Evaluation of nuclear magnetic resonance spectroscopy for determination of deuterium abundance in body fluids: application to measurement of total-body water in human infants.

Nuclear magnetic resonance (NMR) spectroscopy was used to quantitate abundance of 2H in body water of human infants. This method provides precise measurement of total-body water without the extensive sample preparation requirements of previously described methods for determination of 2H content in body fluids. 2H2O (1 g/kg body weight) was administered to infants and saliva and urine were collected for up to 5 h. An internal standard was added directly to the fluid specimen and 2H enrichment in water was measured by NMR spectroscopy. Working range of deuterium abundance was 0.04-0.32 atom %. Coefficients of variation for saliva samples at 0.20 atom % 2H was 1.97%. 2H content in urine and saliva water reached a plateau by 4 h after administration, and amounts in the two fluids were virtually identical. Mean total-body water determination for six infants was 58.3 +/- 5.8% of body weight (range 53-66%).

Zinc absorption in women: comparison of intrinsic and extrinsic stable-isotope labels.

Fractional absorption of extrinsic and intrinsic zinc from milk-based formulated diets was measured in 10 young women by using a simultaneous triple-isotope method based on fecal monitoring of unabsorbed enriched stable-isotope labels. Zinc intake was held nearly constant (mean zinc intake = 1.48 mumol.kg body wt-1.d-1) throughout the 18-d controlled feeding phase of the study by addition of extrinsic 64Zn to all formula feedings, with substitution of the extrinsic 67Zn label for some of the 64Zn when formula intrinsically labeled with 70Zn was given. Fractional absorption values of intrinsic and extrinsic zinc labels, 0.267 +/- 0.092 (mean +/- SD) and 0.282 +/- 0.086, respectively, were highly correlated (r = 0.83, P less than 0.05) and did not differ significantly. The ratio of fractional absorption of the extrinsic to that of the intrinsic label, 1.08 +/- 0.20 (mean +/- SD), was not significantly different from unity. The results show that extrinsic labeling by use of enriched stable isotopes is a valid means of determining zinc absorption from milk-based diets in adult humans.

Use of high-precision gas isotope ratio mass spectrometry to determine natural abundance 13C in lutein isolated from C3 and C4 plant sources.

A method was developed for high-precision stable carbon isotope ratio analysis of lutein isolated from a C3 (marigold flower) and a C4 (corn gluten meal) plant source using gas chromatography-combustion interfaced isotope ratio mass spectrometry. The natural abundance of 13C (expressed as delta 13C versus the international standard, Pee Dee Belemnite, in per mil units, denoted /1000) in lutein isolated from marigold flower and corn gluten meal was determined to be -29.90 +/- 0.20/1000 and -19.77 +/- 0.27/1000 (mean +/- S.D.), respectively. The high precision of gas isotope ratio mass spectrometry is potentially applicable to detect differences of isotopic composition of lutein in the blood, tissues, or excreta of animal models or humans that result from differences in the natural abundance of 13C in C3 and C4 plant foods.

Use of a 13C tracer to quantify the plasma appearance of a physiological dose of lutein in humans.

Increased intake of lutein from vegetables promotes increased density of the macular pigment and therefore may protect against age-related macular degeneration. Our objective was to use a 13C tracer and high-precision gas chromatography-combustion interfaced-isotope ratio mass spectrometry (GC-C-IRMS) to investigate metabolism of a lutein dose equivalent to that absorbed from vegetables. Biosynthetic per-labeled (>99% 13C) lutein was purified from a commercially available extract of algal biomass. Subjects (n = 4) ingested 3 mg of [13C]lutein with a standardized low-carotenoid breakfast. Blood samples were collected at baseline and then hourly for 12 h; additional blood samples were drawn at 16, 24, 48, 72, 96, 192, 360, and 528 h. To produce perhydro-beta-carotene suitable for analysis by GC-C-IRMS, the plasma lutein fraction was hydrogenated on palladium-on-carbon catalyst with acid-catalyzed hydrogenolysis. The stable carbon isotope (13C/12C) ratio measured by GC-C-IRMS was used to calculate the plasma concentration of [13C]lutein. There was a rapid increase in [13C]lutein in plasma until peak enrichment at 16 h followed by a decline to the next measurement at 24 h. At 528 h, small changes in 13C enrichment from baseline could still be measured in plasma lutein. High-precision GC-C-IRMS enables complete definition of the appearance and disappearance of [13C]lutein in plasma after ingestion of a dose similar to that absorbed from foods.

