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A revised colorimetric glutamic-oxalacetic transaminase assay
419
CLINICA CHIMICA ACTA
A REVISED
COLORIMETRIC
GLUTAMIC-OXALACETIC
TRANSAMINASE
ASSAY
ARTHUR
L. BABSON,
The Warner-Lambert (Received
ELSA
Research
G. ARNDT
Institute,
AND
Morris
LAURA
Plains,
J. SHARKEY
N. J. (U.S.A.)
July 16, 1969)
SUMMARY
Calorimetric methods for serum glutamic-oxalacetic transaminase, based on reacting oxalacetic acid with a diazonium salt, have been in widespread use since 1962. A critical evaluation was made of all the modifications introduced since then, and the best of these have been incorporated into a revised procedure. The revised procedure is simpler to use, more linear and more specific than previously described methods.
INTRODUCTION
Several years have elapsed since our initial description of the use of stabilized diazonium salts as reagents for the calorimetric determination of glutamic-oxalacetic transaminasel. During this time our procedure using Fast Violet B%has been modified by ourselveW and others?*. These modifications have involved stabilization of the final color by the addition of acid-Lipal or metabisulfite6, the addition of a-ketoglutarate to the blank reagent to provide a more accurate correction for non-enzymatic color6t6, stopping the enzyme reaction with formaldehyde during the color reaction?, substitution of Fast Ponceau L, which reacts with oxalacetate at an acid pH, for Fast Violet B8ps,and emplo~ent of higher substrate concentrations to improve linearity**@. The work described herein was undertaken with the following objectives: (I) to evaluate the various published modifications, (2) to change some of the volume relationships of our original method which have since proven to be unwieldy, (3) to provide for a true non-enzymatic serum blank correction, and (4) to attempt to increase linearity without sacrificing the sensitivity and specificity of the color reaction. These studies have resulted in a much improved serum glutamic-oxalacetic transaminase procedure which is presented.
C&%. Chim. a’!fCta,26 (1969) 41‘)-423
420
BABSONectd.
Sam+
and reagent vahwtes procedure called for 0.2 ml serum, I ml each of substrate and color reagent and IO ml of diluent. The total volume of 12.2 ml is difficult to mix properly. We therefore arbitrarily decided to use 0.x ml serum and 5 ml of diluent for a total volume of 7.1 ml. Our original
Buffer To assure good pH control we had employed 0.2 M phosphate buffer at pH 7.4. In view of reports that tris is a potent activator of transaminase’O and inorganic phosphate can both activate and inhibit the enzyme l”ell, the buffer was re-evaluated. Versatol-E, assayed with reagents prepared in 0.1 and 0.2 M phosphate and 0.1 and 0.2 M tris, showed relative activities of IOO, 94, 70 and 87 respectively. Since the highest activity was obtained with 0.1 M phosphate, this buffer was used in all subsequent work. Color stabilization
Dilution of the final reaction mixture with water, as initially recommended, does not completely stop the reaction, and the color slowly continues to increase. We subsequently recommended acid-Lipal to stabilize the color completely. Furuno and Sheenas used a diluent containing sodium metabisulfite which achieves color stability by destroying the remaining diazonium salt. While this procedure does indeed work, we do not recommend it, as the metabisulfite solution is not very stable. In our revised procedure we have doubled the original concentration of acid and Lipal, because we are using only one half the original volume. Non-enzymatic serum blank correction Our original procedure employed a blank reagent for the correction of bilirubin interference which contained neither aspartic acid nor cr-ketoglutaric acid. In some modifications636 only aspartic acid was omitted from the blank reagent which is a decided improvement over our initial procedure, as cc-ketoglutarate contributes significantly to the blank. Morgenstern et aLa employed an automated procedure with dialysis to obviate the bilirubin interference. These authors also introduced the use of Fast Ponceau L which reacts with oxalacetate at a lower pH. Sax and Moore* and Paysant et aLla pointed out that dialysis does not eliminate the interference from acetoacetic acid and that individual serum blanks are still necessary. Sax and Moore used Fast