Changes in Choline Metabolites and Ceramides in Response to a DASH-Style Diet in Older Adults.

This feeding trial evaluated the impact of the Dietary Approaches to Stop Hypertension diet on changes in plasma choline, choline metabolites, and ceramides in obese older adults; 28 adults consumed 3oz (n = 15) or 6oz (n = 13) of beef within a standardized DASH diet for 12 weeks. Plasma choline, betaine, methionine, dimethylglycine (DMG), phosphatidylcholine (PC), lysophosphotidylcholine (LPC), sphingomyelin, trimethylamine-N-oxide (TMAO), L-carnitine, ceramide, and triglycerides were measured in fasted blood samples. Plasma LPC, sphingomyelin, and ceramide species were also quantified. In response to the study diet, with beef intake groups combined, plasma choline decreased by 9.6% (p = 0.012); DMG decreased by 10% (p = 0.042); PC decreased by 51% (p < 0.001); total LPC increased by 281% (p < 0.001); TMAO increased by 26.5% (p < 0.001); total ceramide decreased by 22.1% (p < 0.001); and triglycerides decreased by 18% (p = 0.021). All 20 LPC species measured increased (p < 0.01) with LPC 16:0 having the greatest response. Sphingomyelin 16:0, 18:0, and 18:1 increased (all p < 0.001) by 10.4%, 22.5%, and 24%, respectively. In contrast, we observed that sphingomyelin 24:0 significantly decreased by 10%. Ceramide 22:0 and 24:0 decreased by 27.6% and 10.9% (p < 0.001), respectively, and ceramide 24:1 increased by 36.8% (p = 0.013). Changes in choline and choline metabolites were in association with anthropometric and cardiometabolic outcomes. These findings show the impact of the DASH diet on choline metabolism in older adults and demonstrate the influence of diet to modify circulating LPC, sphingomyelin, and ceramide species.

Baseline red blood cell and breast milk DHA levels affect responses to standard dose of DHA in lactating women on a controlled feeding diet.

BACKGROUND: The importance of providing the newborn infant with docosahexaenoic acid (DHA) from breast milk is well established. However, women in the United States, on average, have breast milk DHA levels of 0.20%, which is below the worldwide average (and proposed target) of >0.32%. Additionally, the relationship between maternal red blood cell (RBC) and breast milk DHA levels may provide insight into the sufficiency of DHA recommendations during lactation. Whether the standard recommendation of at least 200 mg/day of supplemental DHA during lactation is sufficient for most women to achieve a desirable RBC and breast milk DHA status is unknown. METHODS: Lactating women (n = 27) at about 5 weeks postpartum were enrolled in a 10-12 week controlled feeding study that included randomization to 480 or 930 mg choline/d (diet plus supplementation). As part of the intervention, all participants were required to consume a 200 mg/d of microalgal DHA. RBC and breast milk DHA levels were measured by capillary gas chromatography in an exploratory analysis. RESULTS: Median RBC DHA was 5.0% (95% CI: 4.3, 5.5) at baseline and 5.1% (4.6, 5.4) after 10 weeks of supplementation (P = 0.6). DHA as a percent of breast milk fatty acids increased from 0.19% (0.18, 0.33) to 0.34% (0.27, 0.38) after supplementation (P<0.05). The proportion of women meeting the target RBC DHA level of >5% was unchanged (52% at baseline and week 10). The proportion of women achieving a breast milk DHA level of >0.32% approximately doubled from 30% to 56% (p = 0.06). Baseline RBC and breast milk DHA levels affected their responses to supplementation. Those with baseline RBC and breast milk DHA levels above the median (5% and 0.19%, respectively) experienced no change or a slight decrease in levels, while those below the median had a significant increase. Choline supplementation did not significantly influence final RBC or breast milk DHA levels. CONCLUSIONS: On average, the standard prenatal DHA dose of 200 mg/d did not increase RBC DHA but did increase breastmilk DHA over 10 weeks in a cohort of lactating women in a controlled-feeding study. Baseline DHA levels in RBC and breast milk affected the response to DHA supplementation, with lower levels being associated with a greater increase and higher levels with no change or a slight decrease. Additional larger, dose-response DHA trials accounting for usual intakes and baseline DHA status are needed to determine how to best achieve target breast milk DHA levels and to identify additional modifiers of the variable breast milk DHA response to maternal DHA supplementation.

