hemoglobin (FeH), the free sulfhydryl of proteins, and other donors is greatly accelerated by these redox'intermediates acting as hydrogen acceptors.

Support for the hypothesis that the hemolytic compounds are both oxidants and reversible oxidation-reduction mediators is gained from the experiments of Brewer (56) . The administration of an oxidant alone (sodium nitrite) produced only equivocal hemolysis and the administration of a redox agent alone (methylene blue) produced only slight hemolysis; when the two were given simultaneously, however, a severe hemolytic process followed in a sensitive volunteer.

Several mechanisms within normal erythrocytes protect them against injury by oxidative drugs. The rate of TPNH regeneration

can be greatly accelerated by increasing the amount of glucose metab-

... olized via the pentose phosphate pathway. Therefore, in normal

erythrocytes under the stress of oxidant drugs, sufficient TPNH is

readily made available for glutathione and methemoglobin reduction

via the respective reductase reactions; reduced glutathione (GSH)

also protects the hemoglobin sulfhydryls and the sulfhydryl contain-

ing enzymes against oxidative destruction; catalase destroys H202 , * a powerful oxidizing agent which can injure the cell.

In contrast to normal erythrocytes, sensitive erythrocytes

are incapable of sufficiently rapid TPNH regeneration because of

the deficiency of G-6-PD. Consequently, 1) they cannot respond

to the stimulus of redox compounds (e. g., methylene blue, oxidant

drugs) and 2) all of the reductive processes within the erythrocyte

dependent upon TPNH are impaired. Most of the abnormalities in

sensitive erythrocytes, including their low GSH and its fall during

drug administration, their vulnerability to methemoglobin accumul-

ation, their short survival, and ultimately the mechanism of their

*Mills has suggested that glutathione peroxidase protects the erythrocyte from oxidation by catalyzing the reaction &Q +2 GSH --> 2 Hai GSSG. The activity of this enzyme in human erythrocytes, however, is very low, and cannot be demonstrated --invitro unless catalase activity is completely inhibited. (118*55)In view of the high activity of catalase in human erythrocytes,(55) the physiological importance of glutathione peroxidase is questionable. The activity of glutathione peroxidase, in intact erythrocytes and in hemolysates is the Sam in drug-sensitive as it is in normal erythrocytes (54). destruction by drug appear to be linked to their relative incapacity

' to regenerate TPNH. ** Their vulnerability to oxidative destruction

appears to be enhanced by deficient catalase activity which may

allow H2 02 to accumulate.

--In vivo experiments conducted in our laboratories indicate

that the steps leading to hemolysis probably are as follows: the

initial dose of drug is followed by a latent interval during which the

drug is metabolized to its active redox form. Thereafter, ferro-

catalase, GSH, ferro-hemoglobin, and globin are oxidized. The final

sign of oxidative destruction of hemoglobin is the appearance of

Heinz bodies. Metabolic processes diminish to levels at which vital

functions can no longer be carried out and alterations in the lipo-

protein membrane occur. The end comes by intravascular lysis.

The _-in vitro studies of Allen ('05) are consistent with the sequence of events we have observed --in Uivo. In their experiments t& initial effect of the redox drug was the oxidation of GSH. A \ poAion of the GSH becomes bound to hemoglobin by forming mixed \: disulfides with the sulfhydryl groups of globin. Then hemoglobin

**The vulnerability of ATP to destruction -__in vitro '95~ 96) (not as yet demonstrated _-in vivo) may also be linked to the inability of sensitive erythrocytes to regenerate TPNH and possibly DPNH. It has been suggested that the destruction of the red cell membrane immediately responsible for lysis, is primarily due to the loss of the potential energy of the high energy phosphate bonds of ATP.

...... c -29-

(Fe++) is oxidized to methemoglobin (Fe4-H) and thereafter the two free sulfhydryl groups of the globin molecule become oxidized,

Subsequently other reactive groups, including the remaining four sulfhydryls of the globin molecule are oxidized and "sulfhemoglobin" is formed. The protective effect of glutathione is lost and denaturative precipitation of hemoglobin into coccoid granules called Heinz bodies occurs. A similar mechanism may operate in destruction (1 10) of the erythrocytic membranes

FAVISM

Acute hemolytic anemia following the ingestion of the broad bean, Vicia fava, occurs only in primaquine-sensitive individuals (1 19,38,120, 121,40,122-126,14) . Favism is most prevalent among the inhabitants of Sardinia in whom the deficiency of G-6-PDis more severe than in sensitive Negroes, but it also is common in most parts of the Mediterranean littoral and in countries to which Mediterr- anean inhabitants have migrated. The same drugs, and possibly additional compounds (Table 11), which will induce hemolysis in primaquine -sensitive Negroes, are hemolytic in individuals susceptible to favism. Other plants will cause a similar syndrome: Pisum sativum (pea), Verbena hybrida, Anagyris --foetida (stinkwood), the bog-

