Biol Trace Elem Res DOI 10.1007/s12011-015-0356-x
Zinc Absorption from Fortified Milk Powder in Adolescent Girls Rosa O. Méndez 1 & Michael Hambidge 2 & Mark Baker 3 & Sergio A. Salgado 3 & Joaquín Ruiz 3 & Hugo S. García 4 & Ana M. Calderón de la Barca 1
Received: 1 November 2014 / Accepted: 26 April 2015 # Springer Science+Business Media New York 2015
Abstract Zinc (Zn) is essential for development, growth, and reproduction. The Mexican government subsidizes micronutrient-fortified milk for risk groups, with positive effect on the targeted groups’ plasma Zn level, inferring a good absorption is achieved although it has not being measured. The aim of this study was to determine the impact of micronutrient-fortified milk intake during 27 days on Zn absorption in adolescent girls from northwest Mexico. Therefore, Zn absorption was evaluated in 14 healthy adolescent girls (14.1 years old) with adequate plasma Zn levels, before and after 27 days of fortified Zn milk intake. Fractional Zn absorption (FZA) was calculated from urinary ratios of stable isotopic Zn tracers administered orally and intravenously on days 0 and 27, and total absorbed Zn (TZA) was calculated. At the beginning, Zn intake was 6.8±0.85 mg/d (mean±SE), and 50 % of the adolescent girls did not achieve their requirement (7.3 mg/d). Additionally, FZA was negatively correlated with Zn intake (r=−0.61, p=0.02), while TZA (1.06 mg/d) was insufficient to cover the physiologic requirements of
* Rosa O. Méndez [email protected] 1
Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a La Victoria km 0.6, PO Box 1735, 83304 Hermosillo, SON, Mexico
2
Center for Human Nutrition and Health Sciences Center, University of Colorado, C-225, 4200 E Ninth Avenue, Denver, CO 80262, USA
3
Department of Geosciences, University of Arizona, 1040 E. 4th Street, Tucson, AZ 85721, USA
4
UNIDA, Instituto Tecnológico de Veracruz, Calz. Miguel Ángel de Quevedo 2779, Formando Hogar, 91860 Veracruz, VER, Mexico
adolescent girls (3.02 mg/d). At the end of the intervention, all the girls reached the Zn intake recommendation and TZA, 3.09 mg/d, which was enough to meet the physiological requirement for 57 % of the adolescent girls. Therefore, the low Zn intake and the Zn status of adolescent girls were positively impacted by Zn-fortified milk intake and its good absorption rate. Keywords Micronutrient-fortified milk . Total absorbed zinc . Physiological requirement . Adolescent girls
Introduction Zinc (Zn) is essential for human development, growth, and reproduction. Adolescent females have high Zn requirements because of its role in growth and sexual maturation, as well as its relevancy for future health during pregnancy [1, 2]. Despite all efforts, Zn deficiency is still present worldwide, and Zn fortification of staple foods can help to achieve adequate Zn intakes in high risk populations [3]. The Mexican government subsidizes by ca. 60 % powdered whole milk through an institutional program (Liconsa) and targeted to risk groups from the lower socioeconomic status. Other criteria are children under 12 years old, 13–15-year-old adolescent girls, pregnant or lactating mothers, elders, as well as other people suffering disabilities. Currently, the program benefits 6,501,760 people, but only 6 % of them are adolescent girls. Since 2002, the milk of the program has been fortified with several micronutrients to help reduce deficiencies: a portion of 500 mL contains 6.6 mg of iron, 6.6 mg of Zn, 60 mg of ascorbic acid, 40 mg of folic acid, 2.5 μg of vitamin D, and 0.54 μg of vitamin B12. The effectiveness of the program on nutritional status, as well as on anemia and iron deficiency in young children and toddlers, has been positively evaluated [4,
Méndez et al.
5]. However, the overall prevalence of low serum Zn is still 28.09 % for 15-year-old adolescent girls [6]. Recently, we evaluated the Liconsa fortified milk effect on Zn intake and plasma concentration in 108 adolescent girls from the northwest area of the country [7]. Their low Zn intake prevalence decreased from 35 to 13 % after 27 days of drinking the fortified milk, and their plasma Zn levels were increased. This result assumes a good Zn absorption from the fortified milk, although the actual value is not known. Therefore, the aim of our study was to determine the impact of micronutrient-fortified milk intake for 27 days on Zn absorption in adolescent girls from northwest Mexico.
