Zinc Stable Isotope Research Articles

Evaluation of zinc metabolism with use of stable-isotope techniques: implications for the assessment of zinc status

Zinc stable isotopes can be applied to the identification of populations at risk for zinc deficiency and to moni-toring the effects of zinc intervention studies designed to improve zinc nutriture. Techniques using these isotopes can provide information on how effectively the intestine is absorbing exogenous dietary zinc and conserving endogenous zinc. They can also yield estimates of the quantity of readily exchangeable zinc in the body. Data derived from stable-isotope studies can provide extensive information on zinc status and the bioavailability of dietary zinc, allowing researchers to relate zinc intake to physiologic and pathologic conditions. Application of these techniques in longitudinal studies can provide quantitative data on the effectiveness of prevention programs such as simple community measures aimed at reducing dietary phytate and zinc fortification and supplementation programs. Further, judicious application of zinc stable-isotope techniques could make an important contribution to progress toward the eradication of zinc deficiency in infants and young children in the developing world. Am J Clin Nutr 1998(suppl);68:410S–3S.

Zinc absorption in women during pregnancy and lactation: a longitudinal study

Zinc is essential for normal fetal growth and development and for milk production during lactation. The metabolic adjustments made in zinc utilization to meet these needs have not been described. The purpose of this study was to determine whether fractional zinc absorption (FZA) is altered during pregnancy and lactation and, if so, to determine whether the change is related to maternal zinc status, specifically, concentrations of zinc in plasma, erythrocytes, urine, and breast milk and dietary zinc intake. Thirteen women were studied at five time points: once preconception; at 8-10, 24-26, and 34-36 wk gestation; and once while they were lactating 7-9 wk postpartum. Zinc intake increased by 35 mumol/d (2.3 mg/d) from preconception to 34-36 wk (P = 0.04); it tended to decrease (P > 0.05) during lactation but did not return to the preconception level. The amount of zinc in breast milk averaged 2.0 mg/d at the lactation time point. FZA measured from urinary enrichments of two stable isotopes of zinc increased from 14% preconception to 25% during lactation (P = 0.023) but the increase to 19% at 34-36 wk gestation was not significant. No increase in FZA occurred in four women who took iron supplements during lactation. FZA was negatively correlated with plasma zinc concentration at 34-36 wk gestation and with urinary zinc excretion at all time points. The nearly twofold increase in zinc absorption during lactation was presumably in response to the demand for zinc to synthesize breast milk.

A compartmental model of zinc metabolism in healthy women using oral and intravenous stable isotope tracers

A mathematical model of zinc metabolism in six healthy women (average age: 30 +/- 11 y) was developed by using stable isotopes of zinc. After equilibration on a constant diet containing 7.0 mg Zn/d, an oral tracer highly enriched in 67Zn and an intravenous tracer highly enriched in 70Zn were administered simultaneously. Multiple plasma and 24-h urine samples were collected for the next 7 d with complete fecal collections for 11 d. Tracer-trace ratios in plasma, urine, and feces were calculated from isotope ratios of 67Zn to 66Zn and 70Zn to 66Zn measured by using inductively coupled plasma-mass spectrometry. An a priori identifiable model composed of seven compartments was developed to describe the kinetics of both tracers as well as that of naturally occurring zinc. The parameters of the model were fitted to the data by using the SAAM-CONSAM modeling software and were estimated with good precision. Several important, not directly measurable zinc variables were estimated (mean +/- SEM) from the model including the fractional absorption from the gastrointestinal tract (0.279 +/- 0.043), the rates of endogenous secretion (2.79 +/- 0.49 mg/d) and excretion (2.01 +/- 0.35 mg/d), the fractional turnover rate of the plasma pool (131 +/- 20/d), and the sizes (7.2 +/- 1.2 and 77.1 +/- 6.4 mg) and fractional turnover rates (22.3 +/- 7.1 and 1.49 +/- 0.18/d) of the fast and slow tissue pools equilibrating with the plasma, respectively.

