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A simple and sensitive method of acetylcholine identification and assay
A Simple and Sensitive Method of Ace~lcholine Identification and Assay Bioassay Combined with Minicolumn Gel Filtration or High-Performance Liquid Chromatography
E. SYLVESTER
VIZI, LAsztt, G. HARSINC JR., DERYCK DUNCALF, HIDEO
NACASHIMA, PAMELAPOTTER, AND FRANCISF. FOLDES
A modified technique of acetylcholine assay on the guinea pig ileum has been combined with either minivoiume gel filtration or high-performance liquid chromatography separation of the samples. In addition, labeled acetylcholine (14CACh) was eluted with unlabeled acetylcholine or with the samples expected to contain acetylcholine, and their elution profiles were compared by bioassay plus radioassay of eluate fractions. When the elution of both biological activity and label occurred in the same eluates, it was concluded that the substance assayed on guinea pig ileum was acetylcholine. The method was sensitive to 0.5 ng f-l.8 pmot) of acetylcholine and its reproducibility was within 5%. It thus represents a substantial improvement in chemical specificity over the previous bioassay method described by Paton and Vizi (1969).
Key Words:
Acetylcholine
assay; Gel filtration;
matography;
Radiochemical
detection
High-performance
liquid chro-
INTRODUCTION There are many methods described for quantitative acetylcholine (ACh) estimation in tissue extracts or solutions. Of the chemical ones, some are relatively insensitive (Stone, 1955, calorimetry; Stavinoha and Ryan, 1965, gas chromatography). Other methods, while often sensitive and specific, are complex, time consuming, and require relatively expensive apparatus (Maslova, 1964, polarography; jenden et al., 1968; Schmidt et al., 1970, gas chromatography; Feigenson and Saelens, 1969, hydrolysis and radioactive labelling; Fellman, 1969, fluorescence; Israel and Lesbats, 1981, chmeluminescence; Potter et al., 1983, high-performance liquid chromatography). Of the numerous bioassay methods available (Dale and Dudley, 1929, isolated rabbit intestine; Feldberg and Gaddum, 1934, cat blood pressure; Chang and Caddum, 1933, eserine-sensitized frog rectus; Macintosh and Perry, 1950, blood pressure of eviscerated cat; Webb, 1950, isolated rabbit auricle; Straughan, 1958, From the Institute of Experimental Medicine Hungarian Academy of Sciences, Budapest, Hungary, and the Departments of Anesthesiology, Montefiore Medical Center and Albert Einstein College of Medicine Bronx, New York. Address reprint requests to ES. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, P.O.B. 67, Hungary. Received May, 1984; revised and accepted September, 1984.
201 burnalof
Pharmacological
Methods
0 1985 Efsevier Science Publishing
13, 201-211 11985) Co., Inc., 52 Vanderbilt
Avenue, New York, NY 10017
202
E. S. Vizi et al.
rat blood pressure; Dudar and Szerb, 1970, leech muscle; Vaapatalo et al., 1976, superfused hamster stomach strip), the guinea pig ileum assay appears to combine sensitivity, reproducibility, and specificity (Ryall et al., 1964) with simplicity. In addition, it has the following advantage: perfused solutions obtained from isolated mammalian nerve-muscle preparations can be assayed directly without the need for dilution or other modification. However, although bioassay techniques used to measure ACh offer accuracy and reproducibility, these techniques are not chemically specific. In the past two decades, several sophisticated methods have been developed that seriously rival the best bioassay methods (Hanin, 19741, but they are time consuming and require relatively expensive equipment and/or chemicals. This paper provides convincing evidence for the specificity of bioassay on guinea pig ileum extended by gel filtration or by high-performance liquid chromatography (WPLC) without a loss in sensitivity. Because the previous descriptions of the guinea pig ileum method in the literature (Paton and Vizi, 1969) were not sufficiently detailed, the preparation could not be set up without additional information. This paper describes the method of ACh assay on the supe~used guinea pig ileum in sufficient detail to avoid this problem. The details of the method are modified from Paton and Vizi (1969) to improve the stability of the preparation and the speed of the assay. METHODS
AND
RESULTS
Assay of Ace~Icholjne
Apparatus The apparatus required for the assay is shown in Figure 1. It was a water-jacketed cylindrical chamber (8 mm internal diameter, 70 mm in length, 3.5 ml volume). The chamber had three side arms and an extension at the bottom. A short piece of latex rubber tubing was attached to the bottom extension, which held the catheter to
Krebe’
soi.
thermostat
FIGURE 1. Water-jacketed glass organ bath in which the guinea pig ileum (or its longitudinal muscle strip) used for the assay of ACh was suspended.
Acetylcholine
identification
and Assay
which the aboral end of the gut is fastened. The lower side arm was also occluded with a clamped latex tube; oxygenation of the chamber occurred through a needle passed through the wall of this tube. The top side arm was the overflow port, which served to keep the fluid volume constant. Krebs solution was kept in a reservoir, and it was continuously bubbled with 5% CO1 in OZ. The reservoir was connected by latex rubber tubing to the middle side arm, which formed a spiral around the chamber and entered it near the bottom. In the spiral, the Krebs solution was warmed to the same temperature as that of the gut chamber. When the clamp on the tubing between the reservoir and the spiral was released, fresh Krebs flowed into the chamber and out through the top side arm. The height of the reservoir was adjusted so that the flow into the chamber was sufficient for three changes of solution in 15 sec. A thermostated water pump was used to maintain constant temperature in the water jackets. The appartatus was completed by a force displacement transducer (Grass FT 03) suitable for the recording of forces from 0.02 to 10 g, an appropriate amplifier, and a chart recorder or polygraph. The upper end of the gut was connected to the strain gauge through a small coil spring that rendered the contractions auxotonic. The compliance of the spring was approximately about 2 cm/gm. The complete assembly is shown in Figure 2. inclusion of a high-frequency filter in the amplifier was not deemed desirable. Solution Used for Pe~usion The preparation was made as described by Paton and Vizi (7969). A modified Krebs solution was used to perfuse the gut. The composition in grams per liter was as follows: NaCl6.60; NaHC03 2.10; KCI 0.35; dextrose 2.00; CaC& 0.28; KH2P04 O.f6; MgS04 x 7 HZ0 0.14. The last three components must always be added in the above order (CaC12 before KH2P04, and MgS04 x 7 Hz0 last) or precipitation will occur. Bubbling the solution with 5% COP in 02 before the addition of CaClz helped the latter to dissolve. Eserine sulphate, 2 ngll (5 x IO-’ mol/l), in the solution increased the sensitivity of the preparation, and morphine sulphate, IO mgil (1.2 x
Bridge Amplifier Strain
FIGURE 2. pig ileum.