Subjects and Methods The subjects consisted of 24 healthy adolescent girls (12– 16 years old) from public high schools in northwest Mexico (Hermosillo City) who agreed to participate in the study. Detailed data for these subjects have been previously described [8]. Briefly, data of age and menarche were collected. The girls were currently consuming daily unfortified milk and instructed to maintain their regular self-selected diet and other lifestyle habits during the study. Six girls dropped from the study after the first part for personal reasons or because of poor compliance and four more because their urine collection was inappropriate. Therefore, data analysis for Zn absorption was performed with 14 participants. On days 0 and 27, subjects were instructed to fast between 10:00 PM of the previous day and the next morning; then, they were transported to our metabolic unit. Weight and height were obtained, and body mass index (BMI) was calculated with AnthroPlus (Version 3.2.2) and World Health Organization (WHO) growth percentile curves [9]. A written informed consent from one of the parents of each participant was obtained. The study protocol and consent forms were approved by the Ethical Committee of the Centro de Investigación en Alimentación y Desarrollo, A.C. Study Design The subjects took 250 mL of Zn-fortified milk at breakfast and 250 mL at dinner each day for 27 days. Previously, the 27-day Zn supplementation period was sufficient to affect the current Zn absorption as evaluated by changes in gene expression of Zn transporters in leucocytes [8, 10]. The subjects were provided with two packets of milk powder weekly, including instructions for preparation, spoons, and cups. Compliance was assessed three times a week in the 1st week by the project’s field workers, then two times a week. The intake was also confirmed by counting the returned empty packages at each visit. Meals were self-selected, except for the
usual two glasses of unfortified milk daily (approx. 400 mL), which was replaced by 500 mL of the Zn-fortified one, providing 6.6 mg Zn/day [8]. Dietary (DZ) and fortified milk (MZ) Zn content was determined using the measured Zn content of duplicate diets from the test days and from the fortified milk, respectively. At baseline, meals were extrinsically labeled with 70Zn (and consumed under supervision), while 68Zn was intravenously administered at breakfast. Fractional Zn absorption (FZA) from meals was calculated by using Zn isotopic ratios of urine samples collected 3–6 days after the tracer administration. Total Zn absorption (TZA) was calculated by multiplying FZA by DZ from each participant duplicate diet. At the end of the study, day 27, FZA from meals and fortified milk were determined by using isotopic ratios of the orally administered 70Zn (each meal) and 67Zn (fortified milk), respectively, to the intravenously administered 68Zn. For each subject, FZA from meals and fortified milk were calculated from urine samples collected on days 31–34. FZA and TZA from meals for each participant was calculated as described before. TZA from fortified milk for each subject was calculated by using FZA from the fortified milk and its Zn content. TZA from total diet (mg/d) is the sum of TZAs from meals and fortified milk. Isotope Solution Preparation Stable isotope dose solutions of 67Zn, 68Zn, and 70Zn were prepared from enriched Zn oxide (Cambridge Isotope Laboratories, Inc., Andover, MA). All of them were dissolved in 0.5 M H2SO4 to prepare a stock solution. For the preparation of orally administered doses of 67Zn and 70Zn, the stock solutions were diluted with deionized water (Milli-Q; Millipore, Billerica, MA) and titrated to pH 5.0 with metal-free ammonium hydroxide. Doses of 70Zn and 67Zn were diluted to a concentration of 0.15 mg/mL and 0.25 mg/mL, respectively. For the intravenously administered 68Zn, the stock solution was adjusted to pH 6.0 with ammonium hydroxide and diluted with sterile isotonic NaCl to 0.2 mg/mL. The Zn concentrations were measured by atomic absorption spectrophotometry (AAS) (Varian SpectrAA-20; Varian Techtron Pty Ltd, VIC, Australia). Accurately weighed quantities were stored at −20 °C until administration in sealed sterile vials for the oral and intravenous doses. The intravenous doses were tested for pyrogens and sterility before administration. Absorption Study Initial Zn Absorption Study On day 0, an aqueous solution of 0.15 mg of 70Zn was orally administered to the subjects at approximately half way through breakfast, lunch, and dinner. Additionally, 2 mg of 68Zn solution was intravenously administered at breakfast to measure Zn absorption from regular
Zinc Absorption from Fortified Milk Powder in Adolescent Girls