Maternal vitamin D biomarkers are associated with maternal and fetal bone turnover among pregnant women consuming controlled amounts of vitamin D, calcium, and phosphorus.

Vitamin D plays a central role in calcium homeostasis; however, its relationship with bone turnover during pregnancy remains unclear due to a lack of studies that have rigorously controlled for vitamin D and other nutrients known to influence bone metabolism. Similarly, prior investigations of the effect of pregnancy on bone turnover relative to the nonpregnant state may have been confounded by varying intakes of these nutrients. Nested within a controlled intake study, the present investigation sought to quantify associations between maternal vitamin D biomarkers and biochemical markers of bone turnover among pregnant (versus nonpregnant) women and their fetuses under conditions of equivalent and adequate intakes of vitamin D and related nutrients. Changes in markers of bone turnover across the third trimester were also examined. Healthy pregnant (26-29 wk gestation; n=26) and nonpregnant (n=21) women consumed 511IU vitamin D/d, 1.6g calcium/d, and 1.9g phosphorus/d for 10weeks while participating in a controlled feeding study featuring two choline doses. Based on linear mixed models adjusted for influential covariates (e.g., BMI, ethnicity, and season), pregnant women had 50-150% higher (P<0.001) concentrations of bone resorption markers than nonpregnant women. Among pregnant women, increases in maternal 25(OH)D across the study period were associated (P<0.020) with lower osteocalcin and deoxypyridinoline at study-end, and higher fetal osteocalcin. In addition, maternal free 25(OH)D, 1,25(OH)2D and 24,25(OH)2D tended to be negatively associated (P≤0.063) with maternal NTx at study-end, and maternal free 25(OH)D and 24,25(OH)2D were positively associated (P≤0.021) with fetal CTx. Similarly, maternal 3-epi-25(OH)D3 was negatively related (P≤0.037) to maternal NTx and deoxypyridinoline at study-end. These declines in bone resorption markers resulting from higher vitamin D biomarker concentrations among pregnant women coincided with increases in their albumin-corrected serum calcium concentrations, indicating that calcium transfer to the fetus was uncompromised. Notably, none of these associations achieved statistical significance among nonpregnant women. Overall, our study findings suggest that achieving higher maternal concentrations of vitamin D biomarkers might attenuate third-trimester bone resorption while ensuring sufficient calcium delivery to the fetus.

Vitamin D Metabolism Varies among Women in Different Reproductive States Consuming the Same Intakes of Vitamin D and Related Nutrients.