gauge
Filter ( hum 8 noise)
Booster amplifier
I
Schema of complete assembly used for the bioassay of ACh on isolated guinea
203
204
E. S. Vizi et al.
10d6 mol/l), reduced intrinsic ACh release, thus eliminating the problem of spontaneous motility. If the samples to be assayed for ACh are expected to contain histamine, the effect of the latter can be blocked by the addition of diphenhydramine in a concentration of 0.2 mg/l (2.8 x lo-’ mol/l). This preparation can also be used for histamine assay; in this assay, atropine, 100 @I (3.4 x IO-’ mol/l), should be added to the basic solution to exclude the effect of ACh on the ileum. Assay Technique The assay was performed at 32-33X. If the temperature is lowered from 37”C, spontaneous motility is reduced with no loss in sensitivity. If the temperature is reduced further, however, an inconveniently slow response and relaxation occur. The assay was performed by a comparison of the response to the unknown sample with responses to known quantities of ACh. A “standard” solution of ACh was prepared in the same solution as the samples to be assayed. The standard ACh was injected in a volume of 0.1-0.3 ml by a 1 ml tuberculin syringe or by a 0.5 ml Hamilton syringe. RESULTS An example of a performed assay is shown in Figure 3. Injections of the unknown solution were made from a 0.5 ml syringe. The difference in the mode of injection between standard and unknown solutions did not affect the accuracy of the method. The working cycle was 60 sec. The injected ACh was allowed to act for IO sec. The preparation was then washed three times with a bath volume of fresh Krebs for 15 set, and it was ready for the next injection when the tracing had returned to the baseline. Regularity of the cycle was important. If the time elapsed between injections varies, the sensitivity of the gut changes.
a52
24@05WllQ4lW5@2ng 0.1
’ 0.25
0.1
ml
I
I
a5
1
;i 2
5
w
FIGURE 3. Results of ACh bioassay on isolated guinea pig ileum. The sample contained endogenous ACh released from eserinized longitudinal muscle strip of guinea pig ileum. Dose-contraction curve is for ACh. The ACh content in the unknown sample was determined by interpolation.
Acetylcholine identification and Assay To determine the ACh content of the unknown sample, the heights of the contractions induced by the two injections of standard were plotted on semilogarithmic paper (log dose versus response), and the quantity of ACh in the unknown solution was read off by interpolation of the height of the response (Figure 3). The concentration of ACh in the unknown sample was then calculated. After some time, spontaneous contractions of the gut may interfere with the assay. The most common cause for this is retention of secretions. These can be cleared by slow injection of IO-20 ml Krebs solution through the catheter to which the gut is tied. If the samples (and the standard) contain an anticholinesterase, this may also cause excessive motility after numerous exposures. Repeated washings for 10 to 15 min are required to reverse this, and the problem often reoccurs after only a few more samples. If this happens, it is best to replace the gut with a fresh piece. However, we have been able to assay over twenty samples, in duplicate, that contained eserine sulphate 2 kg/ml (5 x lop6 mol/liter) without encountering any problems. Samples that contain neostigmine or phospholine have also been assayed successfully. Ambenonium, however, caused prolonged contraction of the gut, and methoxyambenonium appeared to have an atropine-like effect. The response of the gut to ACh is not modified by d-tubocurarine chloride, gallamine triethiodide, or pancuronium bromide used in sufficient concentration to cause total neuromuscular block in the rat phrenic nerve-diaphragm. Decamethonium ioide, however, caused a slight contraction, although it did not interfere with the ACh assay when it was added to the standard. When the effect on ACh release of gastrin-like polypeptides present in the samples was studied and assayed, their interference with the assay ileum was prevented by the use of 0.25 kg/ml (7.8 x IO-’ mol/l) of tetrodotoxin (Vizi, 1973). The effect of ganglionic stimulants (nicotine, DMPP) was studied. To eliminate the possibility of interference from ganglionic stimulants carried over in the samples, hexamethonium (300 kg/ml, 1.09 x lop3 mol/l) was added to the Krebs solution for assay. Although this countered interference from ganglionic stimulants, the sensitivity to ACh was reduced from 7.32 pmol to 36.6 pmol. Identification of the Spasmogenic Substance as Acetylcholine-Accuracy, Sensitivity and Specificity of the Method Table 1 shows different chemical and biological assays for ACh. Bioassay of ACh with isolated superfused guinea pig ileum is the most sensitive; 1 pmole ACh can be assayed. The frog rectus abdominis preparation (Chang and Gaddum, 1933) is also sensitive, but it is not specific to ACh because it is also responsive to other cholinesters believed to be present in different tissues (Table 2). The use of eserinized leech muscle for bioassay of ACh was first described by Fuhner (1918 a,b) and was later recommended by Chang and Gaddum (1933). Later still, Gaddum and Szerb (1961) modified this technique through a reduction of the bath volume to 0.5 ml, thereby increasing the sensitivity of the preparation from 73.2 pmol to 3.66 pmol ACh. In these preparations, the effect of ACh was mediated via nicotinic receptors. This is indicated by the fact that atropine failed to block the effect of ACh, whereas curare inhibited it. Table 2 shows the sensitivity of different
205
206
E. S. Vizi et al. TABLE 1 Sensitivity of Some Biological and Chemical Methods to Acetylcholine” SENSITIVWACH METHOD
(PMOL)
REFERENCES
Bioassayb
75 5-10’
Frog rectus Cat blood pressure Leech dorsal muscle Guinea pig ileum Chemical Radiometry Colorimetry Fluorometry Gas chromatography
Chang and Caddum (1933) Feldberg and Gaddum (1934) Szerb (1961) Paton and Vizi (1969)
15 1
2.5 4 x IO5 200 50 13.7 2
HPLC Bioassay combined with gel filtration
Goldberg and McCamon Stone (1955) Fellman (1969) Hanin et al. (1972) Schmidt et al. (1970) Potter et al. (1983)
5
(1973)
This paper
a For further details, see Hanin (1974) and Eckernas (1977). ’ Per organ bath. ’ Per kilogram body weight.
bioassays to cholinesters. Guinea pig ileum proved to be the most sensitive and selective preparation to ACh. Ryall et al. (1964) found that the only choline ester that affected the gut significantly was acetyl-P-methyl-choline, to which the gut was about one-third as sensitive as it was to ACh. We found this preparation to be 12,000 times less sensitive to choline and 10,000 times less sensitive to phosphorylcholine than to ACh.
