Iron absorption and incorporation into red blood cells by very low birth weight infants: studies with the stable isotope 58Fe.

Measurements of iron absorption and incorporation into RBCs were obtained with the stable isotope 58Fe, administered as a reference dose, in 11 premature infants with birth weights between 780 and 1,520 g and gestational ages between 24 and 33 weeks. Each study included a timed stool and urine collection, nasogastric tube administration of a single dose of about 228 micrograms of 58Fe/kg of body weight (as FeSO4, with 10 mg/kg of vitamin C) between feedings, and blood samples before 58Fe (day 1) and then 2 weeks (day 15) later. Gastrointestinal absorption of the 58Fe dose as measured by fecal isotope balance was 41.6 +/- 17.6% (mean +/- SD). However, only 12.0 +/- 9.6% of the 58Fe dose (28.7 +/- 22.3% of the absorbed 58Fe dose) was incorporated into RBCs on day 15. 58Fe absorption and 58Fe incorporation into RBCs on day 15 were significantly correlated with the hemoglobin concentration and reticulocyte count on day 1. Transfusion history did not affect 58Fe absorption or 58Fe incorporation into RBCs. We conclude that concurrent measurement of 58Fe absorption with fecal monitoring and of 58Fe incorporation into RBCs permits a better understanding of the fate of iron ingested by premature infants than either measurement alone.

Selenium absorption and retention by very-low-birth-weight infants: studies with the extrinsic stable isotope tag 74Se.

Measurements of dietary selenium absorption and retention were obtained after administration of a single dose of the extrinsic stable isotope tag 74Se in 20 appropriate for gestational age premature infants with birth weights between 720 and 1,630 g and gestational ages between 26 and 33 weeks. Infants were assigned randomly to receive a standard premature formula (1.34 microgram of Se/dl) or a selenium-supplemented version of that formula (2.03 micrograms of Se/dl). Each study consisted of one feeding that had been extrinsically labeled with 74Se (1.03 microgram/kg) and a timed stool and urine collection. The percent 74Se absorption was 91.2 +/- 5.4% (mean +/- SD) from the standard formula and 86.2 +/- 3.0% from the selenium-supplemented formula (p less than 0.05), but the percent of the absorbed 74Se retained was not different, i.e., 96.6 +/- 2.1% and 95.0 +/- 2.8%, respectively. The percent net absorption and net retention were also not different between the standard and selenium-supplemented formulas; net absorption was 72.7 +/- 18.1% vs. 67.8 +/- 18.8% and net retention was 57.2 +/- 17.6% vs. 53.3 +/- 20.2%, respectively. The percent 74Se absorption and true selenium absorption were significantly correlated with the percent net selenium absorption and net selenium absorption, respectively. We conclude that an extrinsically administered dose of 74Se can be used to study selenium nutrition in growing premature infants.

Correlation between the size of the selenite-exchangeable metabolic pool and total body or liver selenium in rats.

This study explored the quantitative relationship between the size of the selenite-exchangeable metabolic pool (WSe-EMP) and total body or liver Se in rats of varying age and past Se intake. We performed four experiments. In one, weanling rats were fed either a Se-deficient or Se-supplemented diet for 30 d, followed by measurement of WSe-EMP and total body Se. For the other experiments, rats were fed natural sources of Se without added selenite until adult age and then either subjected to acute Se restriction during the 7 d of measurements or maintained on a Se-sufficient diet. For the animals fed the selenite diet, the 7-d average ratio of WSe-EMP:total body Se (Se(end),0) was 0.370 +/- 0.009, which was not significantly different from the corresponding value (0.350 +/- 0.018, P greater than 0.05) for the Se-deficient group. When the group mean values of WSe-EMP were correlated with the corresponding mean values of Se(end),0 for all experiments, we obtained highly linear relations (r2 greater than 0.96). When WSe-EMP for each animal was correlated with the corresponding value of total body endogenous Se (Se(end)) or liver Se(end) (for t = 1 or 7 d), we found equally strong linear relations (r2 greater than or equal to 0.99). We concluded that WSe-EMP accurately reflected total body Se content or the Se content of such organs as liver, regardless of past Se intake, chemical form of Se or age and size of the animals.

Development of the stable isotope tracer approach for studies of copper turnover in the rat and mouse.

