Determination of biological peptide leukotrienes C4 and D4 by fluorometric detection combined with high-performance liquid chromatography

0952-327&VWOO39-0119/SlO.a)

Prostqbdim Lcukotriaes and Essential Fatty Acids (1990) 39,119-123 @.qmmtGmupUKLtd1990

Determination of Biological Peptide Leukotrienes C4 and D4 by Fluorometric Detection Combined with High-performance Liquid Chromatography Y. Yamashita,

Y. Uchida, A. Nomura, S. Hasegawa and S. Kimura*

Institute of Clinical Medicine, *Institute of Basic Medical Sciences, Ibaraki 305, Japan (Reprint requests to SH)

University of Tsukuba,

Tsukuba,

ABSTRACT.

A fluorometric procedure combined with high-performance liquid chromatography (HPLC) has been developed to separate and quantitate peptide-leukotrienes (p_LTs) C4 and D4. The method is based on pre-column dansylation of the amino terminus of the p-LTs. Its application for the determhtation of the amounts of p_LTs C4 and D4 in human blood and sputum is also described. The lower limit of detection of p-LTs with this method is 1 pmol.

Sweden) was connected to this apparatus and used as an automatic collecting system. Radioactivity of the labelled fractions was counted in a liquid scintillation counter, LS 8000 (Beckman, CA, USA).

INTRODUCTION Peptide-leukotrienes, which are a group of autacoids, are synthesized from arachidonic acid and glutathione in various tissues and are involved in the pathogenesis of a variety of conditions, e.g. anaphylaxis and bronchial asthma. Although the basic structure of p-LTs was determined in 1980, the levels of p-LTs in biological materials have proved difficult to measure, because the amounts of p-LTs present in vivo are lower than those of other autacoids and because p-LTs are chemically unstable. Until now several techniques have been developed for the measurement of p-LTs, for example, bioassay (1, 2) using tracheal or aortic preparations, radioimmunoassay (RIA) (3, 4, 5)) HPLC with ultraviolet (UV) spectrometry (6), and gas chromatography-mass spectrometry (GC-MS) (7, 8). However, the levels of p-LTs obtained by these methods vary too widely for practical use. In this report, we present a new fluorometric method to measure p-LTs by which the content of p-LTs in biological samples was readily measured.

MATERIALS

Chemicals Standard (Std)- LTC4 and Std-LTD, were the gifts of Ono Pharmaceutical Co., Ltd. (Osaka, Japan). The 5-dimethylaminonaphthalene-1-sulphonyl chloride (Dansyl chloride; DNS-Cl) was obtained from Sigma (MO, USA). Acetonitrile, acetone, acetic acid, ethyl alcohol, sodium hydroxide and sodium bicarbonate were all of analytical grade from WAKO (Osaka, Japan). Scintisol EX-H (Dojin, Kumamoto, Japan) was used as a premixed scintillation cocktail.

Radiochemicals [14,15(n)-3H]-LTC, (3H-LTC,) and [14,15 (n)-“HILTD4 (‘H-LTD4) were purchased from Amersham (Buckinghamshire, UK). The purity of the radiochemicals was shown to be greater than 95%.

AND METHODS

Apparatus

Materials

A high-performance liquid chromatograph (HPLC), Model LC-6A (Shimadzu, Kyoto, Japan) was used, consisting of two single-head pumps, a variablevolume injector, (Rheodyn, CA, USA), an ultraviolet spectrophotometric detector, SPD-6A (Shimadzu, Kyoto, Japan), and an HPLC fluorescence minotor, RF535 (Shimadzu, Kyoto, Japan). A fraction collector, Frac 100 (Pharmacia, Uppsala,

Sep-Pak Cl8 cartridges were obtained from Waters USA). The prepacked HPLC Assoc. (MA, columns, Chemcosorb SODS-H (4.6 x 250 mm), were from Chemco Scientific Co., Ltd. (Osaka, Japan). Preparation of biological specimens. Human blood. Blood was drawn from the antecubital vein of a 119

