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A method for measuring haemoglobin at low concentration in the presence of plasma inhibitors
CLINICA
CHIMICA ACTA
279
A METHOD FOR MEASURING HAEMOGLOBIN IN THE PRESENCE
K. R. JOHNSON*,
Department (Received
OF PLASMA INHIBITORS
A. R. INGLE
of Chemacal Pathology, November
AT LOW CONCENTRATION
AND
R. B. PAYNE
Universzty
of Leeds (U.K.)
zr, 1970)
SUMMARY
A method is described for the determination of low concentrations of plasma haemoglobin using o-dianisidine as substrate. The method takes account of the considerable variation in the inhibition of the peroxidase activity of haemoglobin by plasma, and of plasma pigments and turbidity. The recovery of added haemoglobin at 5 mg/roo ml was go-106%.
INTRODUCTION
While using some of the available methods based on the peroxidase activity of haemoglobin to measure plasma haemoglobin concentrations in a healthy youth with haemoglobinuria after cross-country runningl, our attention was drawn to the variability of the recovery of haemoglobin added to normal plasmas. We have confirmed that the peroxidase activity of haemoglobin is inhibited to a variable extent by different plasmas, and that this inhibition is only partially due to haptoglobin2. We have devised a sensitive peroxidase method for measuring plasma haemoglobin using odianisidine (diaminodimethoxydiphenyl) as oxygen acceptor which takes account of the variable inhibition produced by different plasmas. MATERIAL
AND METHODS
Reagents I. 0-Dianisidine
(B.D.H.): 0.5 g/Ioo ml in A.R. ethanol, dissolved by warming at 37”. Store at 2-4”. The greatest care must be taken when weighing o-dianisidine and when handling solutions. It is potentially carcinogenic, and may be absorbed through the lungs and the skin. Solutions must never be pipetted by mouth. 2. Hydrogen Peroxide (B.D.H.): 6 g/Ioo ml (20% v/v). Store at 2-4”. 3. Sod&v phosphate bu..er 0.5 M pH 5.0 Store at 2-4”. * Present address: 7TF (U.K.)
Pathology
Department,
Leeds University
(St. James’s)
Cl&. Chim.
Hospital,
Acta,
Leeds.
LSg
32 (1971) 279-283
JOHNSON et al.
280
4. Stock haemoglobin solution3: I g/100 ml in 0.15 M sodium chloride. Store at in separate small amounts to avoid repeated freezing and thawing. 5. A working haemoglobin solution: 2.5 mg/roo ml. This is freshly prepared by diluting I volume of the stock haemoglobin solution to 400 volumes with sodium phosphate buffer. 6. A mixed reagent: two volumes of hydrogen peroxide solution and I volume of o-dianisidine solution are mixed and made up to roo volumes with sodium phosphate buffer. This must be freshly prepared as auto-oxidation causes progressive darkening. The absorbance in a r-cm cell read against water at 450 nm should not exceed 0.025. The reagent should be protected from sunlight. -15”
Procedure Glassware must be acid cleaned. Plasma, working haemoglobin solution, phospha’te buffer and mixed reagent are mixed in six tubes as follows: Added standard Blank Plasma, ml Working haemoglobin Phosphate buffer Mixed reagent
solution
o
haemwlobin
lmelroo
2.5
5.0
ml blasmal 7.5
IO.0
0.1
0.1
0.1
0.1
0.1
0.0
0.0
0.1
0.2
0.3
0.1 0.4
0.2
0.1
0.0
3.5
3.5
3.5
0.4 3.5
3.9 0.0
0.3 3.5
The tubes are allowed to stand in the dark at room temperature for 20 min. The absorbances of the solutions are read against the blank in I-cm cells at 450 nm and are plotted graphically against the concentrations of added haemoglobin. The line is extrapolated to zero absorbance and the haemoglobin concentration of the test read from the intercept on the haemoglobin axis (Fig. I). The points will fall on a straightline if the haemoglobin-binding capacity of the plasma haptoglobin is greater than the sum of the plasma haemoglobin and the highest standard, or if haptoglobin has been completely depleted. If’the line shows an
!
