Dietary Choline Intake: Current State of Knowledge Across the Life Cycle.

Choline, an essential dietary nutrient for humans, is required for the synthesis of the neurotransmitter, acetylcholine, the methyl group donor, betaine, and phospholipids; and therefore, choline is involved in a broad range of critical physiological functions across all stages of the life cycle. The current dietary recommendations for choline have been established as Adequate Intakes (AIs) for total choline; however, dietary choline is present in multiple different forms that are both water-soluble (e.g., free choline, phosphocholine, and glycerophosphocholine) and lipid-soluble (e.g., phosphatidylcholine and sphingomyelin). Interestingly, the different dietary choline forms consumed during infancy differ from those in adulthood. This can be explained by the primary food source, where the majority of choline present in human milk is in the water-soluble form, versus lipid-soluble forms for foods consumed later on. This review summarizes the current knowledge on dietary recommendations and assessment methods, and dietary choline intake from food sources across the life cycle.

Variations in plasma choline and metabolite concentrations in healthy adults.

BACKGROUND: Plasma concentrations of choline and its metabolites might serve as biomarkers for the health outcomes of several pathological states such as cardiovascular disease and cancer. However, information about the reliability of biomarkers of choline status is limited. We investigated biological variations in repeated measures of choline and metabolites in healthy adults to assess them as biomarkers. METHODS: Blood samples were collected after an overnight fast at three-time points 12 days apart from 40 adults (mean age, 33 y; male, n = 21). A subset (n = 19; [male, n = 8]) provided one additional sample after a breakfast meal. Plasma free choline, betaine and dimethylglycine were measured using liquid chromatography-tandem mass spectrometry, and plasma phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were measured using high-performance liquid chromatography. RESULTS: The biological variations observed for choline and metabolites were ≤ 13% for adult fasting samples. This corresponded to intra-class correlations (ICC) that ranged from 0.593 to 0.770 for fasting values for choline and metabolites. A similar ICC range was also obtained between fasting and post-prandial states. Although most post-prandial concentrations of choline and metabolites were significantly higher (P < .05) than fasting, all fell within a calculated reference interval. The participants were correctly classified in tertiles for fasting and post-prandial states for choline (68%) and metabolites (range = 32% phosphatidylcholine and 79% for sphingomyelin). CONCLUSIONS: These findings indicate that biological variations of choline and metabolites are low in healthy adults and values from a single blood sample can be used as a biomarker. However, choosing phosphatidylcholine as a biomarker is less reliable.

Concentrations of Water-Soluble Forms of Choline in Human Milk from Lactating Women in Canada and Cambodia.

Choline has critical roles during periods of rapid growth and development, such as infancy. In human milk, choline is mostly present in water-soluble forms (free choline, phosphocholine, and glycerophosphocholine). It is thought that milk choline concentration is influenced by maternal choline intake, and the richest food sources for choline are of animal origin. Scarce information exists on milk choline from countries differing in animal-source food availability. In this secondary analysis of samples from previous trials, the concentrations of the water-soluble forms of choline were quantified by liquid chromatography-tandem mass spectrometry in mature milk samples collected from lactating women in Canada (n = 301) and in Cambodia (n = 67). None of the water-soluble forms of choline concentrations in milk differed between Canada and Cambodia. For all milk samples (n = 368), free choline, phosphocholine, glycerophosphocholine, and the sum of water-soluble forms of choline concentrations in milk were (mean (95%CI)) 151 (141, 160, 540 (519, 562), 411 (396, 427), and 1102 (1072, 1133) µmol/L, respectively. Theoretically, only 19% of infants would meet the current Adequate Intake (AI) for choline. Our findings suggest that the concentrations in milk of water-soluble forms of choline are similar in Canada and Cambodia, and that the concentration used to set the infant AI might be inaccurate.

Effect of current emission abatement strategies on air quality improvement in China: A case study of Baotou, a typical industrial city in Inner Mongolia.

