Anal. Chem. 1907, 59, 722-726
722
Application of Thermospray High-Performance Liquid ChromatographylMass Spectrometry for the Determination of Phospholipids and Related Compounds Hee-Yong Kim* and Norman Salem, Jr. Section of Analytical Chemistry, Laboratory of Clinical Studies, DICBR, National Institute on Alcohol Abuse and Alcoholism, Building 10, Room 3C-218, 9000 Rockville Pike, Bethesda, Maryland 20892
The filamenton thermospray LWMS technlque was employed to develop general determlnatlon methods for molecular species of several of the major lipld classes. Detailed structural lnformatlon could be obtalned from the thermospray spectra which contained dlglyceride, monoglycerlde, and molecular ions as well as head group Ions whlch were distlnctlve for each llpld class. New chromatographlc systems which permit rapid separation of molecular specles were also developed for phosphatldyllnosltols, phosphatldylserlnes, sphlngomyellns, trlglycerMes and platelet-activating factor. With thls technlque, complex mlxtures of biologlcal llpM Samples could be separated Into molecular specles and klentlfled within 3 mln. The total sample amount required for these anatyses was usually In the low microgram range.
One of the principal components of biological tissues is phospholipids of which there are several classes and many molecular species within each class (1,2). The physical and chemical properties of cell membranes are critically dependent upon both head group and the fatty acyl composition of phospholipids (3), and therefore the determination of phospholipid molecular species in biological systems is of great importance. However, the lack of quick and efficient analytical methods for these compounds has discouraged characterization of biological systems a t this level. Conventional techniques including thin-layer ( 4 ) and gas chromatography (5) often involve multistep sample preparation procedures which are laborious and time-consuming. Although high-performance liquid chromatography (6) coupled to UV detection has gained growing popularity in rapid determinations of phospholipids, severe limitations are imposed on mobile phase selection due t o the lack of chromophores. The thermospray technique provides a convenient and efficient method of directly analyzing LC effluents without derivatization (7). We have previously described methods for phosphatidylcholine and phosphatidylethanolamine molecular species separation and analysis using this technique in conjuction with reversed-phase HPLC (8). It was apparent from this work that thermospray mass spectrometry offers significant advantages in the analysis of complex lipid mixtures since detailed structural information is obtained with low background and with short analysis times. We now report the generalization of these methods to the other major phospholipid classes including phosphatidylserine, phosphatidylinositol, and sphingomyelin and, in addition, to triglycerides and platelet-activating factor (PAF). New chromatographic systems are presented here for the separation of molecular species within each of these lipid classes. EXPERIMENTAL SECTION Synthetic triglycerides from Supelco (Bellefonte, Pa) and synthetic and naturally occurring phospholipids from Avanti Polar
Lipids, Inc. (Birmingham, AL), were used without further purification. All the samples were dissolved in mobile phase and injected into an Altex 210A injector with a 20-pL sample loop. Chromatography was performed with either an Altex Ultrasphere-ODS (3 wm, 4.6 mm X 7.5 cm) or a Du Pont Zorbax C-18 column (5 pm, 4.6 mm X 25 cm) and Beckman (Model 114M) HPLC pumps. The mobile phase which consists of either 0.1 M ammonium acetate/water or methanol-2-propanol-hexane-0.1 M ammonium acetate/water was employed for separation of molecular species of various phospholipid classes. The HPLC eluent was introduced into an Extrel400-2 quadrupole mass spectrometer via a Vestec thermospray interface (Vestec,Houston, Tx) as described previously (8). Vaporization and ionization were achieved by heating the capillary vaporizer and also by applying an electron-emitting filament current (0.2 A). Without this auxiliary ionizing source, sufficient ionization did not occur since a mobile phase extremely high in organic content was required to dissolve and