Glycoprotein and collagen metabolism in the dermis of hypophysectomized rats.

The effect of hypophysectomy on the concentration of total, salt soluble (0.15 and 0.5M sodium chloride and 0.5M sodium citrate) and insoluble collagen, nitrogen and hexosamine (Hex) in the skin of 60-day-old male rats was investigated. The pituitary glands were ablated at the 30th day of age. The total hydroxyproline (Hyx) concentration in hypophysectomized animals was lowered by about 25 % and that of total Hex increased by 30% as compared to the controls. The Hex/Hyx ratio was 0.047 for controls and 0.081 for hypophysectomized rats. Since the mucopolysaccharide (MPS) concentration was not modified in hypophysectomized rats [Nature (London) 200: 784, 1965], the higher Hex concentration appears due to increased glycoprotein content. Only about one fourth of the total Hex in intact and one fifth in hypophysectomized animals can be accounted "for as MPS. (Endocrinology 78:1255,1966) P hormones have been reported to be involved in mucopolysaccharide (1-3) and hexosamine (3) metabolism. Our studies on skin permeability suggested that the ablation of the pituitary gland increases the degree of polymerization of hyaluronic acid in connective tissue (4). In the present work we investigated the effect of hypophysectomy on the concentration of collagen, glycoproteins and mucopolysaccharides in the dermis. Materials and Methods The studies were performed on young male albino rats of the CFE strain. Animals in the experimental group were hypophysectomized when 30 days old; they weighed 95-100 g. Both hypophysectomized and control rats were purchased from Carworth Farms, Inc., New City, Rockland County, N. Y. They were fed "Wayne Lab Blox mice and rats" (Allied Milling Co.) diet and had free access to tap water. The rats of both groups were sacrificed when 60 days old by exsanguination under light ether anesthesia. At that time, those in the control group weighed an average of 240 g±30, whereas the hypophysectomized rats averaged only 100 g +10. The skin of the animals was shaved, freed from Received December 13, 1965. This work was aided by Grant A-4619 from the National Institute of Arthritis and Metabolic Diseases, USPHS. 1 Present address: Laboratory of Experimental Metabolic Diseases, Veterans Administration Hospital, Long Beach, California 90804. 2 Standard deviation. 3 The animals which increased their body weight after hypophysectomy were eliminated from the experiment. adherent muscles by scraping, ground into paste in a mill (Wiley Laboratory mill, intermediate size) and weighed. One portion of the "wet skin" was defatted with acetone in a Soxhlet extractor, dried at 80 C in a drying oven and used for total hexosamine, collagen and mucopolysaccharide determinations. The other portion of "wet skin" was successively extracted with 0.15M, 0.5M sodium chloride and 0.5 sodium citrate, pH 3.6. These extracts, the insoluble residue, and the portions of dry defatted skin samples were then hydrolyzed with 6N hydrochloric acid in sealed glass tubes at 104 C for 4 hr and analyzed for hexosamine, collagen and nitrogen contents. The hexosamine content was determined by the Elson-Morgan method according to Blix (7). The collagen content was determined by the Stegemann method for hydroxyproline (8). Nitrogen content was determined by the micro-Kjeldahl procedure using Nessler reagent. Acid mucopolysaccharides were extracted from the skin, fractionated and determined according to our previously described method (9). The isolated acid mucopolysaccharides (MPS) were precipitated with cetylpyridinium chloride (CPS) and CPCMPS complex was successively extracted with 0.4M, 1.2M and 2.1M sodium chloride in order to solubilize and separate the hyaluronic acid, chondroitin sulfate and heparin fractions, and then analyzed by the carbazole method according to Bitter and Muir (10) using glucurone as the standard. Results and Discussion The animals with an intact pituitary gland (control) manifested normal growth, whereas 4 Sorensen's citrate buffer (5, 6). 6 Since only small samples of skin could be obtained from hypophysectomized rats, the determination of acid mucopolysaccharides for this group was performed on a pooled sample.