ANALYTICAL BIOCHEMISTRY 99, 421--426 (1979)

Measurement of the Haptoglobin Concentration in Plasma and Other Fluids by a Simple Spectrometric Procedure 1 D O R O T H Y B . C A L H O U N AND S. W A L T E R E N G L A N D E R

Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Received April 2, 1979 A method for determining the concentration of available haptoglobin in plasma and other solutions is described. The procedure involves absorbance measurements in a mixture of plasma and hemoglobin before and after the addition of sodium dithionite. The haptoglobin content of the solution is calculated from the ratio of the absorbances at the Soret peaks of the oxy- and deoxyhemoglobin. The assay can be performed in minutes, and the error of measurement is well under 10%.

Measurement of the haptoglobin content of blood is important because of its physiological role in the removal of extracellular serum hemoglobin and the change in its concentration in various diseases. In addition, a great deal of biochemical work has been done on this protein, and it has been put to use in a number of studies on hemoglobin. Most analytical methods for determining haptoglobin content take advantage of its capacity to form a complex with hemoglobin. A comprehensive review (1) details the various procedures used. These include such techniques as measurement of the peroxidase activity of hemoglobin which is enhanced by the formation of the complex with haptoglobin (2-4), electrophoresis to separate the hemoglobin-haptoglobin complex from excess free hemoglobin (5-9), the precipitation of the complex with rivanol (10), gel filtration (11,12), the protection by the complex of hemoglobin spectral properties in denaturing conditions (13), immunochemical determinations which measure Supported by Research Grant AMl1295 from the National Institutes of Health, U. S. Public Health Service. 421

haptoglobin itself by means of gel diffusion and immunoelectrophoresis (14-17), and radioimmunoassay using 125I-labeled haptoglobin (18). In general, all of these methods involve tedious or elaborate procedures, expensive or uncommon reagents, lengthy assay time, or readings based on personal judgment. This paper describes a simple technique which requires two readings of optical density in a solution containing haptoglobin and a small amount of hemoglobin. Normal tetrameric deoxyhemoglobin shows a Soret peak absorbance (in 0.1 M sodium phosphate, 0.5% sodium chloride buffer, pH 7.4) about 1.07-fold the Soret peak for the same solution oxygenated. It has been shown (19) that when the separated subunits of hemoglobin are deoxygenated, the Soret peak absorbance is about 20% less than for the deoxy tetramer. Since haptoglobin combines only with hemoglobin subunit dimers, the deoxyhemoglobin-haptoglobin complex displays a reduced Soret absorbance similar to that of the separated dimers (20). Thus the deoxyhemoglobin absorbance varies with the haptoglobin present, but the oxyhemoglobin absorbance does not. By comparing the Soret absorb0003-2697/79/160421-06502.00/0 Copyright © 1979by AcademicPress, Inc. All fightsof reproductionin any formreserved.

422

CALHOUN AND ENGLANDER

ance of an oxyhemoglobin-haptoglobin mixture to the Soret absorbance of the same solution deoxygenated, one can calculate the amount of haptoglobin present. The approach taken in this communication is slanted toward the measurement of haptoglobin in raw plasma because that is clearly a most challenging analytical problem, and indeed the method can be put to good use there. The method described can serve also more generally in the biochemical laboratory. We have for example found the assay extremely useful in directly monitoring yields at various stages in the purification of haptoglobin and in determining the amount of haptoglobin necessary for use in measuring the kinetics of deoxyhemoglobin dimer dissociation. MATERIALS Plasma was obtained from the Hospital of the University of Pennsylvania blood bank and also as 10-ml samples of whole blood from several individuals. The plasma from the blood bank was used directly. The individual samples were centrifuged in an International Clinical centrifuge to separate plasma and red cells. Hemoglobin used in the assays was prepared by a standard method (21) involving a sodium chloride wash, lysing of cells by dilution into distilled water, removal of stroma by high speed centrifugation after readdition of sodium chloride, and dialysis against the desired buffer. It was also found possible to use lysed red cells directly without removing the stroma. Freshly prepared hemoglobin was used in order to avoid methemoglobin contamination. Sodium dithionite (sodium hydrosulfite, purified, Fisher Scientific Co.), used to deoxygenate solutions by chemical reduction, was kept in a desiccator. Measurements were made on a recording Cary 118 or a Zeiss PMQII spectrophotometer, though any spectrophotometer capable of measuring absorbance values at the Soret peaks between 0.5 and 1.5 optical density units can be used.