Ponceau L at pH 4.2, where transaminase activity would be effectively inhibited, and added the serum to the blank tube immediately after the diazonium salt. While this procedure gives a true non-enzymatic serum blank correction, we have observed that the blank color is not as stable as Sax and Moore claim. Doumas and Biggs’ used 0.2% formaldehyde in the Fast Violet B solution to inhibit the enzyme. Complete enzyme inhibition would allow for a true serum blank correction by adding the serum immediately after the diazonium salt as is done in the Sax-Moore procedure. However, we have observed that 0.2% formaldehyde significantly interferes with the color reaction. Lesser concentrations of formaldehyde interfere less, but do not inhibit the enzyme completely (TabIe I). C&A Ckim. A&, 26 (xgeg) 419-423
COLORIMETRIC TABLE
TRANSAMINASE
ASSAY
421
I
INHIBITION
OF TRANSAMINASE
Formaldehyde concentration
ACTIVITY
Versatol E Sample
Blank
AND
COLOR
REACTION
BY
FORMALDEHYDE
Inhibition of
Versatol
transaminase
plus added
oxalacetate 0
0.593
0.108
0.025%
0.227
0.088
0.05% O.I%
0.144 O.II3
0.085 0.081
o.2yo
0.086
0.075
TABLE
7:%
no added oxalacetate
Inhibition of color reaction
0.570
0.079
0.535
0.061
88% 94%
0.515 0.475
0.057 0.054
4% 7% 14%
98%
0.432
0.055
23%
0
II
COMPARISON
OF D-ASPARTIC
ACID
AND
FORMALDEHYDE
FOR SERUM
BLANKS
Absorbance of Assay in the Presence of
Sample
Serum + GOT Serum Versatol E Versatol E-N
L-aspartate
D-aspartate
L-aspartate + HCHO
n-asp&ate + HCHO
co 0.303 0.835 0.169
0.159 0.136 0.133 0.122
0.189 0.117 0.118 O.IO7
0.112 0.109 0.105 0.098
Ideally the serum blank should be identical to the incubated sample, except that all enzyme activity should be eliminated. The best procedure we have found is to substitute pure D-aspartic acid in the blank reagent for the L-aspartic acid in the substrate. A comparison of this procedure with formaldehyde inhibition is shown in Table II. In this experiment serum with and without a relatively large amount of purified pig heart transaminase, Versatol E and Versatol E-N (similar except with respect to levels of enzyme activity) were assayed with substrates prepared with D- and L-aspartic acid both with and without 0.2% formaldehyde. The D-aspartic acid was a more effective inhibitor of the GOT added to serum (0.159 - 0.136 = 0.023 vs. 0.189 - 0.117 = 0.072), while in every case the addition of formaldehyde to the D-aspartate substrates caused a significant inhibition of the blank color. Substrate
concentrations
Our original assay conditionsa were selected on the basis of two criteria, to achieve a high degree of sensitivity while maintaining a low blank. More recent modifications have employed higher substrate concentrations in an attempt to improve linearitysp9. We therefore reinvestigated these variables. The diazonium salt reacts with cc-ketoglutarate but at a lesser rate than with oxalacetic acid. The increased blank that will result from higher a-ketoglutarate concentrations can in part be compensated by shortening the color development time. When a 5-min color development time was compared with the previously employed Io-min time less than half of the blank and about two thirds of the sensitivity was obtained. Table III illustrates the effect of increased substrate concentrations on the sensitivity and blank using a 5-min color development time. Concentrations of aspartate and u-ketoglutarate of 20 and IO mM respectively were optimal. Higher concentrations reduced the sensitivity and increased the blank. Fig. I compares the present procedure with the Sax-Moore method and our Clin. Chim. Acta, 26 (1969) 419-423
BABSON
422 TABLE
III
EFFECT OF SUBSTRATE
Aspartate mM
CONCENTRATIONS
ON
a-l~eto&darate mM 5
20 20
IO
20
20
200
et d.
AND
BLANK
Sample A
Blank A
0.560 0.730
0.082 0.114
0.580
0.135
0,577
IO
original method.
SENSITIVITY
0.126
To provide a valid comparison
the first two were diluted to a final
volume of 6.1 ml. (The final volume in our original
procedure
is 12.2 ml, but it em-
ploys a 0.2 ml sample instead of 0.1 ml.) Three facts are apparent from the figure: (I) both the Sax-Moore and the present procedure are about equally nonlinear, but are more linear than our original method, (2) the blank correction of our original method is insufficient, also has a relatively
and (3) while the Sax-Moore larger blank than the present
method has greater
sensitivity
it
procedure.
