Development of a LC-MS/MS method to measure serum 3-sulfate and 3-glucuronide 25-hydroxyvitamin D3 metabolites; comparisons to unconjugated 25OHD in pregnancy and polycystic ovary syndrome.

Vitamin D status is routinely assessed by measuring circulating concentrations of 25-hydroxyvitamin D (25OHD2 or 25OHD3). However as deconjugation is not routinely incorporated into sample treatment prior to analysis, conjugated forms of 25OHD (particularly the more abundant 25OHD3) are often not considered in determining serum concentrations of total 25OHD. Two major circulating conjugated forms of 25OHD3 are 25-hydroxyvitamin D3-3-sulfate (25OHD3-S) and 25-hydroxyvitamin D3-3-glucuronide (25OHD3-G). Incorporating these two conjugated metabolites into the measurement of vitamin D status could improve our understanding of vitamin D status in health, particularly if there are changes in sulfation and glucuronidation activities. The aim of this study was to develop a liquid chromatography tandem-mass spectrometry (LC-MS/MS) targeted method for measurement of 25OHD3-S and 25OHD3-G in serum to enable comparisons with circulating levels of the free 25OHD3 form. We developed and validated a new LC-MS/MS method that measured both 25OHD3-S and 25OHD3-G following a solid phase extraction sample preparation method. Partial separation of analytes by LC, and the separation of analytes by the optimized multiple reaction monitoring transitions enabled the quantitation of both 25OHD3-S and 25OHD3-G in the single method. Serum concentrations of 25OHD3-S (24.7 ± 11.8 ng/mL) and 25OHD3-G (2.4 ± 1.2 ng/mL) were shown to be a significant proportion of circulating vitamin D metabolites in healthy donor serums. These levels of 25OHD3-S and 25OHD3-G closely associated with 25OHD3 concentrations, r = 0.728, p = 0.001 and r = 0.632, p = 0.006 respectively. However in serum from pregnant women and non-pregnant women with polycystic ovary syndrome (PCOS) significant differences in the ratios between conjugated and free 25OHD3 were observed between pregnancy groups (25OHD3/25OHD3-S and 25OHD3/25OHD3-G p < 0.001), and between healthy and PCOS subjects (25OHD3/25OHD3-G p < 0.050). Development of this novel high-throughput LC-MS/MS method indicates that 25OHD3-S and 25OHD3-G are substantial components of circulating vitamin D metabolites. The concentrations of these metabolites relative to conventional 25OHD3 may vary in different physiological and pathophysiological settings, and may therefore play an unrecognized but important role in the actions of vitamin D.

Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells.

Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor ß (TGFß). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.

Defining vitamin D status using multi-metabolite mathematical modelling: A pregnancy perspective.

Vitamin D deficiency is linked to adverse pregnancy outcomes such as pre-eclampsia (PET) but remains defined by serum measurement of 25-hydroxyvitamin D3 (25(OH)D3) alone. To identify broader changes in vitamin D metabolism during normal and PET pregnancies we developed a relatively simple but fully parametrised mathematical model of the vitamin D metabolic pathway. The data used for parametrisation were serum vitamin D metabolites analysed for a cross-sectional group of women (n = 88); including normal pregnant women at 1 st (NP1, n = 25) and 3rd trimester (NP3, n = 21) and pregnant women with PET (n = 22), as well as non-pregnant female controls (n = 20). To account for the effects various metabolites have upon each other, data were analysed using an ordinary differential equation model of the vitamin D reaction network. Information obtained from the model was then also applied to serum vitamin D metabolome data (n = 50) obtained from a 2nd trimester pregnancy cohort, of which 25 prospectively developed PET. Statistical analysis of the data alone showed no significant difference between NP3 and PET for serum 25(OH)D3 and 24,25(OH)2D3 concentrations. Conversely, a statistical analysis informed by the reaction network model revealed that a better indicator of PET is the ratios of vitamin D metabolites in late pregnancy. Assessing the potential predicative value, no significant difference between NP3 and PET cases at 15 weeks gestation was found. Mathematical modelling offers a novel strategy for defining the impact of vitamin D metabolism on human health. This is particularly relevant within the context of pregnancy, where major changes in vitamin D metabolism occur across gestation, and dysregulated metabolism is evidenced in women with established PET.

Serum and urine vitamin D metabolite analysis in early preeclampsia.

