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Editorials |
Creighton University, 601 North 30th Street, Suite 4841, Omaha, NE 68131, Fax 402-280-4751, E-mail rheaney{at}creighton.edu
In the rush to develop "peaceful uses of the atom" in the years following testing of the H-bomb, much interesting work was done with radioactive calcium isotopes (45Ca and 47Ca). Perhaps most important was the finding that the relatively huge quantities of calcium contained in mature bone exchanged only negligibly with calcium either in extracellular fluid (ECF) or in immediately adjacent bone (1). This meant that tracer uptake by bone could be used as a direct measure of new bone mineralization. Although various complicated kinetic models were developed(2)(3), the mathematics were actually quite straightforward. The tracer content of a sample of bone calcium obtained days or weeks after an injection of a calcium isotope into an animal, divided by the average tracer concentration in ECF calcium, directly yields the mass of calcium deposited by bone formation during the interval between tracer injection and the time of the bone sampling.
What was missing from the picture was the ability to measure the other half of the bone remodeling/modeling process, i.e., bone resorption. How could one measure in a bone what was no longer there? Measuring the decrease in bone tracer content was not a feasible approach because the resorptive process removes mainly older bone, which contains little or no tracer. Despite this barrier and because so much of the interest in clinical bone biology was focused on bone resorption, there has for years been a search for a more "direct" quantification of the process. This quest was reinforced by the development of antiresorptive pharmacologic agents for the treatment of osteoporosis, as well as by the demonstration that resorption responds sensitively to environmental or physiologic signals, whereas bone formation tends to be relatively invariant over the short term (4).
Moreover, the investigational focus shifted from in vitro measurement of animal bones to in vivo studies of intact organisms, and from animals to humans. What had been little noted was that this shift had turned the analytic paradigm on its head. We no longer measure bone at all. Calcium kinetics analyses in humans today are based on serial measurements of tracer concentrations in ECF calcium (5). That concentration decreases over time because of dilution: early on from distribution of tracer through a series of progressively more slowly exchanging body compartments, but later because of addition of unlabeled calcium from diet and bony resorption.
Unfortunately, that very decrease in tracer concentration in ECF calcium was sometimes conceptualized to reflect loss of tracer from the system, i.e., into urine, feces, and mineralizing bone (which, because it does not exchange with ECF calcium, is, at least for a time, "lost" to the ECF) (6). Although such loss is indeed occurring, it is not the reason for the dilution, as a simple thought experiment will reveal. Imagine a situation in which bone resorption is zero and the organism is studied on a zero calcium intake. After an injected tracer equilibrates with pool compartments, concentration of the tracer in ECF calcium now remains constant. Total ECF calcium content decreases as calcium is excreted and/or deposited at previously nucleated bone-forming sites, but tracer and carrier decrease in strict parallel; therefore, tracer concentration per unit of carrier does not change. The tracer concentration in serum is decreasing, but the tracer concentration in serum calcium is not. The reason is that no unlabeled calcium is entering the system.
The key point here is that tracer concentration has meaning only with respect to the carrier species for which it functions as a tracer. Calcium isotopes are tracers for calcium, not for ECF water. Under steady-state conditions, the long-term decrease in ECF calcium tracer concentration is attributable to dilution of labeled ECF calcium by entry of unlabeled calcium from the diet and from resorption of unlabeled bone. Thus, the rate of this dilution is, in fact, a direct measure of the rate of bone resorption. By shifting the focus from measurements in bone to measurements in serum, one inverts what can be directly measured (resorption) and what must, instead, be inferred (formation).
