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1 From the Nestlé Water Institute, Vittel, France (MS and MJA); the Western Human Nutrition Research Center, US Department of Agriculture/Agricultural Research Service, University of California, Davis, CA (WRK and JRT); and the Mass Spectrometry Unit, INSERM, Toulouse, France (FP).
2 Portions of the results were presented as an oral communication at the 9th Magnesium International Symposium, Mag 2000, Vichy, France, 10–25 September 2000. 3 Supported by the Nestlé Water Institute and the US Department of Agriculture. 4 Address reprint requests to MJ Arnaud, Nestlé Water Institute, BP 101, Vittel Cedex 04, France. E-mail: maurice.arnaud{at}waters.nestle.com.
ABSTRACT
Background: The double-labeling (DL) method for determining magnesium absorption is less cumbersome than is the fecal monitoring method, which has been used most often, but it has not been validated.
Objective: The aim of this study was to compare methods and several sampling protocols for determining magnesium absorption to establish a simple and reliable alternative to the fecal monitoring approach. Fecal monitoring was used as the standard against which the DL methods based on urine data (DLU), plasma data (DLP), and plasma kinetics with the use of a deconvolution analysis (DP) were compared.
Design: Six healthy adult men received 70 mg 26Mg orally and 30 mg 25Mg intravenously. Multiple blood samples and complete urine and fecal samples were collected over 12 d. Stable-isotope ratios were determined by inductively coupled plasma mass spectrometry.
Results: Results from DLU were not significantly different from the fecal monitoring reference value (0.48 ± 0.05;
Key Words: Magnesium absorption • stable isotopes • mass spectrometry • double-labeling methods • dysprosium • men
INTRODUCTION
In humans, > 300 enzyme systems are dependent on the presence of magnesium. It has been suggested that aging, stress, and various diseases may increase magnesium requirements (1) and that inadequate intake and impaired magnesium absorption (MgA) may contribute to many pathologic conditions (2). Therefore, reliable methods for determining MgA are essential for investigating magnesium metabolism and homeostasis.
Few studies of MgA determination with stable isotopes have been reported, despite their safety and accuracy. One limitation of their use is that the 3 magnesium isotopes are relatively high in abundance (3). Consequently, large amounts of isotope must be administered for reliable isotopic measurements, generating a high cost for such experiments. New generations of mass spectrometers with improved accuracy and requiring smaller doses of isotopes to be administered have been developed, but sample preparation remained time consuming (4–6). The development of inductively coupled plasma mass spectrometry (ICP-MS) brought about higher sensitivity, rapid throughput, and less time-consuming sample preparation, simplifying magnesium isotopic ratio measurements (7).
This instrumentation made the investigation of MgA simpler, allowing use of the double-labeling (DL) method. This method requires the administration of 2 stable-isotope tracers, one orally and one intravenously. For calculations, it is assumed that the oral label, once absorbed, is indistinguishable from the intravenous label. Both tracers are assumed to mix rapidly and completely in the plasma portion of the total-body magnesium pool and are excreted in urine in a way that reflects their abundance in the circulation. Thus, evaluation of MgA can be based on urine (DLU) or plasma (DLP) collections (8). The kinetics of the labels can also be analyzed by deconvolution (DP) (9), another alternative for estimating MgA. In the simplest protocol, MgA could be evaluated from a single blood draw or a short urine collection. Fecal monitoring and the oral administration of one isotope have been used most frequently to determine MgA (10–13). MgA is estimated by determining the disappearance of the label during intestinal passage, which is calculated as the difference between intake and fecal content. However because fecal monitoring does not take into account fecal endogenous excretion (FEE), MgA is underestimated and the result corresponds to the apparent MgA (MgAA). FEE can be estimated with a second isotope administered intravenously and then used to correct MgAA. Thus, fecal monitoring does not provide a direct measurement of MgA. The collection of fecal samples associated with this technique is cumbersome compared with the sampling of urine and blood with the DL techniques. To our knowledge, except in rats (14), no comparisons have been done between these various isotopic techniques. The aim of this human study was to investigate the feasibility of studying MgA with the DL methods and to compare the results with the result from fecal monitoring corrected for FEE. Several sampling protocols were tested to identify the simpler and the most reliable alternative to the fecal monitoring method.
SUBJECTS AND METHODS
Subjects
Six healthy men were recruited for the study. These subjects were a subset of 11 men participating in a copper supplementation study. The anthropometric characteristics of the subjects are summarized in Table 1. Written informed consent was obtained from all subjects. The Institutional Review Board of the University of California, Davis, and the US Department of Agriculture Human Studies Review Committee approved the protocol.