TABLE 2 The Sensitivity of Different Bioassays to Choline Esters-Relative Molecule of Choline and Choline Esters to that of ACh”
Choline Acetylcholine Propionylcholine Butyrylcholine Valerylcholine Phosphorylcholine Carbaminoylcholine
GUINEA PIG
FROG
LEECH
ILEUM~
RECTUS~
MUSCLE'
0.0083
-
0.14
0.015
RABBIT INTESTINES 0.075
RABBIT BLOOD PRESSURES 0.005
Potency per
BLOOD
PRESSURE ON
ATROPINIZED Dot? -
100
100
100
100
100
100
550
45
3
4
25
90
90
0.24
0
10
0.2 -
0 0.2
33 -
0.01
25
25.9 18
0.9 lb 12
80
a The sensitivity of the preparations to ACh was taken as 100. b This paper. ’ Chang and Gaddum (1933). d Sekul and Holland (1961).
15
-
Acetylcholine
Identification
and Assay
Pharmacological Identification of Acetylcholine The following tests have been used to differentiate between effects caused by ACh and those caused by other choline esters. 1) The responses of the ileum must be sensitive to lO-‘j mol/l atropine. 2) The active substance should be unstable in alkaline solution (the sample is mixed with an equal volume of 2 N NaOH, kept for 10 min at room temperature, and then neutralized). Chang and Gaddum (1933) believed that these methods were enough to identify the spasmogenic substance as ACh, although they also claimed that when “the activity of the extract is estimated quantitatively in terms of acetylcholine, using several different pharmacological tests . . . ” this assay is more specific. 3) When a tissue extract or perfusate is added to a known amount of ACh, no potentiation or inhibition should be observed. To enhance the specificity of ACh biossay in guinea pig ileum, two further methods were tested for the separation of ACh from other endogenous compounds possibly present in tissue extracts or superfusates. Gel Filtration and Radiochemical Detection Gel filtration was used to identify the substance obtained from tissue extracts and from superfusate. A Sephadex-C-IO column (4 ml volume 20 cm in length, 5 mm in diameter) was prepared, and the samples collected or extracted were eluted through the column. The void volume measured with dextran blue was 1.5 ml. When a sample had entered the top of the gel bed, the collection of fractions was begun. The eluate fractions of 0.5 ml were then tested on guinea pig ileum, or else their radioactivity was measured when 14C-ACh was eluted and the elution profiles were compared. The elution pattern of 14C-labeled ACh was measured by bioassay plus radioassay in 0.5 ml fractions of eluate. When the elution of both biological activity and label occurred in the same 3 to 4 ml (Figure 4) where the endogenous substance released from incubated human striatal slices appeared, it could be concluded that the substance assayed on guinea pig ileum was ACh. Separation of ACh by HPLC The separation of ACh by HPLC can be also used for identification of ACh, as described by Potter et al. (1983). A Biotronik HPLC system (Biotronik Wissenschaftliche Cerate GmbH, Frankfurt am Main) was made from a high-pressure pump (BT 3020 type) with pulse dampener and a Rheodyne 7125 injection valve (Cotati CA) equipped with a 20 t~l sample loop. A 150 x 4.6 mm stainless steel chromatographic column was prepacked with Nucleosil C 18 reversed-phase resin (particle size 5 t_r.m). The mobile phase was 0.1 mol/l sodium acetate/citric acid buffer (pH 4) that contained lop3 mol/l of tetraethylammonium chloride. The solution was filtered and then degassed in an ultrasonic generator, and the flow rate was maintained at 0.7 ml/min. A fraction collector (Contiflow, Labor MIM Budapest) was coupled to the HPLC system, and the outflow was collected in 0.7 ml/min fractions. AChl as authentic standard was dissolved in 0.2 mol/l perchloric acid and 750 pmol was injected onto the column. Because ACh does not produce a signal with either an UV detector or an electrochemical detector, it was measured in the fractions collected after HPLC separation on guinea pig ileum. As responses of the ileum
207
208
A
ACh
10
4
6
8
10
fractions
12
14
FRACTIONS
FIGURE 4. Sephadex C-IO column chromatography of ACh standard and 14C-ACh (A) and endogenous ACh (B) released from human cortical slices. The superfusates were lyophilized, redissolved in 0.3 ml of Krebs solution, centrifuged, and subjected to gel filtration. The fractions (0.5 ml each) were then assayed for ACh on guinea pig ileum, or their radioactivity was determined.
Acetylcholine
Identification
and Assay
were elicited by standard ACh dissolved in the mobile phase, no disturbance of the assay was observed by the mobile-phase components. Tetraethylammonium, which was used in the mobile phase to elute ACh from the column, markedly reduced the spontaneous motility of the ileum because of its ganglion-blocking property. Addition of atropine sulfate (1.5 x 10e6 mol/l) into the organ bath completely blocked the spasmogenic activity of the fractions on the guinea pig ileum. Figure 5 shows the HPLC elution of authentic ACh and endogenous ACh released by ouabain from incubated striatal slices of the rat. Superfusates were lyophilized and reconstituted in 100 ~1 of 0.2 mol/l perchloric acid, centrifuged, and 20 ~1 of the clear supernatant was then injected onto the column. The elution profile of endogenous ACh released was identical with that of standard ACh.
A 100
60
0 ‘0 5
20 ;,
---
II,,
-8
UY
12
Jl-_--,
n 16
,
20
24
Sample
20
24
Sample
m
c
100
-
60
-
20
L__________ I
4
6
12
16
FIGURE 5. (A) An HPLC elution profile of authentic ACh. AChl (3750 pmol) was dissolved in 100 ~1 of 0.2 molll perchloric acid and 20 ~1 was injected onto the column. See text for HPLC conditions. (6) Identification of ACh from striatal slice superfusate. Two rat striata were incubated in 1 ml of Krebs solution that contained 5 x 1O-6 molll eserine sulphate. The release of ACh was triggered by the addition of 2 x 10e5 mol/l ouabain. The samples were lyophilized, redissolved in 100 ~1 of 0.2 mol/l perchloric acid, and centrifuged. The clear supernantant (20 ~1) was then applied to the column. One-minute fractions were collected and assayed for ACh on isolated guinea-pig ileum.
