393
Clinica Chimica Acta, 66 (1976) 393-403 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
CCA 7547
A KINETIC METHOD FOR THE DETERMINATION OF HAPTOGLOBIN AS HAEMOGLOBIN BINDING CAPACITY
SUSAN STANDING
* and C.P. PRICE **
Department of Clinical Chemistry, East Birmingham Hospital, Bordesley Green East, Birmingham, B9 5ST (U.K.) (Received
August l&1975)
summary A method for the determination of haemoglobin binding capacity which involves kinetic measurement of the peroxidase activity of haptoglobin-bound haemoglobin is described. It is compared with two other methods in common use and the relative merits of the three techniques are discussed. Introduction Methods available for the estimation of haptoglobin in serum are based on its ability to react stoichiometrically with haemoglobin or involve the use of specific immunological techniques. When employing the former procedure to determine haemoglobin binding capacity (HBC) the serum initially is saturated with haemoglobin. The difference in electrophoretic mobility or molecular weight of the haemoglobin-haptoglobin complex and the excess free haemoglobin are then used to separate the two forms. The separation has been achieved by electrophoresis on a variety of media [ 1,2] and by gel filtration [3]. These methods are quantitated by measurement of the peroxidase activity of the bound haemoglobin or by its absorbance. The need for physical separation of the two forms may be eliminated by using the differing peroxidase characteristics of haemoglobin in the free and bound form to permit measurement of the bound in the presence of the free excess. Properties employed to achieve this have been pH optima, inhibition characteristics [4] and differing reaction rates [ 51. Immunological methods, which measure the amount of haptoglobin rather than its binding capacity, have included radial immunodiffusion, Laurel1rocket immunoelectrophoresis [ 61 and enzyme-linked immunoassay techniques [ 71. *
Present address: Biochemistry Department. West Norfolk London Road, King’s Lynn, Norfolk. PE30 SQD, U.K. * * Correspondence to C.P.P.
and
King’s Lynn
General Hospital,
394
None of these methods is entirely satisfactory. Those which involve electrophoresis are time-consuming and can only be used to assay a few specimens in a batch. The gel filtration method will only estimate two or three specimens a day and can give falsely elevated results in the presence of lipaemia or hyperbilirubinaemia. For this reason it is frequently necessary to do both a “test” and “blank” determination on each specimen. Immunologic~ methods are expensive and where radial immunodiffusion is used it is necessary to wait three days for the results. The method described here was developed to make use of the speed and precision of an instrument now available in many laboratories to achieve a rapid, simple and inexpensive assay for the routine laboratory. Materials and methods Unless otherwise Ltd, Poole, U.K.
stated all chemicals
Peroxidase analyses Analyser (LKB Produkter of 12 nm band width.
were obtained
from BDH Chemicals
were performed on the LKB 8600 Reaction Rate AR, Sweden) set at 37°C and using a 470 nm filter
Development of the assay
The peroxidase pH values is shown 95% of its maximum 0.18
activity of free and bound haemoglobin over a range of in Fig. 1. At a pH of 4.0 the bound haemoglobin shows activity whilst the activity of the free is negligible.
r
3.2
3.6
40
4.4
4.8
5.2
5.6
60
PH Fig. 1. Variation in peroxidaseactivity of free ( U) with pH (other conditions as used in the final asa~).
and haptoglobin-bound
(-
) haemoglobin
Electron donor The action of peroxidase is to remove hydrogen ions and electrons from an electron donor to enable hydrogen peroxide to be broken down to form water. The electron donors used to detect peroxidase activity are those which become coloured on oxidation. Many of these compounds are known carcinogens and others suffer from interference by a variety of reducing agents and drugs [8]. In the method described here the most important criteria in the choice of electron donor were the pH and thermal stability of the colour produced . The 4-aminophenazone/phenol system has been used successfully in the estimation of glucose employing glucose oxidase and peroxidase [9] . When the reaction used in this glucose method was carried out at pH 4.0 and 37°C using 6 mM hydrogen peroxide as substrate the colour produced was so unstable that the reaction ceased to give a linear rate after 25 seconds. Alteration of the hydrogen peroxide concentration did not improve the linearity but reducing the temperature to 30°C caused the linearity to be extended to 120 seconds. Fig. 2 shows the reaction curves for the same haemoglobin/serum mixture using 4aminophenazone/phenol at 37 and 30°C. Guaiacol, however, at a concentration of 30 mmol/l and under the final conditions of the assay, gave a linear change in absorbance over a period of approximately lo-70 seconds after addition of substrate. The reaction curve given with guaiacol at 37°C and 3 mM hydrogen peroxide is shown in Fig. 2 for comparison. Guaiacol has the added advantage that it has a peroxidase inhibitory effect that is more marked with free haemoglobin than with the bound form [4]. Should 30°C prove to be a more convenient temperature a more concentrated haemoglobin/serum mixture and a suitable hydrogen peroxide concentration would give sufficient sensitivity and linearity for the 4-aminophenazone/phenol system to be used as electron donor. 0.10-
0.16-
20
40
60
00
100
120
Time(s)
Fig. 2. Peroxidase reaction curves for the same haemoglobin/serum A. 4-Aminopbenazone 3.7 mM, phenol 2.4 mM, hydrogen peroxide Guaiacol 30 mM, hydrogen peroxide 3 mM at 3’7’C.
mixture under various conditions: 6 mM at 37Oc. B. As A at 3O’C. C.