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TABLE 1 . Anthropometric characteristics of the subjects
Protocol
All subjects were studied in the Metabolic Research Unit of the US Department of Agriculture/Agricultural Research Service Western Human Nutrition Research Center in San Francisco. The study was performed over 18 d. After 6 d of adjustment to the diet, subjects received the isotopes. The basic diet was a 3-d rotating menu with an energy level set to meet the energy requirement of the subject with the smallest body weight. A liquid formula drink containing additional minerals to meet the recommended dietary allowances of all nutrients and additional energy to maintain the body weight of each subject was added to the basic diet. The drink was divided into 3 portions and given with each meal. During the day, the subjects had free access to deionized water.
Isotope preparation
The 25Mg (99.58 atom%) and 26Mg (98.82 atom%) stable-isotope solutions were prepared from enriched magnesium oxide powders (Oak Ridge National Laboratory, Oak Ridge, TN). The magnesium oxide powders (500 mg 25MgO and 800 mg 26MgO) were weighed into polytetrafluoroethylene beakers that had been washed overnight in hydrochloric acid (1:1, by vol), rinsed with deionized water, refluxed with ultrapure nitric acid (1:1, by vol; Seastar Chemicals, Sidney, Canada), and rinsed again with deionized water. Inside a laminar flow hood, ultrapure concentrated hydrochloric acid (Seastar) was added and the beakers, covered with acid-washed watch glasses, were warmed on a hot plate and gently swirled from time to time until all powder was dissolved. The solutions were quantitatively transferred to acid-washed polypropylene containers. Sufficient sterile 2 N sodium hydroxide was added to adjust the pH to 2. Deionized water was added to achieve the desired concentrations, and weighed aliquots were transferred into acid-washed polypropylene bottles. Aliquots of the oral solution, containing 70 mg 26Mg, were weighed into acid-washed polypropylene test tubes, capped, and stored frozen until the isotope feeding day.
The 25Mg infusion solution was prepared using sterile water for infusion, and all containers and pipette tips were autoclaved. The infusion solution was filtered through a 0.22-µm filter, the filter was rinsed with sterile water, and the solution was transferred to vials for injection. An aliquot was tested for endotoxins. The exact concentrations of the enriched solutions were determined by isotope dilution with the use of certified magnesium standard solutions.
Tracer administration
On the morning of the seventh day, the subjects received the isotopes intravenously through a catheter inserted into an arm vein. A fasting blood sample was drawn. The oral dose of 70 mg 26Mg was given in 200 mL water (Arrowhead; Nestlé Waters North America Inc, Greenwich, CT) with breakfast. The breakfast comprised a 70-g bagel, 25 g jam, and 250 mL cranberry apple juice. A dysprosium fecal marker (2 mg Dy as DyCl3) was administered along with the stable-isotope tracers to check the completeness of fecal collections. Fifteen minutes later, 30 mg 25Mg was infused into the arm vein.
Sample collection and preparation
A schema of the experiment and sampling is reported in Figure 1. Blood samples were obtained via the catheter at the following times after administration of the intravenous dose: 5, 15, and 30 min and 1, 2, 4, 6, 11, 16, 24, 48, 72, 96, 120, 144, and 168 h. After centrifugation at 4000 rpm (1860 x g) for 10 min at 4 °C, plasma was removed and stored at -20 °C until analyzed. Complete urine and fecal collections were obtained throughout the study. Urine was pooled by 24-h and 3-d periods throughout the study and by 8-h periods for 3 d after isotope administration. Urine samples were weighed and acidified with 1 mL concentrated ultrapure hydrochloric acid (Seastar Chemicals) per 200 mL urine before storage at -20 °C. Fecal samples were pooled and homogenized by 3-d periods. Duplicates of all food items were weighed for each day and homogenized with weighed amounts of ultrapure water in a laboratory blender. Diet and fecal samples were lyophilized, crushed, and stored in plastic containers in desiccators. Before the magnesium analysis, sample aliquots were digested in duplicate in a microwave digestion oven (MDS-2000; CEM Corp, Mathews, NC) by using the appropriate program for each. The digestion was done on a 0.2-g sample and with 5 mL concentrated ultrapure HNO3 added. Samples were diluted with ultrapure water for analysis.
FIGURE 1. . Study design. Oral and intravenous tracers were administered to 6 healthy men on the same day. The feces were pooled by 3-d periods. Urine was pooled by 8-h, 24-h and 3-d periods. Blood was drawn at 5, 15, and 30 min and 1, 2, 4, 6, 11, 16, 24, 48, 72, 96, 120, 144, and 168 h. During the experiment, the diet consumed by the subjects was a 3-d rotating menu.
