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Interference by thiels in acetoacetate determination
.,NALYTIC.,L
BIOCHEMISTRY
Interference
138-143
11,
by
Thiols
(1965)
in Acetoacetate
JOHN From
the Department
of
B. ALLRED
Chemistry,
Received
Determination1
Oklahoma August
City
University,
Oklahoma
18, 1964
The calorimetric reaction described by Walker (1) has been frequently used to quantitate acetoacetate produced in enzymic reactions (2-7). This calorimetric reaction, which depends upon acet’oacetate coupling quantitatively with an excess of p-nitrobenzenediazonium hydroxide, is very sensitive, but is not specific since other met,abolites, including malonate, oxaloacetate, and ascorbate will also react with this reagent (1). This lack of specificity has been considered to be unimportant, however, because the absorbance of the products is small compared to that observed with comparable amounts of acetoacetate (2). In the present paper, evidence is presented showing that relatively Iow levels of reduced glutathione or cysteine, which are routinely added to acetoacet.ate-synthesizing systems (2, 3, 6, 7), will interfere with the acetoacetate determination. This interference can be significant,ly reduced by the addition of mercuric ions in the calorimetric reaction. METHOD
The reaction mixt.ures contained 1 ml of 1 M sodium acetate buffer, pH 5.2, and 3 ml of freshly prepared diazo reagent described by Walker (1). Acetoacetate, thiol compounds, and mercuric chloride were varied and the total volume was made up to 5 ml with distilled water. After a 30-min incubation period at 28”C, 1 ml of 5 N hydrochloric acid was added to stop the reaction. The colored product was then extracted with 4 ml of ethyl acetate and the absorbance was measured at 450 mp. Acetoacetate was prepared by hydrolysis of ethyl acetoacetate (8). RESULTS
9 linear varied from However, in the amount ROl
‘This AM
increase in absorbance was observed as acetoacetate was 0 to 150 mpmoles (Fig. 1) in agreement with Walker (1). the presence of 20 pmoles of reduced glutathione, or cysteine, frequently added to enzyme reaction mixtures (e.g., ref. 2),
investigation 06673, from
was supported in part by Public the National Institute of Arthritis 138
Health Service and Metabolic
Grant No. Diseases.
ACETOACETATE
139
DETERMINATIOS
0.1
0. I
n.. w ”
0. f
z < m e: 0
0. t
m cl
0.‘
< 0.
0. i
0.
I 20
40
60
MILLIMICROMOLE~
FIG.
1. Relationship
between
absorbance
I
80
I
190
r
120
I
110
I
ACETOACETATE
at 450 mp and
acetoncetatc
concentration.
none of these levels of acetoacetate gave an observable absorbance at 450 my. When acctoacetate concentration was held constant at 88 rnpmoles and thiol concentrat.ion was varied, it was found that even very small amounts of either cyst,eine or glutathione reduced the absorbance obtained (Fig. 2). Complete inhibition was observed with 7 to 8 pmoles of thiol, which is approximately stoichiometric with the diazo reagent. Figure 2 also shows that the addition of 20 pmoles of mercuric ion significantly reduces this interference by cysteine and eliminates it in the case of reduced glutathione. Neither mercuric chloride, cystine nor oxidized glutathione alone influenced the diazo-acetoacetate reaction. ‘An absorbance peak appears immediately at 332 my lvhen reduced glutathione is added to the reaction mixture, in the absence of mercuric ion, indicating a reaction between the diazo reagent and glutathione. This .indication is substantiated by the approximate stoichiometric
140
JOHN
B.
ALLRED
0.6 1 w u 0.4z 4 rg go.
A Reduced Glutathione . Reduced Glutathione t20r M
3-
0
b
Cysteine
v)
0
Cysteine t 20rM
m 0.2-
H&l2
H&l2
d
0. l-
2
4 MICROMOLES
8
6
10
12
THIOL
FIG. 2. Effect of reduced glutathione and cysteine, in the presence and absence of mercuric ion, on acetoacetate determination.
