ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 6 Copyright © 1980, Institute for Clinical Science, Inc. Differentiation of Myoglobin and Hemoglobin in Biological Fluids ERNEST C. ADAMS, P h .D. Research Products Division, Miles Laboratories, Inc., Elkart, IN 46515 ABSTRACT The use of several methods to differentiate myoglobin from hemoglobin has been investigated. The immunochemical methods, particularly those of hemagglutination inhibition and radioimmunoassay, are the most useful. This report summarizes work in the Ames Research Laboratory over the past 17 years with the several methods. Introduction haptoglobin-hemoglobin complex is Myoglobin, the oxygen binding pig­ large, so that it is not excreted in the urine. m ent of muscle, and hem oglobin, the oxy­ Thus, no hemoglobin appears in the urine gen binding pigment of erythrocytes, are until the blood level of hemoglobin ex­ alike, yet different. They perform similar ceeds the haptoglobin binding capacity. functions and undergo many of the same Once all the haptoglobin is bound, reactions. There are some physical and hemoglobin appears in the plasma, both chemical differences.3,11 Despite their bound to albumin as methemalbumin and many similarities, myoglobin and hemo­ as free hemoglobin. globin are immunologically different. This Myoglobinuria is often inferred from difference, and some of the physical and the clinical symptoms of muscle weakness chemical differences, can be used to dif­ and pain,11,20'21 when there is a ferentiate one from the other. With the peroxidase-like pigment in the urine with exception of the immunochemical few or no erythrocytes seen on the micro­ methods, this usually requires the pres­ scopic examination. A rise in serum ence of relatively large quantities of creatine phosphokinase (CPK) is often myoglobin and hemoglobin. used to bolster the suspicion.21 If the labo­ Myoglobin is released into the blood ratory is asked to confirm this presump­ plasma as a result of damage to muscle tive diagnosis, a classical method such as tissue. Because of its small size and lack of solubility in 80 percent saturated am­ binding to haptoglobin, it is rapidly re­ monium sulfate might be used. When moved from the blood by the kidney and there is insufficient pigment in the urine excreted into the urine. Hemoglobin, to be clearly visible, there generally is no when released from the red cell by in- suspicion of hemoglobinuria or myo­ travascular hemolysis, is rapidly and globinuria. The conditions would be de­ tightly bound to haptoglobin. The tected only by routinely running occult 493 0091-7370/80/1100-0493 $01.20 © Institute for Clinical Science, Inc. 494 ADAMS blood tests. The differentiation would munization, the portion of gel containing then have to be by the more sensitive im­ the myoglobin bands is electroeluted. munochemical tests. Colored eluted fractions are pooled and the absorbance in the Soret band region is M eth od s measured. After the elution, the myoglo­ bin is in the ferro form; at a concentration N o n -Immunological M e t h o d s of one mg per ml, this has an absorbance of The more classical methods of differ­ 5.8 at 422 nm. From this value and the entiation, such as ammonium sulfate sol­ volume of elute, the total amount of myo­ ubility, ultrafiltration, gel exclusion globin is calculated. The elute, which con­ chromatography, affinity chromatog­ tains buffer matetial as well as myoglobin, raphy, and spectra, are described and dis­ is lyophilized. For immunization, the cussed in the earlier publication.3 In some myoglobin can be either human or of these, the literature method was mod­ monkey. ified by using occult blood tests to in­ The hemoglobin immunogen is pre­ crease the sensitivity. pared from laked erythrocytes as de­ scribed earlier3 and purified by electro­ phoresis. I mmunochemical D ifferentiation The earlier immunization scheme3 has o f M y o g l o b in a n d H e m o g l o b in been modified in dose and route. The ini­ The most definitive differentiation of tial immunization in Freund’s complete hemoglobin and myoglobin in biological adjuvant is given into the four footpads of fluids can be achieved by using relatively rabbits or into the four dew claws of goats. simple immunochemical methods. These The boosters in Freund’s incomplete ad­ methods, which include immunodiffu­ juvant are given subcutaneously or in­ sion, hemagglutination inhibition, and tramuscularly (roughly equal amounts immunoelectrophoresis, are dependent into the four quarters of the animals). The upon the fact that specific antisera will initial dose is 45 fig per animal and boos­ react only with its homologous antigen. ter doses are 22.5 fig per animal. The Moreover, the methods will quantitate the schedule of boosting and bleeding remain hemoglobin or myoglobin present. the same. The antisera are