THE REDUCTION OF METHEMOGLOBIN INHUMAN ERYTHROCYTES INCUBATED WITHPURINE NUCLEOSIDES

Ernst R. Jaffé

J Clin Invest. 1959;38(9):1555-1563. https://doi.org/10.1172/JCI103934.

Research Article

Find the latest version:

http://jci.me/103934/pdf

Pdf

THE REDUCTIONOF METHEMOGLOBININ HUMANERYTHRO-CYTES INCUBATEDWITH PURINE NUCLEOSIDES* t

By ERNSTR. JAFFE

(From the Department of Medicine, Albert Einstein College of Medicine and the BronxMunicipal Hospital Center, New York, N. Y.)

(Submitted for publication March 30, 1959; accepted April 23, 1959)

This study is concerned with the effect of purinenucleosides and related compounds on the reduc-tion of methemoglobin to hemoglobin in humanerythrocytes in vitro. Other investigators havereported that mammalian erythrocytes can re-duce methemoglobin to hemoglobin when thecells are incubated with glucose (2), lactate (3),glyceraldehyde, fructose (4), fumaric acid, malicacid (5), mannose, galactose (6), formaldehyde(7) and other aliphatic and aromatic aldehydes(8). Previous studies in this and other labora-tories have demonstrated that normal humanerythrocytes can metabolize the pentose moiety ofcertain purine nucleosides to lactic acid in vitro(9, 10). This metabolic activity is associated withenhanced resistance to osmotic stress (11, 12), in-creased organic phosphate esters (11), retentionof potassium (11) and extrusion of sodium (13),maintenance of reduced glutathione (14, 15) andsome prolongation of viability of erythrocytesstored in vitro (11, 16, 17).

In the present investigation it was observedthat normal human erythrocytes in which 60 to80 per cent of the hemoglobin was chemicallyoxidized in vitro could reduce a considerable por-tion of the methemoglobin to hemoglobin whenincubated with certain purine nucleosides andsugars. This effect was associated with activemetabolism of the compounds by the cells. Themetabolism of these compounds and their effectson the reduction of methemoglobin in erythro-cytes obtained from a patient with congenitalmethemoglobinemia were also studied.

* This work was presented in part at the Eastern Sec-tion Meeting of the American Federation for Clinical Re-search at Boston, Mass., December 12, 1958 (1).

t This investigation was supported by grants from theNational Heart Institute of the National Institutes ofHealth, United States Public Health Service (GrantH-2803), the Atomic Energy Commission [Contract AT(30-1) 1855], and the Office of Naval Research [Con-tract Nonr-1765 (00)].

MATERIALS AND METHODS

Whole blood was obtained from normal adults and wasanticoagulated with about 1 mg. dry heparin per 10 ml. ofblood. The volume of packed erythrocytes was deter-mined by the method of Wintrobe (18). A portion of theblood was incubated at 370 C. with occasional mixing,after the addition of 0.6 to 0.9 mg. of sodium nitrite perml. of packed erythrocytes. The sodium nitrite was dis-solved in 1 ml. of isotonic sodium chloride solution.After 40 minutes' incubation, the treated blood was cen-trifuged at 1,500 G for seven minutes and the erythrocyteswere washed three times with about three volumes of0.9 per cent sodium chloride solution; the final centrifuga-tion was carried out in a Servall Refrigerated AngleCentrifuge for 10 minutes at 1,900 G and at 4° C.(SS-1 rotor). A 25 per cent suspension of the treatedand washed erythrocytes in isotonic sodium chloride solu-tion was prepared and the actual volume of packed eryth-rocytes was determined. This procedure resulted in theconversion of 60 to 80 per cent of the hemoglobin tomethemoglobin. Four ml. portions of the erythrocytesuspension were added to 25 ml. Erlenmeyer flasks con-taining 2 ml. of isotonic sodium chloride-sodium phos-phate buffer solution (equal volumes of 0.9 per cent so-dium chloride solution and isotonic sodium phosphatebuffer, pH 7.3) in which were dissolved the compoundsto be studied. The pH of these final suspensions was7.3 to 7.4. The flasks were stoppered loosely with cot-ton plugs and incubated at 37° C. in a Dubnoff metabolicshaker-incubator oscillating 92 cycles per minute. Inexperiments where hemolysates were used, 50 per centsuspensions of treated and washed erythrocytes wereprepared and the erythrocytes were hemolyzed by twicefreezing and thawing rapidly. Two ml. of the hemoly-sate was added to flasks containing 3 ml. of distilled water,1 ml. of isotonic sodium phosphate buffer, pH 7.3, andthe dissolved compounds.

