A study of the assay method for the guinea pig antistiffness factor.

In 1941 Wulzen and Bahrs (1) reported a characteristic muscular stiffness at the wrist among guinea pigs on a skim milk diet, which progressed in intensity over a period of months. Autopsies of deficient animals revealed extensive calcification throughout the muscles and internal organs. Raw cream fed by mouth was found to alleviate the wrist stiffness within a few days, although disappearance of calcium phosphate deposits required many months. A procedure for the isolation of a factor present in raw cream which cured an induced stiffness in guinea pigs was described in 1943 (2). 3 mg. of the factor were isolated from 15 gallons of raw cream. The compound was re ported to be an oily ketone (steroid) which was very susceptible to atmospheric oxidation and was active in dosages of 0.1 y. The factor present in cream was later traced to the molasses which had been added to the diet of the cow, increasing thereby the original amount in cream. This led to the use of molasses and finally to unheated cane juice as a source of the factor. The procedure for the isolation from molasses or cane juice has been reported (3). 100 mg. of a factor (pure white leaflets, m.p. 81.5-82”) were isolated from 55 gallons of cane juice, which was very susceptible to atmospheric oxidation and was active in dosages of 0.002 y (3). Three reviews (4) have pointed out the need for the confirmation of this work by other laboratories, as well as information regarding the reliability and limitations of the assay. Experiments of this nature have been published recently by Oleson and coworkers of the Lederle Laboratories (5). These have confirmed the existence of a stiffness syndrome, as well as the curative power of a crystalline material derived from cane juice, when fed in a 5 y dosage. Furthermore, several compounds related to the common sterols were reported which exert a similar curative effect.