amount of glutathione required for complete inhibition (Fig. 2). This reaction is extremely rapid compared to the reaction between the diazo reagent and acetoacetate (Fig. 3). After various time intervals, either hydrochloric acid or reduced glutathione was added to a reaction mixture containing buffer, 88 mpmoles of acetoacetate, and the diazo reagent. Glutathione (20 pmoles) proved to be as effective as hydrochloric acid in stopping the reaction even though no change in pH was detected. To investigate the recovery of acetoacetate from enzyme reaction mixtures, various amounts of acetoacetate were added to a typical enzyme reaction mixture (9), which contained 20 pmoles of reduced glutathione. A chicken liver homogenate (10%) was used as an enzyme source. Immediately after all of the components were added, with a final volume of 2.2 ml, 0.7 ml of 7% perchloric acid was added to stop the reaction. The protein was removed by centrifugation and 0.8 ml of the supernatant (containing 5.9 pmoles of glutathione) was removed for acetoacetate determination in the standard calorimetric reaction in the presence and absence of mercuric chloride. As a control, acetoacetate was added to an enzyme reaction mixture containing all components except glmathione. The same procedure was followed for assay of acetoacetate,
ACETOACETATE
141
DETERMINATION
0.6
0.5 w u
0 Reaction stopped with 1 ml 5 N HCl
0.4
z < * d
. Reaction stopped with ZOrM reduced Glutathione
0.3
0 m 0.i Q < 0.1
3 ,
I 5
I
T
FIG. 3. Comparative nating the calorimetric 0.f
0.5
w v
0.4
.
1 15
10 I
M
2’0 E
I 25
I 30
I 35
(MINUTES)
effect of hydrochloric acid and reduced glutatione reaction for acetoacetate determination. Acetoacetate colorimrtric
0 Contains Clutathione
r 40
in termi-
added to reaction 5.9rM +
2OrM
reduced HgCl2
A No Glutathione.
No
A No Glutathione
+ 20rM
HgCIZ HgCl2
z < Q
l
0.3
Contains 5.9 II M reduced Glutathmne. No H&l2
d 0 m
0.2-
a? 4
O.l-
MILLIMICROMOLES
ACETOACETATE
FIG. 4. Effect of reduced glutathione and mercuric chloride on recovery of added acetoacetate from enzymic reaction mixtures. The uppermost curve (m) is included for comparative purposes and represents results obtained when equivalent amounts of acetoacetate are added directly to the calorimetric reaction.
142
JOHiT
I3.
ALLREI)
in the presence and absence of mercuric chloride. Figure 4 shows a typical result of this type of experiment. When acetoacetate was determined in the presence of glutathione and absence of mercuric ion, recovery was very low and the relationship between optical density and acetoacetate concentration was not linear. If, however, mercuric ion was used in t,he calorimetric reaction, recovery of added acetoacetate was approximately 9370, which is in agreement with previous results (1). Similar results were obtained when glutathione was not included in t.he assay system. DISCUSSIOS
The addition of mercuric chloride to the calorimetric reaction mixture has proved to be an effective and reproducible procedure to overcome the interference of reduced glutathione and cyst.eine in the determination of acetoacetate. Glutathione has been noted previously to cause a moderate reduction in color yield in this reaction, and it was indicated that appropriate corrections were made (3). Under conditions of the recovery experiment reported here, corrections for glutathione interference would be very difficult since a linear relationship was not obtained between acetoacetate present in the reaction mixture and the observed optical density (Fig. 4). One can, of course, vary the shape of this curve, and therefore the recovery, by using different amounts of the enzymic reaction mixture in the calorimetric reaction, and thus influence the extent of interference (Fig. 2). Evidence has been presented indicating that a reaction occurs between the diazo reagent and reduced glutathione. No attempt has yet been made, however, to characterize any product(s) from such a reaction. It is tempting to speculate that such a reaction causes interference in acetoacetate determination because it reduces the amount of diazo reagent available for react,ion with acetoacetate. SUMMARY
Cysteine and reduced glutathione have been shown to interfere in the calorimetric determination of acetoacetate, using the method of Walker. This interference can be significantly reduced in the case of cysteine and eliminated in the case of reduced glutathione if mercuric chloride is added to the calorimetric reaction mixture. ACKNOWLEDGMENT The technical assistance gratefully acknowledged.
of
Donald
Upjohn,
Adel
S.zlman
and
REFERENCES 1. WALKER,
P. G., B&hem. J. 58, 699 (1954). 2. DRUMMOND, G. I., AND STERN, J. R.. J. Bid.
C/tern.
235, 318 (1960).
Robert
Fink
is
.ICETOACEThTE
DETERMINATIOK
143
3. SEGAL, H. I,., AND MENON, G. K. Ii., J. Sol. Chem. 236, 2872 (1961). 4. HIRD, F. J. R., AND SYMOXS, R. H., Biochim. Biophys. Acta 46, 457 (1961). 5. STERK, J. R., ASD MILLER, G. E., Biochim. Biophys. Acta 35, 576 (1959). 6. LYNEN, F., HENNING, U., BUBLITZ, C., SORBO, B., ASD KKOPLIS-RUEFF, L., Biochem. 2.330, 269 (1958). 7. CALDWELL, I. C., AND DRUMMOXD, G. I., J. Biol. C’hem. 238, 64 (1963). 8. SEELY, H. W., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 624. Academic Press, New York, 1955. 9. STERN, J. R., DRUMMOND. G. I.. Coos, M. J., DEL CAMPILLO, A., J. Biol. Chem. 235, 313 (1960).
