An HPLC Ultraviolet Method Using Low Sample Volume and Protein Precipitation for the Measurement of Retinol in Human Serum Suitable for Laboratories in Low- and Middle-Income Countries.

BACKGROUND: Assessing vitamin A status in populations remains a high public health priority for low- and middle-income countries. However, analytical difficulties with serum retinol measurements persist in international laboratories. Nearly all participants in a Centers for Disease Control and Prevention external quality assessment program use HPLC to measure serum retinol, but round-to-round results failing to meet acceptable criteria suggest the need to provide a straightforward stable HPLC ultraviolet (UV) method that can be adopted by these laboratories to improve performance. We present a protein precipitation HPLC-UV method that measures serum retinol below the deficiency cutoff value (<0.7 µmol/L or 20 µg/dL) that is suitable for low- and middle-income countries and uses commercially available materials. METHODS: Serum (25 µL) added to retinyl acetate was precipitated with acetonitrile (125 µL) to extract retinol. Solvent-based calibration solutions required no extraction. Calibration used either single-point (50 µg/dL) or multipoint solutions (0.52-100 µg/dL). C18 column (4.6 × 100 mm) and acetonitrile with 0.1% triethylamine/water (83/17, v/v) as isocratic mobile phase (1.1 mL/min), achieved baseline separation (7 minutes). RESULTS: With only 25 µL of serum, the limit of detection was 0.52 µg/dL. Single- and multipoint calibration generated equivalent results. Over several years, between-run imprecision was ≤7.1% in multiple quality-control materials. Overall mean (CV) method bias for NIST-certified reference materials (e-series) was -0.2% (5.8%). Maximally, 180 samples were processed within 24 h. CONCLUSIONS: This method was robust and stable over years and accurately measured serum retinol with low-volume samples. Thus, it may be of interest to low- and middle-income countries and to pediatric and finger stick applications.

Malian children with moderate acute malnutrition who are treated with lipid-based dietary supplements have greater weight gains and recovery rates than those treated with locally produced cereal-legume products: a community-based, cluster-randomized trial.

BACKGROUND: Moderate acute malnutrition (MAM), defined as weight-for-length z score between -3 and -2 or midupper arm circumference between 11.5 and 12.5 cm, affects ∼33 million children aged <5 y worldwide. OBJECTIVE: The objective was to compare the effects of 4 dietary supplements for the treatment of MAM. DESIGN: Twelve community health centers in rural Mali were randomly assigned to provide to 1264 MAM children aged 6-35 mo one of 4 dietary supplements containing ∼500 kcal/d for 12 wk: 1) ready-to-use, lipid-based supplementary food (RUSF); 2) special corn-soy blend (CSB++); 3) locally processed, fortified flour (Misola); or 4) locally milled flours plus oil, sugar, and micronutrient powder (LMF). RESULTS: In total, 1178 children (93.2%) completed the study. The adjusted mean (95% CI) change in weight (kg) from baseline was greater with RUSF than with the locally processed blends and was intermediate with CSB++ [1.16 (1.08, 1.24) for RUSF, 1.04 (0.96, 1.13) for CSB++, 0.91 (0.82, 0.99) for Misola, and 0.83 (0.74, 0.92) for LMF; P < 0.001]. For length change, RUSF and CSB++ differed significantly from LMF. Sustained recovery rates were higher with RUSF (73%) than with Misola (61%) and LMF (58%), P < 0.0001; CSB++ recovery rates (68%) did not differ from any of the other groups. CONCLUSIONS: RUSF was more effective, but more costly, than other dietary supplements for the treatment of MAM; CSB++ yielded intermediate results. The benefits of treatment should be considered in relation to product costs and availability.

An efficacy study on alleviating micronutrient deficiencies through a multiple micronutrient fortified salt in children in South India.

