The separation of leukotrienes and hydroxyeicosatetraenoic acid metabolites of arachidonic acid by high performance liquid chromatography (HPLC)

PROSTAGLANDINS TIiE SEPARATION OF LEUROTRIENES AIdD HYDROXYEICOSATETRAE.I:OIC ACID METABOLITES OF ARACClDONIC ACID BY HIGH PERFORMANCE LIQUID CIII?Cwk4TOGRAFliY (HPLC)

David

3. Osborne,

Brian .I. Peters

and Christopher

J. Elerde

Lilly Research Centre Limited Erl Wood I,lanor Wind1 rsham Surrey England

ABSTRACT ____. The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metaholites of arachidonic acid: Techsphere 5-Cl8 colurn, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pll 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify liporygenase products released from mouse macrophage cells stimulated with ,~--1l~rPchlorocyclohexane. Detection limits between 1 and 10 np wt‘r-eobtained.

INTRODUClION ____-Many workers have used HPTC to examine, the lipoxygenase products of arachidonic acid (l-8). Column type, eluting solvent and pH Although conditions vary leading to difficulty in comparing data. most lipoxygenase products can he examined on any good quality reverse phase packing, leukotriene's C, D and E usually give poor peak shape and'often‘co-elute with c.thcr products of interest. This study was undertaken to solve these problems and develop an improved HPLC system for anelysis of lipoxygenasc products.

IlATERIALS AND IIETHODS _ ._--__--For the development of an HPLC separation for lipoxygenase products of arachidonic acid, the uecessary standards were obtained as described below. All were purified by HPLC and characterised by UV, NMR and in some. cases GC/MS. LIST OF ABBREVIATIONS (in order of appearance in text) HETCS monohydroxyeicosatetraenoic acids; 5HETI: 5S-6E,8Z,llZ,14Z-eicoeatetrnc:r~~~c acid; LTB leukotriene B 5S,lZR-dihydroxy-6Z,8E,lOE,14Zeicisatetraenoic act:; 5S,l?R-diHETT 5S,12R-dihydrc ,y6E,8E,lOE,14Z--ricosatetracnoir acid: 5S,12S-diHl:T.: SS,lZSdihydroxy-6E,8E,lOC,l4Z-eicoeatetra~:n~~ic acid; 13-hydroxylinoleic acid 13 hydroxy-9Z,llE_octadec:dienoic acid; 4,li-diHNTE (Isomer mixture 1) a mixture of 4S,llR- erd 4R,llSdihydroxy-5S.7E,9E,13Z-nonadec;~tetraenoic acids; 4,11--diHNTE (Isomer mixture 2) a mixture of 4S,liS- and 4R,llR.dihydroxy-5E,7E,9C,13Z-nonadecOtetraenoic acids. On IIPLC the 4S,llR- and 4R,llS- isomers coelute as do the 4S,llS- and 4R,llP isomers.

NOVEMBER

1983 VOL. 26 NO. 5

817

PROSTAGLANDINS

Preparation of 5,8,9,11,12 acids (HETES)

and

15 monohydroxyeicosatetraenoic

These were prepared using the method of Boeynaems et al (9). Arachidonic acid (100 mmol) and cupric chloride (50 mmol) were dissolved in methanol (10 ml). The pH was adjusted to 7 with tris bufter and 3 mm01 of hydrogen peroxide added (50%). After stirring at room temperature for 30 minutes the mixture was reduced with sodium borohydride, acidified to pH 3 and extracted with ethyl acetate. The evaporated extract was dissolved in eluting solvent methanol:water: acetic acid (70:30:0.06 v/v) and purified by reverse phase I!PLC on a Spherisorb S5 ODS-1 column. Preparation

of 5HETE,

LTB,

SS,lZR-diHETE

and 5S,l?S-diHETE-

Arachidonic acid was incubated with rabbit polymorphonuclear leukocytes in the presence of calcium ionophore (10). Methanol quenched incubations were reduced with sodium borohydride, acidified to pH 3 and extracted ujth diethylether. The evaporated extract was dissolved in eluting solvent methanol:water:acetic acid (70:30:0.06) and purified by reverse phase HPLC on a Spherisorb S5 CDS-1 column. Preparation

of Leukotriene

C,

(LTC,)