METHOD For a solution of normal tetrameric hemoglobin, one finds a constant relationship between the Soret peak absorbances before and after the addition of dithionite. In 0.1 M phosphate, 0.5% sodium chloride buffer at pH 7.4, A43o(deoxy)/A4~5(oxy)= 1.07. In our experience this value varies from 1.065 to 1.075. If sufficient haptoglobin is added to complex all the hemoglobin present, the ratio of the Soret peaks drops to 0.85, and will remain at 0.85 regardless of how much excess haptoglobin is added. Between the outside values of 0.85 and 1.07, the Soret ratio varies linearly with the amount of haptoglobin added. For example, when the ratio drops to the midvalue of 0.96, this indicates that the haptoglobin present is sufficient to bind just 50% of the hemoglobin. The method described here is based on this kind of measurement. Figure la shows a typical spectrum of undiluted plasma before and after the addition of sodium dithionite. In a real assay, the plasma present is less concentrated by a factor of 10 or so, and one wants to measure the absorbance due to added hemoglobin. The undesirable background absorbance due to plasma components can be effectively cancelled by placing aliquots of the same plasma sample in both the reference and sample cuvets. This is shown in the bottom curve of Fig. la for undiluted plasma. Figure lb shows spectra obtained when a small volume of hemoglobin was added to diluted plasma and measured in the oxy and deoxy (dithionite added) forms. With different concentrations of plasma haptoglobin, the oxy Soret peak is unchanged, but the deoxy peak varies depending on the fraction of hemoglobin complexed by the haptoglobin. The three deoxy peaks shown (430 nm) are for haptoglobin:hemoglobin ratios of 0 (highest peak), 0.5 (middle), and 1 + (lowest). In the assay, plasma is accurately diluted (usually 1/10 to 1/20) into a phosphate buffer (0.1 M sodium phosphate, 0.5% NaC1, pH 7.4) and mixed. A sufficient volume is placed

423

SORET RATIO METHOD FOR HAPTOGLOBIN CONCENTRATION

oI

b

(3

[ 08

O~

04

02

0

...................................................

400

56o

6~)o

400

~o

660

WQvelength (nm) FIG. 1. (a) Spectra of undiluted blood plasma ( - - - ) before the adition of sodium dithionite; ( ) after the addition of sodium dithionite; ( . . . . . ) equal amounts of plasma in reference and sample cuvettes. (b) Hemoglobin spectra resulting from addition of varying amounts of haptoglobin. All spectra were taken on a recording Cary 118 spectrophotometer at 1.0 absorbance full scale.

in the reference and sample cuvettes. To the sample cuvette is added a small volume (less than 2% by volume) of oxyhemoglobin solution, and this can be stirred by inversion or with a suitable plastic instrument, e.g., a plumper. The precise volume of hemoglobin added is unimportant, but it is well to obtain an optical density at 415 nm of about 1, and the addition of a large volume should be avoided unless the plasma sample in the reference cuvet is to be diluted equally. The optical density at the Soret peak ofoxyhemoglobin (-415 nm) is measured. Then to each cuvette is added about 0.5 mg of dithionite by means of a few grains picked up in the tip of a Pasteur pipet, the solution is stirred, and the optical density of the deoxyhemoglobin Soret peak ( - 430 nm) is measured. With these values, the hemoglobin binding capacity (HbBC) ~ and the plasma haptoglobin content (Hp) can be calculated by use z Abbreviations used: HbBC, hemoglobin binding capacity; Hp, haptoglobin.

of the following equations: HbBC(mg/100 ml) --

100A415 _ _ X m r X _ _ E

=

12.3A415

V v

V

X

--

v

X

Ro - R Ro -

R0 - R X

Ro - R=

Hp(mg/100 ml) = 1.3 HbBC.

R=

,

[1] [2]

The terms used are as follows: A415, measured absorbance of oxyhemoglobin at the Soret peak; E, extinction coefficient of oxyhemoglobin at the Soret peak = 1.31 x l0 s per heme; M,., molecular weight of hemoglobin per heme = 16,170; V/v, dilution factor for plasma in assay = (volume of plasma + diluent)/(volume of plasma). For the term (R0 - R ) / ( R o - R ~ ) , each R value represents a ratio ofdeoxy to oxy absorbance (A430/A415), as follows: R0, with no haptoglobin present = 1.07 (in our hands);R, with the haptoglobin sample to be assayed;

424

CALHOUN AND ENGLANDER TABLE

1

HAPTOGLOBIN ASSAYS ON VARYING AMOUNTS OF ADDED PLASMA a

v(ml) (V = 2 ml)