BACKGROUND: The impact of the reproductive state on vitamin D metabolism and requirements is uncertain in part because of a lack of studies with controlled dietary intakes of vitamin D and related nutrients. OBJECTIVE: We aimed to quantify the impact of the reproductive state on a panel of vitamin D biomarkers among women of childbearing age consuming equivalent amounts of vitamin D and related nutrients. METHODS: Nested within a feeding study providing 2 doses of choline, healthy pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant/nonlactating; n = 21) women consumed a single amount of vitamin D (511 ± 48 IU/d: 311 ± 48 IU/d from diet and 200 IU/d as supplemental cholecalciferol) and related nutrients (1.6 ± 0.4 g Ca/d and 1.9 ± 0.3 g P/d) for 10 wk. Vitamin D biomarkers were measured in blood obtained at baseline and study end, and differences in biomarker response among the reproductive groups were assessed with linear mixed models adjusted for influential covariates (e.g., body mass index, season, race/ethnicity). RESULTS: At study end, pregnant women had higher (P < 0.01) circulating concentrations of 25-hydroxyvitamin D [25(OH)D; 30%], 1,25-dihydroxyvitamin D [1,25(OH)2D; 80%], vitamin D binding protein (67%), and C3 epimer of 25(OH)D3 (100%) than control women. Pregnant women also had higher (P ≤ 0.04) ratios of 25(OH)D to 24,25-dihydroxyvitamin D [24,25(OH)2D; 40%] and 1,25(OH)2D to 25(OH)D (50%) than control women. In contrast, no differences (P ≥ 0.15) in vitamin D biomarkers were detected between the lactating and control groups. Notably, the study vitamin D dose of 511 IU/d achieved vitamin D adequacy in most participants (95%) regardless of their reproductive state. CONCLUSIONS: The higher concentrations of vitamin D biomarkers among pregnant women than among control women suggest that metabolic adaptations, likely involving the placenta, transpire to enhance vitamin D supply during pregnancy. The study findings also support the adequacy of the current vitamin D RDA of 600 IU for achieving serum 25(OH)D concentrations ≥50 nmol/L among women differing in their reproductive state. This trial was registered at clinicaltrials.gov as NCT01127022.

Choline intakes exceeding recommendations during human lactation improve breast milk choline content by increasing PEMT pathway metabolites.

Demand for the vital nutrient choline is high during lactation; however, few studies have examined choline metabolism and requirements in this reproductive state. The present study sought to discern the effects of lactation and varied choline intake on maternal biomarkers of choline metabolism and breast milk choline content. Lactating (n=28) and control (n=21) women were randomized to 480 or 930 mg choline/day for 10-12 weeks as part of a controlled feeding study. During the last 4-6 weeks, 20% of the total choline intake was provided as an isotopically labeled choline tracer (methyl-d9-choline). Blood, urine and breast milk samples were collected for choline metabolite quantification, enrichment measurements, and gene expression analysis of choline metabolic genes. Lactating (vs. control) women exhibited higher (P < .001) plasma choline concentrations but lower (P ≤ .002) urinary excretion of choline metabolites, decreased use of choline as a methyl donor (e.g., lower enrichment of d6-dimethylglycine, P ≤ .08) and lower (P ≤ .02) leukocyte expression of most choline-metabolizing genes. A higher choline intake during lactation differentially influenced breast milk d9- vs. d3-choline metabolite enrichment. Increases (P ≤ .03) were detected among the d3-metabolites, which are generated endogenously via the hepatic phosphatidylethanolamine N-methyltransferase (PEMT), but not among the d9-metabolites generated from intact exogenous choline. These data suggest that lactation induces metabolic adaptations that increase the supply of intact choline to the mammary epithelium, and that extra maternal choline enhances breast milk choline content by increasing supply of PEMT-derived choline metabolites. This trial was registered at clinicaltrials.gov as NCT01127022.

Vitamin B-12 Status Differs among Pregnant, Lactating, and Control Women with Equivalent Nutrient Intakes.

