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A kinetic isotope dilution assay for glycerol
ANALYTICAL BIOCHEMISTRY 139, 305-308 (1984)
A Kinetic Isotope Dilution Assay for Glycerol JOHND.BELL,IAIN Divisions
L.O.BUXTON,ANDLAURENCE
of Pharmacology and Cardiology, San Diego, La Jolla,
MO13H, California
University 92093
L.BRUNTON of California
at
Received October 17, 1983 Using glycerol kinase and [‘HIglycerol, a kinetic isotope dilution assay for glycerol has been developed. Reactant and product are separated by stepwise elution from QAE-Sephadex. This assayis sensitive to as little as 100 pmol of glycerol, avoids numerous drawbacks of the traditional fluorescent assay,and readily detects glycerol production by fewer than 10’ cardiomyocytes. KBY WORDS: glycerol; glycerol kinase; hormone-sensitive lipase; cardiomyocytes.
In the course of studying hormonal regulation of metabolism in adult ventricular cardiomyocytes, we recently attempted to detect the activity of hormone-sensitive lipase by quantifying the appearance of glycerol in the incubation medium surrounding purified ventricular myocytes. Using a fluorometric modification ( 1) of the spectrophotometric assay (2) for glycerol, which relies on successive enzymatic phosphorylation and oxidation to produce glyceraldehyde phosphate and NADH, we obtained small hormonally induced increases in fluorescence (glycerol release) that were not reproducibly detectable over a large background fluorescence obtained with untreated cells. Since murine cardiomyocytes are generally only 65-85% viable and are sensitive to hypoxia, we suspected that the high background fluorescence resulted from leakage of cellular contents into the incubation medium, very likely enzymes and substrates that could use the NAD added for the glycerol assay. Indeed, the background signal depended on exogenous NAD and was greatly reduced by prior deproteinizing of the sample with perchloric acid. Even using deproteinized samples, however, we could obtain a reproducible signal only by employing large numbers of myocytes. To circumvent the problem of high background fluorescence and to facilitate the assay 305
of samples from a smaller number of myocytes, we have devised a radiometric assay for glycerol. The method is an isotope dilution procedure employing glycerol kinase and [3H]glycerol under conditions of near saturation of the enzyme by the 3H-labeled substrate and with a reaction time sufficiently short that the rate of product formation is constant. Thus, the amount of product formed is also constant. The addition of sample glycerol lowers the specific radioactivity of product and hence decreases the amount of 3H-labeled product produced. MATERIALS
Prior to use, we purified [2-3HJglycerol (New England Nuclear, 10 Ci/mmol) over the anion-exchange resin QAE-Sephadex A-25 (Pharmacia; functional group: diethyl-(2-hydroxypropyl)aminoethyl), collecting the first peak eluted with water (peak 1, Fig. 1) and then lyophilizing it and reconstituting in water. Glycerol kinase (Sigma, G4509) was dissolved in 20 mM NaPO,, pH 7, and 1 mM &mercaptoethanol, stored at -20°C in small aliquots and thawed as needed. All other materials were of reagent quality from standard commercial sources. Adult ventricular cardiomyocytes were purified by a collagenase perfusion technique (3). As a scintillation fluid 0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rigMs of reproduction in any form reserved.
306
BELL, BUXTON,
for aqueous samples, we used Monofluor tional Diagnostics).
