Anal. Chem. 1981, 53, 2190-2193
2190
Fluorometric Determination of Thiols by Liquid Chromatography with Postcolumn Derivatization Hiroshi Nakamura" and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku, Tokyo 1 13, Japan
An HPLC method has been developed for the fluorometric determination of L-cystelne (CySH), glutathione (GSH), 3'dephosphocoenzyme A( DP-CoASH), and coenzyme A (CoASH). The compounds are separated by anlon-exchange chromatography with gradient elution, reacted with the postcolumn reagents, o-phthalaldehyde (OPA) and taurine at pH 10, to produce highly fluorescent isoindole fluorophores, and monitored with a fluorescence detector (Aex = 360 nm; ,A > 405 nm). With the optimized conditions for chromatography and the postcolumn derivatlzatlon, 30 pmol of CySH and GSH and 100 pmol of DP-CoASH and CoASH can be determined. The relative standard deviatlons of the method are 4.4, 6.6, 3.2, and 8.2% for the analyses of 500 pmol of CySH and GSH and 1 nmol of DP-CoASH and CoASH, respectively.
Because thiol compounds play a diverse and physiologically important role in living cells ( I ) , a sensitive and specific analytical method is highly desired. High-performance liquid chromatography (HPLC) with fluorescence detection is probably one of the most suitable means for the analysis of biological materials since it provides both efficient separation and selective detection. For the fluorometric determination of thiols by HPLC, only two methods have been reported by using precolumn derivatization with dansylaziridine ( 2 , 3 )or N-(9-acidiny1)maleimide (4). The dansylaziridine method has a disadvantage that the reagent itself is fluorescent and gives a large interfering peak. The other precolumn method using N-(g-acridinyl)maleimidehas been reported to form multiple fluorescent products due t o the hydrolysis of the initial fluorescent product ( 4 ) . Furthermore, these precolumn derivatization methods require incubation at about p H 8 for at least 1h to form fluorescent products. Under such conditions the oxidation of thiols and the reaction of thiols with coexistent disulfides are inevitable. Therefore, we searched for a fluorogenic reaction suitable for the postcolumn derivatization of thiols. In view of this, the o-phthalaldehyde (OPA) reaction for primary amines in the presence of thiol (5, 6) seemed appropriate. The mechanism of the reaction was clarified by Simons and Johnson (7-9) as in Scheme I. In this paper, the separation of biogenic thiols such as L-cysteine (CySH), glutathione (GSH), and coenzyme A (CoASH) and the optimized postcolumn derivatization of them with OPA and a primary amine (taurine) is reported. EXPERIMENTAL SECTION Materials. The packed columns (4.6 mm i.d. X 25 cm) of Partisil-10 SAX (10 pm, microparticulate silica-bonded strong anion exchanger) and AS-Pellionex SAX (polystyrene matrix strong anion exchanger) were purchased from Whatman (Clifton, NJ). Cysteamine-HC1 (guaranteed reagent, GR), glutathione (reduced, GR), coenzyme A (lithium salt from yeast, GR), dithiothreitol (GR), 2-mercaptoethanol (extra pure, EP), DLpenicillamine (GR),thiophenol (GR),thiosemicarbazideHC1 (GR), 2-mercaptobenzimidazole (GR), 6-mercaptopurine hydrate (GR), 6-thioguanine (GR), ~-(-)-cystine-2HCl(GR), m-or-lipoic acid (GR), glutathione (oxidized, GR), thiosalicyclic acid (GR), 0-
Scheme I
aCH0 +
CHo 5-R
@N-R'
RSH
+