meals. For that, 68Zn was administered intravenously into a superficial vein in the forearm over a 4–5 min period with a 10-mL syringe. The butterfly tubing and syringe were flushed with 5 mL of sterile saline solution, twice, to ensure that the entire tracer dose was infused. On days 3–6, two urine samples (morning and afternoon) were collected to measure Zn isotopic ratios. Final Zn Absorption Study On day 27, the subjects drank 0.25 mg of aqueous 67Zn in every glass of milk (250 mL) at breakfast and dinner; 0.15 mg of 70Zn at breakfast, lunch and dinner and 2 mg of 68Zn solution was intravenously administered at breakfast. On days 31–34, two urine samples (morning and afternoon) were collected to measure Zn isotopic ratios. Sample Analyses Dietary Intake Each subject collected a duplicate daily composite of all foods and beverages consumed during days 0 and 27 to assess the dietary Zn intakes. The samples were collected in polyethylene containers, weighed, homogenized in a Waring blender (Blender 700, USA), and dried. All the samples were digested using a MDS 2000 microwave sample preparation system (CEM Corporation, Falcon Instruments, México, DF) according to the manufacturer instructions. Zn contents were quantified by AAS. The National Institute of Standards and Technology (NIST) bovine liver (1577b) was analyzed to check on the accuracy and precision of the AAS procedure for Zn. Additionally, food intake was assessed on two random days by the 24-h recall method, and customary nutrient intake was estimated using nutritive values from our laboratory database [11]. Phytates were determined with a commercial kit (K-PHYT 05/ 07, Megazyme International Ireland Ltd.). Before the first morning feeding, on days 0 and 27, a blood sample was collected into polyethylene tubes containing EDTA (BD Vacutainer®, Becton-Dickinson, Rutherford, NJ) to measure plasma Zn. Blood samples were kept on ice for no more than 1 h before processing. Blood was centrifuged (GSGR, Beckman, USA) on the day of collection (2700 rpm, 20 min, 4 °C). The plasma fraction was transferred into trace metal-free plastic microtubes and stored at −70 °C until analysis by AAS [12]. Analytic accuracy and precision of the plasma Zn analysis was determined using the NIST non-fat milk powder (NIST 1549). Hemoglobin (HemoCue AB, Angelhom, Sweden) was determined. Two urine samples (morning and afternoon) were collected from each participant on days 0, 3–6, and 31–34, in 50 mL Znfree containers, acidified to pH 2.0 with concentrated HCl and kept at −20 °C for determination of Zn content by AAS and quantification of urinary isotopic fractions (67Zn, 68Zn, and 70 Zn) using a multicollector inductively coupled plasma mass spectrometer (MC-ICP-MS) (Isoprobe, Thermo Scientific).
Preparation and Measurement of Isotopic Ratios in Urine Ultrapure acids and bases (Seastar Chemicals, Inc.) were used in the purification process and for dilution of samples before analysis by MC-ICP-MS. Approximately 50 mL of thawed, acidified urine was centrifuged at 1400×g for 10 min at room temperature to remove solids. The pH of the urine was adjusted to 5.3±0.2 with 3.0 M NaOH and then 70 μL of 8.0 M NH4C2H3O2 were added. The columns were prepared with 2.0 mL Chelex 100 resin (Bio-Rad Laboratories, Richmond, CA), equilibrated with 2 M HNO3, and rinsed three times with triply deionized water. The resin in the columns was converted to the ammonium form by adding 10 mL of 2 M NH4OH followed by rinsing with 30 mL of triply deionized water. The buffered urine was applied to the prepared columns and the macrominerals were eluted with 10 mL of 1.0 M NH4C2H3O2. The trace elements were eluted with 15 mL of 2.5 M HNO3. The collected fraction was evaporated to dryness, then solubilized in 10 mL of 2.5 M HCI and applied to 1 mL AG1-X8 columns (Bio-Rad Laboratories) equilibrated with 2.5 M HCI. The resin was rinsed with 10 mL each of 2.5 and 0.5 M HCI and eluted with 5 mM HCl. For the determination of Zn isotope ratios (67Zn/64Zn, 68 Zn/64Zn, and 70Zn/64Zn) and ultimately isotopic abundances, samples were prepared in 2 % (vol/vol) HNO3 to have a final concentration of approximately 50 ppb Zn. The resulting solutions were then analyzed directly by MC-ICPMS and normalized to an in-house isotopic Zn standard solution. FZA was calculated by using the equation of Yeung et al. [13]: iv dose ðmgÞ FZA ¼ %E of iv dose urine %E of oral dose in urine oral dose ðmgÞ where %E is the percentage of isotopic enrichment, which is calculated from the isotopic ratio of the sample. The %E from administered isotopes was determined from enriched (enr) and baseline (base) isotope ratio data as follows (for 67Zn): %E Zn ¼ 67
67
Zn 64 Zn
– enr
67
Zn 64 Zn
%NA64 Zn
base
where NA is the percentage isotopic natural abundance of Zn (48.63 %). Identical calculations were done for %E 70 Zn, substituting for 70Zn/64Zn appropriately. 64
Statistical Analysis Statistical analysis was carried out with the use of NCSS, 2007. Correlation matrix was analyzed by Pearson, and Spearman was employed if data distribution rejected normality.
