Maternal administration of superoxide dismutase and catalase in phenytoin teratogenicity1

Embryonic bioactivation and formation of reactive oxygen species (ROS) are implicated in the mechanism of phenytoin teratogenicity. This in vivo study in pregnant CD-1 mice evaluated whether maternal administration of the antioxidative enzymes superoxide dismutase (SOD) and/or catalase conjugated with polyethylene glycol (PEG) could reduce phenytoin teratogenicity. Initial studies showed that pretreatment with PEG-SOD alone (0.5–20 KU/kg IP 4 or 8 h before phenytoin) actually increased the teratogenicity of phenytoin (65 mg/kg IP on gestational days [GD] 11 and 12, or 12 and 13) (p < .05), and appeared to increase embryonic protein oxidation. Combined pretreatment with PEG-SOD and PEG-catalase (10 KU/kg 8 or 12 h before phenytoin) was not embryo-protective, nor was PEG-catalase alone, although PEG-catalase alone reduced phenytoin-initiated protein oxidation in maternal liver (p < .05). However, time-response studies with PEG-catalase (10 KU/kg) on GDs 11, or 11 and 12, showed maximal 50-100% increases in embryonic activity sustained for 8-24 h after maternal injection (p < .05), and dose-response studies (10–50 KU/kg) at 8 h showed maximal respective 4-fold and 2-fold increases in maternal and embryonic activities with a 50 KU/kg dose (p < .05). In controls, embryonic catalase activity was about 4% of that in maternal liver, although with catalase treatment, enhanced embryonic activity was about 2% of enhanced maternal activity (p < .05). PEG-catalase pretreatment (10-50 KU/kg 8 h before phenytoin) also produced a dose-dependent inhibition of phenytoin teratogenicity, with maximal decreases in fetal cleft palates, resorptions and postpartum lethality at a 50 KU/kg dose (p < .05). This is the first evidence that maternal administration of PEG-catalase can substantially enhance embryonic activity, and that in vivo phenytoin teratogenicity can be modulated by antioxidative enzymes. Both the SOD-mediated enhancement of phenytoin teratogenicity, and the inhibition of phenytoin teratogenicity by catalase, indicate a critical role for ROS in the teratologic mechanism, and the teratologic importance of antioxidative balance.     

Fetal oxidative stress mechanisms of neurodevelopmental deficits and exacerbation by ethanol and methamphetamine

In utero exposure of mouse progeny to alcohol (ethanol, EtOH) and methamphetamine (METH) causes substantial postnatal neurodevelopmental deficits. One emerging pathogenic mechanism underlying these deficits involves fetal brain production of reactive oxygen species (ROS) that alter signal transduction, and/or oxidatively damage cellular macromolecules like lipids, proteins, and DNA, the latter leading to altered gene expression, likely via non‐mutagenic mechanisms. Even physiological levels of fetal ROS production can be pathogenic in biochemically predisposed progeny, and ROS formation can be enhanced by drugs like EtOH and METH, via activation/induction of ROS‐producing NADPH oxidases (NOX), drug bioactivation to free radical intermediates by prostaglandin H synthases (PHS), and other mechanisms. Antioxidative enzymes, like catalase in the fetal brain, while low, provide critical protection. Oxidatively damaged DNA is normally rapidly repaired, and fetal deficiencies in several DNA repair proteins, including oxoguanine glycosylase 1 (OGG1) and breast cancer protein 1 (BRCA1), enhance the risk of drug‐initiated postnatal neurodevelopmental deficits, and in some cases deficits in untreated progeny, the latter of which may be relevant to conditions like autism spectrum disorders (ASD). Risk is further regulated by fetal nuclear factor erythroid 2‐related factor 2 (Nrf2), a ROS‐sensing protein that upregulates an array of proteins, including antioxidative enzymes and DNA repair proteins. Imbalances between conceptal pathways for ROS formation, versus those for ROS detoxification and DNA repair, are important determinants of risk. Birth Defects Research (Part C) 108:108–130, 2016. © 2016 Wiley Periodicals, Inc.     

