Identification of structural determinants of nicotinamide phosphoribosyl transferase (NAMPT) activity and substrate selectivity.

NAD homeostasis in mammals requires the salvage of nicotinamide (Nam), which is cleaved from NAD+ by sirtuins, PARPs, and other NAD+-dependent signaling enzymes. Nam phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in vitamin B3 salvage, whereby Nam reacts with phosphoribosyl pyrophosphate (PRPP) to form nicotinamide mononucleotide. NAMPT has a high affinity towards Nam, which is further enhanced by autophosphorylation of His247. The mechanism of this enhancement has remained unknown. Here, we present high-resolution crystal structures and biochemical data that provide reasoning for the increased affinity of the phosphorylated NAMPT for its substrate. Structural and kinetic analyses suggest a mechanism that includes Mg2+ coordination by phospho-His247, such that PRPP is stabilized in a position highly favorable for catalysis. Under these conditions, nicotinic acid (NA) can serve as a substrate. Moreover, we demonstrate that a stretch of 10 amino acids, present only in NAMPTs from deuterostomes, facilitates conformational plasticity and stabilizes the chemically unstable phosphorylation of His247. Thereby the apparent substrate affinity is considerably enhanced compared to prokaryotic NAMPTs. Collectively, our study provides a structural basis for the important function of NAMPT to recycle Nam into NAD biosynthesis with high affinity.

Pulsatile Subcutaneous Hydrocortisone Replacement in Primary Adrenal Insufficiency.

Pulsatile endogenous cortisol secretion is critical for physiological glucocorticoid gene signaling. Conventional glucocorticoid replacement therapy does not mimic endogenous cortisol pulsing in primary adrenal insufficiency. In an open-labeled, two-week, nonrandomized cross-over study of five patients with adrenal insufficiency (Addison's disease in two, bilateral adrenalectomy in one, and congenital adrenal hyperplasia in two patients) we compared pulsatile and continuous cortisol pump treatment and conventional oral glucocorticoid therapy with respect to 24-h serum corticosteroid levels and plasma adrenocorticotropic hormone (ACTH). Pulsed pump restored ultradian rhythmicity as demonstrated by five peaks of serum (all patients) and subcutaneous tissue cortisol (four patients). Morning subcutaneous cortisol and cortisone were higher in continuous and pulsed pump treatment than in oral therapy despite nearly similar serum cortisol levels in all treatment arms. ACTH was within the physiological range during pulsed pump treatment in all patients except for slightly elevated levels in the morning hours 04:00-08:00 h. During oral therapy, ACTH was very high in patients with Addison's disease and suppressed in patients with congenital adrenal hyperplasia. In conclusions, mimicking endogenous cortisol rhythmicity by ultradian subcutaneous infusion of cortisol is feasible. It was superior to both continuous pump and oral therapy in maintaining normal ACTH levels throughout the 24-h cycle. Our results demonstrate a low free cortisol bioavailability on thrice daily oral replacement therapy compared to both types of subcutaneous infusion.

Steroidogenic factor 1 (NR5A1) induces multiple transcriptional changes during differentiation of human gonadal-like cells.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) encodes steroidogenic factor 1 (SF1), a key regulatory factor that determines gonadal development and coordinates endocrine functions. Here, we have established a stem cell-based model of human gonadal development and applied it to evaluate the effects of NR5A1 during the transition from bipotential gonad to testicular cells. We combined directed differentiation of human induced pluripotent stem cells (46,XY) with activation of endogenous NR5A1 expression by conditionally-inducible CRISPR activation. The resulting male gonadal-like cells expressed several Sertoli cell transcripts, secreted anti-Müllerian hormone and responded to follicle-stimulating hormone by producing sex steroid intermediates. These characteristics were not induced without NR5A1 activation. A total of 2691 differentially expressed genetic elements, including both coding and non-coding RNAs, were detected immediately following activation of NR5A1 expression. Of those, we identified novel gonad-related putative NR5A1 targets, such as SCARA5, which we validated also by immunocytochemistry. In addition, NR5A1 activation was associated with dynamic expression of multiple gonad- and infertility-related differentially expressed genes. In conclusion, by combining targeted differentiation and endogenous activation of NR5A1 we have for the first time, been able to examine in detail the effects of NR5A1 in early human gonadal cells. The model and results obtained provide a useful resource for future investigations exploring the causative reasons for gonadal dysgenesis and infertility in humans.