BIOCHEMISTRY
Interference
138-143
11,
by
Thiols
(1965)
in Acetoacetate
JOHN From
the Department
of
B. ALLRED
Chemistry,
Received
Determination1
Oklahoma August
City
University,
Oklahoma
18, 1964
The calorimetric reaction described by Walker (1) has been frequently used to quantitate acetoacetate produced in enzymic reactions (2-7). This calorimetric reaction, which depends upon acet’oacetate coupling quantitatively with an excess of p-nitrobenzenediazonium hydroxide, is very sensitive, but is not specific since other met,abolites, including malonate, oxaloacetate, and ascorbate will also react with this reagent (1). This lack of specificity has been considered to be unimportant, however, because the absorbance of the products is small compared to that observed with comparable amounts of acetoacetate (2). In the present paper, evidence is presented showing that relatively Iow levels of reduced glutathione or cysteine, which are routinely added to acetoacet.ate-synthesizing systems (2, 3, 6, 7), will interfere with the acetoacetate determination. This interference can be significant,ly reduced by the addition of mercuric ions in the calorimetric reaction. METHOD
The reaction mixt.ures contained 1 ml of 1 M sodium acetate buffer, pH 5.2, and 3 ml of freshly prepared diazo reagent described by Walker (1). Acetoacetate, thiol compounds, and mercuric chloride were varied and the total volume was made up to 5 ml with distilled water. After a 30-min incubation period at 28”C, 1 ml of 5 N hydrochloric acid was added to stop the reaction. The colored product was then extracted with 4 ml of ethyl acetate and the absorbance was measured at 450 mp. Acetoacetate was prepared by hydrolysis of ethyl acetoacetate (8). RESULTS
9 linear varied from However, in the amount ROl
‘This AM
increase in absorbance was observed as acetoacetate was 0 to 150 mpmoles (Fig. 1) in agreement with Walker (1). the presence of 20 pmoles of reduced glutathione, or cysteine, frequently added to enzyme reaction mixtures (e.g., ref. 2),
investigation 06673, from
was supported in part by Public the National Institute of Arthritis 138
Health Service and Metabolic
Grant No. Diseases.
ACETOACETATE
139
DETERMINATIOS
0.1
0. I
n.. w ”
0. f
z < m e: 0
0. t
m cl
0.‘
< 0.
0. i
0.
I 20
40
60
MILLIMICROMOLE~
FIG.
1. Relationship
between
absorbance
I
80
I
190
r
120
I
110
I
ACETOACETATE
at 450 mp and
acetoncetatc
concentration.
none of these levels of acetoacetate gave an observable absorbance at 450 my. When acctoacetate concentration was held constant at 88 rnpmoles and thiol concentrat.ion was varied, it was found that even very small amounts of either cyst,eine or glutathione reduced the absorbance obtained (Fig. 2). Complete inhibition was observed with 7 to 8 pmoles of thiol, which is approximately stoichiometric with the diazo reagent. Figure 2 also shows that the addition of 20 pmoles of mercuric ion significantly reduces this interference by cysteine and eliminates it in the case of reduced glutathione. Neither mercuric chloride, cystine nor oxidized glutathione alone influenced the diazo-acetoacetate reaction. ‘An absorbance peak appears immediately at 332 my lvhen reduced glutathione is added to the reaction mixture, in the absence of mercuric ion, indicating a reaction between the diazo reagent and glutathione. This .indication is substantiated by the approximate stoichiometric
140
JOHN
B.
ALLRED
0.6 1 w u 0.4z 4 rg go.
A Reduced Glutathione . Reduced Glutathione t20r M
3-
0
b
Cysteine
v)
0
Cysteine t 20rM
m 0.2-
H&l2
H&l2
d
0. l-
2
4 MICROMOLES
8
6
10
12
THIOL
FIG. 2. Effect of reduced glutathione and cysteine, in the presence and absence of mercuric ion, on acetoacetate determination.
amount of glutathione required for complete inhibition (Fig. 2). This reaction is extremely rapid compared to the reaction between the diazo reagent and acetoacetate (Fig. 3). After various time intervals, either hydrochloric acid or reduced glutathione was added to a reaction mixture containing buffer, 88 mpmoles of acetoacetate, and the diazo reagent. Glutathione (20 pmoles) proved to be as effective as hydrochloric acid in stopping the reaction even though no change in pH was detected. To investigate the recovery of acetoacetate from enzyme reaction mixtures, various amounts of acetoacetate were added to a typical enzyme reaction mixture (9), which contained 20 pmoles of reduced glutathione. A chicken liver homogenate (10%) was used as an enzyme source. Immediately after all of the components were added, with a final volume of 2.2 ml, 0.7 ml of 7% perchloric acid was added to stop the reaction. The protein was removed by centrifugation and 0.8 ml of the supernatant (containing 5.9 pmoles of glutathione) was removed for acetoacetate determination in the standard calorimetric reaction in the presence and absence of mercuric chloride. As a control, acetoacetate was added to an enzyme reaction mixture containing all components except glmathione. The same procedure was followed for assay of acetoacetate,
ACETOACETATE
141
DETERMINATION
0.6
0.5 w u
0 Reaction stopped with 1 ml 5 N HCl
0.4
z < * d
. Reaction stopped with ZOrM reduced Glutathione
0.3
0 m 0.i Q < 0.1
3 ,
I 5
I
T
FIG. 3. Comparative nating the calorimetric 0.f
0.5
w v
0.4
.