396 TABLE
I
REACTION
RATES
AND DEGREE OF LINEARITY
AT VARIOUS
SUBSTRATE
CONCENTRATIONS
Peroxide concentration in cuvette (mM)
Reaction rate (AA/min)
Extent of linearity {seconds)
0.75 1.5 3.0 4.5 6.0 7.5
0.035 0.059 0.144 0.208 0.215 0.318
94 84 67 48 34 26
It has long been known that peroxidases are inactivated by high concentrations of peroxide [lo] and while increasing the substrate concentration in an attempt to optimise it we observed that although this was giving higher reaction rates they lost their linearity after a shorter period of time (Table I). A substrate concentration of 3.0 mM was chosen for the assay because it gave the highest linear reaction over one minute. Standardisution A convenient way of standardising this type of method is to use pooled human serum and a solution of human haemoglobin (or a derivative) of accurately known concentration. The accuracy of the method hinges on the value assigned to this solution. Other methods [4,11] relied on solutions of haemoglobin derivatives prepared from packed cells and standardised by a variety of methods. The cyanmethaemoglobin standard solution prepared by BDH to B.S. 3985 for such st~d~disations was shown to have suitable stability and peroxidase activity to be used here as the source of haemoglobin. In this way a primary standardisation of each batch was achieved. Final assay procedure Reagents Pooled serum “standards” Approximately 250 ml of haemolysis-free Australian Antigen negative pooled serum from a hospital population is filtered through Whatman No. 1 filter paper and poured into 24132 Visking tubing (The Scientific Instrument Centre Ltd., 1 Leek Street, London, WCl). The tubing is then covered with Aquacide IIA (Calbiochem, Thorpe House, King Street, Hereford) for three to six hours to reduce the volume to approximately one half. The concentrated material is mixed well and aliquots of 0.1,0.2,0.3, etc., to 0.9ml are made up to 1.0 ml with sterile sodium chloride solution (153 mmol/l). The tenth standard is neat concentrated serum. When these, or other dilutions have been shown to give a satisfactory assay range they are prepared in larger quantities, aliquoted and deep frozen. Haptoglobin in serum stored at -15°C is said to be stable for one year [12] ; our experience has shown the “standards” to be stable for at least four months.
397
Cyun~ethue~o~lob~n solution 8.0 ml of the BDH standard is diluted very carefully to 50 ml with saline
to give a concentration of 91.5 mg/l. This is prepared from a fresh ampoule for each batch. (This reagent will now be referred to as haemoglobin solution.) Guaiacol reagent 1.86 g of guaiacol is dissolved in 286 ml of 0.175 M acetic acid (a 1 in 100
dilution of glacial) and made up to approxima~ly 400 ml with distilled water. The pH is then adjusted to 4.0 at room temperature using 1 M sodium hydroxide solution, and the volume made up to 500 ml. The reagent is stable for at least six weeks at 4°C. Hydrogen peroxide solution 0.4 ml of 20 vol hydrogen peroxide is added to 19.6 ml distilled water and
mixed well. This reagent is prepared freshly for each batch. Method
50 ~1 of each “standard” and unknown is diluted with 1.5 ml of haemoglobin solution. After mixing, a O.l-ml aliquot of each dilution is further diluted with 1.0 ml guaiacol reagent into a polystyrene cuvette. The LKB reaction rate analyser is set up as shown below and the analyses performed. The reaction in each cuvette is initiated by addition of hydrogen peroxide trough the pump. Filter Reaction course Pump volume Nozzle Range Time Background absorbance
470 nm Increase 100 /.d White o-0.05 1 minute Zero
Lines are drawn through the reaction rate traces, omitting the short nonlinear portion at the beginning, and the increase in absorbance per minute is calculated. A plot of reaction rate against proportion of pooled serum in each of the “standards” takes the form shown in Fig. 3. The line is slightly curved at the lower end but known to go through zero. A satisfactory line may be constructed by joining zero and the first point and drawing the best straight line through those remaining. The first portion of this graph is then replotted, in a similar way, but with a different scale on the abscissa. The transposition is as follows: The point S on Fig. 3 has the activity of an amount of haptoglobin just saturated with the quantity of haemoglobin in 1.5 ml solution. This corresponds to a haemoglobin binding capacity (HBC) of 2.75 g/l. This figure is obtained from the following calculation: 0.572 X & X mle5 X +$$? . = 2.75 g/l
398
0.16 -f----
Serum concentrate in 1 ml of ‘standard’ (ml) Fig. 3. Plot of peroxidase activity against relative amounts of haptoglobin in the “standard”
solutions.