Vitamin D deficiency is common in pregnant women and may contribute to adverse events in pregnancy such as preeclampsia (PET). To date, studies of vitamin D and PET have focused primarily on serum concentrations vitamin D, 25-hydroxyvitamin D3 (25(OH)D3) later in pregnancy. The aim here was to determine whether a more comprehensive analysis of vitamin D metabolites earlier in pregnancy could provide predictors of PET. Using samples from the SCOPE pregnancy cohort, multiple vitamin D metabolites were quantified by liquid chromatography-tandem mass spectrometry in paired serum and urine prior to the onset of PET symptoms. Samples from 50 women at pregnancy week 15 were analysed, with 25 (50%) developing PET by the end of the pregnancy and 25 continuing with uncomplicated pregnancy. Paired serum and urine from non-pregnant women (n = 9) of reproductive age were also used as a control. Serum concentrations of 25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 24,25(OH)2D3 and 3-epi-25(OH)D3 were measured and showed no significant difference between women with uncomplicated pregnancies and those developing PET. As previously reported, serum 1,25(OH)2D3 was higher in all pregnant women (in the second trimester), but serum 25(OH)D2 was also higher compared to non-pregnant women. In urine, 25(OH)D3 and 24,25(OH)2D3 were quantifiable, with both metabolites demonstrating significantly lower (P < 0.05) concentrations of both of these metabolites in those destined to develop PET. These data indicate that analysis of urinary metabolites provides an additional insight into vitamin D and the kidney, with lower urinary 25(OH)D3 and 24,25(OH)2D3 excretion being an early indicator of a predisposition towards developing PET.

Dysregulation of maternal and placental vitamin D metabolism in preeclampsia.

INTRODUCTION: Epidemiology has linked preeclampsia (PET) to decreased maternal serum 25-hydroxyvitamin D3 (25(OH)D3). However, alterations in systemic and placental/decidual transport and metabolism of 25(OH)D3 during pregnancy suggest that other forms of vitamin D may also contribute to the pathophysiology of PET. METHODS: In a cross sectional analysis of normal pregnant women at 1st (n = 25) and 3rd trimester (n = 21), pregnant women with PET (n = 22), and non-pregnant female controls (n = 20) vitamin D metabolites were quantified in paired maternal serum, placental, and decidual tissue. RESULTS: Serum 25(OH)D3 was not significantly different in sera across all four groups. In normal 3rd trimester pregnant women serum active 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was significantly higher than non-pregnant, normal 1st trimester pregnant, and PET women. Conversely, PET sera showed highest levels of the catabolites 3-epi-25(OH)D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3). Serum albumin was significantly lower in normal 3rd trimester pregnant women and PET relative to normal 1st trimester pregnant women, but there was no change in free/bioavailable 25(OH)D3. In PET placental tissue, 25(OH)D3 and 3-epi-25(OH)D3 were lower than normal 3rd trimester tissue, whilst placental 24,25(OH)2D3 was highest in PET. Tissue 1,25(OH)2D3 was detectable in 1st trimester decidua, which also showed 10-fold higher 25(OH)D3 relative to paired placentae. 3-epi-25(OH)D3 and 24,25(OH)2D3 were not different for decidua and placenta. In normal 3rd trimester pregnant women, total, free and bioavailable maternal 25(OH)D3 correlated with placental 25(OH)D3, but this was not conserved for PET. DISCUSSION: These data indicate that PET is associated with decreased activation, increased catabolism, and impaired placental uptake of 25(OH)D3.

Prenatal therapy in transient abnormal myelopoiesis: a systematic review.

