relation of methemoglobin to hernolysis - blood

Relation of Methemoglobin to Hernolysis

By BYRoN B. CLARK, Pii.D., AND ROBERT W. MORRISSEY, M.I).

Jl�ith the Tech nical A ssistance of DOROTHY BLAIR

IT IS \VELL KNO\VN that a number of chemical agents, w’hieh under certain

circumstances cause intravascular hemolvsis an(l the development of hem-

olytic anemia, also produce methemoglobinemia. Some examples of these agents

are stllfanilam ide, phenylhydrazine, aniline , acetanilid, p-aminophenol , plas-

mochin, etc. Such a correlation may lead to the inference that intracorpu.scular

methemoglobin is related in some way to the process of hemolysis. It has been

observed that. the dosage level of acetanilid and acetophenet.idin causing signifi-

cant anemia also exceeds the threshold for methemoglobin formation.’ The data

of Rimington and Hemmings2 might also he interpreted to lend some additional

support to the view’ that methemoglobin plays some positive role. In studies on

drugs related to sulfanilamide, they observed a close correlation between the

action of aromatic amino compounds to increase porphyrin excretion and to

form met.hemoglohin. Since the increased porphyrin excretion in rodent.s was

due largely to coproporphyrin III, it has been suggested that this represents

an abnormal pathw’ay for the degradation of hemoglobin possibly through met-

hemoglobin.2 ‘ � However, Rimington and Hemmings2 w’ere not certain as to the

role of the methemoglobin in either the increased erythrocyte destruction or the

increased porphyrin excretion since the latter w’as not observed after sodium

nitrite administration, which readily produces methemoglobin. Fox and Otten-

berg4 studying the hemolytic anemias from sulfonamides in man observed a

greater percentage of methemoglohin in the serum than in the erythrocytes and

suggest that it ��‘as t.he cells containing methemoglobin w’hich underwent he-

molysis. This discrepancy betw’een serum and cells could be readily accounted

for by the presence in serum of oxidants w’ith low’ penetrability into the eryth-

rocytes, or by different equilibrium conditions, as in fact the high percentage of

met hemoglobin observed in the urine suggests may have been the case.

On the other hand, there are some reasons to doubt that methemoglohin per

se favors hemolysis. Hemolytic anemias have not l)een reported as a feature

of nitrite toxicity, an(l as will be seen, w’e have not. observed it in experiments

designed to test this point. Also Keilin and Mann5 state that intracorpuscular

hemoglobin may be readily changed to methemoglobin with sodium nitrite with-

out altering the integrity of the cell membrane. It is w’ell known that certain

drugs may produce a hemolytic anemia without the formation of methemo-

globin. Furthermore, the alteration of hemoglobin to form carbon monoxide

hemoglobin has been show’n not to alter the osmotic fragility of erythroeytes.6

However, these are insufficient reasons to decide w’hether methemoglobin

From the Department of Pharmacology, Tufts College Medical School, Boston, Massa-chusetts, and the Department of Physiology and Pharmacology, Albany Medical College,

Union University, Albany, N. Y.

A preliminarY report on this subject appeared in Federation Proceedings, 4: 114, 1945.

532

For personal use only.on January 4, 2019. by guest www.bloodjournal.orgFrom

http://www.bloodjournal.org/

http://www.bloodjournal.org/site/subscriptions/ToS.xhtml

CLARK AND MORRISSEY 533

plays any definitive role in the process of hemolysis. There is a paucity of any

quantitative data on the sul)ject. Therefore a comprehensive study has been

ma(le in order to provide quantitative data with highly sensitive t.echnics. A

comparison has been made in vitn’o of the resistance of oxyhemoglobin and

methemoglobin containing ervthrocytes to hemolysis by saponin, hypotonic

saline, l)ile salts and lysolecithin. Also included are oi)servations on the hemato-

logic effects in vivo of chronic methemoglobinemia produced by sodium nitrite.

