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A sensitive micromethod for the determination of methemoglobin in blood
CLINICA CHIMICA ACTA
679
A SENSITIVE ~ICROMETHOD OF METHEMOGLOBIN
E. HEGESH’,
N. GRUENER2,
FOR THE DETERMINATION
IN BLOOD
S. COHENa,
R. BOCHKOVSKF
AND
H. I. SHUVAL*
Biochemicat Research Laboratory, Kaplan Hospitat, Rehovoth? and ~e~artrne~~ of Medical Ecolitgy, Hebrew ~7~jue~s~ty-Hadassah _~~ed~catSchool, Jer%satemP (Israel) (Received
June 5. 1970)
SUMMARY
A modification of the Evelyn and Malloy method for the determination of methemoglobin in blood is presented. A higher precision of the assay is achieved, and sample stability is increased up to 24 h, thus making the procedure suitable for field surveys aimed at detecting slightly raised methemoglobin levels.
INTRODUCTION
Increasing levels of nitrates in drinking water have raised the need for reevaluation of potential risks of its use for the health of the population. Ingestion of nitrates has been shown to induce methemoglobinemia in newborns and young infantslp2. In connection with a study to determine methemoglobin levels in infants exposed to high nitrate content in water the need arose for a precise and accurate micromethod which would assure adequate stability of the sample to allow a field survey to be conducted in communities distant from the laboratory*. The method of Evelyn and MalloyP, which up to date has been the standard procedure for the assay of methemoglobin in blood, is considered not sufficiently sensitive to determine low concentrations of the pigment, present in normal blood samples and to differentiate between normal and slightly elevated levels which might be indicative of subclinical cases of methemoglobinemia caused by nitrate ingestion. Due to the instability of the methemoglobin in the drawn blood sample it is usually recommended in normal clinical procedures to carry out the test as rapidly as possible. This is generally not feasible under field survey conditions. The procedure presented below is a modification of the Evelyn and Malloy method aimed to augment its precision. The size of the blood sample is kept at a minimum so that finger-tip blood samples from infants should suffice. The procedure also assures sample stability.
Clin.
Chim.
Acta, 30 (1970) 679-682
680
HEGESH
et d.
Methemoglobin is reacted with cyanide and the change in light absorption at 632 nm is measured. Reagents I. K,Fe(CN),-5% (w/v) in water. Store in a dark bottle. Prepare monthly. 2. Phosphate buffer-o.5 M, pH 7.2. 3. Ferricyanide-phosphate mixture: 0.2 ml of K,FefCN), solution (reagent I) and 2.5 ml of phosphate buffer (reagent 2) are made up IO to ml with water. Prepare daily. 4. Sodium cyanide-10% (w/v). Prepare monthly. 5. Acetic acid-rz% (v/v). 6. Neutralized cyanide solution. Mix 1.0 ml of reagent 4 with 0.9 ml of reagent 5. Use within 6 h. Instrwnents I. High speed microcentrifuge, capacity IO ooo rev./min. The Sorvall SS- I centrifuge or the Eppendorf Microfuge with the corresponding 1.5-z ml-microtubes were used. 2. Microcuvettes with a path length of I cm, inside diameter 3-4 mm and a minimal working capacity of 0.6 ml. The Zeiss MT, cuvettes were used. 3. A Zeiss PMQ II or another equivalent sensitive spectrophotometer adaptable for the use of microcuvettes. METHOD
Pipette 200 ~1 of freshly drawn, heparinized blood into a r.5-2.o-ml micro centrifuge tube and hemolyse by adding 550 ,ul of water. Mix. After 3 min add 250 ,ul of phosphate buffer (reagent 2) and mix again. Unless test is completed within 30 min, cool hemolysed and buffered sample rapidly to 2' and keep refrigerated until assayed. Centrifuge at IOOOOrev./min for 15 min. Centrifugation at high speed is absolutely necessary. Transfer the completely clear supernatant into a small tube with the aid of a Pasteur pipette. From this tube transfer 600 ~1 of the supernatant to the first (I) of two microcuvettes. Transfer to the second (2) cuvette 50 ~1 of the supernatant and mix with 550 ,ul of the ferricyanide-phosphate mixture (reagent 3). In this step the total hemoglobin in cuvette No. 2 is oxidized to methemoglobin. NIeasure the absorbances A, and A, at 632 nm against air. Add 20 ~1 of the neutralized cyanide solution (reagent 6) to each of the two cuvettes. Mix gently and let stand for I min. Read again absorbances A, and A,.
