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Copper plays an important role in cardiac and brain function possibly through endocrine and neuroendocrine systems. The syndrome of copper deficiency is worsened by dietary fructose and other trace metals such as zinc. We investigated the effect of a low copper diet on plasma opioid peptides in 11 healthy young volunteers who were fed foods low in copper but adequate in all other nutrients. The study was divided into three dietary periods. Copper was added to the diet so that the diet contained 0.66 mg/day for 24 days (marginal Cu), 0.38 mg/day for 42 days (low Cu) and 2.49 mg/day for 24 days (adequate Cu). The indices of copper status, ceruloplasmin and plasma copper concentrations, declined and were significantly lower (p0.05) at the end of the low Cu period than at the beginning of the study and the end of the marginal Cu period. They increased significantly at the end of the adequate Cu diet to the levels of the marginal Cu diet. Plasma β-endorphin (BEN), Leu-enkephalin (LE), Met-enkephalin (ME) and Adrenocorticotropic hormone (ACTH) were measured by radioimmunoassay at the beginning of the study and at the end of each dietary period. No significant differences were observed in BEN, LE or ME during any of the periods. There were only small increases in LE and ME at the end of marginal and low copper diet periods and no significant changes were observed on copper repletion. Plasma ACTH was significantly lower at the end of low copper compared to baseline value but was not lower after marginal copper. Copper repletion had no significant effect on ACTH. The data show that plasma opioid peptides did not respond significantly to differential copper intake.

Background Numerous studies have examined the effect of low and adequate intakes of copper on absorption and retention, but little information is available on the regulation of absorption and retention of copper when intake is high. Objective A study was conducted in men to determine the effect of long-term high copper intake on copper absorption, retention, and homeostasis. Design Nine men were confined to a metabolic research unit (MRU) for 18 d and were fed a 3-d rotating menu containing an average of 1.6 mg Cu/d. They continued the study under free-living conditions for 129 d, supplementing their usual diets with 7 mg Cu/d. They then returned to the MRU for 18 d and consumed the same diet as during the first period, except that copper intake was 7.8 mg/d. The stable isotope (63)Cu was fed to 3 subjects and infused into the other 6 on day 7 of each MRU period, and complete urine and stool collections were made throughout the study. Total copper and (63)Cu were determined by inductively coupled plasma mass spectrometry. Copper absorption, excretion, and retention were calculated on the basis of dietary, urinary, and fecal copper and (63)Cu. Results Results were as follows when comparing the high copper intake with the usual intake: fractional copper absorption was significantly lower, but the amount absorbed was significantly higher; excretion of the infused (63)Cu was significantly faster; and total retention was significantly higher. Conclusions Homeostatic regulation of copper absorption and retention helped to minimize the amount of copper retained with high copper intake but was not sufficient to prevent retention of >0.6 mg Cu/d.

Background: Short-term high copper intake does not appear to affect indexes of copper status or functions related to copper status, but the effects of long-term high copper intake are unknown. Objective: A study was conducted in men to determine the effect of long-term high copper intake on indexes of copper status, oxidant damage, and immune function. Design: Nine men were confined to a metabolic research unit (MRU) for 18 d and were fed a 3-d rotating menu providing an average of 1.6 mg Cu/d. The men continued the study under freeliving conditions for 129 d and supplemented their usual diets with 7 mg Cu/d. The men then returned to the MRU for 18 d of the same diet as during the first period, except that copper intake was 7.8 mg/d. Plasma copper, ceruloplasmin activity, ceruloplasmin protein, plasma malondialdehyde, benzylamine oxidase activity, erythrocyte superoxide dismutase, hair copper, urinary copper, and urinary thiobarbituric acid–reactive substances were measured during each MRU period. Results: Ceruloplasmin activity, benzylamine oxidase, and superoxide dismutase were significantly higher at the end of the second MRU period than at the end of the first. Urinary copper excretion, hair copper concentrations, and urinary thiobarbituric acid–reactive substances were significantly higher during the second MRU period than during the first. Polymorphonuclear cell count, the percentage of white blood cells, lymphocyte count, and interleukin 2R were affected by copper supplementation. Antibody titer for the Beijing strain of influenza virus was significantly lower in supplemented subjects after immunization than in unsupplemented control subjects. Conclusions: Under highly controlled conditions, long-term high copper intake results in increases in some indexes of copper status, alters an index of oxidant stress, and affects several indexes of immune function. The physiologic implications of these changes are unknown. Am J Clin Nutr 2004;79:1037– 44.

The role of intestinal cells in copper homeostasis and storage has not been studied. Homeostasis is regulated by absorption, excretion, and storage. Copper is excreted via the bile, and it has been assumed that the increased excretion into the gastrointestinal tract when diets are high in copper was due to biliary copper. It seemed possible that some of the absorbed copper is sequestered in the intestinal cells. To test this hypothesis we measured the copper content of mucosal epithelial cells isolated from stool samples of 8 young men when consuming their usual diets and after 129 days of supplementation with 7 mg/day copper. The mean copper content of the cells, expressed as mg copper per gram of cell protein, was 0.78 versus 1.65 mg/g (SEM 0.19) (P < 0.02) when the usual and copper-supplemented diets, respectively, were consumed. In contrast to the copper content of plasma and urine, copper in intestinal cells increased significantly when intake was high. It is likely that copper was sequestered by these cells following absorption and did not get into systemic circulation. Thus, the amount of endogenous copper in stools is probably a combination of biliary copper excretion and copper sequestered in exfoliated intestinal epithelial cells. This suggests that retention of copper by the intestinal cells increases when dietary copper is high and plays a role in copper homeostasis. J. Trace Elem. Exp. Med. 16:105–108, 2003. Published 2003 Wiley–Liss, Inc.

