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Background and objective Whether treatment with vitamin D receptor activators contributes to cardiovascular disease in patients with chronic kidney disease is a matter of debate. We studied mechanisms involved in vitamin D-related vascular calcifications in vivo and in vitro.
Methods Aortic calcifications were induced in subtotally nephrectomized (SNX) rats by treatment with a high dose (0.25 mu g/kg per day) of 1,25-dihydroxyvitamin D-3 (calcitriol) given for 6 weeks. Likewise, primary rat vascular smooth muscle cells (VSMCs) were incubated with calcitriol at concentrations ranging from 10(-11) to 10(-7) mol/l. Immunohistochemistry revealed that the aortic expression of osteopontin, osteocalcin and bone sialoprotein was significantly increased in calcitriol-treated SNX rats compared to untreated SNX controls. In addition, aortic expression of the transient receptor potential vanilloid calcium channel 6 (TRPV6) and calbindin D9k was significantly up-regulated by treatment with calcitriol. Furthermore, calcitriol significantly increased expression of the osteogenic transcription factor osterix. In-vitro studies showed similar results, confirming that these effects could be attributed to treatment with calcitriol.
Conclusions High-dose calcitriol treatment induces an osteoblastic phenotype in VSMC both in SNX rats and in vitro, associated with up-regulation of proteins regulating mineralization and calcium transport, and of the osteogenic transcription factor osterix.
We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N-2-(1-MIM)-dG, N-6-(1-MIM)-dA] in six tissues, as well as protein adducts [tau N-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N-2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N-6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.
This study aimed to estimate the optimal body size, limb segment length, and girth or breadth ratios of 100-m breaststroke performance in youth swimmers. In total, 59 swimmers [male: n= 39, age = 11.5 (1.3) y; female: n= 20, age = 12.0 (1.0) y] participated in this study. To identify size/shape characteristics associated with 100-m breaststroke swimming performance, we computed a multiplicative allometric log-linear regression model, which was refined using backward elimination. Results showed that the 100-m breaststroke performance revealed a significant negative association with fat mass and a significant positive association with the segment length ratio (arm ratio = hand length/forearm length) and limb girth ratio (girth ratio = forearm girth/wrist girth). In addition, leg length, biacromial breadth, and biiliocristal breadth revealed significant positive associations with the 100-m breaststroke performance. However, height and body mass did not contribute to the model, suggesting that the advantage of longer levers was limb-specific rather than a general whole-body advantage. In fact, it is only by adopting multiplicative allometric models that the previously mentioned ratios could have been derived. These results highlighted the importance of considering anthropometric characteristics of youth breaststroke swimmers for talent identification and/or athlete monitoring purposes. In addition, these findings may assist orienting swimmers to the appropriate stroke based on their anthropometric characteristics.
Abdominal and general adiposity are independently associated with mortality, but there is no consensus on how best to assess abdominal adiposity. We compared the ability of alternative waist indices to complement body mass index (BMI) when assessing all-cause mortality. We used data from 352,985 participants in the European Prospective Investigation into Cancer and Nutrition (EPIC) and Cox proportional hazards models adjusted for other risk factors. During a mean follow-up of 16.1 years, 38,178 participants died. Combining in one model BMI and a strongly correlated waist index altered the association patterns with mortality, to a predominantly negative association for BMI and a stronger positive association for the waist index, while combining BMI with the uncorrelated A Body Shape Index (ABSI) preserved the association patterns. Sex-specific cohort-wide quartiles of waist indices correlated with BMI could not separate high-risk from low-risk individuals within underweight (BMI<18.5 kg/m(2)) or obese (BMI30 kg/m(2)) categories, while the highest quartile of ABSI separated 18-39% of the individuals within each BMI category, which had 22-55% higher risk of death. In conclusion, only a waist index independent of BMI by design, such as ABSI, complements BMI and enables efficient risk stratification, which could facilitate personalisation of screening, treatment and monitoring.
