A METHOD FOR DETERMINING METHEMOGLOBIN IN THE PRESENCE OF ITS CLEAVAGE PRODUCTS.

Transcription

1 A METHOD FOR DETERMINING METHEMOGLOBIN IN THE PRESENCE OF ITS CLEAVAGE PRODUCTS. BY JAMES B. CONANT AND LOUIS F. FIESER. (From the Chemical Laboratory of Harvard University, Cambridge.) (Received for publication, October 28, 1924.) Aside from the use of a spectrophotometer, the methemoglobin content of a solution is usually estimated indirectly by determining the total amount of pigmented protein and the reduced hemoglobin, and assuming the difference to represent methemoglobin. Thus, the procedure of Stadie (1) and of Van Slyke and Stadie makes use of the cyanhemoglobin calorimetric method for determining so called total hemoglobin (i.e. the sum of reduced and methemoglobin) and the oxygen capacity for determining the reduced hemoglobin. This procedure for total hemoglobin is very satisfactory if no other colored substances are present, but is not applicable in the presence of the colored cleavage products of hemoglobin (hematin). This became evident to us in the course of some experiments with hemoglobin solutions which had undergone considerable decomposition; we compared the amount of sodium hyposulfite required in an electrometric titration (3) and the percentage of methemoglobin by the Van Slyke-Stadie method, and found the ratio to be very different from that obtained with pure hemoglobin. By adding varying amounts of hematin to solutions of hemoglobin it was further demonstrated that the presence of such cleavage products greatly affected the colorimetric determination of the total hemoglobin content. Spectrophotometric methods would probably not be affected in this manner, although it is possible that the presence of certain colored substances might make even the use of this instrument unreliable for the determination of methemoglobin. As we desired to determine methemoglobin in a variety of solutions and a spectropho- 1 Van Slyke and Stadie (2), p

2 624 Methemoglobin and Cleavage Products tometer was not available, we developed a simple procedure for determining total hemoglobin in the presence of its cleavage products and any other colored substance. This method may be found useful as an adjunct to the spectrophotometric procedure and, as it requires no special apparatus, may recommend itself to others who for one reason or another cannot employ an optical method. The method depends on the fact that methemoglobin is quantitatively changed to reduced hemoglobin by certain powerful reducing agents (3), and the amount of reduced hemoglobin thus formed can be determined by the increase in oxygen capacity of the solution after treatment with the reducing agent. Three reducing agents were tried, sodium hyposulfite, sodium anthrahydroquinone-/?-sulfonate, and disodium anthrahydroquinone-2, 6- disulfonate. All three substances rapidly and quantitatively reduce methemoglobin, and apparently would be equally suitable for the purpose at hand. It was found, however, that the use of sodium hyposulfite was quite out of the question as, on shaking the reduced hemoglobin with oxygen, methemoglobin was rapidly formed again. Apparently the oxidation products of sodium hyposulfite are powerful catalysts for the oxidation of hemoglobin to methemoglobin. This adverse experience with sodium hyposulfite, as well as some experiments on the decomposition of hemoglobin solutions to be described in another paper, made it evident that it would be necessary to employ some reducing agent whose oxidation products would influence slightly, if at all, the rate of formation of methemoglobin by oxygen. By using disodium anthrahydroquinone-2,, 6-disulfonate as the reducing agent and vigorously shaking the solution for only 15 seconds with oxygen, it was possible to obtain fairly satisfactory results. These are recorded in Table I; the detailed procedure need not be described, as eventually this method was superseded by the use of the p-monosulfonate in the manner outlined below. The hemoglobin solution employed was freshly laked corpuscular cream from horse blood, and the oxygen capacities were determined in the usual manner in the Van Slyke apparatus. An inspection of the table shows that the reduced hemoglobin content could be increased by the action of the reducing agent to almost the original value of the solution before treat-

