ANALYTICAL SCIENCES JUNE 1989, VOL. 5 289

Determination of Leucine- and Methionine-Enkephalins in Rat Brains by High Performance Liquid Chromatography with Precolumn Fluorescence Derivatization

Masaaki KAI, Mutsuko NAKANO, Guo-Qing ZHANG and Yosuke OHKURA

Faculty of Pharmaceutical Sciences, Kyushu University, Maidashi, Fukuoka 812, Japan

Fluorometricallyreactive peptides of leucine- and methionine-enkephalins in rat brain tissues such as cortex, striatum and hypothalamus were simultaneously assayed by reversed-phase high performance liquid chromatog- raphy with fluorometric detection, based on precolumn derivatization specific for N-terminal tyrosine-containing peptides. The enkephalin peptides extracted from the tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate in a weakly alkaline aqueous solution (pH 8.5). The fluorescent derivatives of the peptides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 8.5) and tetrabutylammonium chloride, and then determined by fluorometry. The peaks of both the enkephalins in the tissue sample were not observed in the chromatogram after the enzymatic degradation with carboxypeptidase A. The determined concentrations of the leucine- and methionine-enkepalins in the tissues were 20-238 pmol and 80-75 pmol per g of the tissue, respectively. The method was sensitive enough to determine the endogenous enkephalins at concentrations as low as Ca. 6 pmol per g of the brain tissues.

Keywords Enkephalin, rat brain, fluorometry, high performance liquid chromatography, precolumn derivatization

Enkephalins, leucine-enkephalin (LE; Tyr-Gly-Gly- Tyr-containing peptides, by which the peptides yields Phe-Leu) and methionine-enkephalin (ME; Tyr-Gly- intense fluorescences under mild conditions in the Gly-Phe-Met), have opiate-like activity in nervous presence of hydroxylamine, cobalt(II) ion and borate. systems.' Recently, opioid peptides have been found to This reaction provided a single fluorescent product for act in vivo as neurotransmitters via interaction with each of the synthetic peptides and did not permit the opiate receptors.2 Most of the opioid peptides production of fluorescent derivatives for peptides involving LE and ME have a tyrosyl residue at the N- having no N-terminal tyrosyl residue (e.g. Gly-Tyr, terminus in their molecules. Phe-Arg-Gly and Arg-Val-Tyr-Ile-His-Pro-Phe).13 During the studies on the physiological mechanism of However, the chemical structure of the products the opioid peptides, many methods have been developed remains unknown. for the quantitative determination of the opioid peptides In this paper, the above-mentioned fluorescence in biological samples. These methods include bioassay3, reaction selective for N-terminal Tyr-containing peptides radioimmunoassay (RIA)4'5, enzyme-immunoassay6, ra- was applied to a precolumn fluorescence derivatization dioreceptorassay' and mass spectrometry8, either alone method of HPLC with fluorescence detection for the or in combination with high performance liquid sensitive determination of the endogenous enkephalins chromatography (HPLC). in rat brains. [D-A1a2,3]-ME was used as internal HPLC can simultaneously separate many synthetic standard for the quantification of the enkephalins. and biological peptides9"0, and has the advantages of superior reproducibility, practicability and speed in the analysis. However, the quantification of the endogenous Experimental opioid peptides in brain tissues by the current HPLC methods with spectrophotometric'° and electrochemical" Chemicals and solutions detections is hampered by problems of insufficient The following synthetic peptides were purchased sensitivity and selectivity for the peptides. from Sigma (St. Louis, M0, U.S.A.): Tyr-Arg, Tyr- To resolve the problems in the HPLC, we previously Gly, Tyr-Gly-Gly, Tyr-Gly-Gly-Phe, LE, ME, [D- developed a unique chemical reaction'2 for N-terminal A1a2'3]-ME, LE-Arg, ME-Arg-Phe, ME-Arg-Gly-Leu, 290 ANALYTICAL SCIENCES JUNE 1989, VOL. 5 a-endorphin, y-endorphin, a-neoendorphin, /3-neoendor- phin, dynorphin 1-7, dynorphin 1-8, dynorphin 1-9, dynorphin 1-10, dynorphin 1-13 and dynorphin B. Carboxypeptidase A treated with diisopropylfluoro- phosphate was obtained from Funakoshi (Tokyo, Japan). Water was deionized and then distilled before use. Other chemicals were of the highest purity available. The synthetic peptides were used as received and dissolved in water. The solutions were usable for at least 3 weeks when stored at -80° C. The reagent solutions used for fluorescence derivatization were prepared as described previously.13

