ANALYTICAL
75, 604-610 (1976)
BIOCHEMISTRY
An Automated Enzymic Method for the Determination of Glycerol MICHAELG.GORE Department
of Physiology
and Biochemistry, University Southampton, England
of Southampton,
Received February 26, 1976; accepted June 10, 1976 An automated procedure has been described based on the manual method of Hagen and Hagen [(1%2) Canad. J. Biochem. 40, 11291 which will rapidly and reproducibly measure glycerol concentrations. The method was developed primarily for the determination of glycerol in adipose tissue and various incubation media. The glycerol in solution is estimated by its partial conversion to dihydroxyacetone by glycerol dehydrogenase from Aerobacter aerogenes. The range of glycerol concentrations able to be measured is more extensive than with other automated methods.
Current studies in lipolysis using fat-pad incubations have indicated the need for a cheap, rapid, and reproducible assay for glycerol. Many assays for glycerol exist (I-7) although most of them suffer from interference by other metabolites or use of expensive reagents. All of these assays can be or have been automated, which has compounded the difficulties in some cases. In this paper a new automated assay using the enzyme glycerol dehydrogenase is discussed and compared with an automated coupledenzyme assay using glycerokinase and a-glycerophosphate dehydrogenase. Glycerol dehydrogenase from Aerobacter aerogenes has been shown by previous workers (1) to be useful in the estimation of blood glycerol. The enzyme catalyzes the oxidation of glycerol to dihydroxyacetone with the simultaneous reduction of NAD to NADH in sodium carbonate buffer at pH 10.0. METHODS AND MATERIALS Reagents and Enzymes
Nicotinamide (ATP), glycerol dehydrogenase U.K. All other Dorset, U.K.
adenine dinucleotide (NAD), adenosine 5’4phosphate dehydrogenase, glycerokinase, and cr-glycerophosphate were obtained from Sigma Chemical Co., Ltd., London, reagents were obtained from BDH Chemicals, Poole,
604 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction ID any form reserved.
AUTOMATED
GLYCEROL
7
ASSAY NAD
‘-1
Fluorlmeter I
0.1 ml mid
Buffer
1.2 ml mm-’
Sample
0.16 ml rnb
AW Pull
605
O.L2 ml mm” through 1.0 ml mini
I
FIG. 1. A diagram representing the manifold used for the automated assay of glycerol using the enzyme glycerol dehydrogenase. All coils used were 2.3-mm i.d. MCI, a coil to mix the sample with the carbonate buffer for 1.0 min; MC2, a coil for the enzymic reaction to take place for 5.0 min. The fluorimeter was equipped with a primary filter allowing light at 340 nm to pass and a secondary filter allowing light at 450 nm to pass.
Enzymes
(a) Glycerol dehydrogenase, specific activity of 4.17 units/mg of protein. One unit of enzyme will oxidize 1 pmol min-’ of glycerol to dihydroxyacetone at pH 10.0 at 25°C. (b) Glycerokinase, specific activity of 81 units/mg of protein. One unit of enzyme will convert 1 pmol min-’ of glycerol to cY-glycerophosphate at pH 9.8 at 37°C. (c) a-glycerophosphate dehydrogenase, 175 units/mg of protein. One unit of enzyme will reduce 1.0 pmol min-’ of dihydroxyacetone phosphate to cu-glycerophosphate at pH 7.4 at 25°C. Buffers and Solutions
(1) For automated glycerol assays using the enzyme glycerol dehydrogenase: (a) Sixty milliliters of 0.5 M sodium carbonate buffer, pH 10.0 were mixed with 180 ml of 0.04 M (NH&SO4 solution to give the working carbonate buffer containing ammonium ions (8). (b) Seven milligrams of NAD were dissolved in 1 ml of water and the pH of the solution adjusted to neutrality by the addition of solid NaHCO,. (c) One milligram of glycerol dehydrogenase was weighed out and dissolved in 5 ml of water. The solution was made fresh each day and stored in ice. (2) For automated glycerol analyses using the coupled glycerokinasecY-glycerophosphate dehydrogenase enzyme reactions: (a) Hydrazine buffer (0.1 M), pH 9.6, contained 52 g of 96% (w/v) hydrazine hydrate, 15 g of glycine, and 2 ml of 1 M MgCl, made up to 1 liter with distilled water. (b) NAD (147 mg) and ATP (225 mg) were dissolved in 20 ml of water and the pH adjusted to neutrality with 20% NaOH. (c) The working enzyme solution contained 0.1 ml of a-glycerophosphate dehydrogenase
606
MICHAEL
G. GORE
Buffer
1.0 ml min.’