210
E. S. Vizi et al.
DISCUSSION Since Otto Loewi first provided evidence that stimulation of the vagus nerve inhibits the frog’s heart by causing the release of a substance that was identified pharmacologically as a choline ester and chemically as acetylcholine, and which was later shown to be a principal neurotransmitter at many transmission sites, there has been an increasing demand to have an assay technique that is sensitive, accurate, chemically, specific, and not time consuming. The bioassay of acetylcholine on guinea pig ileum (Paton and Vizi, 1969) has generally been used to measure small quantities of acetylcholine. In the last few years, however, it has been criticized because of its chemical unspecificity. We have, in this paper, elaborated a technique that makes the guinea pig ileum bioassay, already known as a sensitive and accurate method, chemically more specific by the extension of the technique through preliminary gel filtration of the samples. Gel filtration (Sephadex-IO) or HPLC separation was used for identification of the spasmogenic substance assayed on guinea pig ileum. In addition, labeled ACh (‘4C-ACh) was eluted with unlabeled ACh or unidentified ACh, and their elution profiles were compared by use of bioassay plus radioassay of fractions of eluate. When the elution of both biological activity and label occurred in the same eluates, it was concluded that the substance assayed on guinea pig ileum was acetylcholine. The samples that were separated by gel filtration or reverse-phase column chromatography might not be expected to contain cholinomimetic or spasmogenic substances that act indirectly (e.g., gastrin, cholecystokinin, substance P, etc.), which have been demonstrated, or suspected to occur, in tissue extracts. Both the concentrations and the potencies of other choline esters are relatively low and are thus unable to affect the assay, which is otherwise relatively sensitive to ACh.
REFERENCES Chang HC, Gaddum JH (1933) Choline esters in tissue extracts. / Physiol 79:255-285. Dale HH, Dudley HW (1929) The presence of histamine and acetylcholine in the spleen of the ox and the horse / Physiol (Land.) 68:97-123. Dudar JD, Szerb JC (1970) Bioassay of acetylcholine on leech muscle suspended in a microbath. Exp Physiol
Biochem
3:341-350.
Eckernas SA (1977) Plasma choline and cholinergic mechanisms in the brain. Acta Physiol Stand (SuppI) 449:1-62. Feigenson MF, Saelens JK (1969) An enzyme assay for acetylcholine. Biochem. Pharmaco/18:14791486.
Feldberg W, Gaddum JH (1934) The chemical transmitter at synapses in a sympathetic ganglion. / Physio/81:305-319.
Fellman JH (1969) A chemical method for the determination of acetylcholine: its application in a study of presynaptic release and a choline acetyltransferase assay, / Neurochem 16:135-143. Fuhner H (1918a) Ein Vorlesungsversuch zur Demonstration der erregbarkeitssteigernden Wirkung des Physostigmins. Arch fxper Path Pharm 82:81-85. Ftihner H (1918b) Biochem Z 92:347-358 cited in Chang and Gaddum (1933). Gaddum JH, Szerb JC (1961) The assay of substance P on gold fish intestine in a microbath. Brl Pharmacol
Chemother
17:451-463.
Goldberg AM, McCamon RE (1973) The determination of picomole amounts of acetylcholine in mammalian brain. / Neurochem 20:1-E. Hanin I (1974) Choline
and Acetylcholine.
Hand-
Acetylcholine book of Chemical Raven Press.
Assay Methods.
New York:
Hanin I, Masarelli R, Costa E (1972) An approach to the in vivo study of acetylcholine turnover in rat salivary glands by radio gas chromatography. / Pharmacol Exp Tber 181 :lO-18. Israel M, Lesbats B (1981) Chemiluminescent determination of acetylcholine, and continuous detection of its release from torpedo-electric synapses and synaptosomes. Neurochem Int 3:8190. Jenden DJ, Hanin I, Lamb SI (1968) Gas chromatographic microestimation of acetylcholine and related compounds Anal Chem 48:125-128. Macintosh FC, Perry WLM (1950) Biological estimation of acetylcholine. Methods Med Res 3:7892. Maslova AF (1964) Quantitative determination of acetylcholine in biological materials by polarographic analysis. Vopr Med Khim 10:311. Paton WDM, Vizi ES (1969) The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig longitudinal muscle strip. Br J Pharmacol35:10-28. Potter PE, Meek JL, Neff NH (1983) Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. j Neurothem 41:188-194. Ryall W, Stone N, Watkins JC (1964) The cholinomimetic activity of extracts of brain nerve terminals. I Neurochem 11:621-637.
Identification
and Assay
Schmidt DE, Szilagyi PIA, Alkon DA, Green JP. (1970) A method for measuring nanogram quantities of acetylcholine by pyrolysis-gas chromatography: The demonstration of acetylcholine in effluents from the rat phrenic nerve-diaphragm preparation. I Pharmacol fxp Ther 174:337-345. Sekul AA, Holland WC (1961) Comparative pressor effect of certain unsaturated acid esters of choline. / Pharmacol fxp Ther 133:313-318. Straughan DW (1958) Assay of acetylcholine on the rat blood pressure preparation. J Pharm (London) 10:783-784. Stavinoha WB, Ryan LC (1965) Estimation of the acetylcholine content of rat brain by gas chromatography, / fharmacol Fxp Ther 150:231-235. Stone WE (1955) Acetylcholine in the brain I. “Free” “bound” and total acetylcholine. Arch Biochem Biophys 59:181-192. Szerb J (1961) The estimation of acetylcholine using leech muscle in a microbath. j Physio/158:8-9P. Vapaatalo H, Linden IB, Parentainen J (1976) A sensitive biological assay for prostaglandin E and acetylcholine. / Pharm Pharmaco/28:188-191. Vizi ES (1973) Acetylcholine release from guinea pig ileum by parasympathetic ganglion stimulants and gastrin like polypeptides. Br J Pharmacol 47~765-777. Webb JL (1950) The action of acetylcholine rabbit auricle. Br J Pharmacol5:335-375.