BACKGROUND: Limited data are available from controlled studies on biomarkers of maternal vitamin B-12 status. OBJECTIVE: We sought to quantify the effects of pregnancy and lactation on the vitamin B-12 status response to a known and highly controlled vitamin B-12 intake. METHODS: As part of a 10-12 wk feeding trial, pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant, nonlactating; n = 21) women consumed vitamin B-12 amounts of ∼8.6 µg/d [mixed diet (∼6 µg/d) plus a prenatal multivitamin supplement (2.6 µg/d)]. Serum vitamin B-12, holotranscobalamin (bioactive form of vitamin B-12), methylmalonic acid (MMA), and homocysteine were measured at baseline and study-end. RESULTS: All participants achieved adequate vitamin B-12 status in response to the study dose. Compared with control women, pregnant women had lower serum vitamin B-12 (-21%; P = 0.02) at study-end, whereas lactating women had higher (P = 0.04) serum vitamin B-12 throughout the study (+26% at study-end). Consumption of the study vitamin B-12 dose increased serum holotranscobalamin in all reproductive groups (+16-42%; P ≤ 0.009). At study-end, pregnant (vs. control) women had a higher holotranscobalamin-to-vitamin B-12 ratio (P = 0.04) with ∼30% (vs. 20%) of total vitamin B-12 in the bioactive form. Serum MMA increased during pregnancy (+50%; P < 0.001) but did not differ by reproductive state at study-end. Serum homocysteine increased in pregnant women (+15%; P = 0.009) but decreased in control and lactating women (-16-17%; P < 0.001). Despite these changes, pregnant women had ∼20% lower serum homocysteine than the other 2 groups at study-end (P ≤ 0.02). CONCLUSION: Pregnancy and lactation alter vitamin B-12 status in a manner consistent with enhanced vitamin B-12 supply to the child. Consumption of the study vitamin B-12 dose (∼3 times the RDA) increased the bioactive form of vitamin B-12, suggesting that women in these reproductive states may benefit from vitamin B-12 intakes exceeding current recommendations. This trial was registered at clinicaltrials.gov as NCT01127022.

Pregnancy and lactation alter biomarkers of biotin metabolism in women consuming a controlled diet.

BACKGROUND: Biotin functions as a cofactor for several carboxylase enzymes with key roles in metabolism. At present, the dietary requirement for biotin is unknown and intake recommendations are provided as Adequate Intakes (AIs). The biotin AI for adults and pregnant women is 30 µg/d, whereas 35 µg/d is recommended for lactating women. However, pregnant and lactating women may require more biotin to meet the demands of these reproductive states. OBJECTIVE: The current study sought to quantify the impact of reproductive state on biotin status response to a known dietary intake of biotin. METHODS: To achieve this aim, we measured a panel of biotin biomarkers among pregnant (gestational week 27 at study entry; n = 26), lactating (postnatal week 5 at study entry; n = 28), and control (n = 21) women who participated in a 10- to 12-wk feeding study providing 57 µg of dietary biotin/d as part of a mixed diet. RESULTS: Over the course of the study, pregnant women excreted 69% more (vs. control; P < 0.001) 3-hydroxyisovaleric acid (3-HIA), a metabolite that accumulates during the catabolism of leucine when the activity of biotin-dependent methylcrotonyl-coenzyme A carboxylase is impaired. Interestingly, urinary excretion of 3-hydroxyisovaleryl-carnitine (3-HIA-carnitine), a downstream metabolite of 3-HIA, was 27% lower (P = 0.05) among pregnant (vs. control) women, a finding that may arise from carnitine inadequacy during gestation. No differences (P > 0.05) were detected in plasma biotin, urinary biotin, or urinary bisnorbiotin between pregnant and control women. Lactating women excreted 76% more (vs. control; P = 0.001) of the biotin catabolite bisnorbiotin, indicating that lactation accelerates biotin turnover and loss. Notably, with respect to control women, lactating women excreted 23% less (P = 0.04) urinary 3-HIA and 26% less (P = 0.05) urinary 3-HIA-carnitine, suggesting that lactation reduces leucine catabolism and that these metabolites may not be useful indicators of biotin status during lactation. CONCLUSIONS: Overall, these data demonstrate significant alterations in markers of biotin metabolism during pregnancy and lactation and suggest that biotin intakes exceeding current recommendations are needed to meet the demands of these reproductive states. This trial was registered at clinicaltrials.gov as NCT01127022.

Pregnancy alters choline dynamics: results of a randomized trial using stable isotope methodology in pregnant and nonpregnant women.