1 15
10 I
M
2’0 E
I 25
I 30
I 35
(MINUTES)
effect of hydrochloric acid and reduced glutatione reaction for acetoacetate determination. Acetoacetate colorimrtric
0 Contains Clutathione
r 40
in termi-
added to reaction 5.9rM +
2OrM
reduced HgCl2
A No Glutathione.
No
A No Glutathione
+ 20rM
HgCIZ HgCl2
z < Q
l
0.3
Contains 5.9 II M reduced Glutathmne. No H&l2
d 0 m
0.2-
a? 4
O.l-
MILLIMICROMOLES
ACETOACETATE
FIG. 4. Effect of reduced glutathione and mercuric chloride on recovery of added acetoacetate from enzymic reaction mixtures. The uppermost curve (m) is included for comparative purposes and represents results obtained when equivalent amounts of acetoacetate are added directly to the calorimetric reaction.
142
JOHiT
I3.
ALLREI)
in the presence and absence of mercuric chloride. Figure 4 shows a typical result of this type of experiment. When acetoacetate was determined in the presence of glutathione and absence of mercuric ion, recovery was very low and the relationship between optical density and acetoacetate concentration was not linear. If, however, mercuric ion was used in t,he calorimetric reaction, recovery of added acetoacetate was approximately 9370, which is in agreement with previous results (1). Similar results were obtained when glutathione was not included in t.he assay system. DISCUSSIOS
The addition of mercuric chloride to the calorimetric reaction mixture has proved to be an effective and reproducible procedure to overcome the interference of reduced glutathione and cyst.eine in the determination of acetoacetate. Glutathione has been noted previously to cause a moderate reduction in color yield in this reaction, and it was indicated that appropriate corrections were made (3). Under conditions of the recovery experiment reported here, corrections for glutathione interference would be very difficult since a linear relationship was not obtained between acetoacetate present in the reaction mixture and the observed optical density (Fig. 4). One can, of course, vary the shape of this curve, and therefore the recovery, by using different amounts of the enzymic reaction mixture in the calorimetric reaction, and thus influence the extent of interference (Fig. 2). Evidence has been presented indicating that a reaction occurs between the diazo reagent and reduced glutathione. No attempt has yet been made, however, to characterize any product(s) from such a reaction. It is tempting to speculate that such a reaction causes interference in acetoacetate determination because it reduces the amount of diazo reagent available for react,ion with acetoacetate. SUMMARY
Cysteine and reduced glutathione have been shown to interfere in the calorimetric determination of acetoacetate, using the method of Walker. This interference can be significantly reduced in the case of cysteine and eliminated in the case of reduced glutathione if mercuric chloride is added to the calorimetric reaction mixture. ACKNOWLEDGMENT The technical assistance gratefully acknowledged.
of
Donald
Upjohn,
Adel
S.zlman
and
REFERENCES 1. WALKER,
P. G., B&hem. J. 58, 699 (1954). 2. DRUMMOND, G. I., AND STERN, J. R.. J. Bid.
C/tern.
235, 318 (1960).
Robert
Fink
is
.ICETOACEThTE
DETERMINATIOK
143
3. SEGAL, H. I,., AND MENON, G. K. Ii., J. Sol. Chem. 236, 2872 (1961). 4. HIRD, F. J. R., AND SYMOXS, R. H., Biochim. Biophys. Acta 46, 457 (1961). 5. STERK, J. R., ASD MILLER, G. E., Biochim. Biophys. Acta 35, 576 (1959). 6. LYNEN, F., HENNING, U., BUBLITZ, C., SORBO, B., ASD KKOPLIS-RUEFF, L., Biochem. 2.330, 269 (1958). 7. CALDWELL, I. C., AND DRUMMOXD, G. I., J. Biol. C’hem. 238, 64 (1963). 8. SEELY, H. W., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 624. Academic Press, New York, 1955. 9. STERN, J. R., DRUMMOND. G. I.. Coos, M. J., DEL CAMPILLO, A., J. Biol. Chem. 235, 313 (1960).