Varying susceptibility of pulmonary cytokine production to lipopolysaccharide in mice

Objectives: The lipopolysaccharide (LPS)-induced acute lung injury (ALI) model has been widely applied for pathophysiological and pharmacological research. The aim of present study is to understand the variation of acute pulmonary inflammation between mouse strains. Methods: The present study investigated the susceptibility of acute production of inflammatory mediators, e.g. cytokines, chemokines and others, to LPS in C57BL/6J, Balb/cJ, DBA/1J, CD-1, NMRI, DBA/2J, A/J, and C3H/HeN mice. Results: The susceptibility to intra-tracheal challenge with LPS varied between measured variables, durations and strains. General lung hyper-reactive susceptibility to LPS-induced pulmonary production of 6–8 inflammatory mediators followed the order NMRI, Balb/cJ, C3H/HeN, A/J, C57BL/6J, DBA/1J, DBA/2J and CD-1 mice at 4 h, and A/J, C3H/HeN, CD-1, NMRI, C57BL/6J, Balb/cJ, DBA/2J and DBA/1J mice at 24 h. Conclusions: Our data provide information for scientists to consider the proper strain of mice for the measurement of specific inflammatory mediators and to select sensitive or resistant mouse strains for understanding genetic variation in the pathogenesis and for the screening of target-oriented drug development.     

Effects of lead exposure on the expression of amyloid β and phosphorylated tau proteins in the C57BL/6 mouse hippocampus at different life stages

The objective of this study was to investigate the effects of lead exposure on spatial learning and memory capacity and the expression of amyloid β and phosphorylated tau proteins in the mouse hippocampus. A total of 24 adult C57BL/6 mice (12 of each sex) were mated at a 1:1 ratio. After delivery, the litters were normalised to 6 pups per litter. During the lactation period, the pups were randomly separated into four groups: control, early exposure, late exposure, or long-term exposure. These groups were not exposed to lead, exposed to lead from birth to week 24, exposed to lead from week 24 to week 48, or exposed to lead from birth to 48 weeks of age, respectively. Lead exposure was induced by providing Pb-contaminated drinking water at a concentration of 0.1%. All of the pups were fed until 72 weeks of age, at which time their spatial learning and memory capacity was evaluated via the Morris water maze test. Then, the lead levels in their blood and hippocampus were measured via graphite furnace atomic absorption spectrometry. The protein expression of amyloid β and phosphorylated tau in the hippocampus was detected via Western blot. The results revealed that the hippocampal and blood lead levels were significantly higher in all of the groups exposed to lead than the control group (P < 0.05). The spatial learning and memory performances of the lead-exposed groups were much poorer than those of the control group (P < 0.05). The expression levels of amyloid β and phosphorylated tau proteins were increased in the lead-exposed groups compared to the control group (P < 0.05). The enhanced expressions of amyloid β and phosphorylated tau proteins might contribute to the impairment in spatial learning and memory in the lead-exposed mice.     

Thromboxane synthase expression and correlation with VEGF and angiogenesis in non-small cell lung cancer

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n = 46) by EIA. TXB₂ levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB₂ levels were correlated with VEGF (p < 0.05). TXS and VEGF were expressed to a varying degree in NSCLC tissue. TXS was associated with VEGF (p < 0.0001) and microvessel density (CD-31; p < 0.05). TXS and VEGF expression levels were higher in adenocarcinoma (p < 0.0001) and female patients (p < 0.05). Stable overexpression of TXS increased VEGF secretion in-vitro. While no significant association with patient survival was observed for either TXS or VEGF in our patient cohort, TXS overexpression significantly (p < 0.05) increased tumor growth in-vivo. TXS overexpression was also associated with higher levels of VEGF, microvessel density, and reduced apoptosis in xenograft tumors. Conclusion: TXS promotes tumor growth in-vivo in NSCLC, an effect which is at least partly mediated through increased tumor angiogenesis.     

Placental antiangiogenic prolactin fragments are increased in human and rat maternal diabetes

