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Technical Briefs |
1 Creighton University, Omaha, NE 68131
2 Teachers College, Columbia University, New York, NY 10027
aaddress correspondence to this author at: Creighton University, 601 N. 30th St., Suite 4841, Omaha, NE 68131; fax 402-280-4751, e-mail rheaney{at}creighton.edu
When both calcium intake and calcium absorption are measured under controlled conditions, variation in absorption efficiency explains more of the interindividual differences in balance than does actual calcium intake (1). Small wonder, therefore, that interest in measuring absorption has remained high for nearly 40 years.
True calcium absorption is defined as the quantitative, unidirectional flux of calcium from intestinal lumen into the blood. It is most accurately measured by a dual-tracer method, with one tracer labeling the oral calcium load and the other labeling the miscible calcium pool into which the absorbed calcium is introduced. This approach was first developed into a practicable human test by deGrazia et al. (2). As described, it is usually time-consuming and expensive. To reduce these barriers for widespread use, Heaney and Recker (3)(4) developed a single-tracer variant for women, requiring only a single blood sample, and calibrated it against a simultaneously performed double-tracer method. The single-tracer method has been used efficiently in thousands of women (5). However, because the calibration is empirical and based on body-size variables, it is not directly suitable for use in men who, with a typically higher proportion of fat-free mass than women, would be expected to distribute absorbed tracer in a larger mass of calcium.
To fill this methodologic gap, we performed a small set of parallel measurements in adult men, using the female-based algorithm together wiketh a modified double-tracer approach.
Participants in the study were 30 Caucasian men (age range, 20–60 years; weight range, 63.5–104 kg; height range, 1.67–1.93 m). All participants were free of known diseases affecting bone remodeling or calcium homeostasis, and tests were not performed if the individual had experienced any gastrointestinal disturbance in the preceding 5 days. Each gave informed consent after the procedures of the study were explained. Both the project and the consent were approved by the Creighton Institutional Review Board. Each volunteer was tested twice. We performed the first test for several unrelated projects, using the standard, single-dose protocol, giving a 45Ca-labeled oral load and obtaining the usual 5-h serum sample for measurement of serum calcium specific activity. The volunteers abstained from all food after the test breakfast until the 5-h blood sample was drawn. The test calcium load (depending on the individual projects) was 300 mg in 25 individuals and 500 mg in 5. Sources were calcium-fortified orange juice in 20 volunteers, skim milk in 5, and precipitated calcium carbonate in 5. The second test, performed 6.2 (± 3) days later, used an intravenous (i.v.) dose of high-specific activity 45Ca, given 2 h after an identical test breakfast that contained the same oral calcium source and the same calcium load as on the first test day. With the second test, serum was obtained 3 h after the i.v. dose for measurement of serum calcium specific activity. This timing reproduces accurately the dosing scheme of a simultaneous double-tracer experiment in which, as originally described (2), the i.v. tracer is given 2 h after the oral tracer. (The 2-h lag introduces the i.v. tracer at the approximate midpoint of absorption of the oral tracer.) Because the same tracer was used to label both the oral load and then subsequently the miscible calcium pool, a baseline serum sample was obtained at the second test to determine the concentration of residual tracer from the earlier oral test. (Mean correction was -9.6% of the total counts in the 3-h blood sample, with the maximum being -25% for the shortest interval and the minimum being -3.1% for the longest.)
Absorption fraction was calculated in two ways. The first consisted of the female-derived algorithm:
 | (1) |
The second method used both tracers, calculating the quotient of the 5-h calcium specific activity after the oral tracer dose and the net 3-h activity after the i.v. tracer. The results with the two methods were then regressed on one another by both standard Pearson and orthogonal regression analyses (6). Additionally, the mean ratio of the within-individual values for the two methods was tested against a null hypothesis value of 1.0. The statistical software was Crunch 4.01 (Crunch Software) and SPSS (SPSS, Inc.) for Windows (Release 10).