3 J. B. Conant and L. F. Fieser 625 ing with ferricyanide. The average of the results obtained (Experiments 3 to 7, Table I), however, is about 0.50 per cent too 10~ in total hemoglobin as compared with the original material; this, in terms of percentage error, is about 3.3 per cent, and is much greater than the errors of the Van Slyke method for determining the oxygen capacity. Evidently some of the reduced hemoglobin TABLE I. Determination of Total Hemoglobin by Reduction with Disodium Anthrahydroq u&one-2, 6-Disulfonate. - - cc. None 3.007: None I rn. per 100 cc cc. per cc. None { min rn. per 1oocc d , Remarks. Blank determination giving Hb content of pure solution. Illustrating effect of prolonging time with 02. Hb content determined by oxygen capacity in the Van Slyke apparatus; correction for physically dissolved oxygen applied as described under Calculation of results. was reconverted to methemoglobin even in the very short time the solution was shaken with oxygen. This was further shown to be the case by carrying out the last four experiments recorded in the table, in which the time of contact with the oxygen was increased; the decrease in reduced hemoglobin content is very evident, particularly in the last experiment.

4 626 Methemoglobin and Cleavage Products While the results presented in Table I show that fairly satisfactory determinations of total hemoglobin can be made by using d&odium anthrahydroquinone-2, 6-disulfonate, we desired to make the method still more accurate. To this end we substituted sodium anthrahydroquinone-fi-sulfonate for the disulfonate, since preliminary experiments seemed to indicate that this substance was somewhat less effective in catalyzing the formation of methemoglobin. Thus, in comparing the action of equal weights of the p-sulfonate and disulfonate on pure hemoglobin solution, it was found that the former produced only 5.7 per cent of methemoglobin, while the latter produced 7.0 per cent in a given time. The reduction potential of the p-sulfonate is even lower than that of the disulfonate (4), so that it is perfectly suitable from this point of view. Experiments in which the p-sulfonate was used as a reducing agent showed that the determination of total hemoglobin with this reagent was very satisfactory. They are recorded in Table II; the exact experimental procedure which is recommended for use as an analytical method is given below. The methemoglobin content of the solution after the addition of the ferricyanide in Experiments 5 and 6 was estimated from our experiments on the ferricyanide reaction described in the preceding paper (3). Similarly the methemoglobin content after the addition of 0.5 equivalent of benzoquinone (Experiment 7), and after shaking with air for 1 hour in the presence of anthraquinone disulfonate (Experiment S), could be approximately stated from work which is soon to be published. The exact amount of methemoglobin was of little importance in showing the validity of the analytical procedure, and the oxygen capacity of the solution was, therefore, not determined at this point. That the success of the procedure is in no way dependent on the reagent used for forming the methemoglobin, is demonstrated by these last two experiments. An examination of the last column of Table II shows that the total hemoglobin content of the solution after considerable methemoglobin had been produced by various reagents agrees well with the blank determinations made with the pure hemoglobin solution (Experiments 1 and 2). The error is not more than 3 parts in 130, and in most cases is considerably less than this.