...... I ; -30-

bilberry or whortleberry, Morchella esculenta (a mushroom) and the m’ale fern (14) \ \ Not all persons having the genetically determined deficiency \$ of G-6-PD experience acute hemolysis after ingestion of fava beans, and individuals suffering acute fava-induced hemolysis may have eaten the beans many times previously without adverse reaction (127, 14)

Hemolysis may result from inhalation of the pollen of the fava plant

(127). The hemolysis is usually extremely acute - within a few hours in cases of inhalation - and often is very severe (127). The evidence for participation of an extracorpuscular, possibly an immune, process is strong (127, 128, 14, 129, 126). The experiments of Panizon (130)and (131) Sartori in Italy have led them to conclude that susceptibility to favism requires the inheritance of two separate metabolic disorders:

1) the G-6-PD deficiency which is sex-linked and 2) an extracorpuscular factor, possibly expressed by differences in absorption or metabolism of the fava bean, which is inherited as an autosomal recessive gene. GENETIC VARIANTS OF THE G-6-PD DEFICIENCY

Decreased activity of an enzyme may arise by several genetic mechanisms: a decreased rate of synthesis of the enzyme,

. -31-

the synthesis of an abnormal enzyme molecule, the absence of activators or co-factors necessary for activation of the enzyme, the presence of inhibitors, and probably others. Evidence is rapidly accumulating that the genetic variants of G-6-PD deficiency, several of which have been described, are produced by the operation of some of these mechanisms, possibly all of them. The difficulty of defining the role of each of these factors is illustrated by the complexity of the conditions --in vitro which can affect the G-6-PD activity from normal erythrocytes. c Glucose-6-phosphate dehydrogenase activity of normal erythrocytes: Glucose-6-phosphate dehydrogenase from erythrocytes (132) has not been crystallized. Kirkman, however, recently purified the enzyme ten-thousand fold and he observed that the normal enzyme may exist in three forms: a monomer free of TPNt which is completely t inactive, 2) a dimer free of TPN which has 20% of full activity, and

3) a dimer with bound TPN’ which is fully active. Furthermore, he inactivated normal G-6-PD by carefully removing the bound TPNt , + and subsequently reactivated the enzyme by adding TPN . Similar studies have not been performed with the enzyme from G-6-PD deficient erythrocytes.

Washington National Record Center Oflice of the Army Surgeon General Record Group 112 Accession #: 6 ;f A - L+B 13 Box #: - 32-

The relationship, when known, of the important findings

of Kirkman to the 1) physiological regulation of G-6-PD activity

-2in vivo to the 2) progressive loss in activity of this enzyme which

occurs as circulating erythrocytes age, and to the 3) accelerated \\ lossi\ of G-6-PD activity which takes place as primaquine-sensitive eryt/ irocytes age ("premature senescence"), will add significantly .^ not only to our knowledge of genetics at the molecular level but to

our understanding of the normal process of aging as well. (133, 134) Carson has shown that the stromata of erythrocytes

can inactivate the G-6-PD of hemolysates; the stromata from normal

and from drug-sensitive erythrocytes act alike in this capacity,

Inactivation occurs with removal of bound coenzyme (TP#) from

G-6-PD and is prevented by adding TP#. The inactivation appears (134) to be due to a TPN-ase on the stromata. (136) In contrast, Rimon (135) and Ramot in Israel using

different techniques from those of Carson, has reported that hemolysate

G-6-PD can be activated by stromata from normal, but not from drug- .. -- ~, .. - sensitive, erythrocytes. This activation of G-6- PD by stromata,

however, could not be confirmed in entirety by Marks (137). Full

evaluation of the stromal factors must await studies using purified

. . I-- preparations of both the enzyme and the stromal components.

Several genetic variants of the G-6-PD deficiency have been reported and at least four must be tentatively accepted. It seems certain that when our knowledge is more complete, other variants, both genotypic and phenotypic, will emerge.

I. Primaquine-Sensitive Negroes: - The 139) activity in males is 10-1570 of normal (9). Kirkman 138, presented strong evidence, confirmed by Marks (137), that the de-

creased activity of G-6-PD is not due to a qualitative abnormality of the enzyme molecule. The survival time of the erythrocytes is diminished to approximately ninety days even before drug is administered

(60)., primaquine-sensitivity, therefore, can be classified as a congenital non-spherocytic hemolytic disorder. There is no anemia unless drug is administered. The defect is inherited as a sex-linked gene of partial dominance. The enzyme deficiency also has demonstrated in (141) the ocular lens ('*') and in the platelets of sensitive Negroes.

