[Show abstract][Hide abstract] ABSTRACT: We developed a direct assay of human bone collagen synthesis using [13C] or [15N] proline and applied it to determine the effects of feeding in young healthy men. Surprisingly, postabsorptive bone collagen synthesis is not sluggish, being approximately 0.07%/h more rapid than that of muscle protein, and capable of being stimulated within 4 h of intravenous feeding by 66 +/- 13%.
All current methods for estimation of bone collagen turnover are indirect, depending on the assay of collagen "markers." Our aim was to develop a direct method for human bone collagen synthesis to be used to study its physiology and pathology, and specifically, in the first instance, the effect of feeding.
We applied, over 2 h, flooding doses of [13C] and [15N] proline to label iliac crest bone collagen in eight young healthy men. The rate of collagen synthesis was determined as the rate of labeling of collagen hydroxyproline (assayed by gas chromatography-combustion-isotope ratio mass spectrometry in collagen extracted by differential solubility) compared with plasma proline labeling (assayed by gas chromatography-mass spectrometry). We also determined (in a second group of eight young healthy men) the effect of intravenous nutrition (glucose, lipid emulsion, and amino acids (in the ratio of 55%:30%:15% energy, respectively).
Free bone proline labeling was 92 +/- 6% of that of plasma proline, supporting the flooding dose assumption. Human iliac crest bone collagen is heterogeneous, with NaCl-EDTA, 0.5 M acetic acid, pepsin-acetic acid, and hot water-extractable pools being responsible for approximately 1%, 3%, 8%, and 81% of content, respectively. The synthetic rates were 0.58 +/- 0.1, 0.24 +/- 0.05, 0.07 +/- 0.02, and 0.06 +/- 0.01%/h, respectively, giving an average rate of approximately 0.066%/h. [13C] and [15N] proline gave identical results. Intravenous nutrition caused the disappearance of proline label from the procollagen pool and its increased appearance in the less extractable pools, suggesting nutritional stimulation of collagen processing.
The results show (1) that iliac crest bone collagen synthesis is faster than generally assumed and of the same order as muscle protein turnover and (2) that feeding increases synthesis by approximately 66%. Given its ability to detect physiologically meaningful responses, the method should provide a new approach to studying the regulation of bone collagen turnover.
Journal of Bone and Mineral Research 07/2005; 20(6):930-7. · 6.13 Impact Factor
[Show abstract][Hide abstract] 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.
Biochemical Society Transactions 05/2002; 30(2):61-5. · 2.59 Impact Factor