To explore the genetic determinants of obesity and Type 2 diabetes (T2D), the German Center for Diabetes Research (DZD) conducted crossbreedings of the obese and diabetes-prone New Zealand Obese mouse strain with four different lean strains (B6, DBA, C3H, 129P2) that vary in their susceptibility to develop T2D. Genome-wide linkage analyses localized more than 290 quantitative trait loci (QTL) for obesity, 190 QTL for diabetes-related traits and 100 QTL for plasma metabolites in the out-cross populations. A computational framework was developed that allowed to refine critical regions and to nominate a small number of candidate genes by integrating reciprocal haplotype mapping and transcriptome data. The efficiency of the complex procedure was demonstrated for one obesity QTL. The genomic interval of 35 Mb with 502 annotated candidate genes was narrowed down to six candidates. Accordingly, congenic mice retained the obesity phenotype owing to an interval that contains three of the six candidate genes. Among these the phospholipase PLA2G4A exhibited an elevated expression in adipose tissue of obese human subjects and is therefore a critical regulator of the obesity locus. Together, our broad and complex approach demonstrates that combined- and comparative-cross analysis exhibits improved mapping resolution and represents a valid tool for the identification of disease genes.
Background Soy protein is effective in lowering plasma cholesterol, LDL cholesterol and triglyceride concentrations. It has not been conclusively answered, whether and to what extent other soy constituents may also contribute to this effect. Objective To investigate the change in blood lipid levels after application of two soy-based supplements containing soy protein either without (SuproSoy(R)) or with (Abacor(R)) soy fiber and phospholipids in a randomized placebo-controlled triple-armed study. Methods 121 hypercholesterolemic adults ( 66 females, 55 males) were recruited and randomly assigned to one of three treatments. Over 8 weeks they received daily either 25 g soy protein ( as a component of the supplements Abacor(R) or SuproSoy(R)) or 25 g milk protein ( as a component of placebo). Serum lipids were measured at baseline and after 4, 6 and 8 weeks. Results After 8 weeks of supplementation total cholesterol levels were reduced by 8.0 +/- 9.6% (Abacor(R)) and 3.4 +/- 8.3% (SuproSoy(R)); LDL cholesterol levels by 9.7 +/- 11.7% ( Abacor(R)) and 5.4 +/- 11.6% ( SuproSoy(R)); and Apolipoprotein B levels by 6.9 +/- 14.6% (Abacor(R)) and 4.0 +/- 12.4 % (SuproSoy(R)). Serum levels of HDL cholesterol and triglycerides remained unchanged. Conclusions A preparation combining isolated soy protein with soy fibers and phospholipids showed twice the lipid-lowering effect of a preparation containing isolated soy protein alone. Therefore, such soy-based supplements can be useful in reducing the cardiovascular risk
Background/Aims: Cortisol plays an important role during pregnancy. It controls maternal glucose metabolism and fetal development. Cortisol metabolism is partially controlled by the 11b-HSD2. This enzyme is expressed in the kidney and human placenta. The activity of the enzyme is partially controlled by functional polymorphisms: the HSD11B2[CA]n microsatellite polymorphism. The impact of this functional gene polymorphism on cortisol metabolism and potential effects on the newborn's is unknown so far. Methods: In the current prospective birth cohort study in southern Asia, we analyzed the association of the HSD11B2[CA]n microsatellite polymorphisms in 187 mothers and their newborn's on maternal and newborn's serum cortisol concentrations. Results: Using multivariable regression analyses considering known confounding ( gestational age, newborn's gender, the labor uterine contraction states and the timing during the day of blood taking), we showed that the fetal HSD11B2[CA]n microsatellite polymorphisms in the first intron was related to maternal cortisol concentration ( R2=0.26, B=96.27, p=0.007), whereas as the newborn's cortisol concentrations were independent of fetal and maternal HSD11B2[CA] n microsatellite polymorphism. Conclusions: Our study showed for the first time that the fetal HSD11B2[CA]n microsatellite polymorphism of the HSD11B2 gene in healthy uncomplicated human pregnancy is associated with maternal cortisol concentration. This indicates that fetal genes controlling cortisol metabolism may affect maternal cortisol concentration and hence physiology in healthy pregnant women.