120

Prostaglandins

Leukotrienes

and Essential

Fatty Acids

healthy, non-allergic adult into a syringe with a No. 18 gauge needle. The blood was promptly transferred to an ice-chilled tube containing 3.8% sodium citrate (0.5 ml/45 ml blood). Then Std-LTC,, StdLTD4, 3H-LTCt and 3H-LTD~ were added to the blood samples to find out the proper conditions for chromatography. 3H-LTCt and $I-LTD4 in amounts lower than 100 fmol were also added as the internal standards. Identification of the DNS-LTC4 and DNS-LTD, peaks was also performed by 3H counting. The sample tube was mixed, and the plasma was separated by centrifugation at 2000 x g for 15 min at 4 “C. The plasma was diluted to 80% (v/v) ethyl alcohol with absolute ethyl alcohol and let stand at 4 “C for 1 hr. The supernatant was separated by centrifugation at 10 000 X g for 15 min at 4 gC, and then evaporated to l-2 ml of the final volume. The resulting preparation was applied to a Sep-Pak Crs cartridge, which was prewashed with 5 ml of 60% acetonitrile and 10 ml of water, and then washed with 10 ml of water and eluted with 5 ml of 60% acetonitrile. The eluate was evaporated to dryness under vacuum, and the sample ‘was subjected to dansylation.

uv at

a

280

Human sputum. Sputum samples were obtained from patients with bronchiectasis (BE), diffuse panbronchiolitis (DPB) and chronic bronchitis. To each sample tube, ethanol was added to final 80% (w/v) and the sample was let stand at 4 “C for 1 h. To determine the best HPLC separation conditions, Std-LTC4 and Std-LTD, were added to the sputum which was obtained from patients with bronchiectasis. 3H-LTC4 and 3H-LTD~ were also added as the internal standards. The procedures of dansylation were similar to those described above. Estimation of the LTC4 and LTD4 content in the sputum from other patients was also undertaken without Std-LTs and 3H-LTs. Dansylation of LTC4 and LTD4 A certain amount of Std-LTC, or LTD4 dissolved in methyl alcohol was evaporated to dryness under vacuum and was dissolved in 20 ml of 0.2 N borate buffer pH 9.0. Then 20 ml of an acetone solution of DNS-Cl (5 mg!‘ml) was added for dansyiation. After incubation at 50 “C for 1 h in the dark, the sample was evaporated and then dissolved in PBS buffer containing 50% MeOH.

nm LT C.

LT Da

I

2g ____.._____--------_______.__._.__..--------__/

,,’

,________...------

0

5 2 A. 10% CHlCN 1% CHlCOOH NaOH pH 4.9 o .-0 S&Jtm 6, 60% CH,CN 1% CHaCOOH NaOH P” 4 9 S&M”

B ccrc.

: O%-50%-loo%-100% IO%lmn

2%/mn

S Flow Rate

x

b

lm(/rm

Fluorescence

gj

Ex

L

: 340

nm

Em

: 530

nm

9 LL

c

RI

counts

I

‘H-LT C, ‘H LTD.

0 Time

Fig. 1

Chromatogram

of Std-LTC,

10

20 Time

(min.1

and Std-LTD,

(min.1

LTC, and LTD., were eluted at 16 min and 18.5 min, respectively.

30

Determination of Biological Peptide Lmkotrienes C4 and D4

Chromatography

of DNS-LTC,, and DNS-LTD4

The sample was applied on a reverse phase column (Chemcosolb SODS-H, 4.6 x 250 mm). The column was eluted with a linear gradient (25 min) from 30 to 60% acetonitorile in 1% acetate solution (pH 4.9, titrated with sodium hydroxide) at a flow rate of 1 mvmin. The column effluent was monitored by measuring UV absorbance at 280 nm and fluorescence (excitation: 340 nm, emission: 530 nm).