Sample
‘1
l-u-l I
Fluorimeter
0.23ml
AW
0.42
Pull
through
mme’
ml mid
1 .O n-f min“
I
FIG. 2. A diagram of the apparatus used to assay glycerol using the coupled-enzyme reactions involving glycerokinase and wglycerophosphate dehydrogenase. The coils and optical filters used are as given in the legend to Fig. 1 and in the text.
suspension in 2.0 M (NH&SO4 (the protein concentration was approximately 10 mg/ml) and 0.075 ml of glycerokinase suspension in 3.2 M (NHJ2S04, pH 6.0 and 1% ethylene glycol (v/v); the protein concentration of this preparation was approximately 5 mg/ml. This mixture was diluted 12-fold by the addition of 1% (v/v) P-mercaptoethanol dissolved in water. This solution was made fresh each day and stored in ice. Instrument
A Technicon AutoAnalyzer II was employed that consisted of the following units: sampler II, proportioning pump II, fluoronephelometer II, recorder II, and a programmer-digital readout device. The assay unit was constructed in this laboratory and was used for both types of glycerol assays. The manifold and pumping rates are described in Figs. 1 and 2. Operating Procedure (1) Automated assay using glycerol dehydrogenase. The glycerol solutions were pumped from the sampler to the manifold at 0.16 ml min-’ and at a rate of 80 hr-’ with a 12-set wash period between samples. The glycerol solution was mixed with a segmented stream of air (0.42 ml min-‘) and buffer (1.2 ml min-‘) in a short mixing coil and the glycerol dehydrogenase (0.05 ml min-I) added immediately prior to the addition of the NAD solution (0.1 ml min-I). The solution was then mixed and incubated at 35°C for 5.0 min in a coil immersed in an open thermostated water bath. The reaction mixture was then pumped into the fluoronephelometer equipped with a primary filter allowing passage of light at 340 nm and a second filter allowing light at 450 nm to pass. The changes in fluorescence were recorded on a Technicon recorder II. The baseline of the
AUTOMATED
GLYCEROL Glycerol
ImM I
5.0
0
-#10.0 I
1.0
0.5 Glycerol
607
ASSAY
lmMl
z
FIG. 3. Calibration curves obtained using either glycerol dehydrogenase (-O-or--C) or glycerokinase + a-glycerophosphate dehydrogenase (0). Note that the top axis describes glycerol concentrations IO-fold higher than the bottom axis.
recordings was set at 5% full scale deflection when a solution NAD, water, and enzyme was passing through the fluorimeter
of buffer, cell.
(2) Automated assay using glycerokinase and wglycerophosphate dehydrogenase. For these assays the glycerol solutions were pumped at
0.23 ml min-’ again at a rate 80 hr-’ with a wash period of 12 set between samples. After the samples joined an air (0.42 ml min-‘) segmented stream TABLE
1
THE PERCENTAGE OF GLYCEROL ADDED TO AN EXPERIMENTAL DETECTED ON REASSAY BY THE GLYCEROL DEHYDROGENASE
Glycerol in sample b-w
Glycerol added to sample m-M
Known glycerol concentration mw
0.92* 0.206 0.92” 0.2ob
1.08 1.08 2.16 2.16
2.0 1.28 3.08 2.36
SOLUTION THAT PROCEDURE
Experimentally determined glycerol concentration mf) 1.95 1.22 3.01 2.20
Is
Added glycerol detected (%I 96 95 97 93
a The experimental solutions were obtained by incubation of 150 mg of mouse (ob-ob) gonadial fat-pad for 1.5 hr at 37°C in 0.5 ml Krebs-Ringer bicarbonate solution (0.05 mM Ca2+) containing 2 mg/ml of bovine serum albumin. * Same as a except 40 mg of mouse gonadial fat-pad were used.
