Recent developments in the determination of acetylcholine and choline

77PS -March

84

about whose formation we all seem to know so much at the molecular level, what with heterosynaptic activity dependent, CAMPmediated interactions and the like, be in any way dependent on plain old sugar? Well. the brain does indeed use glucose as its main scrrrrce of energy, except in conditions of extreme fasting; and recent experiments measuring labelling with 2deoxyglucose do suggest that different brain regions subtly modify their use of glucose depending on the type and/or of the stage of leaming’2,‘3. And then, Gold’s arguments are there; they are every bit as compelling for glucose as his own previous data and those of many others on epinephrine and epinepbtine is indeed widely regarded as a post-trial modulator of memory, both for appetitive and

for aversive tasks2,3*9, if not, as was recently ploposed’l, as an eventual addition to the reinforcements. It will now take as much further experimentation to ascertain the validity of Gold’s recent proposal on the role of ost-training glucose in memo ry5-H, as to reaffirm the older claim by Gold himself1*3, supported by so much evidence’, that epinephrine, or other stress hormones, play a role in memory storage. IVAN

IZQUIERDO

de Memoria. Departamento de Bioquimica, Institufo de Biociencias, UFRGS (cenfro), 90049 Porto Alegre, RS, Brazil. Centro

10 11 12

References 1 Gold, I’. E. and van Buskirk, R. 8. (1975)

Behav. Biol. 13, 145-153 2 McGaugh, J. L. (1983) Annu. Rev. Psychol. 34, 297-323 3 Gold, P. E. and McCarty, R. (1981) Behav.

Recent developments in the determination of acetylcholine and choline Attempted deteminations of acetylcholine (ACh) and choline (Ch) in nervous system tissues and fluids by standard physicochemical techniques have been fraught with difficulties’,‘. The distinguishing structural characteristics of these species (Fig. 1) are represented by the acetyl ester of ACh, the hydroxyl group of Ch and the quatemary amine of both. Acetyl esters and nydroxyl groups are fairly common constituents of many biological samples, but the remaining functionality, the quatemary ammonium group, is relatively unique in nature and would thus appear to be a more appropriate point for the primary focus of attention. Quantitative chemical transf&mation of this group is very demanding, requiring high temperatures or specialized reagents. Thus, it would seem appropriate to leave this group as it is found endogenously. However, there are currently no direct physical methods for the detection of very low levels (s pmols) of quatemary amines and enzymic coiiversion to a more tractable

9

chemical form for direct chemical analysis is usually required. Early bioassays, while providing excellent detection limits and being quite simple, were subject to substantial interference by agonists and antagonists from both endogenous and exogenous sources. The initial chemical assays generally re!ied on enzymes to selectively attach a radiolabelled group which was subjected to direct radiochemical analysis following separation from the source of the labe13. These assays still offer excellent selectivity, primarily through the enzymes employed and moderate to low detection limits. However, they entail considerable sample manipulation. Radioreceptor and radioimmunological approaches to the determination of ACh have also appeared. But these have been shown to suffer from similar disadvantages and, in the case of the latter, from unexpected interferences by selected drugs. Other techniques employed for ACh and Ch determinations include spectrophotometry, liquid

13

1987 [Vol. 81

Neural Biol. 31, 247-260 Minnemann, K. P., Pittman, R. N. and Molinoff, P. B. (i%i) Annr. Rev. Neurosci. 4, 419-461 Gold, P. E. (1986) Behav. Neural BioL 45, 342-349 Gold, P. E.; Vogt, J. and Hall, J. L. (1986) Behav. Neural Biol. 46, 145-155 Ha& J. L. and Gold, P. E. (1986) Behav. Neural Biol. 46, 156-167 Gold, P. E. (1986) in Learning and Memory: Mechanisms of Information Storage in the Nervous System (Matties, H., ed.), pp. 307-314, Pergamon Press Stemberg, D. B., Isaacs, M., Gold, P. E. and McGaugh, J. L. (1985) Behav. Neural Biol. 44,447-453 De Wied, D. (1966) Proc. Sot. Exp. Biol. Med. 122.28-32 Izquierdo, I. (1986) Trends Pharmacol. Sci. 7, 476-07 Gonzalez-Lima, F. and Scheich, H. (19&I) Newosci. Letl. 51, 79-85 Destrade, C., Sif, J., Gauthier, M., GaIey, D. S., Durkin, T. and CaIas. A. (1986) in Learning and Memory: Mechanisms of Informatioir Storage in fhe Nervous System (Matthies, H., ed.), pp. 289-293, Pergamon Press