AND BRUNTON
(Na-
RESULTS
We reasoned that a sensitive isotope dilution assay using [3H]glycerol and glycerol kinase would be possible if substrate (glycerol) and product (glycerol 3-phosphate) could be readily separated and if a significant signal ( [3H]glycerol phosphate) could be generated with small amounts of enzyme and substrate under conditions of significant fractional saturation of the enzyme. The following data establish these and other basic criteria. Initial conditions. We first reacted [3H]glyCerOl(20 PM; -2.7 X lo5 dpm/nmol), ATP (2.25 mM), MgC12 (5 mM), and glycerol kinase (0.2 mu/tube) in T&-Cl (25 mM) at pH 7.6 in 0.1 to 0.2 ml at 30°C. We terminated the reaction by adding unlabeled glycerol (1 ml of 2 M glycerol). This amount of glycerol dilutes the specific radioactivity of the substrate by a factor of lo5 and thus effectively stops the accumulation of signal. To separate reactant and product, we tried several means, including anion-exchange paper disks that could be washed in bulk or filtered individually (4) and small anion-exchange columns. Column chromatography provided the most reproducible signals with the lowest background, as described below. Separation of substrate and product. Since glycerol and glycerol phosphate differ so greatly in charge at neutral pH, we chose the anion-exchange resin QAE Sephadex A-25 for their separation (see details in legend to Fig. 1). [3H]Glycerol does not interact significantly with QAE-Sephadex and runs essentially with the void volume (0 time, Fig. 1). When [3H]glycerol is incubated with ATP-Mg*+ and glycerol kinase, however, there is a progressive decline in the size of the glycerol peak (peak 1) and the residual counts, putatively glycerol phosphate, may be eluted with HCl (peak 2). The background, that is, the appearance of signal in the product peak in the absence of glycerol kinase, is small, ~2.5% of maximal signal at IO min and ~0.25% of total counts
0 0
I
2
3 4 ELUTICN
9 IO II VOLUME, ml
12 I3
I4
FIG. 1. Anion-exchange chromatography of substrate and product. Reactions (100 ~1) were initiated by addition of glycerol kinase (0.2 mu), incubated for various times at 30°C and terminated by addition of 1 ml of 2 M glycerol (in water). Zero-time sample had no glycerol kinase and was equivalent to adding glycerol kinase after addition of excessglycerol. The terminated reaction was added to a QAE-Sephadex A-25 column (1 X 0.7-cm bed in a 0.7 X IO-cm Econo-Column, Bio-Rad). Fractions were eluted directly into scintillation vials with l-ml aliquots of water (fractions l-8) and 1 N HCI (fractions 9-12) and, after addition of fluor, counted in a scintillation spectrometer at -22% efficiency. Reaction times: l ,O min; 0,5 min; A, 15 min; A, 25 min. Inset; Distribution of labeled materials as reaction proceeds. Radioactivity in peak 1 declines to the extent that cpm appear in peak 2 such that total cpm are constant and entirely accounted for.
added. The assay blank as a percentage of maximal signal becomes proportionately smaller as the assay runs longer and the signal increases; one can readily double the signal size and the ratio of signal to blank by incubating for 20 rather than 10 min. All of the radioactivity can be accounted for by the summation of the two peaks, as the inset to Fig. 1 demonstrates. Based on this elution profile (Fig. 1) we routinely use a batchwise elution after adding the sample: 8 ml of water (peak 1, discarded) followed by 3 ml of 1 N HCl (peak 2, collected in scintillation vial). Dependence on time and on concentrations of enzyme and substrate. Collecting peak 2 (Fig. 1) as the signal by batchwise elution (de-
ISOTOPE DILUTION
ASSAY FOR GLYCEROL
tailed above), we find that the phosphorylation of glycerol under our reaction conditions is linear for 25 to 30 min (Fig. 2). We routinely use a lo- to 20-min incubation period. We based our choice of the quantity of enzyme employed on the data of Fig. 3. These data demonstrate that 2 nmol of glycerol/ 100 ~1 reaction are not limiting below 0.5 mU glycerol kinase/lOO ~1. We use 0.2 mU of enzyme per tube as a minimal amount that gives a sufficiently large signal (cpm in peak 2 from the QAE-Sephadex column). This quantity of enzyme used in a lo-min incubation with 20 PM glycerol converts -20% of substrate to product. Using glycerol kinase at 0.2 mU/lOO ~1 reaction tube we have examined the reaction over the range of [‘HIglycerol between 0.1 and 50 PM. Eadie-Hofstee plots of such experiments are linear and yield kinetic constants that agree well with published values (4) with the apparent K, in the range 7 to 1 I PM. Isotope dilution with added glycerol. Based on an apparent K,,, of 7 PM, the concentration of [3H]glycerol routinely employed (2 nmol/ 100 ~1 or 20 FM) gives a fractional saturation of glycerol kinase of -0.75, sufficient saturation to give an isotope dilution assay (5) that is sensitive over the range of 0 to 40 nmol unlabeled glycerol per tube (O-400 PM). A loglog plot of glycerol phosphate production vs added glycerol routinely gives a linear fit with a slope between -0.85 and -1 (Fig. 4). Such
0
20
IO TIME
30
(min)
FIG. 2. Time course of product formation.
