Daniela Hampel

Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC–MS/MS) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

A novel, rapid and sensitive ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin adenine dinucleotide (FAD), nicotinamide and pyridoxal (PL) has been optimized within 2 min using a gradient of 10 mM ammonium formate (aq) and acetonitrile. Thiamin-(4-methyl-¹³C-thiazol-5-yl-¹³C₃) hydrochloride, riboflavin-dioxo-pyrimidine-¹³C₄,¹⁵N₂, and pyridoxal-methyl-d₃ hydrochloride were used as internal standards. A sample-like matrix was found to be mandatory for the external standard curve preparation. ¹³C₃-caffeine was added for direct assessment of analyte recovery. Intra- and inter-assay variability for all analytes ranged from 0.4 to 7.9% and from 2.2 to 5.2%, respectively. Samples were subjected to protein precipitation and removal of non-polar constituents by diethyl ether prior to analysis. Quantification was done by ratio response to the stable isotope labeled internal standards. The standard addition method determined recovery rates for each vitamin (73.0-100.2%). The limit of quantitation for all vitamins was between 0.05 and 5 ppb depending on the vitamin. Alternative approaches for sample preparation such as protein removal by centrifugal filter units, acetonitrile or trichloroacetic acid revealed low recovery and a greater coefficient of variation. Matrix effect studies indicated a significant influence by matrix constituents, showing the importance of stable isotope labeled internal standards for analyte quantitation in complex matrices.

Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk.

Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for serum/plasma (IMMULITE and SimulTRAC-SNB) for B12 analysis in human milk. B12-recovery rates (United States Environmental Protection Agency, 2007) were determined to be 78.9 ± 9.1% with IMMULITE and 225 ± 108% (range 116-553%) using SimulTRAC-SNB, most likely due to the presence of excess HC. HC-interferences were not observed with the IMMULITE assay, rendering previously reported mandatory HC-removal (Lildballe et al., 2009) unnecessary. Linearity continued at low B12-concentrations (24-193 pM; r(2)>0.985). Milk B12 concentrations from Bangladeshi women (72-959 pM) were significantly lower than those from California (154-933 pM; p<0.0001) showing IMMULITEs robustness against the complex milk matrix and its ability to measure low milk B12 concentrations.

Regional, Socioeconomic, and Dietary Risk Factors for Vitamin B-12 Deficiency Differ from Those for Folate Deficiency in Cameroonian Women and Children

BACKGROUND Representative data on folate and vitamin B-12 dietary intake and status in low-income countries are rare, despite the widespread adoption of folic acid fortification. OBJECTIVE The purpose of this study was to evaluate folate and vitamin B-12 intake, status, and risk factors for deficiency before implementation of a national fortification program in Cameroon. METHODS A nationally representative cross-sectional cluster survey was conducted in 3 ecologic zones of Cameroon (South, North, and the 2 largest cities, Yaoundé/Douala), and information on dietary intake was collected from 10 households in each of 30 randomly selected clusters per zone. In a subset of women and their 12- to 59-mo-old children (n = 396 pairs), plasma folate and vitamin B-12, as well as breast milk vitamin B-12, were analyzed. RESULTS Vitamin B-12 and folate dietary intake patterns and plasma concentrations were similar for women and children. In the subsample, 18% and 29% of women and 8% and 30% of children were vitamin B-12 (≤ 221 pmol/L) and folate (< 10 nmol/L) deficient, respectively. Mean dietary folate ranged from 351 μg dietary folate equivalents/d in the North to 246 μg dietary folate equivalents/d in Yaoundé/Douala; plasma folate was negatively associated with socioeconomic status (P = 0.001). Plasma vitamin B-12 deficiency was similar in the South and North, 29% and 40%, respectively, but was only 11% in Yaoundé/Douala, and was positively associated with socioeconomic status. Mean breast milk vitamin B-12 was statistically significantly lower in the North (101 pmol/L) than in the South (296 pmol/L) or Yaoundé/Douala (349 pmol/L). CONCLUSIONS Folate intake and status are inadequate among women and young children in Yaoundé/Douala, whereas low vitamin B-12 intake and status are more common in poor and rural areas, especially in the North. Different strategies may be needed to control deficiency of these nutrients in different regions of Cameroon.

