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1. A method for measuring interstitial ‘fluid’ pressure by using a wick consisting of long-stranded cotton wool at the interface of the tissue is described. 2. A correct measurement of hydrostatic fluid pressure was obtained when the wick, connected to a suitable transducer, was applied to filter paper in which the channels contained fluid at a known subatmospheric pressure. 3. A mean subatmospheric pressure of −1.6 cmH2O was recorded in the subcutaneous tissue of the normally hydrated frog; pressure fell with dehydration and rose with overhydration. 4. A mean subatmospheric pressure of −2.8 cmH2O was recorded in the subcutaneous tissues of the abdominal wall and scalp of the normally hydrated rat. Simultaneous measurements made at symmetrical sites showed a high degree of correlation. 5. A comparison of interstitial ‘fluid’ pressure in the subcutaneous tissues of the scalp (measured by a wick) and the abdominal wall (measured by a Guyton capsule), in both anaesthetized and conscious rats showed some degree of correlation. There was, however, a wide scatter of values. 6. The interstitial ‘fluid’ pressure in the rat, measured by both wick and capsule, became more negative when the animals underwent frusemide diuresis; the capsule pressures fell more rapidly for a given degree of fluid loss. 7. The wick method was applied to the subcutaneous tissues of the arm in normal man; a mean atmospheric pressure of −3.4 cmH2O was recorded in five subjects. There were no untoward sequelae. 8. The forces responsible for the measured pressure have been analysed. The recorded subatmospheric interstitial ‘fluid’ pressure in the rat rose towards atmospheric pressure and sometimes became positive when the wick was removed, soaked in increasing concentrations of hyaluronic acid, and reinserted in the tissues. This did not happen in similar experiments in which the wick was soaked in bovine albumin, rat plasma or saline. The large macromolecules of hyaluronic acid therefore exert a force which opposes the forces responsible for the subatmospheric interstitial pressure. 9. It is suggested that the apparent subatmospheric hydrostatic pressure is due to the osmotic forces developed by the hyaluronic acid molecules in the interstitial tissues trapped by their mutual entanglement in a sieve of collagen fibrils acting as a semipermeable membrane.

Bone and dentine collagens are virtually insoluble in the solvents employed to extract native tropocollagen from soft tissue collagens. The decrease in the ease of extraction of the tropocollagen is generally considered to be due to an increase in the presence of crosslinked components ( see Piez, 1968 ; Bailey, 1968a for reviews ). Bone and dentine collagen must therefore be extensively inter molecularly crosslinked, but the chemistry of the crosslink is unknown. Glimcher and Katz, 1965 , Glimcher et al., 1965 suggested that non-covalent bonds are involved in the stabilization of bone. However a recent report by Miller et al. (1967) clearly demonstrated the presence of stable covalent inter - chain crosslinks in the denatured guanidine-HCl extract from bone. Veis and Schlueter (1964) similarly concluded that dentine was extensively crosslinked by stable covalent bonds, and proposed phosphatemediated ester bonds in addition to a system of periodate sensitive bonds. Our recent studies on the crosslinks of soft tissue collagens have demonstrated the presence of both labile and stable inter molecular crosslinks ( Bailey, 1968b ; Bailey and Peach, 1968 ). The present communication describes the isolation and identification of two covalent inter-chain crosslinks stabilizing bone and dentine.

Two O-hydroxylysyl glycosides, Hyl-Gal-Glc and Hyl-Gal, have been isolated from normal human urine and shown to be identical to two glycosides isolated from alkaline hydrolysates of collagen. A relatively sample and reproducible analytical procedure has been devised to measure the levels of these glycosides in human urine. By the use of this procedure it was shown that a normal diet has only a small effect on 24-hr urinary excretion levels of these glycosides indicating an endogenous origin. Urinary glycoside levels appear to be highest in children, roughly paralleling collagen turnover as indicated by urinary hydroxyproline levels. Collagen turnover equivalents calculated from urinary hydroxylysyl glycoside levels were found to be significantly larger than collagen turnover equivalents calculated from urinary hydroxyproline levels. This suggests that urinary glycosides are more quantitative indicators of collagen metabolism than urinary hydroxyproline. The ratio of Hyl-Gal-Glc to Hyl-Gal was measured in urines of diseased as well as normal individuals and a bimodal distribution was found. Alkaline hydrolysates of different human connective tissue collagens showed that only bone collagen, of the collagens examined, had a low ratio of Hyl-Gal-Glc to Hyl-Gal compared to human urine. Other collagens examined had higher ratios than found in human urine. On the basis of these results it is postulated that the bimodal distribution of glycoside ratios represents two populations of collagen turnover, the lower ratio population having a high bone collagen turnover, the lower ratio population having a high bone collagen turnover relative to the second population. Examination of the types of subjects making up the two populations supports this hypothesis. These data suggest that urinary O-hydroxylysyl glycoside excretion, in addition to providing a more quantitative estimate of collagen turnover than urinary hydroxyproline, may prove to be of value as a specific means of studying the metabolism of bone collagen.