Where 0.572 = original concentration of BDH standard haemoglobin solution (g/l); $, = dilution factor for haemoglobin solution; & = factor to obtain g haemoglobin per 1.5 ml solution; ‘Ooo o.05 = factor to convert HBC g/50 ~1 to g/l. Point S corresponds to a “standard” value of 0.53 for this particular pooled serum. One must take 3/4, l/2, l/4 and l/8 of this value, i.e., 0.435, 0.29, 0.145 and 0.0725 and read off their corresponding reaction rates. These are then plotted against 3/4, l/2, l/4 and l/8 of 2.75. The haemoglobin binding capacity of the unknowns may then be read off directly from this graph (Fig. 4). Both graphs are plotted for each batch as the co-ordinates of point S tend to vary from batch to batch. (The coefficient of variation for the amount of concentrate in the “standard” at point S over 28 batches was 6.4%) The dilutions of pooled serum used for the “standards” and the haemoglobin solution may have to be varied when a new batch of either is used. These adjustments have to be made in order to obtain: (a) a range of activities that will fit the absorbance range of the reaction rate analyser; (b) a suitable number of “standards” on either side of point S and (c) a convenient range of HBC for the unknowns to be estimated. Test sera with a capacity of greater than 2.75 g/l will give an activity similar to that of the plateau, therefore unknowns with a reaction rate within about 10% of the plateau must be repeated after dilution of the serum. Alternatively, one may use 25 ~1 instead of 50 ~1 in the first dilution. Low values may be measured more accurately by using 100 /.11at this stage. Satisfactory results will not be obtained by varying the size of the aliquot added to the guaiacol reagent unless a “standard” curve is prepared in the same way (this is possibly due to the inhibitory effect of guaiacol). Heparinised plasma should not be us& as its
399
cm
r
Haemoglobin
binding capucity (g/l)
Fig. 4. Data from Fig. 3 replotted in terms of haemoglobin binding capacity.
effect on haemoglobin binding and peroxidase both contribute to cause falsely low results [12]. Other methods Gel filtration rn~t~od The method used was that described by Ratcliff and Hardwicke [ 31. Radial imm~n~diffu~ion technique For this method, commerci~ly prepared immunod~fusion plates and standardised human serum (mixed phenotype) were obtained from Behringwerke (U.K. Agents: Hoechst Pharmaceuticals, Hoechst House, Salisbury Road, Hounslow, TW4 6JH) and the analyses performed according to the instructions provided. Phenotype de termination The haptoglobin phenotypes of sera were determined by disc electrophoresis in polyacrylamide gel by the same method as Smith et al. [13 1, except that 7.5% insteady of 5% gel was used. After electrophoresis the gels were placed in a 1 g/l solution of human haemoglobin in sodium chloride solution (153 mmol/l) overnight and were then washed for 48 hours with several changes of the sodium chloride solution. The haemoglobin remaining bound to haptoglobin in the gel was stained by incubation at 37°C in a solution of 5 g/l p-phenylenediamine dihydrochloride in 0.1 M acetate buffer, pH 5.7 containing 0.5 ml 20 vol hydrogen peroxide per 100 ml. This solution was prepared immediately before use. The gels were allowed to stay in the stain until purple bands could clearly be seen (5-3 min) and then they were removed and placed
400
in water immediately. The visualised phenotype could then be rapidly deduced from the pattern of bands according to Engler et al. [ 141. Results New kinetic
method
Precision
The within-batch coefficient of variation calculated from twenty-six pairs of duplicate results from a serum of mean haemoglobin binding capacity of 1.3 g/l was 2.6%. The between-batch coefficient of variation of this specimen estimated once in each of sixteen batches was 7.3%. Specificity
As would be expected this method is not affected by raised bilirubin levels or lipaemia in serum but haemolysis visible to the naked eye will cause falsely elevated results. The maximum amount of the added excess haemoglobin actually in the cuvette contributes less than 0.01 g/l haemoglobin binding capacity to the final result. Sera giving a positive Schumm’s test (i.e. containing methaemalbumin) appear to give higher results by this method than by the Mancini technique. One such specimen gave a value of 0.28 g/l even though no haptoglobin could be detected immunologically. It is necessary, therefore, to screen specimens visually before analysis for haemolysis and the presence of methaemalbumin. Specimens preserved with sodium azide were found to be unsuitable, probably due to the known inhibitory effect of azide on peroxidases [lo] . Speed
and cost
A batch of forty can be estimated in 2-3 h and the cost of reagents and cuvettes is, at present, about f 0.02 per specimen. Correlation
with other methods
Gel filtration procedure
62 sera were estimated in parallel with the gel filtration method and the results are shown in Fig. 5. The slope and intercept as given by the least squares method were 1.0383 and -0.04 g/l, respectively, and the correlation coefficient r = 0.9786. Immunodiffusion
technique
All of the one hundred and thirty four samples studied were phenotyped by the method described. The results for these specimens grouped according to phenotype are shown in Fig. 6. The immunodiffusion method results were calculated using the factors 0.6,1.3, and 1.5 for the l-l, 2-l and 2-2 phenotypes, respectively. (The factors were given by Behringwerke to allow for the differences in behaviour between the “pure” phenotypes and the mixed standard.) The statistical details for Fig. 6 are shown in Table II.
401
HBC (g/l) by gel filtration
method
Fig. 5, Correlation between haemo$lobm binding capacity (HBC) as determined by the reaction rate method (y) and by gel filtration (x): n = 62, y = 1.0383% - 0.04, r= 0.9786.