Developmental changes of selected minerals in Zucker rats.

Effects of obesity and age on copper, iron, zinc, sodium, potassium, and protein were compared in liver, kidney, brain, and muscle of obese (fa/fa) and nonobese (non-fa/fa) male Zucker rats. Blood plasma cerulopasmin, copper, zinc, sodium, and potassium were also determined. Mean brain weight of fa/fa rats was less than that of non-fa/fa rats at 12 weeks of age; mean brain protein concentration was greater in fa/fa than in non-fa/fa at 5 and 12 weeks of age. At 18-19 days of age, mean sodium concentration (mg/g protein) in liver of fa/fa was less than that of non-fa/fa. At 5 weeks of age, mean copper concentration (microgram/g protein) in kidney was greater in fa/fa. Mean total copper, iron, zinc, sodium, and potassium in liver and kidney were greater in fa/fa than in non-fa/fa at 5 weeks because of the larger livers and kidneys of fa/fa. Mean concentrations of copper, zinc, sodium, and potassium per gram of brain protein were slightly (6-10%) less in fa/fa than in non-fa/fa at 5 weeks. By 12 weeks, mean concentrations of copper in liver, kidney, (tibialis) muscle, and blood plasma, ceruloplasmin in blood plasma, zinc in liver and muscle, iron in muscle, and sodium in liver were greater in fa/fa than in non-fa/fa. However, total amount of each mineral in muscle at 12 weeks was less in fa/fa than in non-fa/fa because of the smaller mean muscle weight of fa/fa. Mean concentrations of copper and zinc in brain and of iron in liver and brain were less in fa/fa than in non-fa/fa at 12 weeks. The major age-related changes in fa/fa that were not observed in non-fa/fa were large increases in liver and kidney copper between 5 and 12 weeks of age. It seems that the abnormal mineral metabolism is a consequence of the obesity, but the mechanisms are not identified.

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.

Intrinsic and extrinsic stable isotopic zinc absorption by infants from formulas.

The kinetics of fecal elimination and the extent of fractional absorption of intrinsic and extrinsic zinc from infant formula were studied in 11 human infants by using a direct, simultaneous dual-label method based on fecal monitoring of unabsorbed stable isotope labels. The relative positions of the two stable isotopic tags (67Zn, 70Zn) as extrinsic and intrinsic labels were reversed in two separate determinations of fractional absorption. Administration of doubly labeled formula for 24 h or less permitted elimination of unabsorbed labels before the end of a 72-h metabolic interval. The extent of enrichment and the kinetics of fecal elimination of unabsorbed extrinsic stable isotopic zinc tag were observed to agree closely with the corresponding values for the intrinsic tag. Fractional absorption of intrinsic and extrinsic tags was highly correlated and did not differ significantly. The ratio of fractional absorption of extrinsic tag to that of intrinsic tag was 1.05 +/- 0.19 (mean +/- SD). The results constitute strong support for validation of the use of enriched stable isotopes at low levels (less than or equal to 10%) of extrinsic addition to milk-based diets for studies of zinc absorption by humans.

Interactions of dietary methionine, lead and lindane in rats.