chromatography combined with LJV detection, fluorometry, and indirect determination of ACh using polarography. These generally afford inadequate detection limits for the more demanding samples and the intermediate in the polarographic procedure, iron, is subject to undesirable redox reactions. The procedure which is currently capable of providing the greatest degree of selectivity incorporates gas chromatography mass spectrometric detection (GCMS)4; operation of the mass spectrometer in an alternative mode also allows this procedure to provide the iowes!, or very close to the lowest, detection iimits. However, this is a very demanding

(CH,),N+-“-

II 0

Acetylcholine

(CH$3N+-dH Choline Fig. 1. The chemical struchtres of acetylcholine (ACh) and choline (Ch).

TIPS - March

1987 iVol.81

Waste Fig. 2. LCECsystem fordeterminafion of choline and acetyicholine. A: mobile phase; B: et?zyme solution; C: high-pressure pump; D: pressure gauge; E: injection valve; F: sampling 100~: G: analviical column: H: Tw&ector; I: &action wil; J:. electrochemical eelI( K: potentiostat (amplifier); L: recorder; 4: eluate stream: ---4: electrical signal.

technique, requiring rather extensive purification and chemical or pyrolytic derivatization of the sample prior to chromatographic separation and providing only a modest throughput. In addition, the highest degree of selectivity and the lowest detection limits cannot be simultaneously accessed with this instrumentation. Maximal selectivity, i.e., highly precise and confident identification of the pyrolysis product of either ACh or Ch, requires a complete mass spectral scan covering the entire (m/e) range of mass-to-charge ratios. This is not possible with the very low levels of fragment ions produced at or near the detection limit. The lowest detection limit, conversely, requires the constant monitoring of a single ion from the entire mass spectrum. Thus, the lowest detection limits of GCMS are achieved by relinquishing much of the inherent selectivity and vice versa. Greater selectivity

85 in GCMS can be obtained by using capillary (PP other GC columns with greater resolving capabilities in comparison with the usually employed packed columns’. But, this gain in selectivity must be balanced by consideration of the associated loss in injectable volumes and, it pressed to the limit, the excessive chromatographic retention times that would necessarily result from the use of columns having very large numbers of theoretical plates. Liquid chromatography with electrochemical detection (LCEC) now provides a reasonable alternative to the above mentioned procedures for ACh and Chd-15. Originally introduced by Potter, Meek, and Neff in 1983, it is based upon the same principles incorporated into the chemiluminescent procedure reported by Israel and Lesbats l6 . In the chemiluninescent approach, ACh is converted to acetate and Ch by added acetylcholinesterase. The Ch is then converted by choline oxidase to betaine along with the production of H202. Subsequent detection of the light produced from the reaction of H202 with luminol in the presence of peroxidase is quantitatively related to the amount of ACh originally present in the sample. While the final, light producing reaction initially suffered from interference due to metal. ions and, possibly, other unidentified endogenous substances, this problem has currently been solved by oxidation of the ACh and Ch containing supernatant with KIOS. A detection limit of ~500 fmol for ACh by chemiluminescerce looks very promising for general application, even though this approach requires an additional enzyme and greater