At intervals during the incubation, 0.4 ml. portions ofeach suspension were transferred to 10 ml. of 0.0167 Mphosphate buffer, pH 6.6, and the methemoglobin con-centration was determined by the method of Evelyn andMalloy (f9), adapted for the Coleman Junior Spectro-photometer. The amount of methemoglobin was ex-pressed as the per cent of the total hemoglobin pigment.The methemoglobin concentrations of the whole bloodprior to treatment with sodium nitrite and of the eryth-rocyte suspensions or hemolysates prior to incubationwere also determined. Duplicate samples assayed by this

1555

ERNSTR. JAFFE

spectrophotometric method agreed within + 1.5 per cent.In a few experiments, the methemoglobin concentrationswere also estimated from the difference between the totalhemoglobin content, determined by comparison with acyanmethemoglobin standard (20), and the hemoglobincontent determined by the oxygen binding capacity of theerythrocyte suspensions, determined in the laboratory ofDr. Charles W. Frank. The spectrophotometric and thegasometric methods agreed within ± 4.5 per cent. Thespectrophotometric method described recently by Millsand Randall (21) was employed in some experimentsto permit the calculation of both methemoglobin and cho-leglobin concentrations. The methemoglobin concentra-tions by this method and by the method of Evelyn andMalloy agreed within +2 per cent. Significant chole-globin concentrations were not detected in erythrocytesuspensions during 22 hours of incubation with the variousnucleoside and carbohydrate substrates and only 1.8 percent was present in a suspension incubated with ascorbicacid.

Penicillin G and streptomycin sulfate, 1 mg. of each,were added to the incubation flasks in most of the ex-periments detailed in this paper. Even after 22 hours ofincubation there was no bacterial contamination as evi-denced by the absence of growth when aliquots of thesuspensions were cultured on blood agar and in infusionbroth. Identical results in the reduction of methemoglobinto hemoglobin were observed, however, in experimentsperformed with and without added antibiotics.

Lactic acid determinations were performed by themethod of Barker and Summerson (22) on clear filtratesof perchloric acid extracts prepared by adding one vol-ume of each erythrocyte suspension to six volumes ofcold 2 per cent perchloric acid after the suspensions hadbeen incubated for six hours.

Determinations of the pH of the erythrocyte suspensionsafter 22 hours of incubation were made with a BeckmanModel G glass electrode pH meter. The pH of the con-trol suspensions ranged from 7.2 to 7.5, but the pH ofthe suspensions incubated with the various compoundsdid not differ from that of the control by more than± 0.1 pH unit. These changes in the pH could not becorrelated with the effect of the compounds on the reduc-tion of methemoglobin to hemoglobin.

The degree of spontaneous hemolysis that occurredduring incubation was determined by adding 0.2 ml.aliquots of each suspension to 2 ml. of isotonic sodiumchloride solution and 2 ml. of distilled water. The amountof hemoglobin was determined, after centrifugation anddilution of 1 ml. of the supernatant solution with 4 ml.of distilled water, by measuring the optical density at540 mgu in a Beckman DU spectrophotometer. The percent spontaneous hemolysis was then calculated by relatingthe optical density of the isotonic sodium chloride solu-tion sample to the distilled water sample whose hemolysiswas taken as 100 per cent. Spontaneous hemolysis, evenafter 22 hours of incubation, did not exceed 4 per cent.

Most of the compounds used in these experiments were

obtained commercially. The 2,6-diaminopurine riboside(2,6-diamino-9-p-D-ribofuranosyl purine) and the ade-nine glucoside (9-fi-D-glucopyranosyl adenine) were syn-thesized and provided by Dr. Bertram A. Lowy. Sam-ples of purine riboside (9-p-D-ribofuranosyl purine) andadenosine-1-N-oxide were generously provided by Dr.George B. Brown. Mr. David Schwarz of Schwarz Lab-oratories supplied the adenosine-5'-phosphoramide. Dr.Henry M. Kissman of the Lederle Laboratories Divisionof the American Cyanamid Company kindly supplied thePuromycin Hydrochloride® (6-dimethylamino-9 (3'-p-methoxy-L-phenylalanyl amino-.8-D-ribofuranosyl) purine),dimethyl-adenosine (6-dimethyl-amino-9-,8-D-ribofuranosylpurine), amino-adenosine (6-amino-9 (3'-amino-3'-deoxy-j-D-ribofuranosyl) purine), aminonucleoside (6-dimethyl-amino-9 (3'-amino-3'-deoxy-,-D-ribofuranosyl) purine)and aminoribose (3-amino-3-deoxyribose) hydrochloride.The concentrations of the compounds were expressed asmicromoles per milliliter of packed erythrocytes and werecontained in a final volume of 6 ml.

Case history. The patient (E. D., Hospital No. 30216)was a 30 year old Puerto Rican woman who was seen inthe Prenatal Clinic of the Bronx Municipal HospitalCenter during the eighth month of her fifth pregnancy inNovember, 1957. At that time cyanosis of the hands, lipsand vaginal mucosa was noted. She reported frequentheadaches, slight exertional dyspnea and easy fatigability.Except for these findings, physical examination was en-tirely normal with a normal intrauterine pregnancy.The hemoglobin concentration was 11.6 Gm. per cent, thehematocrit 40 per cent and the erythrocytes appearedslightly hypochromic on the stained blood smear. Thereticulocyte count ranged from 0.8 to 1.5 per cent. Thewhite blood cell count, differential count, urine analysis,serology, roentgenogram of the chest and liver chemis-tries were within normal limits. Spectrophotometric ex-amination of hemolysates prepared from whole blood andfrom washed erythrocytes obtained from a sample ofthe patient's blood revealed the absorption spectrum ofmethemoglobin as characterized by a peak at 630 mAu thatdisappeared on the addition of sodium cyanide. On re-peated examinations the concentration of methemoglobinranged between 23 and 26 per cent, but on one occasionwas 31 per cent. One hour after the intravenous adminis-tration of methylene blue, 1 mg. per Kg., the patient'smucous membranes were of a normal pink color and theconcentration of methemoglobin was 1 per cent. Themethemoglobin concentration was maintained at a levelof 9 to 13 per cent by the daily oral administration of500 mg. of ascorbic acid and was accompanied by re-lief of her symptoms. That the abnormal hemoglobin inthe erythrocytes of this patient was actually normalmethemoglobin and not hemoglobin M was further es-tablished by spectrophotometric analysis of a hemoly-sate treated with potassium ferricyanide (23) and bystarch block electrophoresis of a hemolysate kindly per-formed by Miss Margaret Pease through the cooperationof Dr. Park S. Gerald.

