130
ASSAYS
[15]
release of labeled serotonin. This latter technique uses expensive radioactive compounds which must be appropriately disposed of, necessitates a preincorporation step of the label, and, most importantly, does not yield immediate data. Off-range concentrations of paf cannot be adjusted in real time as is possible with aggregation techniques where results are immediately available, thus delaying the acquisition of valid results by several days. The aggregation method was and still is the basis for most of the studies conducted so far on this mediator, notably on the structure, the origin, the metabolism, and the physiological properties of paf. In addition, most of the paf antagonists have been developed using this test either as a screening procedure or for more complex pharmacological studies.
[15] B i o a s s a y o f P l a t e l e t - A c t i v a t i n g F a c t o r b y R e l e a s e of [3H]Serotonin
By PETER M. HENSON Introduction Platelet-activating factor (PAF) is a highly potent cell communication mediator thought to be involved in a wide variety of pathophysiological processes. Three major approaches have been taken in assaying for the molecule: bioassays, including platelet aggregation, platelet secretion, and neutrophil activation; synthetic approaches, including incorporation of [3HI acetate into the molecule; and physicochemical procedures, including mass spectrometry. Each has advantages and disadvantages. The bioassays are relatively simple, can be applied to a large number of samples, often require little workup, are highly sensitive, and by the use of appropriate standards, give reasonably quantitative results. They can be carried out with minimal equipment and are inexpensive. Their disadvantage, in general, is the potential lack of specificity (necessitating appropriate controls and/or purification), since many agents can activate platelets or neutrophils, the lack of quantitative precision that would be found in physical approaches, and the inability to obtain structural data from the assay. Rabbit platelets are particularly sensitive to stimulation by PAF and are often used as indicator cells. Thus the assay is described for these cells, although it could be adapted to ceils from humans. Platelet aggregation is more sensitive to low concentrations of the mediator than is secretion and METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[15]
BIOASSAY OF P A F : RELEASE OF [3H]SEROTONIN
131
100
80
.=_ t-
~D 60 "1-
u~
40 n"
"E Q..
20
0
. . . . . . . .
.I
I
I
. . . . . . . .
I
. . . . . . . .
10
i
.
.
.
.
100
',
. ,/=al~
1000
PAF [nM] FIG. 1. Dose response of [3H] serotonin release from platelets stimulated with increasing concentrations of hexadecyl-PAF. Mean of five standard curves _+ SEM.
does not require separation of the platelets from the platelet-rich plasma, but does need specialized equipment (an aggregometer), can only be performed one or two assays at a time, and is less easily quantitated. Release of serotonin, on the other hand, is a simple assay, can be performed on multiple samples at once, and is linear over a wide range of PAF concentrations (see Fig. 1).
Reagents and Materials Acid-citrate-dextrose (13.65 g citric acid, 25 g sodium citrate, 20 g dextrose in 1000 ml H20) 1 Tyrode's buffer containing 0.2555 w/v gelatin, but lacking calcium 2 Tyrode's buffer containing gelatin with calcium i R. H. Aster and J. H. Jandl, J. Clin. Invest. 43, 843 (1964). 2 N. G. Ardlie, M. A. Packham, and J. F. Mustard, Br. J. Haematol. 19, 7 (1970).