Can a double isotope method be used to measure fractional zinc absorption from urinary samples?

This study was designed to test whether fractional absorption calculated from urinary enrichment of stable isotopes of zinc accurately reflects true absorption measured by faecal monitoring. The two techniques were directly compared in volunteers, whereby each subject acted as his/her own control. Volunteers were healthy adults, living at home, who were taking part in a larger study on zinc. They attended the local hospital for isotope administration. Fifteen volunteers were recruited, seven of whom had non-insulin dependent diabetes mellitus. All completed the study, but five did not carry out a complete faecal collection. Each subject received an oral and i.v. dose of different stable isotopes of zinc with breakfast. Faecal and urinary enrichment with the isotopes was measured. There was no significant correlation between the two measures of absorption. The double label stable isotope technique for determining zinc absorption from measurements of urinary enrichment does not reliably predict true zinc absorption (measured by faecal and urinary monitoring) in a heterogenous group of adults given relatively low doses of stable isotopes of zinc.

Zinc Metabolism in Non-insulin Dependent Diabetes Mellitus

Low plasma zinc concentrations and increased zinc excretion have been reported in patients with non-insulin dependent diabetes mellitus (NIDDM). This study was designed to compare zinc metabolism in ten NIDDM patients with ten healthy controls, using stable isotopes of zinc. Zinc absorption from a standard breakfast labelled extrinsically with 3 mg 67Zn was measured using a fecal monitoring technique and thermal ionization quadrupole mass spectrometry. One hour after the test meal each volunteer was given 0.5 mg 70Zn (as citrate) intravenously and blood samples taken at intervals for up to 6 days. Isotopic enrichment was measured and the data entered into a simple kinetic model to estimate the size and rate of turnover of exchangeable pools of zinc. Urinary zinc excretion was higher in the male diabetics than controls (p < 0.05), but not in females. Since the efficiency of absorption and endogenous losses of zinc were very variable between individuals, and there was only a limited amount of data, it was not possible to draw conclusion about the effect of NIDDM on zinc absorption and excretion. However, the fact that there were no differences in exchangeable zinc pool sizes suggests that zinc metabolism is not altered in NIDDM.

Digestion and extraction of biological materials for zinc stable isotope determination by inductively coupled plasma mass spectrometry. Invited lecture

A separation method is described for rapidly removing zinc from interfering matrix components, such as chloride, from digested biological samples prior to measurement of zinc stable isotopes by ICP-MS. The method employs chelation of the zinc with trifluoroacetylacetone (TFA), extraction of the chelate with hexane, destruction of the chelate and dissolution of the zinc into nitric acid for determintion. Complete separation of the zinc from matrix salts is achieved, and the method does not employ chlorinated hydrocarbon solvents as used in earlier, similar schemes. Cerium from borosilicate glass tubes used in sample digestion was found to be a potential problem when measuring 70Zn, owing to the presence of 140Ce2+. This potential problem can be avoided by using quartz digestion tubes and/or correcting for any cerium by monitoring m/z 140. Accuracy was verified by an independent method, and examples of the use of stable isotopes of zinc as metabolic tracers in human metabolism are given. The detection limit for the ID method (3s of blank) was 0.7 µg Zn. An in-house urine pool was found to contain 1.18 ± 0.005 µg g–1 of Zn (n= 3) by ID and 1.17 ± 0.006 µg g–1 of Zn (n= 3) by AAS.

Zinc absorption in healthy elderly humans and the effect of diet

Absorption of a zinc stable isotope was measured on two consecutive occasions in nine young and eight elderly healthy men aged 24-40 and 70-83 y, respectively. A zinc stable-isotope label (0.8 mg 70Zn) was added to a test meal of either high or low zinc bioavailability, depending mainly on phytic acid content. Zinc absorption from the high-bioavailability test meal was not significantly different (P > 0.05) in the young (38.9 +/- 9.8%, mean +/- SD) and elderly (35.0 +/- 10.9%) subjects. Zinc absorption from the low-bioavailability test meal was 40% and 43% lower, at 23.4 +/- 10.2% and 19.8 +/- 6.1% in these young and elderly men, respectively. Again, no significant effect of age was found. These results show that aging does not lead to nutritionally relevant changes in zinc absorption and in the effect of dietary inhibitors on zinc absorption. Thus, zinc absorption ability seems to be preserved in healthy elderly people, at least until the age of 80 y.

