142

ASSAYS

[17]

ethanol. A silicic acid solid-phase extractor cartridge (500 mg silica, Supelco) which has been washed with 5 ml ethanol is then connected directly to the end of the ODS cartridge with an adapter (Analytichem) and the PAF is eluted directly from the ODS cartridge onto the silica with 5 ml ethanol. The cartridges are uncoupled and the silica is washed with an additional 5 ml ethanol. The PAF fraction is then eluted into a polyethylene tube with 4 ml of methanol/water (3 : 1). This solution is then taken to dryness on a centrifugal vacuum dryer (Savant). The phosphate ester is cleaved with hydrofluoric acid (48%, 0.5 ml) which is added to the polyethylene tube containing the dried extract and left at room temperature for 4 hr. At the end of this time the diglycerides produced are extracted with 2 ml hexane. The hexane is washed once with water and then taken to dryness with a gentle stream of nitrogen. The dried extracts are then prepared for G C - M S analysis as the TBDMS derivative by reaction with 50/zl MTBSTFA for 15 rain at 60 ° The TBDMS derivative of PAF gives the mass spectrum illustrated in Fig. 3. This derivative gives an intense M - 5 7 ion (m/z 415 for the hexadecylacetyl diglyceride from PAF) for each of the acetyl-containing diglycerides related to PAF. The combination of GC retention time and ion at M - 5 7 gives good identification of the molecular species of compounds related to PAF. By alternately measuring the M - 5 7 ion and its deuterated analog 3 mass units higher, accurate measurement of the amount of PAF can be made. Figure 4 illustrates measurement of hexadecyl-PAF (m/z = 415 and 418) and the palmitoylacetyl analog (m/z = 429 and 432) produced in response to calcium ionophore stimulus of murine mast cells. The two peaks for PAF and the three peaks observed for the palmitoyl analog are from all of the possible positional isomers of the acyl groups produced by intramolecular isomerization.

[ 17] Q u a n t i t a t i v e A n a l y s i s 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-Negative-Ion Chemical Ionization Mass Spectrometry

By WALTER C. PICKETT and CHAKKODABYLUS. RAMESHA Mass spectrometric analysis of the platelet-activating factor (PAF) has been severely limited due to the inherent involatility of this phospholipid. Although valuable structural information has been gathered with respect to the intact molecule utilizing fast atom bombardment mass METHODS IN ENZYMOLOGY, VOL, 187

Copyright © 1990 by Academic Press, inc. All rights of reproduction in any form reserved.

[17]

ANALYSISOF PAF BY GC-NCI-MS

143

spectrometry ~-3 and to a lesser extent with thermo-spray4 techniques, generally these methods have not been sensitive enough for the analysis of PAF derived from physiological fluids or tissue. Analysis of PAF as neutral derivatives has circumvented many of these problems by not only increasing volatility but also enhancing chromatographic characteristics. One of the most promising of these PAF derivatives is the pentafluorobenzoyl (PFB)-diglyceride. It is conveniently derived from PAF by the phospholipase C (PLC)-catalyzed hydrolysis to the diglyceride which is, in turn, esterified with pentafluarobenzoyl chloride. The PFBdiglyceride provides a highly sensitive and molecular species-selective method of PAF analysis)

Reagents 1-O-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) 1-O-Hexadecyl-2-1yso-sn-glycero-3-phosphocholine (lyso-PAF), primulin (Sigma, St. Louis, MO) 1-O-[3H]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (New England Nuclear, Boston, MA) Perdeuteroacetic acid and trideuteroacetyl chloride (MSD Isotopes, St. Louis, MO) Pentafluorobenzoyl chloride, PFB (Aldrich, Milwaukee, WI) Phospholipse C, PLC (EC 3.1.4.3) from Bacilus cereus (CalBiochem, San Diego, CA) Procedure

Synthesis of Deuterium-Labeled PAF. 5 One milligram of lyso-PAF is dissolved in 100 /xl of perdeuteroacetic acid containing 25 mg of trideuteroacetyl chloride. After 1 hr at room temerature and under nitrogen the reaction is complete. [2H3]AcetyI-PAF is purified by thin-layer chromatography (see below) and stored in methanol. It is stable for over a year at - 2 0 °. As assessed by tracer lyso-PAF, recovery of lyso-PAF as [2H3]acetyl-PAF is greater than 98%. Sample Preparation. The overall isolation and derivatization scheme is summarized in Fig. 1. Specifically, the lipids are extracted by a slight 1 K. Clay, D. Stene, and R. C. Murphy, Biomed, Mass. Spectrom. U , 47 (1984). 2 K. Clay, R. C. Murphy, J. Andres, J. Lynch, and P. Henson, Biochem. Biophys. Res. Commun. 121, 815 (1984). 3 S. T. Weintraub, J. Ludwig, G. Mott, L. McManus, C. Lear, and R. N. Pinckard, Biochem. Biophys. Res, Commun. 129, 868 (1985). 4 H. Y. Kim and N. Salem, Anal. Chem. 59, 722 (1987). 5 C. S. Ramesha and W. C. Pickett, Biomed. Mass. Spectrom. 13, 107 (1986).