Recent investigations indicate that primaquine-sensitive

Negroes have a generalized metabolic disorder: 1) the serum cholesterol was significantly higher in primaquine-sensitive Negro males compared to a control group of comparable age (s5),, 2) the serum -34-

esterified cholesterol fell sharply when primaquine was administered to sensitive men (during acute hemolysis) but it re1 ained unchanged in normal men (142), and 3) the rate of glucose oxidation by the whole 14 14 body from both glucose-1-C and glucose-6-C , determined by 14 measuring the C 02 in the expired air, was markedly below normal in drug-sensitive Negro males (143). The deficiency of G-6-

PD is probably present in all tissues to a variable extent but appears to be most severe in non-nucleated cells.

2. Partially Expressed Primaquine-Sensitive Males: The erythrocytes of one sensitive Negro man, of the 80 studied in our laboratory, had an intermediate deficiency of G-6-PD (30% of normal), had intermediate glutathione stability, and gave intermediate results on the methemoglobin reduction test (98). The hemolysate catalase activity of this "partially expressed" man was normal, whereas that 51 of fully expressed men is decreased. The Cr erythrocytic survival time before drug administration was normal and during drug the hemolysis was less severe than that of fully expressed men. The (55) family studies indicated that vhis man was hemizygous; they were compatible with inheritance by a sex-linked gene of intermediate dominance. Three brothers and his mother had complete expression -35-

of the genetic defect, i. e., their erythrocytes, having a 90% de-

crease in G-6-PD activity, were fully susceptible to hemolysis by

drug when the cells were transfused into a normal individual who was taking 30 mg. primaquine daily. Two sisters were intermediate

and his father was normal.

Marks (144) recently reported the occurrence of an inter-

mediate deficiency of G-6-PD (50% of normal) in two Italian male

children; the partially purified enzyme differed in some physical properties from the G-6-PD of normal erythrocytes.

3. Primaquine -Sensitive Caucasians: - Sensitive Caucasians

(Sardinians, Sephardic Jews, Greeks, Iranians) are more severely affected than sensitive Negroes (145). They are susceptible to clinical hemolysis by a number of drugs in addition to those which cause clinical hemolysis in sensitive Negro volunteers (Table II). , e. g., para-aminosalicylic acid, quinine, quinidine, chloramphenicol, and atabrine(14). Erythrocyte G-6-PD activity may be completely (146). absent as was first demonstrated by L6hr 1 n two sensitive

(124) . Iranians and by Larizza in Italians. Furthermore, the deficiency js easily demonstrable in tissues otlr r than the erythrocyte, i. e.,

(147) platelets , leukocytes (148), saliva (149), and liver (150), They

. _._. . -- - 3h

have no anemia unless drug is administered.

Some abnormalities, which have not been found in the red

cells of sensitive Negroes, have been reported in the erythrocytes of sensitive Caucasians, e. g., decreased oxidized glutathione (151), increased activity of transketolase and transaldolase (152), and a (135, 136) G-6-PD activator in the stroma , Other abnormalities

of the erythrocytes of sensitive Caucasians, not as yet evaluated

in the Negro, are a decreased rate of incorporation of glycine into

glutathione (153) , an increased activity of fructokinase (' 54) and an increased activity of methemoglobin reductase (1 55). Electron- microscopic examination of the erythrocyte stroma from sensitive

Sephardic Jews, recently performed by Danan (' 56) indicate that the'majority of cells are morphologically old. \I I\$4. Congenital Non-Spherocytic Anemia: Eight cases of \! hemolytic anemia have been reported in children who have either a

complete, or almost complete, absence of erythrocyte G-6-PD (157, 158, 19, 159, 160) (Table IV) . All of the children were Caucasian males predominantly of Northern European stock. All had mild chronic anemia and they all developed severe, acute hemolytic anemia during infection or during drug administration. Inheritance in five of the children was by a sex-linked gene; no apparent genetic -37-

source could be demonstrated in the families of the other three. Kirkman (159) has reported that the erythrocyte G-6-Pv was qualitatively as well as quantitatively abnormal in two of the children.

C- IJ hlCAI, TESTS FOR PRIMAQUl NE - SENSITIVITY The original test for primaquine-sensitivity, the Heinz (161,162) Body Test (91) has proved non-specific , often inaccurate, and has been superceded. All subsequent tests are accurate when applied to fully expressed individuals. Normal values may be o!Xained in some intermediate females by all tests; the G-6-PD assay and the

RIethemoglobin Reductiorr Test are more reliable in this regard than the others.