elute the phospholipids. Excess solvent was pumped away through a vacuum line via a dry ice/isopropyl alcohol cold trap. The vaporizer tip temperature was maintained at 145-148 “C and the source temperature at 300 “C. All the data were acquired with an Extrel EL-1000 data system. For fatty acid analysis, a Hewlett-Packard 5880 capillary GC system was used after transmethylating phospholipids with boron trifluoride in methanol (9). Fatty acid methyl esters were separated with an OV-351 fused silica capillary column (0.32 mm i.d. X 30 m, Foxboro/Analabs, North Haven, CT) with helium as carrier gas at a linear velocity of 20 cm/s. The oven temperature was programmed from 200 to 230 “ C in 30 min and the injector and detector temperatures were both 230 “C. RESULTS AND DISCUSSION Positive ion thermospray spectra of the lipid classes studied here showed fragmentation patterns similar to those of phosphatidylcholines and phosphatidylethanolamines (8) as they produced diglyceride, monoglyceride, head group, and molecular ion species. The notation of “di-” and “monoglyceride” used in this paper indicates the fragment ions resulting from the loss of head group and from the hydrolysis of a fatty acyl chain from the diglyceride ions, respectively. Our results concerning phosphatidylinositol, phosphatidylserine, sphingomyelin, triglycerides, and platelet-activating factor are presented below. Triglycerides. The spectrum in Figure 1 was obtained from an injection of 5 pg of 16:0, 181,18:O-triglyceride (TG) without a column. The carrier solvent was a methano1:hexane:0.1 M ammonium acetate in water (500:3025) mixture and the flow rate was 1 mL/min. The three possible diglyceride (DG) ions were observed a t m/z 578 (16:0,18:1), m / z 580 (16:0,180),and m/z 606 (180,181). Monoglyceride ions (MG) containing 16:0, 18:1, or 18:O fatty acid were also detected a t m / z 313, 339, or 341, respectively. The molecular ion species shown in this spectrum is the ammonium adduct ion which is usually observed for polyhydroxylated compounds when ammoniated buffers are used in the mobile phase. The addition of the ammonium acetate to this mobile phase led to an increase in sensitivity.
This article not subject to U S . Copyright. Published 1987 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987 M/Z
a
723
REL INS 1
604 605
18:0,18.2
578 579
3.9
(5.3 I 3 7 (33)
160.18 I
REL. INT.
16:0,18~1,18~0 TRIGLYCERIDE (MW =860.6) I
I
in
150
190
--
311
I
I
I
210
230
250
I 270
602 603
I
290
310
3 4 ( 2 31
*
576 577
I
1
I 8 1,182
160.I8:P
100 0 (100 01
330 600 60I
4 5
182,18 2
(2 8 )
i i 574 575
C
I
I
550
530
YL
570
I
590
610
630
I
650
I
670
I
690
I
I6 0,I8 3
I
0
I
4 TIME (MINI
710
879 M NH;
1
I
720
I
I
I
I
760
740
780
800
I
840
820
I
860
1,. 880
900
M/Z
Figure 1. Thermospray mass spectrum of synthetic 16:0,18: 1,18:0triglyceride (TG) obtained by injecting 5 pg without a column. Diglyceride ions (DG)and monoglyceride fragments (MG) resulting from hydrolysis of a fatty acyl chain from diglyceride ions were the major components of the spectrum along with the ammoniated molecule. The m l z values were not corrected for the mass defect which becomes significant above 550 Da. M/Z
552
g:
553
I 1
REL INT.
27'0
18'0 TO
16 16 0 TO
:i
A
I
58.4
58'4
ZOO
300
400
500
600
700
800
900
1000
M/Z 100.0
t.-"---.c--"
234.6
TIC
& -
100
x1
22
24
26
28
30
32
Figure 3. (a) Separation and detection of molecular species of molecular species of plant phosphatidylinosltol (PI) using reversed-phase chromatography and thermospray mass spectrometry. The reconstructed ion chromatograms of diglyceride ions are presented along with the total ion current (105-1005 De). The relative intensity is shown based on both peak height and the area (in parentheses). (b) Thermospray mass spectrum obtained from the chromatographic peak assigned as 16:0, 18:2-PI in Figure 3a.
TIME (MINI
Figure 2. Separation of three synthetic triglyceride standards (10Fg each). The ion chromatograms of diglyceride ions were reconstructed from data acqulred by full mass scanning from 140 to 1040 Da. The relative intensity is shown based on peak height. The intensities of the major '% and 13Cisotope peaks for each fragment were summed and plotted as a chromatogram since it resulted in better signal-to-noise ratlos than that from the 12C isotope alone.
