Further studies on the preparation, purification and nature of irin

Abstract

Distilled water extracts of rabbit iris contain an acid which is physiologically active on certain smooth muscles (Ambache, 1957a). Improvements in the purification and assay of this substance are now described and also an investigation of its chemical nature. First, a number of acids of biological origin have been screened for activity. Although representative of a variety of structuretypes, the acids examined are nearly all inactive or can be otherwise distinguished from irin. Direct evidence is then produced that irin is a long-chain unsaturated hydroxy-fatty acid. Further structure-action studies on available hydroxy-acids and lactones have shown that (+ )-ricinoleic acid is a powerful stimulant of the smooth muscle in the hamster colon, whereas oleic acid which lacks the hydroxyl group of ricinoleic but is otherwise identical with it, is relatively inert. These and other findings suggest that an OH group, a double bond, and the spatial relation between them, determine the irin-like activity of fatty acids. METHODSIrin. The source of active material has been the rabbit's iris throughout; although ox irides are larger and would have been more plentiful they contain less than &-O jO the activity found in rabbits' irides. The procedure for making iris extracts described in the previous paper was modified slightly as follows: (i) unless otherwise stated all extracts were made in distilled water (1 ml./ 100mg tissue) neutralized when necessary with 0.02% NaHCO3; (ii) as the pharmacological activity of'extracts is reduced by boiling, the heating was omitted-instead, the centrifuged extracts were left for some days at -15°C, which precipitates out further impurities removable by a second or third centrifugation; (iii) the irides were excised more rapidly, by direct removal through a slit in the cornea without prior enucleation of the eyes. I am indebted to Dr B. C. Whaler for'four extracts of several hundred irides prepared in this way at Porton. For these, the dried irides were ground, unweighed, in 0*4 ml. of distilled water per iris, i.e. assuming an average weight of 40 mg per iris; the dosage of these extracts is expressed in arbitrary units of' _ mg'; (iv) some extracts were prepared from irides excised a few hours after death. Through the courtesy of Dr M. Sterne the severed heads from rabbits killed at the Wellcome Research Laboratories in the morning were sent here, where the eyes were excised and the irides were extracted in the early afternoon. Such extracts were rather less active than those made from fresh irides.

The effects of prostaglandins on the intraocular pressure of the rabbit

“…The present observations may be related to the increased protein content of the aqueous humour in the eyes which received prostaglandins, since Duke-Elder & Duke-Elder (1931) observed that the IOP in the rabbit became refractory to repeated mechanical stimulation of the iris, and Ambache (1959) showed that protein may interfere with the action of irin on the hamster colon. Thus, the presence of plasma proteins in the aqueous humour may curtail the action of intracamerally administered prostaglandin.…”

PROCEEDINGS OF THE BRITISH PHARMACOLOGICAL SOCIETY, 8TH‐9TH‐10TH July, 1970

“…A poststimulatory inhibition of acetylcholine release in the presence of eserine was also found at higher frequencies (0 1 and 0 5 Hz), but it was not so marked. Ambache (1957Ambache ( , 1959 extracted from iris tissue a pharmacologically active material which he called irin. This material is now thought to contain prostaglandin (Waitzman, Bailey & Kirby, 1967).…”

A Simple Chemical Procedure for Distinguishing E From F Prostaglandins, With Application to Tissue Extracts

“…3, are recovered from the ether phase (Ambache, 1957(Ambache, , 1959. The residues of the evaporated ether phases will be referred to as ether-purified irins, and their doses will be given in -mg of original tissue.…”

“…3, as described previously (Ambache, 1959;Ambache, Reynolds & Whiting, 19%3). The ether phases were evaporated in a stream of nitrogen and the residues obtained in this way were dissolved in 0.9% NaCl solution and subdivided into samples, some of which were treated with the reagent (100 mg/ml.)…”

“…The separated ether phases were evaporated to dryness in a stream of nitrogen; these will be referred to as ether-purified residues. For bio-assay the residues were taken up in a suitable volume of de Jalon's colon solution of the same composition as used previously (Ambache, 1959).…”