Progressive Motor and Non-Motor Symptoms in Park7 Knockout Zebrafish.

DJ-1 is a redox sensitive protein with a wide range of functions related to oxidative stress protection. Mutations in the park7 gene, which codes for DJ-1 are associated with early onset familial Parkinson's disease and increased astrocytic DJ-1 levels are found in pathologic tissues from idiopathic Parkinson's disease. We have previously established a DJ-1 knockout zebrafish line that developed normally, but with aging the DJ-1 null fish had a lowered level of tyrosine hydroxylase, respiratory mitochondrial failure and a lower body mass. Here we have examined the DJ-1 knockout from the early adult stage and show that loss of DJ-1 results in a progressive, age-dependent increase in both motoric and non-motoric symptoms associated to Parkinson's disease. These changes coincide with changes in mitochondrial and mitochondrial associated proteins. Recent studies have suggested that a decline in NAD+ can contribute to Parkinson's disease and that supplementation of NAD+ precursors may delay disease progression. We found that the brain NAD+/NADH ratio decreased in aging zebrafish but did not correlate with DJ-1 induced altered behavior. Differences were first observed at the late adult stage in which NAD+ and NADPH levels were decreased in DJ-1 knockouts. Considering the experimental power of zebrafish and the development of Parkinson's disease-related symptoms in the DJ-1 null fish, this model can serve as a useful tool both to understand the progression of the disease and the effect of suggested treatments.

Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival.

BACKGROUND: Resistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ. METHODS: Cell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan-Meier method. RESULTS: Pre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood-brain barrier, diminished proteasome activity and significantly prolonged animal survival. CONCLUSION: BTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

Drug monitoring of tamoxifen metabolites predicts vaginal dryness and verifies a low discontinuation rate from the Norwegian Prescription Database.

PURPOSE: Tamoxifen is an important targeted endocrine therapy in breast cancer. However, side effects and early discontinuation of tamoxifen remains a barrier for obtaining the improved outcome benefits of long-term tamoxifen treatment. Biomarkers predictive of tamoxifen side effects remain unidentified. The objective of this prospective population-based study was to investigate the value of tamoxifen metabolite concentrations as biomarkers for side effects. A second objective was to assess the validity of discontinuation rates obtained through pharmacy records with the use of tamoxifen drug monitoring. METHODS: Longitudinal serum samples, patient-reported outcome measures and pharmacy records from 220 breast cancer patients were obtained over a 6-year period. Serum concentrations of tamoxifen metabolites were measured by LC-MS/MS. Associations between metabolite concentrations and side effects were analyzed by logistic regression and cross table analyses. To determine the validity of pharmacy records we compared longitudinal tamoxifen concentrations to discontinuation rates obtained through the Norwegian Prescription database (NorPD). Multivariable Cox regression models were performed to identify predictors of discontinuation. RESULTS: At the 2nd year of follow-up, a significant association between vaginal dryness and high concentrations of tamoxifen, Z-4'-OHtam and tam-NoX was identified. NorPD showed a tamoxifen-discontinuation rate of 17.9% at 5 years and drug monitoring demonstrated similar rates. Nausea, vaginal dryness and chemotherapy-naive status were significant risk factors for tamoxifen discontinuation. CONCLUSIONS: This real-world data study suggests that measurements of tamoxifen metabolite concentrations may be predictive of vaginal dryness in breast cancer patients and verifies NorPD as a reliable source of adherence data.

Serum concentrations of active tamoxifen metabolites predict long-term survival in adjuvantly treated breast cancer patients.