The stable isotope tracer approach was explored for long-term investigations of copper turnover in the adult rat and mouse, with inductively coupled plasma mass spectrometry for isotope measurements. The isotopic measurement method permitted precision and accuracy of <1.0%, with an overall sample blank of <0.05 microg copper. Rats were fed a copper-deficient diet and deionized water with (+Cu) or without (-Cu) copper (20 microg/ml). Both groups underwent a single-day replacement of drinking water with 20 microg/ml of (65)Cu. Compared with the baseline isotope ratio ((65)Cu/(63)Cu) of 0.462 +/- 0.002, blood plasma ratios for the +Cu group on days 2, 7, and 14 postdosing were 0.702 +/- 0.021, 0.557 +/- 0.004, and 0.474 +/- 0.001, respectively. The corresponding data for liver were 1.652 +/- 0.018, 0.560 +/- 0.005, and 0.482 +/- 0.001, respectively. For the -Cu group, respective plasma ratios were 1.580 +/- 0.04. 0.917 +/- 0.02, and 0.664 +/- 0.01 for days 2, 7, and 14 postdosing, and the ratios for liver were 0.987 +/- 0.02, 0.876 +/- 0.04, and 0.739 +/- 0.03. Mice previously made copper deficient to varying degrees were given a single-day replacement with the label. When the 24-hour postdosing isotope ratios in the livers of these mice were correlated with the activity of plasma ceruloplasmin, a negative correlation (r = -0.85) was observed. Isotope enrichment in both rats and mice was greater in the copper-deficient animals compared with the controls.

Effect of chronic selenite supplementation on selenium excretion and organ accumulation in rats.

We examined the effect of chronic selenite supplementation on whole body and selected organ selenium (Se) accumulation, urine excretion of total Se and trimethylselenonium ion, and Se balance in adult male rats. Animals were housed in metabolic cages and given either deionized water or water containing 4 micrograms of Se/mL as selenite for 30 d. Absorption of selenite was nearly complete, with only approximately 10% of ingested Se appearing in feces. There was a rapid rise in urinary Se that reached a plateau within a few days and accounted for 54 +/- 2% of the intake. Excretion of trimethylselenonium ion (TMSe) in urine increased rapidly, representing 35-40% of urinary Se in the supplemented animals compared with only 2% for the control group. In one experiment, rats were killed at 30 d and total carcass Se was measured using isotope dilution analysis. Supplemented rats had only a modest increase in whole body Se (94 +/- 4 micrograms Se vs. 66 +/- 3 in controls). Calculation of Se balance in the supplemented rats showed that approximately 35% of ingested Se could not be accounted for by urine plus fecal losses combined with the portion retained in the carcass. The results from this study demonstrate that under the condition of supplementation at 4 micrograms of Se/mL of drinking water, pathways other than urinary and fecal excretion may account for a substantial portion of Se loss.

Comparison of the magnitude of the selenite-exchangeable metabolic pool and whole body endogenous selenium in adult rats.

The quantitative relationship between the size of the selenite-exchangeable metabolic pool (WSe-EMP) and whole body endogenous selenium (Seend) was investigated in adult male rats. Two experiments based on multiple labeling with stable isotopes were performed. One focused on short-term (7 d, Expt. 1) and the other on long-term (60 d, Expt. 2) relationships. Rats were fed a Torula yeast diet and water supplemented with [76Se]selenite at 0.1 micrograms Se/mL; the in vivo [74Se]selenite tracer was administered orally. Groups of three or four animals were killed at timed intervals and whole carcass or selected organs were analyzed for the stable isotopes 74Se, 77Se and 82Se with hydride generation/inductively coupled plasma mass spectrometry. The value of WSe-EMP was determined from plasma or urine isotope ratios. In Experiment 1, with plasma as the sampling compartment, WSe-EMP at 24 h was 36.5 +/- 1.2% of the baseline value of whole body endogenous selenium (Seend) and 36.3 +/- 1.8% at 7 d. When urine was the sampling compartment, the corresponding values were 3.9 +/- 0.3% and 43.1 +/- 2.8%, respectively. In Experiment 2, WSe-EMP (plasma) was 38.9 +/- 1.3% of Seend at 7 d, increasing to 45.5 +/- 1.6% at 60 d. The corresponding values for urine as the sampling compartment were 45.5 +/- 2.0% (7 d) and 61.5 +/- 1.7% (60 d), respectively.

Effect of acute selenium restriction on whole body endogenous selenium and the selenite-exchangeable metabolic pool in the adult rat.

