Sherry A. Tanumihardjo

Background: Malnutrition and micronutrient deficiencies remain significant public health burdens among Indonesian children under five years of age. This study assessed the relationship between growth indicators and micronutrient status of children aged 0–59 months. Methods: A cross-sectional design was performed, utilizing the 2018 Indonesian Basic Health Research (Riskesdas) data and biological specimens, including 550 samples from children aged between 0 and 59 months old. The Riskesdas data used in this study were demographic characteristics, anthropometric measurements, and nutritional status. The biological samples were used to measure micronutrients (iron, zinc, calcium, vitamin A (VA), and vitamin D (VD). Results: Overall, 23.1% of children were stunted, and 10.5% were wasted. This study also revealed that the prevalence of micronutrient deficiency was 34.2%, 38.7%, 8.2%, 7.8%, and 0.4% for ferritin, zinc, calcium, VA, and VD, respectively. Moreover, wasting was significantly higher in girls (13.5%) than in boys (8.2%, p = 0.044). Stunted children had significantly lower ferritin values, while wasted children had lower VA concentrations. In contrast, VD was lower in overweight children. Lastly, this study found that height for age Z-scores (HAZ score) and Body Mass Index (BMI) for age Z-scores (BAZ score) correlated positively with ferritin, zinc, and calcium levels. Conclusions: Stunting, wasting, and multiple micronutrient deficiencies remain prevalent among Indonesian children under five years of age. Strengthening nutrition interventions, in particular for iron, zinc, VA, and VD, is essential to improve child growth and health outcomes in this population.

Vitamin A is critical for health, but assessment of vitamin A status is challenging. Vitamin A circulating in blood is homeostatically controlled and often does not reflect vitamin A deficiency or excess. Use of 13C-vitamin A allows tracing and quantifying the total body vitamin A stores and additional information on pharmacokinetics and metabolism. This protocol describes the extraction, purification, and determination of 13C-isotopic enrichment of vitamin A in serum, breast milk, and tissue samples using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Analysis using GC-C-IRMS provides sensitive and precise enrichment values, particularly for samples with 13C enrichments near natural abundance levels. This enables flexibility in study designs to use smaller tracer doses and longer-term pharmacokinetic sampling. Briefly, to 0.4 to 1 mL serum, ethanol is added to precipitate proteins, C-23 β-apo-carotenol is added as an internal standard, and hexane is used to extract retinoids. The extract is subsequently resuspended in methanol and purified using HPLC. The retinol fraction is then resuspended in hexane and injected into the GC-C-IRMS system. Atom % (At%) is directly calculated in reference to carbon dioxide, which is calibrated against a sucrose standard using an elemental analyzer. Other tissue types that have been analyzed by GC-C-IRMS include breast milk and liver after saponification. Solid phase extraction techniques can be used to assist in the purification of these or other complex matrices. Furthermore, an elemental analyzer can be attached to the IRMS to analyze food samples for total 13C content.

Vitamin A is critical for health, but assessment of vitamin A status is challenging. Vitamin A circulating in blood is homeostatically controlled and often does not reflect vitamin A deficiency or excess. Use of 13C-vitamin A allows tracing and quantifying the total body vitamin A stores and additional information on pharmacokinetics and metabolism. This protocol describes the extraction, purification, and determination of 13C-isotopic enrichment of vitamin A in serum, breast milk, and tissue samples using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Analysis using GC-C-IRMS provides sensitive and precise enrichment values, particularly for samples with 13C enrichments near natural abundance levels. This enables flexibility in study designs to use smaller tracer doses and longer-term pharmacokinetic sampling. Briefly, to 0.4 to 1 mL serum, ethanol is added to precipitate proteins, C-23 β-apo-carotenol is added as an internal standard, and hexane is used to extract retinoids. The extract is subsequently resuspended in methanol and purified using HPLC. The retinol fraction is then resuspended in hexane and injected into the GC-C-IRMS system. Atom % (At%) is directly calculated in reference to carbon dioxide, which is calibrated against a sucrose standard using an elemental analyzer. Other tissue types that have been analyzed by GC-C-IRMS include breast milk and liver after saponification. Solid phase extraction techniques can be used to assist in the purification of these or other complex matrices. Furthermore, an elemental analyzer can be attached to the IRMS to analyze food samples for total 13C content.

The retinol isotope dilution (RID) test is the most sensitive method to assess vitamin A status by estimating total liver reserves, considered the reference standard. For gas chromatography-combustion-isotope ratio mass spectrometry detection, 13C is added to the retinol moiety. The synthetic procedure for 13C-retinyl acetate begins with the naturally occurring β-ionone. Briefly, C18-tetraene ketone is synthesized from β-ionone and purified. If a 13C4-retinyl acetate is desired, the carbanion of 13C2-labeled triethylphosphonoacetate can be used to add two 13C's to the β-ionone moiety before adding acetone's three carbons to synthesize the C18-tetraene ketone. If a 13C3-retinyl acetate is desired, the three carbons of the acetone could be labeled with 13C. For the most commonly used 13C2-retinyl acetate for future analysis by gas-chromatography-combustion isotope ratio mass spectrometry, a modified Wittig-Horner procedure is used, and unlabeled C18 tetraene ketone is reacted with the carbanion of 13C2-labeled triethylphosphonoacetate to add two-13C's to the 14 and 15 positions of the retinol molecule. The resulting ethyl ester is reduced to the alcohol and esterified to 13C2-retinyl acetate with acetic anhydride. The all-trans isomer is purified on 8%-water-deactivated alumina using relatively innocuous, highly volatile, solvents-hexanes and diethyl ether-which are removed by rotary evaporation and sonification of oil doses. The product is characterized by ultraviolet-visible (UV-Vis) spectroscopy, thin layer chromatography (TLC), and high-pressure liquid chromatography (HPLC) against analytical standards. The concentrated dose is stored dissolved in soybean oil at -80 °C. Final doses are diluted in soybean oil (~200 µL/dose) and quantified by UV-Vis spectroscopy against the vehicle soybean oil. Doses are measured for delivery to humans with a positive displacement pipette or tuberculin syringes for the RID test. Using appropriate assumptions, total body stores are calculated, and total liver reserves are estimated.