Analyzing B-vitamins in Human Milk: Methodological Approaches

According to the World Health Organization (WHO), infants should be exclusively breastfed for the first six months of life. However, there is insufficient information about the concentration of nutrients in human milk. For some nutrients, including B-vitamins, maternal intake affects their concentration in human milk but the extent to which inadequate maternal diets affect milk B-vitamin content is poorly documented. Little is known about infant requirements for B-vitamins; recommendations are generally set as Adequate Intakes (AI) calculated on the basis of the mean volume of milk (0.78 L/day) consumed by infants exclusively fed with human milk from well-nourished mothers during the first six months, and the concentration of each vitamin in milk based on reported values. Methods used for analyzing B-vitamins, commonly microbiological, radioisotope dilution or more recently chromatographic, coupled with UV, fluorometric and MS detection, have rarely been validated for the complex human milk matrix. Thus the validity, accuracy, and sensitivity of analytical methods is important for understanding infant requirements for these nutrients, the maternal intakes needed to support adequate concentrations in breast milk. This review summarizes current knowledge on methods used for analyzing the B-vitamins thiamin, riboflavin, niacin, vitamin B-6 and pantothenic acid, vitamin B-12, folate, biotin, and choline in human milk, their chemical and physical properties, the different forms and changes in concentration during lactation, and the effects of deficiency on the infant.

Antiretroviral therapy provided to HIV-infected Malawian women in a randomized trial diminishes the positive effects of lipid-based nutrient supplements on breast-milk B vitamins

BACKGROUND Little information is available on B vitamin concentrations in human milk or on how they are affected by maternal B vitamin deficiencies, antiretroviral therapy, or maternal supplementation. OBJECTIVE The objective was to evaluate the effects of antiretroviral therapy and/or lipid-based nutrient supplements (LNSs) on B vitamin concentrations in breast milk from HIV-infected women in Malawi. DESIGN Breast milk was collected from 537 women recruited within the Breastfeeding, Antiretrovirals, and Nutrition study at 2 or 6 wk and 24 wk postpartum. Women were assigned to receive antiretrovirals and LNSs, antiretrovirals only, LNSs only, or a control. Antiretrovirals and LNSs were given to the mothers from weeks 0 to 28. The antiretrovirals were zidovudine/lamivudine and nelfinavir or lopinavir/ritonavir. LNSs provided 93-118% of the Recommended Dietary Allowances of thiamin, riboflavin, niacin, pyridoxine, and vitamin B-12. Infants were exclusively breastfed. RESULTS LNSs increased milk concentrations of all vitamins except thiamin, whereas antiretrovirals lowered concentrations of nicotinamide, pyridoxal, and vitamin B-12. Although antiretrovirals alone had no significant effect on riboflavin concentrations, they negatively affected the LNS-induced increase in this vitamin. Thiamin was not influenced by the study interventions. Concentrations of all B vitamins were much lower than usually accepted values. CONCLUSIONS All B vitamins were low in milk, and all but thiamin were increased by maternal supplementation with LNSs. Antiretrovirals alone decreased concentrations of some B vitamins in milk. When LNS was given in addition to antiretrovirals, the negative effect of antiretrovirals offset the positive effect of LNSs for all vitamins except thiamin. This trial was registered at clinicaltrials.gov as NCT00164762.

Taxol biosynthesis: Identification and characterization of two acetyl CoA: taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5alpha,13alpha-diacetoxy-9alpha,10beta-diol to generate taxadien-5alpha,10beta,13alpha-tri-acetoxy-9alpha-ol and taxadien-5alpha,9alpha,13alpha-triacetoxy-10beta-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5alpha- or the 13alpha-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.

Perinatal Consumption of Thiamine-Fortified Fish Sauce in Rural Cambodia: A Randomized Clinical Trial