608
MICHAEL TABLE
G. GORE 2
THE EFFECT OF VARJOUS METABOLITES USING THE ENZYME GLYCEROL
ON THE ENZYME DEHYDROGENASE
ASSAY
Glycerol concentration given in assay @M)
Glycerol standard (5mM) in the presence of
-
5.0 4.9 4.9 4.9
2.0 mM lactate 0.5 mM glucose 0.5 mM cu-glycerophosphate 1.1 mM glyceric acid (sodium salt) 1.0 mM pyruvic acid (sodium salt)
4.9 5.0
of buffer (1 .O ml min-‘) the solution passed through a short mixing coil to the point at which a solution of ATP and NAD was added (0.1 ml min-I) to the stream and then the working enzyme solution was added (0.1 ml min-l). The reaction mixture was then incubated at 37°C for 4.3 min in a mixing coil submerged in a thermostated water bath. The changes in fluorescence intensity at 450 nm were recorded as before. Both methods were calibrated by use of a set of glycerol standards containing 0- 10.8 mM glycerol. RESULTS
Standard curves obtained using both enzyme systems are given in Fig. 3; the ordinate displays the relative fluorescence intensity at 450 nm, and the abscissa is calibrated in molarity of glycerol in the sample. Using the enzyme solutions described above, it became clear that the glycerol dehydrogenase system produced a more linear calibration curve than the glycerokinase- a-glycerophosphate dehydrogenase system. No deviation from linearity was detectable using glycerol dehydrogenase when 1.08 mM glycerol was assayed. By increasing the concentration of enzyme used in the assay it was possible to extend the range of this linear response. For general use it was found to be convenient to use the stated concentration of enzyme and to use a diluted sample. However, it can be seen from Fig. 3 (squares) that even using glycerol concentrations in the range of O-10.8 mM the calibration curve obtained was still reasonable. The coupled glycerokinase-a-glycerophosphate dehydrogenase assay did not produce a linear calibration curve at glycerol concentrations above 0.43 mM unless the concentration of enzymes used was at least doubled, which of course increases the cost of the assays.
AUTOMATED
GLYCEROL
ASSAY
609
Recovery experiments were carried out where samples of fat-pad incubations were assayed before and after the addition of known concentrations of glycerol. Typical results are given in Table 1 and show that 96% of the added glycerol is detected by the glycerol dehydrogenase assay, even though the concentration of glycerol present is on the nonlinear part of the calibration curve. Solutions of glycerol of these concentrations need dilution before the coupled glycerokinase- a-glycerophosphate dehydrogenase assay is attempted. Tests were made to determine whether any interference occurred when other metabolites such as lactate, alcohol, glucose, or a-glycerophosphate were present. Table 2 shows values of glycerol readings obtained from standards prepared in saline and containing various metabolites. No deviation from the known glycerol concentration was obtained (outside of experimental error) which indicates that the enzyme glycerol dehydrogenase is very specific for its substrate. Substitution of NH&l for (NH&SO4 had no effect on the activity of the glycerol dehydrogenase, indicating that unlike glycerokinase and a-glycerophosphate (9,10) this enzyme is not affected by sulfate ions. It was shown to be possible to use undiluted incubation fluid if a 12-in. dialyzer were included in the manifold. The sample and buffer were mixed as in Fig. 1 and then passed through the lower half of a 12-in. dialyzer. A second stream of buffer at 0.8 ml min-’ was used to carry any dialyzed glycerol from the upper half of the dialyzer to where the enzyme and NAD solutions were added prior to incubation. The dialyzer effectively diluted the glycerol solutions to concentrations on the linear part of the calibration curve. Unfortunately, blood sera samples contain too little glycerol for the system in the presence of the 12-in. dialyzer unless modifications were made to the manifold. DISCUSSION
The automated assay using the bacterial enzyme glycerol dehydrogenase is a rapid, reproducible, and, at present, economic way of analyzing glycerol in incubation media. Unlike the enzyme system containing glycerokinase and cY-glycerophosphate dehydrogenase (9,lO) it has not been shown to be inhibited by sulfate ions. Activator ions such as NH,+ have been added to the buffer system in a high concentration in order to provide a constant effector concentration. Because the enzymic conversion catalyzed by glycerol dehydrogenase was carried out at pH 10.0, the reaction was virtually unidirectional due to the low concentration of H+ ions. The glycerol dehydrogenase method is therefore able to be used to detect lower concentrations of glycerol than the coupled glycerokinasea-glycerophosphate dehydrogenase assay, since there is a higher concentration of NADH formed from a given glycerol concentration. The assay solutions are technically easier to prepare and handle than
610
MICHAEL
G. GORE
the ones involved when using the coupled glycerokinase- cu-glycerophosphate dehydrogenase system, since only one enzyme and not two is needed. The system could be modified by the incorporation of a 12in. dialyzer. The sample and buffer were mixed as before and then passed through the upper half of a 12-in. dialyzer (Technicon). A second buffer stream (0.8 ml min-‘) was used to pick up dialyzed glycerol from the lower half of the dialyzer and this glycerol solution was then assayed as before. This procedure effectively automatically diluted incubation media to concentrations of glycerol which gave a proportional increase in fluorescence at 450 nm with changes in glycerol concentration. ACKNOWLEDGMENT Dr. M. G. Gore would like to express his gratitude to R. Jewel1 for expert technical assistance.
