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Original Research Communications |
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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Objective: Our objective was to establish whether the molybdenum in foods is metabolized similarly to molybdenum added to the diet.
Design: We first studied whether sufficient amounts of molybdenum stable isotopes could be incorporated into wheat, kale, and soy for use in a human study. Enough molybdenum could be incorporated into soy and kale to study molybdenum absorption and excretion. Two studies were then conducted, one in women and one in men. In the first study, each meal contained 
100 µg Mo from soy, kale, and extrinsic molybdenum. In the second study, soy and extrinsic molybdenum were compared; the meal contained 300 µg Mo.
Results: In the first study, molybdenum was absorbed equally well from kale and an extrinsic source. However, the molybdenum in soy was less well absorbed than the molybdenum in kale or that added to the diet. In the second study, absorption of molybdenum from soy was less than from the extrinsic label. Urinary excretion of soy molybdenum was also lower than urinary excretion of the extrinsic label, but excretion as a percentage of the absorbed dose was not significantly different between treatments.
Conclusions: The molybdenum in soy is less available than molybdenum added to the diet, but the molybdenum in kale is as available as molybdenum added to the diet. Once absorbed, excretion is not significantly different for soy, kale, and extrinsic molybdenum.
Key Words: Molybdenum • absorption • utilization • humans • stable isotope • intrinsic label • soy • kale
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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We conducted stable-isotope-tracer studies to measure absorption, excretion, and retention of molybdenum over a broad range of dietary intakes in young men (5, 6) and to develop a model of molybdenum metabolism (7, 8). These studies showed that molybdenum is well absorbed over a range of intakes, that it is rapidly excreted via the urine, and that total body molybdenum retention is regulated primarily via urinary excretion. With high molybdenum intake, molybdenum absorption increases and excretion is more rapid. However, these studies were all done by adding stable isotopes to the diet. It was thus unknown whether the metabolism of extrinsically labeled molybdenum reflected the metabolism of molybdenum incorporated into foods. The bioavailability of molybdenum from labeled foods had not been investigated previously in humans or animals because short half-lives of molybdenum radioisotopes (
67 h) precluded preparation of endogenously labeled foodstuffs.
In the present study, we labeled plants with stable isotopes of molybdenum for use in human bioavailability experiments. Kale, soybeans, and wheat were selected for labeling to represent a dark-green leafy vegetable, a legume, and a cereal grain, respectively. Two human experiments were conducted to determine whether molybdenum added to the diet was absorbed and retained similarly to molybdenum in food.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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100Mo was added to the hydroponic nutrient solution of soybeans and kale starting 5 wk after germination and to wheat starting 1 wk after transplantation of the seedlings. 100Mo was administered for 4 consecutive weeks in doses of 17.63 or 0.88 µg per bucket weekly for total doses of 70.5 or 3.53 µg per bucket. In addition, the stem of the wheat plant was injected at the milking stage with 0.11 or 2.21 µg per head as described previously (9). The harvested wheat and soybeans from each container were ground into a powder and transferred to individual plastic bottles. The harvested kale leaves were placed in sealed plastic bags. Portions of the plant samples were weighed and 97Mo was added as an isotopic diluent.
Labeling of soybeans and kale for human feeding study
Soybeans (G. max var. Century) were grown hydroponically in pots as described for the previous study. One week before dosing, the nutrient solution was replaced with a molybdenum-depleted solution. Twenty-five buckets of soybeans containing 4 plants each were dosed weekly for 4 wk beginning 19 d after the onset of flowering with a total of 64 µg 100Mo per bucket. After dosing, the plants were returned to the normal Hoagland-Arnon nutrient solution that contained 8 µg Mo/L until harvest. The soybeans from each bucket were stored in separate bags. The total yield was 3303 g. Five grams from each bag was ground into a powder and 94Mo was added as an isotopic diluent. The soybeans from the buckets that had incorporated the most 100Mo were combined, powdered, puréed, and stored frozen.