Separation of triglycerides in this study was achieved by using nonaqueous reversed-phase chromatography since a mobile phase containing ammonium acetate led to very long retention times on the C-18 column. As shown in Figure 2, three synthetic triglyceride standards (14:O-, 16:O-, and 180TG) were well-separated on a Du Pont Zorbax C-18 column (5 Km, 4.6 mm X 25 cm). A linear gradient of 90/10 (methanol/2-propanokhexane 125:25 ratio) to 30170 in 25 min was employed with a flow rate of 1 mL/min. Under these conditions, the protonated molecules were present rather than the ammonium adduct ions. Since the diglyceride ions were the base peaks in the spectra, the appropriate diglyceride ion for each species is presented along with the total ion current. Phosphatidylinositol. Phosphatidylinositols (PI) gave rather intense monoglyceride fragment ions compared to those
of phosphatidylcholines (PC) or phosphatidylethanolamines (PE). In general, the relative intensity of mono- and diglyceride ions seemed to be dependent upon the vaporizer tip temperature, the distance between the tip and the sampling aperture, source temperature, and the inner diameter of the capillary vaporizer. However, under a given set of conditions, responses were reproducible. Figure 3 illustrates the results obtained after injection of 20 gg of plant phosphatidylinositol through a reversed-phase chromatographic column (Altex Ultrasphere-ODS, 3 pm, 4.6 mm X 7.5 cm). The mobile phase was a methanokhexane:O.l M ammonium acetate in water (500:25:25) mixture and the flow rate was 1 mL/min. As shown in Figure 3a, at least six molecular species were separated in 3 min according to differences in their chain length and degree of unsaturation. The assignment of each species was based upon spectral analysis of chromatographic peaks and an example is shown in Figure 3b for the 16:0,182 species of plant PI. The principal fragments observed in this spectrum are the diglyceride ion at m / z 576 and monoglyceride ions containing either 16:O or 1 8 2 fatty acid at mlz 313 or 337. The peak at m / z 198 corresponds to an ammoniated inositol fragment and was therefore characteristic of the PI
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
724
Table I. Fatty Acid Distribution of Phosphatidylinositol from Plant and Bovine Brain" fatty acid
plant
bovine brain
16:O 18:O 18:lw9 18:2w6 18:3w3 20:3w6 204w6 22.4~6 22.5~6
35.1 8.1 5.2 44.9 5.1
3.1 44.8 8.0 6.5 0.3 10.0 16.4 1.8 1.8
REL. INT.
1
IO0
578 Id>O,I8 I DO 18'0 YG
18,1 YO 339
16:O.IS:l
- PS (MW=761.61
50
The results were obtained by fused silica capillary GC analysis after transmethylating plant- and bovine brain-PI. The assignment of 22:4w6 was based upon relative retention times (RRT)and the thermospray data. I?EL.INT
I
100
MeOH:HEXANE:O.IM NH4 OAc
200
300
400
500
600
700
800
900
1000
M/Z
= 500:25:25
Figure 5. Positive ion spectrum obtained from direct injection of synthetic 16:0,18:1-phosphatidylserine. MI2
REL INT
652 653
47 8
604
17 8
6%
0
I
2
I
I
3
4
TIME(MIN1
Fi~ure4. Total ion chromatogram (mass range 105-1005 Da) obtained for 20 pg of phosphatidylinositol from bovine brain using the same chromatographic conditions as in Figure 3a.
class. A small peak at m / z 243 which arose from the loss of water from the protonated phosphoinositol was sometimes detected for PI. In this particular spectrum, the molecular ion species were not observed; however, the identities of the chromatographic peaks were easily confirmed by the head group, diglyceride, and monoglyceride fragments. Plotting diglyceride ions as in Figure 3a enabled the deconvolution of partially resolved or unresolved chromatographic peaks, thus allowing unambiguous assignment of each species. The relative intensity of the various reconstructed ion chromatograms for the diglyceride fragments show that the 16:0,18:2 species is the principal component of plant PI. The distribution of each species obtained here was in good agreement with the results obtained from the GC analysis presented in Table I. This indicates that quantification of each molecular species based upon the diglyceride ion intensity is possible although further methods-development is necessary in order to achieve greater precision. This approach is particularly advantageous since it enables quantification even when separation is incomplete. Another example is shown in Figure 4 for bovine brain phosphatidylinositol. The separation and detection of molecular species were achieved under the same conditions as described for Figure 3a. The total ion current (105-1005 Da) is presented in this chromatogram and seven molecular species were identified by spectral analysis. The distribution of fatty acids in this sample obtained by GC is shown in Table I for comparison with the thermospray data. Phosphatidylserine. Phosphatidybrines (PS)produced intense monoglyceride ions as did PI, although the diglyceride