Racem-ic leukotriene A (LTA4) methyl ester in methanol was reacted with glutathione (4 eq) rn the presence of triethylamine The reaction product was hydrolysed in O.lK aqueous (12 eq). potassium carbonate/methanol (3:l) for 16 hrs at 21'C. The resulting products were dissoived in eluting scivent methanol: water:acetic acid (60:40:0.06) and separated on reverse phase HPLC (11). Preparation

of Leukotriene

0,

(LTD,)

Racemic LTA4 methyi ester (1,) was reacted with N-trifluoroacetyiL-cysteinylglycice methyl ester (7) to produce protected LTD&. The diastereoisomers were separated by HPLC (13) and then carbonate:methanol (i:l) hydrolysed in C.15M aqueous potassium This was dissolved for 18 hrs at 21'C to produce natural LTD . in methanoi:water:acetic acid (70:30:0.06$+ eluting solvent and purified on reverse phase HPLC using a Nucleosil 5 Cl8 column. Preparation

of 13-hydroxylinoieic

acid

1 mg of linoleic acid (Sigma) was dissolved in 10 ml of pI1 9.0 borate buffer. 5,000 units of soya bean lipoxygenase enzyme (Sigma) were added and the total stirred fcr 15 mins at 20°C. After sodium borohydride reduction and acidification to pH 3.0, the reaction was extracted with ethyl acetate. The ethyl acetate was removed in a stream of nitrogen and the residue dissolved in methanol:water:acidic acid (70:30:G.06) elutinp solvent and purified on HPLC using a Spherisorb 5 ODS-1 column.

818

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1983 VOL. 26 NO. 5

PROSTAGLANDINS

Preparation

of 4,11-diHNTES

(Isomer mixtures

1 and 2)

Nor-LTA was prepared by coupling 4,5-(E)-oxido-&oxo-act-6triphenyl (E)eonic acl4 , methyl ester with undec-2Z,5%-dienyl phosphonium bromide in a method analagous to the preparation of LTA4 Nor-LTA methyl ester (1.5 mg) was hydrolysed for 10 minutes in (12). The 2 ml of tetrahy 3 rofuran:water (3:l) containing a trace of acid. resultant 4S,llR+4R,llS and 4S,-llSi-4R,llR-diHNTES methyl esters were separated on HPLC using methanol:water:acetic acid (75:25:0.06 v/v) on a Nucleosil 5 Cl8 column. Each isomer was hydrolysed overnight at 20°C in 0.15EI aqueous potassium carbonate:methanol (3:2 v/v) and further purified by HPLC on a Nucleosil 5 Cl8 column using methanol:water: acetic acid (70:30:0.06 v/v) as eluent to give pure 4,11-dil:NTES (Isomer mixtures 1 and 2). Incubation of mouse peritoneal and 7-hexachlorocyclohexane

macrophage

cells with arachidonic

acid

Resident peritoneal cells (4 x 106/m1) from outbred TO mice were cultured for 3 hrs at 37°C in bicarbonate buffered Eagles MEM supplement@ with 15% heat inactivated foetal caif serum and After 3 hrs non-adherent luCi/ml of [l- Cl-arachidonic acid. cells were discarded and radiolabelled adherent cells were Cells were stimulated as described previously (14,15). incubated for 1 hr at 37°C with 200uM y-hexachlorocyclohexane in heparin containing medium. The samples were centrifuged to remove cell debris and 6 ml aliquots of supernatant medium were subjected to our extraction procedure. Extraction peritoneal