An isotope dilution method was developed to measure total molybdenum and molybdenum stable isotope concentrations in 0.5 mL of human blood plasma by ICP-MS. To minimize contamination, a microwave digestion method was developed without molybdenum separation. Plasma aliquots were weighed into PTFE beakers placed on supporting PTFE beakers inside PFA liners, adding 1.4 mL HNO3 to the plasma and 8 mL HNO3 to the liner base. The samples were digested in five 10-min steps under pressure control from 20 to 100 psi. Repeated microwave digestions of the liners and beakers reduced processing blanks from 6.6 to 0.03 ng Mo. The samples were analyzed by ICP-MS. Analysis of a reference human serum produced an average molybdenum concentration of 1.02 ± 0.12 µg L−1versus the informational value of 1.07 ± 0.04 µg L−1. Total Mo and 97Mo were measured in plasma collected from a human subject following intravenous adminstration of 33 µg 97Mo, with concentration RSDs

Use of stable isotopes in applied nutrition has been a developing field for over 60 years. The past 20 years has seen improvements in computer and mass-spectrometer technology that has opened up even greater possibilities in the understanding of human metabolism. While improvements in technology can bring great opportunities, it can also cause problems if there is no consensus among the stable isotope user-community on standardization of new techniques and methods. Users of stable isotopes have traditionally been split into two groups; those who work with heavy isotopes (e.g., 58Fe, 70Zn) and those who work with light isotopes (e.g., 2H218O). Standardization issues have been addressed by the light isotope users and awareness of this type of problem is starting to emerge within the heavy isotope community.

Stable isotopes are used with increasing frequency to trace the metabolic fate of minerals in human nutrition studies. The precision of the analytical methods used must be sufficient to permit reliable measurement of low enrichments and the accuracy should permit comparisons between studies. Two methods most frequently used today are thermal ionization mass spectrometry (TIMS) and inductively coupled plasma mass spectrometry (ICP-MS). This study was conducted to compare the two methods. Multiple natural samples of copper, zinc, molybdenum, and magnesium were analyzed by both methods to compare their internal and external precision. Samples with a range of isotopic enrichments that were collected from human studies or prepared from standards were analyzed to compare their accuracy. TIMS was more precise and accurate than ICP-MS. However, the cost, ease, and speed of analysis were better for ICP-MS. Therefore, for most purposes, ICP-MS is the method of choice, but when the highest degrees of precision and accuracy are required and when enrichments are very low, TIMS is the method of choice.

Background: Stable-isotope studies of molybdenum metabolism have been conducted in which molybdenum was added to the diet and was assumed to be absorbed and utilized similarly to the molybdenum in foods. 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.

A study was conducted in young men to evaluate the effect of a low-copper diet on copper absorption, excretion, and retention. Eleven young men were confined to a metabolic research unit for 90 d. The study was divided into three periods, with dietary copper as the only variable. Dietary copper intake was 0.66 mg/d for 24 d, 0.38 mg/d for 42 d, and 2.49 mg/d for 24 d. The stable isotope 65Cu was fed to five of the subjects once during the first and last dietary period and twice, early and late, in the second period to determine copper absorption. 65Cu was infused into an arm vein of the other six subjects once during each dietary period to estimate excretion of endogenous copper. Total copper and 65Cu were determined by isotope dilution with thermal-ionization mass spectrometry. Fractional absorption was significantly higher during the low-copper period than in either period with higher dietary copper and excretion of the infused isotope was significantly lower in the low-copper period. Subjects were in negative balance early in the first two periods but achieved balance by the end of those periods. They retained copper during the highest dietary copper period (third period). The results suggest that endogenous copper excretion is a major point of regulation of the body's copper stores. Regulation of absorption and of endogenous excretion in response to dietary copper intake helps to protect against deficiency and toxicity. However, this regulation was not sufficient to maintain copper status at the lowest intake of dietary copper, 0.38 mg/d.

To clarify the role of the intestine, kidney, and bone in maintaining calcium homeostasis during pregnancy and lactation and after the resumption of menses, a longitudinal comparison was undertaken of 14 well-nourished women consuming approximately 1200 mg Ca/d. Measurements were made before conception (prepregnancy), once during each trimester of pregnancy (T1, T2, and T3), early in lactation at 2 mo postpartum (EL), and 5 mo after resumption of menses. Intestinal calcium absorption was determined from the enrichment of the first 24-h urine sample collected after administration of stable calcium isotopes. Bone mineral of the total body and lumbar spine was measured by dual-energy X-ray absorptiometry and quantitative computerized tomography, respectively. Twenty-four-hour urine and fasting serum samples were analyzed for calcium, calcitropic hormones, and biochemical markers of bone turnover. Despite an increase in calcium intake during pregnancy, true percentage absorption of calcium increased from 32.9+/-9.1% at prepregnancy to 49.9+/-10.2% at T2 and 53.8+/-11.3% at T3 (P < 0.001). Urinary calcium increased from 4.32+/-2.20 mmol/d at prepregnancy to 6.21+/-3.72 mmol/d at T3 (P < 0.001), but only minor changes in maternal bone mineral were detected. At EL, dietary calcium and calcium absorption were not significantly different from that at prepregnancy, but urinary calcium decreased to 1.87+/-1.22 mmol/d (P < 0.001) and trabecular bone mineral density of the spine decreased to 147.7+/-21.2 mg/cm3 from 162.9+/-25.0 mg/cm3 at prepregnancy (P < 0.001). Calcium absorption postmenses increased nonsignificantly to 36.0+/-8.1% whereas urinary calcium decreased to 2.72+/-1.52 mmol/d (P < 0.001). We concluded that fetal calcium demand was met by increased maternal intestinal absorption; early breast-milk calcium was provided by maternal renal calcium conservation and loss of spinal trabecular bone, a loss that was recovered postmenses.