EXPERIMENTAL METHODS

In order to carry out. these experiments, it waS necessary to use some chemical agent

to l)roduce intracorpuscular n�ethensoglobin. The requirenients for such an agent are (1)

that it forms n�ethemoglobin in vitro as well as iii vivo, (2) that it can readily penetrate theerytlirocyte niembrane and (3) that it Produces no injury to the nien�branse in the concen-

trrttions required to form relatively high concentrations of niethemoglobins. These require-

nienits eliniiniate many aromatic coniipounds, some of which at least act as sin�l)le h’sins or

accelerate certain lvsin.s. After i1U�iC�OUS �)relirnin1ary experiments sodium nitrite was

chosen as the niost suitable agent availal)le. The nit rite readily penetrates the erythiro-

cyte and fornis n�ethiemoglobin ra�)idly at loss- conicenitrations.7 In the procedure of pre-

�arinsg the erythrocyte suspenisionis, there was no gross evidence of hemolysis :Issociate(iwitl’i the presence of the nitrite ; after repeated washings si-it hi saline no t race of nit rite

could he detected.7’ 8 Furthern�ore, the intentional additions of low ct)nicenltrations of so-

diun� nit rite I 0 the lytic system (under t hose condit ions inn which oxyhenioglobin arid niet-

hen�oglohini cells hemolyzed in tine same time) did not significantly alter the time required

for hennolysis, indicating that this agent did not act to an iniportanit degree as an aced-

erator or inhibitor of the lvsins used. Keilin arid Mann5 have also presented some evi(lencethat siniilar treatn�ent of er�throc�tes does riot n�oclifv their J)ernseability.

Erythi’ocyte suspensions. Dog erythrocytes were eniployedi for all of the data to he re-

ported. However, human erythrocytes were also employedl in a sufficient number of experi-ments to establish the qualitative similarity of the results. The standard cell suspensions

��‘ere prepared ins the folloiving ‘�-:�‘. A sanll)le of 1)100(1, about 4 cc., s�as (Irawn into a ceni-

trifuge tube containing 25 cc. of 0.85 l)d� cent sodium chloride anti 250 mg. of sodium cit-

rate. This s:tline-cit.rate suspension was (livided into two portions; one l)ortion WHS treated

with sodium nitrite, arid 10 mg. �er cc. of blood was allowed! to stand for ten minutes dur-

ing which time about 80 to 90 per cent of the hemoglobin was converted to methemoglobin.

When lower percentages of met hemoglobin were desired proport ionately smaller amounts

of sodium nitrite were added. The dells of both portions were then washed three times by

cent rifugat ions and resuspension irs 30 cc. of saline per cc. of packed cells. The suspensions

were carefully standardized in the final dilution with saline to contain 0.8 Gm. of total

hemoglobin per 100 cc.,9 which corresponded approximately to 250,000 cells per cu.mm.These two cell suspensions will be referred to as “oxyhemoglobin cells” and “methemo-

globin cells.”

All experiments except t.hose with hypot.onic saline were carried out in buffered saline.

Sorensen’s M/l5 J)hiosphate mixtures were used in proportion of 1 l)art in 10 of 0.85 per

cent saline to obtain the desired p11 indicated in the experiments; the initial p11 before

hemolysis for a sufficient number of experiments was checked by the glass electrode.

Saponin. The time required for complete hernolysis was determined for a sUspension of

washieti erythrocytes and saponin ins a saline-phosphate buffer with the tubes in a constant

temperature water bath according to the general method described by Ponder.’#{176}�’t The

hemol�’tic system consisted of 2 cc. of buffered saline, 2 cc. of the saponin dilution in buf-

fered saline and 1 cc. of the standard cell suspension in soft. glass test tubes 12 X 100 mm.

A series of dilutions of saponin were employed to obtain time-dilution curves. The dilu-

tions of saponin indicated in the figures are the final dilutions iii the hemolytic system.Hypotonic Saline. Percentage hemolysis curves were obtained with a series of dilutions

of hypotonic saline using the general technic described by Hunter.” The erythrocyte sus-

pensions were prepared as described above except that the final dilution was that of the




534 RELATION OF METHEMOGLOBIN TO HEMOLYSIS

original volume of blood. One-tenth cc. of such a suspension was ad(ledi to 10 cc. of each

dilution of hypotonsic saline. After standing for one hour at. room tensperature, the series

of tubes were centrifuged and the liberated hemoglobin determined9 in the supernatant.