Glutathione Stability Test: Designed by Beutler, this

-_--I_- test is based on his observation that the GSH of sensitive, but not of normal, erythrocytes decreases markedly when the cells are incubated aerobically with acetylphcnylhydrazine and glucose. The determination of glutathione is not simple. False negative results are obtained in

(17) 30- 50 percent of intermediate females (86) %e Reduction Test:_. This test, designed by Motulsky , is based on :he inabiLity of hcmolysatcs of drug-sensitive individuals to reduce the dye brilliant crcxsyl blue. It has been used widely in field screening studies in Sardinia, Africa, and Asia and has the advantage

. . '. of a visual end point in fully expressed" individuals. However, false negative results were reported in a high percentage of intermediate females during a field study in Africa by Allison (23). In addition, variation in results with different lots of the dye have been observed (163)

Assay of G-6-PD Activity: These procedures (summarized by Carson are very useful in research and identify approximately

eighty percent of the intermediate females. A precise spectro-

photometer is required; in general, the direct assay is not recommend-

ed for routine clinical laboratory application. It is primarily a

research procedure.

A colorimetric assay for G-6-PD activity in hemolysates employing phenazine methosulfate as an electron carrier between

TPNH generated by G-6-PD and the dye dichloroindophenol has been (164) introduced by Ells and Kirkman . This assay requires only a siG'ple spectrophotometer or photoelectric colorimeter and may be \, adaptable to visual estimation of G-6-PD activity. Independently, a y> visuai test employing the same dyes as used by Ells and Kirkman has recently been made available commercially in kit-form with all reagents in a single vial (163). Neither of these two dye tests has 39-

had a thorough evaluation; specifically, their reliability in identifying heterozygous females has not been determined.

Methemoglobin Reduction Test: This test, introduced by (146) Brewer (85) is based on the observations, initially made by Lahr and by Dawson (84), that the rate of methemoglobin reduction by sensitive erythrocytes in the presence of methylene blue is markedly slower than normal. It yields similar results to those obtained with the G-6-PD assay, correlates better with the severity of hemolysis in intermediate females, and is more reproducible than the Gibtathione

Stability Test. StandardizaTion of the procedure was achieved by testing a group of non-anemic Kegro women known to be intermediate on the basis of previous clinical hemolysis to a standard dose of primaquine. Approrimately 80 percent of the heterozygous females can be identified with confidence with this procedure when the results are determined spectrophotometrically. A clinical colorimeter or spectrophotometer such as the Coleman Junior model can be employed for quantitative evaluation of drug-sensitivity because only the determination of methemoglobin is required.

A simplification of the Methemoglobin Reduction Test having a visual end point ran lie used as a practical scre.ening procedure.

Only a fc>w inesp,nsivr, easily pr-c,pared, rpagcnts arr rcquirvd rind -40-

whole blood may be employed. All individuals having the defect of sufficient severity to render them susceptible to hemolysis of clinical significance, i. e., fully cxprcsscd males atid fernalcs, and appro-ximately 30% of heterozygous females, can be detected by the visual test. The clinical test is performed as follows: ~II;?~III-:;\~IOCI~OBINREDUCTION TEST: VISUAL METHOD OF HR12WISR ::<

C’oi?ibine~lSodium Nitrite and Gliicost.~ Solution: Bring 1. 25 gm. sodium nitrite (h,Iercli)and 5. 0 gm. glucosc in the sa=- flask up to 100 ml. with distilled water. (The final concentration in this solution of NAN02 is 0. 18 M and of glucose is 0. 28 ?#I). A:L::hvlcnc-~- Blue (0.0004 M): Bi-i!?z 0. 15 gm. trihydrated methylene blue jlrIal;.inckrodt) up to 1000 ml. with distilled water.

(-;:ass Tube.~. Preparation:~ __ (Screw-top glass vkls or srnall test tubes are conve,iient for this purpose. Plastic tubes should not be used). 1. Sample Tube: Pipette precisely 0. 1 ml. of the sodium nitrite-glucose solution and 0. 1 ml. of the methylene blue solution into this tube. 2. __~__-______Positi..ie Rcfercnce Tube: Pipette 0. 1 ml. of the sodiun; nitrite-glucose solution into this tube (omit methylene blue). 3. Normal Reference___ Tube: No reagents are pipetted into this tube. The test may be performed by pipetting the blood directly into the tubes as preparvd above, or the solutions in tubes 1 and 2 may be allowed to evaporate to dryness at room tem:,.-srature and the blood added to the dried rvagents. The lubes containing the dried reagents may be prepared in advance, stoppi‘red, and stored at room temperature for at least 3 months prior to use.

1ilcm:l:~ ~ T~Ptest may be performed immediately on freshly drawn heparinized blood. Otherwise, the blood should i refrigerated immediately at 40 C. and kept andcr one of the foilowing conditio.;,;: depcnding upon the duration of storage:

P~.~~~~eduri~:Arid 2. 0 in!. of the blood to be tested to the Sample tube. Add 2. 0 ml. ~ of bl~w~i(c>iti:er normal, sensitive, or unknown) to both the Positive Reference a::rl S:,. ’ Itcferpnce__ tubes. Only one positive and one normal reference ~,ub[.are i~ccdecifor each batch of tests performed. Mix well by inversion. Incubatc at 37 --t 1 OC., unstoppered, for 3 hours without shaking. After incubation, add 0. 1 ml. of the test mixture to 10 ml. water and 2 to 10 min- utcs later compare it visually to the similarly diluted references.

