The approximate determination of spermine in single human organs

Abstract

IN the writer's published work [Harrison, 1931] on the distribution of spermine in human tissues it was necessary to pool the smaller organs from a number of subjects in order to make a single estimation by the steam-distillation method. As a development of the subsequent study of Barberio's picric acid test for semen [Harrison, 1932] the method for estimating spermine in organs has been modified, so that approximate determinations of the amount in a single prostate, or in a pair of testes or in a single sample of human semen can be undertaken. The investigation is an extension of the work previously published, in the hope of throwing some light on the significance of the relatively large quantities of spermine in the average human prostate. The modified method. In brief, spermine and other substances are extracted from the dried material by trichloroacetic acid. An aliquot part of the cleared extract is precipitated with picric acid. The crude spermine picrate is converted into the hydrochloride, and then into the characteristic phosphate which is insoluble in 25 % alcohol. The spermine phosphate is separated, washed, dried and weighed. The details are as follows. After mincing and weighing, the organ is heated on the boiling water-bath for several hours, absolute alcohol being added at intervals to hasten the drying process. It is then stored in a vacuum-desiccator as long as is convenient. After weighing, the dried organ is extracted with ether for 2 to 6 hours in a Soxhlet apparatus (when very fatty it is more satisfactory to weigh the organ after ether extraction). The residue is transferred quantitatively from the Soxhlet thimble into a glass-stoppered measuring cylinder of suitable capacity. 2-5 % trichloroacetic acid is added to a total of 10 volumes; i.e. each g. of dried (or of dried and ether-extracted) material is made up to a total of 10-0 cc. by addition of the acid. The mixture is thoroughly shaken at intervals, extraction being allowed to continue for 2 hours. Any contraction in volume is made good by addition of more acid, and, after mixing, the extract is decanted and cleared as much as possible by centrifuging or filtering or both. As a rule the final fluid is clear or only slightly opalescent. Its volume is measured, and an equal volume of saturated aqueous picric acid is added. The mixture is heated on the boiling water-bath till it is clear or only slightly opalescent. The flame is turned out and cooling allowed to proceed slowly to room temperature. (This makes the subsequent separation of the picrate less difficult.) After standing for a few hours (conveniently overnight), the picrate precipitate is separated by decanting and centrifuging (filtration is generally less satisfactory), and is roughly dried in air (e.g. in the incubator at 370 overnight). It is then dissolved in the minimum of boiling acid alcohol (concen-trated HCI 7 cc., water 43 cc., alcohol to 200 cc.), and thrown into at least 10 volumes of acetone. The precipitate of crude spermine hydrochloride is filtered off, most conveniently through a Soxhlet thimble placed in a conical funnel which is mounted in a filter-flask, so that slight suction can be applied at the end of the process. After washing with acetone until free from picric acid, the funnel is transferred without elay to a suitable boiling-tube with side-arm. The precipitate