A,-- A, (A,-A,)x
x IOO =
methemoglobin,
12
C&z. Chim. Acta, 30 (1970) 679-682
in percent of total hemoglobin.
METHEMOGLOBIN
681
IN BLOOD
RESULTS
(a) Precision In Table I data on the precision of the method are presented. Replicate blood samples drawn from three normal adults were assayed. The concentration of methemoglobin was around 0.5% of the total pigment. The standard error was in the range o.org-0.025. (b) Accuracy The accuracy of the method was investigated by recovery experiments. A solution of methemoglobin was prepared from nitrite-treated erythrocytes with a methemoglobin content of 96% (ref. 6). The assay was modified by incorporating into a series of microcentrifuge tubes, each one containing 200 ~1 of normal blood, increasing amounts of the methemoglobinemic solution which substituted part of the 550 ~1 of water used for hemolysis. The results are summarized in Table II. It can be seen that 94-99% of the added “nitrite-methemoglobin” is recovered. TABLE
I
PRECISION
OF METHOD
Subject
Number of replicates
Mean % methemoglobin
S.E.
A B
IO
0.49
0.020
C
6
TABLE RECOVERY
7
0.38
0.019
0.63
0.025
II OF METHEMOGLOBIN
ADDED
TO HEMOLYSATES
Methemoglobin added *
Percent of total pigment
IlOIltZ
0.46
I.2 2.4 4.8 9.6
I .60 2.84 5.23 9.88
Found
* The added methemoglobin
Expected
Recovery %
1.66 2.86 5.26 10.46
96.4 99.4 99.4 94.5
was prepared from nitrite-treated
erythrocytes.
(c) Stability of methemoglobin The stability of methemoglobin in blood samples was investigated in both whole and hemolysed blood from normal adults. Storage of whole blood, kept at room temperature (24“) or refrigerated at 2’ or below, results in the oxidation of part of its hemoglobin to methemoglobin. However, in buffered hemolysates kept at 2’ for not more than 24 h the rate of auto-oxidation of hemoglobin is practically zero. DISCUSSION
The sensitivity of the Evelyn and Malloy method in which the blood is diluted was assessed by the authors to be sufficient to detect a minimum of 0.2 g methemoglobin per IOO ml of blood5. This sensitivity is considered low for the assay I: IOO
Clin. Chim. Acta, 30
(1970)
679-682
682
HEGESH
et al.
of methemoglobin in normal blood samples containing approximately 0.15 g methemoglobin per IOO ml (calculated on the basis of total hemoglobin of 15 g per IOO ml wherein the methemoglobin content is 1%). By using a considerably more concentrated hemolysate with a dilution ratio of I : 5 in the modification presented above, sensitivity was increased to detect approximately 0.02 g of methemoglobin per 100 ml. The stability of the methemoglobin of the hemolysed freshly drawn blood cooled to 2’ allows the carrying out of field surveys in communities distant from the laboratory, providing the samples are assayed within 2.4 h. The pH of the buffered hemolysate should be approximately 7.35 (at zoo). Its alkalinity retards the autooxidation of hemoglobin to methemoglobin. If the samples are stored at room temperature, they should nevertheless be assayed within 30 min. REFERENCES I H. H. COMLY, J. Amer. Med. Assoc., Izg (1945) 112 z 2. KNOTEK AND P. SCHMIDT, Pediatrics, 34 (1964) 78. 3 N. GRUENER AND H. I. SHUVAL, Health Aspects of Nitrates in Drinking Water, Developments in Watev Quality Research, Ann Arbor, Humphrey Science Publishers, Ann Arbor-London, 1970. 4 K. A. EVELYN AND H. T. MALLOY, J. Biol. Chem., 126 (1938) 655. 5 R. I. HENRY, Clinical Chemistry, Harper and Row, Philadelphia, 1964, p. 757. 6 A. ROSSI-FANNELLI, E. ANTONINI AND B. MONDOVI, Clin. Chim. Acta, z (1957) 476. 7 IV. 0. CRUZ, Acta Haematol., 6 (1951) 367.