Méndez et al. Table 1 Physical characteristics, plasma zinc, hemoglobin measurement, and usual dietary intake at baseline and the day 27 of Zn-fortified milk intake from adolescent girls
Baseline Age (years) Age at menarche (years) Weight (kg) Height (m) Body mass index/age Plasma zinc (μg/dL) Hemoglobin (g/dL) Energy intake (kcal/d) Zinc intake (mg/d) Phytate intake (mg/d) Phytate to zinc (molar ratio)
Day 27 of Zn-fortified milk intake
p
14.1±0.21 11.9±0.31 58.7±2.9 159.4±0.9 23.0±1.1
59.0±2.9 159.6±0.9 23.2±1.1
0.08 0.13 0.07
153.2±6.0 12.6±0.34 1744.5±139 6.8±0.85 413.1±83.2 4.4±0.76
153.9±7.1 13.4±0.31 2033±110 15.5±0.81 507.7±68 2.69±0.36
0.93 0.09 0.09
Zinc Absorption from Fortified Milk Powder in Adolescent Girls Rosa O. Méndez 1 & Michael Hambidge 2 & Mark Baker 3 & Sergio A. Salgado 3 & Joaquín Ruiz 3 & Hugo S. García 4 & Ana M. Calderón de la Barca 1
Received: 1 November 2014 / Accepted: 26 April 2015 # Springer Science+Business Media New York 2015
Abstract Zinc (Zn) is essential for development, growth, and reproduction. The Mexican government subsidizes micronutrient-fortified milk for risk groups, with positive effect on the targeted groups’ plasma Zn level, inferring a good absorption is achieved although it has not being measured. The aim of this study was to determine the impact of micronutrient-fortified milk intake during 27 days on Zn absorption in adolescent girls from northwest Mexico. Therefore, Zn absorption was evaluated in 14 healthy adolescent girls (14.1 years old) with adequate plasma Zn levels, before and after 27 days of fortified Zn milk intake. Fractional Zn absorption (FZA) was calculated from urinary ratios of stable isotopic Zn tracers administered orally and intravenously on days 0 and 27, and total absorbed Zn (TZA) was calculated. At the beginning, Zn intake was 6.8±0.85 mg/d (mean±SE), and 50 % of the adolescent girls did not achieve their requirement (7.3 mg/d). Additionally, FZA was negatively correlated with Zn intake (r=−0.61, p=0.02), while TZA (1.06 mg/d) was insufficient to cover the physiologic requirements of
* Rosa O. Méndez [email protected] 1
Centro de Investigación en Alimentación y Desarrollo, A.C. Carretera a La Victoria km 0.6, PO Box 1735, 83304 Hermosillo, SON, Mexico
2
Center for Human Nutrition and Health Sciences Center, University of Colorado, C-225, 4200 E Ninth Avenue, Denver, CO 80262, USA
3
Department of Geosciences, University of Arizona, 1040 E. 4th Street, Tucson, AZ 85721, USA
4
UNIDA, Instituto Tecnológico de Veracruz, Calz. Miguel Ángel de Quevedo 2779, Formando Hogar, 91860 Veracruz, VER, Mexico
adolescent girls (3.02 mg/d). At the end of the intervention, all the girls reached the Zn intake recommendation and TZA, 3.09 mg/d, which was enough to meet the physiological requirement for 57 % of the adolescent girls. Therefore, the low Zn intake and the Zn status of adolescent girls were positively impacted by Zn-fortified milk intake and its good absorption rate. Keywords Micronutrient-fortified milk . Total absorbed zinc . Physiological requirement . Adolescent girls
Introduction Zinc (Zn) is essential for human development, growth, and reproduction. Adolescent females have high Zn requirements because of its role in growth and sexual maturation, as well as its relevancy for future health during pregnancy [1, 2]. Despite all efforts, Zn deficiency is still present worldwide, and Zn fortification of staple foods can help to achieve adequate Zn intakes in high risk populations [3]. The Mexican government subsidizes by ca. 60 % powdered whole milk through an institutional program (Liconsa) and targeted to risk groups from the lower socioeconomic status. Other criteria are children under 12 years old, 13–15-year-old adolescent girls, pregnant or lactating mothers, elders, as well as other people suffering disabilities. Currently, the program benefits 6,501,760 people, but only 6 % of them are adolescent girls. Since 2002, the milk of the program has been fortified with several micronutrients to help reduce deficiencies: a portion of 500 mL contains 6.6 mg of iron, 6.6 mg of Zn, 60 mg of ascorbic acid, 40 mg of folic acid, 2.5 μg of vitamin D, and 0.54 μg of vitamin B12. The effectiveness of the program on nutritional status, as well as on anemia and iron deficiency in young children and toddlers, has been positively evaluated [4,