#’ I
IO.0
_
I
7.5
5.0
2.5
_(
0
2.5
Plasma -,-Added.___hacmoglobln ( mg /
50
7.5
10.0
hacmoglobln
100 ml)
Fig. I. E 450 rim/r cm plotted against concentration of added haemoglobin. The plasma haemoglobm is indicated by the intercept of the extrapolated line on the x axis (m this example, 7.5 mg/roo ml). For greater precision the Intercept may be calculated mathematically (see text). C&z. Chcm. Acta, 32
(1971)
279-283
PLASMA INHIBITORS
IN
Hb
MEASUREMENT
281
upward inflexion, the haemoglobin-binding capacity of the plasma haptoglobin is at the point of inflexion. It may then be necessary to make measurements with additional haemoglobin standards below the point of inflexion to obtain sufficient points to extrapolate a straight line to zero absorbance. For greater precision the intercept may be calculated mathematically, and this method was used in determining the concentrations shown in Tables I and II. If x represents the readings of absorbance for each of a number (n) of final concentrations in plasma of added haemoglobin, y, then the slope of the line of best fit
and the intercept
on the haemoglobin
concentration
axis
a = y--b2 RESULTS
In the presence of hydrogen peroxide, haemoglobin in free solution catalysed the oxidisation of 0-dianisidine to the orange-brown product di-iminodimethoxydiphenyl. This was only sparingly soluble in aqueous solution in the absence of protein, but when sufficient plasma or serum was included in the reaction mixture it remained in solution. The maximum absorbance of di-iminodimethoxydiphenyl in protein solutions was at 450 nm. A concentration of albumin of 50 mg/roo ml was sufficient to maintain in solution the product formed by 20 mg of haemoglobin, and increasing the concentration of albumin did not affect the amount of product formed. Kinetic experiments showed that between pH 4.0 and pH 7.0 the maximum concentration of di-iminodiphenyl had been reached after 20 min in the dark and remained constant for at least a further 30 min. The pH optimum for the peroxidase activity of haemoglobin in plasma or albumin determined using wide ranger buffer4 was 5.0. At pH 5.0 reduction of the ionic strength of the phosphate buffer below 0.05 M reduced the initial velocity of the reaction and the concentration of di-imino compound formed. The reactions were identical in phosphate buffers between 0.1 M and 0.5 M. The peroxidase activity of haemoglobin in plasma was considerably less than that in albumin solution. This is largely due to the formation of a complex between haemoglobin and haptoglobin 518 . Addition of increments of plasma to a solution containing haemoglobin-haptoglobin complex but no free haemoglobin resulted in further small incremental decreases in peroxidase activity. The additional inhibition was not related to albumin concentration. There are, therefore, one or more inhibitors of peroxidase activity in plasma in addition to haptoglobin. Increments of haemoglobin insufficient to saturate haptoglobin were added to a number of specimens of normal plasma, and the peroxidase activities were determined under identical conditions. The relationship between added haemoglobin and peroxidase activity was linear, but the slope of the line varied from plasma to plasma. There are, therefore, variations in the inhibitory activity in normal specimens of plasma under these conditions which make it impossible to determine haemoglobin concentration directly by measuring peroxidase activity. Ctin. Chim. Acta, 32
(1971)
279-283
JOHNSON
282
et al.