High-fiber, high-carbohydrate diets, including foods with low glycemic index, have been associated with prevention and treatment of diseases such as coronary heart disease and diabetes. β-glucan, a soluble, viscous polymer found in oat and barley endosperm cell wall, was incorporated into pasta test meals. Five fasted adult subjects were fed test meals of a barley and durum wheat blend pasta containing 100 g of available carbohydrate, 30 g of total dietary fiber (TDF) and 12 g of β-glucan, or an all durum wheat pasta containing the same amount of available carbohydrate, 5 g of TDF, and negligible β-glucan. The β-glucan and durum wheat pasta resulted in a lower glycemic response as measured by average total area and maximum increment of the blood glucose curves. Lower insulin response to the β-glucan and durum wheat pasta was also indicated by lower average area and increment characteristics of the insulin curves. Barley β-glucans may be an economical and palatable ingredient for processed food pr...

The effect of low dietary copper on copper status and the copper-containing enzyme lysyl oxidase was studied in young men. The study was divided into three dietary periods. During the first period, subjects were fed 0.66 mg/day Cu for 24 days (marginal copper). The level of copper was dropped to 0.38 mg/day for the next 42 days (low copper) and they were repleted with 2.49 mg/day Cu for next 24 days. Skin biopsies were taken at the beginning of the study and at the end of each dietary period and lysyl oxidase was measured enzymatically. There was a 24% drop in activity when the dietary copper level was reduced from 0.66 to 0.38 mg/day. When the subjects were repleted with copper, there was a significant increase in the activity of lysyl oxidase. The activity reached the level observed before the subjects were fed the restricted copper diet. These data show that, in humans, lysyl oxidase activity declines when dietary copper intake is inadequate and suggests that the cross-linking of collagen may be modulated by dietary copper. Lysyl oxidase in healthy young men can serve as a useful indicator of copper status.

A study was conducted in 11 young men to evaluate the effect of a low-copper diet on indexes of copper status and to define an amount of dietary copper at which adequate copper status could not be maintained. The young men were confined to a metabolic research unit for 90 d. The study was divided into three periods, with dietary copper as the only variable. Dietary copper was 0.66 mg/d for 24 d, 0.38 mg/d for 42 d, and 2.49 mg/d for 24 d. Plasma copper, ceruloplasmin activity, ceruloplasmin concentration, and erythrocyte superoxide dismutase (SOD) were measured at selected time points during each dietary copper period. Urine was collected throughout the study. Plasma copper, ceruloplasmin concentration and activity, and urinary copper declined significantly during the lowest dietary copper period. Plasma copper, ceruloplasmin concentration, and urinary copper increased in response to repletion. The average erythrocyte SOD concentration was lower during the depletion period than in the periods before or after depletion, but it did not decline significantly over time in the depletion period. The results suggest that these indexes are sensitive to copper depletion; that 0.38 mg Cu/d is not sufficient to maintain copper status in normal, healthy young men; and that the minimum dietary copper requirement is between 0.4 and 0.8 mg/d.

Enriched stable isotopes were used in nutrition studies of normal, healthy adults to measure zinc, copper, and iron absorption. After obtaining baseline values for zinc, copper, and iron absorption from diets adequate in all nutrients, the effects of age, pregnancy, and deveral dietary variables were studied. Stable isotopes of zinc, copper, and iron were incorporated into diets. Complete fecal samples were collected and the unabsorbed isotopes remaining in the samples were measured by thermal ionization mass spectrometry, the most precise analytical method for the determination of stable mineral isotopes. Stable isotopes were also infused in five young men to evaluate the potential of studying mineral utilization and kinetics with stable isotopes. The results of these studies demonstrate that a number of factors can affect mineral absorption, but the specific effects differ for different minerals. Isotopic enrichments could be measured in urine and blood, so kinetic studies of utilization of essential minerals are now feasible with enriched stable isotopes. Continued use of stable isotopes to determine mineral absorption, combined with stable-isotope studies of mineral utilization, balance data, and biochemical indicators of mineral status, should result in a better understanding of mineral requirements and metabolism under a variety of conditions.