1556

REDUCTIONOF METHEMOGLOBINBY PURINE NUCLEOSIDES

NORMALHUMANERYTHROCYTES

z

0

0

0

Jx

I-z

V

Lai

10 pM per ml. ERYTHROCYTES.EXCEPT20pM LACTATE

WHOLEBLOOD: 0.6% METHEMOGLOBIN

HOURSAT 37"C.FIG. 1. EFFECT OF INCUBATION WITH VARIOUS COM-

POUNDSON THE REDUCTIONOF METHEMOGLOBININ NOR-MAL HUMANERYTHROCYTES

RESULTS AND DISCUSSION

Normal human erythrocytes

Data from a typical experiment are presentedin Figure 1 and Table I, Column A. Incubationwith 10 MuMoles per ml. of erythrocytes of inosine,adenosine, deoxyribose, glucose and deoxyinosine.as well as L (+)-sodium lactate, present in twicethe molar concentration of the other compounds,resulted in the reduction of methemoglobin to

TABLE I

Lactic acid present after six hours of incubation of A) normalhuman erythrocytes and B) erythrocytes of con-

genital methemoglobinemia with purinenucleosides and sugars

A BErythrocytes

Normal of congenitalhuman methemo-

Compound erythrocytes globinemia

,uMoLes/ml.* juMoles/ml.*Glucose 1.09 1.51Inosine 1.02 1.18Adenosine 0.99 1.18Deoxyinosine 0.96 1.07Deoxyribose 0.69 0.73L (+)-sodium lactate 0.68 0.87Ribose 0.25 0.31Ascorbic acid 0 0

* Micromoles of lactic acid per micromole of substrateadded per milliliter of erythrocytes, corrected for control.

hemoglobin at a much greater rate than occurredin the control erythrocyte suspension incubatedwithout substrate. After six hours of incubation,almost identical amounts of lactic acid were pre-sent in the perchloric acid extracts of the eryth-rocyte suspensions incubated with adenosine, ino-sine and deoxyinosine, while perhaps slightlymore was found in the suspension incubated withglucose. Although about 30 per cent less lacticacid had been formed after six hours of incubationwith deoxyribose, the effect on methemoglobinconversion was only slightly less than that of thepurine nucleosides and glucose. The extent ofreduction of methemoglobin by deoxyribose wasindistinguishable from that of the purine nucleo-sides and glucose after 22 hours. Incubationwith ribose resulted in slower reduction of methe-moglobin to hemoglobin than did incubation withthe other compounds, and after six hours onlyabout one quarter as much lactic acid was pres-ent as in the suspensions incubated with thepurine nucleosides and glucose. L ( + ) -sodiumlactate, 20 MLMoles per ml. of erythrocytes, af-fected the reduction of methemoglobin at aboutthe same rate as deoxyribose, 10/lMoles per ml.After 22 hours, however, the effect of lactate wasessentially the same as that of the purine nucleo-sides and sugars. About 30 per cent of the lactateadded had disappeared from the total erythrocytesuspension after six hours of incubation. Al-though initially ascorbic acid had almost the sameeffect as the various purine nucleosides, glucose,deoxyribose and lactate, this compound was theleast effective after 22 hours of incubation. Fromthe data presented in Table II it is apparent thatincubation with guanosine, 2,6-diaminopurineriboside, xanthosine, deoxyadenosine and deoxy-guanosine also had a marked effect on the reduc-tion of methemoglobin to hemoglobin. In everyinstance, after 22 hours of incubation, the effectof the purine deoxyriboside was slightly greaterthan the effect of the corresponding purine ribo-side. The reason for this observed difference ineffect is obscure. Although incubation withfructose and galactose promoted the reductionof methemoglobin to hemoglobin, these hexoseswere less effective than an equimolar amountof glucose. A significant effect from incubationwith fumaric acid could be demonstrated only

1557

ERNSTR. JAFFE

TABLE II

The effect of purine nucleosides, sugars and related com-pounds on the reduction of methemoglobin

in normal human erythrocytes

Methemoglobin afterInitial - incubation for:

Concen- methemo-Compound tration* globin 3 hrs. 6 hrs. 22 hrs.

% % % %Control 70 69 67 62Adenosine 8 47 38 24Deoxyadenosine 8 50 31 18

Control 65 65 62 50Inosine 10 42 28 13Guanosine 10 44 30 12Deoxyguanosine 10 45 24 5

Control 62 59 60 47Adenosine 8 43 32 122, 6-DAPRt 8 44 31 10Xanthosine 8 50 40 13

Control 77 73 73 63Glucose 9 57 43 20Fructose 9 68 56 32Galactose 9 69 63 37Fumaric acid 19 68 64 48

Control 65 65 63 56Adenosine 10 42 29 15Aminoadenosine 10 63 60 39

* Concentration in micromoles per milliliter of erythro-cytes.

t 2,6-Diaminopurine riboside.

when this compound was present in twice themolar concentration of the other sugars. Theseexperiments demonstrated that the compoundseffective in promoting the reduction of methemo-globin to hemoglobin included purine ribosides(adenosine, guanosine, inosine, 2,6-diaminopurineriboside and xanthosine), purine deoxyribosides(deoxyadenosine, deoxyguanosine and deoxy-inosine), hexoses (glucose, galactose and fruc-tose), pentoses (ribose and deoxyribose), fumaricacid, L (+)-sodium lactate and ascorbic acid.