Sphingolipids are major components of the plasma membrane. In particular, ceramide serves as an essential building hub for complex sphingolipids, but also as an organizer of membrane domains segregating receptors and signalosomes. Sphingomyelin breakdown as a result of sphingomyelinase activation after ligation of a variety of receptors is the predominant source of ceramides released at the plasma membrane. This especially applies to T lymphocytes where formation of ceramide-enriched membrane microdomains modulates TCR signaling. Because ceramide release and redistribution occur very rapidly in response to receptor ligation, novel tools to further study these processes in living T cells are urgently needed. To meet this demand, we synthesized nontoxic, azido-functionalized ceramides allowing for bio-orthogonal click-reactions to fluorescently label incorporated ceramides, and thus investigate formation of ceramide-enriched domains. Azido-functionalized C-6-ceramides were incorporated into and localized within plasma membrane microdomains and proximal vesicles in T cells. They segregated into clusters after TCR, and especially CD28 ligation, indicating efficient sorting into plasma membrane domains associated with T cell activation; this was abolished upon sphingomyelinase inhibition. Importantly, T cell activation was not abrogated upon incorporation of the compound, which was efficiently excluded from the immune synapse center as has previously been seen in Ab-based studies using fixed cells. Therefore, the functionalized ceramides are novel, highly potent tools to study the subcellular redistribution of ceramides in the course of T cell activation. Moreover, they will certainly also be generally applicable to studies addressing rapid stimulation-mediated ceramide release in living cells.
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
Wheat is one of the most consumed foods in the world and unfortunately causes allergic reactions which have important health effects. The α-amylase/trypsin inhibitors (ATIs) have been identified as potentially allergen components of wheat. Due to a lack of data on optimization of ATI extraction, a new wheat ATIs extraction approach combining solvent extraction and selective precipitation is proposed in this work. Two types of wheat cultivars (Triticum aestivum L.), Julius and Ponticus were used and parameters such as solvent type, extraction time, temperature, stirring speed, salt type, salt concentration, buffer pH and centrifugation speed were analyzed using the Plackett-Burman design. Salt concentration, extraction time and pH appeared to have significant effects on the recovery of ATIs (p < 0.01). In both wheat cultivars, Julius and Ponticus, ammonium sulfate substantially reduced protein concentration and inhibition of amylase activity (IAA) compared to sodium chloride. The optimal conditions with desirability levels of 0.94 and 0.91 according to the Doehlert design were: salt concentrations of 1.67 and 1.22 M, extraction times of 53 and 118 min, and pHs of 7.1 and 7.9 for Julius and Ponticus, respectively. The corresponding responses were: protein concentrations of 0.31 and 0.35 mg and IAAs of 91.6 and 83.3%. Electrophoresis and MALDI-TOF/MS analysis showed that the extracted ATIs masses were between 10 and 20 kDa. Based on the initial LC-MS/MS analysis, up to 10 individual ATIs were identified in the extracted proteins under the optimal conditions. The positive implication of the present study lies in the quick assessment of their content in different varieties especially while considering their allergenic potential.
A new evidence-based diet score to capture associations of food consumption and chronic disease risk
(2022)
Previously, the attempt to compile German dietary guidelines into a diet score was predominantly not successful with regards to preventing chronic diseases in the EPIC-Potsdam study. Current guidelines were supplemented by the latest evidence from systematic reviews and expert papers published between 2010 and 2020 on the prevention potential of food groups on chronic diseases such as type 2 diabetes, cardiovascular diseases and cancer. A diet score was developed by scoring the food groups according to a recommended low, moderate or high intake. The relative validity and reliability of the diet score, assessed by a food frequency questionnaire, was investigated. The consideration of current evidence resulted in 10 key food groups being preventive of the chronic diseases of interest. They served as components in the diet score and were scored from 0 to 1 point, depending on their recommended intake, resulting in a maximum of 10 points. Both the reliability (r = 0.53) and relative validity (r = 0.43) were deemed sufficient to consider the diet score as a stable construct in future investigations. This new diet score can be a promising tool to investigate dietary intake in etiological research by concentrating on 10 key dietary determinants with evidence-based prevention potential for chronic diseases.