centrifuged Preparation of Antisera. The isolation and processed as soon as a good clot has and purification of the immunogens, the formed. On immunodiffusion or im­ immunization schedules, and the process­ munoelectrophoresis, the antisera to ing of the antisera are essentially the same myoglobin should give a precipitin line as previously reported.3 Myoglobin is iso­ against a myoglobin solution or a urine lated from human or rhesus monkey mus­ containing myoglobin at levels of 40 to 50 cle according to the selective precipita­ fig per ml and no line against human al­ tion method of Luginbuhl.18 Hemoglobin bumin at levels of 1000,100, and 10 fig per and proteins other than myoglobin are ml or against human serum. The antisera precipitated from extracts of muscle at pH to hemoglobin should give a precipitin 8 with 80 percent ammonium sulfate sat­ line against hemoglobin at levels of 40 to uration. Under these conditions, the 50 ¡jug per ml and no lines against albumin myoglobin remains in solution. Myoglo­ or other serum proteins at levels of 1000, bin is precipitated at pH 7 with 100 per­ 100, and 10 fig p er ml. cent ammonium sulfate saturation. The Methods for Detecting Antigen- myoglobin is further purified by elec­ Antibody Reaction. In this laboratory, the trophoresis on acrylamide gel which re­ methods employed for the immunochem­ moves albumin and other serum protein ical reactions of myoglobin and hemoglo­ contaminants. To obtain material for im­ bin are immunodiffusion, immunoelec­ MYOGLOBIN AND HEMOGLOBIN DIFFERENTIATED IN BIOLOGICAL FLUIDS 495 trophoresis, and hemagglutination inhibi­ protein-buffer without antisera. One hori­ tion for urine, radioimmunoassay for zontal row is marked for a negative urine serum myoglobin, and latex agglutination (titer row) one is marked for each standard for special applications. Immunodiffusion urine, and one for each unknown urine. In and immunoelectrophoresis are the same each well of the first vertical row is placed, as described earlier,3 but with modifica­ with a Pasteur pipet (0.025 ml), one drop tions of media and buffer as in a later pa­ of the 1:20 antiserum dilution; in each per.5 Precipitin lines develop within four well of the second vertical row is placed to 16 hours. one drop of the 1:40 dilution. This is con­ Hemagglutination Inhibition. M yoglo­ tinued through the ninth vertical row. In bin is coupled to formalinized sheep red the tenth row is placed one drop of the blood cells as previously described3 with protein-buffer. One drop of negative urine modifications. After dialysis in the carbo­ is added to all the wells of the first hori­ nate buffer, the crude myoglobin solution zontal row; one drop of standard or un­ is diluted so that it has an absorbance of known urine is added to each well of the 0.79 at 410 nm. One half ml of this solution other horizontal rows. One drop of the ap­ is reacted with 6 mg pyrrole-2-carboxylic propriate conjugate is added to all the acid azide at pH 9.5. The packed cells wells. from 7 ml of the 10 percent formalinized The contents of the wells are mixed sheep erythrocytes are reacted with 2 ml either by vigorously rotating the whole bis-diazobenzidine at pH 3.5. The plate or by stirring each well with an myoglobin-pyrrole component is reacted applicator stick or toothpick. The plate is with the diazo-benzidine-cells at pH 7. placed either over a mirror or on a white Hemoglobin is coupled to formalinized background for one hour. A positive is in­ sheep red blood cells.3 dicated by a ring or button of cells in a well For both myoglobin and hemoglobin to the left of the first well with a ring in the conjugates, the stabilized diazonium salt, titer row. The greater the number of wells Fast Black B, can be substituted for the with rings, the greater the concentration bis-diazobenzidine.6 A solution of 1.2 mg of myoglobin or hemoglobin. Fast Black B per ml is equivalent to the The best standard for myoglobin is a prepared bis-diazobenzidine. The prep­ urine containing a large amount of myo­ aration of pyrrole-2 carboxylic acid azide globin but no hemoglobin. The amount of has been described.6 myoglobin can be determined by a The antisera are heated at 56° to destroy peroxidase activity method and then the complement and absorbed with for­ urine can be diluted with negative urine malinized sheep red blood cells as de­ to give appropriate standards. A standard scribed.3 Two fold dilutions of the anti­ for myoglobin can be prepared by sera (starting with 1:20) are made in 0.2 M, chromatography of a muscle extract on pH 8.5 Bicine buffer containing 1 percent Sephadex® G-100. The first peroxidase-, nonimmune rabbit serum. The number of like reactive band is catalase, the second dilutions will depend on the titer, but one is hemoglobin, and the third is myo­ nine dilutions are usually prepared.