The national Air Pollution Prevention and Control Action Plan required significant decreases in PM2.5 levels over China. To explore more effective emission abatement strategies in industrial cities, a case study was conducted in Baotou to evaluate the current national control measures. The total emissions of SO2, NOX, PM2.5 and NMVOC (non-methane volatile organic compounds) in Baotou were 211.2Gg, 156.1Gg, 28.8Gg, and 48.5Gg, respectively in 2013, and they would experience a reduction of 30.4%, 26.6%, 15.1%, and 8.7%, respectively in 2017 and 39.0%, 32.0%, 24.4%, and 12.9%, respectively in 2020. The SO2, NOX and PM2.5 emissions from the industrial sector would experience a greater decrease, with reductions of 37%, 32.7 and 24.3%, respectively. From 2013 to 2020, the concentrations of SO2, NO2, and PM2.5 are expected to decline by approximately 30%, 10% and 14.5%, respectively. The reduction rate of SNA (sulfate, nitrate and ammonium) concentrations was significantly higher than that of PM2.5 in 2017, implying that the current key strategy toward controlling air pollutants from the industrial sector is more powerful for SNA. Although air pollution control measures implemented in the industrial sector could greatly reduce total emissions, constraining the emissions from lower sources such as residential coal combustion would be more effective in decreasing the concentration of PM2.5 from 2017 to 2020. These results suggest that even for a typical industrial city, the reduction of PM2.5 concentrations not only requires decreases in emissions from the industrial sector, but also from the low emission sources. The seasonal variation in sulfate concentration also showed that emission from coal-burning is the key factor to control during the heating season.

Negative Regulation of Human Pregnane X Receptor by MicroRNA-18a-5p: Evidence for Suppression of MicroRNA-18a-5p Expression by Rifampin and Rilpivirine.

Small noncoding microRNAs act as post-transcriptional regulators of gene expression involved in diverse biologic functions. Pregnane X receptor (PXR, NR1I2), a member of the superfamily of nuclear receptors, is a transcription factor governing the transport and biotransformation of various drugs and other chemicals. In the present study, we identified a specific microRNA (miR) involved in regulating the expression and functionality of human PXR (hPXR). According to bioinformatics analysis employing three commonly used algorithms (TargetScan, miRanda, and DIANA-microT-CDS), miR-18a-5p was predicted to be the top candidate microRNA regulator of hPXR. Consequently, this microRNA was selected for detailed experimental investigation. As shown in cell-based dual-luciferase reporter gene assays, functional interaction occurred between miR-18a-5p and the microRNA recognition element of miR-18a-5p in the 3'-untranslated region of hPXR mRNA. Transfection of LS180 human colorectal adenocarcinoma cells with an miR-18a-5p mimic decreased hPXR mRNA and protein expression, whereas transfection of LS180 cells with an miR-18a-5p inhibitor increased hPXR mRNA and protein expression. The decrease in hPXR expression by the miR-18a-5p mimic was associated with a reduction in the extent of hPXR target gene (CYP3A4) induction by rifampin and rilpivirine. Treatment of untransfected LS180 cells with either of these hPXR agonists decreased endogenous expression of miR-18a-5p, and this preceded the onset of CYP3A4 induction. In conclusion, miR-18a-5p is a negative regulator of hPXR expression and the hPXR agonists rifampin and rilpivirine are chemical suppressors of miR-18a-5p expression.

Zinc inhibits magnesium-dependent migration of human breast cancer MDA-MB-231 cells on fibronectin.