Analysis
The plasma volume for each subject was calculated on the basis of age and body mass by using a nomogram (15). The magnesium content of the breakfast was analyzed by using the Nutrition Data System for Research (NDS-R, version 4.01; Nutrition Coordinating Center, University of Minnesota, Minneapolis, 1998).
Plasma, urine, and digested feces and diet composites were diluted with 0.5% lanthanum chloride to a magnesium concentration of 0.1–0.4 ppm (16). Magnesium concentrations were determined by flame atomic absorption spectrophotometry (model 5100; Perkin-Elmer, Norwalk, CT). A certified bovine liver standard was analyzed with each set of samples (SRM 1577; National Institute of Standards and Technology, Gaithersburg, MD).
Urine and digested fecal samples were diluted to 100 ppb with ultrapure 1% HNO3 for magnesium isotopic ratio determinations. Plasma samples were diluted to 50 ppb to decrease the matrix effect.
Dysprosium excreted in the feces was determined by ICP-MS with the use of external calibration with standards of 0.5, 1, 1.5, 2.5, and 5 ppb Dy. The fecal digest was evaporated to dryness, dissolved again, and diluted to give an approximate concentration of 1–5 ppb Dy in 1% HNO3. Rhodium was used as internal standard at a concentration of 1 ppb. The ICP-MS parameters used for the dysprosium and magnesium analyses are shown in Table 2.
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TABLE 2 . Parameters of inductively coupled plasma mass spectrometry used for dysprosium and magnesium analyses
All acids and other chemicals used in sample preparation and dilution were ultrapure. All flasks used for sample collection and manipulations were acid washed in 1 mol HNO3/L for 24 h, followed by rinsing in ultrapure deionized water.
Isotope ratio determinations
Isotopic ratios were determined with an ICP-MS (ELAN 6000; Perkin-Elmer Instruments) equipped with an ultrasonic nebulizer (U-6000AT+; Cetac Technologies Inc, Omaha). The instrument parameters used for magnesium isotope ratio analysis are shown in Table 2. Instrument bias over time was corrected for by measuring an unenriched reference sample appropriate to each kind of matrix with the same concentration. The ratio correction factor was checked every 10 samples.
Within-run precision (5 replicates) was < 1% for 25Mg/24Mg and 26Mg/24Mg ratios. Repeatability, determined by measuring baseline samples several times over 5 h on the same day, was < 0.3% for both ratios in feces, urine, and plasma. Limits of detection (LODs) of 25Mg and 26Mg enrichments in urine and feces were obtained from the same measurements and were calculated by using the definition that the LOD for measuring a change in an isotope ratio is 3 times the SD of the baseline (17). LODs for 25Mg/24Mg and 26Mg/24Mg were 0.62% and 0.62% in urine, 0.89% and 0.92% in fecal samples, and 0.84% and 0.94% in plasma samples, respectively.
Calculations
Magnesium balance
Magnesium balance was calculated for each subject by subtracting the magnesium excreted in urine (U) and feces (F) from the amount ingested (I) as determined by atomic absorption spectrophotometry:
RESULTS
The magnesium content in the diet was 252.9 ± 26.9 mg/d for the 3-d rotating menu. The liquid formula drink added 4.0 ± 1.6 mg Mg/d per subject. Inclusion of the isotopes administered yielded an average total daily intake of 265.3 ± 1.6 mg Mg/d.
The endpoint of 26Mg tracer excretion in feces and the completeness of fecal collection were checked by determining dysprosium recoveries in feces. Individual dysprosium recoveries are presented in Table 3. The mean recovery was 100.5 ± 5.7%, ranging from 89.3% to 104.6% of the administered dysprosium dose, suggesting that collections were complete. The data indicated that the excretion of orally administered isotope was complete after 6–9 d.
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TABLE 3 . Dysprosium recoveries in the fecal samples of the 6 subjects
Magnesium intakes and fecal and urinary excretion during the 12 d after isotope administration are shown in Table 4. For 4 subjects with an average magnesium intake of 265 mg/d, the sum of fecal and urinary excretion exceeded the magnesium intake, resulting in negative balance. However, the average magnesium balance was not significantly different from zero (-16 ± 15 mg/d).