There were no significant differences in the peroxidase activities of oxyhaemoglobin, reduced haemoglobin, methaemoglobin and carboxyhaemoglobin. Methaemalbumin has been reported to have a greater peroxidase activity than haemoglobin’ but we have not tested this pigment. Haemoglobin was added to specimens of plasma of very low initial haemoglobin concentration, and the plasma haemoglobin concentrations before and after the additions were determined. In fifteen experiments recovery ranged from go-106% (Table I). The reproducibility of measurements of five plasma specimens with different degrees of haemolysis is shown in Table II. TABLE
I
RECOVERY
OF HAEMOGLOBIN
ADDED
TO-PLASMA
Concentratzons of added haemoglobzn (mglIo0 ml plasma)
Recoveries of added haemoglobin (mglloo ml plasma)
Range of recovevtes 96
Mean recovery 0, /O
5.0
4.6, 5.2, 5 o. 8.5, 8.7,
go-106
100.4
91-106 97-99
97.5 98.2
8.0
9.0 TABLE PRECISION
5.3, 4.5. 5.3, 7.6, 8.9,
5.2, 4.9. 5.2 7.3 8.9
II OF PLASMA
Plasma number
I
2
3 4 5
HAEMOGLOBIN
DETERMINATIONS
Plasma haemoglobin concentration (mgjroo 1.4, 3.1. 8.8, 9.9, 11.8,
1.9. 2.8, 8.9, 9.5, II
7,
1.9 3.2 8.9 9.2 12.1
ml)
Mean and range (mglroo ml) I.7 k 0.3 3.0 f 0.2 8.9 k 0.1 9.5 + 0.4 II.9
&
0.2
DISCUSSION
Several peroxidase methods of varying degrees of complexity and sensitivity have been described for the measurement of plasma haemoglobin, using benzidine (ref. 5, 8, g), o-tolidinelo and o-dianisidine 6~~1.Although some methods allow for the inhibition of peroxidase activity by haptoglobin, it is assumed that this is the same in different specimens of plasma. However, inhibition of the peroxidase activity of haemoglobin by plasma depends not only on haptoglobin but also-on other constituent9 and we have found that the inhibition can vary considerably from plasma to plasma. Vanzente and Valentela overcame the problem by acidifying and extracting the acid haematin into ether and then ethanol. The final pink oxidation product of benzidine was measured. The method is sensitive (1-3 mg/roo ml) but the authors reported that the recovery of added haemoglobin was always low. The assumption is made in the method described here, as in all similar methods, that all the peroxidase activity of plasma is due to haemoglobin. It has two disadvantages. The first is that benzidine, o-tolidine and o-dianisidine are potentially carcinogenic, even under laboratory conditions, and it is essential that proper precautions Clin. Chim. .4&a, 32 (1971) 279-283
PLASMA INHIBITORS
IN
Hb
MEASUREMENT
283
all the methods for measuring are taken when they are handled 15. Unfortunately plasma haemoglobin at low concentrations which are available at the present time make use of one of these diphenyls. The second disadvantage is that five absorbance measurements have to be made for each determination. However it is the only method other than the extraction method of Vanzente and Valente12, which takes account of haptoglobin and other inhibitors in plasma, and of variations in plasma pigments and turbidity. It is sensitive (0.5-12 mg/roo ml), and shows satisfactory reproducibility and recovery. REFERENCES I R. B. PAYNE, J. Clin. Pathol., 19 (1966) 170. 2 R. J. HENRY, Clanical Chemistry, Harper and Row, New York, 1961, p. 788. 3 W. LEHMANN AND J. A. M. AGER, The Laboratory Detectzon of Abnormal Haemoglobins, Broadsheet No. 33, New Series, Association of Clinical Pathologists, London, 1961. 4 D. A. ELLIS, Nature, 191 (1961) Iogg. 5 W. H. CROSBY AND F. W. FURTH, BZood, II (1956) 380. 6 A. LUPOVITCH AND B. ZAK, CZtn. Chum. Acta, g (1964) 49. 7 H. FLEISCH, HeZv. Med. Acta, 27 (1960) 383. 8 H. WV, Biochem. J.. z (1922) 189. g G. E. HANKS, M. CASSELL, R. N. RAY AND H. CHAPLIN, J. Lab. Clzn. Med., 56 (1961) 486. IO G. P. LEWIS, J. Clin. Pathol., 18 (1965) 235. II P. H. TARUKOSKI, Stand. J. CZin. Lab. Invest., 18 (1966) 80. 12 G. VANZENTE AND D. VALENTE, CZin. Chim. Acta, II (1965) 442. 13 CHESTER BEATTY RESEARCH INSTITUTE, Precautzons for Laboratory Workers who Handle Cavclnogenic Aromatic Amines, Institute of Cancer Research, London, 1966. CZtn. Chim. Acta, 32 (1971) 27g-283
CHIMICA ACTA
279
A METHOD FOR MEASURING HAEMOGLOBIN IN THE PRESENCE
K. R. JOHNSON*,
Department (Received
OF PLASMA INHIBITORS
A. R. INGLE
of Chemacal Pathology, November
AT LOW CONCENTRATION
AND
R. B. PAYNE
Universzty
of Leeds (U.K.)