Bile Salts. The same general technic for 100 per cent hemolysis as describetl for saponin

was used. Both sodium glycocholat.e and taurocholate were tested and yielded qualita-

lively similar results, but only the data on glycocholate will he presented.

Lysolecithin. The lysolecithin used was prepared by the actions of snake venom* on egg

yolk, aS described l)�’ King and Dolan.’2 The material obtained was l)rohahly somewhat

impure, since no attempt was made to separate out the lysocephalin, but nevertheless pro-

vided a preparation of constant activity. For use a stock solution of lysolecithin was pre-

pared each day containing 10 mg. per cc. in absolute nsethyl alcohol from which appropriatedilutions were made with saline and kept in paraffined test tubes.’3 The standiard suspen-

sions of ervthroc�tes were prepared in the same way as described ah)ove.

The time required for Sf) per cent hemolysis was used as the end point for this lysin;

this could he estimated accurately from the time per cent hemolysis curves which were

obtained by an adaptation of the photoelectric method described by Wilbur and Collier.’3

The hemolytic system consisted of 8 cc. of buffered saline, 1 cc. of the standard cell sus-

pension and 1 cc. of the lysolecithin dilution in saline. The dilutions indicated in the data

are the final dilutions of the lysin in the colorimeter tube. The per cent hemolysis curves

were obtained using the Evelyn colorimeter with filter 660.� The galvanometer was set at

100 for the one hundred per cent hemolysis blank containing the above system of the cor-

responding type of cells which were completely hemolyzed with saponin. The initial opac-it)’ reading for zero per cent hemolysis was obtained for both kinds of cells with the above

system in which the lysolecithin solution was replaced by saline. Such control tubes were

prepared for each pH and temperature used. (In most. cases it was impractical to obtain

an initial opacity reading following the addition of the lysolecithin because of the initial

rapid rate of hemolysis with concentrations of lysin yielding fifty per cent hemolysis in a

reasonable time.) In the experimental tubes following the addition of lysolecithin, galva-

nometer readings were recorded at appropriate time intervals. The per cent of hemolysiswas read directly from semi-log graph paper, using the opacity reading of the unhemolyzed

control tube to establish the point for zero hemolysis.

RESULTS

Saponin. Time-dilution curves for 100 per cent hemolysis were determined

with saponin in parallel experiments on both oxyhemoglobin and methemo-

globin cells. The average results in 16 parallel experiments at. pH 7.3 and 38 C.

are show’n in figure IA, and indicate that the presence of methemoglobin in the

erythrocytes has no effect on their resistance to saponin lysis. Qualitatively

similar results were obtained at pH 6.2 and 38 C. and also at 25 C. pH 6.2 and

7.3, and with human eryt.hrocytes. The addition of free oxyhemoglobin and met-

hemoglobin initially to the system had a small hut equal inhibitory effect on the

subsequent saponin hemolysis.

Another series of experiments, using both the methemoglobin and oxyhemo-

globin cells, were performed in an identical manner except. that. chemical agents

know’n to accelerate saponin lysis were included in equal concentrations in both

systems. Thus, each experiment consisted of four time-dilution curves; that is,

oxyhemoglobin and methemoglobin cells each in the presence and absence of

t.he accelerator. The results are illustrated in figure 1B, C, and D. The accelera-

t.ors varied from the highly active quinine to the relatively weak acetanilid.’4

The curves obtained for the oxyhemoglobin and methemoglobin cells were es-

* Crotalus atrox venom kindly supplied by Sharpe and Dohme.




A B

QUININE-O.125’,M/L

z

�

C D

ACETANILID- 12 MM/L

0

(‘LARK AND MORRISSEY 535

sentially identical. Similar data ss’ere obtained w’ith several other accelerators,

such as aniline, acetophenetidin, and p-aminophenol; the latter is the only agent

listed which forms met.hemoglobin in vitro under t.he condit.ions of these experi-

ment.s. (Sulfanilamide ss’as found to be a w’eak inhibitor and to affect both types

of cells to the same degree.) The results clearly indicate that t.he presence of

methemoglobin does not alter the time required for hemolysis of erythrocytes

by saponin alone or combined with an accelerator.

zz0Q.