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TABLE 4 . Magnesium intake, fecal and urinary excretion of magnesium, and magnesium balance in 6 men during the last 12 of 18 d of a controlled diet
Individual FEE and magnesium absorption calculated by the fecal monitoring and DP methods are summarized in Table 5. The average FEE expressed as a percentage of the absorbed dose of isotopic tracer excreted per day was 2.4 ± 0.6%. The average value calculated by fecal monitoring was 0.46 ± 0.05% for MgAA and 0.48 ± 0.05 for MgA after correction for FEE. The results of the DL method were compared with those of the fecal monitoring method after correction for FEE. With the use of the DP calculation from plasma kinetics, the average MgA value was 0.47 ± 0.06, which was not significantly different from the value calculated by fecal monitoring.
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TABLE 5 . Fractional apparent (MgAA) and true (MgA) magnesium absorption values determined in the 6 subjects by the fecal monitoring method and by deconvolution analysis (DP) and fecal endogenous excretion (FEE)
MgA values determined with the DLU method are reported in Table 6. To evaluate the effect of the interval time of collection on MgA values, urine was pooled by 8-h (first 72 h), 24-h, and 3-d periods of time. In the 8-h pools, statistical analysis showed that absorption was significantly different from that determined by fecal monitoring through 48 h after isotope administration. Only values determined from the two 8-h urine pools between 48 and 64 h (0.43 ± 0.09 and 0.54 ± 0.10) were not significantly different from the reference value. The 64–72-h mean (0.57 ± 0.07) was significantly different from the mean determined by fecal monitoring. Except for the 0–8-h (0.14 ± 0.04) and 48–56-h (0.43 ± 0.09) pools, the 8-h means were higher than the fecal monitoring mean.
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TABLE 6 . Fractional magnesium absorption determined with the double-labeling method based on urine data1
For the 24-h urine pools, there were no significant differences between the DLU means and the fecal monitoring means 24–144 h, 192–240 h, and 264–288 h after isotope administration. The 24-h DLU mean was higher than the fecal monitoring mean from 24 to 120 h and then lower than the fecal monitoring mean thereafter. The closest agreements were between 48–72 h (0.49 ± 0.06), 72–96 h (0.51 ± 0.11), and 96–120 h (0.50 ± 0.06). No significant differences were observed between the 3-d urine pools and fecal monitoring after the first 72 h after isotope administration; the closest agreement was during the 216–288-h period (0.46 ± 0.07).
MgA values calculated with the DLP method are shown in Table 7. As for the means in the 8-h urine pools, the means for all time points through 48 h after isotope administration were significantly different from the fecal monitoring means, except at the 4-h time point. No significant differences from the fecal monitoring mean were observed at any time points from 72 to 168 h after isotope administration, although the DLP mean was consistently higher than the fecal monitoring mean. Beginning at the 4-h time point, no significant differences were observed when DLP-determined values were compared with DLU-determined values from the 8-h, 24-h, and 3-d urine pools after the first pool of each.
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TABLE 7 . Fractional magnesium absorption determined in plasma samples with the double-labeling method1
DISCUSSION
After a 6-d adaptation period to the diet, magnesium balance was negative in 2 of 3 of the subjects studied. However, on average, the magnesium balance of the group was not different from zero. The dietary intake of magnesium was below the new dietary reference intake of 400–420 mg Mg/d for men (21). One interpretation of the balance data is that balance does not determine the requirement for a mineral element but is the intake required to maintain the existing pool (22). Similar results were obtained in a previous study after 7 d of adaptation to the experimental diet followed by 14 d of sample collection. The investigators concluded that their study was probably too short (23). It has been suggested that 4 wk are needed to achieve a reliable estimate of magnesium balance (24).
The primary goal of the current study was to compare different techniques for determining MgA to identify a simpler alternative to fecal monitoring. We chose the results of the fecal monitoring method as the reference because until now it was the most commonly used method (10–13). This method evaluates MgAA, or MgA after correction for FEE. The correction requires that a second stable isotope be administered intravenously, and a complete collection of fecal samples for 5 d is required. This method of sampling is time consuming. In our study, DL and several intervals of sample collection were tested. MgA calculated from the first urine pool of 72 h was significantly lower than the value obtained with the reference fecal monitoring method. This finding agrees with previously reported results, showing that MgA was 80% complete at 24 h and 95% complete at 72 h. This finding suggests that absorption was underestimated when based on the 72-h urine collection after isotope administration (25). Nevertheless, investigators concluded that MgA might be measured with the DL method and a complete 72-h urine collection without comparison with another method or different time intervals. When compared with the results of the reference method, our results showed that it was necessary to wait for the second collection of 72 h (between 72 and 144 h) to closely evaluate MgA when urine was pooled by 3-d periods of time.