R'NH~
I
+
2H20
mercaptopropionic acid (GR), thioglycolic acid (GR), a-mercaptopropionic acid (GR), ethanethiol (EP) (Tokyo Kasei, Tokyo, Japan), tri-n-butylphosphine, DL-homocysteine (GR), N-acetylL-cysteine (GR), taurine (GR), @-cyclodextrin(GR), o-phthalaldehyde (GR),cystamine.2HCl (GR), thiomalic acid (EP), K&06 (GR) (Nakarai Chemicals, Kyoto, Japan), citric acid (GR), lithium hydroxide monohydrate (GR), boric acid (GR), L-cysteine (GR), Na2S.9Hz0 (GR), NaHS03 (GR), NazSO3.7Hz0 (GR), Na2SzO3.5Hz0 (GR), NazSzO, (EP), NazSzO, (GR), NazS207(GR), KzSzOB(GR), and ethylenediaminetetraacetic acid disodium salt (EDTA, GR) (Kanto Chemical, Tokyo, Japan) were used as received. 3'-Dephosphocoenzyme A dilithium salt pentahydrate was kindly provided by Daiichi Seiyaku (Tokyo, Japan). Preparation of Thiol Solutions. Ten millimolar solutions of thiols were prepared with 0.2 M lithium citrate buffer (pH 4.0) containing 0.1 mM EDTA. The buffer containing EDTA was used to prepare dilute solutions. HPLC Apparatus. The flow diagram for the HPLC postcolumn derivatization of thiols is shown in Figure 1. All tubings, coils and loops used were made of stainless steel (0.5 mm i.d. X in. 0.d.). The eluent was delivered through a Mini-micro pump (Type KHD-16; Kyowa Seimitsu, Tokyo, Japan). The gradient device (Model GE-2) provided by Toyo Soda (Tokyo, Japan) was used with mode X. A Pyrex glass tube (4 mm i.d. X 5.5 cm) packed with AS-Pellionex SAX was placed before the analytical column and served as the guard column. The column temperature was ambient. In-stream injection of samples was performed with a 10-pL Hamilton syringe through a line sample injector (Type KLS-3T; Kyowa Seimitsu) which was connected to a six-wayvalve (Type KMH-6V; Kyowa Seimitsu) attached with a 55-fiL loop. A 10-cm pulse-damping column (1mm i.d.; Durrum, Palo Alto, CA) filled with 40-pm glass beads was placed between the pump and the six-way valve. The eluate was mixed in a three-way tee (type KYS-16; Kyowa Seimitsu) with 25 mM OPA in 50% methanol, delivered a t a flow rate of 0.5 mL/min with a double plunger-type Mini-micro pump (type WD-1; Kyowa Seimitsu). The outlet of the tee was connected to a 38-cm length of tubing, which was connected to the second three-way tee. The mixture of eluate and OPA was mixed in the second tee with 10 mM taurine in 0.4 M sodium borate buffer (pH 10.0 delivered at a flow rate of 1.2 mL/min with the double plunger type Mini-micro pump. The outlet of the second tee was connected to a 75-cm reaction coil and its end was introduced to a 14-pL quartz flow cell in a Shimadzu fluorescence detector (type FLD-1; Shimadzu Seisakusho, Kyoto, Japan) equipped with a coated low-pressure mercury lamp emitting light a t 300-400 nm(maximum intensity at 360 nm) and an EM-3 secondary filter which cuts off light shorter than 405 nm. The outlet of the flow cell was connected to a 10-m back-pressure coil. The fluorescence intensity was recorded with a Shimadzu recorder (type R-12; Shimadzu Seisakusho).
0003-2700/81/0353-2190$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981
Figure 1. Flow diagram ior the HPLC postcolumn derivatization of thiols: G, gradient device; P, pump; D, pulse-damping column; PG, pressure gauge; S, samplirig device; GC, guard column; AC, analytical column; 0, OPA reagent; T, taurine reagent; F, fluorescence detector; R, recorder; W,waste.