Interrntdiatc Females: The color varies between red and brown in accordance ivitii the degree of expression of the trait.

~. .i.,,‘e i?r.i;.:.;e?’) Cor a dctailud description; several steps in the precise spectro- ;-!.~jtcl~~iic,tricmethod are unnecessary for the visual screening test. SUMMARY

The efforts of many scientific investigators have helped to define

the unilary nature of many drug or vegetable induced hemolytic anemias

previousiy thought to be d;-ierse. The susceptibility to drug-induced

hemolysis is a genetically acquircd error of metabolism which has been

partially defined in biochemical terms and is most accurately charact-

erized by a dcficiency of glucose -6-phosphate dehydrogenase in the

erythrocytes and, to a ;-ariable degree, in other tissues. The abnormal-

ity is inherited by a gene of pirtial dominance carried on the X

ci:romcsome (sex-lin!ied); affected males, therefore, are fully expressed

.. __ (A;.I ,,, v/hile females may have full expression (XX) or, more often, partial expression (e)of tlx trait. The mutant gene usually occurs in

the more dar!:ly pigmented racial and ethnic groups and may reach a

frequency of 50 percent in some populations. The geographic distribution

is broad in tropical and semi-tropical areas. and teiids to parallel the

occurrence of falciparum malaria, against which it may offer a biological

advantage. The number of drugs capable of inducing hemolysis already

exceeds -4i-t~and i:icludes antimalarials, sulfonamides, nitrofurans, antipyretics, analgesics, sulfones, vitamin K analogues, fava beans, and other vegetables. Hemolysis induced by infections and diabetic acid-

osis in affected indi;iduals may eventually prove to be even more

common clinically than that induced by drugs, but the role of these factors cannot be fully evaluated at present. Perhaps of even greater

importance are the recent observations of a very high incidence of

G-6-PD deiiciency in unexplained neonatal jaundice and kernicterus

o c: curri ng in n c wbo rns.

Thc erythroc>?zs ol' drt::-su:;:ceptiblc individuals are deficient in

glucose- 6-phosphat2 deI~.ydrog-nase and, usually, in the reduced form

of glutathione and catalase. Susceptibility of the erythrocytes to

hemolysis is probably due to thcii- inability to regenerate TPNH with

sufficient rapidity to comba? the oxidant effect of the partially degraded

drugs. When drug is administ;.red, thcrefore, ferro-catalase, GSH, fcrro-humoglobin, and globin arc oxidized and the denatured hemoglobin precipitztcs into coccoid granules called Heinz bodies. Intravascular lysis and lic~111 oglo b inu ria follow .

Tlw scv(.rity of hemolysis is dosage-dependent. The hemolysis is sc.2-iinii~,o:lif thc, initial dose is not excessive even when the same

~IOSC~of c!r.ug is rontinut~d,bi?caiise the older and most susceptible erythrocytes are destroyed and the remaining younger erythrocytes arc relatively resistant to hemolysis. However, the severity of hemo- lysi.> can be enhanccd or mitigatcd by many factors and is often unpredictable in clinical practice. Several laboratory methods have been devised for identifying susceptible individuals; the Methemoglobin

Reduction Test is reliable, simple, and inexpensive to perform.

The importance GA ;his type of drug-induced hemolysis is emphasized by the increasing number of drugs and systemic diseases ivliic:l~ may prccipitatc stlvcrc slir:il-lia. Only by alertness on the part of physicians at the bedside will t!lr? danger of the primaquine-type hemolysis bc completely defined. I I ~I ! I 9 ! 169

T A B L E I. Statistical &lysis (chi-square) of the A, B, 0 and Rh blood group distribution among 89 primaquine-sensitive, and 184 normal, American Negro males. The relatively low frequency of E in primaquine-sensitive Negroes is highly significant. If E MOLY TI C C 0 M POU N -DS

LWTI MA LARIA LS ANTIPYRETICS & ANALGESICS Primaquine Aspirin Pamaquine Acetanilid Pcntaquine Acetophenetidin (Phenacetin) Plasmoquine Aminopyrine (Pyramidon) (C) SN 3883 Antipyrine (C) SN 15324 CN 1110 SULFONES CN 11 15 (Quinocide) (52) Sulfoxone (Diasone) Quinacrine (Atabrine) (53) Thiazosulfone (Promizole) Quinine (C) Diaminodiphenyl Sulphone (DDS)(25)

SULFONAMIDES- OTHERS Sulfarli lami de Dimercaprol (BAL) (55) NZ Acctylsulfanilamide Methylene Blue (56) Sulfacetamide (Sulamyd) Naphthalene (Moth Balls) Sulfamethoxypyridazine Para-aminosalicylic Acid (Kynex, Rfidicel) Phenylhydrazine Salicylazosulfapyridine (Azulfidine ) Acetylphenylhydrazine Sulfisoxazole (Gantrisin) Probenecid (Benemid) Vitamin K (Water Soluble Analogues) (57)