BACKGROUND: Controversies exist as to whether the genetic polymorphisms of the enzymes responsible for the metabolism of tamoxifen can predict breast cancer outcome in patients using adjuvant tamoxifen. Direct measurement of concentrations of active tamoxifen metabolites in serum may be a more biological plausible and robust approach. We have investigated the association between CYP2D6 genotypes, serum concentrations of active tamoxifen metabolites, and long-term outcome in tamoxifen treated breast cancer patients. METHODS: From an original observational study comprising 817 breast cancer patients, 99 women with operable breast cancer were retrospectively included in the present study. This cohort of patients were adjuvantly treated with tamoxifen, had provided serum samples suitable for measuring tamoxifen metabolites, and were relapse-free at 3 years after the primary treatment commenced. The median follow-up time from this entry point to breast cancer death was 13.9 years. Patients were CYP2D6 genotyped and grouped into four CYP2D6 phenotype groups (Ultra rapid, extensive, intermediate, and poor metabolizers). Tamoxifen and nine metabolites were quantified in serum (n = 86) and compared with CYP2D6 phenotype groups and outcome. RESULTS: Breast cancer patients with low concentrations of Z-4-hydroxy-tamoxifen (Z-4OHtam; ≤ 3.26 nM) had a breast cancer-specific survival (BCSS) of 60% compared to 84% in patients with Z-4OHtam concentrations > 3.26 nM (p = 0.020, log-rank hazard ratio (HR) = 3.56, 95% confidence interval (CI) = 1.14-11.07). For patients with Z-4-hydroxy-N-desmethyl-tamoxifen (Z-endoxifen) levels ≤ 9.00 nM BCSS was 57% compared to 84% for patients with concentrations > 9.00 nM (p = 0.029, HR = 3.73, 95% CI = 1.05-13.22). Low concentrations of Z-4OHtam and Z-endoxifen were associated with poorer survival also after adjusting for clinically relevant variables (HR = 4.27, 95% CI = 1.35-13.58, and HR = 3.70, 95% CI = 1.03-13.25, respectively). Overall survival analysis showed similar survival differences for both active metabolites. The Antiestrogen Activity Score showed comparable effects, but did not improve the prognostic information. CONCLUSIONS: Patients with Z-4OHtam and Z-endoxifen concentrations lower than 3.26 nM or 9.00 nM, respectively, showed an adverse outcome. Our results suggest that direct measurement of active tamoxifen metabolite concentrations could be of clinical value. Validation in larger study cohorts is warranted.

Menopause as a predictor of new-onset asthma: A longitudinal Northern European population study.

BACKGROUND: There is limited and conflicting evidence on the effect of menopause on asthma. OBJECTIVES: We sought to study whether the incidence of asthma and respiratory symptoms differ by menopausal status in a longitudinal population-based study with an average follow-up of 12 years. METHODS: The Respiratory Health in Northern Europe study provided questionnaire data pertaining to respiratory and reproductive health at baseline (1999-2001) and follow-up (2010-2012). The study cohort included women aged 45 to 65 years at follow-up, without asthma at baseline, and not using exogenous hormones (n = 2322). Menopausal status was defined as nonmenopausal, transitional, early postmenopausal, and late postmenopausal. Associations with asthma (defined by the use of asthma medication, having asthma attacks, or both) and respiratory symptoms scores were analyzed by using logistic (asthma) and negative binomial (respiratory symptoms) regressions, adjusting for age, body mass index, physical activity, smoking, education, and study center. RESULTS: The odds of new-onset asthma were increased in women who were transitional (odds ratio, 2.40; 95% CI, 1.09-5.30), early postmenopausal (odds ratio, 2.11; 95% CI, 1.06-4.20), and late postmenopausal (odds ratio, 3.44; 95% CI, 1.31-9.05) at follow-up compared with nonmenopausal women. The risk of respiratory symptoms increased in early postmenopausal (coefficient, 0.40; 95% CI, 0.06-0.75) and late postmenopausal (coefficient, 0.69; 95% CI, 0.15-1.23) women. These findings were consistent irrespective of smoking status and across study centers. CONCLUSIONS: New-onset asthma and respiratory symptoms increased in women becoming postmenopausal in a longitudinal population-based study. Clinicians should be aware that respiratory health might deteriorate in women during reproductive aging.

Antioxidant profile of strawberry tree honey and its marker homogentisic acid in several models of oxidative stress.

