Absolute quantification of eleven A, D, E and K vitamers in human plasma using automated extraction and UHPLC-Orbitrap MS.

Fat-Soluble Vitamers [FSV] deficiencies and hypervitaminosis are associated with lifestyle diseases such as cardiovascular disease, diabetes, and cancer. Quantification of FSV and their metabolites in plasma has proved to be one of the most demanding analytical chemistry challenges. Current FSV quantification methods are compromises between breadth of coverage and sensitivity across the physiological range. Here, we developed and validated a sensitive, robust, semi-automated method using liquid-liquid extraction coupled with LC-ESI-MS/MS to quantify 11 FSV across their physiological concentrations in plasma. The addition of Phree® phospholipid removal plates as the last step in the extraction process reduced matrix effects, improving precision, recoveries, and the method's final sensitivity. This method can detect and quantify: retinol, retinoic acid, retinyl palmitate, 25 hydroxyvitamin D3 [25-OH-D3], 1-α-25-dihydroxy-D3, α-tocopherol, γ-tocopherol, α-tocotrienol, phylloquinone [K1], Menatetrenone [MK-4], and menaquinone-7 [MK-7].The Instrument Quantitation Limit [IQL]s for retinol (64.1 ng/mL), 25-OH-D3 (10.2 ng/mL), and α-tocopherol (3000 ng/mL) can detect clinical deficiencies. Our automated method will assist in the understanding of the complex interaction between these compounds and their possible role in health and disease.

LC-MS/MS quantification of fat soluble vitamers - A systematic review.

Fat soluble vitamers (FSV) are several biochemically diverse micronutrients essential for healthy development, growth, metabolism, and cell regulation. We cannot synthesize FSV completely or at the required concentrations. Deficiency or excess of FSV can result in many health problems. Plasma is the most accessible sample matrix for the quantification of FSV. However, due to its complexity and other analytical challenges (e.g., FSV sensitivity to light, oxygen, heat, pH, chemical heterogeneity, standard availability), developing a method for the simultaneous quantification of multiple FSV at physiological concentrations has been challenging. In this systematic review, we examine the parameters and criteria used in existing Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) methods for FSV quantification to the extraction method, chromatographic resolution, matrix effects, and method validation as critical to a sensitive and robust method. We conclude that the final FSV method sensitivity is predominantly based on aforementioned criteria and future method development using LC-MS/MS will benefit from the application of this systematic review.

Antenatal sildenafil citrate treatment increases offspring blood pressure in the placental-specific Igf2 knockout mouse model of FGR.

Fetal growth restriction (FGR), where a fetus fails to reach its genetic growth potential, affects up to 8% of pregnancies and is a major risk factor for stillbirth and adulthood morbidity. There are currently no treatments for FGR, but candidate therapies include the phosphodiesterase-5 inhibitor sildenafil citrate (SC). Randomized clinical trials in women demonstrated no effect of SC on fetal growth in cases of severe early onset FGR; however, long-term health outcomes on the offspring are unknown. This study aimed to assess the effect of antenatal SC treatment on metabolic and cardiovascular health in offspring by assessing postnatal weight gain, glucose tolerance, systolic blood pressure, and resistance artery function in a mouse model of FGR, the placental-specific insulin-like growth factor 2 (PO) knockout mouse. SC was administered subcutaneously (10 mg/kg) daily from embryonic day (E)12.5. Antenatal SC treatment did not alter fetal weight or viability but increased postnatal weight gain in wild-type (WT) female offspring (P < 0.05) and reduced glucose sensitivity in both WT (P < 0.01) and P0 (P < 0.05) female offspring compared with controls. Antenatal SC treatment increased systolic blood pressure in both male (WT vs. WT-SC: 117 ± 2 vs. 140 ± 3 mmHg, P < 0.0001; P0 vs. P0-SC: 113 ± 3 vs. 140 ± 4 mmHg, P < 0.0001; means ± SE) and female (WT vs. WT-SC: 121 ± 2 vs. 140 ± 2 mmHg, P < 0.0001; P0 vs. P0-SC: 117 ± 2 vs. 144 ± 4 mmHg, P < 0.0001) offspring at 8 and 13 wk of age. Increased systolic blood pressure was not attributed to altered mesenteric artery function. In utero exposure to SC may result in metabolic dysfunction and elevated blood pressure in later life.NEW & NOTEWORTHY Sildenafil citrate (SC) is currently used to treat fetal growth restriction (FGR). We demonstrate that SC is ineffective at treating FGR, and leads to a substantial increase systolic blood pressure and alterations in glucose homeostasis in offspring. We therefore urge caution and suggest that further studies are required to assess the safety and efficacy of SC in utero, in addition to the implications on long-term health.