The effects of dietary methionine on lead and lindane toxicities in rats were studied in two experiments. Rats were fed methionine-deficient (60% of requirement) or methionine-sufficient soy protein-based diets with lead acetate added (10,000 mg/kg Pb) and treated with a single dose of lindane (25% of LD50 or 88 mg/kg, p.o.) in both experiments. In experiment I, all rats were fed ad libitum. In experiment II, rats fed the methionine-sufficient diet were pair-fed to rats fed the methionine-deficient diet. In experiments I and II, the methionine-sufficient and the methionine-deficient rats had decreased final body weights, increased liver weights, decreased hematocrits, and no changes in glutathione S-transferase activity when compared to a control group. Lead + lindane treatments increased liver glutathione levels in the methionine-sufficient and methionine-deficient rats in both experiments. However, in experiment II (pair-feeding), the methionine-sufficient rats had a much greater level of liver glutathione than the methionine-deficient rats. The methionine status of the animals seems to be an important factor in determining the liver glutathione level of pair-fed rats treated with lead + lindane.

Erythrocyte incorporation of iron is similar in infants fed formulas fortified with 12 mg/L or 8 mg/L of iron.

Although feeding of formulas with iron concentration of 215 mumol/L (12 mg/L) is a reliable means of preventing iron deficiency, high intakes of iron may adversely affect absorption of copper and zinc. Because data are not available to establish whether fortification at a lower level would result in equivalent iron absorption, we tested the hypothesis that iron absorption is greater by infants fed formulas with an iron concentration of 215 mumol/L (12 mg/L) than by those fed formulas with an iron concentration of 143 mumol/L (8 mg/L). Fifty-two normal infants entered the study at 112 +/- 4 d of age, and 46 of these were successfully studied until 196 d of age. Using the stable isotope 58Fe, we determined erythrocyte incorporation of iron by infants fed Formula 8 [iron approximately 143 mumol/L (8 mg/L)] and by infants fed Similac with Iron [iron approximately 215 mumol/L (12 mg/L)]. On each of three test days beginning at 154 d of age, a major portion of the formula was labeled with 58Fe. Geometric mean erythrocyte incorporation of iron adjusted for plasma ferritin concentration at 168 d of age was 4.82 mumol/d (0.269 mg/d) by infants fed Formula 8 and 5.21 mumol/d (0.291 mg/d) by infants fed Similac with Iron. Corresponding values at 196 d of age were 5.12 and 5.41 mumol/d (0.286 and 0.302 mg/d). The differences in quantity of iron incorporated into erythrocytes by infants fed Formula 8 and Similac with Iron were not statistically significant (P = 0.66 at 168 d of age, P = 0.75 at 196 d of age) and were judged to be nutritionally trivial. Because we were unable to provide support for our hypothesis that iron absorption is greater by infants fed formulas providing 215 mumol (12 mg) of iron per liter than by those fed formulas providing 143 mumol (8 mg) of iron per liter, we conclude that, pending the results of further studies, It is reasonable to decrease the iron concentration of iron-fortified infant formulas.

Erythrocyte incorporation of iron by 56-day-old infants fed a 58Fe-labeled supplement.

In an effort to obtain information about absorption of supplemental iron by breast-fed infants during the early months of life, we determined erythrocyte incorporation of a stable iron isotope, administered to 56-d-old breast-fed infants in the form of a 58Fe-labeled vitamin-iron supplement. Infants of similar age fed a milk-based formula low in iron (approximately 4 mg/L) were also studied. The 58Fe-labeled vitamin-iron supplement was given between feedings. Fourteen days after administration of 58Fe, mean erythrocyte incorporation of the isotope was 7.8% of the dose by breast-fed infants and 4.4% of the dose by formula-fed infants. The feeding-related difference was statistically significant, probably reflecting the greater quantities of inhibitors of iron absorption in the intestines of formula-fed infants. With mean iron intake from the 58Fe-labeled vitamin-iron supplement of 7.99 mg for the breast-fed infants, erythrocyte incorporation of 7.8% of the dose corresponded to 0.62 mg, a value in the range of the estimated requirement for absorbed iron. We conclude that 2-mo-old breast-fed infants are able to absorb nutritionally significant amounts of iron from an iron supplement.

Less than 80% of absorbed iron is promptly incorporated into erythrocytes of infants.