Metastasis is the major cause of breast cancer mortality. The strength of cell adhesion to extracellular matrix is critical to cancer cell migration. Integrins, the primary mediators of cell to extra-cellular matrix adhesion, contain distinct divalent cation-binding sites. Binding of manganese and magnesium is vital to integrin-mediated cancer cell adhesion and migration. We hypothesized that zinc, a divalent cation, can modulate breast cancer metastasis through interfering with these divalent cation-dependent integrin-mediated cancer cell adhesion and migration. MDA-MB-231 cells were cultured in a zinc-depleted medium supplemented with 0 (control), 2.5, 5, 10, 25 and 50 µM of zinc to mimic severe zinc-deficiency, moderate zinc-deficiency, adequate zinc and three levels of zinc-supplementation: low-, moderate- and high-levels of zinc-supplementation, respectively. Zinc treatments had no effect on cellular zinc concentration, cell number and cell viability. Zinc at 5-50 µM reduced migration distance of MDA-MB-231 cells on fibronectin by 43-86% and migration rate on fibronectin by 72-90%. Zinc induced a dose-dependent inhibition of cell adhesion to fibronectin (R(2)=-0.98). Zinc at 10-50 µM reduced magnesium-facilitated cell adhesion to fibronectin in a dose-dependent manner (R(2)=-0.90). However, zinc had no effect on manganese-facilitated cell adhesion to fibronectin. Zinc at 5-50 µM caused rounding of the normally elongated, irregular-shaped MDA-MB-231 cells and disappearance of F-actin. Anti-integrin α5- and ß1-subunit blocking antibodies inhibited magnesium-facilitated cell adhesion to fibronectin by 95 and 99%, respectively. In summary, zinc inhibited MDA-MB-231 cell migration on fibronectin by interfering with magnesium-dependent integrin-, likely integrin α5/ß1-, mediated adhesion.

Marginal zinc deficiency increased the susceptibility to acute lipopolysaccharide-induced liver injury in rats.

Lipopolysaccharide (LPS) triggers a global activation of inflammatory responses leading to liver injury in humans. Zinc pretreatment has been shown to prevent LPS-induced hepatic necrosis. In North America, suboptimal zinc status is more common than once realized. However, the effect of inadequate zinc nutrition on the host's susceptibility to LPS-induced liver injury is not known. The objective of this study was to determine whether marginal zinc deficiency would render rats more susceptible to LPS-induced liver injury. Weanling Sprague-Dawley rats were assigned to one of three dietary treatment groups: marginally low zinc ad libitum (Z3; 3 mg zinc/kg diet), adequate zinc ad libitum (Z30; 30 mg zinc/kg diet), or adequate zinc pair-fed (Z30P) group. After 6 weeks, each dietary treatment group was further divided into LPS-control (saline) groups (C-Z3, C-Z30P, C-Z30) and LPS-treatment (1 mg/kg body weight, intraperitoneal, 8 hrs) groups (LPS-Z3, LPS-Z30P, LPS-Z30). LPS reduced the serum zinc concentration and increased the liver zinc concentration regardless of dietary zinc intake. Serum alanine aminotransferase level was higher in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. LPS also induced hepatocyte necrosis and neutrophil infiltration into the liver sinusoids. This LPS-induced liver damage was more severe in the LPS-Z3 rats than in the LPS-Z30P and LPS-Z30 rats. Together these findings have demonstrated that marginal zinc deficiency increased the susceptibility to LPS-induced liver injury in rats. These results indicate that patients with sepsis who have suboptimal zinc nutrition status may be at higher risk of developing greater liver damage.

Increased abundance of labile intracellular zinc during cell proliferation was due to increased retention of extracellular zinc in 3T3 cells.

Platelet-derived growth factor (PDGF)-, epidermal growth factor (EGF)- and insulin-like growth factor I (IGF-I)-stimulated cell proliferation in 3T3 cells was accompanied by increased abundance of labile intracellular pool of zinc (LIPZ). However, the origin and regulation of this cell proliferation-associated increase in the abundance of LIPZ are unknown. Cellular zinc homeostasis involves zinc transporters and metallothionein. The objectives of this study were to determine whether cell proliferation-associated increase in the abundance of LIPZ was a result of an increased zinc uptake and to assess the involvement of zinc transporters and metallothionein in this cell proliferation-associated increase in the abundance of LIPZ in 3T3 fibroblasts. Zinc transporters assessed included both zinc importer (Zip1) and zinc exporters (ZnT1, ZnT2 and ZnT4). Growth factors increased the abundance of LIPZ while total cellular zinc concentration remained unaffected, demonstrating that LIPZ was responsive to the increased needs for zinc during growth factor-stimulated cell proliferation. Growth factors also increased net zinc retention as indicated by higher 65zinc radioactivity and elevated mRNA levels of Zip1, ZnT1 and ZnT4. Although zinc is essential to cell proliferation, excessive cellular zinc accumulation causes cytotoxicity. Collectively, these observations suggest that increase in the abundance of LIPZ during growth factor-stimulated cell proliferation was due to increased net retention of extracellular zinc, which was apparently achieved through a coordinated up-regulation of the expression of transporters involved in both zinc influx and efflux to ensure adequate supply of zinc to sustain cell proliferation, yet to prevent potential zinc cytotoxicity in 3T3 cells.