BACKGROUND: Multiple micronutrient deficiencies are prevalent in India. OBJECTIVE: The study aims to establish the efficacy of multi-micronutrient fortified salt in addressing multiple micronutrient deficiencies among children compared to nutrition education and no intervention in Tamilnadu. METHODS: The study employed a community based randomized controlled trial designed to study the impact of multiple micronutrient salt (micronutrient group) in comparison with nutrition education (education group) and no intervention (control group) on haemoglobin, serum ferritin, soluble transferrin receptor, body iron stores, serum retinol and urinary iodine outcomes over a period of 8 months. The fortified salt contained iron, iodine, vitamin A, vitamin B12 and folic acid. All the children were dewormed at baseline and at the end of the study just before the biochemical measurements. RESULTS: There was a significant improvement in most biochemical parameters studied in the micronutrient group when compared with the control group whereas this was not seen between the education and control. Over 8 months, in the micronutrient group, hemoglobin increased by 0.52 g/dL, retinol by 8.56 µg/dL, ferritin by 10.8 µg/L, body iron stores by 1.27 mg and the decrease in the prevalence of retinol deficiency was from 51.6% to 28.1%, anaemia from 46.0% to 32.6%, iron deficiency from 66.9% to 51.3% and iron deficiency anaemia from 35.2% to 31.0%, while the prevalence of all these deficiencies increased or the changes were not significant in the other two groups. CONCLUSIONS: Multiple micronutrient fortified salt was able to improve iron and vitamin A status, whereas this was not seen in the nutrition education group.

Prevalence and public health relevance of micronutrient deficiencies and undernutrition in pre-school children and women of reproductive age in Côte d'Ivoire, West Africa.

OBJECTIVE: To provide nationally representative data on the prevalence of anaemia, vitamin A and Fe deficiencies among pre-school age children (pre-SAC) and non-pregnant women of reproductive age (WRA), and on vitamin B12 and folate deficiencies in WRA, and the influence of inflammation on their interpretation. DESIGN: A cross-sectional survey to measure anthropometry, malaria parasitaemia and micronutrient status. Specifically, blood samples were analysed for Hb, plasma ferritin, soluble transferrin receptors, C-reactive protein, α1-acid glycoprotein, retinol-binding protein, vitamin B12 and folate. SETTING: Côte d'Ivoire in 2007. SUBJECTS: Nine hundred and twenty-eight WRA and 879 pre-SAC. RESULTS: In WRA, prevalence of Plasmodium parasitaemia (5 %) was low, but inflammation (34 %) was higher. Anaemia was a severe public health problem and prevalence differed by residency and eco-region. Inflammation-adjusted Fe deficiency was highest in urban areas (20 %). Nationally, folate deficiency was 86 %, higher in urban areas and varied by eco-region. Prevalence of vitamin B12 deficiency was low but higher in the rural areas and the north. Inflammation-adjusted vitamin A deficiency was very low (1 %). In pre-SAC, prevalence of inflammation (67 %) and Plasmodium parasites (25 %) was high; the latter was associated with poverty, rural residency and higher ferritin concentrations. Anaemia was classified as a severe public health problem (72 %), and was higher in rural areas (76 %) and the north (87 %). A quarter of pre-SAC suffered from vitamin A deficiency (inflammation-adjusted) and prevalence of undernutrition was high. CONCLUSIONS: Prevalence of inflammation, Plasmodium parasitaemia and micronutrient deficiencies were high in Côte d'Ivoire, particularly in pre-SAC. Nutritional interventions should be accompanied by strategies to reduce exposure to infections.

Plasma ferritin and soluble transferrin receptor concentrations and body iron stores identify similar risk factors for iron deficiency but result in different estimates of the national prevalence of iron deficiency and iron-deficiency anemia among women and children in Cameroon.