Introduction/objectivesThe role of the placenta in diabetic mothers on fetal development and programming is unknown. Prolactin (PRL) produced by decidual endometrial cells may have an impact. Although full-length PRL is angiogenic, the processed form by bone morphogenetic protein-1 (BMP-1) and/or cathepsin D (CTSD) is antiangiogenic.The objectives were to investigate the involvement of decidual PRL and its antiangiogenic fragments in placentas from type-1 diabetic women (T1D) and from pregnant diabetic rats with lower offspring weights than controls.MethodsPRL, BMP-1, and CTSD gene expressions and PRL protein level were assessed in T1D placentas (n = 8) at delivery and compared to controls (n = 5). Wistar rats received, at day 7 of pregnancy, streptozotocin (STZ) (n = 5) or nicotinamide (NCT) plus STZ (n = 9) or vehicle (n = 9). Placental whole-genome gene expression and PRL western blots were performed at birth.ResultsIn human placentas, PRL (p < 0.05) and BMP-1 (p < 0.01) gene expressions were increased with a higher amount of cleaved PRL (p < 0.05) in T1D than controls. In rats, diabetes was more pronounced in STZ than in NCT–STZ group with intra-uterine growth restriction. Decidual prolactin-related protein (Dprp) (p < 0.01) and Bmp-1 (p < 0.001) genes were up-regulated in both diabetic groups, with an increased cleaved PRL amount in the STZ (p < 0.05) and NCT–STZ (p < 0.05) groups compared to controls. No difference in CTSD gene expression was observed in rats or women.ConclusionsAlterations in the levels of the PRL family are associated with maternal diabetes in both rats and T1D women suggesting that placental changes in these hormones impact on fetal development.     

Increased survival of neonatal pigs by supplementing medium-chain triglycerides in late-gestating sow diets

Two experiments were conducted to study the efficacy and causes of medium-chain triglycerides (MCT) in sow diet in improving the survival of neonatal pigs. In Experiment 1, beginning on d84 of gestation and continuing through d28 of lactation, 51 sows were fed corn–soybean meal diet mixed with either soybean oil (SO; n=17), coconut oil (CO; n=18), or MCT (n=16) in a proportion of 9 : 1 by weight. The highest improvement in survival of pigs by sows fed MCT (p<0.01) or CO (p<0.05) was observed during the first three days after birth in pigs weighing <1100 g at birth, compared with sows fed SO. Their three-day survival was 98.6, 80.0 and 47.6%, respectively, for MCT, CO and SO groups. In Experiment 2, beginning on d84 of gestation and continuing through farrowing, 24 sows, 8 sows per treatment, were fed diets as in Experiment 1. Liver glycogen content of pigs 4 h after born from sows fed MCT (p<0.10) and CO (p<0.01), and muscle glycogen of pigs from sows fed MCT (p<0.01) and CO (p<0.10) were increased, compared to those of pigs from sows fed SO. Plasma albumin was increased by MCT and CO (p<0.01), relative to SO. The results suggest that MCT or CO in sow diets may enhance the body glycogen stores and maturity of pigs at birth and, hence, their survival, particularly in pigs with low birth weight during the first three days of life.     

A microsphere-based duplex competitive immunoassay for the simultaneous measurements of aldosterone and testosterone in small sample volumes: Validation in human and mouse plasma

BackgroundThe small blood volumes available in rodent studies often limit adequate quantification of all hormones of interest. We report here the development of two new assays combining an extraction step with multiplex immunoassay (MIA) technology for the simultaneous determination of aldosterone and testosterone in 50 μl sample volume.MethodsFollowing solvent extraction, aldosterone and testosterone competitive immunoassays are performed incorporating biotinylated tracers and antibody-coated beads each having a unique fluorescence. Quantification is via addition of streptavidin–R–phycoerythrin (SA–PE). The assays were validated and compared to established methods. Baseline hormone levels in mice from four different strains, and changes after ACTH and HCG stimulation in CD-1 mice are shown.ResultsThe assays are sensitive (aldosterone 15 pg/ml, testosterone 12 pg/ml), reproducible (intra-/inter-assay imprecision aldosterone 5.1–15.6%/9.9–15.8% and testosterone 9.7–10.9%/7.7–11.4%) and correlate significantly to established assays (r = 0.94–0.95). Baseline aldosterone levels varied between strains, but not between the genders. Testosterone was significantly higher in male of all strains except in C57BL/6× NMRI mice. After ACTH injection, aldosterone (median, interquartile range) rose from 354 (261–396) pg/ml to 2008 (875–2467) in male and from 260 (210–576) to 1120 (734–1528) in female CD-1 mice. HCG injection in the same strain increased testosterone in male mice only (3.5 (0.4–8.3) ng/ml to 31.8 (30.4–33.9) ng/ml, P < 0.01).ConclusionsWe describe a MIA for the simultaneous measurement of aldosterone and testosterone in small volumes after extraction. In addition to presenting a new tool for steroid research in rodent models, our data show strain-dependent differences in steroid hormone metabolism in rodents.     