144

ASSAYS

[ 17]

Cells, Tissue of Biofluids + HCCI~qVIeOH

~

- ' ~ [2H]PAF

TLC

/',, 1. HCl(g)

]

P C

/

2. 0.1 M NaOl-I~

3. AcOOA¢ | 4. [2H]PAF J

Lyso-PAF ' i. HCl(g) 2. 0.1 M NaOH 3. AcOOAc .4. [2H]PAF

PAF ~

Phosphocholine

OR O

Diglyceride

OH3_II_O.. OH 0

F

F

C~O >F F--~F IC HCI ' /---" I ON GIla ~100"i

F

F

Lo3_

PFB

DG-PFB

Negative Ion Chemical Ionization GCMS Fro. 1. Isolation and derivatization of PAF (lyso-PAF and PC) to the PFB-diglyceride. modification o f the B l i g h - D y e r 6 procedure. To one volume o f biological fluids such as urine, serum or cell suspensions, 3.5 volumes o f methanol: chloroform 5 : 2 (v/v) are immediately added after collection or at specified times after stimulation. Tissue samples such as lung preparations are collected on dry ice, minced, and homogenized with a Polytron (Brinkman) in 5 : 2 (v/v) methanol : chloroform. The presence o f solvent is needed to deactivate degradative e n z y m e s particularly the acetylhydrolases. Acid inhibition of hydrolases is avoided especially in samples con6 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).

[17]

ANALYSISOF PAF BY GC-NCI-MS

145

taining erythrocytes due to poor PAF recoveries. Apparently this is due to the increase in the amount of pigments extracted at low pH. At this point, 0.05-10 ng of [2H3]acetyl-PAF is added in 10/.d of MeOH as an internal standard. The samples are then stored or further processed by the addition of 1.5 and 1 volume additions of chloroform and water, respectively. From the resulting two phase system, the chloroform is removed and residual lipid remaining in the aqueous phase partitioned into an additional 2.5 ml of chloroform. PAF, PC, and lyso-PAF are isolated by TLC utilizing activated (100°, 30 min) silica G plates (500-1000 ~m thick) developed with 65/25/0.5/2.5 (v/v/v/v) chloroform:methanol:acetic acid:water yielding R f values of 0.35, 0.5 and 0.2, respectively. Plate thickness and bandwidth of silica receiving lipid extract are adjusted according to the amount of lipid applied in order to provide clear separation of PC, PAF, and lyso-PAF. Standards and sample PC (generally PAF and lyso-PAF) are identified under UV after lightly spraying with primulin (0.5 mg/ml in ethanol). Indentification of these lipids after exposure of the plate to 12 vapors is strictly avoided due to the unexpectedly efficient depletion of unsaturated molecular species of PAF. The band corresponding to PAF and sometimes those also corresponding to standard PC and lyso-PAF are scraped, extracted 6 and stored (less than 2 days) in chloroform. Enzymatic Hydrolysis to 1-O-Hexadecyl-2-acetylglycerol. 7 Purified PAF samples are placed in silanized 13 × 100 mm screw-cap vials with Teflon seals, the solvent removed under a stream of nitrogen, and suspended in 300/zl of 80 mM borate buffer (pH 8.0) and 1 ml of Et20. After the addition of 50 units of PLC, the samples are vigorously shaken 1 hr at 37° in tubes slanted 45 ° from vertical to increase the interfacial reaction area. The ether phase is retained and residual diglyceride is partitioned into two 1-ml portions of ether which are pooled with the original ether phase. Due to acetyl migration of 1,2-diglycerides to the thermodynamically favored 1,3-diglycerides, the samples are directly prepared for reaction with PFB without storage. Although borate stabilizes 8 the acetyl group during hydrolysis and purification prior to derivatization with PFB is not found necessary, it should be pointed out that silica catalyzes this acetyl migration and should be avoided. A chemical alternative (HF) to the PLC-catalyzed hydrolysis has been described. 9 Synthesis of Pentafluorobenzoyl Derioatives. The ether extracts of l-O-alkyl-2-acetylglycerol are immediately and rigorously dried (nitrogen stream with consecutive washes of ethanol and dry acetonitrile). To each tube is added 100/~1 of PFB, which is then flushed with nitrogen, sealed, 7 K. Satouchi and K. Saito, Biomed. Mass Spectrom. 6, 396 (1979). 8 Kito, H. Takamura, H. Narita, and R. Urade, J. Biochem. 98, 327 (1985). 9 p. Haroldsen, K. Clay, and R. C. Murphy, J. Lipid Res. 28, 42 (1987).