of the lipoxygecase macrophage cells

products

released

from mouse

To each sample was added 500 ng of 13-hydroxy linoleic acid and Sample volunes 4,11-diHHTE (Isoser mixture 1) as internal standards. were made up to 20 ml with distilled water and adjusted to pH 8 with A Waters Cl8 sep-pak column was prepared by washing with O.lM NaOH. 20 ml of ethanol followed by 20 ml of water and the sample applied to The column was the column using a syringe with a luer tip fitting. washed with 10 ml of ethanol:water (10:X v/v) and then 5 ml of methanol. The methanol fraction was blown to dryness and made up in HPLC eluting solvent. HPLC Analysis HPLC was performed using either a Constametric II C pump and Pye LC-UV detector or pi Spectraphysics 8700 solvent delivery system and Perkin Elmer IX85 detector. Productc were monitored Ultraviolet spectra were at 280 nm (triene's) or 237 nm (HL?liS). obtained on HP!.C peaks using the scanning facility cf the LC-85 detector. To do this, solvent flow was stopped as a peak reached its maximum, and the UV scan recorded. Alternatively absorbance values at preselected wavelengths were compared with standards. In each case the instrument was calibrated just before the peak eluted and a real'stic background subtracted. HPLC packings Polygosil, Kucleosil, Prrtisil, Hypersil, Spherisorb, Techsil and Techspherc were purchesed frcm HPLC Technology I.td. Eluting solvents were prepared by mixing the appropriate proportions of methanol, water and acetic acid and adjusting the

NOVEMBER

1983 VOL. 26 NO. 5

819

PROSTAGLANDINS

final pH by addition of aqueous ammonia, 35% solution. The pH measured is the apparent pH (pH*) and does not reflect the actual proton concentration in the water (pH). All solvents were HPLC grade (Fisons). They were degassed by bubbling helium through the solvent mixture and automatically filtered before entering the pump.

RESULTS AND DISCUSSION OPTIMISATIOII OF HPLC CONDITIONS

FOR L,IPOXYGENASC PRODUCT

SEPARATION

Choice of column A recent publication (16) has highlighted the variation in selectivity of a wide range of chemically bonded reverse phase packings. Our investigation of the elution characteristics of leukotrienes on seven reverse phase columns has shown column choice to be most important (Table 1). Standard elution conditions of methanol:water:acetic acid (70:30:0.06) with the pH* adjusted to 5.0 were used in each case. The relatively "on-polar HETES and LTB4 's chromatographed well on most 5 micron reverse phase Cl8 columns. LTC4 and LTD4, the more polar zwittcrjonic leukotrienes, gave a poor peak shape on scme columns and failed to elute completely on others, eve" after increasing Of the the methanol concentration in the eluting solvent. columns tested Polygosil, Nucleosil and Techsphere_ showed promise for lipoxygenase product analysis. a

Table

1

Comparison

of Reverse

phase packings

_~__ Relative Packing Material

PGB2

POLYGOSIL

3.3

4.5

5.0

TECHSIL

3.8

5.5

PARTISIL

5.9"

HYPERSIL

Retention

Time

(K') LTC4

LTD4

5.5

4.2

10.1

6.2

6.9

5.1*

13.0

7.7*

8.5*

9.8*

NE

16.6"

4.4

6.3

7.0

7.9

NE

NE

TECHSPHERE

4.2

5.6

6.1

6.6

5.2

27.0

NUCLEOSIL

4.8

7.0

7.8

8.7

5.9

9.0

SPHERISORB

4.8

7.1

8.0

9.4

NE

NE

5S,12R -diHETE

55,125 -diHETE

LTB4

(NE - NOT ELUTED, * POOR PEAK SHAPE)

820

NOVEMBER

1983 VOL. 26 NO. 5

PROSTAGLANDINS

Column efficiency of Nucleosil, Polygosil and Techsphere was reduced with prolonged use. The efficiency could be regenerated by This "settling" may be caused by repacking the top of the column. a high back pressure or dissolution of the packing in the eluting solvent. Despite use of a guard column packed with 80 urn silica, to saturate the fluting solvent, settling is still a problem. Prolonged studies with Techsphere have indicated this problem tc be less severe than with Nucleosil and Polygosil. Choice