Kale (B. oleracea var. Vates Blue Curl) was grown in a circulating hydroponic system containing Hoagland-Arnon nutrient solution (9); 50 plants were grown in each of 3 troughs. 97Mo was added at a rate of 220 µg per trough 6 times over 5 wk. About half of the kale had to be harvested after 3 doses because of wilting. The 2 batches of kale leaves (3526 g) were blended into separate purées. A portion (5.5 g) of each batch was saved for analysis and 94Mo was added. The level of label incorporation between the 2 batches was similar, so the 2 purée batches were combined into a single purée. Three portions of each of the final soy and kale purées were weighed for analysis and 94Mo was added.
Subjects
Two molybdenum absorption studies were conducted. Participants were confined to a metabolic research unit for the duration of the studies. Twelve young women participated in study 1 and 11 young men in study 2. Participants in both studies had been recruited for other studies that were independent of the molybdenum absorption studies (10, 11) but that overlapped them. Characteristics of the participants in the 2 studies are shown in Table 1
. The protocols and consent forms for both studies were reviewed and approved by the Human Subjects Review Committee of the University of California, Davis, and by the US Department of Agriculture, Agricultural Research Service, Human Studies Committee. Details of each study were explained to participants and each participant signed a consent form before the beginning of the study
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On day 4 only, the diets in each study contained casseroles in which the foods were labeled with stable isotopes of molybdenum as follows. In study 1, isotopically enriched soy and kale, an extrinsic label, and natural molybdenum, prepared as described below under "Isotope preparation," were included in the diets of all subjects. The isotopic labels and their amounts are shown in Table 2. Two sources of molybdenum were fed at lunch and the other 2 at dinner, as shown in Table 3. The enriched molybdenum content of each meal was 100 µg and the total molybdenum intake for the day was 207 µg. This design allowed us to compare the absorption and excretion of molybdenum in soy with the absorption and excretion of molybdenum in kale and to determine the effect of each on the absorption and excretion of extrinsic molybdenum. In study 2, the lunch meal on day 4 contained soy with 147 µg Mo enriched in 100Mo and the extrinsic label contained 156 µg 96Mo, for a total of 303 µg labeled Mo. The amounts of soy and molybdenum in study 2 were increased to determine the effect of soy on absorption when the amounts of soy and molybdenum were higher. In study 2, the other meals consumed on day 4 were the usual study meals; the total molybdenum intake for the day was 442 µg.
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The soy casserole for study 1 contained 20 g low-protein, cooked noodles, 85 g soy purée (20 g soybeans cooked in chicken bouillon), 55 g cooked egg white, and 5 g margarine. The kale casserole for study 1 contained 276 g kale purée (120 g raw kale cooked and puréed in chicken bouillon), 230 g low-protein noodles, 55 g cooked egg white, and 5 g margarine. The soy casserole for study 2 contained 230 g low-protein noodles, 15 g margarine, 240 g soy purée (60 g dry soy cooked in 180 g broth), and 55 g cooked egg white.
Isotope preparation
Molybdenum metal powders enriched in 94Mo (93.77 atom%), 96Mo (96.44 atom%), 97Mo (94.25 atom%), and 100Mo (97.42 atom%) (Oak Ridge National Laboratory, Oak Ridge, TN) and unenriched molybdenum were weighed into acid-washed polytetrafluoroethylene beakers, a 3:1 mixture of ultrapure hydrochloric acid and nitric acid (Seastar Grade; Seastar Chemicals, Sidney, Canada) was added, and the solutions were heated in a laminar flow hood until the molybdenum was dissolved. The solutions were transferred to acid-washed polypropylene bottles and diluted with deionized water. The 94Mo solution was diluted to 10 µg/g for use as the isotopic diluent in the analysis. The 96Mo solution was diluted to 45.7 µg/g for use as the extrinsic label and the unenriched molybdenum solution was diluted to 49.5 µg/g. One-gram aliquots of these were pipetted into acid-washed microcentrifuge tubes and frozen for later addition to the meal. The 97Mo and 100Mo solutions were diluted to 8 mg/L to be incorporated as intrinsic labels in the kale and soy. The molybdenum concentrations of the isotope solutions were verified by isotope dilution by using a molybdenum standard solution (Ricca Chemical Co, Arlington, TX) (4). Precautions were taken to avoid trace element contamination during all phases of isotope solution preparation, sample collection and preparation, and analysis as described previously (5).