A novel common variant in DCST2 is associated with length in early life and height in adulthood
(2015)
Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 x 10(-6)) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; beta = 0.046, SE = 0.008, P = 2.46 x 10(-8), explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 x 10(-4)) and adult height (N = 127 513; P = 1.45 x 10(-5)). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
In order to assess the individual trace element status of humans for either medical or scientific purposes, amongst others, blood serum levels are determined. Furthermore, animal models are used to study interactions of trace elements. Most published methods require larger amounts (500-1000 mu L) of serum to achieve a reliable determination of multiple trace elements. However, oftentimes, these amounts of serum cannot be dedicated to a single analysis and the amount available for TE-determination is much lower. Therefore, a published ICP-MS/MS method for trace element determination in serum was miniaturized, optimized and validated for the measurement of Mn, Fe, Cu Zn, I and Se in as little as 50 mu L of human and murine serum and is presented in this work. For validation, recoveries of multiple LOTs and levels from commercially available human reference serum samples were determined, infra- and inter-day variations were assessed and limits of detection and quantification determined. It is shown, that the method is capable of giving accurate and reproducible results for all six elements within the relevant concentration ranges for samples from humans living in central Europe as well as from laboratory mice. As a highlight, the achieved limits of detection and quantification for Mn were found to be at 0.02 mu g/L serum and 0.05 mu g/L serum, respectively, while using an alkaline diluent for the parallel determination of iodine.
Obligate human pathogenic Neisseria gonorrhoeae are the second most frequent bacterial cause of sexually transmitted diseases. These bacteria invade different mucosal tissues and occasionally disseminate into the bloodstream. Invasion into epithelial cells requires the activation of host cell receptors by the formation of ceramide-rich platforms. Here, we investigated the role of sphingosine in the invasion and intracellular survival of gonococci. Sphingosine exhibited an anti-gonococcal activity in vitro. We used specific sphingosine analogs and click chemistry to visualize sphingosine in infected cells. Sphingosine localized to the membrane of intracellular gonococci. Inhibitor studies and the application of a sphingosine derivative indicated that increased sphingosine levels reduced the intracellular survival of gonococci. We demonstrate here, that sphingosine can target intracellular bacteria and may therefore exert a direct bactericidal effect inside cells.
1 Two isoforms of the rat prostaglandin E-2 receptor, rEP3 alpha-R and rEP3 beta-R, differ only in their C- terminal domain. To analyze the function of the rEP3-R C-terminal domain in agonist induced desensitization, a cluster of Ser/Thr residues in the C-terminal domain of the rEP3 alpha-R was mutated to Ala and both isoforms and the receptor mutant (rEP3 alpha-ST341-349A-R) were stably expressed in HEK293 cells. 2 All rEP3-R receptors showed a similar ligand- binding profile. They were functionally coupled to Gi and reduced forskolin-induced cAMP-formation. 3 Repeated exposure of cells expressing the rEP3 alpha-R isoform to PGE(2) reduced the agonist induced inhibition of forskolin-stimulated cAMP-formation by 50% and led to internalization of the receptor to intracellular endocytotic vesicles. By contrast, Gi- response as well as plasma membrane localization of the rEP3 beta-R and the rEP3 alpha-ST341-349A-R were not affected by prior agonist-stimulation. 4 Agonist-stimulation of HEK293-rEP3 alpha-R cells induced a time- and dose-dependent phosphorylation of the receptor most likely by G protein-coupled receptor kinases and not by protein kinase A or protein kinase C. By contrast, upon agonist-stimulation the rEP3 beta-R was not phosphorylated and the rEP3 alpha-ST341-349A-R was phosphorylated only weakly. 5 These results led to the hypothesis that agonist-induced desensitization of the rEP3 alpha-R isoform is mediated most likely by a GRK-dependent phosphorylation of Ser/Thr residues 341 - 349. Phosphorylation then initiates uncoupling of the receptor from Gi protein and receptor internalization
Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions.
