THE QUANTITATIVE MEASUREMENT OF BONE RESORPTION AND BALANCE IN VITRO BY ANALYSIS OF HYDROXYPROLINE METABOLISM

Barry Flanagan and George Nichols, Jr.~ Department of Medicine, Harvard Medical School, Boston and Cambridge City Hospital, Cambridge, Massachusetts, USA

The use of hydroxyproline as an indicator of collagen metabolism is now generally ac- cepted (Prockop and Kivlrikko~ 1967). Metaphyseal bone slices from 40-50 day old rats were prepared as previously described (Borle et a l . , 1960)t and incubated in Krebs- Ringer bicarbonate medium, fortified with glucose 11.1 mM~ prollne 1.5ram and con- taining prollne-u-C-14 l~ Incubations were carried out for periods of up to 6 hours, under 95% 02/5% CO 2. Hydroxyproline is released and accumulates in the in- cubation media. The time course of accumulation revealed at least 2 components: (a) an early exponential release completed by approximately 2 hours, and (b) a linear release rate which was constant for up to 6 hours. Suitable prepared dead samples pro- duced only the initial release (a). We concluded that this represented a solubility phen- omenon, and that (b) represented the combined contribution of new synthesis and release together with resorption.

To separate these contributions quantitatively, a series of experiments were conducted using proline-u-C-14 as an indicator of the newly formed medium hydroxyprollne. It was established that (a) there exists no significant pool of free intracellular prollne so that the specific activity of the intracellular proline pool instantaneously equals that of the applied medium proline, and (b) that the rate of release of labelled hydroxyproline into the medium rises to a maximum by 3 hours, and continues to be linear until at least 6 hours of incubation. With this information, the contribution of synthesis and release to the medium pool can be accurately quantitated~ and the true resorption rate of established col lagen estimated by difference.

The rate of synthesis of hydroxyproline~ together with its rate of deposition in the tissue may be measured at the same time allowing the balance of matrix collagen to be studied directly in vitro.

This approach has been applied to a number of states of altered bone metabolism. During the ageing process the rates of collagen synthesis and resorption decline rapidly, the former to a greater degree than the latter. The solubility of bone collagen is also considerably reduced. A very intriguing discovery was that the fraction of the new synthesized collagen which is actually deposited in matrix also declines progressively with ageing.

The changes demonstrable in vitro following acute exposure to parathyroid hormone, cortisone, thyroid hormone and t-~yrocalcltonin in vivo have been studied and will be presented together with changes produced in vitro ~y varying phosphate concentrations and by thyrocalcitonin applied in vitro.

D.J. Prockop and K.I . Kivlrlkko, Annals Int. Med. 66:1243 (1967).

A. Borle, N. Nichols and G. Nichols, Jr., J. Biol. Chem. 235:1211 (1960).

51 Calc. Tiss. Res. 2, Suppl. (1968)

SOURCES OF MEDIUM HYDROXYPROLINE

PROLINE

kl

OSTEOBLAST INTERSTITIAL FLUID

PRO PRO

P HYPRO

MATRIX

k4~, k 5

MEDIUM HYDROXYPROLINE

Fig. I. A diagrammatic representation of the sources of medium hydroxyproline and of various rate constants involved. Proline transported into the osteoblast (K) is partially transformed into protocollagen hydroxyproline (K2~ K3) and extruded (K4) as a soluble polypeptide chain into the interstitial fluid. It is then either deposited (K5) or escapes into the medium (K6). K 7 represents the rate at which passive solubillzatlon of matrix collagen occurs during incubation~ and K8 ~ the active cell-medlated resorptlve process, is shown here conventionally as being caused by a multinucleate osteoclast.

Calc. Tiss. Res. 2, Suppl. (1968) 51 A

80

70

�9 TOTAL HYDROXYPROLINE

0 LABELLED HYDROXYPROLINE

I . - 60

m h i

I ' - ,,, 50

E o 40 0

0

" 50 12. > - " I-

~ ao

I0

I 2 5 4 5 6 7' B HOURS OF INCUBATION

Fig. 2. Experimental plot to illustrate the three components of medium hydroxypro- line, The upper curve shows the cumulative release of hydroxyproline wlth tlme. Extrapolation of the linear portion of thls curve to the ordinate glves the slze of the passively solubilized fraction. The lower curve shows the release of labelled hydroxyproline into the medium. The parallel dotted line represents the tru release rate of newly synthesized hydroxyproline. The difference between the slopes of the linear portions of both curves give the true resorption of matrix collagen. One SD above and below the line is shown.

51 B Calc. Tiss. Res. 2, Suppl. (1968)