0
0.4 GLYCEROL (mU/iOO~l
0.2
307
0.6 0.8 KINASE tWCti0n)
1.0
FIG. 3. Product formation vs glycerol kinase concentration. Reactions (100 $1, containing 2 nmol [-‘H]glycerol) were initiated by the addition of glycerol kinase and terminated after 10 min.
a line forms an easily employed standard curve for a glycerol assay. The assay can be run equally well in either dilute buffer or isotonic growth medium (the diluent for samples from intact cell experiments). The addition of as little as 100 pm01 of glycerol gives a measurable isotope dilution (see inset, Fig. 4). DISCUSSION
We have developed an assay for glycerol based on a dilution of the specific radioactivity of 3H-labeled substrate for glycerol kinase. Substrate and product are readily separated by anion-exchange chromatography. The assay gives appropriate kinetic constants for the enzyme. The utility of the assay lies in its ease, its freedom from interfering fluorescence (hence avoidance of extensive sample preparation and associated dilution), and its sensitivity. We have used both the fluorometric and isotope dilution assays to study glycerol production by hormonally stimulated cardiomyocytes and have obtained comparable results with the two assays: isoproterenol increases glycerol production by 17.7 f 1.5 nmol/106 cells/h (mean f SE, n = 3) over a basal rate of 12 nmol/ 1O6 cells/h, figures that compare well with recently published values
308
BELL, BUXTON,
‘%
/’
” ’ 34 6 TOTAL GLYCEROL,
I 12 nmoles
I 22 per tube
I 42
I
FIG. 4. Isotope dilution standard curve. Reactions were run as described in Fig. I, with 2 nmol [3H]glycerol in each tube and 0 to 40 nmol of glycerol added as standards. The least-squares regression line fitting the log-log plot isy = 4.6 - 0.88x, r = 0.99 (PC 0.001). Inset: An example of standards between 0 and 400 pmol in excess of [3H]glycerol (points are means of duplicates).
(6). The fluorescent assay requires the medium associated with - lo6 cells to produce a signal at the lower range of the assay. By contrast, the isotope dilution assay readily measures the hourly glycerol release from 10’ cells, with a probable lower limit near lo4 cells. Thus, the isotope dilution assay can permit more judicious use of a valuable cell suspension and also assessment of earlier time points at which glycerol production is proportionately less. The isotope dilution assay has the additional advantages of requiring only one enzymatic step and of using only a tenth as much glycerol kinase. Having developed and used the assay described above and prepared the method for
AND BRUNTON
publication, we found that Newsholme (7) has also described a radiochemical assay for glycerol. By virtue of conditions that allow glycerol kinase to catalyze with a lower apparent K,, the current assay proves more sensitive by about an order of magnitude. The current assay might be made even more sensitive, if necessary, by reducing the pH and increasing the temperature of the reaction, alterations that increase the apparent affinity of glycerol kinase for glycerol (4). In our hands, separation of reactant and product by column chromatography gives a lower blank value than previously used methods (4,7) and further contributes to increased sensitivity. Our plotting of the standard curve in log-log format facilitates both visual and computerized calculation of sample data. These differences provide significant improvements and are, we believe, sufficient to warrant consideration of the new assay. REFERENCES 1. Laurell, S., and Tribling, G. (1966) Clin. Chim. Acta 13,3 17-322.
2. Wieland, 0. (1957) Biochem. Z. 329, 3 13-3 19. 3. Buxton, I. L. O., and Brunton, L. L. (1983) J. Biol. Chem. 258, 10233-10239. 4. Thomer, J. W. (1975) in Methods in Enzymology (Wood, W. A., ed.). Vol. 42, pp. 148-156, Academic Press, New York. 5. Brown, B. L., Ekins, R. P., and Albano, J. D. M. (I 972) Adv. Cyclic Nucl. Rex 2, 25-40. 6. Kryski, A., Jr., and Severson, D. L. (1983) Fed. Proc. 42, 598. 7. Newsholme, E. A. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 14091414, Verlag Chemie, Weinheim.