U,

‘I.

0

B(NZCNE- 0-45 MM/L

2 4 6 8

TIME IN MINUTES

FIG. 1-Effect of methemoglobin on saponin hemolysis at. pH 7.3 and 38 C.

o Oxyhemoglobins cells#{149}Met.hemoglobin cells

L�s Oxyhemoglobin cells plus accelerator

A Methensoglobin cells plus accelerator

Ilypo#{252}inic saline. Percentage hemolysis curves w’ere obtained for a series of

dilutions of hypotonic saline at room temperature (about 25 C.). Ten parallel

experiments on both oxyhemoglohin and methemoglobin cells were made, and

the averaged percentage of hemolysis for each concentration of saline appears in

figure 2. These data indicate that t.he presence of methemoglobin does not

alter the resistance of the erythrocytes t.o hemolysis by hypotonic saline.

In a second series of experiments a sub-lytic concentration of quinine (i.e., a

concent.ration producing no hemolysis in 0.85 per cent. saline in one hour) s�’as

included in the hypotonic saline before the addition of the erythrocytes. As

shown in figure 2, the presence of quinine increases the percentage of hemolysis





for a given concentration of saline, but again no difference is evident. in the per-

centage of hemolysis of the oxyhemoglobin and methemoglobin cells.

Bile salts. The experiments with the bile salts w’ere carried out using the same

technic for 100 per cent hemolysis as s�’as described for saponin. Most of the ex-

periments were made at 38 C. w’ith sodium glycocholate. In preliminary experi-

ments, it was found that the relative rates of hemolysis of the oxyhemoglobin

and methemoglobin cells differed according to the pH of the system. This rela-

tionship is illustrated in figure 3, which represent.s times for hemolysis for the

tw’o types of cells by 1: 2000 glycocholate over a range of pH values. Below’ pH

‘00

80 //

En /

> /-J #{163}0� 60 /

uJ /

I Es’

O 40 #{163}5,,’

/� ./

.62 � .46 .42 1

PER CENT SALT CONCENTRATION

FIG. 2.-Effect of methemoglobin on hypotonic saline hemolysis at 25 C.

o Oxyhemoglobin cells#{149}Methemoglobin cells

� Oxyhemoglohin cells plus quinine

A Methemoglohin cells plus quinine

6.6, hemolysis of the methemoglobin cells takes place at. a slightly iiicreased

rate. On the other hand, above pH 7.0 the time required for the hemolysis of

the methemoglobin cells becomes excessively long as compared to the oxyhemo-

globin cells. The two curves cross at approximately pH 6.7. The general rela-

t.ionship evident at pH 6.2 and 7.3 was also observed to hold for a series of dilu-

tions of both glycocholate and taurocholate. The inhibition of methemoglohin

cells at pH 7.3 and their slight acceleration at pH 6.2 was found to be roughly

proportional to the methemoglobin concentration in the cells.

In view’ of the reversal of the rates of hemolysis of the two types of cells with

respect to pH, it seemed advisable to determine w’hether solutions of free oxy-

hemoglobin and methemoglobin differed in their inhibiton’y activity. A portion

of the buffered saline in the hemolytic system was replaced by buffered hemo-





200

150

00

50

I0

InL�7

z

2z

U2I-

8

6

2

60 62 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 60

hemolysis as influenced by pH con-

wI-

-J

0I000

-I

0LI.