I.21 1.1. 1.0.
///
Sax 8. Moore ’ /
0.9.
E &68 so.7. 8 kO.6. b $0.5.
0.4. 0.30.2
150 200 250 300 Transcminase activity in transAc units
350
Fig. I. Standard curves for three methods for serum glutamic oxalacetic
transaminase.
RECOMMENDED PROCEDURE
Reagent preparation* Substrate. Dissolve 292.2 mg a-ketoglutaric acid, polyvinylpyrrolidone, 0.2 g ethylenediaminetetraacetic * Prepared reagents are available Morris Plains, New Jersey.
commercially
C&Z. Chim. Ada, 26 (1969) 419-423
as TransAc
mg L-aspartic acid, acid, tetrasodium salt,
532.4
from General Diagnostics
g and
2.0
Division,
COLORIMETRIC
TFUNSAMINASE
423
ASSAY
3.48 g K,HPO, in about 180 ml of water. Adjust pH to 7.40 (25”) with I N NaOH, and dilute to 200 ml with water. Store under refrigeration. Blank reagent. Identical to substrate, except use n-aspartic acid. Store under refrigeration. Color reagent. Dissolve 125 mg of 6-benzamido-4-methoxy-m-toluidine diazonium chloride (Fast Violet B) in 25 ml of water. The solution is stable for several days if kept in an amber bottle under refrigeration. Diluent. Dilute 3.4 ml of concentrated hydrochloric acid and IO ml of ethoxylated tridecyl alcohol (Lipal 610) to IOOOml with water. Assay firocedwe
To I ml of substrate at 37’ add 0.1 ml of serum. Similarly add 0.1 ml of serum to I ml of blank reagent. After exactly 20 min of incubation add I ml of color reagent, mix and immediately return to the water bath. Exactly 5 min after adding the color reagent add 5 ml of diluent and mix. Measure the absorbance of the sample at 530 nm after setting the calorimeter at IOO~/~ T with the serum blank. The activity is determined by reference to a standard curve prepared with enzyme reference standards such as Versatol E* and Versatol E-N* similarly treated. Normal range
When the above procedure was used with 65 healthy adults a range of 7 to 34 TransAc units was obtained. The mean normal activity was 19units with a standard deviation of f 6.5 units. The normal range could therefore be considered to be 6 to 32 units. A certain amount of caution is advised in interpreting what is normal. Doumas and Biggs? found an average of 50% more activity in hospitalized patients without conditions which would usually elevate the transaminase activity than they did in healthy adults. It is recommended that each laboratory decide for itself what should be considered the limits of normal. REFERENCES I A. L. BABSON, Clin. Chem., 6 (1960) 394. 2 A. L. BABSON, P. 0. SHAPIRO, P. A. R. WILLIAMS 3
4 5 6 7 8
AND G. E. PHILLIPS, CZin. Chim.
Acta,
(19% 199. A. L. BABSON AND P. 0. SHAPIRO, C&z. Chim. Ada, 8 (1965) 326. A. L. BABSON AND G. E. PHILLIPS, Clin. Chem., II (1965) 533. M. FURUNO AND A. SHEENA, Clin. Chcm., II (1965) 23. R. R. SCHAFPERT, G. R. KINGSLEY AND G. GETCHELL, C&n. Chem., IO (1964) 519. B. DOUMAS AND H. G. BIGGS, Cl&. Chim.Acta, 23 (1969) 75. S. MORGENSTERN, M. OKLANDER, J. AUERBACH, J. KAUFMAN AND B. KLEIN, CZin. Chem.,
(1966) 95. g S. M. SAX AND J. J. MOORE, CZin. Chem., 13 (1967) 175. IO C. TURANO, P. FASELLA AND A. GIARTOSIO, B&him. Biophys. II T. R. C. BOYDE, Biochem. J., 106 (1968) 581.
Ada,
12 P. PAYSANT, P. NABET, F. GENETET AND M. FAIVRE, CZin. Chim. * General
Diagnostics
Division,
Morris Plains,
New
7
IZ
58 (1962) 255. Acta, 23 (1969) 507.
Jersey. CZin. Chim.