REFERENCES 1. Hagen, J. H., and Hagen, P. B. (1%2) Canad. J. Biochem. 40, 1129. 2. Hagen, J. H. (1%1)5. Biol. Chem. 236, 1023. 3. Lynn, W. S., MacLeod, R. M., and Brown, R. H. (l%O) J. Biol. Chem. 235, 1904. 4. Kom, E. D. (1955)J. Biol. Chem. 215, 1. 5. Lambert, M., and Neish, A. C. (1950) Canad. J. Res. 28, 83. 6. Biesold, D., and Strack, E. (1958) Hoppe-Seyler’s Z. Physiol. Chem. 311, 115. 7. Strack, E., and Biesold, D. (1959) Z. Gesamte Exp. Med. 130, 547. 8. Lin, E. C. C., and Magasanik, B. (1960) J. Biol. Chem. 235, 1820. 9. Sellinger, 0. Z., and Miller, 0. N. (1959) Nature (London) 183, 889. 16. Sellinger, 0. Z., and Miller, 0. N. (1958) Biochim. Biophys. Acta 29, 74.
75, 604-610 (1976)
BIOCHEMISTRY
An Automated Enzymic Method for the Determination of Glycerol MICHAELG.GORE Department
of Physiology
and Biochemistry, University Southampton, England
of Southampton,
Received February 26, 1976; accepted June 10, 1976 An automated procedure has been described based on the manual method of Hagen and Hagen [(1%2) Canad. J. Biochem. 40, 11291 which will rapidly and reproducibly measure glycerol concentrations. The method was developed primarily for the determination of glycerol in adipose tissue and various incubation media. The glycerol in solution is estimated by its partial conversion to dihydroxyacetone by glycerol dehydrogenase from Aerobacter aerogenes. The range of glycerol concentrations able to be measured is more extensive than with other automated methods.
Current studies in lipolysis using fat-pad incubations have indicated the need for a cheap, rapid, and reproducible assay for glycerol. Many assays for glycerol exist (I-7) although most of them suffer from interference by other metabolites or use of expensive reagents. All of these assays can be or have been automated, which has compounded the difficulties in some cases. In this paper a new automated assay using the enzyme glycerol dehydrogenase is discussed and compared with an automated coupledenzyme assay using glycerokinase and a-glycerophosphate dehydrogenase. Glycerol dehydrogenase from Aerobacter aerogenes has been shown by previous workers (1) to be useful in the estimation of blood glycerol. The enzyme catalyzes the oxidation of glycerol to dihydroxyacetone with the simultaneous reduction of NAD to NADH in sodium carbonate buffer at pH 10.0. METHODS AND MATERIALS Reagents and Enzymes
Nicotinamide (ATP), glycerol dehydrogenase U.K. All other Dorset, U.K.
adenine dinucleotide (NAD), adenosine 5’4phosphate dehydrogenase, glycerokinase, and cr-glycerophosphate were obtained from Sigma Chemical Co., Ltd., London, reagents were obtained from BDH Chemicals, Poole,
604 Copyright All rights
0 1976 by Academic Press. Inc. of reproduction ID any form reserved.