Procedure for the Examination of Stability of Thiols a t Various pH Levels. A 10 mM thiol (CySH or GSH) solution of 0.1 M buffers (200 p1,) with or without 0.1 mM EDTA was stored at room temperature (ca. 20 "C) in an Eppendorf polypropyrene 1.5-mL microtube tightly capped. A 5-pL aliquot was withdrawn at appropriate intervals for the determination of thiol by the HPLC method described above. As the thiol control, 10 mM thiol (CySH or GSH) solution (200 pL), containing 10 pL of tri-n-butylphosphine, in 0.1 M phosphate buffer (pH 7.0) was stored overnight at room temperature and sampled as described above. The residual amounts of thiol were calculated by using a peak height ratio method. Tri-n-butylphosphine was reported to be an effective reducinig agent of disulfides t o thiols (10, 11). Quantitation of Thiolg. Typically, to 50 pL of sample solution was added 50 p L of a given concentration of N-acetyl-L-cysteine (NacCySH) which was used as an internal standard. The mixture was then vortex-mixed and an aliquot, usually 5 pL, injected. The amount of thiol was calculated from the working curve of the peak height ratio to the internal standard vs. picomoles of the thiol. RESULTS S e p a r a t i o n of Biogenic Thiols. The conditions for the separation of nonprotein thiol were first examined by using unoptimized post,column derivatization. Since the thiols are generally unstable in neutral and alkaline media, the entire procedure was performed under acidic conditions and the separation of thiols by ariion exchange HPLC was examined. The biogenic thiols were well separated on Partisil-10 SAX with a linear gradient elution at a flow rate of 0.7 mL/min, using 10 mM citric acid (solvent A) added with 200 mM lithium citrate, pH 4.66, (solvent B) at a rate of lO%/min to 100%. After elution of the sample, the column was equilibrated for 10 min with solvent A. As shown in Figure 2, cysteamine (CyNH2), CySH, GSH, N-acetyl-L-cysteine (NAcCySH), 3'-dephosphocoenzyme A (DP-CoASH) and CoASH were separated within 18 min. The relative standard deviations (n = 8) of the elution times were 1.31, 1.26, 2.38, 0.41, 0.45, and 0.41% for Cy",, CySH, GSH, NAcCySH, DP-CoASH, and CoASH, respectively. If the determination of DP-CoASH and CoASH was not required, the isocratic elution with solvent A could be used to separate CyNH2, CySH, and GSH. D e t e r m i n a t i o n of Conditions for the P o s t c o l u m n Fluoresceince Derivatization. Taurine was arbitrarily used as a primary amine reagent for the fluorogenic reaction for thiols. OPA and taurine could not be used in a mixture a8 the postcolumn derivatization reagent because of the red color that was generated. The color became intense with time with concomitant decrease in the capability of inducing fluorescence from thiols. Therefore, the column eluate was first mixed with OPA solution and then with taurine solution. T o optimize fluorescence reaction conditions, a 4-pL aliquot of 0.2 M citric
0
2191
IO
MINUTES Figure 2. Elution curve of a synthetic mixture of thiols. Sample size 6 nmol of consisted of a 10-pL aliquot containing 30 nmol of Cy",, CySH, 10 nmol of GSH, 10 nmol of NAcCySH, 40 nmol of DP-CoASH, and 40 nmol of CoASH. Other conditions are the same as described in the Experimental Section. The small peak between NAcCySH and DP-CoASH is an unidentified component in the DP-CoASH reagent.
Figure 3. Effect of the length of reaction coil on the fluorescence intensity. Sample size consisted of a 4-pL aliquot containing 10 nmol of Cy",, 3 nmol of CySH, 7 nmol of GSH, and 20 nmol of CoASH. Other conditions are described in the text.
acid solution containing CyNH2 (10 nmol), GSH (7 nmol), and CoASH (20 nmol) was injected. The flow rate of the eluent was fixed a t 0.7 mL/min. When 37 mM OPA in 50% methanol at a flow rate of 0.3 mL/min and various concentrations of taurine in 0.4 M sodium borate buffer (pII 10.0) at a flow rate of 1.2 mL/min were pumped into the eluate containing thiols, 5-10 mM taurine solution induced maximal fluorescence from CySH, GSH, and CoASH. For Cy",, more than 50 mM taurine was required. When various concentrations of OPA in 50% methanol and 10 mM taurine in 0.4 M sodium borate buffer (pH 10.0) were mixed at flow rates of 0.3 and 1.2 mL/min, respectively, 25 mM OPA solution gave maximal fluorescence from the thiols except CyNH2. In the case of CyNII,, the lowest concentration of OPA tested, Le., 1 mM, gave the maximal fluorescence. Maximal fluorescence was obtained when approximately equal moles of OPA and taurine were reacted with thiols other than Cy",. To acertain this, 25 mM OPA and 10 mM taurine were pumped at various flow rates so that the total flow rates were constant (4.4mL/min). The maximal fluorescence was obtained when the molar ratio of OPA/taurine was ca. 1.25. The reactions of CySH, GSH, and CoASH with OPA and taurine were found to be optimal in the range of pH 8.5-10.5, though the maximal fluorescence was obtained with CyNHz at around pH 7. Thus optimal fluorogenic reaction for CySH, GSH, and CoASH was with 25 mM OPA in 50% methanol (0.5 mL/min) and 10 mM taurine in 0.4 M sodium borate buffer (pH 10.0) (1.2 mL/min). Since it was found that the