Acta,
26 (1969) 419-423
CLINICA CHIMICA ACTA
A REVISED
COLORIMETRIC
GLUTAMIC-OXALACETIC
TRANSAMINASE
ASSAY
ARTHUR
L. BABSON,
The Warner-Lambert (Received
ELSA
Research
G. ARNDT
Institute,
AND
Morris
LAURA
Plains,
J. SHARKEY
N. J. (U.S.A.)
July 16, 1969)
SUMMARY
Calorimetric methods for serum glutamic-oxalacetic transaminase, based on reacting oxalacetic acid with a diazonium salt, have been in widespread use since 1962. A critical evaluation was made of all the modifications introduced since then, and the best of these have been incorporated into a revised procedure. The revised procedure is simpler to use, more linear and more specific than previously described methods.
INTRODUCTION
Several years have elapsed since our initial description of the use of stabilized diazonium salts as reagents for the calorimetric determination of glutamic-oxalacetic transaminasel. During this time our procedure using Fast Violet B%has been modified by ourselveW and others?*. These modifications have involved stabilization of the final color by the addition of acid-Lipal or metabisulfite6, the addition of a-ketoglutarate to the blank reagent to provide a more accurate correction for non-enzymatic color6t6, stopping the enzyme reaction with formaldehyde during the color reaction?, substitution of Fast Ponceau L, which reacts with oxalacetate at an acid pH, for Fast Violet B8ps,and emplo~ent of higher substrate concentrations to improve linearity**@. The work described herein was undertaken with the following objectives: (I) to evaluate the various published modifications, (2) to change some of the volume relationships of our original method which have since proven to be unwieldy, (3) to provide for a true non-enzymatic serum blank correction, and (4) to attempt to increase linearity without sacrificing the sensitivity and specificity of the color reaction. These studies have resulted in a much improved serum glutamic-oxalacetic transaminase procedure which is presented.
C&%. Chim. a’!fCta,26 (1969) 41‘)-423
420
BABSONectd.
Sam+
and reagent vahwtes procedure called for 0.2 ml serum, I ml each of substrate and color reagent and IO ml of diluent. The total volume of 12.2 ml is difficult to mix properly. We therefore arbitrarily decided to use 0.x ml serum and 5 ml of diluent for a total volume of 7.1 ml. Our original
Buffer To assure good pH control we had employed 0.2 M phosphate buffer at pH 7.4. In view of reports that tris is a potent activator of transaminase’O and inorganic phosphate can both activate and inhibit the enzyme l”ell, the buffer was re-evaluated. Versatol-E, assayed with reagents prepared in 0.1 and 0.2 M phosphate and 0.1 and 0.2 M tris, showed relative activities of IOO, 94, 70 and 87 respectively. Since the highest activity was obtained with 0.1 M phosphate, this buffer was used in all subsequent work. Color stabilization
Dilution of the final reaction mixture with water, as initially recommended, does not completely stop the reaction, and the color slowly continues to increase. We subsequently recommended acid-Lipal to stabilize the color completely. Furuno and Sheenas used a diluent containing sodium metabisulfite which achieves color stability by destroying the remaining diazonium salt. While this procedure does indeed work, we do not recommend it, as the metabisulfite solution is not very stable. In our revised procedure we have doubled the original concentration of acid and Lipal, because we are using only one half the original volume. Non-enzymatic serum blank correction Our original procedure employed a blank reagent for the correction of bilirubin interference which contained neither aspartic acid nor cr-ketoglutaric acid. In some modifications636 only aspartic acid was omitted from the blank reagent which is a decided improvement over our initial procedure, as cc-ketoglutarate contributes significantly to the blank. Morgenstern et aLa employed an automated procedure with dialysis to obviate the bilirubin interference. These authors also introduced the use of Fast Ponceau L which reacts with oxalacetate at a lower pH. Sax and Moore* and Paysant et aLla pointed out that dialysis does not eliminate the interference from acetoacetic acid and that individual serum blanks are still necessary. Sax and Moore used Fast Ponceau L at pH 4.2, where transaminase activity