2-l 2-2
1.0
20
3.0
4.0
5x)
Ha~toglobin
60
7.0
concentration
8.0
Fig. 6. Correlation according to phenotype, l-l capacity (HBC) and haptoglobin concentration.
TABLE
9.0
10.0
11.0
(g/It (A) 2-l
(X)
2-2
(e),
between haemoglobin
binding
II
STATISTICAL CENTRATION
Parameter
DATA FOR THE CORRELATION (x) SWOWN IN FIG. 6
Slope Intercept r
HBC (Y) AND
Phenotype l-l
n
BETWEEN
29 0.8312 + 0.22 0.9530
2-1 45 0.6336 - 0.01 0.9697
2-2 60 0.5343 + 0.17 0.9731
HAPTOGLOBIN
CON-
402
I I
I
I
I
1
0.4
0.0
1.2
1.6
Serum
IIIII 20
2.4
2.6
haptoglobin as HBC (g/l)
Fig. 7. Distribution of serum baptoglobin levels in sixty normal subjects.
Haptoglobin levels in the well population Haptoglobin levels were estimated in sixty laboratory staff (thirty males and thirty females) in the age range eighteen to thirty years, using the proposed technique. The distribution is shown in Fig. 7. Presentation of conventional statistical parameters is prevented by the fact that the distribution is neither Gaussian nor log-Gaussian. In view of the known variation of serum haptoglobin levels with age and other factors such as steroid therapy, it is proposed to study a much larger group of well people, covering a wider age range. Discussion This paper describes a simple method for the estimation of the haemoglobin binding capacity (HBC) of serum which is far more economical than the immunodiffusion technique and does not involve a long wait for results. The technique is more rapid than the gel filtration period and does not suffer from interference from increased bilirubin levels or lipaemia. However, visible haemolysis will give falsely elevated results and methaemalbumin may give serum an apparent binding capacity even though haptoglobin cannot be detected immunologically. The method has been shown to correlate well with the gel filtration method. The correlation with the immunodiffusion technique was also good but some comments on the genetic variation in haptoglobin type are necessary to interpret the results obtained. Haptoglobins of the 2-l and 2-2 phenotypes take the form of a series of high molecular weight polymers whereas that of the l-l type occurs as a single molecule. There has been some contradiction in the literature as to whether this variation in molecular size affects the amount of haemoglobin bound per unit weight of haptoglobin [15] . If such an effect does exist the similarity in size of the 2-l and 2-2 types would suggest that the relationships they show between haemoglobin binding
403
capacity (HBC) and haptoglobin concentration would resemble one another and be somewhat different to that shown by the l-l type. The correlation according to phenotype observed here, supports this theory as does other work
WI. The immunological techniques now available will be affected to differing extents by other complications which arise from the effects of polymerisation. In the case of radial immunodiffusion the variation in size causes the diffusion coefficient to vary and hence the size of the precipitin ring [16]. The factors given by Behringwerke should allow for this when one uses their mixed standard to determine haptoglobin of a known type. Immunodiffusion methods are therefore limited by the need to rely on such factors and to know the phenotype before an accurate value can be given. The functional determination of haptoglobin as its haemoglobin binding capacity is essentially free from the problems which complicate the measurement of haptoglobin concentration by immunological techniques and would, therefore, seem to be more satisfactory. The method described gives an opportunity to estimate this binding capacity with a good degree of precision and, furthermore, as it employs a recognised haemoglobin standard, it should be possible to obtain reproducible results between laboratories using the same standard. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Colfs. B and Verheyden. J. (1965) Clii. Chim. Acta 12. 4’70-472 Ferris, T.G.. Easterling, R.E., Nelson, K.J. and Budd, R.E. (1966) Am. J. Clin. Pathol. 46, 386-389 Ratcliff, A.P. and Hardwicke. J. (1964) J. Clii. Path& 17, 676-679 Connell. G.E. and Smithies, 0. (1959) Biochem. J. 72.115-121 Lupovitch, A. and Katase. R.Y. (1965) Clin. Chim. Acta 11. 566-570 Driscoll, M.J. (1973) Ann. Clin. Biochem. 10,4-13 Miedema, K.. Boelhouwer, J. and Otten. J.W. (1972) Clin. Chim. Acta 40, 187-192 Sharp, P. (1972) Clin. Chim. Acta 40,115-120 Trinder. P. (1969) Ann. Clin. Biochem. 6, 24-27 Sumner. J.B. and Somers, G.F. (1943) in Chemistry and Methods of Enzymes, p. 181, Academic Press, New York Owen, J.A., Better, F.C. and Hoban, J. (1960) J. Clin. Pathol. 13,163-164 Nyman, M. (1959) Stand. J. Clin. Lab. Invest. 11. Suppl. 39 Smith, I., Lightstone. P.J. and Perry, J.D. (1968) Clin. Chim. Acta 19, 499-505 Engler, R.. Rondeau. Y.. Pointis, J. and Jayle. M.F. (1973) Clin. Chim. Acta 47.149-152 Giblett, E.R. (1969) in Genetic Markers in Human Blood, P. 77, Blackwell, Oxford and Edinburgh Braun, H.J. and Aly, F.W. (1969) Clin. Chim. Acta 26, 588-590
Clinica Chimica Acta, 66 (1976) 393-403 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
CCA 7547
A KINETIC METHOD FOR THE DETERMINATION OF HAPTOGLOBIN AS HAEMOGLOBIN BINDING CAPACITY
SUSAN STANDING
* and C.P. PRICE **
Department of Clinical Chemistry, East Birmingham Hospital, Bordesley Green East, Birmingham, B9 5ST (U.K.) (Received
August l&1975)