Available iron status indicators reflect different aspects of metabolism. We compared the prevalence and distribution of iron deficiency (ID) and iron-deficiency anemia (IDA) among Cameroonian women and children, as measured by plasma ferritin, and soluble transferrin receptor concentrations, body iron stores (BIS), and hemoglobin, and evaluated the impact of adjustments for inflammation on these measures. In a nationally representative survey, we randomly selected 30 clusters in each of 3 zones (north, south, and large cities) and 10 households/ cluster, each with a child aged 12-59 mo and a woman 15-49 y. Ferritin and BIS were mathematically adjusted for inflammation, using plasma C-reactive protein and α(1)-acid glycoprotein both as continuous and categorical variables. Inflammation was present in 48.0% of children and 20.8% of women and anemia was diagnosed in 57.6% of children and 38.8% of women. Depending on the iron status indicator applied, the prevalence of ID ranged from 14.2 to 68.4% among children and 11.5 to 31.8% among women, and the prevalence of IDA ranged from 12.0 to 47.4% among children and 9.0 to 19.4% among women; the proportion of anemia associated with ID ranged from 20.8 to 82.3% among children and 23.2 to 50.0% among women. The different iron indicators generally identified similar groups at greatest risk of deficiency, using both conventional and derived cutoffs: younger children, pregnant women, and women and children in the north and rural areas. Research is needed to clarify the relationships between iron status indicators, particularly in the presence of inflammation, to harmonize global data on prevalence of ID.

Plasma retinol-binding protein predicts plasma retinol concentration in both infected and uninfected Cameroonian women and children.

Variation in the relationship between plasma retinol-binding protein (RBP) and retinol (ROH) has implications for vitamin A (VA) status assessment using RBP. Our objectives were to identify factors affecting the RBP:ROH relationship and to derive and evaluate population-specific RBP cutoffs for VA deficiency (VAD) in Cameroon. Plasma RBP, C-reactive protein (CRP), α1-acid-glycoprotein (AGP), and ROH concentrations were compared in a subsample of women 15-49 y (n = 121) and children 12-59 mo (n = 123) included in a national survey conducted in 2009. Plasma RBP and ROH were highly correlated (r = 0.94 for women; r = 0.96 for children; P < 0.001). Pregnancy and lactation altered the RBP:ROH relationship in women, but obesity, elevated CRP and AGP, age, and VA status did not. Among children, age altered the RBP:ROH relationship, but sex, stunting, VA status, and elevated CRP and AGP did not. Cutoffs for VAD derived using regression analysis were <1.17 µmol RBP/L for women (corresponding to <1.05 µmol ROH/L) and <0.83 µmol RBP/L for children (corresponding to <0.70 µmol ROH/L). The sensitivity and specificity of derived cutoffs were 81.8 and 93.0% for women and 94.7 and 88.9% for children, respectively. The infection-adjusted prevalence of low VA status (<1.17 µmol RBP/L) was 21.9% (95% CI = 18.7-25.0%) among women. Among children, the infection-adjusted prevalence of VAD (<0.83 µmol RBP/L) was 35.0% (95% CI = 31.1-39.0%). In conclusion, VAD remains a public health problem in Cameroon. The RBP:ROH relationship should be considered in surveys using RBP to assess VA status, and use of population-specific cutoffs may be advisable.

Fermented soyabean and vitamin C-rich fruit: a possibility to circumvent the further decrease of iron status among iron-deficient pregnant women in Indonesia.