would be effectively inhibited, and added the serum to the blank tube immediately after the diazonium salt. While this procedure gives a true non-enzymatic serum blank correction, we have observed that the blank color is not as stable as Sax and Moore claim. Doumas and Biggs’ used 0.2% formaldehyde in the Fast Violet B solution to inhibit the enzyme. Complete enzyme inhibition would allow for a true serum blank correction by adding the serum immediately after the diazonium salt as is done in the Sax-Moore procedure. However, we have observed that 0.2% formaldehyde significantly interferes with the color reaction. Lesser concentrations of formaldehyde interfere less, but do not inhibit the enzyme completely (TabIe I). C&A Ckim. A&, 26 (xgeg) 419-423
COLORIMETRIC TABLE
TRANSAMINASE
ASSAY
421
I
INHIBITION
OF TRANSAMINASE
Formaldehyde concentration
ACTIVITY
Versatol E Sample
Blank
AND
COLOR
REACTION
BY
FORMALDEHYDE
Inhibition of
Versatol
transaminase
plus added
oxalacetate 0
0.593
0.108
0.025%
0.227
0.088
0.05% O.I%
0.144 O.II3
0.085 0.081
o.2yo
0.086
0.075
TABLE
7:%
no added oxalacetate
Inhibition of color reaction
0.570
0.079
0.535
0.061
88% 94%
0.515 0.475
0.057 0.054
4% 7% 14%
98%
0.432
0.055
23%
0
II
COMPARISON
OF D-ASPARTIC
ACID
AND
FORMALDEHYDE
FOR SERUM
BLANKS
Absorbance of Assay in the Presence of
Sample
Serum + GOT Serum Versatol E Versatol E-N
L-aspartate
D-aspartate
L-aspartate + HCHO
n-asp&ate + HCHO
co 0.303 0.835 0.169
0.159 0.136 0.133 0.122
0.189 0.117 0.118 O.IO7
0.112 0.109 0.105 0.098
Ideally the serum blank should be identical to the incubated sample, except that all enzyme activity should be eliminated. The best procedure we have found is to substitute pure D-aspartic acid in the blank reagent for the L-aspartic acid in the substrate. A comparison of this procedure with formaldehyde inhibition is shown in Table II. In this experiment serum with and without a relatively large amount of purified pig heart transaminase, Versatol E and Versatol E-N (similar except with respect to levels of enzyme activity) were assayed with substrates prepared with D- and L-aspartic acid both with and without 0.2% formaldehyde. The D-aspartic acid was a more effective inhibitor of the GOT added to serum (0.159 - 0.136 = 0.023 vs. 0.189 - 0.117 = 0.072), while in every case the addition of formaldehyde to the D-aspartate substrates caused a significant inhibition of the blank color. Substrate
concentrations
Our original assay conditionsa were selected on the basis of two criteria, to achieve a high degree of sensitivity while maintaining a low blank. More recent modifications have employed higher substrate concentrations in an attempt to improve linearitysp9. We therefore reinvestigated these variables. The diazonium salt reacts with cc-ketoglutarate but at a lesser rate than with oxalacetic acid. The increased blank that will result from higher a-ketoglutarate concentrations can in part be compensated by shortening the color development time. When a 5-min color development time was compared with the previously employed Io-min time less than half of the blank and about two thirds of the sensitivity was obtained. Table III illustrates the effect of increased substrate concentrations on the sensitivity and blank using a 5-min color development time. Concentrations of aspartate and u-ketoglutarate of 20 and IO mM respectively were optimal. Higher concentrations reduced the sensitivity and increased the blank. Fig. I compares the present procedure with the Sax-Moore method and our Clin. Chim. Acta, 26 (1969) 419-423
BABSON
422 TABLE
III
EFFECT OF SUBSTRATE
Aspartate mM
CONCENTRATIONS
ON
a-l~eto&darate mM 5
20 20
IO
20
20
200
et d.
AND
BLANK
Sample A
Blank A
0.560 0.730
0.082 0.114
0.580
0.135
0,577
IO
original method.
SENSITIVITY
0.126
To provide a valid comparison
the first two were diluted to a final
volume of 6.1 ml. (The final volume in our original
procedure
is 12.2 ml, but it em-
ploys a 0.2 ml sample instead of 0.1 ml.) Three facts are apparent from the figure: (I) both the Sax-Moore and the present procedure are about equally nonlinear, but are more linear than our original method, (2) the blank correction of our original method is insufficient, also has a relatively
and (3) while the Sax-Moore larger blank than the present
method has greater
sensitivity
it
procedure.