132
ASSAYS
[15]
[3H]Serotonin binoxalate diluted to 1/zCi//.tl (29.7 Ci/mmol, New England Nuclear, Boston, MA) PAF standards (hexadecyl-PAF) EGTA, 0.1 M pH 7.2 9.25% Formaldehyde 10% Triton X-100 Scintiverse II Liquid scintillation cocktail
Equipment Tabletop centrifuge Liquid scintillation counter 37° water bath
Procedure (After Lynch et al. 3) I. Collection and Labeling of Rabbit Platelets. Blood is collected free-flowing from the central ear artery of New Zealand White rabbits into a 1 : 6 volume (final) of acid-citrate-dextrose using a 19-gauge needle. It is centrifuged at room temperature at 300 g for 20 min to obtain platelet-rich plasma (PRP) which then is aspirated off using a transfer pipette. The PRP is incubated at 37° for 45 min with 1/zCi[3H]serotonin binoxalate/ml. The platelets are pelleted by centrifugation at 1200 g for 20 min and washed. 2. Washing of Platelets. 2 Platelets are washed in Tyrode's gelatin buffer without calcium in the presence of 0.1 mM EGTA and pelleted at 1200 g for 20 min at room temperature. Platelets are washed once with Tyrode's gelatin buffer without calcium and no EGTA and again pelleted at 1200 g for 20 min. The platelet pellet is gently resuspended in Tyrode's gelatin buffer without calcium (3 ml for every 60 ml blood originally collected), and diluted to a concentration of 1.25 x 109 platelets/ml as determined from a hemocytometer or Coulter counter or a standard curve of absorbance of platelet suspensions in the spectrophotometer previously derived from known platelet concentrations. 3. [3H]Serotonin Release Reaction. Polystyrene reaction tubes (75 x 12 mm Sarstedt tubes) containing replicate samples or PAF standards in a 0.45-ml volume of Tyrode's gelatin buffer with calcium, each receive a 0.05-ml aliquot (approximately 6.25 x 108) of the labeled platelets. After 90 sec of incubation at room temperature, the reaction is stopped by the addition of 0.02 ml of 9.25% formaldehyde. (Glutaraldehyde could also be used and acts more rapidly. Alternatively, if the tubes are cooled rapidly the cells can be centrifuged without the fixation step). The platelets are pelleted by centrifugation at 2500 g for 15 min at 4 °. 3 J. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979).
[15]
BIOASSAYor PAF: RELEASEOF [3H]sEROTONIN
133
4. Quantitation. Aliquots of the supernatants (0.1 ml) are pipetted into 4-ml polypropylene scintillation vials. Three milliliters of Scintiverse II scintillation cocktail is added and the samples counted in a scintillation counter. The supernatant from the platelets in one set of reaction tubes containing only buffer are used to indicate the spontaneous release. Platelets lysed with 0.01 ml 10% Triton X-100 give the total [3H]serotonin available. The release of serotonin is reported as a percentage of the total available. The amount of PAF present in the unknown samples is determined by comparing the percentage of serotonin released to that released by standard amounts of synthetic PAF. A standard curve of synthetic PAF is run with every assay. The results can then be expressed as nanogram equivalents of synthetic PAF. Problems
This is a straightforward assay when the only platelet stimulant likely to be present in the sample is PAF. Thus it is particularly useful for examining PAF production from cells in serum-free culture. However, this is seldom the case and therefore appropriate controls and/or separatory procedures have to be employed. The assay is even less satisfactory when the PAF is present in complex biological fluids such as plasma or bronchoalveolar lavage fluid that may contain materials that themselves stimulate platelets, that interfere with the platelet stimulation, or that destroy the PAF. To confirm that the material being assayed is PAF it should be sensitive to destruction by phospholipase A2. Much more effective, however, and essential for study of PAF in biological fluids or extracts of whole cells is some degree of purification before assay. Methods for isolating PAF are described by Hanahan in this volume [18]. Lipid extraction, TLC, and/or HPLC separations before assay ensure the accuracy of the results. In such cases, however, standards must be included for assessment of recovery through the separation steps. The degree of purification to employ depends on the nature of the material in which the PAF is being assayed. For extracts of cells, TLC is generally satisfactory. Since PAF is generally found in significant amounts within cells after stimulation (see Ref. 4), it is recommended that the cells be extracted and their PAF content also assayed. For blood or plasma, complete isolation through HPLC is recommended and even then the results may not match those obtained by mass spectrometric analysis (see [16] and [17] in this volume). 4 D. L. Bratton and P. M. Henson, in "Platelet Activating Factor and Human Disease" (P. Barnes, C. Page, and P. Henson, eds.). Blackwell, Oxford, 1989.
134
ASSAYS
[16]
Despite these caveats, the platelet serotonin assay for PAF is quick, simple, inexpensive, and has proved very useful in numerous studies of PAF production and its modulation. Acknowledgments This work was carried out in the F. L. Bryant Jr. Research Laboratory and was supported by National Institutes of Health Grant HL 34303.
