Paper Chromatographic Detection of N-Acylaminosugars in Human Semen

Abstract

In 1945-6, Man1' reported the presence of fructose in bull semen. Identification was effected by the preparation of p-methylphenylosazone of fructose. Goto and Ugami2' used later a different technique, paper chromatography, and found to contain only fructose in bull semen. Kubicek and Santavy13' reported that fructose is accompanied with glucose, xylulose, ribose, ribulose and some polysaccharides in the seminal fluids of men and several other animals. Clemont, Glegg and Leblond4' reported that the acrosome of guinea pig spermatozoon contains galactose, mannose, fucose and hexosamines. However, the presence of N-acylaminosugars in free form in human semen has not yet been clarified.Paper chromatography appears to be a sensitive method for the detection of the free N-acylaminosugars in human semen, and it has been applied for that purpose. Preliminary chromatograms on semens from men failed to reveal the presence of the free N-acylaminosugars. However, when the semens were deionized, concentrated and chromatographed, then several N-acylaminosugars appeared. The present paper describes the methods employed for deionization, paper chromatography, and identification of the free N-acylaminosugars in human semen.ExperimenraL Deionization and concentration. From 3 to 5ml of fresh semen from man was dialysed in a cellophan bag against 300-500ml of distilled water at 4-5°C for 24 hours. After dialysis, the dialysate was passed through a column (12mm in internal diameter and 150mm long) of Amberlite IR-120 resin (hydrogen form), 100-200 mesh, and the column was washed with 30-50ml of distilled water. The effluent and washings are combined. The combined solution was then passed through a column (12mm in internal diameter and 150 mm long) of Amberlite IR-4B resin (hydroxyl form), 100-200 mesh, and the column was washed with 30-50m1 of distilled water. The rate of flow of the effluents was 1ml/minute. The effluent and washings are combined. The combined solution was concentrated to about d 2-3ml at 45-50°C under vacuum and the concentrated solution was *1 . Sanban-cho, Chiyoda-ku, Tokyo.

Chromatography

“…New spray reagents are ammonium ceric nitrate (143), 3,6-dinitrophthalic acid (1070), and titanium chloride (1289). The benzidine (258), periodate (1155), and silver nitrate (202) tests have been improved. Complex sugars may be treated with phosphoric acid on the paper and heated prior to spraying with a composite reagent (925).…”