Méndez et al.
5]. However, the overall prevalence of low serum Zn is still 28.09 % for 15-year-old adolescent girls [6]. Recently, we evaluated the Liconsa fortified milk effect on Zn intake and plasma concentration in 108 adolescent girls from the northwest area of the country [7]. Their low Zn intake prevalence decreased from 35 to 13 % after 27 days of drinking the fortified milk, and their plasma Zn levels were increased. This result assumes a good Zn absorption from the fortified milk, although the actual value is not known. Therefore, the aim of our study was to determine the impact of micronutrient-fortified milk intake for 27 days on Zn absorption in adolescent girls from northwest Mexico.
Subjects and Methods The subjects consisted of 24 healthy adolescent girls (12– 16 years old) from public high schools in northwest Mexico (Hermosillo City) who agreed to participate in the study. Detailed data for these subjects have been previously described [8]. Briefly, data of age and menarche were collected. The girls were currently consuming daily unfortified milk and instructed to maintain their regular self-selected diet and other lifestyle habits during the study. Six girls dropped from the study after the first part for personal reasons or because of poor compliance and four more because their urine collection was inappropriate. Therefore, data analysis for Zn absorption was performed with 14 participants. On days 0 and 27, subjects were instructed to fast between 10:00 PM of the previous day and the next morning; then, they were transported to our metabolic unit. Weight and height were obtained, and body mass index (BMI) was calculated with AnthroPlus (Version 3.2.2) and World Health Organization (WHO) growth percentile curves [9]. A written informed consent from one of the parents of each participant was obtained. The study protocol and consent forms were approved by the Ethical Committee of the Centro de Investigación en Alimentación y Desarrollo, A.C. Study Design The subjects took 250 mL of Zn-fortified milk at breakfast and 250 mL at dinner each day for 27 days. Previously, the 27-day Zn supplementation period was sufficient to affect the current Zn absorption as evaluated by changes in gene expression of Zn transporters in leucocytes [8, 10]. The subjects were provided with two packets of milk powder weekly, including instructions for preparation, spoons, and cups. Compliance was assessed three times a week in the 1st week by the project’s field workers, then two times a week. The intake was also confirmed by counting the returned empty packages at each visit. Meals were self-selected, except for the
usual two glasses of unfortified milk daily (approx. 400 mL), which was replaced by 500 mL of the Zn-fortified one, providing 6.6 mg Zn/day [8]. Dietary (DZ) and fortified milk (MZ) Zn content was determined using the measured Zn content of duplicate diets from the test days and from the fortified milk, respectively. At baseline, meals were extrinsically labeled with 70Zn (and consumed under supervision), while 68Zn was intravenously administered at breakfast. Fractional Zn absorption (FZA) from meals was calculated by using Zn isotopic ratios of urine samples collected 3–6 days after the tracer administration. Total Zn absorption (TZA) was calculated by multiplying FZA by DZ from each participant duplicate diet. At the end of the study, day 27, FZA from meals and fortified milk were determined by using isotopic ratios of the orally administered 70Zn (each meal) and 67Zn (fortified milk), respectively, to the intravenously administered 68Zn. For each subject, FZA from meals and fortified milk were calculated from urine samples collected on days 31–34. FZA and TZA from meals for each participant was calculated as described before. TZA from fortified milk for each subject was calculated by using FZA from the fortified milk and its Zn content. TZA from total diet (mg/d) is the sum of TZAs from meals and fortified milk. Isotope Solution Preparation Stable