[Flow cytometric detection of minimal residual disease in pre-cursor-B-acute lymphoblastic leukemia on the basis of phenotypic aberrancies on minor leukemic cell populations].

To test the European BIOMED-1 Concerted Action proposed technique to detect minimal residual disease (MRD) in the chinese patients with precursor-B-acute lymphoblastic leukemia (precursor-B-ALL) by triple-staining flow cytometry and to define both normal and aberrant phenotypic profiles of precursor B cells, a series of bone marrow samples, 35 from precursor-B-ALL (13 in newly diagnosed cases, 15 at the end of remission induction therapy and 7 at end of the consolidations), and 19 from normal controls, were immunophenotyped with the five triple-staining antibodies (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) recom-mended by the BIOMED-1 using common flow cytometric protocols. Further, with different ratios of the leukemic cells with CD34/CD38/CD19 phenotype and normal mononuclear cells, a serial dilution test was analyzed. The results showed that three major CD19(+) cell subpopulations were identified in the normal controls, representing three consecutive maturation stages. The subpopulations in the precursor-B-ALL cases disappeared and were replaced with a great number of luekemic cells which had different characteristics of phenotypes, and then they reappeared with almost same characteristics as the normal CD19(+) cells after the patients achieved complete remission. When the five triple-staining antibody combinations were used, the phenotypic aberrancies could be identified in 12/13 (92.3%) cases with newly diagnosed precursor-B-ALL, at least one triple-labeling per case at the level of 0.01% or more. The frequencies of phenotypic aberrations detected with the triple-staining were 8/13 (61.5%) for CD10/CD20/CD19, 5/13 (38.5%) for CD34/CD38/CD19, 4/13 (30.8%) for CD10/TdT/CD19, 3/13 (23.1%) for CD34/CD22/CD19, and 2/13 (15.4%) for CD34/CD45/CD19. At the end of remission induction, the phenotypic aberrancies could be detected in 5/15 (33.3%), of which, 3/8 (37.5%) cases with the leukemic phenotypes detected both at the newly diagnosis and at the end of induction. The dilution test indicated that the cells with CD34/CD38/CD19 detected by flow cytometry correlated well with the leukemic cells added (r = 0.85, P < 0.05) over 1:1 to 1:400,000. It is concluded that the flow cytometric detection of precursor-B-ALL-MRD proposed by BIOMED-1 Concerted Action were well realized in this study. The one precursor-B-ALL cell can be effectively detected out of 10(4) normal bone marrow cells.

Low zinc intake suppressed N-methyl-N-nitrosourea-induced mammary tumorigenesis in Sprague-Dawley rats.