A Kinetic Isotope Dilution Assay for Glycerol JOHND.BELL,IAIN Divisions
L.O.BUXTON,ANDLAURENCE
of Pharmacology and Cardiology, San Diego, La Jolla,
MO13H, California
University 92093
L.BRUNTON of California
at
Received October 17, 1983 Using glycerol kinase and [‘HIglycerol, a kinetic isotope dilution assay for glycerol has been developed. Reactant and product are separated by stepwise elution from QAE-Sephadex. This assayis sensitive to as little as 100 pmol of glycerol, avoids numerous drawbacks of the traditional fluorescent assay,and readily detects glycerol production by fewer than 10’ cardiomyocytes. KBY WORDS: glycerol; glycerol kinase; hormone-sensitive lipase; cardiomyocytes.
In the course of studying hormonal regulation of metabolism in adult ventricular cardiomyocytes, we recently attempted to detect the activity of hormone-sensitive lipase by quantifying the appearance of glycerol in the incubation medium surrounding purified ventricular myocytes. Using a fluorometric modification ( 1) of the spectrophotometric assay (2) for glycerol, which relies on successive enzymatic phosphorylation and oxidation to produce glyceraldehyde phosphate and NADH, we obtained small hormonally induced increases in fluorescence (glycerol release) that were not reproducibly detectable over a large background fluorescence obtained with untreated cells. Since murine cardiomyocytes are generally only 65-85% viable and are sensitive to hypoxia, we suspected that the high background fluorescence resulted from leakage of cellular contents into the incubation medium, very likely enzymes and substrates that could use the NAD added for the glycerol assay. Indeed, the background signal depended on exogenous NAD and was greatly reduced by prior deproteinizing of the sample with perchloric acid. Even using deproteinized samples, however, we could obtain a reproducible signal only by employing large numbers of myocytes. To circumvent the problem of high background fluorescence and to facilitate the assay 305
of samples from a smaller number of myocytes, we have devised a radiometric assay for glycerol. The method is an isotope dilution procedure employing glycerol kinase and [3H]glycerol under conditions of near saturation of the enzyme by the 3H-labeled substrate and with a reaction time sufficiently short that the rate of product formation is constant. Thus, the amount of product formed is also constant. The addition of sample glycerol lowers the specific radioactivity of product and hence decreases the amount of 3H-labeled product produced. MATERIALS
Prior to use, we purified [2-3HJglycerol (New England Nuclear, 10 Ci/mmol) over the anion-exchange resin QAE-Sephadex A-25 (Pharmacia; functional group: diethyl-(2-hydroxypropyl)aminoethyl), collecting the first peak eluted with water (peak 1, Fig. 1) and then lyophilizing it and reconstituting in water. Glycerol kinase (Sigma, G4509) was dissolved in 20 mM NaPO,, pH 7, and 1 mM &mercaptoethanol, stored at -20°C in small aliquots and thawed as needed. All other materials were of reagent quality from standard commercial sources. Adult ventricular cardiomyocytes were purified by a collagenase perfusion technique (3). As a scintillation fluid 0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rigMs of reproduction in any form reserved.
306
BELL, BUXTON,
for aqueous samples, we used Monofluor tional Diagnostics).