OBJECTIVE: We evaluated the effect of a long-term, high-copper intake on plasma total homocysteine, folate, vitamin B12, and pyridoxal-5'-phosphate concentrations in humans. METHODS: Nine healthy young men were confined in a metabolic research unit for 18 d and fed 3-d rotation diets supplying an average of 1.6 mg of copper per day followed by 129 d of free-living conditions when they received 7 mg of copper per day in addition to their usual diets. The subjects returned to the metabolic research unit for the second 18-d period and were given the same diets as during the first 18 d with the exception that the copper intake was 7.8 mg/d. There was no apparent biochemical indication that the subjects were deficient in copper before the large-dose copper intake. Blood samples were obtained at the end of the first and second 18-d periods at the metabolic research unit, and plasma concentrations of total homocysteine, folate, vitamin B12, and pyridoxal-5'-phosphate were measured. RESULTS: The long-term, high-copper intake resulted in small but significant decreases in plasma concentrations of total homocysteine and folate. There was no effect of the high-copper intake on plasma concentrations of vitamin B12 and pyridoxal-5'-phosphate. CONCLUSIONS: These findings can be explained by our previous observation in rats suggesting that methionine synthase is copper dependent and that the metabolism of homocysteine and folate is regulated in part by copper nutriture. It may be necessary to consider copper nutriture for the interpretation of plasma concentrations of total homocysteine in humans.

A study of molybdenum absorption, excretion, and balance was conducted in four young men fed five amounts of dietary molybdenum, ranging from 22 to 1490 micrograms/d, for 24 d each. The study was conducted to obtain scientific data on which to base a recommendation on dietary molybdenum intake for healthy young men. Stable isotopes of molybdenum were used as tracers. 100Mo was fed five times during the study and 97Mo was infused three times. 94Mo was used to quantify the molybdenum isotopes and total molybdenum in urine, fecal collections, and diets by isotope dilution. Adverse effects were not observed at any of the dietary intakes. Molybdenum was very efficiently absorbed, 88-93%, at all dietary molybdenum intakes, and adsorption was most efficient at the highest amounts of dietary molybdenum. The amount and percentage of molybdenum excreted in the urine increased as dietary molybdenum increased, suggesting that molybdenum turnover is slow when dietary molybdenum is low and increases as dietary molybdenum increases. We conclude from these results that dietary intakes between 22 and 1500 micrograms/d by adult men are safe for > or = 24 d and that molybdenum retention is regulated by urinary excretion. Molybdenum is conserved at low intakes and excess molybdenum is rapidly excreted in the urine when intake is high.

We examined the effects of low-copper diets on indexes of immune response of 11 healthy men (aged 21-32 y) during a 90-d metabolic suite study. Daily copper intake for the first 24 d, next 42 d, and the last 24 d of the study was 0.66, 0.38, and 2.49 mg, respectively. Feeding the diet with 0.38 mg Cu/d was associated with a significant (P < or = 0.05) decrease in the proliferation of peripheral blood mononuclear cells cultured with phytohemagglutinin, Concanavalin A, or pokeweed, and an increase in the percentage of circulating B cells (CD 19+), but had no effect on the concentration of serum interleukin 2 receptor, the percentage of peripheral monocytes, neutrophils, CD3+, CD4+, or CD8+ T cells; or on the neutrophil phagocytic activity. Feeding 2.49 mg Cu/d for 24 d prevented further decreases in the indexes affected by the low-copper diet but did not restore them to the prestudy concentrations, even though plasma copper and ceruloplasmin concentrations were restored to normal.

A compartmental model of copper metabolism in adult men was developed from stable isotope data obtained in a 90-day study during which three levels of dietary copper (1.68, 0.785, 7.52 mg/d) were fed. The tracer 65 Cu was administered intravenously (i.v.) three times and orally four times during the study period. The model contains five compartments, two delay components, and two excretion pathways interpreted as two plasma compartments, two liver compartments, an other-tissue compartment, and fecal and urinary excretion routes. Dietary copper level influences the flow from the second liver compartment to the second plasma compartment and from the second plasma compartment to the other-tissues compartment. The model suggests that the tissue uptake of oral and i.v. copper is different, with flow from plasma to the first liver compartment varying with route of isotopic administration. The model-predicted masses of copper within the compartments and delay components follow a pattern of expected masses within the body. The major storage sites predicted are the second liver compartment, the delay after the other-tissue compartment, and the delay in the fecal excretion pathway. The model predicts that 65% of plasma copper is bound to ceruloplasmin, compared with 56 to 68% calculated from the data. Approximately 4.1 ± 0.8% of total body copper was predicted to be in plasma compared with 2 to 6% expected.

Methods were developed to separate and purify Mo from biological samples and to measure isotopic ratios in 1 microgram of Mo. A magnetic sector, thermal ionization mass spectrometer was used with simultaneous collection of five isotopes. Isotopic ratios were corrected for mass fractionation by iterative normalization using the 96/98 ratio. Ion beam intensity was enhanced by using a double-filament configuration, loading samples onto evaporation filaments with silica gel and boric acid. A triple-isotope-dilution approach was used, so the method could be applied to two-tracer studies of Mo metabolism in human subjects. 94Mo was added to samples prior to purification to quantify the total Mo content of samples and to determine the amounts of enriched 97Mo and 100Mo appearing in urine and fecal samples of study participants. The three ratios, 94/98, 97/98, and 100/98, were determined with within-run precision of from 0.06 to 0.10% (RSD). Precision of the ratios between replicates was from 0.05 to 0.08%.