0z0

I-

-J

5 0

FIG. 3.-Effect of nsethensoglobini on glycocholate

centration of glycocholate 1:2,000 at 38 C.

o Oxyhemoglohin cells

#{149}Methemoglobin cells

TIME IN MINUTES

FIG. 4.-Relative inhihitor�’ effect of free oxyhemoglobin arid met hensoglohin on hennol-

ysis by glycocholate at p11 7.3 arid 38 C.

o Oxyhennoglobin cells aloneD Oxyhennoglobi ni cells plus free oxyhemoglobi n

� Oxyhemoglobi n cells plus free met hemoglobin





glohin solutions equivalent to 10 per cent of the hemoglobin contained in the

cells in the system. It was found as show’n in figure 4 that at pH 7.3, where the

methemoglobin cells were apparently resistant to hemolysis, that t.he addition

of a solution of methemoglobin to the oxyhemoglohin cells caused a much greaterinhibition than the addition of oxyhemoglohin in a similar system. At. pFI 6.2

the tw’o types of hemoglobin had very little inhibitory effect. The same qualita-

tive results for pH 6.2 and 7.3 were also obtained at 25 C. It therefore seems

probable that the apparent increased resistance of the methemoglobin contain-

200

80

60

UI 140

-j

0

120

I

U� 100

0

0 80z0U

60

2

I- 40

20

20 25 30 35 38

TEMPERATURE

FIG. 5.-Effect of methemoglobin on lysolecithin hemolysis as influenced by pH and tern-

l)erature.

o Oxyhernoglobin cells at pH 7.3

L�s Oxyhemoglobin cells at pH 6.2

#{149}Methemoglobin cells at pH 7.3

A Methernoglohin cells at pH 6.2

ing cells at pH 7.3 is due to the greater inhibitory effect of the init.ially released

methemoglobin on further hemolysis.

Lysolecithin. In these experiments, the time required for fifty per cent he-

molysis was determined as described in the section on methods. The results with

lysolecithin were found to be influenced in a rather complex way by pH and tem-

perature, w’ith t.hese factors determining whether the oxyhemoglobin and met-

hemoglobin cells hemolyzed in the same or different times.

The influence of varying the pH from 6.0 to 8.0 at 25 and 38 C. was deter-

mined for a 1:200,000 dilution of lysolecithin. At 25 C. and between pH 6.8 to 7.6





the oxyhemoglobin and methemoglohin cells hemolyzed at. the same times. How’-

ever, at pH 6.0 to 6.8 at. 25 C. and at all pHs tested at 38 C. the methemoglobin

cells consistently hemolyzed more rapidly than the oxyhemoglobin cells. From

this data pH 6.2 and 7.3 were selected for a further study of the effect of tem-

perature; an example of the results obtained with a 1 :200,000 dilution of lysolec-

it.hin are illustrated in figure 5. From the above data and figure 5, it. may be said

that around pH 7.3 and 25 C. 1)0th the oxyhemoglobin and methemoglobin

cells hemolyzed in approximately the same time, and more extensive data show

that there is no significant. difference. How’ever, under several other conditions

of pH and temperature, the methemoglobin cells hemolyzed more rapidly.

In other experiments these differences in the hemolysis times of oxyhemoglobin

and methemoglohin cells w’ere found to he proportional to the methemoglohin

concentration. As might have been predicted, at pH 7.3 and 25 C., varying the

methemoglol)in concentration resulted in no significant difference in the he-

molysis time. However, for the other conditions tested (pH 6.2 at. 25 C. and pH

TABLE 1 -Effect of Free Henioqlobin on Lijsolecithin Heinolysis

(Lvsolecithini dilutions 1 : 200,000)

25U 38USystem � � � � �. � . ________

_______________________________ I pH 6.2 � pH 7.3 � pH 6.2 � pH 7.3

Hb02 Cells � 77 � 24 � 47 � 90

11l)O� Cells + 11h00 120 35 � 80 � 180

Hb02 Cells + MlIb � 65 � 35 I 45 � 100

MIIb Cells � 22 � 28 � 13 � 20

Time ins seconds for 50 per cent hemolysis.

6.2 and 7.3 at 38 C.), increasing the methemoglobin concentration caused a

proport ional acceleration of hemolysis.