NITROW KANS Chloramphenicol (C) Quinidine (C) (14) Nitrofurantoin (Furadantin) Trinitrotoluene (14) Furazolidone (Furoxone) Fava Beans and other Vegetables (C) (14) Furaltadone (Altafur) Nitrofurazone (Furacin) (54)

TABLE 11; Specific references other than those noted, can be obtained from the recent reviews by Kellermeyer (58) and Beutler (59). The (C) denotes drugs whLch cause hemolysis in sensitive Caucasians (I4) bat which are not, or am only k,lightly, hrmolytic in healthy, sensitive, Negro males. The susceptibility of pl-imacjuinc-s(’IlsitiVt’ Ainericaii Negroes to hemolysis by fava beans is unknown. NORMAL NEGRO MEN II SENSITIVE NEGRO MEN

Wintrobe Micro Wintrobe Micro True True

Hct. % Hct. =% Hct. % 70 Hct‘ 1 Deviation Deviation Hct’ 1 Deviation Hct’ 1 Deviation I I48.5 + 1.7 46. 5 - 2.5 45.3 48. 5 + 7.1 46. a + 3.3 47.7

46.5 48.0 +3.2 46. 3 - 0.4 48.4 50. 5 f4.3 ~ 48.8 f0.8

50.7 52.0 + 2.6 49. a - 1.8 43.2 46.5 +7.6 45.2 + 4. 6

I MEAN DEVIATIONS -+ 2.5 1.6- 11 MEAN DEVIATIONS -+ 6. 3 -+2.9 I

TABLE I11

A greater than normal volume of plasma was trapped intercellularly by erythrocytes from three primaquine-sensitive men during hematocrit determinations compared to the true hematocrit determined by the I 131-

albumin method of Owen (’i7). The Wintrobe tubes were centrifuged for one hour at 4* C. with a centrtfugal force of 2200 x g at the distal tip. The micro-capillary tubes were centrifuged for three and one-half minutes at room temperature with a centrifugal force of 12,000 x g at the distal tip. T .A B 1- E I \-. 1IEI’ABOLIC CHARACTERISTICS OF THE ERYTHROCYTES

1. DERCIEKT GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY (9)., primar: 11. OTHER ABNORMALITIES OF THE PENTOSE PHOSPHATE PATHWAY; impaired A. DIR.IThTSHED TPNH, INCREASED TPX, CONTENT (81,64) B. DIMINISHED RESPONSIVENESS TO REDOX DYES

.rlrnN?30

IV. ABNORMALITIES OF TIE EMBDEN-MEYERHOF PATHWAY A. INCREASED ALDOLASE ACTIVITY (93)., ? compensatory, or a reflection B. DECREASED DPNH AND INCREASED -DPN CONTENT (81,641 C. FALL IN ATP CONTENT- IN VITRO WITH ACETYLPHENYLHYDRAZINE \ V. DECREASED CATALASE ACTIVITY AND FURTHER FALL DURING DRUG-INDU NORAWL METABOLIC CHARACTERISTICS I?. - .-

$1 - PFJOSPHCGLUCONIC DEHYDROGENASE (9). PENTOSE CONTENT (83’.<- PU TK.4NSA1,DOLASE (83), PHOSPHOHEXOSE ISOMERASE (82), ISOCITRIC DEH CHOLINESTERASE (55), GLYCERALDEHYDE -PHOSPHATE DEHYDROGENA! LACTIC DEHYDROGENASE (82) and GLUTATHIONE PEROXIDASE (55)

*For a complete discussion of the metabolic characteristics see the rccent rex .- TOTAL RBC LIPIDS -3 U GM. x 10 per GM. HEMOGLOBIN

PRIMAQUINE NORMAL SENSITIVE

18.88 19. 14 17. 91 19. 57 16. 93 18.18 18. 60 17. 29 16. 20 17. 83 18.77 18.12 16. 11 18.80 17. 56 19.14 15. 92 16. 78 18.38 18.59

18. 39----MEAN----17. 38 16. 78-19. 14--- RANGE ---15. 92-18. 77

t = 2.3 p = <.os

1 I

T Ai3 LE V. The total lipid content of the erythrocytes (expressed per gram hemoglobin) of 9 primaquine-sensitive and 11 normal Negro males was determined gravimetrically according to the method of Reed (92). Each value represents the average of at least 3 determinations on different days (maxtmum variation +4%). When expressed as grams lipid per milliliter of packed RBC the values for primaquine-sensitive and normal, respectively, were as follows: mean = ,0054, .0058; range = ,0051-. 0058, .0056-. 0062; t = 3.98, p = <. 0001 C L U C OS E - 6 - 1' HOS P HA T E D E H Y D R0G EN AS F: D E F IC [E N C Y LV IT H C 0 3 C E N IT A 1, N 0 NSP H E R 0 CY T IC C H R 0 N IC H E hI 0 L Y 'r IC AN E M I A TABLE VI