BACKGROUND: Although biomarkers of choline metabolism are altered by pregnancy, little is known about the influence of human pregnancy on the dynamics of choline-related metabolic processes. OBJECTIVE: This study used stable isotope methodology to examine the effects of pregnancy on choline partitioning and the metabolic activity of choline-related pathways. DESIGN: Healthy third-trimester pregnant (n = 26; initially week 27 of gestation) and nonpregnant (n = 21) women consumed 22% of their total choline intake (480 or 930 mg/d) as methyl-d9-choline for the final 6 wk of a 12-wk feeding study. RESULTS: Plasma d9-betaine:d9-phosphatidylcholine (PC) was lower (P ≤ 0.04) in pregnant than in nonpregnant women, suggesting greater partitioning of choline into the cytidine diphosphate-choline (CDP-choline) PC biosynthetic pathway relative to betaine synthesis during pregnancy. Pregnant women also used more choline-derived methyl groups for PC synthesis via phosphatidylethanolamine N-methyltransferase (PEMT) as indicated by comparable increases in PEMT-PC enrichment in pregnant and nonpregnant women despite unequal (pregnant > nonpregnant; P < 0.001) PC pool sizes. Pregnancy enhanced the hydrolysis of PEMT-PC to free choline as shown by greater (P < 0.001) plasma d3-choline:d3-PC. Notably, d3-PC enrichment increased (P ≤ 0.011) incrementally from maternal to placental to fetal compartments, signifying the selective transfer of PEMT-PC to the fetus. CONCLUSIONS: The enhanced use of choline for PC production via both the CDP-choline and PEMT pathways shows the substantial demand for choline during late pregnancy. Selective partitioning of PEMT-PC to the fetal compartment may imply a unique requirement of PEMT-PC by the developing fetus.

Choline intake influences phosphatidylcholine DHA enrichment in nonpregnant women but not in pregnant women in the third trimester.

BACKGROUND: Phosphatidylcholine (PC) produced via the S-adenosylmethionine-dependent phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is enriched with docosahexaenoic acid (DHA). DHA plays a critical role in fetal development and is linked to health endpoints in adulthood. It is unknown whether choline, which can serve as a source of S-adenosylmethionine methyl groups, influences PC-DHA or the PC:PE ratio in pregnant and nonpregnant women. OBJECTIVE: This study tested whether choline intake affects indicators of choline-related lipid metabolism, including erythrocyte and plasma PC-DHA and PC:PE ratios, in pregnant women in the third trimester and nonpregnant women. DESIGN: Pregnant (n = 26) and nonpregnant (n = 21) women consumed 480 or 930 mg choline/d and a daily DHA supplement for 12 wk. Blood was collected at baseline and at the midpoint and end of the study. PC-DHA was analyzed as the proportion of total PC fatty acids. RESULTS: Pregnant women had greater (P = 0.002) PC-DHA concentrations than did nonpregnant women at baseline. The proportion of erythrocyte and plasma PC-DHA increased (P ≤ 0.002) in pregnant and nonpregnant women regardless of choline intake. However, in nonpregnant women, consumption of 930 mg choline/d led to greater (P < 0.001) erythrocyte PC-DHA and a more rapid increase (P < 0.001) in plasma PC-DHA. Lower (P = 0.001-0.024) erythrocyte and plasma PC:PE in pregnant women was not modified by choline intake. CONCLUSIONS: A higher choline intake may increase PEMT activity, resulting in greater PC-DHA enrichment of the PC molecule in nonpregnant women. Increased production of PC-DHA during pregnancy indicates elevated PEMT activity and a higher demand for methyl donors. This trial was registered at clinicaltrials.gov as NCT01127022.

A higher maternal choline intake among third-trimester pregnant women lowers placental and circulating concentrations of the antiangiogenic factor fms-like tyrosine kinase-1 (sFLT1).