An attempt. to explain these varying results suggested the possibility that free

oxyhemoglobin and methemoglobin initially released might cause differing (IC-

grees of inhibition of subsequent hemolysis, especially since an example of this

had already been found in the case of the bile salts. This possibility was tested

by comparing the relative inhibitory effects produced by replacing a portion of

the buffered saline in the system by buffered hemoglobin solutions equivalent

to 20 per cent of the hemoglobin contained in the cells in the syst.em. Represen-

tative results are show’n in table 1. It is evident that at 25 C. and pH 7.3, where

the oxyhemoglobin and methemoglobin cells hemolyze at approximately the same

rate, the addition of free oxyhemoglohin and methemoglohin caused the same

degree of inhibition. However, for the other conditions under which the methemo-

globin cells hemolyzed more rapidly, free oxyhemoglobin caused a much greater

degree of inhibiton than did free met.hemoglobin; in fact., the latter at times

caused some acceleration, especially at 25 C. and pH 6.2. These differences in

the inhibitory action of the tw’o kinds of hemoglobin were more easily demon-

strat.e(I using oxyhemoglobin cells, although t.hey were evident in a certain num-

her of the experiments using methemoglobin cells. These data suggest that the

observed differences in the inhibitory action of the two kinds of hemoglobin





may account for the difference noted in the rates of hemolysis of the oxyhemo-

globin and methemoglobin cells.

Some experiments were also made with lysolecithin prepared from dog blood

as described by Singer’5 using the method w’e have outlined for “synthetic”

lysolecithin. In a general sense the results w’ere similar, but. the conditions under

which they occurred w’ere different. At 38 C. and pH 6.2 and 7.3, there ss’as no

definite difference in the rate of hemolysis of oxyhemoglohin and methemoglobin

:: �

04

F-LUa 0

� : ________________ ________ ____

40 #{149}1��

0 0 20 30

DAYS

FIG. 6.-Flematologic effects of chronic niethemoglohinemia maintainned by the daily

administration of sodium nitrite.

Each character represents an individual dog. The vertically arranged characters at the

left are control determinations.

cells. However, at 25 C. and both pH 6.2 and 7.3, the methemoglobin cells

hemolyzed more rapidly than the oxyhemoglobin cells.

J7lernak3logic effects of methemoglobinem.ia in rico. Chronic methemoglobinemia

was maintained in 5 dogs over a period of thirty-five days by the administration

of sodium nit.rite subcutaneously as a 5 per cent solution once daily. The hemo-

globin pigments w’ere measured by the method of Evelyn and Malloy.9 The

dosage of nit.rite ��‘as adjusted to obt.ain a maximal met.hemoglobinemia ranging

between 50 and 70 per cent each day. These maximal values w’hich occurred

about tw’o hours after the administration of nitrite are represented in figure 6;





thereafter, the methemoglobinemia progressively decreased so that none was

present. the following morning when the blood samples were obtained for the

other ol)servations recorded. The data summarized in figure 6 indicate that no

significant alterations occurred in the values for total hemoglobin, erythrocyt.es,

and leukocvtes. There s�’as a small l)ut significant increase in ret.iculocytes, and

in some cases a small increase in cell volume. The reticulocytosis indicates an

increased erythropoiesis that may be a response to increased erythrocyte (lestruc-

tion. If so, it must have been so small as to be covered by an increased erythro-

(yte l)rO(Iuctiofl, for there is no evidence of anemia. An alternative explanation

for the reticulocytosis appears more likely. It has been previously observed in

this lahorator’�’16 that this degree of methemoglobinemia causes some reduction

in the venous oxygen tension, and this could act as a stimulus to the hone mar-

l�O\\’. Ibis view is strengthened i�y the observation that an additional increase

in l)��)th total hemoglobin and reticulocytes occurs shortly after the nitrite ad-

ministration when the methemoglobinemia is greatest. Furthermore, Rimington

and Hemmings2 have ol)served no increased porphyrin excretion follow’ing nitrite

a(lminist ration . �I�If , t hese (lata indicate that chronic methemoglobinemia

maintained by nitrite cause no significant anemia, and suggest that no significant

decrease in erythrocyte resistance to the natural in vivo hemolysins occurred.

DIscussIoN

rI�le l)niflcilMtl l)UEI)OSC of this paper was to determine whether the change in

the composition of inti�acorpuscular hemoglobin to methemoglobin may influence

the response of the erythrocyte to hemolysins. As outlined in the introduction,

the reasons for assuming that met.hemoglobin may have some influence are chiefly

circumstantial ; it s�’as also indicated that there were insufficient data on which

to base a (lecision.