summary A method for the determination of haemoglobin binding capacity which involves kinetic measurement of the peroxidase activity of haptoglobin-bound haemoglobin is described. It is compared with two other methods in common use and the relative merits of the three techniques are discussed. Introduction Methods available for the estimation of haptoglobin in serum are based on its ability to react stoichiometrically with haemoglobin or involve the use of specific immunological techniques. When employing the former procedure to determine haemoglobin binding capacity (HBC) the serum initially is saturated with haemoglobin. The difference in electrophoretic mobility or molecular weight of the haemoglobin-haptoglobin complex and the excess free haemoglobin are then used to separate the two forms. The separation has been achieved by electrophoresis on a variety of media [ 1,2] and by gel filtration [3]. These methods are quantitated by measurement of the peroxidase activity of the bound haemoglobin or by its absorbance. The need for physical separation of the two forms may be eliminated by using the differing peroxidase characteristics of haemoglobin in the free and bound form to permit measurement of the bound in the presence of the free excess. Properties employed to achieve this have been pH optima, inhibition characteristics [4] and differing reaction rates [ 51. Immunological methods, which measure the amount of haptoglobin rather than its binding capacity, have included radial immunodiffusion, Laurel1rocket immunoelectrophoresis [ 61 and enzyme-linked immunoassay techniques [ 71. *
Present address: Biochemistry Department. West Norfolk London Road, King’s Lynn, Norfolk. PE30 SQD, U.K. * * Correspondence to C.P.P.
and
King’s Lynn
General Hospital,
394
None of these methods is entirely satisfactory. Those which involve electrophoresis are time-consuming and can only be used to assay a few specimens in a batch. The gel filtration method will only estimate two or three specimens a day and can give falsely elevated results in the presence of lipaemia or hyperbilirubinaemia. For this reason it is frequently necessary to do both a “test” and “blank” determination on each specimen. Immunologic~ methods are expensive and where radial immunodiffusion is used it is necessary to wait three days for the results. The method described here was developed to make use of the speed and precision of an instrument now available in many laboratories to achieve a rapid, simple and inexpensive assay for the routine laboratory. Materials and methods Unless otherwise Ltd, Poole, U.K.
stated all chemicals
Peroxidase analyses Analyser (LKB Produkter of 12 nm band width.
were obtained
from BDH Chemicals
were performed on the LKB 8600 Reaction Rate AR, Sweden) set at 37°C and using a 470 nm filter
Development of the assay
The peroxidase pH values is shown 95% of its maximum 0.18
activity of free and bound haemoglobin over a range of in Fig. 1. At a pH of 4.0 the bound haemoglobin shows activity whilst the activity of the free is negligible.
r
3.2
3.6
40
4.4
4.8
5.2
5.6
60
PH Fig. 1. Variation in peroxidaseactivity of free ( U) with pH (other conditions as used in the final asa~).
and haptoglobin-bound
(-
) haemoglobin
Electron donor The action of peroxidase is to remove hydrogen ions and electrons from an electron donor to enable hydrogen peroxide to be broken down to form water. The electron donors used to detect peroxidase activity are those which become coloured on oxidation. Many of these compounds are known carcinogens and others suffer from interference by a variety of reducing agents and drugs [8]. In the method described here the most important criteria in the choice of electron donor were the pH and thermal stability of the colour produced . The 4-aminophenazone/phenol system has been used successfully in the estimation of glucose employing glucose oxidase and peroxidase [9] . When the reaction used in this glucose method was carried out at pH 4.0 and 37°C using 6 mM hydrogen peroxide as substrate the colour produced was so unstable that the reaction ceased to give a linear rate after 25 seconds. Alteration of the hydrogen peroxide concentration did not improve the linearity but reducing the temperature to 30°C caused the linearity to be extended to 120 seconds. Fig. 2 shows the reaction curves for the same haemoglobin/serum mixture using 4aminophenazone/phenol at 37 and 30°C. Guaiacol, however, at a concentration of 30 mmol/l and under the final conditions of the assay, gave a linear change in absorbance over a period of approximately lo-70 seconds after addition of substrate. The reaction curve given with guaiacol at 37°C and 3 mM hydrogen peroxide is shown in Fig. 2 for comparison. Guaiacol has the added advantage that it has a peroxidase inhibitory effect that is more marked with free haemoglobin than with the bound form [4]. Should 30°C prove to be a more convenient temperature a more concentrated haemoglobin/serum mixture and a suitable hydrogen peroxide concentration would give sufficient sensitivity and linearity for the 4-aminophenazone/phenol system to be used as electron donor. 0.10-
0.16-
20
40
60
00
100
120
Time(s)
Fig. 2. Peroxidase reaction curves for the same haemoglobin/serum A. 4-Aminopbenazone 3.7 mM, phenol 2.4 mM, hydrogen peroxide Guaiacol 30 mM, hydrogen peroxide 3 mM at 3’7’C.
mixture under various conditions: 6 mM at 37Oc. B. As A at 3O’C. C.