The relationship between fetal and maternal selenium concentrations in sheep and goats

In this study, biological samples (slaughterhouse material) were collected from 30 sheep and 36 goats and classified according to gestational stage into either early or late gestation. Samples consisted of allantoic fluid, amniotic fluid, fetal liver, fetal kidney, fetal thyroid gland, maternal plasma and liver to determine selenium (Se) concentrations throughout gestation. The Se concentrations in the allantoic fluid, fetal liver and kidney increased significantly (p < 0.01) during late gestation. Concurrently, the Se concentrations in amniotic fluid, maternal plasma and liver decreased significantly (p < 0.01) over time. Significant (p < 0.01) positive relationships were recorded between the age of the fetus and Se concentrations in the allantoic fluid (r = 0.57–0.75), fetal liver (r = 0.43–0.59) and kidney (r = 0.80–0.81) in both sheep and goats. A significant (p < 0.05) positive relationships were also recorded between the Se concentrations in the allantoic fluid and fetal liver (r = 0.35–0.37), the maternal plasma and liver Se concentrations (r = 0.37–0.57) between sheep and goats. A significant (p < 0.05) negative correlation was recorded between the Se concentrations in the allantoic fluid with maternal plasma of sheep (r = −0.41) as well as between the fetal liver and maternal liver Se (r = −0.22 to 0.50) and a negative correlation (r = −0.42 to 0.43) (p < 0.01) between Se concentrations in the fetal liver and amniotic fluid in both sheep and goats, respectively. Se concentration in the fetal liver was significantly (p < 0.01) higher than that of the kidney and thyroid. In the thyroid gland no morphological differences were noted. Strong fetal–maternal relationships in Se concentration were evident throughout the gestational period and dams seem to sacrifice Se levels in order to maintain that in the fetus. Se concentrations in the amniotic and allantoic fluids could be used as a possible indicator of the Se status of the fetus throughout gestation.     

Thyroxine administration prevents matrilineal intergenerational consequences of in utero ethanol exposure in rats

The neurodevelopmental fetal alcohol spectrum disorder (FASD) is characterized by cognitive and behavioral deficits in the offspring. Conferring the deficits to the next generation would increase overall FASD disease burden and prevention of this transmission could be highly significant. Prior studies showed the reversal of these behavioral deficits by low dose thyroxine (T4) supplementation to the ethanol-consuming mothers. Here we aim to identify whether prenatal ethanol (PE) exposure impairs hippocampus-dependent learning and memory in the second-generation (F2) progeny, and whether T4 administration to the ethanol-consuming dam can prevent it. Sprague-Dawley (S) dams received control diets (ad libitum and nutritional control) or ethanol containing liquid diet with and without simultaneous T4 (0.3 mg/L diet) administration. Their offspring (SS F1) were mated with naive Brown Norway (B) males and females generating the SB F2 and BS F2 progeny. Hippocampus-dependent contextual fear memory and hippocampal expression of the thyroid hormone-regulated type 3 deiodinase, (Dio3) and neurogranin (Nrgn) were assessed. SS F1 PE-exposed females and their SB F2 progeny exhibited fear memory deficits. T4 administration to the mothers of F1 females reversed these deficits. Although SS F1 PE-exposed males also experienced fear memory deficit, this was neither transmitted to their BS F2 offspring nor reversed by prenatal T4 treatment. Hippocampal Dio3 and Nrgn expression showed similar pattern of changes. Grandmaternal ethanol consumption during pregnancy affects fear memory of the matrilineal second-generation progeny. Low dose T4 supplementation prevents this process likely via altering allele-specific and total expression of Dio3 in the hippocampus.     