Although the precise mechanisms by whichmethemoglobin in intact erythrocytes is reducedto hemoglobin are still unknown, there is con-siderable evidence that one or more enzyme sys-tems are involved. The normal process of methe-moglobin reduction is dependent upon the integ-rity of the erythrocyte (2, 24), is associated withcarbohydrate metabolism (4, 25) and requires theregeneration of reduced pyridine nucleotides (4,26). Recently, partial purification, identificationand characterization of an enzyme from humanerythrocytes whose existence was implied by the

investigations of Warburg and Christian (24)and which was named methemoglobin reductase(Himiglobinreduktase) by Kiese (4) have beenreported (27-30). It was postulated (28, 30)that this enzyme has two prosthetic groups: 1)an unknown carrier, perhaps ionic iron, which isdetached upon hemolysis and purification andthat can be substituted for by methylene blue orother autoxidizable dyes, and 2) a tightly boundiron porphyrin moiety. This enzyme requiresreduced triphosphopyridine nucleotide (TPNH),but it can also utilize reduced diphosphopyridinenucleotide (DPNH) at a slower rate. There mayexist, however, in normal human erythrocytestwo separate reductases, one dependent uponTPNHand the other dependent upon DPNH(4,31, 32). It has been suggested that the systemthat utilizes DPNH is the one that reducesmethemoglobin to hemoglobin in normal humanerythrocytes in the absence of methylene blue (32).

Metabolic pathways for the regeneration ofeither of the two reduced pyridine nucleotides arepresent within normal human erythrocytes. Forexample, reduction of triphosphopyridine nucleo-tide (TPN) to TPNH can occur via thehexose-monophosphate shunt upon metabolism ofglucose-6-phosphate to 6-phosphogluconate and6-phosphogluconic acid to ribulose-5-phosphate.Reduction of diphosphopyridine nucleotide (DPN)to DPNH can occur by way of the Embden-Meyerhof pathway upon the oxidation of glycer-aldehyde-3-phosphate to 1,3-diphosphoglycerateand by the oxidation of lactate to pyruvate.

The results of the experiments described heremay be explained by the metabolism of the riboseor deoxyribose portion of the purine nucleosidesby way of the hexose-monophosphate shunt andthe Embden-Meyerhof pathways with the regen-eration of reduced pyridine nucleotides. Phos-phorolytic cleavage of inosine or guanosine byerythrocyte purine nucleoside phosphorylase (33-35) results in the formation of ribose-1-phosphatethat can be isomerized to ribose-5-phosphate.This latter compound can enter the hexose-mono-phosphate shunt with the formation of hexosephosphate and triose phosphate (36) which maybe metabolized further by way of the Embden-Meyerhof pathway. Adenosine is deaminated toinosine by an active erythrocyte adenosine deami-nase (37), but the reactions involved in the

1558

REDUCTIONOF METHEMOGLOBINBY PURINE NUCLEOSIDES

utilization of 2,6-diaminopurine riboside andxanthosine are not yet known. Since erythrocytepurine nucleoside phosphorylase can apparentlyalso cleave deoxyinosine (33), deoxyribose fromthe deoxyribosides could also enter a metabolicpathway the details of which have not been eluci-dated in the erythrocyte. The observation thatribose and deoxyribose were effective in promot-ing the reduction of methemoglobin to hemo-globin and that lactic acid accumulated when eryth-rocytes were incubated with these pentoses is ofsome interest. Other investigators have stated thatribose is not metabolized by erythrocytes in vitro(38), although increased oxygen uptake by rabbiterythrocytes incubated with ribose, arabinose andxylose plus methylene blue has been reported(39). The pathways by which ribose and deoxy-ribose are metabolized are unknown, but theymight involve phosphorylation and entry intothe hexose-monophosphate shunt and Embden-Meyerhof pathway. Glucose, fructose and galac-tose are metabolized by these pathways with theregeneration of reduced pyridine nucleotides.Fumaric acid may be metabolized to malic acid byfumarase and the latter can be oxidized to oxalo-acetate by malic acid dehydrogenase with the re-

duction of DPN to DPNH. These two enzymes

of the tricarboxylic acid cycle are known to bepresent in mature mammalian erythrocytes (38).Since the metabolism of these various substratescan lead to the regeneration of DPNH, the re-

ported observations are consistent with the conceptthat the DPNH-dependent mechanism is the one

that normally reduces methemoglobin to hemo-globin in normal human erythrocytes. Differ-ences in the effectiveness of these various com-

pounds may reflect differences in the permeabilityof the erythrocyte or differences in the rates atwhich the metabolic reactions proceed within thecell.