CZin. Chim. Acta, 30 (1970) 679-682
679
A SENSITIVE ~ICROMETHOD OF METHEMOGLOBIN
E. HEGESH’,
N. GRUENER2,
FOR THE DETERMINATION
IN BLOOD
S. COHENa,
R. BOCHKOVSKF
AND
H. I. SHUVAL*
Biochemicat Research Laboratory, Kaplan Hospitat, Rehovoth? and ~e~artrne~~ of Medical Ecolitgy, Hebrew ~7~jue~s~ty-Hadassah _~~ed~catSchool, Jer%satemP (Israel) (Received
June 5. 1970)
SUMMARY
A modification of the Evelyn and Malloy method for the determination of methemoglobin in blood is presented. A higher precision of the assay is achieved, and sample stability is increased up to 24 h, thus making the procedure suitable for field surveys aimed at detecting slightly raised methemoglobin levels.
INTRODUCTION
Increasing levels of nitrates in drinking water have raised the need for reevaluation of potential risks of its use for the health of the population. Ingestion of nitrates has been shown to induce methemoglobinemia in newborns and young infantslp2. In connection with a study to determine methemoglobin levels in infants exposed to high nitrate content in water the need arose for a precise and accurate micromethod which would assure adequate stability of the sample to allow a field survey to be conducted in communities distant from the laboratory*. The method of Evelyn and MalloyP, which up to date has been the standard procedure for the assay of methemoglobin in blood, is considered not sufficiently sensitive to determine low concentrations of the pigment, present in normal blood samples and to differentiate between normal and slightly elevated levels which might be indicative of subclinical cases of methemoglobinemia caused by nitrate ingestion. Due to the instability of the methemoglobin in the drawn blood sample it is usually recommended in normal clinical procedures to carry out the test as rapidly as possible. This is generally not feasible under field survey conditions. The procedure presented below is a modification of the Evelyn and Malloy method aimed to augment its precision. The size of the blood sample is kept at a minimum so that finger-tip blood samples from infants should suffice. The procedure also assures sample stability.
Clin.
Chim.
Acta, 30 (1970) 679-682
680
HEGESH
et d.
Methemoglobin is reacted with cyanide and the change in light absorption at 632 nm is measured. Reagents I. K,Fe(CN),-5% (w/v) in water. Store in a dark bottle. Prepare monthly. 2. Phosphate buffer-o.5 M, pH 7.2. 3. Ferricyanide-phosphate mixture: 0.2 ml of K,FefCN), solution (reagent I) and 2.5 ml of phosphate buffer (reagent 2) are made up IO to ml with water. Prepare daily. 4. Sodium cyanide-10% (w/v). Prepare monthly. 5. Acetic acid-rz% (v/v). 6. Neutralized cyanide solution. Mix 1.0 ml of reagent 4 with 0.9 ml of reagent 5. Use within 6 h. Instrwnents I. High speed microcentrifuge, capacity IO ooo rev./min. The Sorvall SS- I centrifuge or the Eppendorf Microfuge with the corresponding 1.5-z ml-microtubes were used. 2. Microcuvettes with a path length of I cm, inside diameter 3-4 mm and a minimal working capacity of 0.6 ml. The Zeiss MT, cuvettes were used. 3. A Zeiss PMQ II or another equivalent sensitive spectrophotometer adaptable for the use of microcuvettes. METHOD
Pipette 200 ~1 of freshly drawn, heparinized blood into a r.5-2.o-ml micro centrifuge tube and hemolyse by adding 550 ,ul of water. Mix. After 3 min add 250 ,ul of phosphate buffer (reagent 2) and mix again. Unless test is completed within 30 min, cool hemolysed and buffered sample rapidly to 2' and keep refrigerated until assayed. Centrifuge at IOOOOrev./min for 15 min. Centrifugation at high speed is absolutely necessary. Transfer the completely clear supernatant into a small tube with the aid of a Pasteur pipette. From this tube transfer 600 ~1 of the supernatant to the first (I) of two microcuvettes. Transfer to the second (2) cuvette 50 ~1 of the supernatant and mix with 550 ,ul of the ferricyanide-phosphate mixture (reagent 3). In this step the total hemoglobin in cuvette No. 2 is oxidized to methemoglobin. NIeasure the absorbances A, and A, at 632 nm against air. Add 20 ~1 of the neutralized cyanide solution (reagent 6) to each of the two cuvettes. Mix gently and let stand for I min. Read again absorbances A, and A,.