on the
211
E. SYLVESTER
VIZI, LAsztt, G. HARSINC JR., DERYCK DUNCALF, HIDEO
NACASHIMA, PAMELAPOTTER, AND FRANCISF. FOLDES
A modified technique of acetylcholine assay on the guinea pig ileum has been combined with either minivoiume gel filtration or high-performance liquid chromatography separation of the samples. In addition, labeled acetylcholine (14CACh) was eluted with unlabeled acetylcholine or with the samples expected to contain acetylcholine, and their elution profiles were compared by bioassay plus radioassay of eluate fractions. When the elution of both biological activity and label occurred in the same eluates, it was concluded that the substance assayed on guinea pig ileum was acetylcholine. The method was sensitive to 0.5 ng f-l.8 pmot) of acetylcholine and its reproducibility was within 5%. It thus represents a substantial improvement in chemical specificity over the previous bioassay method described by Paton and Vizi (1969).
Key Words:
Acetylcholine
assay; Gel filtration;
matography;
Radiochemical
detection
High-performance
liquid chro-
INTRODUCTION There are many methods described for quantitative acetylcholine (ACh) estimation in tissue extracts or solutions. Of the chemical ones, some are relatively insensitive (Stone, 1955, calorimetry; Stavinoha and Ryan, 1965, gas chromatography). Other methods, while often sensitive and specific, are complex, time consuming, and require relatively expensive apparatus (Maslova, 1964, polarography; jenden et al., 1968; Schmidt et al., 1970, gas chromatography; Feigenson and Saelens, 1969, hydrolysis and radioactive labelling; Fellman, 1969, fluorescence; Israel and Lesbats, 1981, chmeluminescence; Potter et al., 1983, high-performance liquid chromatography). Of the numerous bioassay methods available (Dale and Dudley, 1929, isolated rabbit intestine; Feldberg and Gaddum, 1934, cat blood pressure; Chang and Caddum, 1933, eserine-sensitized frog rectus; Macintosh and Perry, 1950, blood pressure of eviscerated cat; Webb, 1950, isolated rabbit auricle; Straughan, 1958, From the Institute of Experimental Medicine Hungarian Academy of Sciences, Budapest, Hungary, and the Departments of Anesthesiology, Montefiore Medical Center and Albert Einstein College of Medicine Bronx, New York. Address reprint requests to ES. Vizi, Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1450 Budapest, P.O.B. 67, Hungary. Received May, 1984; revised and accepted September, 1984.
201 burnalof
Pharmacological
Methods
0 1985 Efsevier Science Publishing
13, 201-211 11985) Co., Inc., 52 Vanderbilt
Avenue, New York, NY 10017
202
E. S. Vizi et al.
rat blood pressure; Dudar and Szerb, 1970, leech muscle; Vaapatalo et al., 1976, superfused hamster stomach strip), the guinea pig ileum assay appears to combine sensitivity, reproducibility, and specificity (Ryall et al., 1964) with simplicity. In addition, it has the following advantage: perfused solutions obtained from isolated mammalian nerve-muscle preparations can be assayed directly without the need for dilution or other modification. However, although bioassay techniques used to measure ACh offer accuracy and reproducibility, these techniques are not chemically specific. In the past two decades, several sophisticated methods have been developed that seriously rival the best bioassay methods (Hanin, 19741, but they are time consuming and require relatively expensive equipment and/or chemicals. This paper provides convincing evidence for the specificity of bioassay on guinea pig ileum extended by gel filtration or by high-performance liquid chromatography (WPLC) without a loss in sensitivity. Because the previous descriptions of the guinea pig ileum method in the literature (Paton and Vizi, 1969) were not sufficiently detailed, the preparation could not be set up without additional information. This paper describes the method of ACh assay on the supe~used guinea pig ileum in sufficient detail to avoid this problem. The details of the method are modified from Paton and Vizi (1969) to improve the stability of the preparation and the speed of the assay. METHODS
AND
RESULTS
Assay of Ace~Icholjne
Apparatus The apparatus required for the assay is shown in Figure 1. It was a water-jacketed cylindrical chamber (8 mm internal diameter, 70 mm in length, 3.5 ml volume). The chamber had three side arms and an extension at the bottom. A short piece of latex rubber tubing was attached to the bottom extension, which held the catheter to
Krebe’
soi.
thermostat
FIGURE 1. Water-jacketed glass organ bath in which the guinea pig ileum (or its longitudinal muscle strip) used for the assay of ACh was suspended.
Acetylcholine
identification
and Assay
which the aboral end of the gut is fastened. The lower side arm was also occluded with a clamped latex tube; oxygenation of the chamber occurred through a needle passed through the wall of this tube. The top side arm was the overflow port, which served to keep the fluid volume constant. Krebs solution was kept in a reservoir, and it was continuously bubbled with 5% CO1 in OZ. The reservoir was connected by latex rubber tubing to the middle side arm, which formed a spiral around the chamber and entered it near the bottom. In the spiral, the Krebs solution was warmed to the same temperature as that of the gut chamber. When the clamp on the tubing between the reservoir and the spiral was released, fresh Krebs flowed into the chamber and out through the top side arm. The height of the reservoir was adjusted so that the flow into the chamber was sufficient for three changes of solution in 15 sec. A thermostated water pump was used to maintain constant temperature in the water jackets. The appartatus was completed by a force displacement transducer (Grass FT 03) suitable for the recording of forces from 0.02 to 10 g, an appropriate amplifier, and a chart recorder or polygraph. The upper end of the gut was connected to the strain gauge through a small coil spring that rendered the contractions auxotonic. The compliance of the spring was approximately about 2 cm/gm. The complete assembly is shown in Figure 2. inclusion of a high-frequency filter in the amplifier was not deemed desirable. Solution Used for Pe~usion The preparation was made as described by Paton and Vizi (7969). A modified Krebs solution was used to perfuse the gut. The composition in grams per liter was as follows: NaCl6.60; NaHC03 2.10; KCI 0.35; dextrose 2.00; CaC& 0.28; KH2P04 O.f6; MgS04 x 7 HZ0 0.14. The last three components must always be added in the above order (CaC12 before KH2P04, and MgS04 x 7 Hz0 last) or precipitation will occur. Bubbling the solution with 5% COP in 02 before the addition of CaClz helped the latter to dissolve. Eserine sulphate, 2 ngll (5 x IO-’ mol/l), in the solution increased the sensitivity of the preparation, and morphine sulphate, IO mgil (1.2 x
Bridge Amplifier Strain
FIGURE 2. pig ileum.