The antioxidant activity of several honeys was evaluated considering the different contribution of entire samples. The strawberry tree honey emerged as the richest in total phenols and the most active honey in the DPPH and FRAP tests, and could protect cholesterol against oxidative degradation (140°C). Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA), the main phenolic compound from strawberry tree honey, showed interesting antioxidant and antiradical activities, and protective effect against thermal-cholesterol degradation, comparable to those of well known antioxidants. Moreover, the pre-treatment with HGA significantly preserved liposomes and LDL from Cu(2+)-induced oxidative damage at 37°C for 2h, inhibiting the reduction of polyunsaturated fatty acids and cholesterol and the increase of their oxidative products. This phenol had no toxic effect in human intestinal epithelial Caco-2 cells within the concentration range tested (5-1000µM). HGA was able to pass through the Caco-2 monolayers, the apparent permeability coefficients (Papp) in the apical-to-basolateral and basolateral-to-apical direction were 3.48±1.22×10(-6) and 2.18±0.34×10(-6)cm/s, respectively, suggesting a passive diffusion pathway as the dominating process. The results of the work qualify HGA as natural antioxidant, able to exert a significant in vitro protective effect and to contribute to the strawberry tree honey antioxidant activity.

Biodiversity of Salix spp. honeydew and nectar honeys determined by RP-HPLC and evaluation of their antioxidant capacity.

Rare unifloral willow (Salix spp.) honeys obtained from nectar or honeydew were investigated by direct RP-HPLC-DAD method in order to identify and quantify compounds that can be used as possible markers of their origin. Antioxidant and antiradical activities of willow honeys were evaluated using FRAP (=ferric reducing antioxidant assay) and DPPH (=1,1-diphenyl-2-picrylhydrazyl radical) tests, respectively. Also HMF (=5-(hydroxymethyl)furfural), diastase activity, and CIE L*a*b*C*h* chromatic coordinates were evaluated. Abscisic acids (ABA) are typical of willow nectar honey, with a predominance of (Z,E)-ABA on (E,E)-ABA (98.2 and 31.7 mg/kg, resp.). Kinurenic acid and salicylic acid are useful to mark willow honeydew honey. The proposed HPLC-DAD method proved to be easy and reliable to identify the two different Salix spp. honeys, being not affected from any sample preparation artifact. Total antioxidant activity measured with the FRAP assay ranged from 3.2 to 12.6 mmol Fe(2+) /kg, and the antiradical activity measured with the DPPH assay ranged from 0.6 to 3.0 mmol TEAC (=Trolox equivalent antioxidant capacity)/kg in nectar and honeydew honeys, respectively. Salix spp. nectar and honeydew honeys proved to be two completely different honeys, because, besides color attributes, they show different antioxidant properties and specific compounds.

Association of CYP2D6 genotype and tamoxifen metabolites with breast cancer recurrence in a low-dose trial.

Low-dose tamoxifen halves recurrence in non-invasive breast cancer without significant adverse events. Some adjuvant trials with tamoxifen 20 mg/day had shown an association between low endoxifen levels (9-16 nM) and recurrence, but no association with CYP2D6 was shown in the NSABP P1 and P2 prevention trials. We studied the association of CYP2D6 genotype and tamoxifen metabolites with tumor biomarkers and recurrence in a randomized phase III trial of low-dose tamoxifen. Median (IQR) endoxifen levels at year 1 were 8.4 (5.3-11.4) in patients who recurred vs 7.5 (5.1-10.2) in those who did not recur (p = 0.60). Tamoxifen and metabolites significantly decreased C-reactive protein (CRP, p < 0.05), and a CRP increase after 3 years was associated with higher risk of recurrence (HR = 4.37, 95% CI, 1.14-16.73, P = 0.03). In conclusion, endoxifen is below 9 nM in most subjects treated with 5 mg/day despite strong efficacy and there is no association with recurrence, suggesting that the reason for tamoxifen failure is not poor drug metabolism. Trial registration: ClinicalTrials.gov, Identifier: NCT01357772 .

Low Z-4OHtam concentrations are associated with adverse clinical outcome among early stage premenopausal breast cancer patients treated with adjuvant tamoxifen.