5 J. B. Conant and L. F. Fieser 627 Procedure. Preparation of Sodium Anthrahydroquinone-&%lfonate.-The commercial sodium anthraquinone+-sulfonate is usually about 90 per cent pure and is satisfactory for the preparation of the anthrahydroquinone compound. This is accomplished by shaking a solution of the anthraquinone sulfonate with platinized asbestos and hydrogen, preferably under slight pressure. A solution of convenient strength is prepared by dissolving 1.5 gm. of the sodium anthraquinone+sulfonate in 100 cc. of a M mixture of disodium and monosodium phosphates of ph about 6.4. This, together with a small amount of platinized asbestos, is placed in a small bottle provided with a hydrogen inlet entering below the surface of the TABLE Determination o.f Total, Hemoglobin with the Aid of Sodium Anthrahydroquilzone-P-Sulfonate. T Experi- Reagent employed for producing ment No. methcmoglobin. None. I 0.5 equivalent of K,Fe(CN)a. 0.7 ( 0.5 benzoquinone. 1.0 anthraquinone-2, 6- disulfonate and 1 hr. with air. II. - 1% :: Drone cc. None Hb content after redurtion. gm. per 100ce The hemoglobin solution employed was freshly laked corpuscular cream from horse blood. - liquid, a gas outlet provided with a stop-cock, and a tube extending to the bottom of the vessel; this tube ends in a small inverted funnel provided with a Witt filter plate covered with cotton gauze. On the upper end is a piece of vacuum rubber tubing closed by a pinch-cock, and into which the tip of a very small burette (of a few cc. capacity)2 could be fitted for removing the anthrahydroquinone solution. After saturating the solution with hydrogen, the outlet is closed and the bottle shaken for 1 to 2 hours, which completes the reduction. If desired, the amount of hydrogen absorbed can be measured by a suitable measuring apparatus, and the 2 Conveniently made by sealing a glass stop-cock on the bottom of the usual graduated 2 or 5 cc. pipette (uniform bore).

6 628 Methemoglobin and Cleavage Products progress of the reduction followed in this way. The resulting highly colored solution is molar or normal in respect to its reducing power, which may be determined by electrometric titration against potassium ferricyanide. It is very sensitive to air, and must be handled rapidly and protected against the action of oxygen by a layer of xylene in the burette from which it is measured. Determination of Total Hemoglobin.-A carefully measured 4 cc. sample of the hemoglobin solution is freed from oxygen in a 250 cc. tonometer by evacuation and filling with nitrogen in the usual manner. The anthrahydroquinone solution is then forced from the bottle in which it was prepared, through the tube previously described, into the micro burette by the hydrogen pressure, a small amount of xylene being previously introduced into the burette. From 0.5 to 1.0 cc. of this anthrahydroquinone solution (the amount depending on the concentration of methemoglobin present) is then rapidly introduced into the nitrogen-filled tonometer, which is then evacuated. The tonometer is then given a few rotations to insure complete mixing, filled rapidly with oxygen (at atmospheric pressure), shaken vigorously for 5 to 10 seconds, and a measured sample withdrawn by means of an Ostwald-Folin pipette and at once introduced into the Van Slyke apparatus for determining oxygen capacity. The determination of the oxygen combined in the sample is carried out exactly as described by Van Slyke and Neil1 (5). Even using the /3-sulfonate, it is necessary to analyze the sample immediately after it has been shaken with oxygen, and to restrict the shaking with this gas to 5 to 10 seconds of vigorous agitation. Experiments showed that the time during which the reducing agent is in contact with the solution is of no significance and may be as long as 20 minutes. The only point at which especial pains must be taken to work rapidly is during the equilibration with oxygen, and subsequent removal of a sample to thk Van Slyke apparatus. Calculation of Results.-The calculation of the oxygen content of the sample from the difference in pressure is made according to the formula given by Van Slyke. Because of the fact that the hemoglobin is equilibrated with pure oxygen and not air, the correction for dissolved oxygen is different from that usually employed. We have taken the amount of oxygen physically dissolved in hemoglobin equilibrated with pure oxygen at 38 and 760 mm. (710 mm. partial pressure) as 1.89 cc. per cc., by extrapolation of data given by Van Slyke and Stadie.3 Using this value and Bohr s solubility data (CZ = cc. of gas at O, 760 mm., dissolved by 1 cc. of water), we have calcblated the correction for the dissolved oxygen at different temperatures from the following formula: 760-aqueous tension (Cc. of O2 per 100 cc.)i = 1.89 X og X Van Slykr and Stadie (2). p. 17.