Sample collection and preparation
Two food composites were prepared for study 1 containing all foods fed on day 4. For one composite, the lunch contained the soy casserole and the natural molybdenum solution and the dinner contained the kale casserole and the extrinsic 96Mo solution. For the other composite, the lunch contained the kale casserole and the natural molybdenum solution and the dinner contained the soy casserole and the extrinsic 96Mo solution. Two portions of each of the 2 daily food composites were weighed for analysis and 94Mo was added. Two food composites of the lunch meal alone and 2 composites of the entire day's meals were prepared for study 2. Two aliquots of each composite were weighed for analysis and 94Mo was added.
Complete fecal and urine collections were made throughout the study and pooled, beginning with an initial 3-d pool before the isotope feeding day (day 4) and followed by 4-d pools beginning on the feeding day. Urine samples were frozen until analyzed. Diet composites and fecal pools were homogenized, refrozen, lyophilized, crushed to a fine powder, and stored in desiccators as described previously (5).
For fecal analysis, 3-g portions of the dried fecal pools were weighed with 4 µg 94Mo added as an isotopic diluent. For urinalysis, 100-g portions of the urine pools were weighed with 5 µg 94Mo added as an isotopic diluent. The diet, fecal, and urine samples were dried and ashed in a muffle furnace. The ashed samples were dissolved in 6 mol ultrapure HCl/L and the molybdenum was separated and purified by ion-exchange chromatography as described previously (4, 5).
Sample analysis and calculations
Molybdenum isotope ratios were determined with a computer-controlled, 90° magnetic sector, thermal ionization mass spectrometer (model 261; Finnigan MAT, Bremen, Germany) as described previously (5). The same quality-control procedures were followed as described previously (4). The total molybdenum and molybdenum isotope contents of the labeled plants and of the diet, urine, and fecal samples were determined by isotope dilution. Triple-isotope dilution, with the intrinsic 100Mo from soy, extrinsic 96Mo solution, and 94Mo isotopic diluent, was used in study 2. These calculations were described previously (4, 5). Quadruple-isotope dilution calculations, with the additional intrinsic 97Mo from kale, were used in study 1 as shown in Appendix A.
Molybdenum absorption was calculated by subtracting the amount of each enriched source of molybdenum appearing in the feces during the 8-d period after feeding from the amount fed. This value was divided by the amount fed, multiplied by 100, and expressed as percentage absorbed. Urinary excretion of the isotope labels was determined over the same 8-d period and calculated both as a percentage of the fed dose and a percentage of the absorbed dose.
Statistics
The isotope dilution calculations were carried out and summarized and statistical analysis was performed with SAS (personal computer version 6.10; 12, 13). PROC GLM was used to perform an analysis of variance (ANOVA) on the absorption and urinary excretion of molybdenum from the intrinsic and extrinsic labeled dietary sources. If a significant effect was found, the Student-Neuman-Keuls test was used to determine which means differed. For study 1, ANOVA models were used to test for any significant effect on absorption and urinary excretion of molybdenum due to the meal (lunch or dinner) in which the soy, kale, or extrinsic label was fed or to whether the extrinsic label was fed with soy or kale. When no significant difference between the 4 feeding groups was observed, the data for the groups were combined. For both studies, ANOVA models with the Student-Neuman-Keuls test were used to determine the effect of dietary molybdenum source on molybdenum absorption (% of fed dose) and on urinary molybdenum excretion (both % of fed dose and % of absorbed dose). A significance level of 0.05 was used in all statistical tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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) were low because the plants were grown in a greenhouse during the winter months. The fresh yield per bucket averaged 13 g for soybeans, 73 g for kale, and 4 g for wheat. Accumulation of 100Mo was high for soybeans, moderate for kale, and low for wheat. Stem injection of wheat resulted in a high rate of isotope incorporation, but the amount of molybdenum that could be incorporated per stem was low. The total concentration of 100Mo incorporated in soybeans and kale from the higher dose (4.56 and 2.71 µg/g dry wt, respectively) was sufficient for a human feeding study. The low accumulation of 100Mo by wheat (<0.75 µg per bucket) suggested that intrinsic labeling of this food would not be feasible.