Background/Aims: Impaired pregnancy outcomes, such as low birth weight are associated with increased disease risk in later life, however little is known about the impact of common infectious diseases during pregnancy on birth weight. The study had two aims: a) to investigate risk factors of influenza virus infection during pregnancy, and b) to analyze the impact of influenza virus infection on pregnancy outcome, especially birth weight.
Methods: Prospective and retrospective observational studies found in PubMed, MEDLINE, Embase, Google Scholar, and WangFang database were included in this meta analysis. Data of included studies was extracted and analyzed by the RevMan software.
Results: Pregnant women with anemia (P=0.004, RR=1.46, 95% CI: 1.13-1.88), obesity (P<0.00001, RR=1.35, 95% CI: 1.25-1.46) and asthma (P<0.00001, RR=1.99, 95% CI: 1.67-2.37) had higher rates of influenza virus infection. Regarding birth outcomes, influenza A virus infection did not affect the likelihood for cesarean section. Mothers with influenza had a higher rate of stillbirth (P=0.04, RR=2.36, 95% CI: 1.05-5.31), and their offspring had low 5-minute APGR Scores (P=0.009, RR=1.39, 95% CI: 1.08-1.79). Furthermore, the rate for birth weight < 2500g (P=0.04, RR=1.71, 95% CI: 1.03-2.84) was increased.
Conclusion: Results of this study showed that anemia, asthma and obesity during pregnancy are risk factors influenza A virus infection during pregnancy. Moreover, gestational influenza A infection impairs pregnancy outcomes and increases the risk for low birth weight, a known risk factor for later life disease susceptibility.
Extra-cellular matrix (ECM) components are important and their stabilization is significant in maintaining normal healthy joint environment. In osteoarthritis (OA), ECM components are altered and indicate disease progression. The joint ECM is composed of proteoglycans (aggrecan, perlecan,inter α-trypsin inhibitor), glycoproteins (fibronectin, lubricin, COMP) and collagen types (most abundantly collagen type II) which represent structural and functional transformation during disease advancement. ECM investigation revealed significant biomarkers of OA that could be used as a diagnostic and therapeutic tool in different canine orthopedic diseases. This review deliberates our current findings of how the components of ECM change at the molecular level during disease progression in canine OA.
ABCB1/4 gallbladder cancer risk variants identified in India also show strong effects in Chileans
(2020)
Background: The first large-scale genome-wide association study of gallbladder cancer (GBC) recently identified and validated three susceptibility variants in the ABCB1 and ABCB4 genes for individuals of Indian descent. We investigated whether these variants were also associated with GBC risk in Chileans, who show the highest incidence of GBC worldwide, and in Europeans with a low GBC incidence.
Methods: This population-based study analysed genotype data from retrospective Chilean case-control (255 cases, 2042 controls) and prospective European cohort (108 cases, 181 controls) samples consistently with the original publication.
Results: Our results confirmed the reported associations for Chileans with similar risk effects. Particularly strong associations (per-allele odds ratios close to 2) were observed for Chileans with high Native American (=Mapuche) ancestry. No associations were noticed for Europeans, but the statistical power was low.
Conclusion: Taking full advantage of genetic and ethnic differences in GBC risk may improve the efficiency of current prevention programs.
As our climate changes, plant mechanisms involved for dormancy release become increasingly important for commercial orchards. It is generally believed that abscisic acid (ABA) is a key hormone that responds to various environmental stresses which affects bud dormancy. For this reason, a multi-year study was initiated to obtain data on plant metabolites during winter rest and ontogenetic development in sweet cherry buds (Prunus avium L.). In this paper, we report on metabolites involved in ABA synthesis and catabolism and its effect on bud dormancy in the years 2014/15-2016/17. In previous work, the timings of the different phases of para-, endo-, ecodormancy and ontogenetic development for cherry flower buds of the cultivar ‘Summit’ were determined, based on classical climate chamber experiments and changes in the bud’s water content. Based on these time phases, we focused now on the different aspects of the ABA-metabolism. The results show that there is a continual synthesis of ABA about 5 weeks before leaf fall, and a degradation of ABA during ecodormancy and bud development until the phenological stage ‘open cluster’. This is confirmed by relating the ABA content to that of the total precursor carotenoids, neoxanthin and violaxanthin. The tentative monitoring of individual intermediate metabolites revealed that dihydroxyphaseic acid is the most abundant catabolite of ABA and ABA glucosyl ester is in terms of mass intensity, the most abundant ABA metabolite observed in this study. The results suggest that the direct route for ABA biosynthesis from farnesyl pyrophosphate may also be relevant in cherry flower buds.