AND BRUNTON
(Na-
RESULTS
We reasoned that a sensitive isotope dilution assay using [3H]glycerol and glycerol kinase would be possible if substrate (glycerol) and product (glycerol 3-phosphate) could be readily separated and if a significant signal ( [3H]glycerol phosphate) could be generated with small amounts of enzyme and substrate under conditions of significant fractional saturation of the enzyme. The following data establish these and other basic criteria. Initial conditions. We first reacted [3H]glyCerOl(20 PM; -2.7 X lo5 dpm/nmol), ATP (2.25 mM), MgC12 (5 mM), and glycerol kinase (0.2 mu/tube) in T&-Cl (25 mM) at pH 7.6 in 0.1 to 0.2 ml at 30°C. We terminated the reaction by adding unlabeled glycerol (1 ml of 2 M glycerol). This amount of glycerol dilutes the specific radioactivity of the substrate by a factor of lo5 and thus effectively stops the accumulation of signal. To separate reactant and product, we tried several means, including anion-exchange paper disks that could be washed in bulk or filtered individually (4) and small anion-exchange columns. Column chromatography provided the most reproducible signals with the lowest background, as described below. Separation of substrate and product. Since glycerol and glycerol phosphate differ so greatly in charge at neutral pH, we chose the anion-exchange resin QAE Sephadex A-25 for their separation (see details in legend to Fig. 1). [3H]Glycerol does not interact significantly with QAE-Sephadex and runs essentially with the void volume (0 time, Fig. 1). When [3H]glycerol is incubated with ATP-Mg*+ and glycerol kinase, however, there is a progressive decline in the size of the glycerol peak (peak 1) and the residual counts, putatively glycerol phosphate, may be eluted with HCl (peak 2). The background, that is, the appearance of signal in the product peak in the absence of glycerol kinase, is small, ~2.5% of maximal signal at IO min and ~0.25% of total counts
0 0
I
2
3 4 ELUTICN
9 IO II VOLUME, ml
12 I3
I4
FIG. 1. Anion-exchange chromatography of substrate and product. Reactions (100 ~1) were initiated by addition of glycerol kinase (0.2 mu), incubated for various times at 30°C and terminated by addition of 1 ml of 2 M glycerol (in water). Zero-time sample had no glycerol kinase and was equivalent to adding glycerol kinase after addition of excessglycerol. The terminated reaction was added to a QAE-Sephadex A-25 column (1 X 0.7-cm bed in a 0.7 X IO-cm Econo-Column, Bio-Rad). Fractions were eluted directly into scintillation vials with l-ml aliquots of water (fractions l-8) and 1 N HCI (fractions 9-12) and, after addition of fluor, counted in a scintillation spectrometer at -22% efficiency. Reaction times: l ,O min; 0,5 min; A, 15 min; A, 25 min. Inset; Distribution of labeled materials as reaction proceeds. Radioactivity in peak 1 declines to the extent that cpm appear in peak 2 such that total cpm are constant and entirely accounted for.
added. The assay blank as a percentage of maximal signal becomes proportionately smaller as the assay runs longer and the signal increases; one can readily double the signal size and the ratio of signal to blank by incubating for 20 rather than 10 min. All of the radioactivity can be accounted for by the summation of the two peaks, as the inset to Fig. 1 demonstrates. Based on this elution profile (Fig. 1) we routinely use a batchwise elution after adding the sample: 8 ml of water (peak 1, discarded) followed by 3 ml of 1 N HCl (peak 2, collected in scintillation vial). Dependence on time and on concentrations of enzyme and substrate. Collecting peak 2 (Fig. 1) as the signal by batchwise elution (de-
ISOTOPE DILUTION
ASSAY FOR GLYCEROL
tailed above), we find that the phosphorylation of glycerol under our reaction conditions is linear for 25 to 30 min (Fig. 2). We routinely use a lo- to 20-min incubation period. We based our choice of the quantity of enzyme employed on the data of Fig. 3. These data demonstrate that 2 nmol of glycerol/ 100 ~1 reaction are not limiting below 0.5 mU glycerol kinase/lOO ~1. We use 0.2 mU of enzyme per tube as a minimal amount that gives a sufficiently large signal (cpm in peak 2 from the QAE-Sephadex column). This quantity of enzyme used in a lo-min incubation with 20 PM glycerol converts -20% of substrate to product. Using glycerol kinase at 0.2 mU/lOO ~1 reaction tube we have examined the reaction over the range of [‘HIglycerol between 0.1 and 50 PM. Eadie-Hofstee plots of such experiments are linear and yield kinetic constants that agree well with published values (4) with the apparent K, in the range 7 to 1 I PM. Isotope dilution with added glycerol. Based on an apparent K,,, of 7 PM, the concentration of [3H]glycerol routinely employed (2 nmol/ 100 ~1 or 20 FM) gives a fractional saturation of glycerol kinase of -0.75, sufficient saturation to give an isotope dilution assay (5) that is sensitive over the range of 0 to 40 nmol unlabeled glycerol per tube (O-400 PM). A loglog plot of glycerol phosphate production vs added glycerol routinely gives a linear fit with a slope between -0.85 and -1 (Fig. 4). Such
0
20
IO TIME
30
(min)
FIG. 2. Time course of product formation.