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Absorption
The highest fraction of the enriched isotopes appeared in the first 4-d fecal pool; the fraction in the second 4-d pool was 20% of that in the first. By the third 4-d pool, from 0.1% to 0.5% of the dose appeared in the feces per day, with less appearing after that. The 8-d collection period was therefore used to calculate absorption.
In study 1, there were no significant differences in absorption due to the order in which the labeled meals were fed. Therefore, data were combined for statistical tests. The mean (±SE) absorption of molybdenum was 87.5 ± 1.5% from the extrinsic molybdenum, 86.1 ± 1.5% from kale, and 56.7 ± 1.5% from soy (Figure 1). There was no significant difference in absorption between kale molybdenum and the extrinsic molybdenum, but absorption of molybdenum from soy was significantly less than absorption of extrinsic molybdenum and kale molybdenum. Absorption of the extrinsic molybdenum was not significantly different when it was combined with soy or kale. In study 2, in which more soy and molybdenum were fed, absorption was again significantly less from soy (58.3 ± 1.2%) than from the extrinsic molybdenum (92.8 ± 1.2%) (Figure 2).
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). Excretion was not significantly different between the extrinsic molybdenum and the kale molybdenum, but was significantly lower for the soy molybdenum than for the extrinsic molybdenum and the kale molybdenum. However, when calculated as a percentage of the absorbed dose excreted in the urine, there were no significant differences between the sources of molybdenum. Mean excretion, as a percentage of the absorbed dose, was 60.8 ± 3.3% for extrinsic molybdenum, 56.6 ± 3.3% for kale, and 63.9 ± 3.3% for soy. In study 2, results were similar to study 1. Excretion of molybdenum in the urine was significantly lower for soy (39.6 ± 0.8%) than for the extrinsic molybdenum (62.6 ± 0.8%) (Figure 2). However, the percentage of the absorbed dose excreted in the urine was not significantly different between treatments: 67.8 ± 1.3% from the extrinsic molybdenum and 68.8 ± 1.3% from the soy. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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67 h). Very short-term studies showed rapid accumulation of molybdenum by tomato roots and subsequent rapid translocation to upper plant parts, but no accumulation of molybdenum in stem tissue (14). Soybeans have also been shown to accumulate high quantities of zinc (27.5% of dose) and iron (12.4% of dose), although uptake was less efficient than for molybdenum (15). As with molybdenum, uptake of iron into the edible seeds was greater for soybeans than for wheat (5.5% of dose), but uptake of zinc by wheat (25.5% of dose) was not significantly different from uptake by soybeans. In contrast with molybdenum, calcium accumulation by leaves is much greater than for any seeds (15).
In calculating absorption, we assumed that the isotope appearing in the stools >8 d after the feeding reflected excretion of the absorbed isotope. This conclusion was based on an earlier study in which a stable isotope of molybdenum was infused. In that study, we found that only 2–4% of the infused dose appeared in the feces in the first 6 d after infusion (0.3–0.7%/d) and that excretion was gradually less after this time (5, 6). This is consistent with the excretion of 0.1–0.5%/d in the feces during the 8 d after isotope feeding in the present study. The results show that molybdenum absorption from soy, but not from kale, was less efficient than absorption of molybdenum added to the diet. Additionally, molybdenum absorption from soy was less efficient than that from the extrinsic label at both intakes, suggesting that the effect was not dose dependent. Once molybdenum is absorbed, it appears to be handled similarly regardless of the source, as shown by urinary excretion after correction for absorption. However, the presence of soy in the meal does not alter the absorption of other molybdenum in the meal. The absorption of extrinsic molybdenum was the same whether it was combined with kale or with soy. The lower absorption of molybdenum from soy has also been observed for other minerals. Soy protein, even after phytate has been removed, inhibits iron absorption (16). The bioavailability of zinc is also less from soy food products than from animal-derived foods (17).