Importance Infantile beriberi, a potentially fatal disease caused by thiamine deficiency, remains a public health concern in Cambodia and regions where thiamine-poor white rice is a staple food. Low maternal thiamine intake reduces breast milk thiamine concentrations, placing breastfed infants at risk of beriberi. Objective To determine if consumption of thiamine-fortified fish sauce yields higher erythrocyte thiamine diphosphate concentrations (eTDP) among lactating women and newborn infants and higher breast milk thiamine concentrations compared with a control sauce. Design, Setting, and Participants In this double-blind randomized clinical trial, 90 pregnant women were recruited in the Prey Veng province, Cambodia. The study took place between October 2014 and April 2015. Interventions Women were randomized to 1 of 3 groups (n = 30) for ad libitum fish sauce consumption for 6 months: control (no thiamine), low-concentration (2 g/L), or high-concentration (8 g/L) fish sauce. Main Outcomes and Measures Maternal eTDP was assessed at baseline (October 2014) and endline (April 2015). Secondary outcomes, breast milk thiamine concentration and infant eTDP, were measured at endline. Results Womens mean (SD) age and gestational stage were 26 (5) years and 23 (7) weeks, respectively. April 2015 eTDP was measured among 28 women (93%), 29 women (97%), and 23 women (77%) in the control, low-concentration, and high-concentration groups, respectively. In modified intent-to-treat analysis, mean baseline-adjusted endline eTDP was higher among women in the low-concentration (282nM; 95% CI, 235nM to 310nM) and high-concentration (254nM; 95% CI, 225nM to 284nM) groups compared with the control group (193nM; 95% CI, 164nM to 222M; P < .05); low-concentration and high-concentration groups did not differ (P = .19). Breast milk total thiamine concentrations were 14.4 μg/dL for the control group (95% CI, 12.3 μg/dL to 16.5 μg/dL) (to convert to nanomoles per liter, multiply by 29.6); 20.7 μg/dL for the low-concentration group (95% CI, 18.6 μg/dL to 22.7 μg/dL ); and 17.7 μg/dL for the high-concentration group (95% CI, 15.6 μg/dL to 19.9 μg/dL). Mean (SD) infant age at endline was 16 (8) weeks for the control group, 17 (7) weeks for the low-concentration group, and 14 (8) for the high-concentration group. Infant eTDP was higher among those in the high-concentration group (257nM; 95% CI, 222nM to 291nM; P < .05) compared with the low-concentration (212nM; 95% CI, 181nM to 244nM) and control (187nM; 95% CI, 155nM to 218nM) groups. Conclusions and Relevance Compared with women in the control group, women who consumed thiamine-fortified fish sauce through pregnancy and early lactation had higher eTDP and breast milk thiamine concentrations and their infants had higher eTDP, which was more pronounced in the high group. Thiamine-fortified fish sauce has the potential to prevent infantile beriberi in this population. Trial Registration Clinicaltrials.gov Identifier: NCT02221063.

Vitamin B-12 concentrations in breast milk are low and are not associated with reported household hunger, recent animal-source food, or Vitamin B-12 intake in women in rural Kenya

BACKGROUND Breast milk vitamin B-12 concentration may be inadequate in regions in which animal-source food consumption is low or infrequent. Vitamin B-12 deficiency causes megaloblastic anemia and impairs growth and development in children. OBJECTIVE We measured vitamin B-12 in breast milk and examined its associations with household hunger, recent animal-source food consumption, and vitamin B-12 intake. METHODS In a cross-sectional substudy nested within a cluster-randomized trial assessing water, sanitation, hygiene, and nutrition interventions in Kenya, we sampled 286 women 1-6 mo postpartum. Mothers hand-expressed breast milk 1 min into a feeding after 90 min observed nonbreastfeeding. The Household Hunger Scale was used to measure hunger, food intake in the previous week was measured with the use of a food-frequency questionnaire (FFQ), and vitamin B-12 intake was estimated by using 24-h dietary recall. An animal-source food score was based on 10 items from the FFQ (range: 0-70). Breast milk vitamin B-12 concentration was measured with the use of a solid-phase competitive chemiluminescent enzyme immunoassay and was modeled with linear regression. Generalized estimating equations were used to account for correlated observations at the cluster level. RESULTS Median (IQR) vitamin B-12 intake was 1.5 μg/d (0.3, 9.7 μg/d), and 60% of women consumed <2.4 μg/d, the estimated average requirement during lactation. Median (IQR) breast milk vitamin B-12 concentration was 113 pmol/L (61, 199 pmol/L); 89% had concentrations <310 pmol/L, the estimated adequate concentration. Moderate or severe hunger prevalence was 27%; the animal-source food score ranged from 0 to 30 item-d/wk. Hunger and recent animal-source food and vitamin B-12 intake were not associated with breast milk vitamin B-12 concentrations. Maternal age was negatively associated with breast milk vitamin B-12 concentrations. CONCLUSION Most lactating Kenyan women consumed less than the estimated average requirement of vitamin B-12 and had low breast milk vitamin B-12 concentrations. We recommend interventions that improve vitamin B-12 intake in lactating Kenyan women to foster maternal health and child development. The main trial was registered at clinicaltrials.gov as NCT01704105.