Peptide extraction Male Sprague-Dawley rats (220 - 280 g, 7 weeks) were anesthetized with diethyl ether and killed by dehematization. Each one's brain was removed quickly, Fig. 1 Chromatogram of a standard mixture of synthetic fourteen N-terminal Tyr-containing peptides. Peaks (pmol and the tissues of cortex, striatum and hypothalamus injected): 1, Tyr-Arg (40 pmol); 2, dynorphin 1-7(40 pmol); 3, were separated. The tissues were stored at -80° C until Tyr-Gly (20 pmol) and Tyr-Gly-Gly (20 pmol); 4, LE-Arg use. The tissues were quickly washed with saline and (40 pmol); 5, Tyr-Gly-Gly-Phe (20 pmol) and ME-Arg-Gly- weighed after removing the saline with filter-paper. A Leu (20 pmol); 6, dynorphin 1-8 (40 pmol); 7, ME-Arg-Phe portion (0.2 - 0.4 g) of the tissue was homogenized at (40 pmol); 8, ME (40 pmol); 9, LE (40 pmol);10, a-endorphin 0 - 4° C with 3 ml of 0.1 M hydrochloric acid, then a 50- (40 pmol); 11, [D-A1a2'3]-ME (40 pmol); 12, y-endorphin µl portion of 2.0 µM [D-A1a2'3]-MEas internal standard (40 pmol). was added to the homogenate. The homogenate was transferred into a centrifuge tube, rinsing with 1 ml of 0.1 M hydrochloric acid. The homogenate was depro- teinized with 0.5 ml of 2 M perchloric acid. After Enzymatic degradation centrifugation at 2450g for 10 min, the precipitate was For the identification of peptide peaks in chromato- suspended with 2.0 ml of 0.2 M perchloric acid and gram, a portion (40 µl) of the sample eluted from the centrifuged again. The combined supernatant (ca. cartridge was mixed with 10 µl of 15 unit ml-' carboxy- 6.5 ml) was neutralized at pH 7 - 8 with ca. 2 ml of 1 M peptidase A in 50 mM phosphate buffer (pH 7.5). The sodium hydrogencarbonate solution. The supernatant mixture was incubated at 37° C for 30 min. The final was then applied to a cartridge (Bond Elut C18; reaction mixture was used for the above fluorescence Analytichem International, Harbor City, CA, U.S.A.). derivatization. Before this procedure, each cartridge was washed with 3 ml of water and of methanol. After loading the tissue Apparatus and HPLC conditions extract, a series of liquids: 1 ml of water, 2 ml of The HPLC system consisted of a Hitachi 655 high- dichloromethane to remove strongly hydrophobic sub- pressure pump, which has a programmed electronic stances, 1 ml of water, 3 ml of 0.1 M hydrochloric acid, controller of the electronic valves for various gradient 1 ml of water, 3 ml of 0.1 M borate buffer (pH 8.5) and elutions, a Rheodyne 7125 syringe-loading sample 1 ml of water were successively passed through the injector (100-µ1 loop) and a Hitachi F-1100 fluo- cartridge. Finally, the enkephalin-rich fraction was rescence spectrophotometer fitted with a 12-µ1flow-cell. obtained by the elution with 1.0 ml of aqueous 90% A reversed-phase column (150X4 mm i.d.) packed with methanol. After evaporation in vacuo at ca. 30° C, the TSKgel ODS-120T (particle size, 5 µm; Tosoh, Tokyo, residue was dissolved in ca. 200 µl of water. The Japan) was used. The column temperature was solution was used for fluorescence derivatization. ambient (24±4° C). For the separation of the fluorescent derivatives of peptides on the column, gradient elution Fluorescence derivatization between 2 and 26% (v/ v) acetonitrile in the mobile A 50-µ1 portion of the sample solution was placed in phase containing 45% (v/ v) of 0.1 M sodium borate a test-tube (50X5 mm i.d.), to which were added 50 µl buffer (pH 8.5) and 15% (v/ v) of 10 mM tetrabutyl- each of a mixture of 10 mM hydroxylamine and ammonium chloride was carried out during 25 min at a 0.5 mM cobalt(II) acetate, and 0.4 M borate buffer constant flow-rate of 1.0 ml min-'. The acetonitrile (pH 8.5). The mixture was heated in a bath of boiling gradient is indicated in Fig. 1. When the tissue sample water for 3 min, and then a 25-µ1 portion of 20 mM 2- was analyzed, the column was washed with the mobile mercaptoethanol was added to stabilize the derivatives phase containing 40% acetonitrile for 5 min to remove produced. A 100-µ1 portion of the final reaction strongly hydrophobic substances. The fluorescence mixture was used for HPLC. intensity in the column eluate was monitored at 440 nm (emission maximum) and 330 nm (excitation maximum). ANALYTICAL SCIENCES JUNE 1989, VOL. 5 291