Erythrocyte incorporation of an administered iron isotope has been used as a surrogate for iron retention on the assumption (validated in normal and iron-deficient adults) that 80-100% of the retained isotope is promptly incorporated into circulating erythrocytes. This assumption has not been validated in infants or children. The purpose of our study was to determine concurrently in normal infants absorption and erythrocyte incorporation of the stable isotope, (58)Fe. In a preliminary study (Study 1), we demonstrated that fecal excretion of ingested isotope occurs predominantly during the first 4 d after administration but continues beyond 7 d after ingestion, that is, beyond the point at which isotope in feces can be explained either by excretion of isotope that failed to enter enterocytes or by exfoliation of isotope-enriched enterocytes. In Study 2, we administered (58)Fe to nine younger (age 20-69 d) and nine older (age 165-215 d) term infants and collected feces for 11 d. Geometric mean retention of (58)Fe by the younger infants was 31.2% of intake at 4 d and 26.9% at 11 d, and by the older infants, 35.0% at 4 d and 32.5% at 11 d. Erythrocyte incorporation of (58)Fe 14 d after ingestion was 5.2% of the dose by the younger infants and 12.5% by the older infants. Utilization of retained (11 d) isotope thus was 19.8% by the younger infants and 38.3% by the older infants. We conclude that far less than 80% of retained isotope is promptly incorporated into erythrocytes (utilized) by infants.

Time course of and effect of dietary iron level on iron incorporation into erythrocytes by infants.

As a part of our effort to explore various aspects of ferrokinetics in infancy, the present study was designed to determine the timing of entry of an orally ingested iron isotope into circulating erythrocytes, and the effect of the level of dietary iron [0.3 mg/100 kcal (418.4 kJ) vs. 1.8 mg/100 kcal] after isotope administration on erythrocyte incorporation of the isotope. We administered the stable isotope, (58)Fe, orally to 56-d-old and 168-d-old infants. All infants were fed a low-iron formula (LF) before and until 5 h after isotope administration. Thereafter, half the infants were fed a formula high in iron (HF group) while the remaining infants continued to receive the LF (LF group) for an additional 28 d. The quantity of (58)Fe in circulating erythrocytes increased from 14 to 28 d after isotope administration was nearly constant from 28 through 84 d of age (plateau value) and decreased between 84 and 112 d. Erythrocyte incorporation of (58)Fe was greater by the 168-d-old infants than by the 56-d-old infants, presumably because of the lesser iron stores of the older infants. In the 56-d-old infants, erythrocyte incorporation of (58)Fe was greater by the LF than by the HF group, but this difference was not significant in the 168-d-old infants. Thus, at least in younger infants, the level of iron intake after administration of an iron isotope affects erythrocyte incorporation of the isotope. The fact that less isotope was present in erythrocytes 112 d than 84 d after administration indicates that the life span of erythrocytes of infants, even beyond the immediate newborn period, is less than the 120-d life span of erythrocytes in the adult.

Effect of low zinc intake on absorption and excretion of zinc by infants studied with 70Zn as extrinsic tag.

The effect of low dietary intake of zinc was studied in six normal infants with the use of 70Zn as an extrinsic tag. Of the two study formulas, one provided a zinc intake similar to that of customary infant formulas ("high" intake), whereas the other provided a "low" zinc intake. Two zinc absorption studies were performed with each formula (sequence: high-low-low-high). Extrinsically labeled formula was fed for 24 h and excreta were collected for 72 h. Zinc isotope ratios were determined by inductively coupled plasma mass spectrometry (ICP/MS). When zinc intake was high, net zinc absorption was 9.1 +/- 8.7% (mean +/- SD) of intake and net zinc retention was 74 +/- 91 micrograms/(kg.d). True zinc (70Zn) absorption was 16.8 +/- 5.8% of intake and fecal excretion of endogenous zinc was 78 +/- 56 micrograms/(kg.d). When zinc intake was low, net absorption of zinc increased significantly (P less than 0.001) to 26.0 +/- 13.0% of intake, but net retention was not significantly different at 42 +/- 33 micrograms/(kg.d). True absorption of zinc also increased significantly (P less than 0.001) to 41.1 +/- 7.8% of intake, whereas fecal endogenous zinc decreased (P less than 0.05) to 34 +/- 16 micrograms/(kg.d) during low zinc intake. Thus, infants maintain zinc balance in the face of low zinc intake through increased efficiency of absorption and decreased excretion of endogenous zinc.