I.21 1.1. 1.0.
///
Sax 8. Moore ’ /
0.9.
E &68 so.7. 8 kO.6. b $0.5.
0.4. 0.30.2
150 200 250 300 Transcminase activity in transAc units
350
Fig. I. Standard curves for three methods for serum glutamic oxalacetic
transaminase.
RECOMMENDED PROCEDURE
Reagent preparation* Substrate. Dissolve 292.2 mg a-ketoglutaric acid, polyvinylpyrrolidone, 0.2 g ethylenediaminetetraacetic * Prepared reagents are available Morris Plains, New Jersey.
commercially
C&Z. Chim. Ada, 26 (1969) 419-423
as TransAc
mg L-aspartic acid, acid, tetrasodium salt,
532.4
from General Diagnostics
g and
2.0
Division,
COLORIMETRIC
TFUNSAMINASE
423
ASSAY
3.48 g K,HPO, in about 180 ml of water. Adjust pH to 7.40 (25”) with I N NaOH, and dilute to 200 ml with water. Store under refrigeration. Blank reagent. Identical to substrate, except use n-aspartic acid. Store under refrigeration. Color reagent. Dissolve 125 mg of 6-benzamido-4-methoxy-m-toluidine diazonium chloride (Fast Violet B) in 25 ml of water. The solution is stable for several days if kept in an amber bottle under refrigeration. Diluent. Dilute 3.4 ml of concentrated hydrochloric acid and IO ml of ethoxylated tridecyl alcohol (Lipal 610) to IOOOml with water. Assay firocedwe
To I ml of substrate at 37’ add 0.1 ml of serum. Similarly add 0.1 ml of serum to I ml of blank reagent. After exactly 20 min of incubation add I ml of color reagent, mix and immediately return to the water bath. Exactly 5 min after adding the color reagent add 5 ml of diluent and mix. Measure the absorbance of the sample at 530 nm after setting the calorimeter at IOO~/~ T with the serum blank. The activity is determined by reference to a standard curve prepared with enzyme reference standards such as Versatol E* and Versatol E-N* similarly treated. Normal range
When the above procedure was used with 65 healthy adults a range of 7 to 34 TransAc units was obtained. The mean normal activity was 19units with a standard deviation of f 6.5 units. The normal range could therefore be considered to be 6 to 32 units. A certain amount of caution is advised in interpreting what is normal. Doumas and Biggs? found an average of 50% more activity in hospitalized patients without conditions which would usually elevate the transaminase activity than they did in healthy adults. It is recommended that each laboratory decide for itself what should be considered the limits of normal. REFERENCES I A. L. BABSON, Clin. Chem., 6 (1960) 394. 2 A. L. BABSON, P. 0. SHAPIRO, P. A. R. WILLIAMS 3
4 5 6 7 8
AND G. E. PHILLIPS, CZin. Chim.
Acta,
(19% 199. A. L. BABSON AND P. 0. SHAPIRO, C&z. Chim. Ada, 8 (1965) 326. A. L. BABSON AND G. E. PHILLIPS, Clin. Chem., II (1965) 533. M. FURUNO AND A. SHEENA, Clin. Chcm., II (1965) 23. R. R. SCHAFPERT, G. R. KINGSLEY AND G. GETCHELL, C&n. Chem., IO (1964) 519. B. DOUMAS AND H. G. BIGGS, Cl&. Chim.Acta, 23 (1969) 75. S. MORGENSTERN, M. OKLANDER, J. AUERBACH, J. KAUFMAN AND B. KLEIN, CZin. Chem.,
(1966) 95. g S. M. SAX AND J. J. MOORE, CZin. Chem., 13 (1967) 175. IO C. TURANO, P. FASELLA AND A. GIARTOSIO, B&him. Biophys. II T. R. C. BOYDE, Biochem. J., 106 (1968) 581.
Ada,
12 P. PAYSANT, P. NABET, F. GENETET AND M. FAIVRE, CZin. Chim. * General
Diagnostics
Division,
Morris Plains,
New
7
IZ
58 (1962) 255. Acta, 23 (1969) 507.
Jersey. CZin. Chim.
Acta,
26 (1969) 419-423