This study investigated the influence of maternal choline intake on the human placental transcriptome, with a special interest in its role in modulating placental vascular function. Healthy pregnant women (n=26, wk 26-29 gestation) were randomized to 480 mg choline/d, an intake level approximating the adequate intake of 450 mg/d, or 930 mg/d for 12 wk. Maternal blood and placental samples were retrieved at delivery. Whole genome expression microarrays were used to identify placental genes and biological processes impacted by maternal choline intake. Maternal choline intake influenced a wide array of genes (n=166) and biological processes (n=197), including those related to vascular function. Of special interest was the 30% down-regulation (P=0.05) of the antiangiogenic factor and preeclampsia risk marker fms-like tyrosine kinase-1 (sFLT1) in the placenta tissues obtained from the 930 vs. 480 mg/d choline intake group. Similar decreases (P=0.04) were detected in maternal blood sFLT1 protein concentrations. The down-regulation of sFLT1 by choline treatment was confirmed in a human trophoblast cell culture model and may be related to enhanced acetylcholine signaling. These findings indicate that supplementing the maternal diet with extra choline may improve placental angiogenesis and mitigate some of the pathological antecedents of preeclampsia.

Folate-status response to a controlled folate intake in nonpregnant, pregnant, and lactating women.

BACKGROUND: Folate dose-response studies in women of childbearing age who consumed a folic acid (FA)-containing multivitamin in the era of FA fortification are lacking. OBJECTIVE: We sought to investigate folate-status response to a known folate dose comprising an FA-containing prenatal supplement (750 µg/d) plus natural food folate (400 µg/d) in third-trimester pregnant women, lactating women 5-15 wk postpartum, and nonpregnant women. DESIGN: Pregnant (n = 26), lactating (n = 28), and nonpregnant (n = 21) women consumed the study folate dose under controlled intake conditions for 10-12 wk. Blood, urine, and breast milk were collected at baseline, study midpoint, and study end. RESULTS: Study-end serum total folate concentrations averaged ~30 ng/mL and did not differ by physiologic group (P = 0.876). Study-end urinary folate excretion represented ~9-43% of total folate intake and ranged from 100 to 500 µg/d. Third-trimester pregnant women excreted less urinary folate than did lactating (P = 0.075) and nonpregnant (P < 0.001) women. Lactating women excreted less (P < 0.001) urinary FA than did nonpregnant women. Breast-milk total folate concentrations remained constant (P = 0.244; 61.8 ng/mL at study end), whereas breast-milk FA concentrations increased (P = 0.003) to 24.1 ng/mL at study end. CONCLUSIONS: The consumption of the study folate dose yielded a supranutritional folate status regardless of the physiologic state. Based on urinary folate excretion, folate use was greatest to least: pregnant > lactating > nonpregnant women. Breast-milk folate species were responsive to maternal folate intake, and FA made up ~40% of breast-milk total folate at study end. These findings warrant revisiting prenatal supplement FA formulation in populations exposed to FA-fortification programs.

Predisposition to late-onset obesity in GIRK4 knockout mice.

G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels mediate the inhibitory effects of many neurotransmitters on excitable cells. Four Girk genes have been identified (Girk1-4). Whereas GIRK4 is associated with the cardiac GIRK channel, Girk4 expression has been detected in a few neuron populations. Here, we used a transgenic mouse expressing enhanced green fluorescent protein (EGFP) under the control of the Girk4 gene promoter to clarify the expression pattern of Girk4 in the brain. Although small subsets of EGFP-positive neurons were evident in some areas, prominent labeling was seen in the hypothalamus. EGFP expression was most pronounced in the ventromedial, paraventricular, and arcuate nuclei, neuron populations implicated in energy homeostasis. Consistent with a contribution of GIRK4-containing channels to energy balance, Girk4 knockout -/- mice were predisposed to late-onset obesity. By 9 months, Girk4-/- mice were approximately 25% heavier than wild-type controls, a difference attributed to greater body fat. Before the development of overweight, Girk4-/- mice exhibited a tendency toward greater food intake and an increased propensity to work for food in an operant task. Girk4-/- mice also exhibited reduced net energy expenditure, despite displaying elevated resting heart rates and core body temperatures. These data implicate GIRK4-containing channels in signaling crucial to energy homeostasis and body weight.