OBJECTIVE: Increasing the consumption of Fe-rich foods and thus improving Fe bioavailability without significantly increasing diet cost is the most sustainable intervention for improving Fe intake. We assessed the effect of supplementary food consisting of fermented soyabean (tempeh) and vitamin C-rich fruit consumed during pregnancy on maternal iron deficiency (ID). DESIGN: Pregnant women were randomly allocated by village into optimized diet and control groups. Supplementary food was given 6 d/week at home. The average weekly food provided comprised 600 g of tempeh, 30 g of meat, 350 g of guava, 300 g of papaya and 100 g of orange. Hb, ferritin and transferrin receptor (TfR) concentrations were measured at 12-20 and at 32-36 weeks of gestation. SETTING: Thirty-nine villages in Indonesia. SUBJECTS: Pregnant women (12-20 weeks of gestation, n 252). RESULTS: At baseline, mean Hb, ferritin and TfR concentrations and body Fe concentration were within the normal range and did not differ between groups. At near term, mean Hb, ferritin and body Fe decreased, whereas mean TfR increased significantly in both groups. The mean changes in Fe status were similar in both groups. In Fe-deficient women, consumption of an optimized diet was associated with smaller decreases in Hb (1·02 (95% CI 0·98, 1·07) g/l; P = 0·058), ferritin (1·42 (95% CI 1·16, 1·75) µg/l; P = 0·046) and body Fe (2·57 (95% CI 1·71, 3·43) mg/kg; P = 0·073) concentrations, compared with a state of no intervention. Fe-deficient women at baseline benefited more from supplementary food compared with Fe-replete women. CONCLUSIONS: Daily supplementary food containing tempeh and vitamin C-rich fruits during pregnancy might have positive effects on maternal ID.

Impact of a multiple-micronutrient fortified salt on the nutritional status and memory of schoolchildren.

OBJECTIVE: This study was conducted to test the efficacy of a multiple micronutrient-fortified cooking salt. METHODS: A randomized controlled trial with a pre- and post-test design was used to study children 5 to 18 years of age, with an experimental (n=213) and control group (n=189). The children were sampled from 3 residential schools and were studied for 9 months. The experimental group received a multiple micronutrient-fortified salt containing vitamins A, B(1), B(2), B(6), B(12), as well as folic acid, niacin, iron, iodine, and zinc. The control group received iodized salt. Biochemical measurements [hemoglobin, serum ferritin (SF), serum transferrin receptor (sTfR), C-reactive protein (CRP), alpha-1 acid glycoprotein (AGP), serum retinol, serum vitamin B(12), serum folic acid, serum zinc, and urinary iodine (UI)] were measured at baseline and post-intervention. Hemoglobin was measured in all the children three times during the study period, while the remaining biochemical measurements were performed in a subsample of children. Children between 11 and 18 years of age were given cognitive tests to assess memory and attention. RESULTS: There was a significant improvement (p<0.05) in all the biochemical measurements and memory tests in the experimental group when compared with the control group. Post-intervention in the experimental group, the increase in hemoglobin was 0.67 g/dL (p<0.05). Iron status and body iron stores increased significantly (p<0.05) in the experimental group compared to the control group, while serum zinc increased by 50 mug/dL (p<0.05), and the prevalence of retinol deficiency decreased from 57.1 % at baseline to 16 % post-intervention (p<0.05). CONCLUSION: The multiple micronutrients from the multiple micronutrient-fortified cooking salt were absorbed in the children and helped in combating micronutrient deficiencies.

Effect of daily or weekly multiple-micronutrient and iron foodlike tablets on body iron stores of Indonesian infants aged 6-12 mo: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: There is still uncertainty about the best procedure to alleviate iron deficiency. Additionally more reliable methods are needed to assess the effect of iron intervention. OBJECTIVE: We examined the efficacy of daily iron (10 mg), daily and weekly multiple-micronutrient supplementation (10 and 20 mg Fe, respectively) in improving body iron stores of Indonesian infants. DESIGN: Infants aged 6-12 mo were randomly allocated to 1 of 4 groups: daily multiple-micronutrients (DMM) foodlike tablets (foodLETs), weekly multiple-micronutrient (WMM) foodLETs, daily iron (DI) foodLETs, or daily placebo. Hemoglobin, ferritin, transferrin receptors, and C-reactive protein data were obtained at baseline and 23 wk. RESULTS: Body iron estimated from the ratio of transferrin receptors to ferritin was analyzed for 244 infants. At baseline, mean iron stores (0.5 +/- 4.1 mg/kg) did not differ among the groups, and 45.5% infants had deficits in tissue iron (body iron < 0). At week 23, the group DI had the highest increment in mean body iron (4.0 mg/kg), followed by the DMM group (2.3 mg/kg; P < 0.001 for both). The iron stores in the WMM group did not change, whereas the mean body iron declined in the daily placebo group (-2.2 mg/kg; P < 0.001). Compared with the daily placebo group, the DMM group gained 4.55 mg Fe/kg, the DI group gained 6.23 mg Fe/kg (both P < 0.001), and the WMM group gained 2.54 mg Fe/kg (P = 0.001). CONCLUSIONS: When compliance can be ensured, DI and DMM foodLETs are efficacious in improving and WMM is efficacious in maintaining iron stores among Indonesian infants.