MODE OF CLrNNICAL C.UE SEX AGE NATIONALITY G-6-PD REFERFNCF INHERITANCE CHARACTERISTICS

Neonatal Jaundice Marked 1 d 4 It. None Apparent Acute Hemolysis hr- Decrease -~ ing Infection or Drug Neonatal Jaundice Marked (1 57,1581 2 d - Not Given None Apparent Acute Hemolysis Dur- N Decrease wt ing Infection or Drug

Neonatal Jaundice Marked 3 d - Not Given None Apparent Acute Hemolysis Dur- Decrease ing Infection or Drug

Neonatal Jaundice E. R. d 6 Ger., Eng. Sex-Linked Acute Hemolysis Dur- Absent ing Infection or Drug

Acute Hemolysis Dur- (19) Ed. d 2 1/2 Ger., Eng. Absent Zinkham R. 1 Sex-Linked ing Infection or Drug

Icterus in Infancy A.K. d 10 Eng., Fr., Ger. Sex-Linked Acute Hemolysis Dur- Absent ing Infection or Drug

Karked Quantita- Acute Hemolysis Dur- 1 2/3 Ir., Du., Eng. Sex- Linked .ive and Qualita- in2 Infection or Drug tive Differences :irkman (159.1 60 Neonatal Anemia !larked Quantita- 1\- - 2 3 Ir., Du., Eng. Sex- Linked Acute Hemolysis Dur- ive and Qualita- T is2 Jnfection oi- Dmg :ive Oi.fierrences REFERENCES

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144. Marks, P.A.; Gross, R. T., and Banks, J.: Evidence for Heterogeneity ' Among Subjects with Glucose-6-Phosphate Dehydrogenase Deficiency., (Abst.) J. Clin. Invest. 40: 1060-1061 (Jnne) 1961.

145. Marks. P. A,, and Gross, R.T.: Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency: Evidence of Differences Between Negrczes and Caucasians with Respect to this Genetically Determined Trait, J. Clin. Invest. 38: 2253-2262 (Dec.) 1959.

.. .. 166. Bowman, J. E. and Walk.r, D. G. : Virtual Absence of Glutathione Instability of the Erythrocytes Among Armenians in Iran. Nature, 191: 221-222 (July 15) 1961.

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Fig. I. The typical course of the acute hemolysis indu, ed by 30 mg. smaquine- base daily in a drug-sensitive negro male At the peak of hemolysis the urine is usually dark, sometimes black, and may give a positivc bemidin<:test. The acute blood destruction is self-limited, even with continued drug administration, and clinical recovery follows because the remaining young erythrocytes are relativcly rcsistant to drug. The life-span of drug- sitive erythrocytes is shorter tka n normal even before(bqyg '"'and is further diminished during the equilibrium phase even though clinical hematological recovery is complete.

Fig. 2. -The severity of drug-induced hemolysis in relation to the daily -do*

Drug was begun on day zero. The Cr 51 RBC survival is expressed as the percent of labeled cells remaining, uncorrected for isotope elution. (Data of Kellermeyer and Tarlov).

Fig, 3 __Mitigation of the hemolytic effect of primaquine by intermittent weekly drug administration- The hemolysis induced by primaquine can be decreased, without loss of therapeutic effectiveness against vivax malaria by administering large doses once a wcek (weekly scale on '1 the right) rather than samll doses daily (daily scale on the left) (52) . \' Fig. 4i\ The increased resistance of primaquine-sensitive erythrocytes \: to o-tdc lysis \,. The increased resistance occurs throughout the entire range of saline concentrations (lcft). The difference between the mean values of percent hemolysis in 0.425% saline of 14 drug-sensitive, and 15 normal, Negro males ri ht) is highly significant (P=,00001). Parpart's method 1755 was employed at 4 OC using saline solutions buffered with phosphate to pH 7.4. ?! \',