396 TABLE
I
REACTION
RATES
AND DEGREE OF LINEARITY
AT VARIOUS
SUBSTRATE
CONCENTRATIONS
Peroxide concentration in cuvette (mM)
Reaction rate (AA/min)
Extent of linearity {seconds)
0.75 1.5 3.0 4.5 6.0 7.5
0.035 0.059 0.144 0.208 0.215 0.318
94 84 67 48 34 26
It has long been known that peroxidases are inactivated by high concentrations of peroxide [lo] and while increasing the substrate concentration in an attempt to optimise it we observed that although this was giving higher reaction rates they lost their linearity after a shorter period of time (Table I). A substrate concentration of 3.0 mM was chosen for the assay because it gave the highest linear reaction over one minute. Standardisution A convenient way of standardising this type of method is to use pooled human serum and a solution of human haemoglobin (or a derivative) of accurately known concentration. The accuracy of the method hinges on the value assigned to this solution. Other methods [4,11] relied on solutions of haemoglobin derivatives prepared from packed cells and standardised by a variety of methods. The cyanmethaemoglobin standard solution prepared by BDH to B.S. 3985 for such st~d~disations was shown to have suitable stability and peroxidase activity to be used here as the source of haemoglobin. In this way a primary standardisation of each batch was achieved. Final assay procedure Reagents Pooled serum “standards” Approximately 250 ml of haemolysis-free Australian Antigen negative pooled serum from a hospital population is filtered through Whatman No. 1 filter paper and poured into 24132 Visking tubing (The Scientific Instrument Centre Ltd., 1 Leek Street, London, WCl). The tubing is then covered with Aquacide IIA (Calbiochem, Thorpe House, King Street, Hereford) for three to six hours to reduce the volume to approximately one half. The concentrated material is mixed well and aliquots of 0.1,0.2,0.3, etc., to 0.9ml are made up to 1.0 ml with sterile sodium chloride solution (153 mmol/l). The tenth standard is neat concentrated serum. When these, or other dilutions have been shown to give a satisfactory assay range they are prepared in larger quantities, aliquoted and deep frozen. Haptoglobin in serum stored at -15°C is said to be stable for one year [12] ; our experience has shown the “standards” to be stable for at least four months.
397
Cyun~ethue~o~lob~n solution 8.0 ml of the BDH standard is diluted very carefully to 50 ml with saline
to give a concentration of 91.5 mg/l. This is prepared from a fresh ampoule for each batch. (This reagent will now be referred to as haemoglobin solution.) Guaiacol reagent 1.86 g of guaiacol is dissolved in 286 ml of 0.175 M acetic acid (a 1 in 100
dilution of glacial) and made up to approxima~ly 400 ml with distilled water. The pH is then adjusted to 4.0 at room temperature using 1 M sodium hydroxide solution, and the volume made up to 500 ml. The reagent is stable for at least six weeks at 4°C. Hydrogen peroxide solution 0.4 ml of 20 vol hydrogen peroxide is added to 19.6 ml distilled water and
mixed well. This reagent is prepared freshly for each batch. Method
50 ~1 of each “standard” and unknown is diluted with 1.5 ml of haemoglobin solution. After mixing, a O.l-ml aliquot of each dilution is further diluted with 1.0 ml guaiacol reagent into a polystyrene cuvette. The LKB reaction rate analyser is set up as shown below and the analyses performed. The reaction in each cuvette is initiated by addition of hydrogen peroxide trough the pump. Filter Reaction course Pump volume Nozzle Range Time Background absorbance
470 nm Increase 100 /.d White o-0.05 1 minute Zero
Lines are drawn through the reaction rate traces, omitting the short nonlinear portion at the beginning, and the increase in absorbance per minute is calculated. A plot of reaction rate against proportion of pooled serum in each of the “standards” takes the form shown in Fig. 3. The line is slightly curved at the lower end but known to go through zero. A satisfactory line may be constructed by joining zero and the first point and drawing the best straight line through those remaining. The first portion of this graph is then replotted, in a similar way, but with a different scale on the abscissa. The transposition is as follows: The point S on Fig. 3 has the activity of an amount of haptoglobin just saturated with the quantity of haemoglobin in 1.5 ml solution. This corresponds to a haemoglobin binding capacity (HBC) of 2.75 g/l. This figure is obtained from the following calculation: 0.572 X & X mle5 X +$$? . = 2.75 g/l
398
0.16 -f----
Serum concentrate in 1 ml of ‘standard’ (ml) Fig. 3. Plot of peroxidase activity against relative amounts of haptoglobin in the “standard”
solutions.