OBJECTIVE: To systematically review current evidence regarding prenatal diagnosis and management of transient abnormal myelopoiesis (TAM) in fetuses with trisomy 21. A novel case of GATA1-positive TAM, in which following serial in utero blood transfusion clinical improvement and postnatal remission were observed, is included. SEARCH STRATEGY AND DATA COLLECTION: A systematic search of electronic databases (inception to October 2014) and reference lists, hand-searching of journals and expert contact. All confirmed cases of prenatal TAM were included for analysis. Data on study characteristics, design and quality were obtained. RESULTS: Of 73 potentially relevant citations identified, 22 studies were included, describing 39 fetuses. All studies included comprised single case or small cohort studies; overall quality was 'very low'. Fetal/neonatal outcome was poor; 12 stillbirths (30.8%), 4 neonatal deaths (10.2%) and 7 infant deaths (17.9%). In two cases, the pregnancy was terminated (5.1%). TAM was primarily detected in the third trimester (79.4%), and in 14 a retrospective diagnosis was made postpartum. Ultrasound features indicative of TAM included hepatomegaly±splenomegaly (79.5%), hydrops fetalis (30.8%), pericardial effusion (23.1%) and aberrant liquor volume (15.4%). When performed, liver function tests were abnormal in 91.6% of cases. CONCLUSIONS: Prenatal TAM presents a challenging diagnosis, and prognosis is poor, with consistently high mortality. A low threshold to measure haematological and biochemical markers is advised when clinical features typical of TAM are detected in the context of trisomy 21. Larger prospective studies are warranted to accurately ascertain the role of GATA1 analysis and potential value of prenatal therapy.

Immunological role of vitamin D at the maternal-fetal interface.

During pregnancy, immune activity is tightly regulated so that antimicrobial protection of the mother and fetus is balanced with the need for immune tolerance to prevent fetal rejection. In this setting, the maternal-fetal interface, in the form of the uterine decidua, provides a heterogeneous immune cell population with the potential to mediate diverse activities throughout pregnancy. Recent studies have suggested that vitamin D may be a key regulator of immune function during pregnancy, with the fetal-maternal interface representing a prominent target. Among its non-classical actions are potent immunomodulatory effects, including induction of antibacterial responses and modulation of T-lymphocytes to suppress inflammation and promote tolerogenesis. Thus, vitamin D may play a pivotal role in normal decidual immune function by promoting innate responses to infection, while simultaneously preventing an over-elaboration of inflammatory adaptive immunity. Research to date has focused upon the potential role of vitamin D in preventing infectious diseases such as tuberculosis, as well as possibly suppressing of autoimmune disease. Nevertheless, vitamin D may also influence facets of immune function not immediately associated with primary innate responses. This review summarises our current understanding of decidual immune function with respect to the vitamin D metabolism and signalling, and as to how this may be affected by variations in maternal vitamin D status. There has recently been much interest in vitamin D supplementation of pregnant women, but our knowledge of how this may influence the function of decidua remains limited. Further insight into the immunomodulatory actions of vitamin D during pregnancy will help shed light upon this.

Association and prediction of amniotic fluid measurements for adverse pregnancy outcome: systematic review and meta-analysis.

BACKGROUND: Measurements of amniotic fluid volume are used for pregnancy surveillance despite a lack of evidence for their predictive ability. OBJECTIVE: To evaluate the association and predictive value of ultrasound measurements of amniotic fluid volume for adverse pregnancy outcome. SEARCH STRATEGY: Electronic databases (inception to October 2011), reference lists, hand searching of journals, contact with experts. SELECTION CRITERIA: Studies comparing measurements of amniotic fluid volume with adverse outcome, excluding pre-labour ruptured membranes or congenital/structural anomalies. DATA COLLECTION: Data on study characteristics, design, quality. Random effects meta-analysis to estimate summary odds ratios (prognostic association) and summary sensitivity, specificity and likelihood ratios (predictive ability). MAIN RESULTS: Forty-three studies (244,493 fetuses) were included demonstrating a strong association between oligohydramnios (varying definitions) and birthweight <10th centile (summary odds ratio [OR] 6.31, 95% confidence interval [95% CI] 4.15-9.58; high-risk population [author definition] n = 6 studies, 28,510 fetuses), and mortality (neonatal death any population summary OR 8.72, 95% CI 2.43-31.26; n = 6 studies, 55,735 fetuses; and perinatal mortality high-risk population summary OR 11.54, 95% CI 4.05-32.9; n = 2 studies, 27;891 fetuses). There was a strong association between polyhydramnios (maximum pool depth >8 cm or amniotic fluid index ≥25 cm) and birthweight >90th centile (OR 11.41, 95% CI 7.09-18.36; n = 1 study, 3960 fetuses). Despite strong associations, predictive accuracy for perinatal outcome was poor. AUTHOR'S CONCLUSION: Current evidence suggests that oligohydramnios is strongly associated with being small for gestational age and mortality, and polyhydramnios with birthweight >90th centile. Despite strong associations with poor outcome, they do not accurately predict outcome risk for individuals.

The immunological basis of villitis of unknown etiology - review.