\ Fig. 5. 'Pathways-- of carbohydrate metabolism in mature human erythrogd(.s__ Ten percent of the glucose utilized by normal erythrocytes under physiological conditions is metabolized via the pentose - phosphate pathway (79). The pentose pathway is defective in dmg- sensitive erythrocytes because of a marked deficiency of the enzyme glucose-6-phosphate dehydrogenase. Abbreviations: ADP, adenosine diphosphate; ATP, adenosine triphosphate: DPNH, DPN, reduced and oxidized diphosphopyric~ne nucleotide; GAPD, glyceraldehyde phosphate dehydrogenase; GSH, GSSG, reduced and o~dizedglutathione; Hb+t-, hemoglobin; Met%+++, methemoglobin; LD, lactic dehydrogenase; PHI, phosphohexose isomerase; TPNH, TPN, reduced and oxidized triphosphopyridine nucleotide. fig. 6. Methylene blue decreases the rate of glucose utilization by primaquine-sensitive erythrocytes in contrast to its effect on normal erytiirocytes (55). The erythrocytes (defibrinated blood) were prepared free of leukocytes and platelets by four successive centrifugations in plasma after each of which the top 10% of the cellular trass was discarded. The final RBC preparation, therefore, was relatively enrJ (fi ed with older cells, the younger cells having been discarded. 'I'he RBC were incubated in plasma at a hematocrit of approximately 40% at 37°C for 3 hours with constant gentle shaking in air (PH bctwcen 7. 7 - 8.1) with supplemental glucose to an initial concentration of approximately 300 mg. %. In control stddies without methylene blue the rate of glucose utilization for 9 primaquine-sensitive RBC preparations was mean- 6.7 p moles/ml. RBC/hct. 40%, range 4. 9 - 9.4, and for 8 normal RBC preparations mean = 6.0, range 3.8 - 7.6. This difference is not significant (t = 1.03, p = .3).

Fig. 7. Biochemical changes in the erythrodytes of primaquine-sensitive Negro males during the course of acute hemolysis induced by 30 mg. primaquine base daily

The catalase activity is expressed as mEq sodium perborate decomposed by 1.0 ml. of a 0.015 gm. 70 Hb. emolysate solution in a final volume of 10 ml. (98). The reducedk glutathione (GSH) was calculated as mg. GSH per 100 ml. RBC The total RBC Lipids were determined gravimetrically b the method of Reed (92) and are expressed as grams of lipid x 10 -'per gram of hemoglobin. The G-6-PD acti 't was assayed by the method of Kornberg adapted to he moly satesy9il) and is expressed as the change in optical density at 340 m p (rate of TPNH formation). Fig. 8. -_The ___ anti-catalase activity in the plasma of a normal individual duringingestion of single daily doses of 120 mg. primaquine base .-__ - The relationship of the initial portion of this curve to that of drug degradation suggests that the anti-catalase factor is a v: v: degradation product of primaquine rather than primaquine itself. The fall in the slope of the curve indicates that an alteration in drug metabolism occurs during continued drug administration. The first portion of the curve (to the break) represents the first '24 hours after a single dose given at zero time. Subsequently, the p!asma samplcs were collected 24 hours after the preceding dose, but prior to the next dose of drug. The anti-catalase activity was assayed as follows: Aliquots of the plasma were stored at -200 C until assayed (identical results were obtained after 10, 40, and 70 days of storage). Seven ml. of the plasma were incubated with 3 ml. of packed primaquine- sensitive erythrocytps at 37O C in air for 240 minutes without shaking with glucose supp1ementc.d to 200 mg. 70. The anti-catalase units express the percrnt inhibition of the erythrocyte catalase activity. Catalase was determined by the sodium perborate method as described (98).

Fig. 9. Postulated bio-degradation of primaquine in man

Indirect evidence indicates that during the degradation of primaquine to a quinoriimine oneor more intermediate products (quinones) are formed which are resonating and, therefore, potentially can undergo reversible oxidation- reduction (indicated above by reversible arrows). The above theoretical scheme for the dcgradation of primaquine is modeled after the degradation of pentaquine in monkeys as proposed by Smith who confirmed the fundamental concepts of Schonhofer 14) for the degradation of pamaquine. W I- >- u 0 a I >t- K W 1 I I I I A- 7 14 21 2a 7 14 21 28 DAYS'

Figure 2 HEMOLYTIC EFFECT OF PRIMAQUINE

i b0 YO WIfXlY i Figure 5 GLUCOSE UTILIZATION BY ERYTHROCYTES (FREE OF LEUKOCYTES1 I t60 -

W In2 +45- a V +30 - Normal- // s +I5 -

--- 0- Y In U w -J5 - a u W Sensitive - -30 - 'S I I I I I

0 10-6 IO+ -w-4 METHYLENE BLUE - MOLAR CONCENTRATION

Figure 6 46

HEMATOCRIT 41 36

,300 CATA-ASE ,200

53 GSH 43

21 Tow RBC LIPIDS 18 15

.250 I I G-6-PO .OJO I I 9

I 1 1 I I 1 I I DAYS --5 0 5 10 15 20 25 30 35 I I

Figure 7 ANT I - C ATAL AS E ACT I V I T Y I N PL AS M A

I '"1 I c r -51 I "

0 4 8 12 210 30 50 70 -HOURS +I- DAYS

Figure 8 3

I II: P r i moquine 6-hydroxy - C

N-R N-R .. : >. -3 0”0”til p /

5,6-di hydroxy- 5,6-quinonk- q u inonimine -

Figure 9