Where 0.572 = original concentration of BDH standard haemoglobin solution (g/l); $, = dilution factor for haemoglobin solution; & = factor to obtain g haemoglobin per 1.5 ml solution; ‘Ooo o.05 = factor to convert HBC g/50 ~1 to g/l. Point S corresponds to a “standard” value of 0.53 for this particular pooled serum. One must take 3/4, l/2, l/4 and l/8 of this value, i.e., 0.435, 0.29, 0.145 and 0.0725 and read off their corresponding reaction rates. These are then plotted against 3/4, l/2, l/4 and l/8 of 2.75. The haemoglobin binding capacity of the unknowns may then be read off directly from this graph (Fig. 4). Both graphs are plotted for each batch as the co-ordinates of point S tend to vary from batch to batch. (The coefficient of variation for the amount of concentrate in the “standard” at point S over 28 batches was 6.4%) The dilutions of pooled serum used for the “standards” and the haemoglobin solution may have to be varied when a new batch of either is used. These adjustments have to be made in order to obtain: (a) a range of activities that will fit the absorbance range of the reaction rate analyser; (b) a suitable number of “standards” on either side of point S and (c) a convenient range of HBC for the unknowns to be estimated. Test sera with a capacity of greater than 2.75 g/l will give an activity similar to that of the plateau, therefore unknowns with a reaction rate within about 10% of the plateau must be repeated after dilution of the serum. Alternatively, one may use 25 ~1 instead of 50 ~1 in the first dilution. Low values may be measured more accurately by using 100 /.11at this stage. Satisfactory results will not be obtained by varying the size of the aliquot added to the guaiacol reagent unless a “standard” curve is prepared in the same way (this is possibly due to the inhibitory effect of guaiacol). Heparinised plasma should not be us& as its
399
cm
r
Haemoglobin
binding capucity (g/l)
Fig. 4. Data from Fig. 3 replotted in terms of haemoglobin binding capacity.
effect on haemoglobin binding and peroxidase both contribute to cause falsely low results [12]. Other methods Gel filtration rn~t~od The method used was that described by Ratcliff and Hardwicke [ 31. Radial imm~n~diffu~ion technique For this method, commerci~ly prepared immunod~fusion plates and standardised human serum (mixed phenotype) were obtained from Behringwerke (U.K. Agents: Hoechst Pharmaceuticals, Hoechst House, Salisbury Road, Hounslow, TW4 6JH) and the analyses performed according to the instructions provided. Phenotype de termination The haptoglobin phenotypes of sera were determined by disc electrophoresis in polyacrylamide gel by the same method as Smith et al. [13 1, except that 7.5% insteady of 5% gel was used. After electrophoresis the gels were placed in a 1 g/l solution of human haemoglobin in sodium chloride solution (153 mmol/l) overnight and were then washed for 48 hours with several changes of the sodium chloride solution. The haemoglobin remaining bound to haptoglobin in the gel was stained by incubation at 37°C in a solution of 5 g/l p-phenylenediamine dihydrochloride in 0.1 M acetate buffer, pH 5.7 containing 0.5 ml 20 vol hydrogen peroxide per 100 ml. This solution was prepared immediately before use. The gels were allowed to stay in the stain until purple bands could clearly be seen (5-3 min) and then they were removed and placed
400
in water immediately. The visualised phenotype could then be rapidly deduced from the pattern of bands according to Engler et al. [ 141. Results New kinetic
method
Precision
The within-batch coefficient of variation calculated from twenty-six pairs of duplicate results from a serum of mean haemoglobin binding capacity of 1.3 g/l was 2.6%. The between-batch coefficient of variation of this specimen estimated once in each of sixteen batches was 7.3%. Specificity
As would be expected this method is not affected by raised bilirubin levels or lipaemia in serum but haemolysis visible to the naked eye will cause falsely elevated results. The maximum amount of the added excess haemoglobin actually in the cuvette contributes less than 0.01 g/l haemoglobin binding capacity to the final result. Sera giving a positive Schumm’s test (i.e. containing methaemalbumin) appear to give higher results by this method than by the Mancini technique. One such specimen gave a value of 0.28 g/l even though no haptoglobin could be detected immunologically. It is necessary, therefore, to screen specimens visually before analysis for haemolysis and the presence of methaemalbumin. Specimens preserved with sodium azide were found to be unsuitable, probably due to the known inhibitory effect of azide on peroxidases [lo] . Speed
and cost
A batch of forty can be estimated in 2-3 h and the cost of reagents and cuvettes is, at present, about f 0.02 per specimen. Correlation
with other methods
Gel filtration procedure
62 sera were estimated in parallel with the gel filtration method and the results are shown in Fig. 5. The slope and intercept as given by the least squares method were 1.0383 and -0.04 g/l, respectively, and the correlation coefficient r = 0.9786. Immunodiffusion
technique
All of the one hundred and thirty four samples studied were phenotyped by the method described. The results for these specimens grouped according to phenotype are shown in Fig. 6. The immunodiffusion method results were calculated using the factors 0.6,1.3, and 1.5 for the l-l, 2-l and 2-2 phenotypes, respectively. (The factors were given by Behringwerke to allow for the differences in behaviour between the “pure” phenotypes and the mixed standard.) The statistical details for Fig. 6 are shown in Table II.
401
HBC (g/l) by gel filtration
method
Fig. 5, Correlation between haemo$lobm binding capacity (HBC) as determined by the reaction rate method (y) and by gel filtration (x): n = 62, y = 1.0383% - 0.04, r= 0.9786.