isotope dose solutions of 67Zn, 68Zn, and 70Zn were prepared from enriched Zn oxide (Cambridge Isotope Laboratories, Inc., Andover, MA). All of them were dissolved in 0.5 M H2SO4 to prepare a stock solution. For the preparation of orally administered doses of 67Zn and 70Zn, the stock solutions were diluted with deionized water (Milli-Q; Millipore, Billerica, MA) and titrated to pH 5.0 with metal-free ammonium hydroxide. Doses of 70Zn and 67Zn were diluted to a concentration of 0.15 mg/mL and 0.25 mg/mL, respectively. For the intravenously administered 68Zn, the stock solution was adjusted to pH 6.0 with ammonium hydroxide and diluted with sterile isotonic NaCl to 0.2 mg/mL. The Zn concentrations were measured by atomic absorption spectrophotometry (AAS) (Varian SpectrAA-20; Varian Techtron Pty Ltd, VIC, Australia). Accurately weighed quantities were stored at −20 °C until administration in sealed sterile vials for the oral and intravenous doses. The intravenous doses were tested for pyrogens and sterility before administration. Absorption Study Initial Zn Absorption Study On day 0, an aqueous solution of 0.15 mg of 70Zn was orally administered to the subjects at approximately half way through breakfast, lunch, and dinner. Additionally, 2 mg of 68Zn solution was intravenously administered at breakfast to measure Zn absorption from regular
Zinc Absorption from Fortified Milk Powder in Adolescent Girls
meals. For that, 68Zn was administered intravenously into a superficial vein in the forearm over a 4–5 min period with a 10-mL syringe. The butterfly tubing and syringe were flushed with 5 mL of sterile saline solution, twice, to ensure that the entire tracer dose was infused. On days 3–6, two urine samples (morning and afternoon) were collected to measure Zn isotopic ratios. Final Zn Absorption Study On day 27, the subjects drank 0.25 mg of aqueous 67Zn in every glass of milk (250 mL) at breakfast and dinner; 0.15 mg of 70Zn at breakfast, lunch and dinner and 2 mg of 68Zn solution was intravenously administered at breakfast. On days 31–34, two urine samples (morning and afternoon) were collected to measure Zn isotopic ratios. Sample Analyses Dietary Intake Each subject collected a duplicate daily composite of all foods and beverages consumed during days 0 and 27 to assess the dietary Zn intakes. The samples were collected in polyethylene containers, weighed, homogenized in a Waring blender (Blender 700, USA), and dried. All the samples were digested using a MDS 2000 microwave sample preparation system (CEM Corporation, Falcon Instruments, México, DF) according to the manufacturer instructions. Zn contents were quantified by AAS. The National Institute of Standards and Technology (NIST) bovine liver (1577b) was analyzed to check on the accuracy and precision of the AAS procedure for Zn. Additionally, food intake was assessed on two random days by the 24-h recall method, and customary nutrient intake was estimated using nutritive values from our laboratory database [11]. Phytates were determined with a commercial kit (K-PHYT 05/ 07, Megazyme International Ireland Ltd.). Before the first morning feeding, on days 0 and 27, a blood sample was collected into polyethylene tubes containing EDTA (BD Vacutainer®, Becton-Dickinson, Rutherford, NJ) to measure plasma Zn. Blood samples were kept on ice for no more than 1 h before processing. Blood was centrifuged (GSGR, Beckman, USA) on the day of collection (2700 rpm, 20 min, 4 °C). The plasma fraction was transferred into trace metal-free plastic microtubes and stored at −70 °C until analysis by AAS [12]. Analytic accuracy and precision of the plasma Zn analysis was determined using the NIST non-fat milk powder (NIST 1549). Hemoglobin (HemoCue AB, Angelhom, Sweden) was determined. Two urine samples (morning and afternoon) were collected from each participant on days 0, 3–6, and 31–34, in 50 mL Znfree containers, acidified to pH 2.0 with concentrated HCl and kept at −20 °C for determination of Zn content by AAS and quantification of urinary isotopic fractions (67Zn, 68Zn, and 70 Zn) using a multicollector inductively coupled plasma mass spectrometer (MC-ICP-MS) (Isoprobe, Thermo Scientific).