The retinol isotope dilution (RID) test is the most sensitive method to assess vitamin A status by estimating total liver reserves, considered the reference standard. For gas chromatography-combustion-isotope ratio mass spectrometry detection, 13C is added to the retinol moiety. The synthetic procedure for 13C-retinyl acetate begins with the naturally occurring β-ionone. Briefly, C18-tetraene ketone is synthesized from β-ionone and purified. If a 13C4-retinyl acetate is desired, the carbanion of 13C2-labeled triethylphosphonoacetate can be used to add two 13C's to the β-ionone moiety before adding acetone's three carbons to synthesize the C18-tetraene ketone. If a 13C3-retinyl acetate is desired, the three carbons of the acetone could be labeled with 13C. For the most commonly used 13C2-retinyl acetate for future analysis by gas-chromatography-combustion isotope ratio mass spectrometry, a modified Wittig-Horner procedure is used, and unlabeled C18 tetraene ketone is reacted with the carbanion of 13C2-labeled triethylphosphonoacetate to add two-13C's to the 14 and 15 positions of the retinol molecule. The resulting ethyl ester is reduced to the alcohol and esterified to 13C2-retinyl acetate with acetic anhydride. The all-trans isomer is purified on 8%-water-deactivated alumina using relatively innocuous, highly volatile, solvents-hexanes and diethyl ether-which are removed by rotary evaporation and sonification of oil doses. The product is characterized by ultraviolet-visible (UV-Vis) spectroscopy, thin layer chromatography (TLC), and high-pressure liquid chromatography (HPLC) against analytical standards. The concentrated dose is stored dissolved in soybean oil at -80 °C. Final doses are diluted in soybean oil (~200 µL/dose) and quantified by UV-Vis spectroscopy against the vehicle soybean oil. Doses are measured for delivery to humans with a positive displacement pipette or tuberculin syringes for the RID test. Using appropriate assumptions, total body stores are calculated, and total liver reserves are estimated.

Excessive vitamin A (VA) negatively impacts bone. Interactions between VA and vitamin D (VD) in bone health are not well-understood. This study used a traditional two-by-two factorial design. Pigs were weaned and randomized to four treatments (n = 13/group): −A−D, −A+D, +A−D, and +A+D for 3 and 5 wk. Serum, liver, kidney, adrenal glands, spleen, and lung were analyzed by ultra-performance LC. Growth was evaluated by weight measured weekly and BMD by DXA. Weights were higher in −A+D (18.1 ± 1.0 kg) and +A+D (18.2 ± 2.3 kg) at 5 wk than in −A−D (15.5 ± 2.1 kg) and +A−D (15.8 ± 1.5 kg). Serum retinol concentrations were 0.25 ± 0.023, 0.22 ± 0.10, 0.77 ± 0.12, and 0.84 ± 0.28 µmol/L; and liver VA concentrations were 0.016 ± 0.015, 0.0065 ± 0.0035, 2.97 ± 0.43, 3.05 ± 0.68 µmol/g in −A−D, −A+D, +A−D, and +A+D, respectively. Serum 25(OH)D₃ concentrations were 1.5 ± 1.11, 1.8 ± 0.43, 27.7 ± 8.91, and 23.9 ± 6.67 ng/mL in −A−D, +A−D, −A+D, +A+D, respectively, indicating a deficiency in −D and adequacy in +D. BMD was highest in +D (p < 0.001). VA and the interaction had no effect on BMD. Dietary VD influenced weight gain, BMD, and health despite VA status.

The mass ratio of urinary 2-hydroxyestrone to 16-α-hydroxyestrone (2:16) is hypothesized as a biomarker of breast cancer risk in premenopausal women, with higher ratios being theoretically protective. Cruciferous vegetable intake has been associated with higher urinary 2:16 in some studies. We investigated whether a whole-food supplement made from dried Brussels sprouts and kale would increase urinary 2:16 in comparison with placebo or cruciferous vegetables in women. This randomized, parallel arm, placebo-controlled, partly blinded study included 78 healthy premenopausal women (38–50 y) with screening urinary 2:16 ≤3.0. Subjects received either six capsules containing 550 mg dried Brussels sprouts and kale per capsule, 40 g daily alternating broccoli or Brussels sprouts, or placebo for eight weeks. Urinary 2:16 and creatinine were measured at baseline, four, and eight weeks. Intent-to-treat repeated measures-ANOVA with multiple imputation ( n=100) for missing values identified no treatment effect ( P=0.9) or treatment-by-time interaction ( P=0.6); however, a significant time effect was noted ( P=0.02). Per-protocol analyses including complete cases found no treatment effect ( P=1) or treatment-by-time interaction ( P=0.6); however, the significant time effect remained ( P=0.03). Restricting analysis to subjects with >80% compliance maintained the time effect ( P=0.02). Using Pearson correlations, android-pattern and android:gynoid fat were predictive of change ( P≤0.05). In conclusion, neither cruciferous supplements nor an added vegetable serving altered urinary 2:16 in premenopausal women with eight weeks treatment. This ratio did vary with time, which is important for designing future trials.