\Vhen a hemolysate of normal human erythro-cytes rather than intact erythrocytes was incubatedwith some of the effective compounds, only as-

corbic acid had an effect on the concentration ofmethemoglobin after three hours of incubation(Table III). It should be noted that no attemptwas made to inhibit the destruction of pyridinenucleotides that is known to occur when animaltissues are disrupted (40). The observation thatincubation with ascorbic acid leads to the reduc-

TABLE III

The efect of various compounds on the reduction ofmethemoglobin in a hemolysate of normal

human erythrocytes *

Methemoglobinafter incubation

Compound for 3 hrs.

Control 72Glucose 74Glucose-6-phosphate 71Adenosine 72Deoxyadenosine 73Ribose 75Deoxyribose 70Ascorbic acid 34

* Initial concentration of methemoglobin was 72 percent. Concentration of compounds was 19 A&Moles per ml.of erythrocytes.

tion of methemoglobin in a hemolysate, as well asin intact erythrocytes, is compatible with the be-lief that ascorbic acid acts directly on methemo-globin (41). This experiment with a hemoly-sate confirms the importance of an intact erythro-cyte for methemoglobin reduction in the absenceof methylene blue or other autoxidizable dyes.In addition, it demonstrates that the compoundseffective with intact erythrocytes did not exerttheir effect by reducing methemoglobin to hemo-globin directly.

The effect of methylene blue on the reductionof methemoglobin may be due to the ability of thisdye to serve in an electron transport system (2).It may, however, also be due, in part, to the acti-vation or stimulation of the hexose-monophos-phate shunt pathway (4, 42, 43). The incuba-tion of normal human erythrocytes containingmethemoglobin with glucose, inosine or deoxyino-sine plus methylene blue resulted in marked ac-celeration in the reduction of methemoglobin tohemoglobin (Table IV, Part A). The failureof methylene blue to enhance greatly the effect ofribose and deoxyribose is not readily explained.It is conceivable that the permeability of theerythrocyte to ribose is so limited that no accelera-tion from addition of methylene blue could bedemonstrated. If deoxyribose is metabolized byway of the Embden-Meyerhof pathway, only alimited increase in the rate of methemoglobin re-

duction would be expected since the methemo-globin reduction system that utilizes DPNH isnot enhanced markedly by this dye (4, 32, 42).

1559

1560 ERNS

TABLE IV

The effect of methylene blue on the ability of some com-pounds to enhance the reduction of methemo-

globin in human erythrocytes

Methemoglobinafter incubation

for 3 hrs.

Initial Without WithConcen- methemo- meth. meth.

Compound tration* globin blue bluet

%% %A. Normal human erythrocytes

Control 68 64 59Inosine 10 44 11Glucose 10 49 3L (+)-sodium lactate 19 48 34

Control 67 65 62Ribose 10 58 51Deoxyribose 10 52 47

Control 65 62 57Inosine 10 40 9Deoxyinosine 10 44 14

B. Erythrocytes of congenital methemoglobinemiaControl 68 77 72Glucose 10 74 4Inosine 10 74 10L (+)-sodium lactate 20 75 69

* Concentration in micromoles per milliliter of erythro-cytes.

t Concentration of methylene blue was 0.12 jAMoles perml. of erythrocytes.

;T R. JAFFE

This latter effect was also shown by the minimalincrease in the effect of L (+ )-sodium lactatewhen methylene blue was added to the erythrocytesuspension.

The specificity of the effect on the reduction ofmethemoglobin to hemoglobin is demonstrated bythose compounds that did not increase the rate ofreduction of methemoglobin above that observed inthe control suspensions (Table V). Compoundsclosely related in structure to the effective purinenucleosides were ineffective. Incubation withpurine riboside, adenosine-1-N-oxide, adenineglucoside, dimethyl-adenosine, aminonucleosideand Puromycin Hydrochloride®, as well as withthe purine, adenine, did not result in either en-hanced reduction of methemoglobin to hemoglobinor in the production of lactic acid. A slight effecton methemoglobin reduction was observed withaminoadenosine (Table II) and about 13 per centas much lactic acid was present after six hours ofincubation as with an equimolar amount of adeno-sine. It remains to be determined if this findingrepresented true metabolism of the amino-ad-enosine or if there occurred a small amount of non-enzymatic hydrolysis of the compound with theformation of adenosine. When the purine portion

TABLE V

Compounds ineffective in enhancing the reduction of methemoglobin to hemoglobin whenincubated with normal human erythrocytes

Compound Compound

Pyrimidine ribosidesCytidineUridine

Pyrimidine deoxyribosidesDeoxycytidineDeoxyuridineThymidine

NucleotidesAdenosine-5'-phosphateAdenosine diphosphateAdenosine triphosphateAdenosine-5'-phosphoramide

Related compoundsAdenineAdenosine-1-N-oxideDimethyl-adenosineAminonucleosideAdenine glucosidePurine ribosidePuromycin Hydrochloride®

pMoles*

88

888

11111110

78,10

1010

8,108

10

Intermediates of carbohydratemetabolism andrelated compounds

Sucrose

Glucose-6-phosphateFructose-1,6-diphosphateRibose-5-phosphate2,3-diphosphoglycerate

Aminoribose

D (-)-sodium lactateSodium pyruvate

Miscellaneous compoundsAspartic acidCysteineGlucuronic acidGlucuronolactoneGlutathione, reducedSodium thiosulfate

* Concentration in micromoles per milliliter of erythrocytes.