Preparation and Measurement of Isotopic Ratios in Urine Ultrapure acids and bases (Seastar Chemicals, Inc.) were used in the purification process and for dilution of samples before analysis by MC-ICP-MS. Approximately 50 mL of thawed, acidified urine was centrifuged at 1400×g for 10 min at room temperature to remove solids. The pH of the urine was adjusted to 5.3±0.2 with 3.0 M NaOH and then 70 μL of 8.0 M NH4C2H3O2 were added. The columns were prepared with 2.0 mL Chelex 100 resin (Bio-Rad Laboratories, Richmond, CA), equilibrated with 2 M HNO3, and rinsed three times with triply deionized water. The resin in the columns was converted to the ammonium form by adding 10 mL of 2 M NH4OH followed by rinsing with 30 mL of triply deionized water. The buffered urine was applied to the prepared columns and the macrominerals were eluted with 10 mL of 1.0 M NH4C2H3O2. The trace elements were eluted with 15 mL of 2.5 M HNO3. The collected fraction was evaporated to dryness, then solubilized in 10 mL of 2.5 M HCI and applied to 1 mL AG1-X8 columns (Bio-Rad Laboratories) equilibrated with 2.5 M HCI. The resin was rinsed with 10 mL each of 2.5 and 0.5 M HCI and eluted with 5 mM HCl. For the determination of Zn isotope ratios (67Zn/64Zn, 68 Zn/64Zn, and 70Zn/64Zn) and ultimately isotopic abundances, samples were prepared in 2 % (vol/vol) HNO3 to have a final concentration of approximately 50 ppb Zn. The resulting solutions were then analyzed directly by MC-ICPMS and normalized to an in-house isotopic Zn standard solution. FZA was calculated by using the equation of Yeung et al. [13]: iv dose ðmgÞ FZA ¼ %E of iv dose urine %E of oral dose in urine oral dose ðmgÞ where %E is the percentage of isotopic enrichment, which is calculated from the isotopic ratio of the sample. The %E from administered isotopes was determined from enriched (enr) and baseline (base) isotope ratio data as follows (for 67Zn): %E Zn ¼ 67
67
Zn 64 Zn
– enr
67
Zn 64 Zn
%NA64 Zn
base
where NA is the percentage isotopic natural abundance of Zn (48.63 %). Identical calculations were done for %E 70 Zn, substituting for 70Zn/64Zn appropriately. 64
Statistical Analysis Statistical analysis was carried out with the use of NCSS, 2007. Correlation matrix was analyzed by Pearson, and Spearman was employed if data distribution rejected normality.
Méndez et al. Table 1 Physical characteristics, plasma zinc, hemoglobin measurement, and usual dietary intake at baseline and the day 27 of Zn-fortified milk intake from adolescent girls
Baseline Age (years) Age at menarche (years) Weight (kg) Height (m) Body mass index/age Plasma zinc (μg/dL) Hemoglobin (g/dL) Energy intake (kcal/d) Zinc intake (mg/d) Phytate intake (mg/d) Phytate to zinc (molar ratio)
Day 27 of Zn-fortified milk intake
p
14.1±0.21 11.9±0.31 58.7±2.9 159.4±0.9 23.0±1.1
59.0±2.9 159.6±0.9 23.2±1.1
0.08 0.13 0.07
153.2±6.0 12.6±0.34 1744.5±139 6.8±0.85 413.1±83.2 4.4±0.76
153.9±7.1 13.4±0.31 2033±110 15.5±0.81 507.7±68 2.69±0.36
0.93 0.09 0.09