Effect of vitamin A supplementation with BCG vaccine at birth on vitamin A status at 6 wk and 4 mo of age.

BACKGROUND: The effect of vitamin A supplementation (VAS) at birth on subsequent vitamin A status has not been studied. OBJECTIVE: The objective was to study the effect of 50,000 IU vitamin A administered with BCG vaccine at birth on vitamin A status in both sexes. DESIGN: Within a randomized placebo-controlled trial of VAS, we obtained blood from 614 children at 6 wk of age and from 369 mother-infant pairs at 4 mo of age. We assessed vitamin A status on the basis of serum retinol-binding protein (RBP) and measured serum C-reactive protein to monitor for concurrent infections. RESULTS: RBP concentrations indicated vitamin A deficiency in 32% of the children at age 6 wk and in 16% at age 4 mo. VAS was not associated with higher RBP concentrations overall or in either sex. However, the effect of VAS varied with maternal education (P for interaction = 0.004): At age 6 wk, VAS was associated with higher (9%; 95% CI: 2, 17%) RBP concentrations in children of noneducated mothers but not in children of educated mothers. Overall, RBP concentrations increased between 6 wk and 4 mo of age. The increase correlated inversely with the number of diphtheria-tetanus-pertussis (DTP) vaccines received in the interval (P = 0.009), particularly in girls (P for interaction = 0.01) and in vitamin A recipients (P = 0.01). CONCLUSIONS: Overall, VAS at birth had no effect on vitamin A status. However VAS may temporarily improve vitamin A status in the subgroup of children of noneducated mothers. In vitamin A recipients, subsequent DTP vaccines affected vitamin A status negatively. The main trial was registered at clinicaltrials.gov as NCT00168597.

Combined measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein by an inexpensive, sensitive, and simple sandwich enzyme-linked immunosorbent assay technique.

The measurement of vitamin A (VA) and iron status is very important in the assessment of nutritional deficiencies. The objective of this research was to develop a sandwich ELISA technique for the simultaneous measurement of ferritin, soluble transferrin receptor, retinol binding protein, and C-reactive protein (CRP) as indicators for VA and iron status. The inclusion of CRP as marker of infection allows for more accurate interpretation of VA and iron status. This is accomplished in a 30-microL serum or plasma sample using an ELISA with different capture and detection antibodies and different dilutions of the sample. Commercially available clinical serum controls were used for calibration purposes. The developed assays were compared to commercially available traditional tests. Regression coefficients comparing both assays were better than 0.84 (P < 0.001). Using a limited sample set, the sandwich ELISA assay produced very similar specificity and sensitivity compared to traditional methods when common cutoff values were applied. Intra- and interassay variability was between 5 and 14% for all tests. The cost of the materials for all 5 measurements decreases to less than $1/sample if a large number of samples is analyzed. Due to the low cost, high throughput, and comparability to traditional tests, this procedure has several advantages for assessing VA and iron status in population surveys.

Lycopene, beta-carotene, and colorectal adenomas.