The time course of changes in whole body endogenous selenium (Se(end)) was investigated during a short-term (7-day) selenium restriction study in the adult rat. The method of continuous feeding with a stable isotope of selenium was used to permit normal intake of selenium while distinguishing between the dietary and endogenous components of body selenium. Additionally, the effect of short-term selenium restriction on the time course of the selenite-exchangeable metabolic pool (Se-EMP) was investigated. Two groups of adult male rats were intubated with the in vivo stable isotope (74)SeO(3)(2-), then fed a Torula yeast diet (selenium <0.02 microg/g) and either deionized water (-Se group) or deionized water containing selenium as (76)SeO(3)(2-) (0.1 microg selenium/ml) (+Se group). Three animals from each group were killed at 24-hour intervals. Whole body Se(end) and the estimated size of Se-EMP (W(Se-EMP)) were determined using hydride generation-inductively coupled plasma mass spectrometry for isotopic measurements. Whole body Se(end) decreased linearly in the +Se group (Se degrees (end): 54.4 microg; Se(end) at 3 days: 49.3 +/- 2.1; Se(end) at 7 days: 45.2 +/- 2.2). The decrease was exponential for the -Se group (Se degrees (end): 54.4 microg; Se(end) at 3 days: 42.9 +/- 0.3; Se(end) at 7 days: 42.2 +/- 0.7). The value of W(Se-EMP,pl) (microg) was 19.8 +/- 0.6 at 1 day and 19.7 +/- 1.0 at 7 days for the +Se group. The corresponding values for the -Se group were 15.7 +/- 1.5 and 18.8 +/- 0.4. All respective values of W(Se-EMP,pl) for the -Se group were significantly smaller than for the +Se group (P < 0.05), with the exception of values at days 6 and 7. The value of W(Se-EMP,urine) (microg) was 2.1 +/- 0.2 at 1 day, increasing rapidly to 23.5 +/- 1.5 at 7 days for the +Se group. The corresponding values for the -Se group were 3.0 and 23.1.

Zinc and copper nutritional studies in very low birth weight infants: comparison of stable isotopic extrinsic tag and chemical balance methods.

Measurements of dietary zinc and copper absorption obtained after administration of a single dose of the extrinsic stable isotopic tags 70Zn and 65Cu were compared to measurements made with standard chemical balance methods in 41 appropriate for gestational age premature infants [body wt 1267 +/- 258 g, gestational age 29.8 +/- 1.9 wk (mean +/- SD), 4 to 83 postnatal d of age]. Fifty studies were performed; 33 with premature formula, five with term formula, seven with preterm human milk (PTHM), and five with fortified-PTHM. The percentages of net zinc and 70Zn absorption were found to be significantly greater from PTHM (66.4 +/- 15.2, 68.6 +/- 9.8) than from premature formula (14.0 +/- 29.9, 31.6 +/- 22.4), and term formula (23.6 +/- 18.5, 17.6 +/- 5.6). The percentages of net copper and 65Cu absorption were also found to be significantly greater from PTHM (61.5 +/- 14.0, 69.8 +/- 14.0) than from premature formula (16.6 +/- 20.6, 39.6 +/- 21.6) and term formula (20.6 +/- 24.1, 26.5 +/- 6.9). The percentages of net zinc and 70Zn absorption (35.9 +/- 29.1, 48.4 +/- 9.6) and net copper and 65Cu absorption (38.7 +/- 10.2 and 57.4 +/- 13.1) from fortified PTHM were similar to values from PTHM. Absorption of zinc and copper determined with extrinsic stable isotopic tag and standard nutrient balance methods were significantly correlated. Estimates of endogenous fecal losses of zinc and copper were substantial with each diet, but lower with PTHM. Stepwise, multiple linear regression analysis accounted for, at most, 58% of the variability in the measures of zinc and copper availability. We conclude that extrinsic 70Zn and 65Cu tags can be used to study absorption of dietary zinc and copper by very low birth wt infants.

Isotopic determination of selenium in biological materials with inductively coupled plasma mass spectrometry.