Zinc has been shown to be accumulated in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors. Zinc is required for cell proliferation and tumorigenesis is characterized by dysregulation of cell proliferation. An accumulation of zinc in mammary tumors perhaps indicates a reliance on zinc to sustain tumor growth. Limiting zinc supply by means such as reduced zinc intake should negatively modulate mammary tumorigenesis. Our objective was to determine the effects of zinc status on MNU-induced mammary tumorigenesis in sexually mature female rats. Twenty-one-day-old Sprague-Dawley rats were assigned to low-zinc (3 mg zinc/kg diet) or adequate-zinc (12 mg zinc/kg diet) ad libitum or pair-fed control group (n = 25-33 rats/group). On day 50 of age, all rats were intraperitoneally injected with MNU (50 mg/kg body wt) to induce mammary tumorigenesis. Rats were further maintained on their assigned diet until 14 weeks post-MNU injection. Total food intake and overall body weight gain were lower in low-zinc rats than in adequate-zinc ad libitum control rats, but were similar to adequate-zinc pair-fed control rats. Plasma zinc concentration was lower in low-zinc rats than in adequate-zinc ad libitum and pair-fed control rats, confirming moderately low-zinc status in low-zinc rats. Tumor incidence (46 versus 84 and 80%; P < 0.05) and tumor multiplicity (0.8 versus 5.0 and 2.6 tumors/rat; P < 0.05) and tumor number (28 versus 123 and 66 tumors) were reduced in low-zinc rats compared with that in adequate-zinc ad libitum and pair-fed control rats, respectively. Tumor latency in low-zinc and adequate-zinc pair-fed control rats was not significantly different, but was longer than in adequate-zinc ad libitum control rats (P < 0.05), suggesting that reduced food intake associated with low-zinc intake prolonged tumor latency. Tumor burden and size were not affected by zinc intake. Overall, our observations showed that moderately low-zinc status suppressed MNU-induced rat mammary tumorigenesis.

Role of metallothionein in regulating the abundance of histochemically reactive zinc in rat tissues.

The objectives of this study were (i) to investigate the modulating effects of zinc nutrition on histochemically reactive zinc in the rat intestine and liver and (ii) to assess the relationship between histochemically reactive zinc and metallothionein-bound zinc in these tissues under varying zinc nutrition. Male Wistar rats were fed a zinc-deficient (3 mg zinc/kg), adequate-zinc (30 mg zinc/kg, ad libitum or pair-fed), or zinc-supplemented (155 mg zinc/kg) diet for 2 or 6 weeks. Plasma N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc reflected dietary zinc intake. Abundance of the intestine histochemically reactive zinc was correlated with dietary zinc intake after 2 weeks of dietary treatment. Dietary zinc intake had no effect on the abundance of the intestine histochemically reactive zinc after 6 weeks of dietary treatment and the hepatic histochemically reactive zinc after both 2 and 6 weeks of dietary treatment. This lack of effect of dietary zinc intake on the abundance of histochemically reactive zinc was associated with a higher level of metallothionein. The molecular-mass distribution profile revealed that N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide-reactive zinc and metallothionein-bound zinc represented two different, but interrelated, pools of zinc. Overall, these results suggested that the abundance of histochemically reactive zinc was homeostatically regulated, which was partially achieved through the regulation of metallothionein levels in rats.

Growth factor stimulated cell proliferation is accompanied by an elevated labile intracellular pool of zinc in 3T3 cells.

Growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and insulin-like growth factor-I (IGF-I) are required for quiescent 3T3 cells to proliferate, but zinc deprivation impairs IGF-I-induced DNA synthesis. We recently showed that labile intracellular pool of zinc is involved in cell proliferation. Our objective was to determine whether the labile intracellular pool of zinc plays a role in growth factor (PDGF, EGF, and IGF-I)-stimulated proliferation of 3T3 cells. Quiescent 3T3 cells were cultured in DMEM with or without growth factors. Labile intracellular pool of zinc, DNA synthesis, and cell proliferation were assessed using fluorescence microscopy, 3H-thymidine incorporation, and total cell number counts, respectively. After 24 h, growth factors stimulated DNA synthesis (24%) but not cell proliferation. After 48 h, growth factors stimulated both DNA synthesis (37%) and cell proliferation (89%). In response to growth factor stimulation, the labile intracellular pool of zinc was also elevated after 24 or 48 h of treatment. In summary, growth factor (PDGF, EGF, and IGF-I)-stimulated increase in DNA synthesis and cell proliferation were accompanied by an elevated labile intracellular pool of zinc in 3T3 cells. Since elevation of the labile intracellular pool of zinc occurred along with increased DNA synthesis, but cell proliferation remained unchanged, the elevation of the labile intracellular pool of zinc likely occurred during the S phase to provide the zinc needed to support DNA synthesis and ultimately cell proliferation.