The in VitV() experiments with saponin and hypotonic saline indicate that

intracorpuscular methemoglobin does not influence hemolysis by these lysins.

Neither (lid methemoglobin appear to alter the susceptibility of the erythrocytes

to the (oml)ined action of these lysins plus chemical accelerators.

The in vitro results w’ith bile salts and lysolecithin hemolysis w’ere more com-

plex; it s�’as found that the presence of methemoglobin either did or did not in-

fluence hemolysis depending upon the experimental conditions of pH and tem-

perature. It seems significant, how’ever, that. for 1)0th lysins there are conditions

(pH and temperature) under which the times required for hemolysis of oxy-

hemoglobin and methemoglobin containing eryt.hrocytes are the same. Also

significant are the findings that for those conditions under which the hemolysis

times were different., free oxhemoglobin and methemoglobin (extracorpuscular

hemoglobin added initially to the hemolyt.ic system) had different inhibitory

effects on sul)sequeflt hemolysis, and that these differences �s’ere directly cor-

related with the differences in the behavior of the oxyhemoglobin and methemo-

globrn cells. It is difficult to quantitate these effects. However, qualitatively such

results do permit. the reasonable assumption that the difference in hemolysis

of the oxyhemoglobin and methemoglobin containing erythrocytes may be ex-

plained On the basis of the different degrees to which the originally released

oxyhemoglobin and methemoglobin inhibits subsequent. hemolysis. If this is an





adequate explanation, then the observed data with bile salts and lysolecithin

could be accounted for; and the interpretation is permitted that intracorpuscular

met.hemoglobin has not altered the resistance of the erythrocytes to hemolysis

by bile salts and lysolecithin. A full explanation of the different degrees of in-

hibitory effect of oxyhemoglobin and methemoglobin for these lysins under cer-

tam experimental conditions cannot be given at present.

rFhe in vitro results w’ith saponin and hypotonic saline seem to indicate very

clearly that intracorpuscular methemoglobin does not. alter the resistance of

erythrocytes to hemolysis by these lysins. The results with bile salts and lyso-

lecithin are capable of a similar interpretation for the reasons outlined in the

preceding paragraph, although admittedly the results are more complex, and

there may be other interpretations, but the one given seems the most reasonable

from a consideration of the experimental data as a whole. The in vivo experiments

with chronic met.hemoglobinemia produced by the daily administration of sodium

nitrite did not produce a significant anemia, and this together w’ith the reported

absence of increased porphyrin excretion2 suggests that no significant decrease

in erythrocyte resistance to the intravascular lysins occurred.

rfhe experiments present strong prima-facie evidence that int.racorpuscular

methemoglobinemia per se is not primarily concerned in the process of int.ra-

vascular hemolysis. It is suggested that in those cases ��‘here methemoglobin

formation is associated w’ith the action of chemical agents producing hemolysis

or acceleration of lysins that methemoglobin formation is not an integral part

of the hemolytic action, but is an independent effect indicating the presence of

oxidants capable of penetrating the erythrocyt.e. Several such substances have

been studied in this laboratory and found to he active accelerators of variouslysins; hsow’ever, under specified conditions there �s’as no difference in the degree

of action on erythrocytes containing principally oxyhemoglobin as compared to

erythrocytes containing 80 to 90 per cent methemoglobin.

SUMMARY

Experiments are described which attempt to define the relation of met.hemo-

globin to hemolysis. A comparison w’as made in vitro of t.he resistance of eryth-

rocytes containing chiefly oxyhemoglobin or methemoglobin to hemolysis l)y

saponin, hypotonic saline, bile salts and lysolecithin. These data are interpreted

to indicate that int.racorpuscular methemoglobin does not alter the resistance

of erythrocytes to hemolysis by these lysins, although there are certain peculi-

arities in the results with bile salts and lysolecithin.

In the case of bile salts and lysolecithin, oxyhemoglobin or methemoglobin

may have an equal or different inhibitory effect on hemolysis depending upon

the conditions of pH and temperature.