Influence of a meal and incremental doses of zinc on changes in zinc absorption

The objective of this study was to determine the effect of increasing quantities of zinc, administered with and without a meal, on zinc absorption. Fractional absorption of incremental quantities of zinc in four normal adults was determined by measuring fecal excretion of unabsorbed isotope on 3 consecutive days by using three different stable isotopes of zinc (67Zn, 68Zn, and 70Zn). Isotopes were administered in the post-absorptive state and, on a subsequent occasion, with a standard zinc-free breakfast. In the postabsorptive state, fractional absorption was not affected by the quantity of zinc ingested until this exceeded 5 mg. When the zinc was administered with a meal, however, fractional absorption of 3 and 5 mg was less than for 1 mg. These results are compatible with the hypothesis that exogenous dietary zinc has to compete for absorption with endogenous zinc that has been secreted into the lumen of the gastrointestinal tract in response to a meal.

Determination of zinc stable isotopes in biological materials using isotope dilution inductively coupled plasma mass spectrometry

A method is described for using isotope dilution to determine both the amount of natural zinc and enriched isotopes of zinc in biological samples. Isotope dilution inductively coupled plasma mass spectrometry offers a way to quantify not only the natural zinc found in a sample but also the enriched isotope tracers of zinc. Accurate values for the enriched isotopes and natural zinc are obtained by adjusting the mass count rate data for measurable instrumental biases. Analytical interferences from the matrix are avoided by extracting the zinc from the sample matrix using diethylammonium diethyldithiocarbamate. The extraction technique separates the zinc from elements which form interfering molecular ions at the same nominal masses as the zinc isotopes. Accuracy of the method is verified using standard reference materials. The detection limit is 0.06 μg Zn per sample. Precision of the abundance ratios range from 0.3–0.8% R.S.D. for natural zinc concentrations of about 200–600 μ g −1. The accuracy and precision of the measurements make it possible to follow enriched isotopic tracers of zinc in biological samples in metabolic tracer studies.

The analysis of stable isotopes in urine to determine the fractional absorption of zinc

We measured isotopic enrichment in urine after oral and intravenous administration of stable isotopes of zinc to determine fractional absorption (FA). 68Zn and 70Zn were administered orally and intravenously to four normal adults. Subsequently, urine and fecal samples were collected for 7 and 14 d, respectively, ashed, and passed through ion-exchange columns to separate zinc from other elements. Samples were analyzed by fast-atom-bombardment mass spectrometry. From 32 h onwards the enrichment of 68Zn and 70Zn in urine declined proportionately so that FA could be determined as follows: FA = enrichment (oral/iv) x dose (iv/oral). FA determinations from urine and feces (cumulative excretion) were, respectively, for subject ZK1, urine 0.79 +/- 0.03 and feces 0.70 +/- 0.01; ZK2, 0.79 +/- 0.05 and 0.69 +/- 0.02; ZK3, 0.26 +/- 0.01 and 0.25 +/- 0.01; and ZK4, 0.41 +/- 0.02 and 0.37 +/- 0.02. ZK1 and ZK2 received the oral isotope while fasting whereas ZK3 and ZK4 received the oral isotope with meals. FA of zinc can be determined by measurement of isotope enrichment in urine.

The study of trace metal absoption using stable isotopes and mass spectrometry

The absorption and excretion of zinc stable isotopes have been followed in more than 120 human subjects. The isotope enrichment determinations were made using a standard VG 7070E HF mass spectrometer. A fast atom gun (FAB) was used to form the ions from a dry residue on a pure silver probe tip. Isotope ratio measurements were found to have a precision of better than 2% (relative standard deviation) and required a sample size of 1–5 μg. The average true absorption of zinc was found to be 73 ± 12% (2σ) when the metal was taken in a fasting state. This absorption figure was corrected for tracer that had been absorbed and secreted into the gastrointestinal (GI) tract over the time course of the study. The average time for a majority of the stable isotope tracer to pass through the GI tract was 4.7 ± 1.9 (2σ) days.