Accumulation of retinol in the liver after prolonged hyporetinolemia in the vitamin A-sufficient rat
(2005)
We assessed the effects of prolonged reduction of plasma retinol concentrations (hyporetinolemia) on the distribution of tissue vitamin A (VA) and of its active compounds using a model of continuous recombinant human interleukin-6 (rhIL-6) infusion via osmotic minipumps in VA-sufficient male rats. Plasma retinol and retinol-binding protein (RBP) concentrations remained decreased and lower in rhIL-6- treated rats compared with controls from 7.5 h throughout 7 days of infusion (P < 0.001). This reduction was accompanied by a 68% increase in hepatic retinol concentration by 7 days (P < 0.05). Hepatic and renal retinyl palmitate and retinoic acid concentrations did not change, and renal megalin content remained unchanged; hepatic RBP concentrations were 41% lower in rhIL-6-treated rats compared with controls (P < 0.05). These results indicate that instead of being lost, retinol accumulated in the liver during inflammation and that hyporetinolemia was attributable to a decrease in the availability of hepatic RBP. A plausible consequence of the effect of rhIL-6-induced hyporetinolemia is that by 7 days tissues that are dependent on plasma retinol may become deprived of VA. These results have important implications in understanding the mechanism by which measles infection induces hyporetinolemia and VA deficiency of extrahepatic tissues
Background: Patients with severe forms of cancer are reported to have reduced concentrations of micronutrients in plasma due to the chronic reduction of food intake and an increased metabolism of these components. The purpose of this study was to evaluate if an accumulation of carotenoids, alpha-tocopherol and retinol in malignant ascitic fluid in women with ovarian cancer might contribute to a loss of these components from plasma. Methods: Blood and ascitic fluid samples obtained from 21 women with ovarian carcinomas and 17 healthy controls were analyzed for retinol, retinol- binding protein (RBP), alpha-tocopherol and carotenoids. Results: Plasma concentrations of all micronutrients were lower in cancer patients compared to controls. Ascitic fluid concentration of all investigated components was comparable (73- 110%) to plasma. While the mean concentration of retinol in malignant ascites represented 73% of that in plasma, the concentration of RBP was less than 10% resulting in an increased mean molar ratio of retinol to RBP from 1.18 to 10.5. Conclusions: The results suggest that lower plasma concentrations of micronutrients in women suffering from ovarian carcinoma are not only caused by a cachexia-induced decrease of food intake and a higher rate of metabolic utilization, but also by a substantial yet not considered transfer from plasma into ascitic fluid possibly associated with plasma lipoproteins. This raises questions with regard to the protective function of these plasma components in ascitic fluid, the consequences of paracentesis on an additional supplementation and finally the possibility to use one or a combination of these components as an additional marker to discriminate between benign and malignant ascites. Copyright (C) 2004 S. Karger AG, Basel
Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1(-/-) mice results in replication of HSV-1 and Asah1(-/-) mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.
Objective: We aimed to identify the role of the enzyme acid sphingomyelinase in the aging of stored units of packed red blood cells (pRBCs) and subsequent lung inflammation after transfusion.
Summary Background Data: Large volume pRBC transfusions are associated with multiple adverse clinical sequelae, including lung inflammation. Microparticles are formed in stored pRBCs over time and have been shown to contribute to lung inflammation after transfusion.
Methods: Human and murine pRBCs were stored with or without amitriptyline, a functional inhibitor of acid sphingomyelinase, or obtained from acid sphingomyelinase-deficient mice, and lung inflammation was studied in mice receiving transfusions of pRBCs and microparticles isolated from these units.