Erythrocyte incorporation and absorption of 58Fe in premature infants treated with erythropoietin.

We hypothesized that treatment of very low birth weight premature infants with r-HuEPO would increase erythrocyte incorporation and gastrointestinal absorption of iron. Infants with birth weights < or = 1.25 kg and gestational ages < 31 wk were randomized to receive 6 wk of 500 U of r-HuEPO/kg/wk (epo group, n = 7) or placebo (placebo group, n = 7). All infants received daily enteral supplementation with 6 mg of elemental iron per kg. An enteral test dose of a stable iron isotope, 58Fe, was administered after the 1st ("early dosing") and 4th ("late dosing") wk of treatment. Mean (+/-SD) erythrocyte incorporation of the dose of 58Fe administered determined 2 wk after early dosing was significantly greater in the epo group compared with the placebo group (4.4% +/- 1.6 versus 2.0 +/- 1.4%, p = 0.013). In contrast, after late 58Fe dosing, there was no difference between groups in incorporation (3.8 +/- 1.6% versus 5.5 +/- 2.7%). Within the epo group, percentage erythrocyte incorporation of 58Fe did not differ between early and late dosing, whereas in the placebo group it increased 3-fold (p < 0.01). Percentage absorption of 58Fe was not different between the epo and placebo groups after both early dosing (30 +/- 22% versus 34 +/- 8%) and late dosing (32 +/- 9% versus 31 +/- 6%). Absorption of nonlabeled elemental iron and 58Fe were significantly correlated with one another. The percentage of the absorbed 58Fe dose incorporated into Hb was not different between groups. We conclude that, although erythropoietin treatment stimulates erythrocyte iron incorporation in premature infants, it has no effect on iron absorption at the r-HuEPO dose studied.

Elevated intakes of zinc in infant formulas don not interfere with iron absorption in premature infants.

BACKGROUND: Zinc and iron may share common pathways for absorption and compete for uptake into mucosal cells. We determined whether elevated ratios of zinc to iron would interfere with erythrocyte incorporation of iron in premature infants both during and between feeds. METHODS: In the first experiment, five premature infants (<2500 g birth weight) were enrolled, once receiving full oral feeds by nasogastric tube. They received either high (1200 microg/kg, ratio 4:1) or low (300 microg/kg, ratio 1:1) doses of oral zinc sulfate, together with 300 microg/kg oral 58Fe as chloride in saline with 10 mg/kg vitamin C, between designated feeding periods. Each infant served as its own control and randomly received either high or low doses of zinc or iron and then the alternate dose after 2 weeks. In the second experiment, nine additional premature infants were assigned to the same zinc:iron intake protocol except zinc and iron were given with usual oral feeds (premature formula or human milk) equilibrated before feeding. Iron absorption was measured by the erythrocyte incorporation of 58Fe. RESULTS: High doses of zinc given between feeds significantly inhibited erythrocyte incorporation of iron. 58Fe incorporation (%) with the 1:1 ratio of zinc:iron intake was 7.5 (5.7, 10; geometric mean, -I SD, +1 SD). The percentage of 58Fe incorporation on the 4:1 ratio of zinc:iron intake was 3.6 (2.6, 5.1). Given with feeds, the percentage of 58Fe incorporation on low zinc:iron intake was 7.0 (2.6, 19). Finally, the percentage of 58Fe incorporation on high zinc:iron intake was 6.7 (2.5, 19). CONCLUSION: Elevated intakes of zinc do not interfere with erythrocyte incorporation of iron in premature formulas.