Isotope shifts of Zn neutral atoms measured by two-photon Doppler-free laser-induced fluorescence spectroscopy

Two-photon Doppler-free laser-induced fluorescence spectroscopy has been used for the first time to measure isotope shifts between the three most abundant stable isotopes of zinc (64, 66 and 68). A high-resolution laser system, consisting of an argon ion-pumped cw ring dye laser, a three-stage pulsed amplifier chain, and a doubling crystal, was used to generate tunable uv radiation for the experiment. Gas-phase Zn atoms were generated by laser-ablating a ZnS crystal. The Doppler-free spectrum was produced for the two-photon allowed 4s 2 1 S 0 −4d 1 D 2 transition (λ = 160 nm) of Zn by reflecting the laser beam back on itself. The measured isotope shifts were 976 ± 39 MHz for 64 Zn− 66 Zn and 888 ± 24 MHz for 66 Zn− 68 Zn. The technique of producing counter-propagating light by back-reflecting the laser beam has been used in situ to determine both the zero velocity frequency of particles in Doppler-shifted velocity distribution experiments and to measure the bandwidth of frequency-doubled laser light.

Intrinsic labeling of bovine milk with enriched stable isotopes of zinc.

Bovine milk was labeled intrinsically with enriched stable isotopic zinc for human bioavailability studies. Intrajugular administration of zinc isotopes temporarily increased the plasma zinc concentration of Ayrshire cows by as much as 76%, but milk zinc concentration and the distribution of zinc between casein and whey did not change appreciably. Milk zinc isotopic enrichment reached 105 and 613 atom % excess for 67Zn and 70Zn, respectively within 4-12 hr of zinc administration and decreased gradually over several days. This degree of isotopic enrichment is sufficient for testing bioavailability to infants of intrinsic zinc from milk-based formulas.

Fast atom bombardment mass spectrometry for the determination of zinc stable isotopes in biological samples.

Fast atom bombardment mass spectrometry (FAB MS) has been used to measure the relative abundances of zinc stable isotopes in biological samples. Modification of the standard FAB sample target, moderate MS resolution, and ion exchange column chromatography have been used to eliminate interfering ions at the nominal masses of zinc isotopes. Precision of 1.0% coefficient of variation was obtained on fecal samples at a 1% level of isotopically enriched (polyisotopic) preparation. The detection limit has been established as 0.05% of the polyisotopic preparation in the sample (3sigma). In addition, this work corroborated the previous suggestion that isotope fractionation occurs with FAB MS of metals. After correction for this fractionation, a plot of measured vs. calculated isotope ratios exhibited a slope of 1.012 and an r of 0.998. With the techniques developed, isotope studies have been conducted on adult volunteers, and these demonstrated a 34-36% excretion of the administered zinc isotope preparation.

Zinc, copper, and iron nutrition studied with enriched stable isotopes

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.

Stable Isotope Studies of Zinc Absorption and Retention in Young and Elderly Men

Zinc absorption was determined with 67Zn and 70Zn, stable isotopes of zinc, in six young men and six elderly men who were confined to a metabolic unit for 12 wk. Their purified formula diets, supplemented with select food items, contained 15 mg of zinc per day. Zinc absorption was determined twice for each subject by combining zinc enriched with either 70Zn or 67Zn with the formula diet. Serum, urinary, and fecal zinc and zinc balance were determined by atomic absorption spectrometry. Zinc absorption averaged 17% in elderly men, significantly less than average zinc absorption of 31% in young men. Serum zinc was also lower in elderly men and increased in both groups during the course of the study. Zinc balance did not differ between groups, and endogenous zinc losses were less in the elderly than in the young men. The results suggest that while zinc absorption is less in elderly men than in young men, the lower absorption may reflect a lower requirement for absorbed zinc by the elderly. Alternatively, less efficient zinc absorption could result in decreased endogenous losses.