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981
Table 11. Excitation and Emission Maximaa of Fluorophores in the Absence or Presence of 0-Cyclodextrinb
Table I. Relative Fluorescence Intensities of Various Thiols and Related Compounds Obtained b y Flow Injection Analysisa compound p-mercaptopro-
RFI 285
pionic acid
2-mercaptoethano1 ethanethiol N-acetyl-L-cysteine dithiothreitol DL-homocysteine L-cysteine coenzyme A glutathione 3'-dephosphocoenzyme A cysteamine thiophenol wmercaptopropionic acid thioglycolic acid DL-penicillamine Na,S Na,S*O,
225
155 154 139
119 112 101 100
64.9 31.3
26.4 9.33 4.18 1.85 1.23 1.02
compound thiomalic acid NaHSO, Na, SO
Na2S20, glutathione, oxidized L-(- )-cystine DL-a-lipoic acid cystamine thiosalicylic acid Na,S,O, K2S,0, 6-thioguanine 6-mercaptopurine 2-mercaptobenzimidazole thiosemicarbazide Na,S,O, KlS208
without p-CD
RFI 0.82 0.77 0.70 0.68
0.43 0.28 0.07
0.04 0.02 0.02 0.01 0 0 0 0 0 0
a Apparatus: the HPLC postcolumn derivatization system (Figure 1)from which the guard column and the analytical column are removed. Sample size: a 10-pL aliquot containing 2-1000 nmol of a test compound. b Glutathione is arbitrarily taken as 100.
reaction was completed within several seconds after mixing the taurine reagent (Figure 3), a 1-m length of the reaction coil, which corresponded to a residence time of 6 s, was chosen without substantial band broadening. The time between the mixing of the OPA solution and the addition of the taurine solution did not influence on the final fluorescence intensity. The relative fluorescence responses of various thiols and related compounds using these conditions are summarized in Table I. The fluorogenic reaction gave intense fluorescence for aliphatic thiols while aromatic thiols exhibited no fluorescence except for thiophenol. Inorganic sulfur acids gave weak or no fluorescence. Effect of t h e Addition of (3-Cyclodextrin on t h e Fluorescence Intensity. When P-cyclodextrin (0-CD) was added in the taurine solution, the fluorescence originated from CoASH was enhanced by 80% in concentrations higher than ca. 5 mM p-CD. Similar results were obtained with DPCoASH. However, the addition of (3-CD slightly lowered in the fluorescence intensities induced from CySH and GSH. The excitation and emission maxima of various thiols in the presence and absence of p-CD are summarized in Table 11. It is noteworthy that the excitation maxima in the presence of p-CD showed blue shift with most of thiols while the emission maxima were not influenced. Stability of Thiols at Various pH Values. The oxidation of 10 mM of CySH and GSH was negligible for a t least 16 days a t room temperature in media ranging from p H 2 to 5 . At p H 6 and above, the thiols were oxidized with time. Although the presence of 0.1 mM EDTA was somewhat effective to prevent thiols from oxidation a t pH 6, it could not provide marked effect on the stabilization of thiols a t higher pHs. Quantitation of Biogenic Thiols. On the basis of the above findings, an HPLC system for the postcolumn derivatization of biogenic thiols was established as described in the Experimental Section. The lower limits of determination were 30 pmol for CySH and GSH and 100 pmol for DP-CoASH and CoASH as injected amounts. The precision of the method was evaluated by analyzing the mixture of CyNH2, CySH,
-
thiol
hex,
hem,
hex,
nm
nm
349 347
404 406
337 337
338
407
336
348 344 344 349 346 352
407 421 421 422 429 430
339
nm
cysteamine L-cysteine Dl-penicillamine dit hiothreitol 2-mercaptoethanol DL-homocysteine glutathione coenzyme A IV-acetyl-l-cysteine
with O-CD I_-___
hen,,
nm
339
337 338
339 340
406 408 410 426
426 409 418 428 424
a The excitation and emission maxima are not corrected. Twenty microliters of 0.1 mM thiol in 0.2 M citric acid, 1.1mL of 1 0 mM citric acid, and 1.0 mL of 10 mM taurine in 0.4 M sodium borate buffer (pH 10.0) with or without 1 0 mM 0-CD were mixed together, the solution was rapidly mixed with 0.5 mL of 25 mM QPA in 50% methanol with vigorous stirring for 5 s, and then the fluorescence was measured immediately. __-____.