jsMoles*

9

101111'11

10

1917

101010101010

REDUCTIONOF METHEMOGLOBINBY PURINE NUCLEOSIDES

of the effective compounds was replaced by an-other group, there was no effect on the reductionof methemoglobin and no lactic acid productionwas observed (10). The pyrimidine ribosides(cytidine and uridine) and the pyrimidine deoxy-ribQsides (deoxycytidine, deoxyuridine and thy-midine) were ineffective. The nucleotides of ad-enosine (adenosine-5'-phosphate, adenosine di-phosphate, adenosine triphosphate and adenosine-5'-phosphoramide) were also without effect onmethemoglobin reduction. Incubation with adisaccharide (sucrose), sodium pyruvate, amino-ribose and the optical isomer of the natural formof sodium lactate, i.e., D (-)-sodium lactate, didnot result in increased reduction of methemo-globin to hemoglobin. Several phosphorylatedintermediates of carbohydrate metabolism (glu-cose-6-phosphate, fructose-i,6-diphosphate, ribose-5-phosphate and 2,3-diphosphoglycerate) were in-effective, probably as a result of the impermeabil-ity of the erythrocyte to phosphorylated sugars.Compounds unrelated in structure to the purinenucleosides were studied and were ineffective.These compounds included aspartic acid, sodiumthiosulfate, glucuronic acid and glucuronolactone.Although it has been reported that reduced glu-tathione and cysteine will reduce methemoglobinin hemolysates (44), these compounds were in-effective in the intact erythrocyte system used inthese studies.

Erythrocytes of congenital methemoglobinemiaData from an experiment performed with the

erythrocytes from the patient with congenitalmethemoglobinemia are presented in Figure 2and Table I, Column B. It is apparent that incu-bating these erythrocytes with the purine nucleo-sides and sugars that promoted methemoglobin re-duction in normal human erythrocytes did notresult in significant reduction of methemoglobinto hemoglobin. Incubation with ascorbic acid,which is thought to act directly on methemoglobin(41), did lead to a decrease in the methemoglobinconcentration. The quantity of lactic acid pres-ent after six hours of incubation was comparableto that observed with normal erythrocytes, exceptthat perhaps slightly more lactate was producedfrom glucose. The significance of this apparentincreased glucose utilization remains to be investi-gated. The data on lactic acid production con-

CONGENITAL METHEMOGLOBINEMIAERYTHROCYTES

70

zs 60

0o 5D

I&I 40

30U

"J 20

10

------------------------------CONTROL- L (+1-Na LACTATE

DEXREOROSEYRRIBOSE

*\\IJNOSINE\\ADENOSINE\'EOXYINOSINEGLUCOSE

ASCORBICACID

10p Mper of. ERYTHROCYTES.EXCEPT 2OpMLACTATEWHOLEBLOOD: 31 % METHEMOGLOBIN

o 3 6HOURSAT 37"C.

22

FIG. 2. EFFECT OF INCUBATION WITH VARIOUS COM-POUNDSON THE REDUCTION OF METHEMOGLOBININ THEERYTHROCYTESOF CONGENITAL METHEMOGLOBINEMIA

firm the observation that the erythrocytes of con-genital methemoglobinemia can metabolize glucose(45) and they also demonstrate that these cellscan metabolize ribose, deoxyribose and the pen-tose moiety of certain purine nucleosides.

The nature of the defect in the erythrocytes ofcongenital methemoglobinemia is unknown, butmay involve the absence of an unknown electroncarrier system between reduced pyridine nucleo-tides and methemoglobin (30, 42, 46). That thedeficiency can be corrected by methylene blue hasbeen reported by a number of investigators (32,45, 47) and is further supported by the data inTable IV, Part B. The concentration of methemo-globin in the erythrocytes from the patient withcongenital methemoglobinemia was increased byincubating the cells with sodium nitrite. Incuba-tion of these treated erythrocytes with glucose orinosine plus methylene blue resulted in the reduc-tion of methemoglobin to an extent equal to thatobserved in normal erythrocytes. Only a slighteffect from L (+)-sodium lactate and methyleneblue was noted. Since the pathways by whichnucleosides and sugars are metabolized in thecongenital methemoglobinemia erythrocytes wouldappear to be normal, it may be that methyleneblue serves in a substitute electron transport sys-

1561

80F

ERNSTR. JAFFE

tem between reduced pyridine nucleotides andmethemoglobin in these cells (4, 42).

SUMMARYAND CONCLUSIONS

The incubation of normal human erythrocytesthat contained 60 to 80 per cent methemoglobinwith the purine nucleosides that were activelymetabolized to lactic acid resulted in the reductionof a considerable portion of the methemoglobin tohemoglobin. The effective nucleosides includedadenosine, guanosine, inosine, 2,6-diaminopurineriboside, xanthosine, deoxyadenosine, deoxyguano-sine and deoxyinosine, and a slight effect was ob-served with aminoadenosine. The pentoses, riboseand deoxyribose, were also able to enhance the re-duction of methemoglobin. In addition, the abilityof glucose, galactose, fructose,-fumaric acid andL (+)-sodium lactate to promote methemoglobinreduction was confirmed.