of pH

Nucleosil has been widely used in leukotriene and lipoxygenase analysis and it has been shown that adjustment of the eluting solvent pH is critical (17). Despite careful pH adjustment it has been difficult to obtain a satisfactory separation on Nucleosil between LTC4, LTD4, LTB4 and the 5S,12R and 5S,12S-diJ1ETT.S with reasonable analysis times. Figure 1 illustrates this result. The most suitable isocratic eluting solvent used with the Techsphere Column was found to be methanol:wnter:acetic acid (65:35:0.06). Adjustment of the pH* over the range 4.0 -- 6.0 had only a minor effect on the non-zwitterionic solutes causing a slight reduction in retention time. Slight changes in the pJl* dramatically altered the fluting properties of LTC4 and LTD . A pH* of 5.2 - 5.4 Adjustment of the was found to be optimum for the compounds studled. pH* beyond this narrow band enabled LTC4 and LTD4 to be "slotted" into different "windows" according to the separation require,?. Figure 2 illustrates the results obtained. Choice

of internal

standard -

The criteria used for selection of an internal standard were (i) resemblance in structure to the materials being analysed and (ii) absence of significant amounts of endogenous compound in material for biological analysis. 13-hydroxylinolcic acid was selected as one internal standard. Its structure is closely related to that of It does not occur in significant quantitites in most samples HETES. from mammalian tissues (one exception discovered was guinea pig lung extracts). For diHETE and leukotriene quantitation 4,11-diHBTE (Isomer mixture 1) was selected as internal standard. We have not observed this compound endogenously in any of our mammalian tissue extracts. 4,11-diHNTE (Isomer mixture 2) was also examined but proved less useful. Using the standard conditions described below it coeluted with 5S,12R-diHETE. PGB2 has been used by many workers as the internal standard for HPLC analysis of leukotrienes. We therefore give data for retention of this compound, although structurally it does not closely resemble lipoxygenase products and it may occur endogenously in small quantities in worked-up extracts. Final conditions

selected

The final IIPLC separation, including 13-hydroxylinoleic acid and 4,11-diHNTE (Isomer mixtulc 1) is shown in Figure 3. The leukotrienes were monitored at 280 nm and the HETES at 237 nm. It was necessary to change the wavelength to 237 nm soon after LTD had eluted if HHT was to be monitored. Purified standards could be detected at levels down to 1 ng.

NOVEMBER

1983 VOL. 26 NO. 5

821

PROSTAGLANDINS

Figure

1.

Effect of pHkon retention of some lipoxy enase products on Nucleosil 5 Cl8 (12.5cm x 4.9mm ID) e 4uted with methanol:water:acetic acid, (70:30:0.06). pH*adjusted with ammonia.

A

LTD4

0

LTC4

0

5S,lZR,dihydroxy-6Z,8E,lOE,14Z-eicosatetraenoic

acid

0

5S,lZS,dihydroxy-6E,8E,lOE,14Z-eicosatetraenoic

acid

n

5S,lZR,dihydroxy-6E,8E,lOE,l4Z-eicosatetraenoic

acid

0

PGB2

7.

6.

5.

4.

3.

2

-

1

-

4.4

4.7

5.0

5.3

5.6

5.9

6.2

PH*

822

NOVEMBER

1983 VOL. 26 NO. 5

PROSTAGLANDINS

Figure 2

Effect of pH*on retention of some lipoxygenase products on Techsphere 5 Cl8 (12.5cm x 4.9mm ID) eluted with methanol:water:acetic acid (65:35:0.06) pH*adjusted with ammonia.

A

LTD4

0

LTC4

0

SS,12R,dihydroxy-6Z,8E,lOE,14Z-eicosatetraenoic

0

5S,12S,dihydroxy-6E,8E,lOE,14Z-eicosatetraenoic

acid

5S,12R,dihydroxy-6E,8E,lOE,14Z-ejcosatetraenoic

acid

n 0 A

35 _

30

acid

PGB2 4S,llR+4R,llS-dihydroxy-5E,7E,9E,13Z-nonadecatetraenoic acid

-

25.

-

H 2 .r kt:

g 5 B z ‘S m 2

20

-

15.

10

_

5.