Nevertheless, there has continued to be interest in somehow measuring tracer movement out of bone, as was done, early on, for tracer movement into bone. To do that requires that the skeleton contain the tracer. Then, in individuals ingesting a tracer-free diet, tracer excretion in urine, for example, would reflect bone resorption (as well as measure the proportions of urine calcium contributed by bone and diet). That much is straightforward. Here another thought experiment may be helpful, however. Imagine two streams flowing together to form a larger river. One carries a heavy load of silt, and the other is largely clear. Downstream of the confluence, silt concentration will be intermediate between the concentrations in the two streams. In fact, the silt concentration allows direct calculation of the relative contributions of the two source streams. It is as if one of the two streams were tracer-labeled. But which one? Is it the silt-laden stream (with silt serving as tracer), or is it the clear stream (with the absence of silt serving as tracer)? The truth is: it makes no difference. The calculations are the same either way. So too with ECF calcium and bone calcium. Which source tributary contains the label? Is it the presence of extra isotope in the ECF or the lack thereof in bone and diet? As with the river analogy, it makes no difference. The mathematics are the same either way.
There is also a technical problem with trying to label a skeleton by adding a tracer. Although more than one half of the tracer incorporated into bone today will ultimately get redistributed throughout the skeleton, the process is slow. Cancellous bone turns over at a rate of 20%–30% per year, but cortical bone turnover is an order of magnitude slower. Hence, the time required to develop an even approximately homogeneously labeled skeleton must be measured in years. Until recently, none of the available tracers had the requisite properties to permit their detection over such long periods of time. The radioactive tracers would long since have decayed to background and the quantity of the stable isotopes that could be administered to give a signal above their own natural background abundance years later would have been massive, not to mention prohibitively expensive.
The development of an assay method for 41Ca has changed the rules in this regard. This issue of the Journal describes just such a method and shows, in a simple clinical application, that it reliably detects physiologically interesting differences in the calcium economy (7).
Technically, 41Ca is a radioactive isotope, but its half-life is so long that, for all practical purposes, it behaves as a stable isotope. Its radioactive character means that it ultimately decays and thus has zero natural percent abundance. Therefore, unlike with the true stable isotopes, its measurement involves a zero blank. The development of accelerator mass spectroscopy (AMS) and the perfecting of the specimen preparation chemistry leading up to it have made it possible to detect vanishingly small amounts of this interesting tracer. What utility might this new tool bring to the exploration of bone biology?
One interesting example is found in the existence of a population whose skeletons are prelabeled with 41Ca. Freeman et al. (8) had earlier called attention to the existence of this population, whose 41Ca content is a consequence of the administration of 45Ca and 47Ca to many thousands of adults (in this country and abroad) over the past 50 years. The process of making the radioactive isotopes is basically neutron bombardment of calcium sources, producing a full spectrum of calcium isotopes ranging from 41Ca to 48Ca. The proportions of each depend on previous source enrichment and on the nuclear cross section of the immediate precursor isotope, but all irradiated sources contained some 40Ca. Hence, all products contained some 41Ca, and thus all persons received 41Ca along with the intended 45Ca or 47Ca. The quantity was so small as to be undetectable by then-available methods and, thus, was treated as if it were not there. But it was there, of course, and today essentially all individuals who have been dosed with the radioactive or stable isotopes of calcium have a skeleton that is to some extent prelabeled with 41Ca. AMS makes it possible to detect that tracer in samples obtained from body fluids, from urine, or from bone biopsies. Given sufficient elapsed time, one may assume, as at least a first approximation, that the 41Ca will be to some extent uniformly distributed through the skeleton, at least through its more active components (such as central trabecular bone mass). Whether this fortuitous opportunity can be exploited to provide uniquely useful insights into bone biology remains to be seen.
That is one potential application. Additionally, individuals with no previous calcium isotope exposure can today be given 41Ca, as such, in doses that are both plausible and safe, with the assurance that the tracer will be detectable in their tissues and fluids for years to come. Thus, it is easy to see why interest in 41Ca has increased with the availability of technology permitting its measurement.
There is a wry irony in all this, reflecting a certain asymmetry in human ability to conceptualize these issues. It remains true, but is somehow unsatisfying, that the most uniformly labeled skeleton is not one that was exposed to tracer years ago, but the one that was never exposed at all and is now residing in a body with a freshly labeled ECF. After all, it is not the location of the tracer, but the difference in tracer concentrations between bone and ECF calcium that is important (and informative).
References
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