The erythrocytes from a patient with congenitalmethemoglobinemia were unable to reduce methe-moglobin to hemoglobin when incubated with thenucleosides and sugars that promoted the reduc-tion of methemoglobin in normal erythrocytes,despite the ability of the congenital methemo-globinemia erythrocytes to. produce comparableamounts of lactic acid. These observations areconsistent with the concept that the defect incongenital methemoglobinemia lies in a failure inelectron transport to methemoglobin.

Incubation with ascorbic acid resulted in thereduction of methemoglobin in normal erythro-cytes and in hemolysates, as well as in the erythro-cytes of congenital methemoglobinemia.

The addition of methylene blue to normal eryth-rocyte suspensions containing added glucose orpurine nucleosideg accelerated the rate of reduc-tion of methemoglobin and permitted the erythro-cytes of congenital methemoglobinemia to reducemethemoglobin in a normal manner.

The studies presented here suggest that com-pounds that can be metabolized by human eryth-rocytes by pathways that can lead to the reduc-tion of pyridine nucleotides will promote the re-duction of methemoglobin to hemoglobin, providedthe necessary electron transport mechanism isintact.

ACKNOWLEDGMENT

The author is indebted to Doctors Sheldon Spielman,Sherman Karpen and Selig Neuhardt of the Department

of Obstetrics and Gynecology of the Bronx MunicipalHospital Center who made the patient with congenitalmethemoglobinemia available for study.

REFERENCES

1. Jaffe, E. R. The effect of purine nucleosides on thereduction of methemoglobin in human erythrocytes(abstract). Clin. Res. 1959, 7, 12.

2. Warburg, O., Kubowitz, F., and Christian, W. Vberdie katalytische Wirkung von Methylenblau inlebenden Zellen. Biochem. Z. 1930, 227, 245.

3. Wendel, W. B. Oxidation of lactate by methemo-globin in erythrocytes with regeneration of hemo-globin. Proc. Soc. exp. Biol. (N. Y.) 1931, 28,401.

4. Kiese, M. Die Reduktion des Hamiglobins. Biochem.Z. 1944, 316, 264.

5. Kiese, M., and Schwartzkopff-Jung, W. Die Re-duktion des Haimiglobins. III. Reduktion desHimiglobins und Stoffwechsel in roten Zellen.Naunyn-Schmiedeberg's Arch. exp. Path. Phar-mak. 1947, 204, 267.

6. Spicer, S. S., Hanna, C. H., and Clark, A. M. Stud-ies in vitro on methemoglobin reduction in dogerythrocytes. J. biol. Chem. 1949, 177, 217.

7. Matthies, H. Methamoglobinriuckbildung in Reticu-locyten. Naunyn-Schmiedeberg's Arch. exp. Path.Pharmak. 1956, 229, 331.

8. Matthies, H. Die Wirkung von Aldehyden auf dieMethamoglobinruckbildung in Erythrocyten. Bio-chem. Z. 1957, 329, 341.

9. Rubinstein, D., Kashket, S., and Denstedt, 0. F.Studies on the preservation of blood. IV. The in-fluence of adenosine on the glycolytic activity ofthe erythrocyte during storage at 4° C. Canad. J.Biochem. 1956, 34, 61.

10. Lowy, B. A., Jaffe, E. R., Vanderhoff, G. A., Crook,L., and London, I. M. The metabolism of purinenucleosides by the human erythrocyte in vitro. J.biol. Chem. 1958, 230, 409.

11. Gabrio, B. W., Donohue, D. M., and Finch, C. A.Erythrocyte preservation. V. Relationship betweenchemical changes and viability of stored bloodtreated with adenosine. J. clin. Invest. 1955, 34,1509.

12. Jaffe, E. R., Lowy, B. A., Vanderhoff, G. A., Aisen,P. and London, I. M. The effects of nucleosides onthe resistance of normal human erythrocytes toosmotic lysis. J. clin. Invest. 1957, 36, 1498.

13. Harris, E. J., and Prankerd, T. A. J. The effect ofadenosine on the movement of sodium betweenerythrocytes and the suspension medium (ab-stract). Biochem. J. 1955, 61, xix.

14. Beutler, E., Robson, M., and Buttenwieser, E. Themechanism of glutathione destruction and protectionin drug-sensitive and non-sensitive erythrocytes.In vitro studies. J. clin. Invest. 1957, 36, 617.

15. Klebanoff, S. J. Glutathione metabolism. 2. Theoxidation and reduction of glutathione in intacterythrocytes. Biochem. J. 1957, 65, 423.

1562

REDUCTIONOF METHEMOGLOBINBY PURINE NUCLEOSIDES

16. Prankerd, T. A. J. Revival of stored blood withguanosine and its successful transfusion. Lancet1956, 1, 469.

17. Lange, R. D., Crosby, W. H., Donohue, D. M., Finch,C. A., Gibson, J. G., II, McManus, T. J., andStrumia, M. M. Effect of inosine on red cell pre-

servation. J. clin. Invest. 1958, 37, 1485.18. Wintrobe, M. M. Clinical Hematology, 4th ed.

Philadelphia, Lea & Febiger, 1956, p. 367.19. Evelyn, K. A., and Malloy, H. T. Microdetermina-

tion of oxyhemoglobin, methemoglobin, and sulf-hemoglobin in a single sample of blood. J. biol.Chem. 1938, 126, 655.