Villitis of unknown etiology (VUE) represents a common placental inflammatory lesion, primarily, but not exclusively, identifiable T lymphocytes at term. Despite considerable evidence to contest that this simply represents a benign pathological finding, VUE remains a significantly undervalued diagnosis. Given its association with adverse pregnancy outcomes; including fetal growth restriction, preterm birth, and recurrent pregnancy loss, an increased awareness amongst clinician obstetricians is certainly warranted. The underlying immunopathogenesis of VUE remains uncertain. Despite initial theories that this represents an infectious placental lesion of undiagnosed pathogenic source, a more complex sequence of events involving the "breakdown" of maternal-fetal tolerance is emerging. Characterization of a unique inflammatory phenomenon in which both maternal and fetal T lymphocytes and Höfbauer cells interact has captivated particular research interest and has generated analogies to both the problems of allograft rejection and graft-versus-host disease (GvHD). Within the context of VUE, this review evaluates how disruption of the multidimensional immunological mechanisms underlying feto-maternal tolerance may permit abnormal lymphocyte infiltration into placental villi. We shall review the existing evidence for these events in VUE and outline areas of certain future interest.

Microinjection of urocortin 2 into the dorsal raphe nucleus activates serotonergic neurons and increases extracellular serotonin in the basolateral amygdala.

The intra dorsal raphe nucleus (DRN) administration of corticotropin releasing hormone (CRF) inhibits serotonergic (5-HT) activity in this structure, an effect blocked by antagonists selective for the type 1 CRF receptor (CRF1). The DRN has a high density of the type 2 receptor (CRF2), and so the present experiments explored the impact of CRF2 activation within the DRN on 5-HT function. The intra-DRN administration of the selective CRF2 agonist urocortin 2 (Ucn 2) dose dependently increased 5-HT efflux in the basolateral amygdala, a projection region of the DRN. Intra-DRN Ucn 2 also increased c-fos expression in labeled 5-HT neurons. Both of these effects of Ucn 2 were completely blocked by intra-DRN antisauvagine-30 (ASV-30), a relatively selective CRF2 antagonist. These data suggest that CRF1 and CRF2 activation within the DRN affect 5-HT neurons in opponent fashion. Implications of these results for understanding the behavioral effects of CRF and other CRF-like ligands are discussed.

The pharmacokinetics of ketorolac enantiomers following intramuscular administration of the racemate.

A single dose of racemic ketorolac (30 mg of tromethamine salt, Toradol) was administered by bolus intramuscular injection to four young, healthy volunteers. The concentrations of total (bound plus unbound) (R)- and (S)-ketorolac were measured in plasma for 9 h after dosing. The mean +/- s.d. clearance of (S)-ketorolac (45.9 +/- 10.1 ml h-1 kg-1) exceeded (P = 0.0032) that of the (R)-enantiomer (19.0 +/- 5.0 ml h-1 kg-1). The mean +/- s.d. AUC ratio for (S)-ketorolac:(R)-ketorolac (0.442 +/- 0.043) was significantly different from unity (P = 0.0001). The steady-state volume of distribution of (S)-ketorolac (0.135 +/- 0.022 l kg-1) was significantly different (P = 0.0013) from that of its optical antipode (0.075 +/- 0.014 l kg-1) and the half-lives of (S)- and (R)-ketorolac (2.35 +/- 0.23 h and 3.62 +/- 0.79 h, respectively) were also significantly different (P = 0.026). These data indicate that the disposition of ketorolac in man is subject to marked enantioselectivity and, because of possible differences in biological activity of (S)- and (R)-ketorolac, emphasize the need to monitor separate stereoisomer concentrations of the drug if pharmacological data are to be interpreted correctly.

Stereoselective analysis of ketorolac in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) analytical method is described for the quantification of the (R)- and (S)-enantiomers of ketorolac when present together in human plasma. The method involves derivatization with thionyl chloride/(S)-1-phenylethylamine and subsequent reversed-phase chromatography of the diastereomeric (S)-1-phenylethylamides of (R)- and (S)-ketorolac. The method is suitable for the analysis of large numbers of plasma samples and has been applied in this report to a pharmacokinetic study of ketorolac enantiomers upon intramuscular administration of racemic drug to a human subject. The limit of quantification for each enantiomer of ketorolac is 50 ng/ml (signal-to-noise ratio > 10).