Milk ejection in mice LG/J x SM/J

In mammals, milk provision is crucial to offspring survival and growth from birth to weaning. Milk deficiency early in life may cause death or changes in the progeny metabolism that later may lead to obesity and metabolic disorders. This study investigates milk ejection (ME) the first day after birth (D1) in F₂ females from the intercross of LG/J and SM/J inbred mice strains. The absence of milk in F₃ pups?? stomach at D1 is directly associated with their survival (p?<?0.001) and growth pattern (p?<?0.001) in the early stages of life. Furthermore, late growth pattern is also affected by this lack of nutrients at D1 because pups that survive this absence, mostly males, are heavier at weaning (p?<?0.001) which, after necropsy, is shown to be due to significant higher total fat deposition (p?<?0.01). We performed QTL analysis for ME at D1 in these F₂ females. Maternal performance of ME revealed a complex genetic architecture which even though it contains only a single QTL (accounting for 8?% of the variation in ME), it is totally context-dependent on the genetic background. We discovered many regions involved in epistatic interactions that together with the single QTL explain 19?% of the genetic variation for this trait. Milk ejection is an important component of maternal care, and understanding the mechanisms modulating its variation, along with other maternal features, may help to disentangle the complexity that is the mother/offspring relationship.     

Genetic trends estimation for body weights of Kermani sheep at different ages using multivariate animal models

The objective of the present study was to estimate genetic trends for body weight traits at different ages in Kermani lambs. Data collected from 1993 to 2006 by the Kermani Breeding Station were analyzed. Genetic trends were estimated for birth weight (BW), weaning weight (WW), 6-month weight (6MW), 9-month weight (9MW) and yearling weight (YW). Maximum number of data was 2654 at birth, but only 1124 at yearling. Different appropriate models for investigation of each trait using multivariate analysis were applied. Variance component were estimated using Simplex procedure and breeding values of animals were predicted with Best Linear Unbiased Prediction (BLUP) methodology under multi-trait animal models. Genetic trends of studied traits were estimated by regressing mean of breeding values on birth year. Direct genetic trends were positive and significant for BW (p < 0.05) WW, 6MW, 9MW and YW (p < 0.01) and were 2, 125, 91, 81 and 156 g/year, respectively. Also, maternal trend for BW was positive and highly significant (p < 0.01) and was 3 g/year.     

Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR−/− mice

BackgroundBone marrow of blotchy mouse (blotchy marrow) reflects the function of transmembrane domain and relevant intramembrane sites of ATP7A in myeloid cells. By chronic infusion of angiotensin II, we previously found that blotchy marrow plays a minor role in regulating plasma copper. Moreover, the recipients of blotchy marrow presented a moderate reduction of plasma lipids and inflammatory mediator production. Little is known about whether these changes are a specific response to angiotensin II or reveal a more general role of ATP7A.Objective and designWe investigated if blotchy marrow reduces plasma lipids and inflammatory mediators induced by high-fat diets. To test this hypothesis, blotchy and control marrows were reconstituted to the recipient mice (irradiated male LDLR−/− mice), followed by high-fat-diet feeding for 4 months. At the end points, plasma metals (copper, zinc and iron), lipid profiling (cholesterol, triglyceride, phospholipids and lipoprotein) and six inflammatory mediators (lymphotacin, MCP3, MCP5, TIMP1, VEGF-A and IP-10) were measured. Parallel experiments were performed using male LDLR−/− mice fed either high-fat diets or chow diets for 4 months.ResultsIn addition to hyperlipidemia and low-grade inflammation, high-fat diets selectively increased plasma copper concentration compared to chow diets in LDLR−/− mice. After high-fat-diet feeding, the recipients with blotchy marrow showed a decrease in plasma copper (p < 0.01) and an increase in plasma iron (p < 0.05). The recipients with blotchy marrow also presented decreases in cholesterol (p < 0.01) and phospholipids (p < 0.05) in plasma. Surprisingly, plasma levels of MCP3 (p < 0.05), MCP5 (p < 0.05), TIMP1 (p < 0.01), VEGF-A (p < 0.01) and IP-10 (p < 0.01) were significantly increased in the recipients with blotchy marrow compared to controls; the increased levels of MCP3, MCP5 and TIMP1 were more than 50%.ConclusionOur studies showed that blotchy marrow counteracts the increased copper levels induced by high-fat diets, indicating that circulating myeloid cells can regulate blood copper levels via ATP7A. Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. Overall, blotchy marrow mediates divergent responses to angiotensin II and high-fat diets in vivo.     

Altered cellular redox status,sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH

BackgroundWe have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear.AimsSince the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life.MethodsFemale mice were fed either a control (C, 7% kcal fat) or HF (45% kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbβ, RORα, and Srebp1c) were measured in offspring livers.ResultsOffspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD⁺/NADH (p < 0.05, HF/HF vs C/C), Sirt1 (p < 0.001, HF/HF vs C/C), Sirt3 (p < 0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p < 0.05, C/HF and HF/HF vs C/C).ConclusionOur results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.     