AUTOMATED
GLYCEROL
7
ASSAY NAD
‘-1
Fluorlmeter I
0.1 ml mid
Buffer
1.2 ml mm-’
Sample
0.16 ml rnb
AW Pull
605
O.L2 ml mm” through 1.0 ml mini
I
FIG. 1. A diagram representing the manifold used for the automated assay of glycerol using the enzyme glycerol dehydrogenase. All coils used were 2.3-mm i.d. MCI, a coil to mix the sample with the carbonate buffer for 1.0 min; MC2, a coil for the enzymic reaction to take place for 5.0 min. The fluorimeter was equipped with a primary filter allowing light at 340 nm to pass and a secondary filter allowing light at 450 nm to pass.
Enzymes
(a) Glycerol dehydrogenase, specific activity of 4.17 units/mg of protein. One unit of enzyme will oxidize 1 pmol min-’ of glycerol to dihydroxyacetone at pH 10.0 at 25°C. (b) Glycerokinase, specific activity of 81 units/mg of protein. One unit of enzyme will convert 1 pmol min-’ of glycerol to cY-glycerophosphate at pH 9.8 at 37°C. (c) a-glycerophosphate dehydrogenase, 175 units/mg of protein. One unit of enzyme will reduce 1.0 pmol min-’ of dihydroxyacetone phosphate to cu-glycerophosphate at pH 7.4 at 25°C. Buffers and Solutions
(1) For automated glycerol assays using the enzyme glycerol dehydrogenase: (a) Sixty milliliters of 0.5 M sodium carbonate buffer, pH 10.0 were mixed with 180 ml of 0.04 M (NH&SO4 solution to give the working carbonate buffer containing ammonium ions (8). (b) Seven milligrams of NAD were dissolved in 1 ml of water and the pH of the solution adjusted to neutrality by the addition of solid NaHCO,. (c) One milligram of glycerol dehydrogenase was weighed out and dissolved in 5 ml of water. The solution was made fresh each day and stored in ice. (2) For automated glycerol analyses using the coupled glycerokinasecY-glycerophosphate dehydrogenase enzyme reactions: (a) Hydrazine buffer (0.1 M), pH 9.6, contained 52 g of 96% (w/v) hydrazine hydrate, 15 g of glycine, and 2 ml of 1 M MgCl, made up to 1 liter with distilled water. (b) NAD (147 mg) and ATP (225 mg) were dissolved in 20 ml of water and the pH adjusted to neutrality with 20% NaOH. (c) The working enzyme solution contained 0.1 ml of a-glycerophosphate dehydrogenase
606
MICHAEL
G. GORE
Buffer
1.0 ml min.’
Sample
‘1
l-u-l I
Fluorimeter
0.23ml
AW
0.42
Pull
through
mme’
ml mid
1 .O n-f min“
I
FIG. 2. A diagram of the apparatus used to assay glycerol using the coupled-enzyme reactions involving glycerokinase and wglycerophosphate dehydrogenase. The coils and optical filters used are as given in the legend to Fig. 1 and in the text.
suspension in 2.0 M (NH&SO4 (the protein concentration was approximately 10 mg/ml) and 0.075 ml of glycerokinase suspension in 3.2 M (NHJ2S04, pH 6.0 and 1% ethylene glycol (v/v); the protein concentration of this preparation was approximately 5 mg/ml. This mixture was diluted 12-fold by the addition of 1% (v/v) P-mercaptoethanol dissolved in water. This solution was made fresh each day and stored in ice. Instrument
A Technicon AutoAnalyzer II was employed that consisted of the following units: sampler II, proportioning pump II, fluoronephelometer II, recorder II, and a programmer-digital readout device. The assay unit was constructed in this laboratory and was used for both types of glycerol assays. The manifold and pumping rates are described in Figs. 1 and 2. Operating Procedure (1) Automated assay using glycerol dehydrogenase. The glycerol solutions were pumped from the sampler to the manifold at 0.16 ml min-’ and at a rate of 80 hr-’ with a 12-set wash period between samples. The glycerol solution was mixed with a segmented stream of air (0.42 ml min-‘) and buffer (1.2 ml min-‘) in a short mixing coil and the glycerol dehydrogenase (0.05 ml min-I) added immediately prior to the addition of the NAD solution (0.1 ml min-I). The solution was then mixed and incubated at 35°C for 5.0 min in a coil immersed in an open thermostated water bath. The reaction mixture was then pumped into the fluoronephelometer equipped with a primary filter allowing passage of light at 340 nm and a second filter allowing light at 450 nm to pass. The changes in fluorescence were recorded on a Technicon recorder II. The baseline of the
AUTOMATED
GLYCEROL Glycerol
ImM I
5.0
0
-#10.0 I
1.0
0.5 Glycerol
607
ASSAY
lmMl
z
FIG. 3. Calibration curves obtained using either glycerol dehydrogenase (-O-or--C) or glycerokinase + a-glycerophosphate dehydrogenase (0). Note that the top axis describes glycerol concentrations IO-fold higher than the bottom axis.