Low steady-state levels of active tamoxifen metabolites have been associated with inferior treatment outcomes. In this retrospective analysis of 406 estrogen receptor-positive breast cancer (BC) patients receiving adjuvant tamoxifen as initial treatment, we have associated our previously reported thresholds for the two active metabolites, Z-endoxifen and Z-4-hydroxy-tamoxifen (Z-4OHtam), with treatment outcomes in an independent cohort of BC patients. Among all patients, metabolite levels did not affect survival. However, in the premenopausal subgroup receiving tamoxifen alone (n = 191) we confirmed an inferior BC -specific survival in patients with the previously described serum concentration threshold of Z-4OHtam ≤ 3.26 nm (HR = 2.37, 95% CI = 1.02-5.48, P = 0.039). The 'dose-response' survival trend in patients categorized to ordinal concentration cut-points of Z-4OHtamoxifen (≤ 3.26, 3.27-8.13, > 8.13 nm) was also replicated (P-trend log-rank = 0.048). Z-endoxifen was not associated with outcome. This is the first study to confirm the association between a published active tamoxifen metabolite threshold and BC outcome in an independent patient cohort. Premenopausal patients receiving 5-year of tamoxifen alone may benefit from therapeutic drug monitoring to ensure tamoxifen effectiveness.

Ultraviolet radiation as a predictor of sex hormone levels in postmenopausal women: A European multi-center study (ECRHS).

BACKGROUND: Solar ultraviolet radiation (UVR) affects the body through pathways that exhibit positive as well as negative health effects such as immunoregulation and vitamin D production. Different vitamin D metabolites are associated with higher or lower concentrations of estrogens and may thus alter the female sex hormone balance. OBJECTIVE: To study whether exposure to UVR, as a modifiable lifestyle factor, is associated with levels of sex hormones (17ß-estradiol, estrone, estrone 3-sulfate, testosterone, dehydroepiandrosterone sulfate), gonadotropins (follicle stimulating hormone, luteinizing hormone) as well as sex hormone binding globulin in postmenopausal women, and thus investigate whether managing UVR exposure can influence the hormone balance, with potential benefits for the biological aging process. METHODS: The study included 580 postmenopausal women from six European countries, participating in the European Community Respiratory Health Survey (2010-2014). Average UVR exposure during the month before blood sampling was estimated based on personal sun behavior and ambient levels. Hormone concentrations were measured in serum using state-of-the-art methods. Subsequently we applied linear mixed-effects models, including center as random intercept, hormone concentrations (one at a time) as outcome and UVR, age, skin type, body mass index, vitamin D from dietary sources, smoking, age at completed full-time education and season of blood sampling as fixed-effect predictors. RESULTS: One interquartile range increase in UVR exposure was associated with decreased levels of 17ß-estradiol (-15.6 pmol/L, 95 % Confidence Interval (CI): -27.69, -3.51) and estrone (-13.36 pmol/L, 95 % CI: -26.04, -0.68) and increased levels of follicle stimulating hormone (9.34IU/L, 95 % CI: 2.91, 15.77) and luteinizing hormone (13.86 IU/daL, 95 % CI: 2.48, 25.25). CONCLUSIONS: Exposure to UVR is associated with decreased estrogens and increased gonadotropins in postmenopausal women, a status associated with osteoporosis, lung function decline and other adverse health effects. This study indicates that managing UVR exposure has potential to influence the hormone balance and counteract adverse health conditions after menopause.

Cytotoxic tirucallane triterpenoids from Melia azedarach fruits.

The phytochemical investigation of the dichloromethane-soluble part of the methanol extract obtained from the fruits of Melia azedarach afforded one new tirucallane-type triterpene, 3-alpha-tigloylmelianol and three known tirucallanes, melianone, 21-beta-acetoxy-melianone, and methyl kulonate. The structure of the isolated compounds was mainly determined by 1D and 2D NMR experiments as well as HPLC-Q-TOF mass spectrometry. The cytotoxicity of the isolated compounds toward the human lung adenocarcinoma epithelial cell line A549 was determined, while no activity was observed against the phytonematode Meloidogyne incognita.

First-in-human pharmacokinetics of tamoxifen and its metabolites in the milk of a lactating mother: a case study.