BACKGROUND: Epidemiologic studies found that high tomato intakes reduce the risk of colorectal cancers. This beneficial effect is assumed to be caused by high intakes of lycopene, a carotenoid with strong antioxidant activity that is present predominantly in tomatoes. OBJECTIVE: We assessed the relation between plasma lycopene concentrations and colorectal adenomas, the precursors for most colorectal cancers. In addition, the concentrations of 2 other antioxidants, beta-carotene and alpha-tocopherol, were measured. DESIGN: White subjects undergoing a complete colonoscopy were included in the study (73 with adenomas, 63 without any polyps, and 29 with hyperplastic polyps). A detailed dietary history and information on alcohol consumption and smoking habits were collected from all subjects. Plasma lycopene, beta-carotene, and alpha-tocopherol concentrations were measured by using HPLC. RESULTS: Patients with adenomas and control subjects without polyps did not differ significantly in body mass index; intakes of energy, fat, protein, carbohydrates, fiber, beta-carotene, and alcohol; or prevalence of smoking, but patients with adenomas were slightly older. The median plasma lycopene concentration was significantly lower in the adenoma group than in the control group (-35%; P = 0.016). The median plasma beta-carotene concentration also tended to be lower in the adenoma group (-25.5%), but the difference was not significant. In the multiple logistic regression, only smoking (odds ratio: 3.02; 95% CI: 1.46, 6.25; P = 0.003) and a plasma lycopene concentration < 70 microg/L (odds ratio: 2.31; 1.12, 4.77; P = 0.023) were risk factors for adenomatous polyps. Patients with hyperplastic polyps did not differ significantly from control subjects in any variable. CONCLUSION: Our findings support the hypothesis that lycopene contributes to the protective effect of high tomato intakes against the risk of colorectal adenomas.

Linear programming: a mathematical tool for analyzing and optimizing children's diets during the complementary feeding period.

During the complementary feeding period, children require a nutrient-dense diet to meet their high nutritional requirements. International interest exists in the promotion of affordable, nutritionally adequate complementary feeding diets based on locally available foods. In this context, two questions are often asked: 1) is it possible to design a diet suitable for the complementary feeding period using locally available food? and 2) if this is possible, what is the lowest-cost, nutritionally adequate diet available? These questions are usually answered using a "trial and error" approach. However, a more efficient and rigorous technique, based on linear programming, is also available. It has become more readily accessible with the advent of powerful personal computers. The purpose of this review, therefore, is to inform pediatricians and public health professionals about this tool. In this review, the basic principles of linear programming are briefly examined and some practical applications for formulating sound food-based nutritional recommendations in different contexts are explained. This review should facilitate the adoption of this technique by international health professionals.

Rapid and simple measurement of retinol in human dried whole blood spots.

We describe an improved method for the measurement of retinol in dried blood spots (DBS) on filter paper. Retinol in human DBS on filter paper was analyzed by normal phase HPLC after a simple extraction method. Retinol associated with its binding protein was eluted from the paper into aqueous solution facilitated by ultrasonic agitation. Retinol associated with retinol binding protein was denatured with acetonitrile, and then retinol was isolated in a single hexane extract and analyzed directly by HPLC. When analyzing DBS, the individual plasma volume of the spots was calculated by measuring the sodium content or by weighing the blood spots. The described method yielded low intra- and interassay variability (<6%), with sufficient sensitivity (detection limit, 0.1 micromol/L) and good recovery (97% spike). Compared with matching plasma samples, DBS retinol consistently decreased 18-23% during the 1st wk of storage. After 1 wk, retinol remained stable in the blood spots at 23 degrees C for >3 mo. In conclusion, the analysis of retinol in DBS by HPLC is comparable to retinol analysis in serum. The variability of the method was reduced by using sodium concentration to estimate sample volume. Collection of DBS for retinol analysis is appropriate under field conditions, where it is difficult to centrifuge or freeze blood samples.