Zinc Utilization in Young Men Fed Adequate and Low Zinc Intakes

Zinc absorption and balances were measured in six young men who were confined while participating in a 75-d metabolic study. A diet of conventional food providing either 16.5 or 5.5 mg Zn/d was fed. Apparent absorption of zinc was calculated from the zinc balance data and from the absorption of a zinc stable isotope added to the diet. The apparent zinc absorption calculated from the isotope data was about 25% when 16.5 mg zinc was fed; it increased to about 53 and 49% after 13 and 42 d, respectively, when 5.5 mg zinc was fed. Total zinc absorption fell from 4.1 mg/d to 2.7–2.9 mg/d during the low zinc period, however. This zinc absorption was sufficient to maintain crude zinc balances in five of the six subjects. Neither serum nor urinary zinc levels changed significantly during the period of feeding low levels of zinc. It appears that zinc absorption readily responds to changes in dietary zinc and that an intake of 5.5 mg Zn/d for about 8 wk does not cause serum or urinary zinc level to fall.

Absorption of stable70 Zn in healthy young men in relation to zinc intake

Zinc absorption was measured in two groups of four healthy young men using the method of stable tracer isotope neutron activation analysis. All absorption measurements were made using a test dose of ZnCl2 in subjects who participated in an overnight fast and also in subjects from whom food was withheld for 5 h after ingestion of tracer. In one group of experimental subjects, dietary zinc intake was held constant (approximately 15 mg/day), and three different test doses of the tracer were administered at weekly intervals. Fractional absorption was 81% from a 4.52-mg dose, 67% from a 6.47 mg dose and 61% for a 24.52-mg dose. In the other group of subjects, dietary zinc was reduced from approximately 15 mg to less than 2 mg/day. This was accompanied by a significant increase in fractional zinc absorption of a fixed tracer zinc dose (1.19 mg), from 81 to 92%. This increase, which occurred within 6 days, was also measurable when the tracer zinc dose was increased to 4.76 mg. These data indicate that there is a significant effect of the dose level of stable zinc isotope on its fractional absorption. They also demonstrate that a restriction in dietary zinc intake results in a prompt increase in the absorption of a fixed pulse dose of zinc in healthy young men.

Labeling of Soybeans with the Stable Isotope 70Zn for Use in Human Metabolic Studies

Radiozinc (65Zn) was used to assess the efficiency of incorporation of the label into soybean seeds by means of a noncycling hydroponic system. It was found that 21.3 and 27.6% of the applied dose was incorporated into the mature seeds when the tracer was applied during vegetative or flowering stages of growth cycle, respectively. The latter method was adapted to labeling with the stable isotope 70Zn, and quantitative incorporation of this isotope was investigated within and among hydroponic culture pots each containing four plants. It was found that on the average 17.7% ± 3.0% of the applied dose was recovered in the mature seeds harvested from each pot. The mass isotope enrichment ratio for 70Zn:68Zn in these seeds was 2.93 ± 0.38 (mean ± SD) compared with the natural ratio of 0.0343, indicating a high degree of stable isotope enrichment. Results of soybean fractionation studies showed that the enrichment was somewhat higher in the defatted meal fraction as compared with the full-fat, the hull, and the whole seed, indicating somewhat preferential translocation of the applied stable isotope in the protein. Despite this small but observable difference, all fractions were highly enriched with 70Zn. The data from this study indicated that noncycling hydroponic culture may be effectively employed to intrinsically label soybeans with the stable isotopes of zinc for use in human feeding trials dealing with the issues of zinc availability. A 100-g portion of such labeled seeds provided 38–43 g protein and 490–620 µg 70Zn in various processed products for use in human feeding studies.

Use of enriched stable isotopes to determine bioavailability of trace elements in humans

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.