recordings was set at 5% full scale deflection when a solution NAD, water, and enzyme was passing through the fluorimeter
of buffer, cell.
(2) Automated assay using glycerokinase and wglycerophosphate dehydrogenase. For these assays the glycerol solutions were pumped at
0.23 ml min-’ again at a rate 80 hr-’ with a wash period of 12 set between samples. After the samples joined an air (0.42 ml min-‘) segmented stream TABLE
1
THE PERCENTAGE OF GLYCEROL ADDED TO AN EXPERIMENTAL DETECTED ON REASSAY BY THE GLYCEROL DEHYDROGENASE
Glycerol in sample b-w
Glycerol added to sample m-M
Known glycerol concentration mw
0.92* 0.206 0.92” 0.2ob
1.08 1.08 2.16 2.16
2.0 1.28 3.08 2.36
SOLUTION THAT PROCEDURE
Experimentally determined glycerol concentration mf) 1.95 1.22 3.01 2.20
Is
Added glycerol detected (%I 96 95 97 93
a The experimental solutions were obtained by incubation of 150 mg of mouse (ob-ob) gonadial fat-pad for 1.5 hr at 37°C in 0.5 ml Krebs-Ringer bicarbonate solution (0.05 mM Ca2+) containing 2 mg/ml of bovine serum albumin. * Same as a except 40 mg of mouse gonadial fat-pad were used.
608
MICHAEL TABLE
G. GORE 2
THE EFFECT OF VARJOUS METABOLITES USING THE ENZYME GLYCEROL
ON THE ENZYME DEHYDROGENASE
ASSAY
Glycerol concentration given in assay @M)
Glycerol standard (5mM) in the presence of
-
5.0 4.9 4.9 4.9
2.0 mM lactate 0.5 mM glucose 0.5 mM cu-glycerophosphate 1.1 mM glyceric acid (sodium salt) 1.0 mM pyruvic acid (sodium salt)
4.9 5.0
of buffer (1 .O ml min-‘) the solution passed through a short mixing coil to the point at which a solution of ATP and NAD was added (0.1 ml min-I) to the stream and then the working enzyme solution was added (0.1 ml min-l). The reaction mixture was then incubated at 37°C for 4.3 min in a mixing coil submerged in a thermostated water bath. The changes in fluorescence intensity at 450 nm were recorded as before. Both methods were calibrated by use of a set of glycerol standards containing 0- 10.8 mM glycerol. RESULTS
Standard curves obtained using both enzyme systems are given in Fig. 3; the ordinate displays the relative fluorescence intensity at 450 nm, and the abscissa is calibrated in molarity of glycerol in the sample. Using the enzyme solutions described above, it became clear that the glycerol dehydrogenase system produced a more linear calibration curve than the glycerokinase- a-glycerophosphate dehydrogenase system. No deviation from linearity was detectable using glycerol dehydrogenase when 1.08 mM glycerol was assayed. By increasing the concentration of enzyme used in the assay it was possible to extend the range of this linear response. For general use it was found to be convenient to use the stated concentration of enzyme and to use a diluted sample. However, it can be seen from Fig. 3 (squares) that even using glycerol concentrations in the range of O-10.8 mM the calibration curve obtained was still reasonable. The coupled glycerokinase-a-glycerophosphate dehydrogenase assay did not produce a linear calibration curve at glycerol concentrations above 0.43 mM unless the concentration of enzymes used was at least doubled, which of course increases the cost of the assays.