GSH, DP-CoASH, and CoASH eight times. The standard deviations were 0.113 nmol for 2 nmol of CyNH2, 0.022 nmol for 0.5 nmol of CySH, 0.033 nmol for 0.5 nmol of GSH, 0.032 nmol for 1 nmol of DP-CoASH, and 0.082 nmol for 1 nmol of CoASH, respectively.
DISCUSSION CyNHz gave markedly different results from those of CySH, GSH, and CoASH in the examination of conditions for the fluorescence derivatization. The behavior of Cy", cannot be explained by the difference in basicities of the reactive functional groups since the pK, values of NH2 group and SH group of CyNHz are similar to those of CySH and GSH ( I ) . Our results indicate that the reaction of the NH2 group of CyNHz with OFA leads to the decreased formation of fluorophore. In fact, Cy", and CySH which contain NH2 and S H groups and should produce isoindole-type fluorophores (A) according to Scheme I gave weak or no fluorescence upon reacting with OPA in the absence of other primary amines. This may be explained by either the occurrence of side reaction(s) to form nonfluorescent product(s) or nonfluorescent property of A due probably to the strain resulting from the five-membered ring as compared to the 10-membered ring fluorophore (B) obtained by the reaction of GSH with OPA (12).
9 dN
HOOC-CH2-NH-CO..
&C
A>(cooH)
cooti
\
A
\/
B
In the present procedure, penicillamine (P$-dimethylcysteine) gave only a fraction of fluorescence intensity of CySH, which is obviously due to the steric indrance of the SH group by the two methyl groups. Steric hindrance caused by the methyl groups has been observed in the reduction of penicillamine-S-sulfonic acid by dithiothreitol (13, 14). Many HPLC methods have been reported for the determination of thiols. The HPLC methods using ultraviolet detection (15-20) lack both specificity and sensitivity. Although various electrochemical detectors (21-23) have been developed for increased sensitivity, they were not necessarily specific for thiols. They showed similar response to other compounds, e.g., Agf, Fe(CN)6", S2-,SO$-, Sz032-,and organic
ANALYTICAL CHEMISTRY, VOL. 53, NO. 14, DECEMBER 1981
sulfur compounds such as thiourea (21). When the present investigation was initiated, there was no HPLC method for thiols with post column fluorescence derivatization. During the preparation of thici manuscript, two papers on the POstcolumri fluorescence derivatization of thiols appeared. In the first paper (241, thiols were fluorometrically detected by the postcolumn ligand-exchange reaction between the nonfluoresceiit pdadium(II)-calcein complex and thiols liberating fluorescent calcein. However, the reaction is not specific for thiols and many sulfur.containing compounds yield fluorescence upon reacting with the reagent. The second H P I X method ((25)involves the separation of thiols on an anion exchange column and successive derivatization with N-(9acridiny1)maleimide. The determination limits of the latter method are reported to be 50 pmol of CySH and GSH and 100 pmoll of CoASH by the use of the variable-wavelength fluoresceince detector; the sensitivity is slightly lower than the present method. The fluorescence enhancement by cyclodextrins of a variety of organic compounds has been reported (26-29). Our results that the fluorescence enhancement by 0-CD was observed with CoASH and DP-CoASW but not with CySH and GSW suggest that the interaction of 0-CD with the adenine moieties but not with the isoindole ones is responsible for the above phenomena. The main purpose of the present investigation was to demonstrate the applicability o f the QPA reaction to the postcolumn fluorescence derivatization of thiols. We used taurine arbitrarily as the primary amine reagent in the labeling reaction, but the use of other primary amines may lead to the increased sensitivity. The use of a variable-wavelength fluoresceince detector nnstead of the filter-type used in the present work will be also advantageous to increase sensitivity as well as selectivity. The relative standard deviations of the present rnethod were somewhat high (3-8%). Since the relative standard deviations of the elution times were below 2.4%, the majority of tlhe error seems to be ascribed to the postcolunnn reaction. In that case, use of dampers, which weaken pulsating flows of OPA and taurine reagents, will result in increased reproducibility.