BackgroundBreast cancer represents the most frequent neoplasm diagnosed in women of childbearing age. When the tumour is oestrogen receptor-positive, tamoxifen is among the recommended endocrine treatments. Lactating women are advised not to breastfeed while receiving tamoxifen. However, information about tamoxifen transfer into breast milk is lacking.MethodsWe measured the concentration of tamoxifen and its metabolites by liquid chromatography-tandem mass spectrometry in the milk of a nursing mother that was treated for pregnancy-associated breast cancer diagnosed a few months after delivery. She was advised not to breastfeed her child and she collected milk samples for 23 days while the baby was fed with formula.ResultsTamoxifen concentrations in milk increased reaching a maximum of 214 nM. The two active metabolitesZ-4-hydroxy-tamoxifen and Z-endoxifen, could not be quantified in milk the first days after tamoxifen intake, but increased over time and reached clinically significant levels after day 18.ConclusionThis study demonstrates for the first time in human that tamoxifen and its metabolites transfer into milk. Since tamoxifen has a complete oral bioavailability, a long half-life (>7 days) and may interfere with the normal development of the infant, mothers should not breastfeed during tamoxifen treatment.

Exogenous female sex steroids may reduce lung ageing after menopause: A 20-year follow-up study of a general population sample (ECRHS).

OBJECTIVES: Menopause involves hypoestrogenism, which is associated with numerous detrimental effects, including on respiratory health. Hormone replacement therapy (HRT) is often used to improve symptoms of menopause. The effects of HRT on lung function decline, hence lung ageing, have not yet been investigated despite the recognized effects of HRT on other health outcomes. STUDY DESIGN: The population-based multi-centre European Community Respiratory Health Survey provided complete data for 275 oral HRT users at two time points, who were matched with 383 nonusers and analysed with a two-level linear mixed effects regression model. MAIN OUTCOME MEASURES: We studied whether HRT use was associated with the annual decline in forced vital capacity (FVC) and forced expiratory volume in one second (FEV1). RESULTS: Lung function of women using oral HRT for more than five years declined less rapidly than that of nonusers. The adjusted difference in FVC decline was 5.6 mL/y (95%CI: 1.8 to 9.3, p = 0.01) for women who had taken HRT for six to ten years and 8.9 mL/y (3.5 to 14.2, p = 0.003) for those who had taken it for more than ten years. The adjusted difference in FEV1 decline was 4.4 mL/y (0.9 to 8.0, p = 0.02) with treatment from six to ten years and 5.3 mL/y (0.4 to 10.2, p = 0.048) with treatment for over ten years. CONCLUSIONS: In this longitudinal population-based study, the decline in lung function was less rapid in women who used HRT, following a dose-response pattern, and consistent when adjusting for potential confounding factors. This may signify that female sex hormones are of importance for lung ageing.

Temporomandibular Disorders Related to Stress and HPA-Axis Regulation.

Temporomandibular disorders (TMDs) are characterized by pain and dysfunction in the masticatory apparatus and the temporomandibular joint (TMJ). Previous trauma, stress symptoms, psychosocial impairment, and catastrophizing have been related to TMD. To assess if the hypothalamic-pituitary-adrenal (HPA) axis is upregulated in TMD patients, we performed a cross-sectional study with saliva from 44 TMD patients and 44 healthy sex- and age-matched controls for cortisol (F) and cortisone (E) with liquid chromatography-tandem mass spectrometry. Furthermore, we calculated the F/E ratio for the evaluation of 11ß-hydroxysteroid dehydrogenase activity. We also assessed anxiety/depression and pain catastrophizing scores from a questionnaire that participants completed prior to the examination. We found that F (P=0.01), E (P=0.04), the F/E ratio (P=0.002), and the sum of glucocorticoids (E + E) in saliva (P=0.02) were significantly higher in the TMD group. Anxiety/depression and catastrophizing scores were also significantly higher in the TMD group (P < 0.0001). Our findings indicate that patients with TMDs may have an upregulated HPA axis with higher F secretion from the adrenal cortex. Anxiety/depression and pain catastrophizing scores were significantly higher in the TMD group, and psychological factors may contribute to chronic upregulation of the HPA axis.

Antioxidant capacity and vasodilatory properties of Mediterranean food: the case of Cannonau wine, myrtle berries liqueur and strawberry-tree honey.