A number of minerals contained in foods are essential nutrients for humans, animals, and/or plants. While most vitamins are very well absorbed, most essential minerals are not. Usual absorption of minerals ranges from less than 1% to over 90%. The bioavailability of dietary minerals must be considered when determining whether the diet contains enough, too little, or too much. By using stable isotope tracers as labels, the metabolic fate of minerals in a specific day's diet, a specific meal, or a food can be distinguished from minerals from other sources and followed. A number of mass spectrometric methods have been used to measure stable isotopes. Magnetic sector, thermal ionization mass spectrometry (TIMS) is used routinely in our laboratory to study bioavailability of Zn, Cu, and Fe. Other mass spectrometric methods that are less precise, but useful for many applications requiring isotopic determinations include quadrupole TIMS, inductively coupled plasma mass spectrometry (ICP/MS), and fast atom bombardment mass spectrometry (FAB/MS). One of the major advantages of stable isotope studies is that multiple isotopes of the same mineral can be used simultaneously and multiple minerals can be studied simultaneously. The use of stable isotopes for studies of bioavailability of minerals in foods has gained widespread interest in recent years. The approach is expected to be applied to an increasing number of food science and nutrition problems in the future.

In this study we developed an expanded compartmental model of molybdenum (Mo) kinetics to determine rates of molybdenum distribution during molybdenum depletion and repletion. The model was based on a clinical study in which 4 men consumed a low-molybdenum diet of 22 microg/d (0.23 micromol/d) for 102 d, followed by a high molybdenum diet of 467 microg/d (4.9 micromol/d) for 18 d. Stable isotopes 100Mo and 97Mo were administered orally and intravenously, respectively, at several time points during the study, and serial samples of plasma, urine, and feces were analyzed for 100Mo, 97Mo, and total Mo. Based on plasma, urine, and fecal molybdenum levels, kinetic parameters of distribution and elimination were determined. The rates of molybdenum distribution and elimination were different during depletion and repletion. During high intake, urinary molybdenum excretion was greater than during low intake. In addition, fractional tissue storage of molybdenum was lower during high intake than during low intake. This suggests that low intake results in an adaptation to conserve body Mo, and that high intake results in an adaptation to eliminate Mo. The model also suggested that food-bound molybdenum was approximately 16% less bioavailable than purified Mo. Finally, under the conditions of this study, the model suggested that an intake of 43 microg/d (0.45 micromol/d) would be sufficient to maintain plasma molybdenum levels at steady state. This is a minimum estimate because subjects in this study were in a molybdenum-sparing state. These findings provide an understanding of the adaptations in molybdenum metabolism that take place during depletion and repletion.

The search for a reliable, convenient indicator of Zn status was the focus of research for several decades. Plasma Zn concentration is still the most widely used clinical measurement, despite the known problems of interpretation. More recently, researchers suggested that isotopically determined kinetic parameters, such as the exchangeable Zn pool (EZP), may more accurately and reliably reflect body Zn status. The objective of this study was to examine the relationship between net body Zn loss and gain during acute changes in dietary Zn intake with biochemical and kinetic indices of Zn status. Five men participated in an 85-d Zn depletion/repletion study. Net body Zn loss and gain were determined from the difference between dietary plus intravenously administered Zn and Zn excretion. Biochemical indicators of Zn status included plasma Zn, plasma alkaline phosphatase activity, and plasma retinol binding protein concentration. Following intravenous administration of (70)Zn or (67)Zn, a compartmental model was used to determine EZP mass, fractional Zn absorption, endogenous zinc excretion (EZE), and plasma Zn flux. The changes in total body zinc correlated best with changes in plasma Zn (r(2) = 0.826, P < 0.001), EZE (r(2) = 0.773, P < 0.001), and plasma Zn flux (r(2) = 0.766, P < 0.001). This study confirms that plasma Zn concentration is a valid indicator of whole-body Zn status in the absence of confounding factors; however, further research is needed to determine how kinetic parameters respond to conditions where plasma Zn concentration is known to be unreliable.

The relationship between plasma molybdenum (Mo) and dietary intake has not been investigated in humans. We developed an isotope dilution method to determine molybdenum in 0.5 mL blood plasma by ICP-MS and conducted a study to determine the effect of dietary intake on plasma molybdenum. Twelve young men consumed a very low Mo diet (22 microg/day) for 24 days while confined to the WHNRC metabolic research unit and plasma molybdenum was monitored. (97)Mo was infused in four of the subjects (Group 1) to follow its clearance from the blood. The other eight remained in unit for 120 days (an additional 96 days). Four consumed the 22 microg/day molybdenum diet for 102 days followed by 467 microg/day for 18 days (Group 2). and four consumed five levels of dietary molybdenum for 24 days each (Group 3). (100)Mo was added to the diet one or more times at each dietary level. Total plasma molybdenum and (100)Mo were monitored throughout the study. Plasma molybdenum in the 12 subjects decreased from 8.2 +/- 0.5 to 6.1 +/- 0.5 nmol/L after 13 days of low molybdenum intake and was 5.1 +/- 0.5 nmol/L after 24 days. In Group 2, average plasma molybdenum was 7.8 +/- 0.9 nmol/L at the beginning of the study, 5.4 +/- 0.4 nmol/L during the 102 days low molybdenum period, and 16.5 +/- 0.6 nmol/L during the high molybdenum period. Plasma molybdenum in Group 3 was 4.2 +/- 2.1 nmol/L at 22 microg/day; 5.8 +/- 2.5 nmol/L at 72 microg/day; 6.6 +/- 2.3 nmol/L at 121 microg/day; 19.7 nmol/L +/-2.1 at 467 microg/day; and 43.9 +/- 2.1 nmol/L at 1490 microg/day. The results demonstrate that, in contrast to most other essential minerals, plasma molybdenum reflects low and high dietary molybdenum intakes within 14 days and may a useful indicator of low and high dietary intakes.