(cI+)~N+-‘~

+

WJ

amounts of sample manipulation in comparison to the LCEC technique. In the LCEC procedure, shown in Fig. 2, the original homogenization mixture. after addition of ethylhomocholine as an internal standard, is centrifuged, the suprrnatant is filtered, and the resultant filtrate is directly injected into the liquid chromatograph. Samples which yield substantial overiap of the resulting chromatographic solvent front peak with the Ch peak may be further purified by extraction of the supematant with diethyl ether, precipitation of the quatemary amine functionality or isolation by rapid, short column chromatographic techniques prior to injection6p9. The LC column in the LCEC setup provides separation of Ch, ethylhomocholine, and ACh from each other as well as from the vast majority of other sample components. Requiring only six to seven minutes per chromatogram, a throughput of approximately nine to ten samples per hour or 70-80 samples in an eight hour day is obtained using an automated system. The eluted components in the flow stream are subsequently passed through a short postcolumn reactor on which both acetylcholinesterase and choline reviously been oxidase have immobilized8*13*P6 as shown in Fig. 3. The Hz02 produced is then simply monitored at a platinum eiectrode, placed just downstream from the postcolumn outlet, with the potential set at +0.50 V v. Ag/AgCl (Fig. 4). The cqmbi_n.ation nf the I.C separation, the two enzymes and the electrochemical detection of H202 provide the substantial enhancement in selectivity associated with the method. While

Acetylcholinesterase -

!C”;)3N+,-oH

f

CH&OOH

+

ZHZOL

Ch

i-

Ch

W

Choline oxidase -

W,).+ +ficw

202

beta&

Fig. 3. Enzymicreactions of ACh and Ch in the postwlumn leading to electrochemical detection as &OS

86 surprisingly few chemicals are electrochemically active at the electrode under these conditions and, simultaneously, present in sufficient amounts in common samples to possibly interfere with the 1-1202 detection, one could, if desired, run a ‘background’ by injecting the sample into a system from which the postcolumn was temporarily removed. However, such interference has not been observed to be a problem in our experience. Likewise, hydrodynamic vJ+~m~ograms “..“I..*. of the detected H202 could be constructed to increase the selectivity. Detection limits for ACh and Ch using this procedure have been reported to be as low as 100 fmol’-. The linear dynamic range for both compounds is at least 104, which is completely adequate for virtually anv, samole encountered without necessititing either dilution or Pt

7

+0.5 v

I

I

O2 + 2H+ + 2e’ I

concentration of the sample. Overall, the procedure may be considered to be comparable to cr just shy of the selective capabilities of the GCMS when both are operated at or near the detection limits. The detection limits for LCEC are slightly better than or comparable to those of GCMS. Yet, the sample throughput of the LCEC procedure is approximately twice that of the GCMS one, and the cost of the required instrumentation for LCEC is only one-half to one-tenth that of KMS, depending upon the particular choices for each. Also, the technical expertise required for routine operation, maintenance and repair is considerably less in the case of the LCEC. The many laboratories which have already employed LCEC for the determination(s) of catecholamines, indoleamines, and related enzymes*’ should find the establishment of the LCEC procedure for ACh and Ch to be quite simple. A major advantage of the LCEC approach is afforded by the immobilization of the required enzymes in the postcolumn. These

exhibit reagents immobi!ized stability for two to three months or longer, thus minimizing frequent and expensive replacement. In the future, this approach wilI almost certainly be incorporated into the radioenzymatic and radioimmunological procedures. The effects of interferences should be minimized by the use of substantially larger amounts of enzymes facili tatrd by lower costs. It is also relatively easy to imagine unique enzymic approaches for ACh and Ch incorporating novel reactions, perhaps even enzymereactions, amplifying coupled which would lead to a substantial lowering of detection limits. As far as the LCEC procedure is concerned, we anticipate the use of LC columns with even greater resolving power than those now employed, enhancing both the selectivity and sens’tivity of this approach. But, these advances will necessitate much greater attention to the associated dead volume of the chromatographic systemI’. The LC column employed will almost certainly have to be constructed with the enzyme containing material located at the very bottom of the current analytical separation column. Multiple electrode detection could be employed to enhance both the selectivity and detection limit of the LCEC technique. A dual parallel LC arrangement”, similar to that of Damsma, Westerink, and Horn*‘, could allow the simultaneous determination of ACh, Ch, catecholamines and indoleamines. The GCMS and closely related procedures will, undoubtedly, experience advances in the near future in their application to the determination of ACh and Ch. The existing techniques of GC-MS/MS, CC-high resolution MS, and MS/ MS can already be employed to provide completely specific detection of the commonly obtained demethylated derivatives of these compounds. MS/MS and, perhaps, MS/MS/MS may provide gains in sample throughput and detection limits while maintaining a very high degree of selectivity. It appears that LCMS techniques will shortly be employed for ACh and Ch19, although the detection limits for such procedures will almost certainly never match those provided by LCEC or GCMS. It also appears that ion selective

