Remodelling of the Bone Matrix 25 



from the lightest fraction (1.7) to the one of density 2.2 An almost identical pro- 

 portion of collagen and calcium is located in the heaviest particles. The ratio of 

 calcium to collagen is low in the first three fractions. It increases to a maximum in 

 the fraction with the greatest volume, (2.2), but it becomes lower again in the densest 

 fraction. 



To consider if these fractions are related to the metabolism of the collagen In 

 bone we have labelled the collagen with a radioactive tracer and measured the radio- 

 activity in the bone fractionated by gradient density. 



Sixty day old rats were injected with ^H proline and killed at increasing times 

 after injection of the tracer. The long bones were collected and immediately cooled 

 on ice. Diaphyses cleaned of marrow were ground and separated into fractions of 

 different densities. After hydrolysis in 6 N HCl, the amount of hydroxyproline was 

 measured and this amino acid purified by thin layer chromatography in the presence 

 of cold carrier. The radioactivity of the samples was measured in a liquid scintillation 

 counter. The results are illustrated in Table 2 for dift'erent time points up to 48 

 hours. The S.A. of the hydroxyproline of the unfractionated bone is tabulated in 

 dpm///M. The values for the hydroxyproline of each fraction are multiples of the 

 S.A. of the hydroxyproline of the unfractionated bone. This method of computing 

 the data allows us to locate the migration of the label. The only variable is the time 

 elapsed since the synthesis of the collagen. 



The S.A. of the hydroxyproline in the unfractionated bone is increasing with 

 time, faster during the first hours than later. It is the expression of the overall 

 turnover of the collagen in the bone. The location of the maximum S.A. of the 

 collagen In the fractions gives more information. We see immediately that the S.A. 

 of the collagen varies widely according to the density of the fractions and also to the 

 time elapsed since the Injection of the tracer. Half an hour after the Injection of 

 the labelled precursor, the lightest fraction (1.7) has the highest specific activity. 

 There is however some label in all the fractions. "With Increasing time, up to 

 3 hours, the collagen of the lightest fraction (1.7) keeps the highest S.A. In the mean- 

 time the S.A. of the other fractions decreases. These data would indicate that the 

 newly synthesized collagen Is contained in the 1.7 fraction. The radioactivity in the 

 other fraction could represent some contamination of the samples by labelled non 

 protein bound hydroxyproline disappearing progressively by dilution. Six hours after 

 the Injection of the label, the S.A. of the collagen in the fraction 1.8 increases, 

 indicating the appearance of the labelled molecules coming from the 1.7 fraction. 

 Twelve hours are required to see the same change in the 1.9 and 2.0 fractions and 

 24 hours In the 2.1. At that time the relative S.A. of the 1.7 fraction drops because 

 more labelled molecules are then located In the other fractions. The same appears 

 after 36 hours for the 1.8 and 1.9 fractions. The labelled collagen seems to move to 

 the 2.2 fraction only after 48 hours. More than two days are required for the label 

 to reach the last fraction. The progressive reduction of the relative S.A. of the 

 collagen when It passes from one fraction to the other Is due to the dilution of the 

 newly formed bone collagen in fractions of increasing size. 



The general meaning of this metabolic study Is Illustrated in Table 2 by the con- 

 formation of the area between the dark lines. It represents the location of the 

 maximum relative specific activity of the collagen for each fraction in relation to 

 time. Mainly located In the lightest fraction It moves progressively toward the heaviest 