The aim of this study was to build a compartmental model of magnesium (Mg) kinetics by using data collected from six healthy adult men after oral administration of26Mg and intravenous administration of25Mg. Blood, urine, and feces were collected for 12 days after administration of the isotopes. Isotopic ratios were determined by inductively coupled plasma-mass spectrometry. Data were analyzed for each subject using SAAMII. We began with a compartmental model previously proposed (Avioli LV and Berman M. J Appl Physiol 21: 1688-1694, 1966) and developed an alternative approach to resolve the discrepancy between model-predicted curves and experimental data. This analysis enables the exploration of 25% of total body Mg that exchanges rapidly from plasma compartment with two extraplasma pools. One of the extraplasma compartments contains 80% of the exchangeable Mg with a transport rate of 48 ± 13 mg/h. The second exchanges 179 ± 88 mg of Mg/h. The model permitted estimation of kinetic parameters as well as fractional Mg absorption and fecal endogenous excretion.

Use of stable isotopes in applied nutrition has been a developing field for over 60 years. The past 20 years has seen improvements in computer and mass-spectrometer technology that has opened up even greater possibilities in the understanding of human metabolism. While improvements in technology can bring great opportunities, it can also cause problems if there is no consensus among the stable isotope user-community on standardization of new techniques and methods. Users of stable isotopes have traditionally been split into two groups; those who work with heavy isotopes (e.g., 58Fe, 70Zn) and those who work with light isotopes (e.g., 2H218O). Standardization issues have been addressed by the light isotope users and awareness of this type of problem is starting to emerge within the heavy isotope community.

Background Zinc homeostasis and normal plasma zinc concentrations are maintained over a wide range of intakes. Objective The objective was to identify the homeostatic response to severe zinc depletion by using compartmental analysis. Design Stable zinc isotope tracers were administered intravenously to 5 men at baseline (12.2 mg dietary Zn/d) and after 5 wk of acute zinc depletion (0.23 mg/d). Compartmental modeling of zinc metabolism was performed by using tracer and mass data in plasma, urine, and feces collected over 6-14 d. Results The plasma zinc concentration fell 65% on average after 5 wk of zinc depletion. The model predicted that fractional zinc absorption increased from 26% to essentially 100%. The rate constants for zinc excretion in the urine and gastrointestinal tract decreased 96% and 74%, respectively. The rate constants describing the distribution kinetics of plasma zinc did not change significantly. When zinc depletion was simulated by using an average mass model of zinc metabolism at baseline, the only change that accounted for the observed fall in plasma zinc concentration was a 60% reduction in the rate constant for zinc release from the most slowly turning over zinc pool. The large changes in zinc intake, excretion, and absorption-even when considered together-only explained modest reductions in plasma zinc mass. Conclusion The kinetic analysis with a compartmental model suggests that the profound decrease in plasma zinc concentrations after 5 wk of severe zinc depletion was mainly due to a decrease in the rate of zinc release from the most slowly turning over body zinc pool.

In this study we tested the effect of zinc (Zn) on muscle function in humans. After receiving 12 mg Zn/day for 17 days. 8 male subjects received 0.3 mg Zn/day tor either 33 or 41 days. Subjects were divided into two groups for repletion. Group A subjects received overnight infusions of 66 mg Zn on Days 1 and 10 and then were fed 12 mg Zn/day for another 16 days. Group B subjects were fed 12 mg Zn/day for 3 weeks. Peak force and total work capacity of the knee and shoulder extensor and flexor muscle groups were assessed using an isokinetic dynamometer at baseline, at two points during depletion, and at repletion. Plasma Zn declined significantly during depletion and remained below baseline levels after repletion. The peak force of the muscle groups tesied was not affected by acute Zn depletion: however, total work capacity for the knee extensor muscles and shoulder extensor and flexor muscles declined significantly. The data suggest that acute Zn depletion alters the total work capacity of skeletal muscle.

Whole-body copper metabolism is difficult to study in human subjects. However, the use of isotopic tracers and kinetics modeling has added a dimension beyond what can be learned in humans by direct measurement. Mechanisms regulating total body copper seem to be strong, given the relatively small and constant body pool, but they are not yet well understood. The efficiency of copper absorption varies greatly, depending on dietary intake. Changes in efficiency of absorption help to regulate the amount of copper retained by the body. In addition, endogenous excretion of copper into the gastrointestinal tract depends heavily on the amount of copper absorbed. When dietary copper is high and more is absorbed, endogenous excretion increases, protecting against excess accumulation of copper in the body. When intake is low, little endogenous copper is excreted, protecting against copper depletion. Regulation is not sufficient with very low amounts of dietary copper (0.38 mg/d) and appears to be delayed when copper intake is high. The use of isotopic tracers and kinetic modeling should aid in elucidating the regulatory mechanisms.