606 607
100 0
658 659
70
I2 I
634
E35 73
660 66 I
222 6
TIC
I7
37
57
77
TIME(MIN)
Figure 6. Separation of molecular species of bovine brain-PS (50 pg). Chromatograms of diglyceride ions were extracted from data acquired by full mass range scanning (104-1004 Da). ion was still the base peak in most of the spectra. Figure 5 presents the thermospray mass spectrum of synthetic 16:O-, 181-phosphatidylserineobtained from a 2-pg injection without a column. The carrier solvent was a methanol:hexane:O.l M ammonium acetate in water (5W3025) mixture. Diglyceride ions (mlz 578) formed the base peak and the presence of monoglyceride ions at m / z 313 and 339 confirmed the fatty acyl assignment of a 160 and 18:l species. Fragments at m/z 105 and 114 were characteristic for the PS class and both are believed to be derived from the head group (e.g., m / z 105 is due to serine). The intact molecule was detected as the sodium adduct ion in this spectrum since the sample was provided as the disodium salt. In conjunction with reversed-phase chromatography, fast separation and detection of PS molecular species were achieved for biological samples. An example is shown for bovine brain PS in Figure 6. Six molecular species were separated on an Altex Ultrasphere-ODS column (5 pm, 4.6 mm x 7.5 cm) using a methano1:hexane:O.l M ammonium acetate in water (500:3025) mixture as the mobile phase at a flow rate of 1 mL/min. This preparation contained the 180,181 and 180,226 species as the major components. The assignments of the molecular species of bovine brain PS
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
633
24aO
634 A
63 I
632 605 606 577 578
06
5 0
22 0
34
22
20 0
548 549
loo 0
1830
h
TIC
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JL 0
2
4
8 10 TIME(MIN)
6
i
A
I
I
I
I
12
14
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,n ,c=c,
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bH
c
itl
b-
(CHd3
R-C=O
B
REL.INT
IO0
50
200
300
400
500
600
700
SO0
Table 11. Minor Sphingomyelin Components Identified in a Bovine Brain Preparation
.
24 I
900
1000
M/Z
Figure 7. (a) Separation of molecular species of bovine brain sphingomyelin (20 pg). Ceramide ions of five major species are displayed in the ion chromatogram along with the total ion current (130-930 Da). (b) Thermospray mass spectrum of the coeluting chromatographic peaks assigned as 22:O- and 24:l-sphingomyelin in Figure 7a. Peaks denoted by A, B, or C on the spectrum represent the ions from the phospho-head group, ceramide, or fatty acyl amide ions, respectively.
presented here are consistent with previous results based on argentation-thin-layer chromatography (10, 11). Sphingomyelin. A fast and convenient method to separate and identify the molecular species of sphingolipids was similarly developed by using reversed-phase chromatography and thermospray mass spectrometry. The chromatogram in Figure 7a was obtained by injecting 20 pug of bovine brain sphingomyelin (SM) through a C-18 column (Altex Ultrasphere-ODS, 3 pm, 4.6 mm X 7.5 cm) using a methanol:2-propanol:hexane:0.1 M ammonium acetate in water (100:10:3:5) mixture as mobile phase at a flow rate of 1 mL/min. At least nine molecular species were separated and the structures were confirmed by spectral analysis. Ceramide ions were the base peaks in the spectra and therefore reconstructed ion chromatograms for these ions are presented in Figure 7a along with the total ion current. In this chromatogram, only five major species are presented and all of them appeared to be sphingomyelins containing CIs-sphingenine as the sphingoid base. Although most natural sphingomyelins have a CIS-sphingenine backbone, they may also contain an 18:O or 20:1 sphingoid moiety and this complicates the assignment of chromato-
725
fatty acyl group 16:O 180 200 22:o
sphingoid backbone
ceramide ions ( m / z )
retention time, min
181 180 180
520 551 579 607
4.9 7.5
18:O
11.0 15.8
graphic peaks (12). Thermospray mass spectrometric analysis does, however, allow unambiguous confirmation of peak identity. As an example, the thermospray spectra of the coeluting 22:O- and 24:l-SM species in Figure 7a is shown in Figure 7b. The ceramide peaks were observed at m/z 605 and 631 as base peaks for each species and those for the fatty acyl amide moiety (RC(=O)NH,)+ a t m / z 340 and 366 for 220and 24:1-SM, respectively. There is sufficient information in the fatty acyl amide peaks in combination with a consideration of the ceramide fragment in order to determlne the fatty acyl composition as well as the sphingoid backbone structure. For instance, the peak at m / z 631 could represent the ceramide ion of either the 24:l species with the Cissphingenine or the 22:l species