On the measurement of water compartments,pH, and gradients in calvaria

Summary The concept of fluid compartmentalization in bone has emphasized the need for and lack of suitable methodology for the quantitation of water “spaces” in bone, in particular intracellular water, ICF, and extracellular water, BECF. Over a dozen commonly used marker substances were studied intensively. Small columns of hydroxyapatite crystals were employed to reveal physiochemical interactions with the mineral phase, and isotope distributions in live and dead calvaria (rat pup, and adult mice) were employed to evaluate interactions with the combined organic-inorganic matrix of bone itself. The results were most discouraging. For example, for total water space, only water itself (either by direct weight or by³H₂O exchange) is a reliable measure in all instances. All markers studied were passively concentrated or excluded to varying degrees. In the end, it was necessary to measure intracellular space “by difference.” An extracellular marker, polyethylene glycol (¹⁴C-, mol. wt. 4000), was incubated with viable calvariae and also with comparable calvariae having lysed cells (0.1% Triton X-100). The intracellular space thus determined by difference agreed well with correlative data obtained on mixed cell-isolates from similar specimens. In neonatal calvariae, the intracellular space was 30% of the total water present; that of adult mouse tibia, 18%; and that of adult mouse calvariae, 14.5%. Measurements of dimethyl-2,4-oxazolidinedione (DMO) distribution in live and lysed calvaria revealed an overall pH differential (inside bone vs. medium) of 0.1 unit or less. Using the best available data for ICF, BECF, and the passive concentrating effects of matrix-K⁺ interactions, there still remained an unexplained excess of K⁺ in the BECF.