Copper and molybdenum are essential nutrients for humans. Both trace elements are required in the diet in small amounts, are toxic in excess, and body stores are low. But there are major differences in their metabolism. Metabolic studies were conducted in the human nutrition suite of the Western Human Nutrition Research Center. Young men were confined to the unit for 2 to 90 days in studies of copper metabolism and for 120 days for studies of molybdenum metabolism. Stable isotopes were used as tracers to follow the metabolic fate of these elements. 65Cu was administered orally and intravenously in the copper studies. 100Mo was administered orally and 97Mo was administered intravenously in the molybdenum studies.

We determined the concentrations of copper, the activities of ceruloplasmin and peptidylglycine alpha-amidating monooxygenase (PAM), and the stimulation index of PAM by the in vitro addition of copper in plasma samples obtained from three male patients with occipital horns and a milder Menkes disease phenotype, having severe copper deficiency due to the defect in copper transport. We found a decreased plasma ceruloplasmin activity and an increased copper stimulation index of plasma PAM in these patients compared with healthy control subjects. The combination of these two determinations may provide a means for the assessment of copper nutriture in humans using blood samples obtained in a single microhematocrit tube. Further investigation is warranted to evaluate whether these noninvasive measurements can be used for the diagnosis of mild copper deficiency in humans with sufficient specificity and sensitivity.

The background of the current dietary recommendations for manganese and molybdenum are described. This article reviews how the previous and current estimated safe and adequate daily dietary intakes (ESADDI) were set, shortcomings in the methods used, concerns about the current recommendations, and brief summaries of new research reports. New approaches, endpoints and paradigms to use for the development of useful recommendations are given.

The aim of this study was to develop a compartmental model of molybdenum metabolism based on stable isotope excretion patterns. Molybdenum (Mo) is an essential trace element in humans, with an estimated safe and adequate daily dietary intake (ESADDI) of 75-250 micrograms Mo/d. Four adult men were fed low molybdenum diets, 22 micrograms Mo/d for a period of 102 d. 97Mo+ and 100Mo stable isotopes, in intravenous and oral doses, respectively, were administered at selected intervals. The resulting 6-d cumulative urinary and fecal isotope excretion data were used to model molybdenum metabolism using SAAM/CONSAM software. A kinetic model, including gastrointestinal (GI), plasma, slow-turnover tissue and fast-turn-over tissue compartments, accurately simulated the observed pattern of urinary and fecal excretion for both stable isotopes in all four subjects. Residence time for molybdenum in the GI tract was estimated at 1.7 +/- 0.4 d. Predicted residence time for plasma molybdenum was 22 +/- 4 min, whereas slow-turnover tissue (possible hepatic) retention averaged 58 +/- 16 d. The model thus permitted estimation of kinetic parameters for molybdenum metabolism in tissues not readily accessible or measurable in humans.

We read with interest the paper of Verhas et al (2002), who studied magnesium (Mg) bioavailability from mineral water. The test was carried out on 300 ml of water containing 1.2 mmol of Mg. Using the radioisotope 28Mg, administered orally and intravenously on two separate sessions, Mg absorption (MgA) was estimated to be 59.113.6% (means.d.). We recently worked on the comparison of stable isotopic methods for MgA determination in men and have some comments to make regarding the method used by Verhas et al. Secondly, we would like to underline that MgA is mainly dose dependent, making comparisons between studies difficult.

The amount of an element needed to prevent frank deficiency may not be sufficient to support optimal nutrition, but amounts to support optimal nutrition have not been established. Minerals and trace elements are toxic in excess and the interval between the required and toxic amount of some elements is narrow. Thus, lower and upper limits of an optimal range must be established. Before establishing dietary recommendations to support optimal nutriture for minerals, we need (1) sensitive and reliable methods for assessing status of most elements and (2) a better understanding of the influence of nutrient and non-nutrient components of diets upon requirements. Functions such as immune function, anti-oxidant status, muscle strength, glucose metabolism, and blood clotting can be affected by inadequate or excessive amounts of an element and may be more sensitive than specific status indices. Since such functions are not specific, studies must be designed so that a cause and effect relationship between the mineral and the functional index can be established. Two approaches to mineral status assessment may be both sensitive and specific: (1) tests of metalloenzyme function and (2) tracer studies using stable isotopes of minerals. Not only can stable isotopes be used to follow the metabolic fate of a mineral without exposure to radioactivity, they can be used in conjunction with compartmental modeling to predict kinetics and pool sizes in tissues not accessible in humans.

Eleven young men were confined to a metabolic research unit for 90 d to determine the effect of the amount of dietary copper on copper nutriture. The study was divided into three metabolic periods (MP): 1) with an adequate-copper diet (1.68 mg/d) for 24 d, 2) with a low-copper diet (0.79 mg/d) for 42 d, and 3) with a high-copper diet (7.53 mg/d) for 24 d. Three indices of copper status, urinary copper, and salivary copper were determined at intervals throughout the study. Neither copper status, urinary copper, nor salivary copper differed among MPs. Sweat collections from three subjects suggested that losses of copper through sweat were very low and would not contribute significantly to copper balance. These results suggest that an amount of dietary copper slightly less than 0.8 mg/d is adequate to maintain copper status for greater than or equal to 42 d in normal, healthy men and that neither urinary nor salivary copper is affected by the amount of Cu in the diet.