Chronic methemoglobinemia in dogs maintained by the administration of

sodium nitrite does not produce an anemia.

These data suggest that the formation of methemoglobin is an independent

effect. and not a part of the hemolytic action of certain chemical agents.

REFERENCES

1 VAN Loox, E. J. AND CLARK, B. B.: Observations on the hernatological actions of acetani-

lid and acet.ophenetidin in the dog. J. Lab. & Clin. Med. �9: 942, 1944.





2 Rn�nrNGToN, C. AND HEMMINGS, A. W. : CXVIII. Porphyrinuric action of drugs related to

sulfanilamide. Comparison with reported toxicity, therapeutic efficiency and causations

of methemoglobinemia. Definition of the structure responsible for l)orl)h�’rinuric act ion.Biochem. J. 38: 960, 1939.

3 BROWNLEE, G. : The role of the aromatic amino group in deranged l)igflieflt metal)olism.

Biochem. J. 33: 697, 1939.

4 Fox, C. L. AND OTTENBERG, R. : Acute hemolytic anemia from the sulfonsamides. J. Clin.Investigation 20: 593, 1941.

5 KEILIN, D. AND MANN, T. : Activity of carbonic anihydrase within red blood corpuscles.

Nature, Lonidon 148: 493, 1941.

6 MAYERS, M. R., RIVKIN, H. AND KRASNOW, F.: The effect of chemically pure carbon

monoxide, illuminating gas, and automobile exhaust gas �poni the fragility of the

red blood cells. J. Indust. Hyg. & Toxicol. 12: 300, 1930.

GREENBERG, L. A., LESTER, D. AND hAGGARD, 11. W.: The reaction of hemoglobin withnitrite. J. Biol. Chem. 151: 665, 1943.

8 STIEGLITZ, E. J. AND PALMER, A. E.: A colorimetric method for the determination of

nitrite in blood. J. Pharmacol. & Exper. Therap. 51: 398, 1934.

� EVELYN, K. A. AND MALLOY, 11. T.: Microdetermination of oxyhennoglobin, methemoglo-

bin and sulfhemoglobin in a single sample of blood. J. Biol. Chem. 126: 655, 1938.

ha PONDER, E.: The mammalian red cell and the properties of haernolytic systems, Berlin,

Gebrssder Borntraeger, 1934.

b -: Acceleration of hemolysis in relation to chemical structure. I. Benzene dierivatives.

J. Exper. Biol. 16: 38, 1939.

“HUNTER, F. T.: A photoelectric method for the quantitative determination of erythrocyte

fragility. J. Curs. Investigation, 19: 691, 1940.

12 KING, E. J. AND DOLAN, M.: LIX. Enzynsic hydrolysis of phosphatides. II. Lysoleci-thins. Biochem. J. 27: 403, 1933.

‘� Win�nua, K. M. AND COLLIER, H. B.: A comparison of the hemolytic actions of lysolecithin

and saponin. J. Cell. & Comp. Physiol. 22: 233, 1943.

14 CLARK, B. B., MORRISSEY, R. W. AND BLAIR, D.: Some observations on the mechanism

of hemolytic anemias produced by drugs. Federation Proc. 4: 114, 1945.

‘� SINGER, K.: The lysolecithin fragility test. Am. J. M. Sc. 199: 466, 1940.

16 CLARK, B. B., VAN LOON, E. J. AND ADAMS, W. L.: Respiratory anti circulatory responsesto acute methemoglobinemia produced by aniline. Am. J. Physiol. 189: 64, 1943.




1951 6: 532-543

BYRON B. CLARK, ROBERT W. MORRISSEY and DOROTHY BLAIR Relation of Methemoglobin to Hemolysis

http://www.bloodjournal.org/content/6/6/532.full.htmlUpdated information and services can be found at:

Articles on similar topics can be found in the following Blood collections

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://www.bloodjournal.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://www.bloodjournal.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of


http://www.bloodjournal.org/content/6/6/532.full.html

http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests

http://www.bloodjournal.org/site/misc/rights.xhtml#reprints

http://www.bloodjournal.org/site/subscriptions/index.xhtml





relation of methemoglobin to hernolysis - blood

Documents