Uncorrected fluorescence excitation and emission In addition, the present derivatization method did spectra of the eluate were measured with a Hitachi not give fluorescent products of the following biogenic MPF-4 spectrofluorometer in quartz cells (optical path substances: L-a-amino acids other than Tyr, purine and length, l OX10 mm); spectral band widths of 10 nm were pyrimidine bases (adenine, guanine, uracil, thymine and employed for both the excitation and emission monochro- cytosine), sugars (glucose, lactose and sucrose) and mators. steroids (estrone and cholesterol). Therefore, the derivatization was effectively selective for the N- terminal Tyr-containing peptides. Results and Discussion Figure 2A shows a chromatogram of cortex tissue of rat brains (n=6) obtained by the recommended procedure. Figure 1 shows a chromatogram obtained by precolumn The fluorescent derivatives corresponding to ME, LE fluorescence derivatization of a standard mixture of and [D-A1a2'3]-MEas the internal standard in the tissue fourteen synthetic N-terminal Tyr-containing peptides sample were separated and fluorometrically detected. involving the known opioid peptides, under the HPLC These three peaks were not formed by the same conditions recommended for the determination of procedure but by omitting hydroxylamine in the enkephalins in rat brain. The synthetic LE, ME and [D-A1a2'3]-MEwere mutually separated from the other tested peptides within 25 min, though Tyr-Gly and Tyr- Gly-Gly-Phe were not separated from Tyr-Gly-Gly and ME-Arg-Gly-Leu, respectively. Table 1 shows the detection limits and the retention times of these peptides. The lower limits (S/ N=3) of detection for the peptides were in the range 220 - 810 fmol on the column. The present HPLC conditions did not permit the elution of relatively large molecular peptides such as a- and /3-neoendorphins and dynorphins 1-9,1-10,1-13 and B within 25 min. The basic conditions of the HPLC separation and the peptide derivatization with hydroxylamine, cobalt and borate reagents were described in the previous reports.12"3

Table 1 Detection limits and retention times of synthetic Fig. 2 Chromatograms of cortex tissue of rat brains obtained by (A) the recommended procedure and (B) the same onioid nentides and related nentides procedure but without the fluorescence derivatization. Peaks: 1, ME; 2, LE; 3, internal standard. The arrows in chromatogram B indicate the retention times of the corresponding peptides.

Fig. 3 Chromatograms of (A) striatum tissue and (B) a. Defined as the amount in the injection volume giving a hypothalamus tissue of rat brains. Peaks: 1, ME; 2, LE; signal-to-noise ratio of 3. 3, internal standard. The shaded areas correspond to the ND, not eluted. peaks produced by the fluorescence derivatization. 292 ANALYTICAL SCIENCES JUNE 1989, VOL. 5 fluorescence derivatization (Fig. 2B). Figure 3 also [D-A1a2'3]-MEadded to the homogenate were 52 - 61%, shows the detections of the endogenous enkephalin-like (n=3, each instance). The relatively low recoveries for peptides in other tissue regions of the striatum and these peptides were mainly caused by the derivatization hypothalamus of the brains (n=12, each instance). disturbed with some coexistent substances in the However, several fluorescent peaks other than peaks sample. When the brain extract was not treated with produced by the fluorescence derivatization were observed the cartridge, the peptides in the sample were not in the chromatograms of the brain tissues. These peaks derivatized. The variation of their recoveries was also were due to some fluorescent compounds present in the observed depending on the treatment with each mini- tissues. Under the present HPLC conditions, the native cartridge. However, the constant ratio of the recovery fluorescences of the aromatic amino acids such as Phe, of each enkephalin to that of the internal standard, [D- Trp and Tyr in peptides were not detected, because they A1a2'3]-MEwas attained with each treatment. Thus, the have different emission and excitation maximum wave- intertnal standard was used for a precise and facile lengths from those of the peptide derivatives. quantification of the enkephalins in the brain tissue. The peptide peaks of LE and ME in the chromato- A linear relationship was achieved between the ratio grams of the brain tissues were identified by chromato- of the peak height of each enkephalin to that of the graphic techniques based on retention times, co- internal standard and the amount of the enkephalin chromatography with their synthetic peptides, and also added to the tissue homogenate in the range 10 - by comparison with fluorescence spectra of the eluates 800 pmol per g of the tissue. The correlation coefficients from the peaks. For the further identification of the (r) of the calibration graphs for LE and ME were both peptides, the tissue samples were treated with carboxy- greater than 0.998 (n=3 each plot). The lower limits of peptidase A before the fluorescence derivatization detection for LE and ME in the brain tissues were both (Fig. 4). The peptide peaks produced by the fluorescence approximately 6 pmol per g of the tissue, respectively, derivatization were apparently erased by the enzymatic degradation, as compared with Figs. 2 and 3. The extraction of the enkephalin peptides from the Table 2 Regional concentrations of LE and ME in rat brain tissue was accomplished by the procedures of brains homogenization and deproteinization. The extracts were passed through an ODS mini-cartridge (Bond Elut C18) for clean-up, because many biological substances in the sample interfered with the present fluorescence derivatization of the peptides. This technique has often used for the partial purification14 of bioactive peptides in complex samples. The use of the cartridge facilitated not only the removal of large amounts of the interfering substances but also the concentration of the target peptides in a small sample size. The recoveries of 100 pmol each of LE, ME,

