Sequential extracts of human bone show differing collagen synthetic rates

Abstract

Type I collagen is the major bone protein. Little is known quantitatively about human bone collagen synthesis in vivo, despite its importance for the understanding of bone formation and turnover. Our aim was to develop a method that could be used for the physiological and pathophysiological investigation of human bone collagen synthesis. We have carried out preliminary studies in patients undergoing hip replacement and in pigs to validate the use of the flooding dose method using (13)C- or (15)N-labelled proline and we have now refined our techniques to allow them to be used in a normal clinical or physiological setting. The results show that the application of a flooding dose causes bone free-proline labelling to equilibrate with that of blood in pigs and human beings, so that only 150 mg of bone will provide enough sample to prepare and measure the labelling of three fractions of bone collagen (dissolved in NaCl, acetic acid and pepsin/acetic acid) which have the same relative labelling (1.0:0.43:0.1) as measured by GC-combustion-isotope ratio MS. The rates of incorporation were substantially faster than in skeletal muscle samples taken at the same time. The results suggest that different fractions of human bone collagen turnover at markedly higher rates than had been previously considered. This approach should allow us to discover how growth and development, food, activity and drugs affect bone collagen turnover and to measure the effects on it of ageing and bone disease.

Effect of estrogen on tendon collagen synthesis, tendon structural characteristics, and biomechanical properties in postmenopausal women

“…Fluid collected from the interstitial fluid surrounding the patellar tendon by using a microdialysis technique was analyzed for a marker of CTX-I synthesis, the amino terminal propeptide of CTX-I (PINP) (31). The remaining dialysate was used for analyses of IGF-I and IGF-binding proteins (IGFBPs) (1)(2)(3)(4). After previous preparation of incision sites with local anesthetic (lidocaine 1%), ethylene oxide sterilized catheters with high molecular mass cut-off (3,000 kDa, membrane length 30 mm, inner diameter 0.50 mm) were inserted under ultrasound guidance in the peritendinous spaces of patellar tendons, as previously described (31).…”

Effect of administration of oral contraceptives in vivo on collagen synthesis in tendon and muscle connective tissue in young women

“…Another more methodological explanation for the discrepancy between the effect of OC on tendon and muscle collagen FSR might be related to the different preparation procedures of the tissue samples before GC-C-IRMS analysis. Although seemingly sparse (3,4) some contamination by other proteins cannot be ruled out in the muscle preparations, whereas the risk of this error is minimal in tendon since collagen is the far most abundant protein in tendons.…”

“…Plasma proline was prepared as previously described and analyzed as its t-butyldimethylsilylation (MTBSTFA ϩ 1% TBDMS) derivative by gas-chromatography massspectrometry (GC, Trace GC 2000 series) (3,55). We used the plasma [ 13 C]proline enrichment since the flooding technique is assumed to equilibrate all free amino acid pools, including the true precursor pool, prolyl-tRNA (3).…”

GH and IGF1 levels are positively associated with musculotendinous collagen expression: experiments in acromegalic and GH deficiency patients

“…Measurement of collagen protein FSR was carried out according to previous techniques (31). Skin biopsy was used to establish the natural abundance of [1-13 C] proline in collagen protein, thereby eliminating the need for a pre-infusion basal tissue biopsy.…”