gauge
Filter ( hum 8 noise)
Booster amplifier
I
Schema of complete assembly used for the bioassay of ACh on isolated guinea
203
204
E. S. Vizi et al.
10d6 mol/l), reduced intrinsic ACh release, thus eliminating the problem of spontaneous motility. If the samples to be assayed for ACh are expected to contain histamine, the effect of the latter can be blocked by the addition of diphenhydramine in a concentration of 0.2 mg/l (2.8 x lo-’ mol/l). This preparation can also be used for histamine assay; in this assay, atropine, 100 @I (3.4 x IO-’ mol/l), should be added to the basic solution to exclude the effect of ACh on the ileum. Assay Technique The assay was performed at 32-33X. If the temperature is lowered from 37”C, spontaneous motility is reduced with no loss in sensitivity. If the temperature is reduced further, however, an inconveniently slow response and relaxation occur. The assay was performed by a comparison of the response to the unknown sample with responses to known quantities of ACh. A “standard” solution of ACh was prepared in the same solution as the samples to be assayed. The standard ACh was injected in a volume of 0.1-0.3 ml by a 1 ml tuberculin syringe or by a 0.5 ml Hamilton syringe. RESULTS An example of a performed assay is shown in Figure 3. Injections of the unknown solution were made from a 0.5 ml syringe. The difference in the mode of injection between standard and unknown solutions did not affect the accuracy of the method. The working cycle was 60 sec. The injected ACh was allowed to act for IO sec. The preparation was then washed three times with a bath volume of fresh Krebs for 15 set, and it was ready for the next injection when the tracing had returned to the baseline. Regularity of the cycle was important. If the time elapsed between injections varies, the sensitivity of the gut changes.
a52
24@05WllQ4lW5@2ng 0.1
’ 0.25
0.1
ml
I
I
a5
1
;i 2
5
w
FIGURE 3. Results of ACh bioassay on isolated guinea pig ileum. The sample contained endogenous ACh released from eserinized longitudinal muscle strip of guinea pig ileum. Dose-contraction curve is for ACh. The ACh content in the unknown sample was determined by interpolation.
Acetylcholine identification and Assay To determine the ACh content of the unknown sample, the heights of the contractions induced by the two injections of standard were plotted on semilogarithmic paper (log dose versus response), and the quantity of ACh in the unknown solution was read off by interpolation of the height of the response (Figure 3). The concentration of ACh in the unknown sample was then calculated. After some time, spontaneous contractions of the gut may interfere with the assay. The most common cause for this is retention of secretions. These can be cleared by slow injection of IO-20 ml Krebs solution through the catheter to which the gut is tied. If the samples (and the standard) contain an anticholinesterase, this may also cause excessive motility after numerous exposures. Repeated washings for 10 to 15 min are required to reverse this, and the problem often reoccurs after only a few more samples. If this happens, it is best to replace the gut with a fresh piece. However, we have been able to assay over twenty samples, in duplicate, that contained eserine sulphate 2 kg/ml (5 x lop6 mol/liter) without encountering any problems. Samples that contain neostigmine or phospholine have also been assayed successfully. Ambenonium, however, caused prolonged contraction of the gut, and methoxyambenonium appeared to have an atropine-like effect. The response of the gut to ACh is not modified by d-tubocurarine chloride, gallamine triethiodide, or pancuronium bromide used in sufficient concentration to cause total neuromuscular block in the rat phrenic nerve-diaphragm. Decamethonium ioide, however, caused a slight contraction, although it did not interfere with the ACh assay when it was added to the standard. When the effect on ACh release of gastrin-like polypeptides present in the samples was studied and assayed, their interference with the assay ileum was prevented by the use of 0.25 kg/ml (7.8 x IO-’ mol/l) of tetrodotoxin (Vizi, 1973). The effect of ganglionic stimulants (nicotine, DMPP) was studied. To eliminate the possibility of interference from ganglionic stimulants carried over in the samples, hexamethonium (300 kg/ml, 1.09 x lop3 mol/l) was added to the Krebs solution for assay. Although this countered interference from ganglionic stimulants, the sensitivity to ACh was reduced from 7.32 pmol to 36.6 pmol. Identification of the Spasmogenic Substance as Acetylcholine-Accuracy, Sensitivity and Specificity of the Method Table 1 shows different chemical and biological assays for ACh. Bioassay of ACh with isolated superfused guinea pig ileum is the most sensitive; 1 pmole ACh can be assayed. The frog rectus abdominis preparation (Chang and Gaddum, 1933) is also sensitive, but it is not specific to ACh because it is also responsive to other cholinesters believed to be present in different tissues (Table 2). The use of eserinized leech muscle for bioassay of ACh was first described by Fuhner (1918 a,b) and was later recommended by Chang and Gaddum (1933). Later still, Gaddum and Szerb (1961) modified this technique through a reduction of the bath volume to 0.5 ml, thereby increasing the sensitivity of the preparation from 73.2 pmol to 3.66 pmol ACh. In these preparations, the effect of ACh was mediated via nicotinic receptors. This is indicated by the fact that atropine failed to block the effect of ACh, whereas curare inhibited it. Table 2 shows the sensitivity of different
205
206
E. S. Vizi et al. TABLE 1 Sensitivity of Some Biological and Chemical Methods to Acetylcholine” SENSITIVWACH METHOD
(PMOL)
REFERENCES
Bioassayb
75 5-10’
Frog rectus Cat blood pressure Leech dorsal muscle Guinea pig ileum Chemical Radiometry Colorimetry Fluorometry Gas chromatography
Chang and Caddum (1933) Feldberg and Gaddum (1934) Szerb (1961) Paton and Vizi (1969)
15 1
2.5 4 x IO5 200 50 13.7 2
HPLC Bioassay combined with gel filtration
Goldberg and McCamon Stone (1955) Fellman (1969) Hanin et al. (1972) Schmidt et al. (1970) Potter et al. (1983)
5
(1973)
This paper
a For further details, see Hanin (1974) and Eckernas (1977). ’ Per organ bath. ’ Per kilogram body weight.
bioassays to cholinesters. Guinea pig ileum proved to be the most sensitive and selective preparation to ACh. Ryall et al. (1964) found that the only choline ester that affected the gut significantly was acetyl-P-methyl-choline, to which the gut was about one-third as sensitive as it was to ACh. We found this preparation to be 12,000 times less sensitive to choline and 10,000 times less sensitive to phosphorylcholine than to ACh.