4.0

4.4

5.2

4.8

5.6

6.0

PH*

NOVEMBER

1983VOL.26NO.5

823

PROSTAGLANDINS

824

NOVEMBER

1983 VOL. 26 NO. 5

PROSTAGLANDINS

Recovery of products We also exarlined the recovery of some lipoxygenase products and PGB2 from thr, Techsphere HPLC colurm~ using the optimum conditions for resolution i.e. m~thanol:wnter:acetic acid (65:35:0.06) at pH* 5.4. The data is shown in Table 2. Recoveries between 74 and 87% were obtained. Table 2 ~_._

Componefit

% Recovery

-____

4,11-djMNTE

LTC4

74

PGB2

84

(Isomer mixture

1)

5S,12R-diHETE

79

76

LTD4

a7

11 HETE

85

13 HYDROXY LINOLEIC ACID

84

It must be stressed that the HPLC separation was primarily developed for ~__ resolution of products, for which the pH* of 5.4 is optimal. A different pH* may be necessary to obtain the best -retof products from the column. EXTRACTION

OF LIPOXYGENASE

PRODUCTS FROM BIOLOGICAL

SANPLES

To obtain a satisfactory HPLC analysis of lipoxygenase products in a sample of biological origin a quick, efficient and reproducible extraction process is necessary. Conventional solvent extraction yields good recoveries of HETES and LTB4 but poor recoveries of LTC4 and LTD . XAD extractions work well but resin preparation time is lengthy4(17). We have used Waters sep-pak columns for lipoxygenase product extraction. These are small columns packed with reverse phase Cl8 material or conventional silica. The reverse phase After columns provide an excellent means of sample extraction. adjustment to the appropriate pH the sample is applied to the sep-pak column using a syrjnge. The column is washed with ethanol:water (1:9) and the lipoxygenase and cycle-oxygenase products eluted with methanol. It is possible to process many samples rapidly using this technique.

NOVEMBER

1983 VOL. 26 NO. 5

825

PROSTAGLANDINS

Table 3 shows the optimisation of the method by adjusting the pH of the solution prior to sep-pak extraction. This data was obtained on pure reference standards, pH 8 gives the most efficient extraction with recoveries of 62% and 73% for LTC 4 and LTD. and over 80% for all other products. The comparative study of HPLC4column packings has indicated the wide variations which exist between apparently equivalent materials. 'Sep-paks' have the advantage of being commercially available, but we do not know whether the column packing in sep-paks is optimal for extraction. Table 3

Percentage recoveries of standard material from aqueous solution using Sep-pak extraction method Extraction pH

Component 2

LTC 4

-

PGB 2

43

4,11-diHNTE (Isomer mixture i)

5S,12R-diHETE

-

5S,12S-diHETE

-

LTB 4

-

LTD 4

-

13 HYDROXY LINOLEIC ACID

4

5

6

7

8

9

5

24

57

56

60

62

30

75

78

81

84

86

97

85

3

6]

75

80

89

82

79

4

68

81

85

93

95

94

69

77

81

86

87

88

54

69

77

82

84

79

22

43

67

58

73

54

8

45

62

92

86

99

72

34

15 HETE

-

21

42

54

81

77

95

88

1 1 H E T E

-

24

42

56

81

80

91

73

-

36

44

57

86

83

98

73

32

42

52

85

80

92

78

30

47

55

92

83

i00

96

8,12 HETE

9 HETE

5 HETE

826

-

3

NOVEMBER 1983 VOL. 26 NO. 5

PROSTAGLANDINS

APPLlCATlON __--

OF METUOD

Optimal conditions for extraction and HPLC of lipoxygenase products, as described above, were applied to the analysis of lipoxygenase products The cells were stimulated produced by mouse peritoneal macrophage cells. Control samples were not exposed to with y-hexachlorocyclohexane. Lipoxygenase metabolite identity was confirmed y--hexachlorocyclohexane. by correlation of HPLC retention times with authentic reference standards. In addition UV spectra were obtained on all major HPLC peaks. Figure 4 shows the elution profile of products released from >-hexachlorocyclohexane stimulated cells. Detection