zr, 1970)
SUMMARY
A method is described for the determination of low concentrations of plasma haemoglobin using o-dianisidine as substrate. The method takes account of the considerable variation in the inhibition of the peroxidase activity of haemoglobin by plasma, and of plasma pigments and turbidity. The recovery of added haemoglobin at 5 mg/roo ml was go-106%.
INTRODUCTION
While using some of the available methods based on the peroxidase activity of haemoglobin to measure plasma haemoglobin concentrations in a healthy youth with haemoglobinuria after cross-country runningl, our attention was drawn to the variability of the recovery of haemoglobin added to normal plasmas. We have confirmed that the peroxidase activity of haemoglobin is inhibited to a variable extent by different plasmas, and that this inhibition is only partially due to haptoglobin2. We have devised a sensitive peroxidase method for measuring plasma haemoglobin using odianisidine (diaminodimethoxydiphenyl) as oxygen acceptor which takes account of the variable inhibition produced by different plasmas. MATERIAL
AND METHODS
Reagents I. 0-Dianisidine
(B.D.H.): 0.5 g/Ioo ml in A.R. ethanol, dissolved by warming at 37”. Store at 2-4”. The greatest care must be taken when weighing o-dianisidine and when handling solutions. It is potentially carcinogenic, and may be absorbed through the lungs and the skin. Solutions must never be pipetted by mouth. 2. Hydrogen Peroxide (B.D.H.): 6 g/Ioo ml (20% v/v). Store at 2-4”. 3. Sod&v phosphate bu..er 0.5 M pH 5.0 Store at 2-4”. * Present address: 7TF (U.K.)
Pathology
Department,
Leeds University
(St. James’s)
Cl&. Chim.
Hospital,
Acta,
Leeds.
LSg
32 (1971) 279-283
JOHNSON et al.
280
4. Stock haemoglobin solution3: I g/100 ml in 0.15 M sodium chloride. Store at in separate small amounts to avoid repeated freezing and thawing. 5. A working haemoglobin solution: 2.5 mg/roo ml. This is freshly prepared by diluting I volume of the stock haemoglobin solution to 400 volumes with sodium phosphate buffer. 6. A mixed reagent: two volumes of hydrogen peroxide solution and I volume of o-dianisidine solution are mixed and made up to roo volumes with sodium phosphate buffer. This must be freshly prepared as auto-oxidation causes progressive darkening. The absorbance in a r-cm cell read against water at 450 nm should not exceed 0.025. The reagent should be protected from sunlight. -15”
Procedure Glassware must be acid cleaned. Plasma, working haemoglobin solution, phospha’te buffer and mixed reagent are mixed in six tubes as follows: Added standard Blank Plasma, ml Working haemoglobin Phosphate buffer Mixed reagent
solution
o
haemwlobin
lmelroo
2.5
5.0
ml blasmal 7.5
IO.0
0.1
0.1
0.1
0.1
0.1
0.0
0.0
0.1
0.2
0.3
0.1 0.4
0.2
0.1
0.0
3.5
3.5
3.5
0.4 3.5
3.9 0.0
0.3 3.5
The tubes are allowed to stand in the dark at room temperature for 20 min. The absorbances of the solutions are read against the blank in I-cm cells at 450 nm and are plotted graphically against the concentrations of added haemoglobin. The line is extrapolated to zero absorbance and the haemoglobin concentration of the test read from the intercept on the haemoglobin axis (Fig. I). The points will fall on a straightline if the haemoglobin-binding capacity of the plasma haptoglobin is greater than the sum of the plasma haemoglobin and the highest standard, or if haptoglobin has been completely depleted. If’the line shows an
!