146

ASSAYS

[ 17]

and heated at 120° for 45 ° min. After removal of the excess reagent under a nitrogen stream, the derivative is dissolved in hexane and purified over a short silica-CC4 column (Pasteur pipette). The sample is quantitatively applied to the column with hexane, washed with an additional 4 ml, and eluted with 6 ml 25% diethyl ether in hexane. A 90% overall yield from aqueous sample to PFB derivative is obtained. For whole blood, plasma, or serum, the yield is reduced to 80%. The PFB derivative after evaporation is taken up and stored in tetradecane. Because of the high boiling point of tetradecane, reisolation of the PFB-diglyceride for purpoes of sample concentration requires additional chromatography. Analysis of PAF Precursors and Metabolites. The above methodology is also applicable to the analysis of PC and lyso-PAF molecular species after conversion to PAF. Knowledge of these precursors is of great value in establishing substrate-product relationships necessary to determine specificity of those enzymes responsible for PAF homeostasis. 2 Specifically, PC or lyso-PC is isolated by TLC under the conditions described above, exposed to HCI gas for 5 min to remove plasmalogens, and then partitioned into chloroform. 6 After evaporation of the solvent, the extract (PC or lyso-PAF) is treated with mild base (0.1 M NaOH, 37 ° for 30 min) to remove sn-1 or sn-2 esters to yield 1-O-alkyl-2-1yso-snglycero-3-phosphocholine (lyso-PAF). After extraction, the lyso-PAF is converted to PAF by acetylation, which is complete overnight at 37° after dissolving the sample in 100/zl of acetic anhydride. Internal standard is included and the PC and lyso-PAF (now present as PAF) are derivatized to the PFB-DG exactly as described as above. Mass Spectrometry. A Finnigan Model 4023-T modified with a PPINICI module interfaced with the INCOS data system is used. The mass spectrometry conditions are: Em 1.3 kV, emission current 0.44 mA, conversion dinode +3.1 kV, ionizer and analyzer temperatures 210° and 100% respectively. The mass spectrometer is operated in the negative-ion chemical ionization mode with methane (0.3 tort) as the reagent gas. Spectra are obtained by scans of m/z from I00 to 650. In the selected-ion mode, the molecular anion of l-O-hexadecyl-PAF, m/z 552 (or other molecular species) is quantitated by comparison with m/z 555, the deuterated internal standard with a 0.25-0.5 amu window. Generation of a stable molecular anion with improved chromatographic characteristics is the major requirement for improving the quantitation of PAF. As shown in Fig. 2, the DG-PFB derivative is unusually stable, carrying greater than 92% of the ion current as the molecular anion. Apparently the DG-PFB molecular anion is more stable than analogous PFB adducts because characteristic anions such as [M-PFB]-,pentafluorobenzoylate anion (m/z 211) and pentafluorotolu-

ANALYSISoF PAF BY GC-NCI-MS

[17]

147

SS~M]'"

100311'qP~ H

0

I

il

H2C 14-O - - {CH2)~'~CH3

I

.. , ,/.~-oSO

F

F

c~ ~ / ~ F

"--- - I ~ 493

F

F

(W3 + 1)

(311-1) t:

slo ~oo '

2~o

~o

I

40O '

I

I

~o

o II

2.

"E

I

--C--

rr

"2c~ o--(c"2)i~c"3

I

o

/ X 2 ~~ - - O -

C

C - - O-/-CH

50 ~

(

"---4~493

~:

l:

F

F

(314-1) 100

I

150

(493 + 1)

~ls

i

200

2S0

~

6~0

ssr~M]'"

314"4"-,

o .>

I

soo

- 0I

(4e4)

,

do

.o

s.

m/z FIG. 2. The NCI mass spectra of the pentafluorobenzoyl (PFB) derivative of {a) 1-Ohexadecyl-2-acetylglycerol and (b) l-O-hexadecyl-2-[2H3]acetylglycerol.

ene (m/z 181) are completely absent in the spectrum. Yet minor ions at m/z 311 and 493 provide useful structural information. Also indicated in Fig. 2 is the high degree of isotopic purity of the internal standard, [2H3]acetyl-PAF, necessary for maximal sensitivity. Gas Chromatography. Despite the high molecular weight, the PFB derivatives are volatilized at temperatures compatible with moderately polar capillary columns. For this purpose, we employ DB5 fused silica columns (15 m, 0.25 mm ID; J & W Scientific, Rancho Cordova, CA) placed directly into the ion source. The column is held at an initial temperature of 220° for 1 min and programmed to 280° at 10°/rain. The injection temperature is 290° and the GC-MS transfer lines are held between 285300. Two to four microliters of sample are injected in the splitless mode with helium (9 psi) as the carrier gas. Sensitivity. Under these conditions, a standard curve linear from greater than 10 pg to 100 fg of PAF is generated (Fig. 3). As mentioned