limits

in biological

samples

Ln order to determine detection limits in the scan mode we spiked extracts from unstimulated cells (which produce only small amounts of lipoxygenase products) with known amounts of reference standards. Figure 5a shows LJV scans on an extracted sample of products released from unstimulated cells spiked with 10 ng of PGB2, 4,11-diHNTE (Isomer l), 5S,lZS-diHETE and LTB4. The scan detection limit for LTC4 and LTD4 was approximately 20 ng. Identification ---_-

of unknown

peaks

in extracts

from stimulated

macrophages

Extracts from stimulated macrophages gave major peaks of UV abscsrption corresponding in retention time to LTC4, LTB , 5s lZ?zdiHETE, and 12 HCTE (Figure 4). When the cells were prelabelle 2 with [ .C]arachidonic acid, major peaks of radioactivity were observed corresponding Approximately 6% of total in position to the UV absorption peaks. extracted radioactivity coeluted with LTC4, 13% with 12-HETE and 6% with the 5,12-diHETES. Figure 5b shows scans of the peaks corresponding in elution time to I,TC4, 5S,12S-diHETE and 12 HETE. They resemble the scans The fraction corresponding to the J,TC4 elution of authentic standards. Biological activity was position was bioassayed on guinea pig ileum. detected. The response was antagonised by the Fison's compound FPL55712. Triplicate LTC4 was quantitated by comparison to synthetic standards. determinations gave the mean LTC4 in the fraction as 55 ng by UV absorption and 45 ng by bioassay. In conclusion, the described method provides a quick and reproducible extraction technique which can be applied to most samples of biological origin. Examination of the extracted sample by HPLC using a Techsphere column coupled to the Perkin Elmer LC-85 detector enables sensitive assay of lipoxygenase products. The scanning facility of the LC-85 detector and bioassay of fractions add further specificity to the method.

NOVEMBER

1983 VOL. 26 NO. 5

827

PROSTAGLANDINS

VSO'O J

V80'O

z, -

-

VSO'O

WO'O ~ E=~ o I

0

J

~.-3:

0-~

E

o

tsnm o.1=

q-

E

~

1

m

tOXo -~Eg 5

828

NOVEMBER 1983 VOL. 26 NO. 5

O

PROSTAGLANDINS

Figure 5a.

Scans o f some r e f e r e n c e an e x t r a c t from control A B C D

-

standards (IOng of each) mouse m a c r o p h a g e c e l l s .

Spiked

PGB2 4S,llR+4R,lIS-dihydroxy-5E,7E,9E,13Z-nonadecatetrae~oic 5S,12S,dihydroxy-6E,8E,lOE,14Z-eicosatetraenoic acid 5S,12R,dihydroxy-6Z,8E,lOE,14Z-eicosatetraenoic acid

into

acid.

A

B

r-~--, 310

, -r-, nm

,

,

'

220

r, 330

, ~--~--r , nm

D

r ~ 320

,

,

. nm . . . . .

,

,

220

C

2"2o

NOVEMBER 1983 VOL. 26 NO. 5

' 310

.

.

.

.

. nm.

.

.

2"o2

829

PROSTAGLANDINS

_i? cd

;I:::-:-

E

-_Z

-3i _x m

830

NOVEMBER

1983VOL.26NO.S

PROSTAGLANDINS ACKNOWLEDGMENTS -----We wish to acknowledge: Dr. S.R. Baker for providing synthetic leukotrielles,Dr. 3. Walker for lipoxygenase products from rabbit cells, Dr. S. McKay and Ilrs. P. Shrubsall for separation of the diastereoisomers of protected LTD4, ar.dMrs 0. Sloper for bioassay of Ieukotrienes.

REFERENCES --1.

Eorgeat, P. and B. Samuelsson. Arachidonic acid metabolism in polyrlorphonuclearleukocytes: Effects of ionophore k23187. Proc. Matl. Acad. Sci. U.S.A. -76: 2148. 1579.

2.