In vivo evidence that Agxt2 can regulate plasma levels of dimethylarginines in mice

Elevated plasma concentrations of the asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse cardiovascular clinical outcomes. Both dimethylarginines can be degraded by alanine–glyoxylate aminotransferase 2 (Agxt2), which is also the key enzyme responsible for the degradation of endogenously formed β-aminoisobutyrate (BAIB). In the present study we wanted to investigate the effect of BAIB on Agxt2 expression and Agxt2-mediated metabolism of dimethylarginines. We infused BAIB or saline intraperitoneally for 7 days in C57/BL6 mice via minipumps. Expression of Agxt2 was determined in liver and kidney. The concentrations of BAIB, dimethylarginines and the Agxt2-specific ADMA metabolite α-keto-δ-(N(G),N(G)-dimethylguanidino)valeric acid (DMGV) was determined by LC–MS/MS in plasma and urine. As compared to controls systemic administration of BAIB increased plasma and urine BAIB levels by a factor of 26.5 (p < 0.001) and 25.8 (p < 0.01), respectively. BAIB infusion resulted in an increase of the plasma ADMA and SDMA concentrations of 27% and 31%, respectively, (both p < 0.05) and a 24% decrease of plasma DMGV levels (p < 0.05), while expression of Agxt2 was not different.Our data demonstrate that BAIB can inhibit Agxt2-mediated metabolism of dimethylarginines and show for the first time that endogenous Agxt2 is involved in the regulation of systemic ADMA, SDMA and DMGV levels. The effect of BAIB excess on endogenous dimethylarginine levels may have direct clinical implications for humans with the relatively common genetic trait of hyper-β-aminoisobutyric aciduria.     

Analysis of hydroxylation and nitration products of d-phenylalanine for in vitro and in vivo radical determination using high-performance liquid chromatography and photodiode array detection

d-Phenylalanine is capable of trapping reactive oxygen species (ROS) and reactive nitrogen species (RNS) by forming three major hydroxylation (o-, m-, p-tyrosine) and two major nitration products (nitrophenylalanine, nitrotyrosine). Here, we show how a method for the analysis of these phenylalanine derivatives was established using isocratic HPLC (Nucleosil120, C18 column) coupled with photodiode array detection and validated for cell-free in vitro and in vivo determination of radical formation. An ideal separation was achieved using a mobile phase consisting of 5% acetonitrile, 50 mM KH₂PO₄, pH 3.0, a column temperature of 35 °C and a flow rate of 1.0 mL/min. Limits of detection were in the range of 5–100 nM. Linearity was given within 5 nM–100 μM (correlation coefficient >0.999). Retention times as well as peak heights exhibited a high precision (RSD: ≤0.1% and <1.5%, respectively). The feasibility of d-phenylalanine for ROS/RNS measurement was demonstrated in a cell-free in vitro assay using peroxynitrite and by analysis of brain samples of mice treated with the dopaminergic neurotoxin 6-hydroxydopamine.     

Serum zinc levels of cord blood: Relation to birth weight and gestational period

BackgroundZn-deficiency has been associated with numerous alterations during pregnancy including low birth weight; however, the research relating neonatal zinc status and birth weight has not produced reliable results.ObjectiveTo compare the serum Zn-levels of cord blood in healthy newborns and low birth weight newborns, and to assess a possible relationship between zinc concentration and neonatal birth weight and gestational age.Material and methods123 newborns divided in “study group” (n = 50) with <2500 g birth weight neonates and “control group” (n = 73) with ≥2500 g birth weight neonates were enrolled. Study group was subdivided according to gestational age in preterm (<37 weeks) and full-term (≥37 weeks). Serum cord blood samples were collected and the Zn-levels were analyzed using flame Atomic Absorption Spectrophotometry method and the result was expressed in μmol/L. The Zn-levels were compared between the groups (Mann–Whitney-U test) and the Zn-levels were correlated with the birth weight and gestational age (Spearman's rank correlations).ResultsStatistically significant low positive correlation between Zn-levels and birth weight (ρ = 0.283; p = 0.005) was found. No statistically significant difference between Zn-levels of study and control groups [17.00 ± 0.43 vs. 18.16 ± 0.32 (p = 0.053)] was found. Statistically significant low positive correlation between Zn-levels and gestational age (ρ = 0.351; p = 0.001) was found. No statistically significant difference between Zn-levels of preterm as compare to full-term newborns [16.33 ± 0.42 vs. 18.43 ± 0.93 (p = 0.079)] was found. Zn-level of preterm subgroup was significantly lower compared to control group (p = 0.001).ConclusionsDespite low birth weight preterm neonates had significantly lower serum zinc levels of cord blood than healthy term neonates, the correlation between cord blood zinc levels and birth weight and gestational age was lower. The results are not enough to relate the change in cord blood zinc concentration to the birth weight values or gestational period. In relation to complicated pregnancies, further studies regarding zinc levels in blood in our population are required.     