with the CzO-sphingenine structure. However, the peak at m / z 366 which corresponds to the 241- amide ion excludes the latter possibility. This peak may therefore be unambiguously assigned as a SM species containing C18-sphingenineand a 241 fatty acid group. Phospholipid head group information was obtained from the peak at m / z 142 which represents the loss of trimethylamine from the ammonium adduct of phosphocholine as was previously observed for phosphatidylcholines (8). The molecular ion species were present in the protonated and/or ammoniated forms. Water loss from the ceramide ions was also observed at m / z 587 and 613 for the 220 and 24:l species, respectively. In addition to the peaks illustrated in Figure 7a, other components were observed at a lower intensity in this particular sample preparation and they are listed in Table 11. Platelet-Activating Factor. Platelet-activating factor (PAF) is a potent mediator of cellular function including antihypertensive activity, platelet aggregation, anaphylaxis, and inflammation (13-15). Its potent biological activity is specific for structures which include the 0-alkyl moiety at the sn-1 position and an acetyl or propionyl ester a t the sn-2 position (16,17). The length of the 1-0-alkyl chain in the sn-1 position can have significant effects on the potency of its biological effects (18). Therefore, methods for separating and analyzing PAF molecular species are of great utility. A fast method for the screening of PAF molecular species was developed by using thermospray HPLC/MS employing a short column (Altex Ultrasphere-ODS, 3 pm, 4.6 mm x 7.5 cm) and a methanol:2-propanol:hexane:O.l M ammonium acetate in water (100:10:2:5) mixture as the mobile phase. Figure 8 shows a chromatogram obtained from 20 pg of a commercial PAF preparation from beef heart. The mass fragmentation pattern of PAF molecules was similar to that of other phospholipids in that the diglyceride fragment was the base peak. The reconstructed ion chromatograms based on these diglyceride fragments showed separation of five molecular species in only 3 min (Figure 8). Among these peaks, we observed those which did not appear to be alkylated PAF's since their major fragment (base peak) and molecular ions were 14 amu higher than expected. These compounds appear to be l-acyl-2-acetylphosphocholines, since they eluted from the C-18 column earlier than the alkylated species of corresponding chain length; thus, they are not PAF's either with a longer chain length or containing a propionyl ester group at the sn-2 position. This was further confirmed by spectral analysis of chromatographic peaks eluted with a
ANALYTICAL CHEMISTRY, VOL. 59, NO. 5, MARCH 1, 1987
726
REL INT
M/Z
369 370
1
I
18 0 ALKYL
383 384
I8 0 ACYL
341 342
100 0
1610 ALKYL
us
16:O ACYL
356 327 328
92 7
1510 ALKYL
0
66
2
I
3
TIME I M I N )
Flgure 8. Separation of the components of a commerical preparation of platelet-activating factor from beef heart. Reconstructed ion chromatograms are presented at the masses indicated along with the total ion current (130-630 Da). The assignment of each species is based on spectral analysis of the chromatographic peaks. C%- 0- ICH2 l,z,CH3
R
6
l
CHjCOCH
;%OB
a
O - C K j C 5 - Nz(CH313
REL INT
o-
B
I
IO0 369
18 :0 - PA F
t 100
150
200
250
300
350
400
I
I
450
500
50
600
M/Z
c F I
LITERATURE CITED
CHf 0,C-CCH&CH3 O I A C H j E-0-CH I
P
CHf O - P O - C k - CH;
N.( CH&
0-
b
B
REL INT
100
183 B
1-18 0-2-ACETYL-PHOSPHOCHOLINE
t
l
100
I50
200
250
300
350 M/Z
400
CONCLUSION The thermospray technique has proven particularly useful for analyzing phosphatidylcholines and phosphatidylethanolamines since it provides fragment ions which carry detailed structural information as well as molecular ion species (8). In this study, we found that similar information can be obtained from the other major classes of phospholipid and also neutral lipids such as triglycerides. This detection system alleviates many of the difficulties associated with traditional HPLC of lipids using UV detectors as it is a universal detector. For this reason, it is a powerful HPLC method development tool as evidenced by the five fast HPLC systems developed in the course of this work. The sensitivity of the technique is compound-dependent; however, the total amount of lipid mixture needed is usually in the low microgram range with full mass scanning and the detection limit of each component by selected ion monitoring is in the range of tens of nanograms. We believe that this study provides a framework for the thermospray LC/MS analysis of most major lipid classes and this should prove invaluable particularly for compositional and metabolic studies of biological systems. ACKNOWLEDGMENT The authors thank Linda Jacobson and Nina Holden for preparation of the manuscript.