RESULTS AND DISCUSSION. Figure la represents typical reversed phase HPLC of Std-LTC, and Std-LTD,, chromatograms measured by UV absorption at 280 nm. The radioactivities of 3H-LTCr and 3H-LTD,, that were added as the internal standards showed the same retention times as Std-LTC, (16 min) and Std-LTD, (18.5 min), respectively. There were no natural fluorescent peaks.

at 280

a

‘J’/

b

Fluorescence

Figure 2 shows a chromatogram of DNS-LTC,, and DNS-LTD4. The retention times for DNS-LTC4 and DNS-LTDJ were 26 min and 38 min respectively. Several peaks due to the reaction by products (DNS-Cl, DNS-OH, DNS-NH2,, etc.) were observed. As shown in Figure 1, the UV peaks and the 3H counts showed that almost none of the unlabeled Std-LTs remained and that almost all the p-LTs present were dansylated. As shown in Figure 3, the peak heights of DNSLTC4 and DNS-LTD4 increased linearly with increasing amounts of Std-LTC, and Std-LTD,. The lower limit of detection was approximately 1 pmol. The detection limit was lower than that of UV abafter sorption separation by HPLC. The chromatogram of human biood is shown in Figure 4. Fluorometry shows less contaminant peaks than UV absorbance. The peaks of DNS-LTC4 and DNSLTD4 were identified by the retention times of the radioisotopes DNS-3H-LTC, and DNS-3H-LTD4. The recovery of Std-LTC, and STD-LTD4 was about 60%. A chromatogram of human sputum is

“i-n

!

Dt4s -LTD.

I

DNS-WLTC. DNSVHLTD.

__...

L-

0

to

20

-_

30 Time

(min.1

40

50

0

10

20

-._-‘_-.-_-.---L

Time

30

40

(min.1

2 Chrbmatogram of DNS-LTC, and DNS-LTD+ The retention time. of DNS-LTC, was 26 min and that of DNS-LTD, was 38 min. respectively.

Fig.

121

122 Prostaglandins Leukotrienes and Essential Fatty Acids a

High

pmol

-

300 -

; ol i I 1

Analysis

a. UV at 280 nm

pmol 1

( 100-500

200 .

a

0

100

200 DNS

b

Low

pmol 200

_

LTC,

II b

m LT Do

Analysis

- Std

300

400

LTs

( pmol )

Fluorescent

500

(5-3OaTloll

t

L I -c . RI counts

V. 0

5

10 DNS

Fig. 3 (6-6).

20 - Std

LTs

30 ( Pm01 )

Standard curves for DNS-LTCI (&-&) and DNS-LTD4 0

10

20

30

Time

shown in Figure 5 and the amounts of LTC4 and LTD4 in the samples are shown in Figure 6. We demonstrated that the p-LTs, LTC4 and LTD4, were almost completely dansylated at the derivatization step prior to separation by HPLC. Linear standard curves were obtained in the range from 5 to 500 pmol for each Std-LT used. LTs are bioactive compounds with roles as important mediators of inflammation and allergy. They are derived from arachidonic acid by oxidative metabolism through the lipoxygenase pathway. Although a broad base of knowledge concerning their synthesis and metabolism has been acquired in vitro, because of the small amounts of material available and its marked chemical lability, to assess the concentrations of the LTs in vivo and to evaluate

their

role

in human

disease

has proven

to

be very difficult. Several techniques - bioassay, GC-MS, RIA, and HPLC with the detection by UV absorbance - have been developed for the measurement of LTs in biological materials. Such techniques, however, have certain disadvantages. Bioassay is sensitive, yet the assay is only semiquantitive, with large variations in response occurring between different tissues, and it is not specific. GC-

40

50

(min.1

Fig. 4 Chromatogram of a dansylated blood sample. LTC4, LTD,. 3H-LTC4 and 3H-LTD4 were added before pretreatment.

MS requires complex procedures and special apparatus. Although RIA. is often perfomed combined with HPLC (9), the effluent containing organic solvents needs to be pretreated in order not to disrupt the antigen-antibody reaction. HPLC assay of LTs with UV absorption is interfered with by many contaminants, since the content of LTs is much lower than that of other substances present in the extracts (10). There remains a need for a simple but specific and sensitive chemical technique for the quantitative estimation of LTs in biological materials. As shown in this study, human sputum has been confirmed to contain LTs (ll), and the content is considered to reflect the pathological state of the lung. Our data also shows the presence of LTC4 and LTD4 in human sputum. It will be necessary in the future to assess the concentrations of LTs in biological specimens which are obtained from patients with various diseases to prove possible pathological role of LTs. A dansylation method has been developed for determination of the N-terminus of peptides or