[16] Q u a n t i t a t i o n o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Mass
Spectrometry
B y K E I T H L . CLAY
Introduction Quantitative analysis of platelet-activating factor (PAF, 1-O-alkyl-2-Oacetylglycero-3-phosphocholine) is complicated by the chemical nature of the molecule and by the necessity to measure it at very low concentrations (picomolar or less) in extremely complex biological mixtures. Measurement of PAF has frequently been accomplished by the use of its biological activities, through which this molecule was initially described. 1 Measurement ofplatelet aggregation 2 or of release ofplatelet serotonin content s has been used to estimate the amount of PAF present in biological mixtures. These procedures can give very valuable information and one should not underestimate the importance of biological assays in working with a substance that was initially described as a biological activity. Since the elucidation of the exact chemical nature of PAF, however, it has become possible to develop specific physical techniques for its measurement. 4 Gas chromatography-mass spectrometry has been used as the primary method of measuring PAF. Procedures have been described for the measurement of this molecule in both the positive-ion electron-impact ionization mode 5 and in the negative-ion chemical ionization mode. 6 These procedures have the advantage over bioassay procedures of better I j. Benveniste, P. M. Henson, and C. G. Cochrane, J. Exp. Med. 136, 1356 (1972). 2 G. Camussi, M. Aglietta, F. Malavasi, C. Tetta, W. Piacibeilo, F. Sanavio, and F. Bussolino, J. Immunol. 131, 2397 (1983). 3 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979). 4 R. C. Murphy and K. L. Clay, Am. Rev. Respir. Dis. 136, 207 (1987). 5 K. Satouchi, M. Oda, K. Yasunaga, and L. Saito, J. Biochem. 94, 2067 (1983). 6 C. S. Ramesha, and W. C. Pickett, Biomed. Environ. Mass Spectrom. 13, 107 (1986).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
ASSAYS
[15]
release of labeled serotonin. This latter technique uses expensive radioactive compounds which must be appropriately disposed of, necessitates a preincorporation step of the label, and, most importantly, does not yield immediate data. Off-range concentrations of paf cannot be adjusted in real time as is possible with aggregation techniques where results are immediately available, thus delaying the acquisition of valid results by several days. The aggregation method was and still is the basis for most of the studies conducted so far on this mediator, notably on the structure, the origin, the metabolism, and the physiological properties of paf. In addition, most of the paf antagonists have been developed using this test either as a screening procedure or for more complex pharmacological studies.
[15] B i o a s s a y o f P l a t e l e t - A c t i v a t i n g F a c t o r b y R e l e a s e of [3H]Serotonin
By PETER M. HENSON Introduction Platelet-activating factor (PAF) is a highly potent cell communication mediator thought to be involved in a wide variety of pathophysiological processes. Three major approaches have been taken in assaying for the molecule: bioassays, including platelet aggregation, platelet secretion, and neutrophil activation; synthetic approaches, including incorporation of [3HI acetate into the molecule; and physicochemical procedures, including mass spectrometry. Each has advantages and disadvantages. The bioassays are relatively simple, can be applied to a large number of samples, often require little workup, are highly sensitive, and by the use of appropriate standards, give reasonably quantitative results. They can be carried out with minimal equipment and are inexpensive. Their disadvantage, in general, is the potential lack of specificity (necessitating appropriate controls and/or purification), since many agents can activate platelets or neutrophils, the lack of quantitative precision that would be found in physical approaches, and the inability to obtain structural data from the assay. Rabbit platelets are particularly sensitive to stimulation by PAF and are often used as indicator cells. Thus the assay is described for these cells, although it could be adapted to ceils from humans. Platelet aggregation is more sensitive to low concentrations of the mediator than is secretion and METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[15]
BIOASSAY OF P A F : RELEASE OF [3H]SEROTONIN
131
100
80
.=_ t-
~D 60 "1-
u~
40 n"
"E Q..
20
0
. . . . . . . .
.I
I
I
. . . . . . . .
I
. . . . . . . .