2-l 2-2
1.0
20
3.0
4.0
5x)
Ha~toglobin
60
7.0
concentration
8.0
Fig. 6. Correlation according to phenotype, l-l capacity (HBC) and haptoglobin concentration.
TABLE
9.0
10.0
11.0
(g/It (A) 2-l
(X)
2-2
(e),
between haemoglobin
binding
II
STATISTICAL CENTRATION
Parameter
DATA FOR THE CORRELATION (x) SWOWN IN FIG. 6
Slope Intercept r
HBC (Y) AND
Phenotype l-l
n
BETWEEN
29 0.8312 + 0.22 0.9530
2-1 45 0.6336 - 0.01 0.9697
2-2 60 0.5343 + 0.17 0.9731
HAPTOGLOBIN
CON-
402
I I
I
I
I
1
0.4
0.0
1.2
1.6
Serum
IIIII 20
2.4
2.6
haptoglobin as HBC (g/l)
Fig. 7. Distribution of serum baptoglobin levels in sixty normal subjects.
Haptoglobin levels in the well population Haptoglobin levels were estimated in sixty laboratory staff (thirty males and thirty females) in the age range eighteen to thirty years, using the proposed technique. The distribution is shown in Fig. 7. Presentation of conventional statistical parameters is prevented by the fact that the distribution is neither Gaussian nor log-Gaussian. In view of the known variation of serum haptoglobin levels with age and other factors such as steroid therapy, it is proposed to study a much larger group of well people, covering a wider age range. Discussion This paper describes a simple method for the estimation of the haemoglobin binding capacity (HBC) of serum which is far more economical than the immunodiffusion technique and does not involve a long wait for results. The technique is more rapid than the gel filtration period and does not suffer from interference from increased bilirubin levels or lipaemia. However, visible haemolysis will give falsely elevated results and methaemalbumin may give serum an apparent binding capacity even though haptoglobin cannot be detected immunologically. The method has been shown to correlate well with the gel filtration method. The correlation with the immunodiffusion technique was also good but some comments on the genetic variation in haptoglobin type are necessary to interpret the results obtained. Haptoglobins of the 2-l and 2-2 phenotypes take the form of a series of high molecular weight polymers whereas that of the l-l type occurs as a single molecule. There has been some contradiction in the literature as to whether this variation in molecular size affects the amount of haemoglobin bound per unit weight of haptoglobin [15] . If such an effect does exist the similarity in size of the 2-l and 2-2 types would suggest that the relationships they show between haemoglobin binding
403
capacity (HBC) and haptoglobin concentration would resemble one another and be somewhat different to that shown by the l-l type. The correlation according to phenotype observed here, supports this theory as does other work
WI. The immunological techniques now available will be affected to differing extents by other complications which arise from the effects of polymerisation. In the case of radial immunodiffusion the variation in size causes the diffusion coefficient to vary and hence the size of the precipitin ring [16]. The factors given by Behringwerke should allow for this when one uses their mixed standard to determine haptoglobin of a known type. Immunodiffusion methods are therefore limited by the need to rely on such factors and to know the phenotype before an accurate value can be given. The functional determination of haptoglobin as its haemoglobin binding capacity is essentially free from the problems which complicate the measurement of haptoglobin concentration by immunological techniques and would, therefore, seem to be more satisfactory. The method described gives an opportunity to estimate this binding capacity with a good degree of precision and, furthermore, as it employs a recognised haemoglobin standard, it should be possible to obtain reproducible results between laboratories using the same standard. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Colfs. B and Verheyden. J. (1965) Clii. Chim. Acta 12. 4’70-472 Ferris, T.G.. Easterling, R.E., Nelson, K.J. and Budd, R.E. (1966) Am. J. Clin. Pathol. 46, 386-389 Ratcliff, A.P. and Hardwicke. J. (1964) J. Clii. Path& 17, 676-679 Connell. G.E. and Smithies, 0. (1959) Biochem. J. 72.115-121 Lupovitch, A. and Katase. R.Y. (1965) Clin. Chim. Acta 11. 566-570 Driscoll, M.J. (1973) Ann. Clin. Biochem. 10,4-13 Miedema, K.. Boelhouwer, J. and Otten. J.W. (1972) Clin. Chim. Acta 40, 187-192 Sharp, P. (1972) Clin. Chim. Acta 40,115-120 Trinder. P. (1969) Ann. Clin. Biochem. 6, 24-27 Sumner. J.B. and Somers, G.F. (1943) in Chemistry and Methods of Enzymes, p. 181, Academic Press, New York Owen, J.A., Better, F.C. and Hoban, J. (1960) J. Clin. Pathol. 13,163-164 Nyman, M. (1959) Stand. J. Clin. Lab. Invest. 11. Suppl. 39 Smith, I., Lightstone. P.J. and Perry, J.D. (1968) Clin. Chim. Acta 19, 499-505 Engler, R.. Rondeau. Y.. Pointis, J. and Jayle. M.F. (1973) Clin. Chim. Acta 47.149-152 Giblett, E.R. (1969) in Genetic Markers in Human Blood, P. 77, Blackwell, Oxford and Edinburgh Braun, H.J. and Aly, F.W. (1969) Clin. Chim. Acta 26, 588-590