TABLE 2 The Sensitivity of Different Bioassays to Choline Esters-Relative Molecule of Choline and Choline Esters to that of ACh”
Choline Acetylcholine Propionylcholine Butyrylcholine Valerylcholine Phosphorylcholine Carbaminoylcholine
GUINEA PIG
FROG
LEECH
ILEUM~
RECTUS~
MUSCLE'
0.0083
-
0.14
0.015
RABBIT INTESTINES 0.075
RABBIT BLOOD PRESSURES 0.005
Potency per
BLOOD
PRESSURE ON
ATROPINIZED Dot? -
100
100
100
100
100
100
550
45
3
4
25
90
90
0.24
0
10
0.2 -
0 0.2
33 -
0.01
25
25.9 18
0.9 lb 12
80
a The sensitivity of the preparations to ACh was taken as 100. b This paper. ’ Chang and Gaddum (1933). d Sekul and Holland (1961).
15
-
Acetylcholine
Identification
and Assay
Pharmacological Identification of Acetylcholine The following tests have been used to differentiate between effects caused by ACh and those caused by other choline esters. 1) The responses of the ileum must be sensitive to lO-‘j mol/l atropine. 2) The active substance should be unstable in alkaline solution (the sample is mixed with an equal volume of 2 N NaOH, kept for 10 min at room temperature, and then neutralized). Chang and Gaddum (1933) believed that these methods were enough to identify the spasmogenic substance as ACh, although they also claimed that when “the activity of the extract is estimated quantitatively in terms of acetylcholine, using several different pharmacological tests . . . ” this assay is more specific. 3) When a tissue extract or perfusate is added to a known amount of ACh, no potentiation or inhibition should be observed. To enhance the specificity of ACh biossay in guinea pig ileum, two further methods were tested for the separation of ACh from other endogenous compounds possibly present in tissue extracts or superfusates. Gel Filtration and Radiochemical Detection Gel filtration was used to identify the substance obtained from tissue extracts and from superfusate. A Sephadex-C-IO column (4 ml volume 20 cm in length, 5 mm in diameter) was prepared, and the samples collected or extracted were eluted through the column. The void volume measured with dextran blue was 1.5 ml. When a sample had entered the top of the gel bed, the collection of fractions was begun. The eluate fractions of 0.5 ml were then tested on guinea pig ileum, or else their radioactivity was measured when 14C-ACh was eluted and the elution profiles were compared. The elution pattern of 14C-labeled ACh was measured by bioassay plus radioassay in 0.5 ml fractions of eluate. When the elution of both biological activity and label occurred in the same 3 to 4 ml (Figure 4) where the endogenous substance released from incubated human striatal slices appeared, it could be concluded that the substance assayed on guinea pig ileum was ACh. Separation of ACh by HPLC The separation of ACh by HPLC can be also used for identification of ACh, as described by Potter et al. (1983). A Biotronik HPLC system (Biotronik Wissenschaftliche Cerate GmbH, Frankfurt am Main) was made from a high-pressure pump (BT 3020 type) with pulse dampener and a Rheodyne 7125 injection valve (Cotati CA) equipped with a 20 t~l sample loop. A 150 x 4.6 mm stainless steel chromatographic column was prepacked with Nucleosil C 18 reversed-phase resin (particle size 5 t_r.m). The mobile phase was 0.1 mol/l sodium acetate/citric acid buffer (pH 4) that contained lop3 mol/l of tetraethylammonium chloride. The solution was filtered and then degassed in an ultrasonic generator, and the flow rate was maintained at 0.7 ml/min. A fraction collector (Contiflow, Labor MIM Budapest) was coupled to the HPLC system, and the outflow was collected in 0.7 ml/min fractions. AChl as authentic standard was dissolved in 0.2 mol/l perchloric acid and 750 pmol was injected onto the column. Because ACh does not produce a signal with either an UV detector or an electrochemical detector, it was measured in the fractions collected after HPLC separation on guinea pig ileum. As responses of the ileum
207
208
A
ACh
10
4
6
8
10
fractions
12
14
FRACTIONS
FIGURE 4. Sephadex C-IO column chromatography of ACh standard and 14C-ACh (A) and endogenous ACh (B) released from human cortical slices. The superfusates were lyophilized, redissolved in 0.3 ml of Krebs solution, centrifuged, and subjected to gel filtration. The fractions (0.5 ml each) were then assayed for ACh on guinea pig ileum, or their radioactivity was determined.
Acetylcholine
Identification
and Assay
were elicited by standard ACh dissolved in the mobile phase, no disturbance of the assay was observed by the mobile-phase components. Tetraethylammonium, which was used in the mobile phase to elute ACh from the column, markedly reduced the spontaneous motility of the ileum because of its ganglion-blocking property. Addition of atropine sulfate (1.5 x 10e6 mol/l) into the organ bath completely blocked the spasmogenic activity of the fractions on the guinea pig ileum. Figure 5 shows the HPLC elution of authentic ACh and endogenous ACh released by ouabain from incubated striatal slices of the rat. Superfusates were lyophilized and reconstituted in 100 ~1 of 0.2 mol/l perchloric acid, centrifuged, and 20 ~1 of the clear supernatant was then injected onto the column. The elution profile of endogenous ACh released was identical with that of standard ACh.
A 100
60
0 ‘0 5
20 ;,
---
II,,
-8
UY
12
Jl-_--,
n 16
,
20
24
Sample
20
24
Sample
m
c
100
-
60
-
20
L__________ I
4
6
12
16
FIGURE 5. (A) An HPLC elution profile of authentic ACh. AChl (3750 pmol) was dissolved in 100 ~1 of 0.2 molll perchloric acid and 20 ~1 was injected onto the column. See text for HPLC conditions. (6) Identification of ACh from striatal slice superfusate. Two rat striata were incubated in 1 ml of Krebs solution that contained 5 x 1O-6 molll eserine sulphate. The release of ACh was triggered by the addition of 2 x 10e5 mol/l ouabain. The samples were lyophilized, redissolved in 100 ~1 of 0.2 mol/l perchloric acid, and centrifuged. The clear supernantant (20 ~1) was then applied to the column. One-minute fractions were collected and assayed for ACh on isolated guinea-pig ileum.