10
i
.
.
.
.
100
',
. ,/=al~
1000
PAF [nM] FIG. 1. Dose response of [3H] serotonin release from platelets stimulated with increasing concentrations of hexadecyl-PAF. Mean of five standard curves _+ SEM.
does not require separation of the platelets from the platelet-rich plasma, but does need specialized equipment (an aggregometer), can only be performed one or two assays at a time, and is less easily quantitated. Release of serotonin, on the other hand, is a simple assay, can be performed on multiple samples at once, and is linear over a wide range of PAF concentrations (see Fig. 1).
Reagents and Materials Acid-citrate-dextrose (13.65 g citric acid, 25 g sodium citrate, 20 g dextrose in 1000 ml H20) 1 Tyrode's buffer containing 0.2555 w/v gelatin, but lacking calcium 2 Tyrode's buffer containing gelatin with calcium i R. H. Aster and J. H. Jandl, J. Clin. Invest. 43, 843 (1964). 2 N. G. Ardlie, M. A. Packham, and J. F. Mustard, Br. J. Haematol. 19, 7 (1970).
132
ASSAYS
[15]
[3H]Serotonin binoxalate diluted to 1/zCi//.tl (29.7 Ci/mmol, New England Nuclear, Boston, MA) PAF standards (hexadecyl-PAF) EGTA, 0.1 M pH 7.2 9.25% Formaldehyde 10% Triton X-100 Scintiverse II Liquid scintillation cocktail
Equipment Tabletop centrifuge Liquid scintillation counter 37° water bath
Procedure (After Lynch et al. 3) I. Collection and Labeling of Rabbit Platelets. Blood is collected free-flowing from the central ear artery of New Zealand White rabbits into a 1 : 6 volume (final) of acid-citrate-dextrose using a 19-gauge needle. It is centrifuged at room temperature at 300 g for 20 min to obtain platelet-rich plasma (PRP) which then is aspirated off using a transfer pipette. The PRP is incubated at 37° for 45 min with 1/zCi[3H]serotonin binoxalate/ml. The platelets are pelleted by centrifugation at 1200 g for 20 min and washed. 2. Washing of Platelets. 2 Platelets are washed in Tyrode's gelatin buffer without calcium in the presence of 0.1 mM EGTA and pelleted at 1200 g for 20 min at room temperature. Platelets are washed once with Tyrode's gelatin buffer without calcium and no EGTA and again pelleted at 1200 g for 20 min. The platelet pellet is gently resuspended in Tyrode's gelatin buffer without calcium (3 ml for every 60 ml blood originally collected), and diluted to a concentration of 1.25 x 109 platelets/ml as determined from a hemocytometer or Coulter counter or a standard curve of absorbance of platelet suspensions in the spectrophotometer previously derived from known platelet concentrations. 3. [3H]Serotonin Release Reaction. Polystyrene reaction tubes (75 x 12 mm Sarstedt tubes) containing replicate samples or PAF standards in a 0.45-ml volume of Tyrode's gelatin buffer with calcium, each receive a 0.05-ml aliquot (approximately 6.25 x 108) of the labeled platelets. After 90 sec of incubation at room temperature, the reaction is stopped by the addition of 0.02 ml of 9.25% formaldehyde. (Glutaraldehyde could also be used and acts more rapidly. Alternatively, if the tubes are cooled rapidly the cells can be centrifuged without the fixation step). The platelets are pelleted by centrifugation at 2500 g for 15 min at 4 °. 3 J. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979).