#’ I
IO.0
_
I
7.5
5.0
2.5
_(
0
2.5
Plasma -,-Added.___hacmoglobln ( mg /
50
7.5
10.0
hacmoglobln
100 ml)
Fig. I. E 450 rim/r cm plotted against concentration of added haemoglobin. The plasma haemoglobm is indicated by the intercept of the extrapolated line on the x axis (m this example, 7.5 mg/roo ml). For greater precision the Intercept may be calculated mathematically (see text). C&z. Chcm. Acta, 32
(1971)
279-283
PLASMA INHIBITORS
IN
Hb
MEASUREMENT
281
upward inflexion, the haemoglobin-binding capacity of the plasma haptoglobin is at the point of inflexion. It may then be necessary to make measurements with additional haemoglobin standards below the point of inflexion to obtain sufficient points to extrapolate a straight line to zero absorbance. For greater precision the intercept may be calculated mathematically, and this method was used in determining the concentrations shown in Tables I and II. If x represents the readings of absorbance for each of a number (n) of final concentrations in plasma of added haemoglobin, y, then the slope of the line of best fit
and the intercept
on the haemoglobin
concentration
axis
a = y--b2 RESULTS
In the presence of hydrogen peroxide, haemoglobin in free solution catalysed the oxidisation of 0-dianisidine to the orange-brown product di-iminodimethoxydiphenyl. This was only sparingly soluble in aqueous solution in the absence of protein, but when sufficient plasma or serum was included in the reaction mixture it remained in solution. The maximum absorbance of di-iminodimethoxydiphenyl in protein solutions was at 450 nm. A concentration of albumin of 50 mg/roo ml was sufficient to maintain in solution the product formed by 20 mg of haemoglobin, and increasing the concentration of albumin did not affect the amount of product formed. Kinetic experiments showed that between pH 4.0 and pH 7.0 the maximum concentration of di-iminodiphenyl had been reached after 20 min in the dark and remained constant for at least a further 30 min. The pH optimum for the peroxidase activity of haemoglobin in plasma or albumin determined using wide ranger buffer4 was 5.0. At pH 5.0 reduction of the ionic strength of the phosphate buffer below 0.05 M reduced the initial velocity of the reaction and the concentration of di-imino compound formed. The reactions were identical in phosphate buffers between 0.1 M and 0.5 M. The peroxidase activity of haemoglobin in plasma was considerably less than that in albumin solution. This is largely due to the formation of a complex between haemoglobin and haptoglobin 518 . Addition of increments of plasma to a solution containing haemoglobin-haptoglobin complex but no free haemoglobin resulted in further small incremental decreases in peroxidase activity. The additional inhibition was not related to albumin concentration. There are, therefore, one or more inhibitors of peroxidase activity in plasma in addition to haptoglobin. Increments of haemoglobin insufficient to saturate haptoglobin were added to a number of specimens of normal plasma, and the peroxidase activities were determined under identical conditions. The relationship between added haemoglobin and peroxidase activity was linear, but the slope of the line varied from plasma to plasma. There are, therefore, variations in the inhibitory activity in normal specimens of plasma under these conditions which make it impossible to determine haemoglobin concentration directly by measuring peroxidase activity. Ctin. Chim. Acta, 32
(1971)
279-283
JOHNSON
282
et al.