A method for the isotopic determination of selenium in biological matrices is described. The method is based on hydride generation inductively coupled plasma mass spectrometry (ICP-MS). The development is specifically related to the requirements of stable isotope tracer studies in human subjects. The method is based on isotope dilution using 82Se as the in vitro spike and can quantify the 74Se and 77Se contents of samples. It involves wet oxidation (HNO3 - H2O2 or HNO3 - HClO4) of the 82Se-spiked matrix, reduction to selenite by boiling with HCl followed by measurement of the isotope ratios (82Se/77Se and 74Se/77Se) in the gas stream (H2Se) generated from on-line reduction of the sample selenite with NaBH4. Compared with the isotopic signal resulting from a selenite solution containing 5 ng ml-1 of Se, the total sample blank contributions at m/z = 74, 77 and 82 were less than 5% of the respective isotope signal. Worst-case absolute detection limits were 0.2-0.9 ng of Se, depending on the isotope used. Ion beam intensity ratios were measured with an over-all precision [relative standard deviation (RSD)] of 1% for both isotope pairs. Measured ratios (MRa/b) were stable during a given day's operation within the expected precision of the measurements but varied for different days. The magnitude of MRa/b was generally independent of the nature of the matrix. Highly linear relationships were found between ion beam intensity ratios (MRa/b) and the corresponding true isotope ratios for calibration solutions whose isotope ratios had been altered by as much as one order of magnitude.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of pneumatic nebulization and hydride generation inductively coupled plasma mass spectrometry for isotopic analysis of selenium.

A comparative investigation between pneumatic nebulization and continuous hydride generation as sample introduction methods for inductively coupled plasma mass spectrometry was carried out for isotopic analysis of selenium in biological samples of interest to human metabolic studies. Experimental parameters known to affect the analytical performance of the system were evaluated: instrument operating parameters, analyte solution/NaBH4 flow rate, and NaBH4 concentration. Signal-to-background ratio was examined for the three stable isotopes 74Se, 77Se, and 82Se. While background count rates for the hydride system were 3-5 times larger than those for the nebulization method, the signal-to-background ratios, normalized for Se concentration, were 30-50 times greater for the hydride system. Absolute detection limits (3 sigma) for the two systems were 20-60 (nebulization) and 0.6-1.8 (hydride) ng of Se. Overall memory of the hydride system was evaluated. Measurable effects were observed within 400 s from switching to analyte solution with differing isotopic composition, only if the sequence of analysis was from high to low ratio (1-4% bias). However, if the sequence was from low to high ratio, precise and linear calibration plots could be obtained over the isotope ratio range of an order of magnitude or higher. While further improvements might lead to potential enhancement of sensitivity and precision of as much as an order of magnitude, the present performance of the hydride system was satisfactory in relation to the requirements of isotopic analysis for metabolic investigations employing 74Se as the in vivo stable isotope tracer.

Erythrocyte incorporation of ingested 58-iron by infants.

The least abundant stable isotope of iron, 58Fe (natural abundance 0.322 weight %), was administered orally to infants to explore the feasibility of using a stable rather than a radioisotope in studies of iron absorption. The dose of 58Fe was given between feedings at age 126 days. The mass isotope ratio, 58Fe/57Fe, was determined in blood by inductively coupled plasma mass spectroscopy and at ages 140, 168, and 196 days. The percentage of the 58Fe dose entering the circulation (3.2 to 16.0%) was inversely correlated with serum ferritin concentration (r = -0.867, p less than 0.01). For individual infants the SD of the percentage of administered dose of iron appearing in the circulation ranged from 0.22 to 1.28. We conclude that the method is likely to be suitable for within-subject comparisons of iron availability from foods. Because of the large between-subject variation, we are pessimistic for this age group about the usefulness of study designs based on group comparisons.

Use of the stable isotope, 58Fe, for determining availability of nonheme iron in meals.

Because of reluctance to use radioisotopes for studies of iron absorption in children, we have explored the feasibility of using the least abundant stable isotope of iron, 58Fe (natural abundance, 0.322 weight %) in a study of nonheme iron absorption. With a balanced cross-over design, each of 16 school-age children was fed a standardized lunch on 3 consecutive days and, 28 days later, an alternate standardized lunch on 3 consecutive days. The lunch included either a beef patty or a beef-soy patty. The mass isotope ratio, 58Fe/57Fe (MIR58/57), was measured in blood by inductively coupled plasma mass spectroscopy before and 14 days after (i.e. study day 15) consuming the three lunches. The MIR58/57 on study day 15 was used as a baseline value for lunches fed on study days 29, 30, and 31. Incorporation of 58Fe into erythrocytes was greater from the lunch with beef patty than from the lunch with beef-soy patty (geometric mean values 2.02 and 1.05% of the dose, p less than 0.03). Based on the similarity of our results with those obtained in adults with radioisotopes, we conclude that 58Fe is a satisfactory tag for studies of nonheme iron absorption from meals.

