Methods

Preparation of Tissue Samples-Adult male white Swiss mice, 6 to 8 weeks old (weight 30 to 50 g), were killed by decapitation, exsanguinated, and both knee joints were dissected out. The metaphyses about the articulations were freed from tendinous attachments, and the epiphyseal cartilage carefully removed. Each metaphysis was then cut longitudinally into two pieces and the marrow carefully scraped out with the point of a scalpel. Samples consisting of spongiosal bone surrounded by some cortical bone from the femur and the tibia were then washed in iced Krebs-Ringer medium, quickly blotted dry with filter paper and weighed on a Roller-Smith balance. They were then placed in incubating flasks containing iced medium and stored on ice until the start of the incubation. For each incubation flask, two animals were pooled, each sample then consisting of 8 metaphyses split in two.

Since the mass of these bone samples was largely mineral, a measure of their cellular content was needed to compare their metabolic behavior with other tissues such as liver. The cellular nitrogen content of the samples (cell N) was therefore estimated, with the use of a modification of the noncollagenous nitrogen method of Lilienthal (8). The procedure used was as follows: 2 ml of 0.2 N NaOH were added to the incubation flask containing 2 ml of medium and the sample, at the end of the experiment. The flasks were then shaken for 16 hours at room temperature on a mechanical shaker. Aliquots of the supernatant fluid were digested by the micro-Kjeldahl technique; cooled; Nessler’s reagent was added; and the color compared to nitrogen standards with the use of a Coleman junior spectrophotometer at a wave length of 440 p.