A high-precision method was developed for automated multiple-collector determination of 46Ca enrichment using magnetic sector, thermal ionization mass spectrometry (TIMS). Calcium was separated from biological samples by precipitation as calcium oxalate. The calcium oxalate was washed, then dissolved in nitric acid, and isotope ratios were determined in 10 µg samples. The 44Ca, 46Ca and 48Ca isotopes were collected simultaneously. The 46Ca:48Ca ratios were measured and corrected for fractionation by iterative normalization, using the 44Ca:48Ca ratio, in order to achieve the required precision. Blocks of ten ratios were measured with an internal or within-run precision of 0.14% relative standard deviation (RSD) in urine samples and 0.10% RSD in faecal samples. The external or between-run precision for nine replicates was 0.07% RSD for urine and 0.09% RSD for faecal samples. Enrichment of samples collected following the feeding of 46Ca to pre-term infants ranged from 8 to 179 Δ% excess in faecal samples and from 19 to 91 Δ% excess in urine samples. If another isotope in addition to 46Ca is to be enriched, the identical analytical method can be applied by collecting 42Ca, 43Ca, 44Ca and 46Ca simultaneously, using the two unenriched isotopes to correct for fractionation.

Extract: A sixty-three day study was conducted with young men confined to a metabolic unit to study the effects of alpha-cellulose and phytate on copper absorption. Copper absorption was determined with 65Cu, a stable isotope of copper, during each of 3 dietary treatments (basal diet, basal diet + alpha-cellulose, or basal diet + phytate). The addition of alpha-cellulose or phytate to the basal diet did not affect copper absorption. Average copper absorption was 35.0% from the basal diet, 34.1% from the diet with 0.5 g alpha-cellulose per kg body weight added, and 31.4% from the diet with 2.34 g of phytate as sodium phytate added to the diet. Copper absorption was significantly different between subjects and averaged 44.1%, 26.8%, 33.4%, and 29.5% in individual subjects. The results suggest that high levels of either alpha-cellulose or phytate do not have marked effects on copper absorption, but copper absorption differs between individuals. (author)

Use of stable isotopes with analysis by thermal ionization mass spectrometry can determine trace element availability from dietary sources safely and with a high degree of accuracy. Stable isotopes of zinc (70Zn), copper (65Cu) and iron (58Fe) were fed with semipurified diets to humans. Excretion of isotopes was determined by isotope dilution in fecal composites collected for 15 days following the feedings. A thermal ionization, magnetic sector mass spectrometer was used to measure isotopic ratios. Total mineral content of fecal composites was determined by atomic absorption spectrophotometry. Mean absorption of zinc, iron and copper in six elderly men was 17.3, 8.7 and 26.0% respectively. Results agreed closely with absorption determined simultaneously in the same subjects using radioisotopes.

Serum, hair and urinary zinc levels were measured in 51 pregnant Lebanese women during the last 6 weeks of pregnancy and in 50 nonpregnant controls. Mean serum zinc was significantly lower and mean urinary zinc was significantly higher in pregnant women than in nonpregnant women. Pregnant women gave birth at term to infants who averaged 3449 g (range 2700 and 4300 g); no birth defects were noted. The six women who smoked 0.5 or more packages of cigarettes per day gave birth to infants who were significantly smaller than the other infants (2940 g). Infant birth weight was not correlated with hair, urinary, or plasma zinc, parity or maternal blood pressure. The zinc nutriture of the Lebanese pregnant women seemed to be adequate to support fetal growth.

Stable isotopes are valuable tools for research on mineral bioavailability and metabolism. They can be used as tracers with no exposure to radiation and they do not decay over time. Attempts to use stable isotopes of minerals as metabolic tracers were first described only 25 years ago. There were relatively few reports of their use over the next 15 years, but interest in stable isotopes has expanded markedly in the last 10 years. The advantages of stable isotope tracers are so great that scientists have been willing to accept the laborious and costly nature of mineral isotope analysis, and substantial progress has been made in the field. New applications for stable isotopes and new analytical methods have been introduced recently. However, limitations to the approach and methodological problems remain to be resolved. This review describes early work in the field and discusses the advantages and disadvantages of stable isotope tracers and of the various methods of analysis. Information discovered with stable isotopes is reviewed, and probable future applications are discussed.

Male weanling rats were fed diets containing less than 1, 3, 6 oor 13 ppm zinc or stock rat diet for 3 weeks. Each dietary group was subdivided into a control group and a prednisolone-treated group (5 mg pred/kg body weight). Femur and liver zinc contents were determined by flame atomic absorption spectrophotometry. Prednisolone caused growth retardation at all levels of dietary zinc. A marked increase was seen in femur zinc content of prednisolone-treated rats fed zinc-deficient diets. No such increase was seen in rats fed diets adequate in zinc. No significant change was seen in liver zinc content of zinc-deficient rats due to prednisolone. By comparing femur zinc in rats fed zinc-deficient diets with femur zinc in weanling rats, it appears that prednisolone causes an increase in femur zinc in rats fed deficient diets by (a) preventing bone resorption, and (b) increasing femur uptake of dietary zinc. Because the effects of glucocorticoids in rats appear to differ from effects in man, similar studies in other species are recommended.