0
0.4 GLYCEROL (mU/iOO~l
0.2
307
0.6 0.8 KINASE tWCti0n)
1.0
FIG. 3. Product formation vs glycerol kinase concentration. Reactions (100 $1, containing 2 nmol [-‘H]glycerol) were initiated by the addition of glycerol kinase and terminated after 10 min.
a line forms an easily employed standard curve for a glycerol assay. The assay can be run equally well in either dilute buffer or isotonic growth medium (the diluent for samples from intact cell experiments). The addition of as little as 100 pm01 of glycerol gives a measurable isotope dilution (see inset, Fig. 4). DISCUSSION
We have developed an assay for glycerol based on a dilution of the specific radioactivity of 3H-labeled substrate for glycerol kinase. Substrate and product are readily separated by anion-exchange chromatography. The assay gives appropriate kinetic constants for the enzyme. The utility of the assay lies in its ease, its freedom from interfering fluorescence (hence avoidance of extensive sample preparation and associated dilution), and its sensitivity. We have used both the fluorometric and isotope dilution assays to study glycerol production by hormonally stimulated cardiomyocytes and have obtained comparable results with the two assays: isoproterenol increases glycerol production by 17.7 f 1.5 nmol/106 cells/h (mean f SE, n = 3) over a basal rate of 12 nmol/ 1O6 cells/h, figures that compare well with recently published values
308
BELL, BUXTON,
‘%
/’
” ’ 34 6 TOTAL GLYCEROL,
I 12 nmoles
I 22 per tube
I 42
I
FIG. 4. Isotope dilution standard curve. Reactions were run as described in Fig. I, with 2 nmol [3H]glycerol in each tube and 0 to 40 nmol of glycerol added as standards. The least-squares regression line fitting the log-log plot isy = 4.6 - 0.88x, r = 0.99 (PC 0.001). Inset: An example of standards between 0 and 400 pmol in excess of [3H]glycerol (points are means of duplicates).
(6). The fluorescent assay requires the medium associated with - lo6 cells to produce a signal at the lower range of the assay. By contrast, the isotope dilution assay readily measures the hourly glycerol release from 10’ cells, with a probable lower limit near lo4 cells. Thus, the isotope dilution assay can permit more judicious use of a valuable cell suspension and also assessment of earlier time points at which glycerol production is proportionately less. The isotope dilution assay has the additional advantages of requiring only one enzymatic step and of using only a tenth as much glycerol kinase. Having developed and used the assay described above and prepared the method for
AND BRUNTON
publication, we found that Newsholme (7) has also described a radiochemical assay for glycerol. By virtue of conditions that allow glycerol kinase to catalyze with a lower apparent K,, the current assay proves more sensitive by about an order of magnitude. The current assay might be made even more sensitive, if necessary, by reducing the pH and increasing the temperature of the reaction, alterations that increase the apparent affinity of glycerol kinase for glycerol (4). In our hands, separation of reactant and product by column chromatography gives a lower blank value than previously used methods (4,7) and further contributes to increased sensitivity. Our plotting of the standard curve in log-log format facilitates both visual and computerized calculation of sample data. These differences provide significant improvements and are, we believe, sufficient to warrant consideration of the new assay. REFERENCES 1. Laurell, S., and Tribling, G. (1966) Clin. Chim. Acta 13,3 17-322.
2. Wieland, 0. (1957) Biochem. Z. 329, 3 13-3 19. 3. Buxton, I. L. O., and Brunton, L. L. (1983) J. Biol. Chem. 258, 10233-10239. 4. Thomer, J. W. (1975) in Methods in Enzymology (Wood, W. A., ed.). Vol. 42, pp. 148-156, Academic Press, New York. 5. Brown, B. L., Ekins, R. P., and Albano, J. D. M. (I 972) Adv. Cyclic Nucl. Rex 2, 25-40. 6. Kryski, A., Jr., and Severson, D. L. (1983) Fed. Proc. 42, 598. 7. Newsholme, E. A. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), Vol. 3, pp. 14091414, Verlag Chemie, Weinheim.