electrode techniques for ACh and Ch are beginning to approach the levels necessary fur in-ciao measurements2“. Thus, as has been the case in the past, the methods for the determination of ACh and Ch will surely continue to be refined and provide enhanced capabilities in the future. YUJI IKARASHI*

MARUYAMA,

YASUSHI

AND C. LEROY

BLANKt

Research Laboratories, Nippon Kayaku Co. Ltd., 3-31-12 Shimo, Kita-ku, Tokyo 115, Japan; l Research Laboratories for Applied Toxicoloau, Nivvon Kawaku Co. l.td.. 239 lwahann-machi, Takasaii, Gunma 370-12, Japan; t Department ofChemistry,University 0; Oklchoma, 620 Parring:on Oval, Norman, OK 73019, USA.

References 1 Hanin, I. (1982) in Modern Methods in 2 3

4 5

Pharmncologyy (Spector, S. and Back, N., eds). DD. . . 29-38 Hanin, I. (1974) Choline and Acehdcholine: Handbook of Chemical Assay Me:hods, Raven Press’ McCaman, R. E. (1985) in Methods in Enzymntic Annlysis (Bergmeyer, H. U., ed.), 3rd edn, Vol. 8, pp. 462-473 VerlageseIIschaft Newton, M. W., Ringdahl, B. and Jenden, D. J. (1983) Annl. Biochem. 130, 88-94 Find&, I’. M. and Farwell, S. 0. (1984) J. Hi@ Resofut. Chromatogr. Commun. 7, 19-24

6 Potter, P. E., Meek, J. L. and Neff, N. H. (1983) 1. Neurochem. 41, 188-194 7 Potter,. I-‘. E., Hadjiconstantinou, M., Meek, J. L. and Neff, N. H. (1984) J. Neurochem. 43,288-290 6 Eva, t., Hadjiconstantinou, M., Neff, N. H. and Meek, J. L. (1984) Anal. Biochem 143.320-324 9 Ikarasm, Y., and Sasahara, T. Maruyama, Y. (1985) J. Chromatogr. 322, 191-199 10 Meek, J. L. and Eva, C. (1984) J. ChromntroRr. 317, 343-347 11 Damsma, Gr, Westerink, B. H. C. and Horn, A. S. (1985) J. Neurochem. 45, 1649-1652 12 Bvmaster. F. I’., Perrv. K. W. and Wane. d. T. (1985) Life Sci.‘ti, 1775-1781 “. 13 Asano, M., Mivauchi, T., Kato, T., Fujimori, k. and Yamemoto, k. (1986) J. Liq. Chromatogr. 9, 199-215 14 Yao. T.. Sato. M. and Wasa. T. (1985) Nippon.Kagnku Kaishi 7,15Oi-1503 ’ 15 Yao, T. and Sato, M. (1985) Anal. Chim. Arta 172, 371375 16 Israel, M. and Lesbats, B. (1985) in Biohtminescence nnd Chemiluminescence: Instrumentation and Applications (Van Dyke, K., ed.), Vol. 2,pp. l-11, Chemical Rubber Co 17 Lin, P. Y. T., Bulawa, M. C., Wong, I’., Lin, L.. Scott, 1. and Blank, C. L. (19841 1. Lrq. Chromntogr. 7,509-538 18 Blank, C. L. (1976) J. Chromatogr. 117, 35-46 19 Liberato, D. J. and Yergey, A. L. (1986) Anal. Chem. 58,6-9 20 Jaramillo, A., Lopez, S., Justice, J. B., Salamone, J. D, and Neill, D. 8. (1983) Anal. Chim. Acla 146,149-159