Determination a

UV

a1 2.30 nm

2 8

of Biological Peptide Leukotrienes

C, and D4

proteins (12), and it is also used to separate and measure histamine (13, 14). LTC4, LTD, and LTE4 are referred to as the sulphidopeptide leukotrienes because each contains a thioether-linked peptide at the 6 position. In this paper, we described the determination of p-LTs by dansylation and its application to biological specimens. This method is relatively simple and shows good reproducibility and high sensitivity, which enabled us to quantitate the biological p-LTs in human specimens.

References

lnts

0

DNS-WLTIJ.

10

20

30

50

40

Time (min.) Fii. 5 Chromatogram of a dansylated sample of sputum LTD., ,nd 3H-LTD4 were added before pretreatment.

C4

D4

C4

D4

C4

D4

I

%

6.E

D.P.6

Bronchitis

Fig. 6 The amounts of LTC, and _TDd measured in samples from patients with respiratory disease.

123

1. Orange R P, Austen K F. Slow reacting substance of anaphylaxis. Advan. Immuno. 10: 105-144, 1%9 2. Drazen J M, Austen K F, Lewis R A, Clark D A, Goto G, Marfat A, Corey E J. Comparative airway and vascular activities of leukotrienes C-l and D in vivo and in vitro Proc. Natl. Acad. Sci. USA. 77: 4354-4358,1980 3. Levine L, Morgan R A, Lewis R A, Austen K F, Clark D A, Marfat A, Corey E J. Radioimmunoassay of the leukotrienes of slow reacting substance of anaphylaxis. Proc. Natl. Acad. Sci. USA. 78: 7692-76%, 1981 4. Aehringhaus V, Wolbling R H, Konig W, Patron0 C, Peskar B M, Peskar B A. Release of leukotriene C., from human polymorphonuclear leucocytes as determined by radioimmunoassay. FEBS LE’lT. 146: 111-114, 1982 5. Lindgren J A. Hammarstrom S, Goetzl E J. A sensitive and specific radioimmunoassay for leukotriene C,, FEBS LE’IT. 152: 83-88, 1983 6. Mathews W R, Rokach J, Murphy R C. Analysis of leukotrienes by high-pressure liquid chromatography. Anal. B&hem. 118: 96-101, 1981 7. Orange R P, Murphy R C, Kamovsky M L, Austen K F. The physicochemical characteristics and purification of slow-reacting substance of anaphylaxis. J. Immunol. 110: 760-770, 1973 8. Jubiz W. Biochem. Biophys. Res. Commun. A reevaluation of the chemotactic potency of leukotriene B,. 110: 842-850, 1983 9. Zijlstra F J, Vincent J E. Determinz&on of leukotrienes and prostaglandins in [ C] arachidonic acid labelled human lung tissue by high-performance liquid chromatography and radioimmunoassay. J. Chromatogr. 311: 39-50, 1984 10. Olson N C, Dobrowsky R T, Fleisher L N. Assessment of bronchoalveolar lavage fluid and plasma for sulfidopeptide leukotrienes during endotoxemia in pigs. Prostaglandins Leuko. Med. 28: 203-214,1987 Il. Cromwell 0, Walport M J. Morris H R, Taylor G W, Hodson M E, Batten J, ‘Kay A B. Identification of leukotrienes D and B in sputum from cystic fibrosis patients. Lancet. July 25: 164-165,1981 12. Hartley B S, Massey V. The active centre of chymotrypsin I. Biochem. Biophys. Acta. 2 1: 58-70,1956 13. Yamatodani A, Seki T, Taneda M, Wada H. Determination of histamine and methylhistamines by dansylation and its application to biological specimens. J. Chromatogr. 144: 141-145. 1977 14. Bontemps J, Laschet J, Dandrifosse G. Analysis of dansyl derivatives of di- and polyamines in mouse brain, human serum and duodenal biopsy specimens by high-performance liquid chromatography on a standard reversed-phase column. J. Chromatogr. 311: 59-67, 1984