' (180

148

ASSAYS

[ 17]

o w

if:

0

~ 0.8

1.0

1.5

2.0

2.5

3.0

~H-l- O-Hexadecyl-2-acetyl-glycerol-PFB |n]ected (Pg) FIG. 3, Standard curve for 1-O-hexadecyl-2-acetylglycerol-PFB with 1 pg of 1-Ohexadecyl-2-[2H3]acetylglycerol as internal standard.

above, the increase in sensitivity is related to the stability of the molecular anion generated under these "soft" ionization conditions. Compared to other methods of PAF detection, this method is many orders of magnitudes more sensitive. For example, liquid chromatographic methods utilizing chromogenic m and fluorogenic It derivatives have detection limits of approximately 0.5 and 0.05 ng, respectively. Selective ion-monitoring assays of tert-butyldimethylsilyl derivatives of PAF have limits of approximately 0.25 ng 12 while serotonin release bioassays utilizing rabbit platelets have limits of 0.1 to I ng. The least sensitive but structuraly most informative is the FAB assay requiring greater that a nanogram of PAF. ! l0 M. L. Blank, M. Robinson, V. Fitzgerald, and F. Snyder, J. Chromatog. 298, 473 (1984). 11 C. S. Ramesha, W. C. Pickett, and D. Murthy, J. Chromatog. 491, 37 (1989). 12 K. Satouchi, M. Oda, K. Yasunaga, and K. Saito, J. Biochem. 94, 2067 (1983).

[17]

ANALYSISOF PAF BY GC-NCI-MS

149

Interfering Compounds. Acyl analogs of PAF (e.g., l-O-hexadecyl-2acetyl-sn-glycerophosphocholine) have been identified by Oda et al. ~3 in cells stimulated with ionophore. Although biologically inactive, the "1acyl-PAF" species can present an analysis problem. The sn-1 esters and those PAF molecular species with one additional carbon in the ether chain are isobaric. This is particularly problematic in the analysis of PAF derived from the guinea pig polymorphonuclear leukocytes (PMN) due to the high percentage of 1-O-heptadecyl molecular species. One must verify that the relatively large amount of the heptadecyl ether is not merely due to contamination with the hexadecanoyl ester. One of two approaches have been taken to solve this problem: (1) a mild hydrolysis followed by an acetylation step has been incorporated into the isolation scheme immediately following the initial TLC (Fig. 1); (2) mildly polar columns (DB5) have been utilized to resolve the alkyl and acyl species. 14Although hydrolysis completely eliminates the acyl species, its also removes the internal standard and, therefore, requires a second incorporation of [2H3]acetylPAF partially through the isolation scheme. Measurements o f P A F Derived from Biological Sources. The high sensitivity of this assay has not only permitted the measurements of PAF from stimulated cells where PAF is abundant ~4-~5but also from less abundant sources such as unstimulated cells ~4(Table I) or from tissue available only in small amounts such as psoriatic scales ~6 (Table I). As indicated in Table I, PAF can be measured in a variety of inflammatory cells and tissues ranging from 8 (guinea pig) to 32 (rat PMN) ng/107 cells. In all cases, the hexadecyl ether is the most abundant molecular species for PAF and alkyl-PC. The degree of heterogeneity increases in the basal versus the stimulated cells. Molecular Species Analysis. Equaly important to the high sensitivity of t h i s method is the ability to analyze molecular-species of PAF and lysoPAF as well as the sn-1 chain length of alkyl-PC. Molecular species analysis permits identification of substrate-product relationships which reflect the specificity of key enzymes necessary to PAF homeostasis. As indicated in Fig. 4, analysis of selected anions corresponding to the various molecular species of PAF has revealed in the human PMN one major (l-O-hexadecyl, 96%) and a number of minor molecule species ranging in chain length from 14 to 19. The high degree of homogeneity is in agreement with Clay et al. 2 who concluded that highly specific biosynthetic enzymes 13 M. Oda, K. Satouchi, K. Yasunaga, and K. Saito, ,I. l m m u n o l . 134, 1090 (1985). 14 C. S. Ramesha and W. C. Pickett, J. Biol. C h e m . 2,61, 7592 (1986). ~5 C. S. Ramesha and W. C. Pickett. J. I m m u n o l . 138, 1558 (1987). 16 C. S. Ramesha, N. Soter, and W. C. Pickett, A g e n t s Actions 21, 382 (1987).

s.

0 O~

o .


~3

0

oo

m

eq

"2.

m

,,.a a', ,-.a

o "~ .~