7 J. B. Conant and L. F. Fieser 629 using c\! = at 15, at 20, at 25, and at 38. It is convenient to express this in terms of percentage of hemoglobin, and the corrrection can then be directly subtracted from the uncorrected hemoglobin content calculated directly from the readings of the Van Slyke apparatus. It is very probable that later work may change the value of this correction for physically dissolved oxygen, and we consider the correction we have employed merely as a suitable approximation. The corrected hemoglobin content of the sample must then be converted into percentage (gm. per 100 cc.) of total hemoglobin in the original solution. This is done by multiplying by the factor -> l + 1 m where m is the cc. of reducing agent employed per cc. of hemoglobin solution. In the experiments given in Tables I and II, since we also added varying amounts (n) of ferricyanide solution, etc., the factor $- y n was employed to reduce all the results to a comparable basis. Knowing the total hemoglobin content of the solution, and having determined the oxygen capacity of the original solution and thus the reduced hemoglobin content (corrected, of course, for physically dissolved oxygen), the percentage of methemoglobin is obtained by difference. Or, if one prefers, the results may be expressed in terms of oxygen capacities before and after treatment with the reducing agent, and the change in oxygen capacity then converted to percentage of methemoglobin. This treatment of the data makes it evident that the correction for physically dissolved oxygen affects the value of the methemoglobin content but slightly; indeed, if no increase in volume attended the addition of the reducing agent, the correction would completely cancel. As it is, it amounts to mz, where m is the cc. of reducing agent per cc., and Z the correction for physically dissolved oxygen expressed in percentage of hemoglobin; since m is 0.1 to 0.3 and Z not more than 2 per cent, the value of Z might be considerably in error without influencingverymuch the methemoglobin content. Whichever of the methods of calculation is employed, it is apparent that what one is really measuring is the amount of material which by itself has no oxygen capacity, but which after treatment with a reducing agent combines with oxygen reversibly. As far as is known, the only substance which has this very peculiar property is methemoglobin.

8 630 Methemoglobin and Cleavage Products A few experiments were carried out to see whether the presence of the oxidized form of the reagent employed (i.e. the anthraquinone sulfonate) influenced appreciably the amount of physically dissolved oxygen. The results given in Table III show that the correction we have employed is applicable to dilute solutions and those containing the organic and inorganic substances used in our work; the oxygen capacities were corrected as outlined above, the solutions being equilibrated with pure oxygen, and the amount of hemoglobin in the original solution calculated by use of the factor q, where w is the cc. of diluent per cc. of hemoglobin. TABLE Hemoglobin Content of Solutions Diluted with Various Diluents. None... Diluent. 30percentHz L reducing agent H,O M phosphate buffer... SUMMARY. III. UnCOrrected Percentage Zorrected for 3hysically dissoolred Hb..n original solution corrected) A method for determining methemoglobin in the presence of its cleavage products or other colored material has been developed. It consists in measuring the increase in oxygen capacity, resulting from the action of a powerful reducing agent which quantitatively converts the methemoglobin to hemoglobin. Sodium anthrahydroquinone$-sulfonate is a satisfactory reducing agent for this purpose, although the oxygen capacity after reduction must be determined rapidly, and the equilibration with pure oxygen must be of very short duration. Disodium anthrahydroquinone- 2, 6-disulfonate is a somewhat less satisfactory reagent, while sodium hyposulfite cannot be used at all because its oxidation products catalyze so greatly the reformation of methemoglobin.

9 J. B. Conant and L. F. Fieser 631 BIBLIOGRAPHY. 1. Stadie, W. C., J. Biol. Chem., 1920, xli, Van Slyke, D. D., and Stadie, W. C., J. Biol. Chem., 1921, xlix, Conant, J. B., and Fieser, L. F., J. Biol. Chem., , Ixii, Conant, J. B., Kahn, H. M., Fieser, L. F., and Kurtz, S. S., Jr., J. Am. Chem. Xoc., 1922, xliv, Van Slyke, D. D., and Neill, J. M., J. Biol. Chem., 1924, lxi, 523.

10 A METHOD FOR DETERMINING METHEMOGLOBIN IN THE PRESENCE OF ITS CLEAVAGE PRODUCTS James B. Conant and Louis F. Fieser J. Biol. Chem. 1925, 62: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1