Excretion of trimethylselenonium ion in human urine.

A method to permit isolation and measurement of trimethylselenonium ion [TMSe, (CH3)3Se+] from 1 liter of human urine was developed. The method was based on precipitation of TMSe with ammonium reineckate, preseparation with anion-exchange resin, and final thermal decomposition and collection of the product in HNO3. It was tested for recovery and separation from other selenium moieties present in urine using both in vivo-labeled rat urine and human urine spiked with unlabeled TMSe. Recoveries from the former were in the range 76.8-87.0% (mean +/- SD: 81.8 +/- 3.7%, n = 5), while for the latter they were in the range 72.0-93.0% (mean +/- SD for three occasions (%): 80.9 +/- 5.5, 81.4 +/- 7.8, and 78.9 +/- 1.0). The reliability of the method was tested against an HPLC procedure using in vivo-labeled rat's urine. The mean (+/- SD) percentage of urine radioactivity appearing as TMSe was 36.0 +/- 5.7% for the present and 36.2 +/- 6.6% for the HPLC method. The mean of deviations, as percentage of the HPLC method, was -0.03 +/- 8.8%. The linear regression equation for the two methods was y = -0.805 + 1.029x (r2 = 0.81). Excretion of TMSe was measured in urine samples from several persons (range: 0.18-0.37 micrograms Se/liter; mean +/- SD: 0.26 +/- 0.07, n = 9). One subject consumed three separate doses of unlabeled selenite on alternate days (Day 1, 197 micrograms Se; Day 3, 395; and Day 5, 592). For the first 24 h of each period, TMSe excretions (micrograms Se/24 h) were 0.24, 0.53, and 0.97, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-response relations in urinary excretion of trimethylselenonium in the rat.

75Se-labeled selenite was administered to fasting rats by orogastric intubation (1.5-3000 micrograms/kg body wt). Urine was collected and characterized for total radioactivity as well as for radiolabeled trimethylselenonium (TMSe). At lower doses of selenite (up to 500 micrograms/kg body wt), 30% of the administered dose was excreted. At higher doses of selenite, fractional urine excretion decreased as a function of the dose. The observed decrease in fractional urine excretion was not caused by changes in the absorption of the administered radiolabel. There was a direct relationship between the amount of the administered dose of selenite (up to 1500 micrograms/kg body wt) and the proportion of urinary [75Se] excreted as TMSe. Pretreatment with seleno compounds (10 or 100 micrograms Se/kg body wt as selenite, or selenomethionine) for 35 d before a challenge dose of [75Se]selenite did not influence the excretion of total [75Se] or of [75Se]TMSe in urine. Ingestion of a choline-deficient diet, which should deplete the availability of methyl groups, did not have any effect on excretion of total [75Se] or of [75Se]TMSe in urine after a challenge dose of [75Se]selenite (500 micrograms/kg body wt). The data presented here permit the following conclusions: 1) Production of TMSe is dose dependent, 2) production of TMSe from a single acute dose does not depend on the history of selenium intake and 3) rats fed a methyl-deficient diet are able to eliminate Se via formation of TMSe.

Erythrocyte incorporation of ingested stable isotope of iron (58Fe).

Because of a possible hazard from the use of radioisotopes to determine iron absorption by infants, the use of stable isotopes for this purpose has much appeal. We have applied the method of inductively coupled plasma mass spectrometry (ICP/MS) to determine the mass ratio, 58Fe/57Fe, in blood before and after oral administration of 58Fe. From the increase in erythrocyte enrichment with 58Fe, we have calculated percentage absorption of iron. We have shown that the coefficient of variation of measured mass isotope ratio is 0.1-1.0%, depending on the conditions of the measurement. The method has been applied to a feasibility study involving four infants. Each infant was given 58Fe either as a single dose or as one dose on each of two consecutive days. Each dose provided 1.945 mg iron and 1.440 mg 58Fe. Samples of blood were obtained before isotope administration and at 14, 42, and 60 days thereafter. Isotopic analysis of the samples demonstrates that this approach results in a sufficiently large isotope enrichment to permit satisfactory measurement of iron availability. It is concluded that this new method is highly promising for studies of iron availability in infants and children.