A41s

R

0.00 0.05 0.075 0.1 0.1 0.15 0.2 0.225 0.25 0.3 0.4 0.6 1.0

-0.973 0.852 0.752 0.993 1.044 0.786 0.937 0.781 0.826 0.840 0.576 0.820

1.070 1.045 1.016 0.989 1.005 0.985 0.916 0.930 0.881 0.869 0.856 0.845 0.855

HbBC Hp (mg/100ml) (rag/100 ml)

as just indicated. Pertinent data are as follows: V/v = (2.00 + 0.250)/0.250 = 9.0; A415(oxy) = 0.781; A4~0(deoxy) = 0.688. These values were handled according to Eqs. [1] and [2]: H b B C = 12.3 x 0.781 x 9.0 x 0.859

-56 71 72 76 71 74 73 74 71 a a a

-72b 93 93 99c 93 97 94 96 93 a a a

a The data points cover the range from -25 to 90% of the hemoglobin bound. Calculated values for mg haptoglobin/100 ml plasma range from 93 to 99 with a mean of 95. The standard error of measurement is 2.2 and the standard error of the mean is 0.8 (i.e., 1% error). Symbols used are those in Eqs. [1] and [2]. b Not included in average. c Assay performed following day. a Hp excess, Hp not measurable. R=, with excess haptoglobin = 0.85 (in our hands). Haptoglobin levels are usually presented as milligrams/100 ml or as the hemoglobin binding capacity, abbreviated H b B C , the amount of hemoglobin in milligrams that can be bound by the haptoglobin present in 100 ml serum or other solution. The conversion factor in Eq. [2] is 1.3 because 1 mg hemoglobin is bound by 1.3 mg haptoglobin, regardless of the haptoglobin type (22-24).

RESULTS Here we present examples of some haptoglobin determinations using the method described.

Assay I. Haptoglobin in a Plasma Sample To 2.00 ml buffer (volumetric pipet) was added 0.250 ml plasma. This was dispensed into reference and sample cuvets and treated

= 74.3 mg/100 ml, Hp = 1.3 x 74.3 = 96.6mg/i00ml.

Assay H. Haptoglobin in a Blood Sample Using the Sample Hemoglobin Itself A 10-ml sample of whole blood was centrifuged in an International clinical centrifuge. Most of the plasma was removed (centrifuged again if cloudy). A volume of packed red cells was taken from the pellet (Pasteur pipet) and diluted 10-fold into deionized water. The solution was vortexed, allowed to stand 15 rain, and vortexed again. The red cells lysed. The amount of hemoglobin needed to give an optical density of about 1 was determined (about 3/zl in 1 ml). Assays were done as described above. There was a small background absorbance, about 0.03, due to the stroma, but this did not interfere with the measurements.

Assay IlL Multiple Determinations on a Given Sample Multiple determinations of the haptoglobin content in a single unit of blood plasma were performed in which varying amounts of plasma were mixed with an approximately constant concentration of hemoglobin. Results are given in Table 1. In Fig. 2, the data are plotted to show the relationship of the absorbance ratio to the volume of plasma added, and the fraction of hemoglobin complexed. For the data points shown, which cover the range from - 2 5 to 90% of the hemoglobin bound, calculated values for milligrams haptoglobin/100 ml plasma range from 93 to 99. The point to point variation seen in Fig. 2 is largely due to differences in the concentration of added hemoglobin present in the different assay mixtures. When

SORET RATIO METHOD FOR HAPTOGLOBIN CONCENTRATION the results are calculated as in Eq. [1], the hemoglobin concentration present is taken into account and this source of variation is r e m o v e d (Table 1).

425

0

~.o~ o E

5o °

0.95 DISCUSSION

The method described here can measure the available haptoglobin content in any solution including raw plasma. The method is fast; if a satisfactory sample of the necessary oxyhemoglobin is available, an assay can be completed in a few minutes. Solutions of oxyhemoglobin cannot be kept on hand very long since they spontaneously oxidize to form methemoglobin which has an altered Soret absorbance. As indicated above, a small volume of freshly lysed red cells can supply the hemoglobin required for the assay. Alternatively, it is also possible to perform a similar assay using methemoglobin, which can be obtained commercially as a dry powder, or a solution o f c y a n m e t h e moglobin which does have a very long shelf life (when refrigerated and tightly capped) can be used. These forms, like oxyhemoglobin, are reduced by dithionite to deoxyhemoglobin. Obviously it would be necessary in these cases to use the appropriate Soret extinction coefficients (25), and appropriately calibrated values for R0 and R=. The method described yields unusually high accuracy. Best accuracy is obtained from mixtures that give Soret ratios near the 50% value, and in general the ratio of haptoglobin binding sites to hemoglobin dimers should be kept in the 0.2 to 0.8 range. With an optical density of 1 and a measured ratio of 0.96 (50% hemoglobin bound by haptoglobin) an error of 0.01 in the optical density leads to an error of 10% in the estimated haptoglobin content. If highest accuracy is desired, it is advisable to perform several assays with varying amounts of added haptoglobin test solution, though for most purposes, one or two determinations should yield an accuracy at least equal to any other available method. Finally it may be emphasized that the