210
E. S. Vizi et al.
DISCUSSION Since Otto Loewi first provided evidence that stimulation of the vagus nerve inhibits the frog’s heart by causing the release of a substance that was identified pharmacologically as a choline ester and chemically as acetylcholine, and which was later shown to be a principal neurotransmitter at many transmission sites, there has been an increasing demand to have an assay technique that is sensitive, accurate, chemically, specific, and not time consuming. The bioassay of acetylcholine on guinea pig ileum (Paton and Vizi, 1969) has generally been used to measure small quantities of acetylcholine. In the last few years, however, it has been criticized because of its chemical unspecificity. We have, in this paper, elaborated a technique that makes the guinea pig ileum bioassay, already known as a sensitive and accurate method, chemically more specific by the extension of the technique through preliminary gel filtration of the samples. Gel filtration (Sephadex-IO) or HPLC separation was used for identification of the spasmogenic substance assayed on guinea pig ileum. In addition, labeled ACh (‘4C-ACh) was eluted with unlabeled ACh or unidentified ACh, and their elution profiles were compared by use of bioassay plus radioassay of fractions of eluate. When the elution of both biological activity and label occurred in the same eluates, it was concluded that the substance assayed on guinea pig ileum was acetylcholine. The samples that were separated by gel filtration or reverse-phase column chromatography might not be expected to contain cholinomimetic or spasmogenic substances that act indirectly (e.g., gastrin, cholecystokinin, substance P, etc.), which have been demonstrated, or suspected to occur, in tissue extracts. Both the concentrations and the potencies of other choline esters are relatively low and are thus unable to affect the assay, which is otherwise relatively sensitive to ACh.
REFERENCES Chang HC, Gaddum JH (1933) Choline esters in tissue extracts. / Physiol 79:255-285. Dale HH, Dudley HW (1929) The presence of histamine and acetylcholine in the spleen of the ox and the horse / Physiol (Land.) 68:97-123. Dudar JD, Szerb JC (1970) Bioassay of acetylcholine on leech muscle suspended in a microbath. Exp Physiol
Biochem
3:341-350.
Eckernas SA (1977) Plasma choline and cholinergic mechanisms in the brain. Acta Physiol Stand (SuppI) 449:1-62. Feigenson MF, Saelens JK (1969) An enzyme assay for acetylcholine. Biochem. Pharmaco/18:14791486.
Feldberg W, Gaddum JH (1934) The chemical transmitter at synapses in a sympathetic ganglion. / Physio/81:305-319.
Fellman JH (1969) A chemical method for the determination of acetylcholine: its application in a study of presynaptic release and a choline acetyltransferase assay, / Neurochem 16:135-143. Fuhner H (1918a) Ein Vorlesungsversuch zur Demonstration der erregbarkeitssteigernden Wirkung des Physostigmins. Arch fxper Path Pharm 82:81-85. Ftihner H (1918b) Biochem Z 92:347-358 cited in Chang and Gaddum (1933). Gaddum JH, Szerb JC (1961) The assay of substance P on gold fish intestine in a microbath. Brl Pharmacol
Chemother
17:451-463.
Goldberg AM, McCamon RE (1973) The determination of picomole amounts of acetylcholine in mammalian brain. / Neurochem 20:1-E. Hanin I (1974) Choline
and Acetylcholine.
Hand-
Acetylcholine book of Chemical Raven Press.
Assay Methods.
New York:
Hanin I, Masarelli R, Costa E (1972) An approach to the in vivo study of acetylcholine turnover in rat salivary glands by radio gas chromatography. / Pharmacol Exp Tber 181 :lO-18. Israel M, Lesbats B (1981) Chemiluminescent determination of acetylcholine, and continuous detection of its release from torpedo-electric synapses and synaptosomes. Neurochem Int 3:8190. Jenden DJ, Hanin I, Lamb SI (1968) Gas chromatographic microestimation of acetylcholine and related compounds Anal Chem 48:125-128. Macintosh FC, Perry WLM (1950) Biological estimation of acetylcholine. Methods Med Res 3:7892. Maslova AF (1964) Quantitative determination of acetylcholine in biological materials by polarographic analysis. Vopr Med Khim 10:311. Paton WDM, Vizi ES (1969) The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig longitudinal muscle strip. Br J Pharmacol35:10-28. Potter PE, Meek JL, Neff NH (1983) Acetylcholine and choline in neuronal tissue measured by HPLC with electrochemical detection. j Neurothem 41:188-194. Ryall W, Stone N, Watkins JC (1964) The cholinomimetic activity of extracts of brain nerve terminals. I Neurochem 11:621-637.
Identification
and Assay
Schmidt DE, Szilagyi PIA, Alkon DA, Green JP. (1970) A method for measuring nanogram quantities of acetylcholine by pyrolysis-gas chromatography: The demonstration of acetylcholine in effluents from the rat phrenic nerve-diaphragm preparation. I Pharmacol fxp Ther 174:337-345. Sekul AA, Holland WC (1961) Comparative pressor effect of certain unsaturated acid esters of choline. / Pharmacol fxp Ther 133:313-318. Straughan DW (1958) Assay of acetylcholine on the rat blood pressure preparation. J Pharm (London) 10:783-784. Stavinoha WB, Ryan LC (1965) Estimation of the acetylcholine content of rat brain by gas chromatography, / fharmacol Fxp Ther 150:231-235. Stone WE (1955) Acetylcholine in the brain I. “Free” “bound” and total acetylcholine. Arch Biochem Biophys 59:181-192. Szerb J (1961) The estimation of acetylcholine using leech muscle in a microbath. j Physio/158:8-9P. Vapaatalo H, Linden IB, Parentainen J (1976) A sensitive biological assay for prostaglandin E and acetylcholine. / Pharm Pharmaco/28:188-191. Vizi ES (1973) Acetylcholine release from guinea pig ileum by parasympathetic ganglion stimulants and gastrin like polypeptides. Br J Pharmacol 47~765-777. Webb JL (1950) The action of acetylcholine rabbit auricle. Br J Pharmacol5:335-375.
on the
211