Intranasal administration of a combination of choline chloride,vitamin C,and selenium attenuates the allergic effect in a mouse model of airway disease

Respiratory allergic disease is an inflammatory condition accompanied by oxidative stress. Supplementation of an anti-inflammatory agent with antioxidants may have a therapeutic effect. In this study, the effects of choline chloride in combination with antioxidants were evaluated via the intranasal route in a mouse model of allergic airway disease. Balb/c mice were sensitized on days 0, 7, and 14 and challenged on days 25–30 with cockroach extract (CE) and with a booster challenge on day 38. They were treated with choline chloride (ChCl; 1 mg/kg), vitamin C (Vit C; 308.33 mg/kg), and selenium (Se; 1 mg/kg) alone or in combination via the intranasal route on days 31, 33, 35, 37, and 39. The mice were sacrificed on day 40 to collect blood, bronchoalveolar lavage fluid, lungs, and spleen. Mice immunized with CE showed a significant increase in airway hyperresponsiveness (AHR), lung inflammation, Th2 cytokines, and the oxidative stress markers intracellular reactive oxygen species and 8-isoprostanes compared to the phosphate-buffered saline control group. A significant decrease was observed in these parameters with all the treatments (p<0.01). The highest decrease was noticed in the ChCl+Vit C+Se-treated group, with AHR decreased to the normal level. This group also showed the highest decrease in airway inflammation (p<0.001), IL-4 and IL-5 (p<0.001), IgE and IgG1 (p<0.001), NF-κB (p<0.001), and 8-isoprostane levels (p<0.001). Glutathione peroxidase activity, which was decreased significantly in CE-immunized mice, was restored to normal levels in this group (p<0.001). IL-10 level was decreased in CE-immunized mice and was restored to normal by combination treatment. The combination treatment induced FOXP3⁺ cells in splenocyte culture, responsible for the upregulation of IL-10. In conclusion, the combination of choline chloride, vitamin C, and selenium via the intranasal route reduces AHR, inflammation, and oxidative stress, probably by causing IL-10 production by FOXP3⁺ cells, and possesses therapeutic potential against allergic airway disease.     

Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity

AimImbalance of T-helper-cell (TH) subsets (TH1/TH2/TH17) and regulatory T-cells (Tregs) is suggested to contribute to the pathogenesis of Systemic lupus erythematosus (SLE). Therefore, we evaluated their cytokine secretion profile in SLE patients and their possible association with disease activity.MethodsSixty SLE patients, 24 rheumatoid arthritis (RA) patients and 24 healthy volunteers were included in this study. Demographic, clinical, disease activity and serological data were prospectively assessed. Plasma cytokines levels of TH1 (IL-12, IFN-γ), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-β) were measured by enzyme linked immunosorbent assays (ELISA).ResultsSLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL-12 (p < 0.001) and IL-10 (p < 0.05) but comparable levels of IL-23 and IL-4 and slight reduction (but statistically insignificant) of TGF-β levels compared to controls. IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) with disease activity. The RA group exhibited significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-β (p < 0.5) and lower IFN-γ (p < 0.001) and IL-10 (p < 0.01) than those of healthy subjects.ConclusionOur study showed a distinct profile of cytokine imbalance in SLE patients. Reduction in IFN-γ (TH1) and TGF-β1 (Treg) with the elevation in IL-6 and IL-17 (TH17) could imply skewing of T-cells toward TH17 cells. Breaking TH17/Treg balance in peripheral blood may play an important role in the development of SLE and could be responsible for an increased pro-inflammatory response especially in the active form of the disease.