Nurphy, R.C., S. Hammarstrom and B. Samuelsson. Leukotriene C: A slow-reacting substance from murine mastocytoma cells. Proc. Natl. Acad. Sci. U.S.A. -76: 4275. 1979.

3.

Morris, H.R., G.W. Taylor, P.J. Piper, M.N. Samhoun and J.R. Tippins. Slow reacting substances (SRSs): The structure identification of SRSs from rat basophil leukaemin (KBL-1) cells. Prostaglandins -19: 185. 1980.

4.

Orning, L., S. Hammarstrom and B. Samuelsson. Leukotriene D: A slow reacting substance from rat basophil leukemia cells. Proc. Natl. Acad. Sci. U.S.A. -77: 2014. 1980.

5.

Hammarstron, S. and B. Samuelsson. Stereochemistry of Leukotriene C-l. Biochem. Biophys. Res. Commun. -. 92: 946.

1980.

6.

Bach, M.K., and J.R. Brashler. Identification of a component of rat mononuclear cell SRS as leukotriene D. Biochem. Biophys. Res. Commun. -93: 1121. 1980.

7.

Lewis, R.A., K.F. Austen, J.M. Drazen, D.A. Clark, A. Marfat and E.J. Corey. Slow reacting substances of anaphylaxis: Identification of leukotrienes C-l and D from human and rat sources. Proc. Nstl. Acad. Sci..U.S.A. -77: 3710. 1980.

8.

Morris, H.R., G.W. Taylor, J. Rokach, Y. Girard, P.J. Piper, J.R. Tippins, and M.N. Samhoun. Slow reacting substance of anaphylaxis SRS-A: Assignment of the stereochemistry. Prostaglandins -20, 601. 1980.

9.

Boeynaems, J.M., J.A. Oates and W.C. Hubbard. Preparation and characterization of hydroperoxy-eicosatetraenoic acids (HPETES). Prostaglandins -19: 87. 1980.

10

Walker, J.R., and W. Dawson. Inhibition of rabbit PMN lipoxygenase activity by benoxaprofen. J. Pharm. Pharmacol. -31: 778. 1979.

11.

Baker, S.R., W. Jamieson, D.J. Osborne and W.J. Ross. Svnthesis of the 92 and 9Z,llE isomers of leukotriene C Tet. Leits. 22: 4' 2505. 1981.

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1983 VOL. 26 NO. 5

831

PROSTAGLANDINS

12.

Baker, S.R., W.B. Jamieson, S.W. McKay, S.E. Morgan, D.M. Rackham, W.J. Ross and P.R. Shrubsall. Synthesis, separation and NMR spectra of three double bond isomers of leukotriene A methyl ester. Tet. Letts. -21: 4123. 1980.

i3.

McKay, S.W., D.N.B. Mallcn, P.R. Shrubsall, J.M. Smith, S.R. Baker and R.U. Koenigsberger. Separation of the N-trifl.uoroacetyldi-methylesters of leukotriene D and C isomers by semi-preparative high-performance liquid chromatography. J. of Chrom. (Biomed. Appl.). -. 219: 325. 1981.

14.

Meade, C.J., J. Harvey, J.R. Boot, G.A. Turner, P.E. Bateman and D.J. Osborne. 3-hexachlorocyclohexane stimulation of macrophage phospholipid hydrolysis and leukotriene production. Biochem. Pharmacol. (in press)

15.

Phagocytosis Bretz, V., B. Dewald, T. Payne and J. Schnyder. stimulates the release of an SRS in cultured macrophages. Brit. J. Pharmac. -71: 631. 1980.

16.

Goldberg, A.P. Comparison of columns for reversed-phase liquid chromatography. Anal. Chem. -54: 342. 1982.

17.

Mathews, W.R., J. Rokach and R.C. Murphy. Analysis of leukotriene by high-pressure liquid chromatography. Anal. Biochem. 118: 96. 1981.

Editor:

832

Elisabeth

Granstrom

Received:

3-22-82

NOVEMBER

Accepted:

g-30-83

1983 VOL. 26 NO. 5