AUTOMATED
GLYCEROL
ASSAY
609
Recovery experiments were carried out where samples of fat-pad incubations were assayed before and after the addition of known concentrations of glycerol. Typical results are given in Table 1 and show that 96% of the added glycerol is detected by the glycerol dehydrogenase assay, even though the concentration of glycerol present is on the nonlinear part of the calibration curve. Solutions of glycerol of these concentrations need dilution before the coupled glycerokinase- a-glycerophosphate dehydrogenase assay is attempted. Tests were made to determine whether any interference occurred when other metabolites such as lactate, alcohol, glucose, or a-glycerophosphate were present. Table 2 shows values of glycerol readings obtained from standards prepared in saline and containing various metabolites. No deviation from the known glycerol concentration was obtained (outside of experimental error) which indicates that the enzyme glycerol dehydrogenase is very specific for its substrate. Substitution of NH&l for (NH&SO4 had no effect on the activity of the glycerol dehydrogenase, indicating that unlike glycerokinase and a-glycerophosphate (9,10) this enzyme is not affected by sulfate ions. It was shown to be possible to use undiluted incubation fluid if a 12-in. dialyzer were included in the manifold. The sample and buffer were mixed as in Fig. 1 and then passed through the lower half of a 12-in. dialyzer. A second stream of buffer at 0.8 ml min-’ was used to carry any dialyzed glycerol from the upper half of the dialyzer to where the enzyme and NAD solutions were added prior to incubation. The dialyzer effectively diluted the glycerol solutions to concentrations on the linear part of the calibration curve. Unfortunately, blood sera samples contain too little glycerol for the system in the presence of the 12-in. dialyzer unless modifications were made to the manifold. DISCUSSION
The automated assay using the bacterial enzyme glycerol dehydrogenase is a rapid, reproducible, and, at present, economic way of analyzing glycerol in incubation media. Unlike the enzyme system containing glycerokinase and cY-glycerophosphate dehydrogenase (9,lO) it has not been shown to be inhibited by sulfate ions. Activator ions such as NH,+ have been added to the buffer system in a high concentration in order to provide a constant effector concentration. Because the enzymic conversion catalyzed by glycerol dehydrogenase was carried out at pH 10.0, the reaction was virtually unidirectional due to the low concentration of H+ ions. The glycerol dehydrogenase method is therefore able to be used to detect lower concentrations of glycerol than the coupled glycerokinasea-glycerophosphate dehydrogenase assay, since there is a higher concentration of NADH formed from a given glycerol concentration. The assay solutions are technically easier to prepare and handle than
610
MICHAEL
G. GORE
the ones involved when using the coupled glycerokinase- cu-glycerophosphate dehydrogenase system, since only one enzyme and not two is needed. The system could be modified by the incorporation of a 12in. dialyzer. The sample and buffer were mixed as before and then passed through the upper half of a 12-in. dialyzer (Technicon). A second buffer stream (0.8 ml min-‘) was used to pick up dialyzed glycerol from the lower half of the dialyzer and this glycerol solution was then assayed as before. This procedure effectively automatically diluted incubation media to concentrations of glycerol which gave a proportional increase in fluorescence at 450 nm with changes in glycerol concentration. ACKNOWLEDGMENT Dr. M. G. Gore would like to express his gratitude to R. Jewel1 for expert technical assistance.
REFERENCES 1. Hagen, J. H., and Hagen, P. B. (1%2) Canad. J. Biochem. 40, 1129. 2. Hagen, J. H. (1%1)5. Biol. Chem. 236, 1023. 3. Lynn, W. S., MacLeod, R. M., and Brown, R. H. (l%O) J. Biol. Chem. 235, 1904. 4. Kom, E. D. (1955)J. Biol. Chem. 215, 1. 5. Lambert, M., and Neish, A. C. (1950) Canad. J. Res. 28, 83. 6. Biesold, D., and Strack, E. (1958) Hoppe-Seyler’s Z. Physiol. Chem. 311, 115. 7. Strack, E., and Biesold, D. (1959) Z. Gesamte Exp. Med. 130, 547. 8. Lin, E. C. C., and Magasanik, B. (1960) J. Biol. Chem. 235, 1820. 9. Sellinger, 0. Z., and Miller, 0. N. (1959) Nature (London) 183, 889. 16. Sellinger, 0. Z., and Miller, 0. N. (1958) Biochim. Biophys. Acta 29, 74.