The results of the paired measurements are presented graphically in Fig. 1
and in tabular form, individual-by-individual, in the data supplement available at Clinical Chemistry Online (http://www.clinchem.org/content/vol48/issue5). The similarity of the values obtained by the two methods was extremely close (r2 = 0.904). However, the slope of the double-tracer value on the female-derived value was greater than unity, with a point estimate of 1.085. The mean quotient of the two test values was, of necessity, also 1.085, with a SE of 0.016. This value differs significantly from the null hypothesis value of 1.0 (P <0.001; 95% confidence interval, 1.054–1.117). This quotient was constant across the full range of absorption values. The set of quotients was also independent of both height and weight. Alternatively, when the data were evaluated by orthogonal regression, the equation became y = 1.149x - 0.0173. This slope was significantly different (P <0.05) from 1.0 (the value under H0, the null hypothesis).
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Because the intercept with the standard Pearson regression was virtually zero (and not significantly different from zero), it is simpler to represent the relationship as y = 1.087x (the equation when the line is forced through the origin), where y is the true absorption in men, and x is the calculated value with the parameters derived from women. Using this equation, we obtained the best estimate of absorption fraction in men by multiplying the value derived from the female algorithm by 1.087. Alternatively, the factor could be introduced directly into Eq. 1
, which then becomes:
 | (2) |
This work provides a firm basis for estimating the true calcium absorption fraction in men from nutritionally meaningful, labeled loads. The coefficient of determination from the regression of the two methods on one another was essentially the same as reported previously for the same regression in a much greater number of tests in women (3). Although, in that earlier study, the 5-h sampling time was selected empirically, it makes good intuitive sense because absorption is virtually complete by 5 h (7); in addition, pool turnover is typically small relative to pool size, and hence early time points reflect mainly dilution.
The adjustment factor we found (1.087) is somewhat less than might have been predicted from body-water differences between men and women, where it is generally considered that, per unit of weight, men have 
15% more water than women (8). However, calculations of calcium absorption after an oral tracer based on body water have never seemed very plausible because it is known from tracer distributional studies that, by 3 h after an i.v. injection, tracer is distributed in a mass of calcium that is more than twice the amount of calcium contained in the entire extracellular fluid volume (9)(10). This point is shown clearly in Eq. 2
when one substitutes arbitrary height and weight values. Thus, for a height of 1.65 m and a weight of 75 kg, the apparent volume of distribution at 5 h in women is 2.30 g and 2.50 g in men.
Thirteen years ago, Favus (11) editorialized that enough had been learned about calcium absorption measurement to warrant making a calcium-tracer-based absorption test routinely available. To date, that challenge has not been accepted by any major laboratory. Instead, several strontium-based tests have been used (12)(13). Although they exhibit pragmatic utility in detecting large departures from normal, these strontium-based tests cannot provide the desired quantification of mass transfer of calcium from gut to blood. Moreover, they resist empirical calibration because the active transport process in both kidney and gut discriminates against strontium ions relative to calcium (14)(15). These relationships tend to be nonlinear. A calcium isotope obviates this difficulty.
The tests described here used 45Ca, but they could have been performed just as well with a stable calcium isotope. 45Ca is easier and less expensive to measure. The test we describe can be performed with 3–7 µCi (0.11–0.26 MBq) of 45Ca, which, at typically incomplete absorption fractions (30%), produce a trivial increase in total body radiation (less, for example, than that experienced from cosmic ray sources during a vacation in the mountains or a transcontinental airplane trip). Hence, there would seem to be no substantive objection to implementing the suggestion by Favus (11) with 45Ca as the tracer.
However, whether a stable or a radioactive tracer is used, there is now a suitable algorithm for both men and women, requiring only a single serum sample and providing results within 1 day.
Acknowledgments
This study was supported in part by an agreement with the University of Pittsburgh, Graduate School of Public Health, by contracts with Roots, Inc. and DepoMed, Inc., by a grant from Health Future Foundation, and by Creighton University funds.
References
The following articles in journals at HighWire Press have cited this article:
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  M. D. Parra, B. E. Martinez de Morentin, J. M. Cobo, I. Lenoir-Wijnkoop, and J. A. Martinez Acute Calcium Assimilation from Fresh or Pasteurized Yoghurt Depending on the Lactose Digestibility Status J. Am. Coll. Nutr., June 1, 2007; 26(3): 288 - 294. [Abstract] [Full Text] [PDF]
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