2193
LITERATURE CITED (1) Jocelyn, P. C. "Biochemistry of the SH Group"; Academic Press: New York, 1972. (2) Lankmayr, E. P.; Budna, K. W.; Muller, K.; Nachtmann, F. Fresenius' Z. Anal. Chem. 1979, 295, 371-374. (3) Lankmyr, E. P.; Budna, K. W.; Mulier, K.; Nachtmann, F. J . Chromatogr. 1981, 222, 249-255. (4) Takahashi, H.; Nara, Y.; Meguro, H.; Tuzirnura, K. Agric. Blol. Chsm. 1979, 43, 1439-1445. (5) Rath, M. Anal. Chem. 1971, 43, 660-882. (6) Benson, J. R.; Hare, P. E. Proc. Natl. Acad. Sci. U.S.A. 1975, 72, 619-622. (7) Simons, S. S.,Jr.; Johnson, D. F. J . Am. Chem. SOC. 1978, 98, 7098-7099. (6) Simons, S. S. Jr.; Johnson, D. F. J . Chem. Soc., Chem. Commun. 1977, 1 1 , 374-375. (9) Simons, S. S.; Johnson, D. F. J . Org. Chem. 1978, 43. 2886-2891. ( I O ) Humphrey, R. E.; Potter, J. L. Anal. Chem. 1985, 37, 164-165. (11) Kirkpatrick, A.; Maciaren, J. A. Anal. Blochem. 1973, 56, 137-139. (12) Simons, S. S.; Johnson, B. F. Anal. Blochem. 1978, 90, 705-725. (13) Nakamura, H.; Tamura, Z. J . Chromatogr. 1975, 104, 389-398. (14) Nakamura, H.; Tamura, 2 . Chem. Pharm. Bull. 1977, 25, 2369-2377. (15) Sakai, J.; Mizuta, H. Bunseki Kagaku 1978, 27, 744-748. (16) Baker, F. C.; Schooiey, D. A. Anal. Blochem. 1979, 94, 417-424. (17) Ingebretsen, 0. C.; Normann, P. T.; Flatmark, T. Anal. Blochem. 1979, 96, 181-168. (16) Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Eiiis, W. W.; Potter, D. W. Anal. Biochem. 1980, 106, 55-62. (19) Haivorsen, 0.; Skrede, S. Anal. Biochem. 1980, 107, 103-108. (20) Reeve, J.; Kuhienkamp, J.; Kaplowitz, N. J . Chromatogr. 1980, 194, 424-426. (21) Rabenstein, D. L.; Saetre, R . Anal. Chem. 1977, 49, 1036-1039. (22) Saetre, R.; Rabenstein, D. L. Anal. Chem. 4978, 50, 276-260. (23) Rabenstein, D. L.; Saetre, R. Clin. Chem. (Wlnston-Salem, N . C,) 1978, 24, 1140-1 143. (24) Werkhoven-Goewie, C. E.; Niessen, W. M. A.; Brinkman, U. A. Th.; Frei, R. W. J . Chromatogr. 1981, 203, 165-172. (25) Takahashi, H.; Yoshida, T.; Meguro, H. Bunsekl Kagaku 1981, 30, 339-341. (26) Cramer, F.; Saenger, W.; Spatz, H.-Ch. J . Am. Chem. SOC. 1987, 89, 14-20. (27) Seiiskar, C. J.; Brand, L. Science 1971, 171, 799-800. (28) Kinoshita, T., Iinuma, F.; Tsuji, A. Biochem. Blophys. Res. Commun. 1973, 51,666-671. (29) Kondo, H.; Nakatani, H.; Hiromi, K. J . Blochem. (Tokyo) 1976, 79, 393-405.
RECEIVED for review June 23,1981. Accepted September 15, 1981. Presented in part at the 10lst Annual Meeting of the Pharmaceutical Society of Japan, Kumamoto, April 2-4,1981. The authors are grateful to the Pharmacological Research Foundation for partial support of this work.