We found previously that molybdenum absorption was high over a broad range of intakes and that absorption was higher with the highest intakes (6). When daily molybdenum intake ranged from 25 to 122 µg/d, absorption averaged 89%, but when it ranged from 466 to 1488 µg/d, absorption was significantly higher, averaging 93%.
There were several differences between the 2 studies reported here. Study 1 was conducted in women and study 2 was conducted in men and participants lived in the metabolic unit at different times. The labeled meal in study 2 contained 3 times as much molybdenum as the meal in study 1 and absorption tended to be higher in study 2. The meals contained amounts in the low (study 1) and high (study 2) ranges of our earlier work and results are consistent with those results. Average absorption in study 2, when the molybdenum content of the meal was higher, tended to be higher than in study 1: 92.8 ± 1.2% compared with 87.5 ± 1.5% for the extrinsic molybdenum and 58.3 ± 1.2% compared with 56.7 ± 1.5% for the soy molybdenum.
Molybdenum turnover in our earlier study was faster with higher dietary molybdenum intakes (6). This was reflected by urinary excretion of labeled molybdenum, both from infusions and feedings. The results of the current study support this interpretation of higher turnover when molybdenum intake is higher. More of the absorbed molybdenum tended to be excreted in the urine in study 2 (when molybdenum intake was higher) than in study 1: 67.8 ± 1.3% compared with 60.8 ± 3.3% of the absorbed extrinsic label and 68.8 ± 1.3% compared with 63.9 ± 3.3% of the soy molybdenum.
The current dietary recommendations for molybdenum are from 75 to 250 µg/d (18). We estimated the minimum requirement to be 25 µg/d for men on the basis of studies with an extrinsic stable-isotope label (5, 6). The dietary intake of the US population was estimated to average 180 µg/d in 1980 (19) and 76 µg/d for women and 109 µg/d for men in 1987 (20). Thus, even if the molybdenum in some foods, such as soy, is not as available as an extrinsic label, it appears that risk of inadequate dietary molybdenum is unlikely.
In conclusion, as shown by the intrinsically labeled soy, not all food sources of molybdenum are absorbed as well as molybdenum added to the diet. However, the intrinsically labeled kale showed that the molybdenum in some foods is as available as extrinsically added molybdenum. Despite this difference, molybdenum is better absorbed than many minerals and inadequate dietary molybdenum is unlikely.
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APPENDIX A |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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| (1) |
Equations for isotope dilution calculations:
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| (2) |
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| (3) |
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| (4) |
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| (5) |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Address reprint requests to JR Turnlund, USDA, ARS, PO Box 29997, Western Human Nutrition Research Center, Presidio of San Francisco, CA 94129. E-mail: jturnlun{at}whnrc.usda.gov.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAPPENDIX AREFERENCES |
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This article has been cited by other articles:
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  J. A. Novotny and J. R. Turnlund Molybdenum Intake Influences Molybdenum Kinetics in Men J. Nutr., January 1, 2007; 137(1): 37 - 42. [Abstract] [Full Text] [PDF]
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J. A. Novotny and J. R. Turnlund Molybdenum Kinetics in Men Differ during Molybdenum Depletion and Repletion J. Nutr., April 1, 2006; 136(4): 953 - 957. [Abstract] [Full Text] [PDF]
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 J. R. Turnlund Mineral bioavailability and metabolism determined by using stable isotope tracers J Anim Sci, April 1, 2006; 84(13_suppl): E73 - E. [Abstract] [Full Text] [PDF]
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 K. Y. Patterson and C. Veillon Stable Isotopes of Minerals as Metabolic Tracers in Human Nutrition Research Experimental Biology and Medicine, April 1, 2001; 226(4): 271 - 282. [Abstract] [Full Text]
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