Robert E. Serfass

Isotope ratio determinations by inductively coupled plasma/mass spectrometry for zinc bioavailability studies

A method is described for the measurement of 67Zn/6*Zn and 70ZN/68Zn ratios by inductively coupled plasma/mass spectrometry with ultrasonic nebulization. The method provides sufficient accuracy and precision for zinc bioavailability studies that use samples of human feces or blood plasma. Extraction of zinc from ashed samples yields aqueous solutions sufficiently devoid of matrix ions that could affect count rates and isotope ratios. Effects of sodium matrix, zinc concentration, and instrumental parameters on the precision of isotope ratio determinations are documented. Additions of spikes enriched in 67Zn and 70Zn to natural-abundance fecal samples verify that ratios can be determined on solutions 30 μM in zinc (<300 nmol of zinc per sample) with relative accuracies of <1% and relative standard deviations (r.s.d.) of ⩽1% over the range from natural abundance to 370 atom% excess of 70Zn and to 84 atom% excess of 67Zn.

Measurement of boron concentration and isotope ratios in biological samples by inductivey coupled plasma mass spectrometry with direct injection nebulization

A method for the determination of boron in a variety of biological samples is described. Sample material is fused with sodium carbonate and boron is separated from matrix components by using Amberlite IRA-743 boron selective ion-exchange resin. Boron is eluted with 1% HNO3 and samples are introduced to an inductively coupled plasma mass spectrometer with a direct injection nebulizer. This nebulizer provides a fast sample cleanout of ca. 15 s. The 10B/11B ratio is determined with a relative standard deviation (RSD) of 0.4–1.5%, and the detection limit for boron is approximately 1 ng g−1 in these samples. Stable isotope dilution methodology for quantitation of boron shows that: (1) fusion of sample with sodium carbonate avoids volatilization of boron from samples; (2) approximately 80% of submicrogram amounts of boron from samples can be recovered from the resin with insignificant isotopic fractionation; (3) results for biological reference materials are in agreement with certified values; and (4) the boron concentration of pooled human blood plasma is 24 ± 4 μg l−1 (95% confidence interval).

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-β-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.

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 ≤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 of58 Fe 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.

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

ABSTRACT: 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.

Elevated intakes of zinc in infant formulas do 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.

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.

Developmental changes of selected minerals in Zucker rats

Abstract 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 (μg/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.

Iron absorption and oxidant stress during erythropoietin therapy in very low birth weight premature infants: a cohort study

BackgroundIron supplementation may be associated with oxidative stress particularly in premature infants. Our purpose was to examine 1) early supplemental iron during treatment with erythropoietin (EPO) and oxidative stress; 2) enhanced iron absorption during EPO in those infants receiving human milk. Therefore, we determined the effect of erythropoietin plus supplemental iron intakes (4 mg/kg/d) on antioxidant status and iron incorporation.MethodsTen very-low-birth-weight infants who were enterally fed and receiving either human milk or formula were followed for 4 weeks during erythropoietin therapy; blood and urine were collected at 3 times; baseline, 2 and 4 weeks later. Once oral feeds commenced the study protocol was initiated. After baseline blood collection, a dose of Fe57 was administered. Two weeks later, a dose of Fe58 was administered as ferrous chloride to determine the effect of human-milk or formula on iron incorporation into RBCs.ResultsInfants started the study at 35 ± 13 days. Incorporation of isotope into RBCs did not differ between formula fed for Fe57 (mean incorporation 8 ± 2.9 n = 3) compared to human-milk fed infants (8.7 ± 5 n = 7) nor for Fe58 (6 ± 2.7 n = 3 vs. 8.6 ± 5 n = 7). Tissue damage measured by malondialdehyde in plasma and F-2 – isoprostanes in urine, did not differ by feed or over time. Neither ability to resist oxidative stress/nor RBC superoxide dismutase differed according to feed or over time.ConclusionData suggest that during erythropoietin therapy antioxidant defence in VLBW infants are capable of dealing with early supplemental iron during treatment with EPO.

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

Abstract 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.