The relationship between maternal 25-hydroxyvitamin D status in pregnancy and childhood adiposity and allergy: an observational study.

BACKGROUND: Vitamin D insufficiency (defined as <75 nmol l-1) is widespread among pregnant women around the world and has been proposed to influence offspring outcomes in childhood and into adult life, including adiposity and allergy. Disorders, including asthma and eczema, are on the rise among children. Our aim was to investigate the relationship between maternal 25-hydroxyvitamin D status in pregnancy and offspring adiposity, asthma and eczema in childhood. SUBJECTS AND METHODS: Maternal 25-hydroxyvitamin D concentrations were analysed in serum samples collected at 15 weeks' gestation from 1710 participants of the prospective Screening for Pregnancy Endpoints cohort study. The offspring of 1208 mothers were followed up at age 5-6 years. Data collected included height, weight, percentage body fat (PBF, measured by bioimpedance) and history of asthma and eczema. Multivariable analysis controlled for maternal body mass index (BMI), age and sex of the child and season of serum sampling. RESULTS: Complete data were available for 922 mother-child pairs. Each 10 nmol l-1 increase in maternal 25-hydroxyvitamin D concentration at 15 weeks' gestation was associated with a decrease in offspring PBF of 0.2% (95% confidence interval 0.04-0.36%, P=0.01) after adjustment for confounders but was not related to child BMI z-score. Maternal mean (±s.d.) 25-hydroxyvitamin D concentration was similar in children who did and did not have asthma (71.7±26.1 vs 73.3±27.1 nmol l-1, P=0.5), severe asthma (68.6±28.6 vs 73.3±26.8 nmol l-1, P=0.2) and eczema (71.9±27.0 vs 73.2±27.0 nmol l-1, P=0.5). CONCLUSIONS: The finding of a relationship between maternal vitamin D status and adiposity in childhood is important, particularly because vitamin D insufficiency in pregnancy is highly prevalent. The association between maternal vitamin D supplementation in pregnancy and adiposity in the offspring merits examination in randomised controlled trials.

Older Adults Have Delayed Amino Acid Absorption after a High Protein Mixed Breakfast Meal.

OBJECTIVES: To measure the postprandial plasma amino acid appearance in younger and older adults following a high protein mixed meal. DESIGN: Cross-sectional study. SETTING: Clinical research setting. PARTICIPANTS: Healthy men and women aged 60-75 (n=15) years, and young controls aged 20-25 years (n=15) matched for body mass index and insulin sensitivity based on the homeostatic model assessment of insulin resistance. INTERVENTION: High protein mixed meal of complete food products. MEASUREMENTS: Circulating amino acid concentrations were determined hourly before and for 5 hours after meal ingestion. RESULTS: There was no difference between cohorts in postprandial appearance of non-essential amino acids, or area under the curve of any individual amino acid or amino acid class. However, older adults had higher baseline concentrations of aspartic acid, glutamic acid, glycine, ornithine, threonine and tyrosine and lower baseline concentrations of hydroxyproline, isoleucine, leucine, methionine and valine compared to younger adults. Younger adults showed peak essential (EAA) and branched-chain amino acid (BCAA) concentrations at 1 hour post meal while older adults' peak EAA and BCAA concentration was at 3 hours. Similarly, peak total amino acid concentrations were at 3 hours in older adults. CONCLUSION: Older adults digested and absorbed the protein within a mixed meal more slowly than younger adults. Delayed absorption of AA following a mixed meal of complete food products may suppress or delay protein synthesis in senescent muscle.