[15]
BIOASSAYor PAF: RELEASEOF [3H]sEROTONIN
133
4. Quantitation. Aliquots of the supernatants (0.1 ml) are pipetted into 4-ml polypropylene scintillation vials. Three milliliters of Scintiverse II scintillation cocktail is added and the samples counted in a scintillation counter. The supernatant from the platelets in one set of reaction tubes containing only buffer are used to indicate the spontaneous release. Platelets lysed with 0.01 ml 10% Triton X-100 give the total [3H]serotonin available. The release of serotonin is reported as a percentage of the total available. The amount of PAF present in the unknown samples is determined by comparing the percentage of serotonin released to that released by standard amounts of synthetic PAF. A standard curve of synthetic PAF is run with every assay. The results can then be expressed as nanogram equivalents of synthetic PAF. Problems
This is a straightforward assay when the only platelet stimulant likely to be present in the sample is PAF. Thus it is particularly useful for examining PAF production from cells in serum-free culture. However, this is seldom the case and therefore appropriate controls and/or separatory procedures have to be employed. The assay is even less satisfactory when the PAF is present in complex biological fluids such as plasma or bronchoalveolar lavage fluid that may contain materials that themselves stimulate platelets, that interfere with the platelet stimulation, or that destroy the PAF. To confirm that the material being assayed is PAF it should be sensitive to destruction by phospholipase A2. Much more effective, however, and essential for study of PAF in biological fluids or extracts of whole cells is some degree of purification before assay. Methods for isolating PAF are described by Hanahan in this volume [18]. Lipid extraction, TLC, and/or HPLC separations before assay ensure the accuracy of the results. In such cases, however, standards must be included for assessment of recovery through the separation steps. The degree of purification to employ depends on the nature of the material in which the PAF is being assayed. For extracts of cells, TLC is generally satisfactory. Since PAF is generally found in significant amounts within cells after stimulation (see Ref. 4), it is recommended that the cells be extracted and their PAF content also assayed. For blood or plasma, complete isolation through HPLC is recommended and even then the results may not match those obtained by mass spectrometric analysis (see [16] and [17] in this volume). 4 D. L. Bratton and P. M. Henson, in "Platelet Activating Factor and Human Disease" (P. Barnes, C. Page, and P. Henson, eds.). Blackwell, Oxford, 1989.
134
ASSAYS
[16]
Despite these caveats, the platelet serotonin assay for PAF is quick, simple, inexpensive, and has proved very useful in numerous studies of PAF production and its modulation. Acknowledgments This work was carried out in the F. L. Bryant Jr. Research Laboratory and was supported by National Institutes of Health Grant HL 34303.
[16] Q u a n t i t a t i o n o f P l a t e l e t - A c t i v a t i n g F a c t o r b y Gas Chromatography-Mass
Spectrometry
B y K E I T H L . CLAY
Introduction Quantitative analysis of platelet-activating factor (PAF, 1-O-alkyl-2-Oacetylglycero-3-phosphocholine) is complicated by the chemical nature of the molecule and by the necessity to measure it at very low concentrations (picomolar or less) in extremely complex biological mixtures. Measurement of PAF has frequently been accomplished by the use of its biological activities, through which this molecule was initially described. 1 Measurement ofplatelet aggregation 2 or of release ofplatelet serotonin content s has been used to estimate the amount of PAF present in biological mixtures. These procedures can give very valuable information and one should not underestimate the importance of biological assays in working with a substance that was initially described as a biological activity. Since the elucidation of the exact chemical nature of PAF, however, it has become possible to develop specific physical techniques for its measurement. 4 Gas chromatography-mass spectrometry has been used as the primary method of measuring PAF. Procedures have been described for the measurement of this molecule in both the positive-ion electron-impact ionization mode 5 and in the negative-ion chemical ionization mode. 6 These procedures have the advantage over bioassay procedures of better I j. Benveniste, P. M. Henson, and C. G. Cochrane, J. Exp. Med. 136, 1356 (1972). 2 G. Camussi, M. Aglietta, F. Malavasi, C. Tetta, W. Piacibeilo, F. Sanavio, and F. Bussolino, J. Immunol. 131, 2397 (1983). 3 j. M. Lynch, G. Z. Lotner, S. J. Betz, and P. M. Henson, J. Immunol. 123, 1219 (1979). 4 R. C. Murphy and K. L. Clay, Am. Rev. Respir. Dis. 136, 207 (1987). 5 K. Satouchi, M. Oda, K. Yasunaga, and L. Saito, J. Biochem. 94, 2067 (1983). 6 C. S. Ramesha, and W. C. Pickett, Biomed. Environ. Mass Spectrom. 13, 107 (1986).
METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