The analysis of the 24-h urine pools showed that the results agreed with those of the reference method 24 h after isotope administration. Results from the 8-h urine pools showed that only 2 time intervals (48–56 and 56–64 h) were adequate for the MgA determination. A similar study carried out in rats found lower MgA with the DL method than with a method based on fecal data (14). The explanation for this difference is that it is assumed that the 2 tracers behave the same once they are administered; however, the 2 tracers enter the circulation by different routes and may not have behaved the same. Because plasma magnesium homeostasis is controlled by the kidney (26), the intravenous dose may lead to transient hypermagnesemia, which is responsible for a rapid initial urinary excretion of the tracer. This hypothesis would explain an underestimation of MgA when compared with the fecal data. The disagreement between urinary and fecal values obtained in rats could also be linked to the protocol of isotope administration or to the administered amounts (14). A period of 2 h was observed between the oral and the intravenous administration in the rat study compared with an interval of 15 min in the current study. The ratio between the intravenously administered dose and the volume of distribution of magnesium was higher in the rat study than the one calculated for this human study: 21% compared with 15%. In both cases the volume of distribution was calculated by using a compartmental model (M Sabatier, F Pont, MJ Arnaud, JR Turnlund, unpublished observations, 2002; 27). Thus, the injected dose could explain the difference between the results of the fecal monitoring and DL methods in rats. Furthermore, the dose administered orally in rats was 5.4 times greater than the amount administered intravenously. The ratio of administered amounts of tracers in this study was 2.3, as recommended previously (25). Those observations tend to show that the results are method dependent. The unusable initial fractions of time for MgA determination represent the equilibration period required for both tracers to behave in a similar way. Furthermore, diurnal variation of magnesium excretion (28) could have contributed to invalid results of urine pooled by 8 h during the first 48-h period of time. Three-day urine pools and early 24-h pools are usable for the MgA determination when collected after the equilibration period. Good agreement between the DL and fecal monitoring methods was observed with the 24-h urine pools collected 24 h after isotope administration. The closest agreement in the current study was obtained between 48 and 144 h.
MgA values determined with the DLP method were not significantly different from the values determined with the DLU method or with the values determined by fecal monitoring 72 h after isotope administration. Therefore, MgA can be determined from a simple blood draw with the DL method. This method has the disadvantage of being more invasive than urine collections. The DP method is also invasive and, in addition, requires multiple blood draws for kinetics modeling of the evolution of both tracer concentrations. Even though the results obtained from the DP analysis were not significantly different from the fecal data, this method is cumbersome and necessitates the use of special software. However, the result of the DP analysis could be more valuable in studies that compare populations. When using the DL method, magnesium absorption is calculated from a short urine collection or blood sample. Consequently, the calculation is based on the assumption that the rate of absorption is invariant between persons with respect to physiologic state and diet history and the assumption that the distribution of the intravenous labels is invariant with age or physiologic state. This method cannot, however, be excluded for use in studies of changes of response within similar groups. Another study, which compared methods for determining zinc absorption, recommended the use of urine collected 2 d after tracer administration when the DLU method is used (19).
The rate of MgAA, when administered in water and consumed with a breakfast containing 101 mg Mg (isotope included), was 0.46 ± 0.05 in men. An experiment studying the MgAA from water showed fractional absorption of 0.52 ± 0.04 in women when water was consumed with a breakfast containing 87 mg Mg (isotopes included) (11). The absolute amount absorbed in both cases was the same. The results from studies that compared MgAA from water are similar. The composition of the breakfasts, although different in both studies, did not seem to have an effect on MgAA.
In summary, the DL method was successful in measuring MgA—as it has been for calcium (29, 30) and zinc (19, 31). The DLU and DLP methods were validated by comparing their results with those of the fecal monitoring method. The simplest and least invasive procedure was the DLU method. The most similar results were obtained from the 24-h urine pools collected between 48 and 144 h after isotope administration. An additional advantage of the DLU method over the fecal monitoring method is that the measurement of magnesium isotopic ratios in urine by ICP-MS is less cumbersome than is that in fecal samples; these results are valid under the conditions of this study. The DP method is probably more appropriate for comparison of absorption across populations. To decrease the cost of such experiments, additional studies should be done to optimize the doses of oral and intravenous tracers and to bring the amounts administered closer to tracer doses.
ACKNOWLEDGMENTS
MS contributed to the data collection, sample and data analysis, and writing of the manuscript. WRK contributed to the sample and data collection and analytic methods. FP contributed to the deconvolution analysis. MJA contributed to the study design and writing of the manuscript. JRT contributed to the experimental design, data collection and analysis, and writing of the manuscript. MS was supported by the Nestlé Water Institute. MJA is the director of the Nestlé Water Institute. FP is employed by INSERM (France). JRT and WRK are employed by the US Department of Agriculture.
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