Fig. 4 Chromatograms of (A) cortex, (B) striatum and (C) hypothalamus samples after the enzymatic degradation with carboxypeptidase A. The arrows (1- 3) indicate the retention times of ME, LE and [D-A1a2,3]-ME,respectively. ANALYTICAL SCIENCES JUNE 1989, VOL. 5 293 at a signal-to-noise ratio of 3. Fothergill, B. A. Morgan and H. R. Morris, Nature The precision of the method was established by [London], 258, 577 (1975). repeated determination (n=7) of the same cortex 2. M. Knight and W. A. Klee, J. Biol. Chem., 253, 3843 sample. The relative standard deviations were 4.2 and (1978). 3.6% for 20 pmol LE and 153 pmol ME per g of the 3. C. Bailey and I. Kitchen, J. Pharmacol. Methods, 13, 235 tissue, respectively. The concentrations of LE and ME (1985). 4.. C. Gros, P. Pradelles, C. Rouget, 0. Bepoldin and F. in various tissues of the rat brain were determined by Dray, J. Neurochem., 31, 29 (1987). the present method (Table 2). The results for the 5. A. Cupo, M. Eybalin, G. Patey, J. Rossier and T. Jarry, determination were in good agreement with the published Neuropeptides, 4, 389 (1984). data.4,5 6. G. Zamboni, C. A. Jones and J. Hughes, Anal. Biochem., The sensitivity of the present HPLC method is com- 130, 83 (1983). parable with other HPLC methods combined with mass 7. R. Simatov, S. R. Childers and S. H. Synder, Brain Res., spectrometric detection8 and electrochemical detection", 135, 358 (1977). though it is nearly one order of magnitude lower than 8. D. M. Deiderio, M. Kai, F. S. Tanzer, J. Trimble and C. that of RIA.4'5 However, this method has the necessary Wakelyn, J. Chromatogr., 297, 245 (1984). selectivity and sensitivity for the quantification of the 9. T. Sawagawa, T. Okuyama and D. C. Teller, J. enkephalin peptides in various tissues of the rat brain. Chromatogr., 240, 329 (1982). 10. J. Rivier, R. McClintock, R. Galyean and H. Anderson, This study provided the first practical HPLC method J. Chromatogr., 288, 303 (1984). with fluorescence detection for the simultaneous deter- 11. S. Mousa and D. Couri, J. Chromatogr., 267, 191 (1983). mination of the endogenous LE and ME in the rat 12. M. Kai and Y. Ohkura, Anal. Chim. Acta,182,177 (1986). brain. 13. M. Nakano, M. Kai, M. Ohno and Y. Ohkura, J. Chromatogr., 411, 305 (1987). This work was partly supported by a Grant-in-Aid for 14. H. P. J. Bennett, C. A. Browne and S. Solomon, Scientific Research from the Ministry of Education, Science Biochemistry, 20, 4530 (1981). and Culture, Japan. (Received January 13, 1989) References (Accepted April 28, 1989)

1. J. Hughes, T. W. Smith, H. W. Kosterlitz, L. A.