E

0.85

o

t

o:z

0'.4

o:6

o18

I00

I.'o

Plasma Added (ml)

FIG. 2. The ratio of the Soret peak of deoxyhemoglobin to the Soret peak of oxyhemoglobin plotted against the volume of plasma added to 2 ml buffer. A scale for percentage of hemoglobin complexed is also indicated. The apparent variation is largely due to differences in the hemoglobin concentration from one assay to another which is accounted for in the data calculation (Table 1).

present method measures specifically the concentration of available haptoglobin. If prior hemolysis has occurred owing to any cause including the blood drawing itself, some haptoglobin will be present as the complex and thus will be masked. The concentration of serum hemoglobin, and therefore of cryptic haptoglobin, can be obtained from the serum spectrum (Fig. la) by the method of Cripps (26). In the normal serum used for Fig. la, the hemoglobin concentration, estimated in this way, is less than 3 mg/100 ml. Other estimates of extracellular serum hemoglobin can be obtained from the spectra in Fig. la by assuming that all the difference between the two spectra is due to the oxyto deoxyhemoglobin transition. The readings at 430 nm then indicate a hemoglobin concentration of 0.8 mg/100 ml (he430deoxy - oxy = 8 × 104 M-1 heme) and the 575-nm readings suggest 3 mg/100 ml (Ae~7~oxy - deoxy = 5 x 103 M- l h e m e ) . REFERENCES

1. Pintera, J. (1971)Ser. Haematol. 4 (2), 1. 2. Jayle, M. F. (1951)Bull Soc. Chim. Biol. (Paris). 33, 876. 3. Connell, G. E., and Smithies, O. (1959)Biochem. J. 72, 115.

426

CALHOUN AND ENGLANDER

4. Owen, J. A., De Gruchy, G., and Smith, H. (1960) J. Clin. Pathol. 13, 478. 5. Laurell, C. B., and Nyman, M. (1957) Blood 12, 493. 6. Javid, J., and Horowitz, H. J. (1960) J. Clin. Invest. 47, 2290. 7. Ferris, T. G., Easterling, R. E., Nelson, K. J., and Budd, R. E. (1966) Amer. J. Clin. Pathol. 46, 385. 8. Bernier, G. M. (1967) Clin. Chim. Acta 18, 309. 9. Malin, S. F., Baker, R. P., Jr., and Edward, J. R. (1972)Biochem. Med. 6, 205. 10. Korinek, J., and Bohatova, J. (1963) Folia Biol. (Praha) 9, 375. 11. Hodgson, R., and Sewell, P. (1965)J. Med. Lab. Technol. 22, 130. 12. Hallowes, K. H. (1970)J. Med. Lab. Technol. 27, 102. 13. Roy, R. B., Shaw, R. W., and Connell, G. E. (1969) J. Lab. Clin. Med. 74, 698. 14. Kluthe, R., Faul, J., and Heimpel, H. (1965)Nature (London) 205, 93. 15. Storiko, K. (1968)Blut 16, 200.

16. Becker, W., Rapp, W., Schwick, H. G., and Storiko, K. (1968)Z. Klin. Chem. 6, 113. 17. Mancini, G., Carbonara, A. O., and Heremans, J. F. (1965) Immunochemistry 2, 235. 18. Javid, J., and Yingling, W. (1968) J. Clin. Invest. 47, 2290. 19. Brunori, M., Antonini, E., Wyman, J., and Anderson, S. R. (1968)J. Mol. Biol. 34, 357. 20. Chiancone, E., Wittenberg, J. B., Wittenberg, B. A., Antonini, E., and Wyman, J. (1966)Biochim. Biophys. Acta 117, 379. 21. Rossi-Fanelli, A., Antonini, E., and Caputo, A. (1961) J. Biol. Chem. 236, 391. 22. Moretti, J., and Waks, M. (1962) Nouv. Rev. Ft'. Hematol. 2, 433. 23. Hamaguchi, H. (1968) Proc. Japan Acad. 44, 733. 24. Jayle, M. F., and Moretti, J. (1962) Progr. Hematol. 3, 342. 25. Van Assendelft, O. W. (1970) Spectrophotometry of Haemoglobin Derivatives, Van Gorcum, Assem, Netherlands. 26. Cripps, C. M. (1968)J. Clin. Pathol. 21, 110.