Vitamin Concentrations in Human Milk Vary with Time within Feed, Circadian Rhythm, and Single-Dose Supplementation

Background: Human milk is the subject of many studies, but procedures for representative sample collection have not been established. Our improved methods for milk micronutrient analysis now enable systematic study of factors that affect its concentrations. Objective: We evaluated the effects of sample collection protocols, variations in circadian rhythms, subject variability, and acute maternal micronutrient supplementation on milk vitamin concentrations. Methods: In the BMQ (Breast-Milk-Quality) study, we recruited 18 healthy women (aged 18–26 y) in Dhaka, Bangladesh, at 2–4 mo of lactation for a 3-d supplementation study. On day 1, no supplements were given; on days 2 and 3, participants consumed ∼1 time and 2 times, respectively, the US-Canadian Recommended Dietary Allowances for vitamins at breakfast (0800–0859). Milk was collected during every feeding from the same breast over 24 h. Milk expressed in the first 2 min (aliquot I) was collected separately from the remainder (aliquot II); a third aliquot (aliquot III) was saved by combining aliquots I and II. Thiamin, riboflavin, niacin, and vitamins B-6, B-12, A, and E and fat were measured in each sample. Results: Significant but small differences (14–18%) between aliquots were found for all vitamins except for vitamins B-6 and B-12. Circadian variance was significant except for fat-adjusted vitamins A and E, with a higher contribution to total variance with supplementation. Between-subject variability accounted for most of the total variance. Afternoon and evening samples best reflected daily vitamin concentrations for all study days. Acute supplementation effects were found for thiamin, riboflavin, and vitamins B-6 and A at 2–4 h postdosing, with 0.1–6.17% passing into milk. Supplementation was reflected in fasting, 24-h postdose samples for riboflavin and vitamin B-6. Maximum amounts of dose-responding vitamins in 1 feeding ranged from 4.7% to 21.8% (day 2) and 8.2% to 35.0% (day 3) of Adequate Intake. Conclusions: In the milk of Bangladeshi mothers, differences in vitamin concentrations between aliquots within feedings and by circadian variance were significant but small. Afternoon and evening collection provided the most-representative samples. Supplementation acutely affects some breast-milk micronutrient concentrations. This trial was registered at clinicaltrials.gov as NCT02756026.

Thiamin and Riboflavin in Human Milk: Effects of Lipid-Based Nutrient Supplementation and Stage of Lactation on Vitamer Secretion and Contributions to Total Vitamin Content

While thiamin and riboflavin in breast milk have been analyzed for over 50 years, less attention has been given to the different forms of each vitamin. Thiamin-monophosphate (TMP) and free thiamin contribute to total thiamin content; flavin adenine-dinucleotide (FAD) and free riboflavin are the main contributors to total riboflavin. We analyzed milk collected at 2 (n = 258) or 6 (n = 104), and 24 weeks (n = 362) from HIV-infected Malawian mothers within the Breastfeeding, Antiretrovirals and Nutrition (BAN) study, randomly assigned at delivery to lipid-based nutrient supplements (LNS) or a control group, to investigate each vitamer’s contribution to total milk vitamin content and the effects of supplementation on the different thiamin and riboflavin vitamers at early and later stages of lactation, and obtain insight into the transport and distribution of these vitamers in human milk. Thiamin vitamers were derivatized into thiochrome-esters and analyzed by high-performance liquid-chromatography-fluorescence-detection (HPLC-FLD). Riboflavin and FAD were analyzed by ultra-performance liquid-chromatography-tandem-mass-spectrometry (ULPC-MS/MS). Thiamin-pyrophosphate (TPP), identified here for the first time in breast milk, contributed 1.9–4.5% to total thiamin. Free thiamin increased significantly from 2/6 to 24 weeks regardless of treatment indicating an active transport of this vitamer in milk. LNS significantly increased TMP and free thiamin only at 2 weeks compared to the control: median 170 versus 151μg/L (TMP), 13.3 versus 10.5μg/L (free thiamin, p<0.05 for both, suggesting an up-regulated active mechanism for TMP and free thiamin accumulation at early stages of lactation. Free riboflavin was consistently and significantly increased with LNS (range: 14.8–19.6μg/L (LNS) versus 5.0–7.4μg/L (control), p<0.001), shifting FAD:riboflavin relative amounts from 92–94:6–8% to 85:15%, indicating a preferred secretion of the free form into breast milk. The continuous presence of FAD in breast milk suggests an active transport and secretion system for this vitamer or possibly formation of this co-enymatic form in the mammary gland.