The aim of this work was to use different assays to evaluate the antioxidant and vasodilatory properties of three typical food products from the Mediterranean area and to correlate these activities with their phenolic content. For this purpose, red wines Cannonau, liqueurs obtained by cold maceration of myrtle (Myrtus communis L.) berries and bitter honeys obtained from strawberry-tree flowers (Arbutus unedo L.) were analysed. The total phenolic (TP) content was measured spectrophotometrically with a modified Folin-Ciocalteau method and phenolic compounds were identified and dosed by HPLC-DAD and LC-MS/MS. Antioxidant activities were evaluated with DPPH, FRAP and ABTS assays and the in vitro vasodilatory effects were assessed using norepinephrine precontracted rat aortic rings. Cannonau wines and myrtle liqueurs showed high levels of TP (1978±279 and 1741±150mg GAE/L, respectively), linearly correlated to the results of FRAP, ABTS, and DPPH assays. Their maximal vasodilatory activity was 61.7±4.1% and 53.0±3.0%, respectively. Although strawberry-tree honey contained relatively high levels of phenolic compounds (922±38mg GAE/kg), it did not induce vasodilation, even at the highest dose tested (0.206g/L). These results indicate that foods with high levels of phenolic compounds should be studied using several different biological assays before being recommended to the general public as functional foods.

Riboflavin and lumichrome in Dalmatian sage honey and other unifloral honeys determined by LC-DAD technique.

Riboflavin (vitamin B(2)) and its metabolite lumichrome were quantified in 117 samples from 11 unifloral honeys types (Arbutus unedo L., Asphodelus microcarpus Salzm. et Viv., Citrus spp., Eucalyptus spp., Hedysarum coronarium L., Castanea sativa L. honeydew, Mentha spp., Paliurus spina-christi., Salix spp., Salvia officinalis L., Satureja spp.). The quantification of these two compounds was performed by LC-DAD method which does not require sample purification. The proposed method in our study has low limits of detection and quantification, very good linearity in a large concentration range and very good precision. It allows simultaneous determination of 5-hydroxymethylfurfural (HMF) and known chemical biomarkers of unifloral honeys such as abscisic acid diastereomers, homogentisic acid, methyl syringate and kynurenic acid. No statistical correlation was observed between riboflavin and lumichrome content. Although, the concentration of vitamin B(2) in honey may be too low (<6.1mg/kg) to generate interest in the field of nutrition, the presence of its main metabolite lumichrome may be useful to determine the botanical origin of certain unifloral honeys. In fact, the analysis of 11 unifloral honey types showed that Dalmatian sage (S. officinalis L.) honey is characterised by unusual high levels of lumichrome (20.2±2.6mg/kg). The botanical origin of lumichrome from sage flower was assessed by analysing bee-stomach extracts. Other analytical parameters, such as total phenols, antioxidant and antiradical activities, HMF and diastase activity were studied in Dalmatian sage honey.

Lumichrome and phenyllactic acid as chemical markers of thistle (Galactites tomentosa Moench) honey.

HPLC-DAD-MS/MS chromatograms of thistle (Galactites tomentosa Moench) unifloral honeys, previously selected by sensory evaluation and melissopalynological analysis, showed high levels of two compounds. One was characterized as phenyllactic acid, a common acid found in honeys, but the other compound was very unusual for honeys. This compound was extracted from honey with ethyl acetate and purified by SPE using C(18), SiOH, and NH(2) phases. Its structure was elucidated on the basis of extensive 1D and 2D NMR experiments as well as HPLC-MS/MS and Q-TOF analysis, and it was identified as lumichrome (7,8-dimethylalloxazine). Lumichrome is known to be the main product of degradation obtained in acid medium from riboflavin (vitamin B(2)), and this is the first report of the presence of lumichrome in honeys. Analysis of the G. tomentosa raw honey and flowers extracts confirmed the floral origin of this compound. The average amount of lumichrome in thistle honey was 29.4 ± 14.9 mg/kg, while phenyllactic acid was 418.6 ± 168.9 mg/kg. Lumichrome, along with the unusual high level of phenyllactic acid, could be used as a marker for the botanical classification of unifloral thistle (G. tomentosa) honey.