There were no significant differences in the peroxidase activities of oxyhaemoglobin, reduced haemoglobin, methaemoglobin and carboxyhaemoglobin. Methaemalbumin has been reported to have a greater peroxidase activity than haemoglobin’ but we have not tested this pigment. Haemoglobin was added to specimens of plasma of very low initial haemoglobin concentration, and the plasma haemoglobin concentrations before and after the additions were determined. In fifteen experiments recovery ranged from go-106% (Table I). The reproducibility of measurements of five plasma specimens with different degrees of haemolysis is shown in Table II. TABLE
I
RECOVERY
OF HAEMOGLOBIN
ADDED
TO-PLASMA
Concentratzons of added haemoglobzn (mglIo0 ml plasma)
Recoveries of added haemoglobin (mglloo ml plasma)
Range of recovevtes 96
Mean recovery 0, /O
5.0
4.6, 5.2, 5 o. 8.5, 8.7,
go-106
100.4
91-106 97-99
97.5 98.2
8.0
9.0 TABLE PRECISION
5.3, 4.5. 5.3, 7.6, 8.9,
5.2, 4.9. 5.2 7.3 8.9
II OF PLASMA
Plasma number
I
2
3 4 5
HAEMOGLOBIN
DETERMINATIONS
Plasma haemoglobin concentration (mgjroo 1.4, 3.1. 8.8, 9.9, 11.8,
1.9. 2.8, 8.9, 9.5, II
7,
1.9 3.2 8.9 9.2 12.1
ml)
Mean and range (mglroo ml) I.7 k 0.3 3.0 f 0.2 8.9 k 0.1 9.5 + 0.4 II.9
&
0.2
DISCUSSION
Several peroxidase methods of varying degrees of complexity and sensitivity have been described for the measurement of plasma haemoglobin, using benzidine (ref. 5, 8, g), o-tolidinelo and o-dianisidine 6~~1.Although some methods allow for the inhibition of peroxidase activity by haptoglobin, it is assumed that this is the same in different specimens of plasma. However, inhibition of the peroxidase activity of haemoglobin by plasma depends not only on haptoglobin but also-on other constituent9 and we have found that the inhibition can vary considerably from plasma to plasma. Vanzente and Valentela overcame the problem by acidifying and extracting the acid haematin into ether and then ethanol. The final pink oxidation product of benzidine was measured. The method is sensitive (1-3 mg/roo ml) but the authors reported that the recovery of added haemoglobin was always low. The assumption is made in the method described here, as in all similar methods, that all the peroxidase activity of plasma is due to haemoglobin. It has two disadvantages. The first is that benzidine, o-tolidine and o-dianisidine are potentially carcinogenic, even under laboratory conditions, and it is essential that proper precautions Clin. Chim. .4&a, 32 (1971) 279-283
PLASMA INHIBITORS
IN
Hb
MEASUREMENT
283
all the methods for measuring are taken when they are handled 15. Unfortunately plasma haemoglobin at low concentrations which are available at the present time make use of one of these diphenyls. The second disadvantage is that five absorbance measurements have to be made for each determination. However it is the only method other than the extraction method of Vanzente and Valente12, which takes account of haptoglobin and other inhibitors in plasma, and of variations in plasma pigments and turbidity. It is sensitive (0.5-12 mg/roo ml), and shows satisfactory reproducibility and recovery. REFERENCES I R. B. PAYNE, J. Clin. Pathol., 19 (1966) 170. 2 R. J. HENRY, Clanical Chemistry, Harper and Row, New York, 1961, p. 788. 3 W. LEHMANN AND J. A. M. AGER, The Laboratory Detectzon of Abnormal Haemoglobins, Broadsheet No. 33, New Series, Association of Clinical Pathologists, London, 1961. 4 D. A. ELLIS, Nature, 191 (1961) Iogg. 5 W. H. CROSBY AND F. W. FURTH, BZood, II (1956) 380. 6 A. LUPOVITCH AND B. ZAK, CZtn. Chum. Acta, g (1964) 49. 7 H. FLEISCH, HeZv. Med. Acta, 27 (1960) 383. 8 H. WV, Biochem. J.. z (1922) 189. g G. E. HANKS, M. CASSELL, R. N. RAY AND H. CHAPLIN, J. Lab. Clzn. Med., 56 (1961) 486. IO G. P. LEWIS, J. Clin. Pathol., 18 (1965) 235. II P. H. TARUKOSKI, Stand. J. CZin. Lab. Invest., 18 (1966) 80. 12 G. VANZENTE AND D. VALENTE, CZin. Chim. Acta, II (1965) 442. 13 CHESTER BEATTY RESEARCH INSTITUTE, Precautzons for Laboratory Workers who Handle Cavclnogenic Aromatic Amines, Institute of Cancer Research, London, 1966. CZtn. Chim. Acta, 32 (1971) 27g-283