I
550
species were well-separated. The spectra obtained from these two chromatographic peaks are shown in Figure 9. Besides the major fragment peaks resulting from the head group loss, protonated molecules and ions from the head group (m/z 142 and 184) were present in both spectra. However, in Figure 9b, additional fragments were observed at m / z 117 and 341. The peak at m/z 117 resulted from hydrolysis of the fatty acyl chain a t the sn-1 position of the diglyceride ion and is characteristic of ester-linked species. The peak at m / z 341 represents the monoglyceride ion containing the 18:O fatty acyl moiety. These ions confirm the identity of this unkown peak as l-acyl-18:0-2-acetylphosphocholine.Since under the conditions used in Figure 8, a 2-propanol dimer produced a large background at m / z 121, responses at m / z 117 could not be monitored.
, 450
1
I
500
50
550
600
Figure 9. Thermospray spectra of 18:O alkyl (a) and 18:O acyl (b) species after the separation of beef heart PAF. Peaks denoted by A, B, and C In the spectra represent fragments derived from phosphocholine, diglyceride, and monoglyceride ions, respectively.
second mobile phase which contained a methanol:hexane:O. 1 M ammonium acetate in water (500:25:25) mixture. With this HPLC system, the separation of 150 alkyl, 16:O acyl, and 160 alkyl species was poor. However, 18:O acyl and 18:O alkyl
(1) Rouser, G.; Nelson, G. J.; Flelscher, S.; Simon, G. I n 6io/ogIca/Membranes, Physical Fact and Function; Chapman, D., Ed.; Academlc: New York, 1968; pp 1-69. (2) Holub, B. J.; Kuksls, A. I n Adv8nCeS in LipM Ressarch; Paoletti, R., Kritchevsky, D., Eds.; Academic: New York, 1978; Vol. 16, pp 1-125. (3) Stubbs, C. D.; Smith, A. D. Biochim. Blophys. Acta 1984, 779, 89-137. (4) Arvldson, G. A. E. J. Lipid Res. 1985, 6, 574-577. (5) Myher, J. J.; Kuksis, A. Can. J. Blochem. 1982, 60, 638-650. (6) Patton, G. M.; Fasulo, J. M.; Robins, S. J. J . Lipid Res. 1982, 2 3 , 190-196. (7) Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750-752. (8) Kim, H. Y.; Salem, N., Jr. Anal. Chem. 1988, 58, 9-14. (9) Morrison, W. R.; Smith, L. M. J. Lipid Res. 1984. 5 , 600-608. (IO) Salem, N., Jr.; Abood, L. G.; Hoss, W. P. Anal. Biochem. 1978, 76, 408-4 15. (11) Salem, N., Jr.; Serpentino, P.; Puskin, J. S.; Abood, L. G. Chem. Phys. Lipids 1980, 2 7 , 289-304. (12) Jungalwala, F. 6.; Hayssen, V.; Pasquini, J. M.; McCluer, R. H. J . Lipid Res. 1979, 20, 579-587. (13) Blank, M. L.; Snyder, F.; Byers, L. W.; Brooks, 6.; Muirhead. E. E. Biochim. Biophys. Res. Commun. 1979, 90, 1194-1200. (14) Roubin, R.; Tence, M.; Mencla-Huerta, J. M.; Arnoux, B.; Nlnio, E.; Benveniste, J. I n Lymphokines: Pick, E., Ed.; Academic: New York, 1983; Vol. 8, pp 249. (15) Vargaflig, B. 8.; Chignard, M.; Benveniste, J.; Lefort, J.; Wal. F. Ann. N.Y. Acad. Sei. 1981, 370, 119-137, (16) Hanahan, D. J.; Demopoulos, C. A,; Liehr, J.; Pinckard, R. N. J. Bioi. Chem. 1980, 255, 5514-5516. (17) Clay, K. L.; Murphy, R. C.; Andres, J. L.; Lynch, J.; Henson. M. Biochim. Biophys. Res. Commun. 1984, 121, 815-825. (18) Satouchl, K.; Pinckard, R. N.; Hanahan, D. J. Arch. Biochem. 610PhyS. 1981, 27 1 , 683-688.
RECEIVED for review June 23, 1986. Accepted November 6, 1986.