A,-- A, (A,-A,)x
x IOO =
methemoglobin,
12
C&z. Chim. Acta, 30 (1970) 679-682
in percent of total hemoglobin.
METHEMOGLOBIN
681
IN BLOOD
RESULTS
(a) Precision In Table I data on the precision of the method are presented. Replicate blood samples drawn from three normal adults were assayed. The concentration of methemoglobin was around 0.5% of the total pigment. The standard error was in the range o.org-0.025. (b) Accuracy The accuracy of the method was investigated by recovery experiments. A solution of methemoglobin was prepared from nitrite-treated erythrocytes with a methemoglobin content of 96% (ref. 6). The assay was modified by incorporating into a series of microcentrifuge tubes, each one containing 200 ~1 of normal blood, increasing amounts of the methemoglobinemic solution which substituted part of the 550 ~1 of water used for hemolysis. The results are summarized in Table II. It can be seen that 94-99% of the added “nitrite-methemoglobin” is recovered. TABLE
I
PRECISION
OF METHOD
Subject
Number of replicates
Mean % methemoglobin
S.E.
A B
IO
0.49
0.020
C
6
TABLE RECOVERY
7
0.38
0.019
0.63
0.025
II OF METHEMOGLOBIN
ADDED
TO HEMOLYSATES
Methemoglobin added *
Percent of total pigment
IlOIltZ
0.46
I.2 2.4 4.8 9.6
I .60 2.84 5.23 9.88
Found
* The added methemoglobin
Expected
Recovery %
1.66 2.86 5.26 10.46
96.4 99.4 99.4 94.5
was prepared from nitrite-treated
erythrocytes.
(c) Stability of methemoglobin The stability of methemoglobin in blood samples was investigated in both whole and hemolysed blood from normal adults. Storage of whole blood, kept at room temperature (24“) or refrigerated at 2’ or below, results in the oxidation of part of its hemoglobin to methemoglobin. However, in buffered hemolysates kept at 2’ for not more than 24 h the rate of auto-oxidation of hemoglobin is practically zero. DISCUSSION
The sensitivity of the Evelyn and Malloy method in which the blood is diluted was assessed by the authors to be sufficient to detect a minimum of 0.2 g methemoglobin per IOO ml of blood5. This sensitivity is considered low for the assay I: IOO
Clin. Chim. Acta, 30
(1970)
679-682
682
HEGESH
et al.
of methemoglobin in normal blood samples containing approximately 0.15 g methemoglobin per IOO ml (calculated on the basis of total hemoglobin of 15 g per IOO ml wherein the methemoglobin content is 1%). By using a considerably more concentrated hemolysate with a dilution ratio of I : 5 in the modification presented above, sensitivity was increased to detect approximately 0.02 g of methemoglobin per 100 ml. The stability of the methemoglobin of the hemolysed freshly drawn blood cooled to 2’ allows the carrying out of field surveys in communities distant from the laboratory, providing the samples are assayed within 2.4 h. The pH of the buffered hemolysate should be approximately 7.35 (at zoo). Its alkalinity retards the autooxidation of hemoglobin to methemoglobin. If the samples are stored at room temperature, they should nevertheless be assayed within 30 min. REFERENCES I H. H. COMLY, J. Amer. Med. Assoc., Izg (1945) 112 z 2. KNOTEK AND P. SCHMIDT, Pediatrics, 34 (1964) 78. 3 N. GRUENER AND H. I. SHUVAL, Health Aspects of Nitrates in Drinking Water, Developments in Watev Quality Research, Ann Arbor, Humphrey Science Publishers, Ann Arbor-London, 1970. 4 K. A. EVELYN AND H. T. MALLOY, J. Biol. Chem., 126 (1938) 655. 5 R. I. HENRY, Clinical Chemistry, Harper and Row, Philadelphia, 1964, p. 757. 6 A. ROSSI-FANNELLI, E. ANTONINI AND B. MONDOVI, Clin. Chim. Acta, z (1957) 476. 7 IV. 0. CRUZ, Acta Haematol., 6 (1951) 367.
CZin. Chim. Acta, 30 (1970) 679-682