20. Crosby, W. H., and Houchin, D. N. Preparing stand-ard solutions of cyanmethemoglobin. Blood 1957,12, 1132.

21. Mills, G. C., and Randall, H. P. Hemoglobin catabo-lism. II. The protection of hemoglobin from oxi-dative breakdown in the intact erythrocyte. J. biol.Chem. 1958, 232, 589.

22. Barker, S. B., and Summerson, W. H. The colori-metric determination of lactic acid in biological ma-

terial. J. biol. Chem. 1941, 138, 535.23. Gerald, P. S. The electrophoretic and spectroscopic

characterization of hgb M. Blood 1958, 13, 936.24. Warburg, O., and Christian, W. tber Aktivierung

der Robisonschen Hexose-Mono-Phosphorsaure inroten Blutzellen und die Gewinnung aktivierenderFermentl6sungen. Biochem. Z. 1931, 242, 206.

25. Drabkin, D. L. Maintenance of active hemoglobin-a function of erythrocytes (abstract). Fed. Proc.1946, 5, 132.

26. Gutmann, H. R., Jandorf, B. J., and Bodansky, 0.

The r6le of pyridine nucleotides in the reductionof methemoglobin. J. biol. Chem. 1947, 169, 145.

27. Kiese, M., Schneider, C., and Waller, H. D. Himi-globinreduktase. Naunyn-Schmiedeberg's Arch.exp. Path. Pharmak. 1957, 231, 158.

28. Huennekens, F. M., Caffrey, R. W., Basford, R. E.,Gabrio, B. W. Erythrocyte metabolism. IV. Iso-lation and properties of methemoglobin reductase.J. biol. Chem. 1957, 227, 261.

29. Lonn, L., and Motulsky, A. G. Electrophoretic dem-onstration of a non-hemoglobin protein (methe-moglobin reductase) in hemolysates (abstract).Clin. Res. Proc. 1957, 5, 157.

30. Huennekens, F. M., Caffrey, R. W., and Gabrio, B. W.The.electron transport sequence of methemoglobinreductase. Ann. N. Y. Acad. Sci. 1958, 75, 167.

31. Wendel, W. B. Oxidations by erythrocytes and thecatalytic influence of methylene blue. I. Theoxidation of lactate to pyruvate. J. biol. Chem.1933, 102, 373.

32. Gibson, Q. H. The reduction of methaemoglobin inred blood cells and studies on the cause of idio-

pathic methaemoglobinaemia. Biochem. J. 1948,42, 13.

33. Sandberg, A. A., Lee, G. R., Cartwright, G. E., andWintrobe, M. M. Purine nucleoside phosphorylaseactivity of blood. I. Erythrocytes. J. clin. Invest.1955, 34, 1823.

34. Huennekens, F. M., Nurk, E., and Gabrio, B. W.Erythrocyte metabolism. I. Purine nucleoside phos-phorylase. J. biol. Chem. 1956, 221, 971.

35. Tsuboi, K. K., and Hudson, P. B. Enzymes of thehuman erythrocyte. I. Purine nucleoside phos-phorylase; isolation procedure. J. biol. Chem. 1957,224, 879.

36. Dische, Z. Synthesis of hexosemono- and diphos-phate from adenosine and ribose-5-phosphate inhuman blood in Phosphorus Metabolism, W. D.McElroy and B. Glass, Eds. Baltimore, The JohnsHopkins Press, 1951, vol. I, p. 171.

37. Schaedel, M. L., and Schlenk, F. Adenosine andadenosine deaminase. Tex. Rep. Biol. Med. 1948,6, 176.

38. Denstedt, 0. F. The enzymology of the erythrocytein Blood Cells and Plasma Proteins, J. L. Tullis,Ed. New York, Academic Press Inc., 1953, p. 223.

39. Nossal, P. M. The metabolism of erythrocytes. I.Respiration in the absence and presence of methyl-ene blue. Aust. J. exp. Biol. med. Sci. 1948, 26,123.

40. Handler, P., and, Klein, J. R. The inactivation ofpyridine nucleotides by animal tissues in vitro. J.biol. Chem. 1942, 143, 49.

41. Barcroft, H., Gibson, Q. H., Harrison, D. C., andMcMurray, J. Familial idiopathic methaemoglo-binaemia and its treatment with ascorbic acid.Clin. Sci. 1945, 5, 145.

42. Gibson, Q. H. Methaemoglobin and sulphaemoglobinin The Chemical Pathology of Animal Pigments,R. T. Williams, Ed. Biochemical Society SymposiaNo. 12, Cambridge, University Press, 1954, p. 55.

43. Brin, M., and Yonemoto, R. H. Stimulation of theglucose oxidative pathway in human erythrocytesby methylene blue. J. biol. Chem. 1958, 230, 307.

44. Morrison, D. B., and Williams, E. F., Jr. Methemo-globin reduction by glutathione or cysteine. Sci-ence 1938, 87, 15.

45. Eder, H. A., Finch, C., and McKee, R. W. Congeni-tal methemoglobinemia. A clinical and biochemi-cal study of a case. J. clin. Invest. 1949, 28, 265.

46. Scott, E. M., and Hoskins, D. D. Hereditary methe-moglobinemia in Alaskan Eskimos and Indians.Blood 1958, 13, 795.

47. King, E. J., White, J. C., and Gilchrist, M. A caseof idiopathic methaemoglobinaemia treated by as-corbic acid and methylene blue. J. Path. Bact.1947, 59, 181.

1563