Body zinc distribution profile during N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats at various levels of dietary zinc intake.

Zinc distribution is apparently altered in breast cancer patients. It is unclear if this apparent zinc redistribution is a consequence of altered zinc nutrition or tissue-specific response to breast cancer. Our objectives were to assess effects of N-methyl-N-nitrosourea-treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis on body zinc-distribution profile in rats and to assess effects of dietary zinc intake on the body zinc-distribution profile during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis in rats. Female Sprague-Dawley rats were assigned to zinc-deficient (3 mg/kg diet) or zinc-adequate (31 mg/kg diet) ad libitum or pair-fed group. Rats were sham treated or N-methyl-N-nitrosourea treated (50 mg/kg body weight; Experiment 1 or 40 mg/kg body weight; Experiment 2) (n = 6). In both experiments, the zinc concentration was significantly higher (6-19 times) in mammary tumor than in mammary gland. Tissue zinc concentration was essentially unaffected by N-methyl-N-nitrosourea treatment and tumor bearing, but was reduced by zinc deficiency in the bone, kidney, and liver. Overall, higher mammary tumor zinc concentration and absence of zinc redistribution during N-methyl-N-nitrosourea treatment and N-methyl-N-nitrosourea-induced mammary tumorigenesis, regardless of zinc intakes, indicates zinc accumulation in mammary tumors. Because zinc is essential for growth and cancer is characterized by uncontrolled growth, this zinc accumulation suggests an involvement of zinc in N-methyl-N-nitrosourea-induced rat mammary tumorigenesis.

Influence of chelation and oxidation state on vanadium bioavailability, and their effects on tissue concentrations of zinc, copper, and iron.

Today, vanadium compounds are frequently included in nutritional supplements and are also being developed for therapeutic use in diabetes mellitus. Previously, tissue uptake of vanadium from bis(maltolato)oxovanadium(IV) (BMOV) was shown to be increased compared to its uptake from vanadyl sulfate (VS). Our primary objective was to test the hypothesis that complexation increases vanadium uptake and that this effect is independent of oxidation state. A secondary objective was to compare the effects of vanadium complexation and oxidation state on tissue iron, copper, and zinc. Wistar rats were fed either ammonium metavanadate (AMV), VS, or BMOV (1.2 mM each in the drinking water). Tissue uptake of V following 12 wk of BMOV or AMV was higher than that from VS (p < 0.05). BMOV led to decreased tissue Zn and increased bone Fe content. The same three compounds were compared in a cellular model of absorption (Caco-2 cells). Vanadium uptake from VS was higher than that from BMOV or AMV at 10 min, but from BMOV (250 microM only, 60 min), uptake was far greater than from AMV or VS. These results show that neither complexation nor oxidation state alone are adequate predictors of relative absorption, tissue accumulation, or trace element interactions.

Labile intracellular zinc is associated with 3T3 cell growth.

Increasing evidence shows that labile intracellular zinc is metabolically important. Depletion of labile intracellular zinc using chelators suppresses DNA synthesis. In this study, we tested the hypothesis that labile intracellular zinc could be modulated via varying zinc nutrition. This could result in an altered availability of labile intracellular zinc, which, in turn, could influence zinc-dependent cellular events involved in cell proliferation and ultimately suppress growth. Labile intracellular zinc was detected by using N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), a membrane-permeable fluorescence probe. After 48 h culture in a zinc-depleted medium, labile intracellular zinc in 3T3 cells was diminished along with a suppressed DNA synthesis and cell proliferation. In contrast, supplementation of zinc to the zinc-depleted medium increased the labile intracellular zinc and promoted DNA synthesis and cell proliferation. Furthermore, growth factor-dependent stimulation of DNA synthesis and cell proliferation was also accompanied by increased labile intracellular zinc. Together, our data showed an association between the labile intracellular zinc, detected using TSQ, and 3T3 cell growth, suggesting that labile intracellular zinc could be an important cellular link between zinc nutrition and growth.