Periconceptional undernutrition suppresses cortisol response to arginine vasopressin and corticotropin-releasing hormone challenge in adult sheep offspring.

Poor maternal nutrition during pregnancy can result in increased disease risk in adult offspring. Many of these effects are proposed to be mediated via altered hypothalamo-pituitary-adrenal axis (HPAA) function, and are sex and age specific. Maternal undernutrition around the time of conception alters HPAA function in foetal and early postnatal life, but there are limited conflicting data about later effects. The aim of this study was to investigate the effect of moderate periconceptional undernutrition on HPAA function of offspring of both sexes longitudinally, from juvenile to adult life. Ewes were undernourished from 61 days before until 30 days after conception or fed ad libitum. HPAA function in offspring was assessed by arginine vasopressin plus corticotropin-releasing hormone challenge at 4, 10 and 18 months. Plasma cortisol response was lower in males than in females, and was not different between singles and twins. Periconceptional undernutrition suppressed offspring plasma cortisol but not adrenocorticotropic hormone responses. In males, this suppression was apparent by 4 months, and was more profound by 10 months, with no further change by 18 months. In females, suppression was first observed at 10 months and became more profound by 18 months. Maternal undernutrition limited to the periconceptional period has a prolonged, sex-dependent effect on adrenal function in the offspring.

Transplacental transfer and biotransformation of genistein in human placenta.

OBJECTIVE: To study transplacental transfer and biotransformation of genistein in the human placenta. STUDY DESIGN AND OUTCOMES: Human placentae obtained from healthy term singleton pregnancies were utilised in a dual re-circulating model of ex-vivo placental perfusion. Four placentae were perfused for 180min following addition of genistein (10ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate integrity of the circuits. Concentrations of genistein and its conjugates were determined by liquid chromatography-mass spectrometry (LC-MS). RESULTS: The transfer percentage for antipyrine and genistein was 25.6+/-1.40% and 22.1+/-1.61% respectively and the transfer index for genistein was 0.90+/-0.04 after 180min of perfusion. 12.0+/-2.40% of genistein in the fetal compartment and 7.36+/-4.73% of genistein in the maternal compartment were in the conjugated form. CONCLUSIONS: Genistein can transfer across the human placenta at environmentally relevant levels. Placental metabolizing enzymes conjugate a small fraction of genistein into the glucuronide/sulphate form, which is devoid of estrogenic action.

Effects of twinning and periconceptional undernutrition on late-gestation hypothalamic-pituitary-adrenal axis function in ovine pregnancy.

The relationships between reduced size at birth, increased activity of the hypothalamic-pituitary-adrenal (HPA) axis, and increased risk of disease in adulthood are well described in singletons but are much less clear in twins. This may be because the physiological processes underlying reduced size at birth are different in singletons and twins. Periconceptional undernutrition can cause altered activity of the fetal and postnatal HPA axis without altering size at birth. However, the independent effects of periconceptional undernutrition and twinning on activity of the maternal and fetal HPA axes are not well described. We therefore studied maternal and fetal HPA axis function during late gestation in twin and singleton sheep pregnancies, either undernourished around conception or fed ad libitum. We found that twinning led to suppressed baseline HPA axis function and decreased adrenal sensitivity to ACTH stimulation but increased fetal pituitary ACTH response both to direct stimulation by CRH (ACTH area under the curve response: 29.7 +/- 2.2 vs. 17.1 +/- 1.6 ng/min x ml, P < 0.01) and to decreased cortisol negative feedback. In contrast, periconceptional undernutrition resulted in a decreased pituitary response (ACTH area under the curve response: 19.4 +/- 1.6 vs. 26.1 +/- 2.2 ng/min x ml, P = 0.02) but no difference in adrenal response. Thus, the HPA axis function of twin sheep fetuses in late gestation is very different from that of control and undernourished singletons. If the HPA axis is an important mediator between fetal adaptations and adult disease, these data may help explain why the relationship between fetal growth and postnatal physiology and disease risk is inconsistent in twins.