Results: Acid sphingomyelinase activity in pRBCs was associated with the formation of ceramide and the release of microparticles. Treatment of pRBCs with amitriptyline inhibited acid sphingomyelinase activity, ceramide accumulation, and microparticle production during pRBC storage. Transfusion of aged pRBCs or microparticles isolated from aged blood into mice caused lung inflammation. This was attenuated after transfusion of pRBCs treated with amitriptyline or from acid sphingomyelinase-deficient mice.
Conclusions: Acid sphingomyelinase inhibition in stored pRBCs offers a novel mechanism for improving the quality of stored blood.
Acid sphingomyelinase mediates murine acute lung injury following transfusion of aged platelets
(2017)
Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion-related acute lung injury is mediated by antibodies or bioactive mediators, yet underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two-hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) 2 h before transfusion of 10 ml/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase-deficient mice, which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase-deficient platelets. Compared with wild-type platelets, transfusion of ARC39-treated or acid sphingomyelinase-deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.
Selective adenosine A1 receptor antagonists targeting renal microcirculation are novel pharmacologic agents that are currently under development for the treatment of acute heart failure as well as for chronic heart failure. Despite several studies showing improvement of renal function and/or increased diuresis with adenosine A1 antagonists, particularly in chronic heart failure, these findings were not confirmed in a large phase III trial in acute heart failure patients. However, lessons can be learned from these and other studies, and there might still be a potential role for the clinical use of adenosine A1 antagonists. We review the role of adenosine A1 receptors in the regulation of renal function, and emerging data regarding the safety and efficacy of A1 adenosine receptor antagonists based on all available completed and reported clinical trials using A1 adenosine receptor antagonists. The majority of trials were done in heart failure patients. However, there is clear clinical evidence for a role of this new class in hepatorenal syndrome, hypotension on dialysis, and radiocontrast media-induced nephropathy.
Asymmetric dimethylarginine (ADMA) is a competitive inhibitor of the nitric oxide (NO)-synthase and a biomarker of endothelial dysfunction (ED). ED plays an important role in the pathogenesis of contrast-induced nephropathy (CIN). The aim of our study was to evaluate serum ADMA concentration as a biomarker of an acute renal damage during the follow-up of 90 days after contrast medium (CM) application. Blood samples were obtained from 330 consecutive patients with diabetes mellitus or mild renal impairment immediately before, 24 and 48 hours after the CM application for coronary angiography. The patients were followed for 90 days. The composite endpoints were major adverse renal events (MARE) defined as occurrence of death, initiation of dialysis, or a doubling of serum creatinine concentration. Overall, ADMA concentration in plasma increased after CM application, although, there was no differences between ADMA levels in patients with and without CIN. ADMA concentration 24 hours after the CM application was predictive for dialysis with a specificity of 0.889 and sensitivity of 0.653 at values higher than 0.71 mu mol/L (area under the curve: 0.854, 95% confidential interval: 0.767-0.941, P<0.001). This association remained significant in multivariate Cox regression models adjusted for relevant factors of long-term renal outcome. 24 hours after the CM application, ADMA concentration in plasma was predictive for MARE with a specificity of 0.833 and sensitivity of 0.636 at a value of more than 0.70 mu mol/L (area under the curve: 0.750, 95% confidence interval: 0.602-0.897, P=0.004). Multivariate logistic regression analysis confirmed that ADMA and anemia were significant predictors of MARE. Further analysis revealed that increased ADMA concentration in plasma was highly significant predictor of MARE in patients with CIN. Moreover, patients with CIN and MARE had the highest plasma ADMA levels 24 hours after CM exposure in our study cohort. The impact of ADMA on MARE was independent of such known CIN risk factors as anemia, pre-existing renal failure, pre-existing heart failure, and diabetes. ADMA concentration in plasma is a promising novel biomarker of major contrast-induced nephropathy-associated events 90 days after contrast media exposure.
Background Advanced glycation end-products are proteins that become glycated after contact with sugars and are implicated in endothelial dysfunction and arterial stiffening. We aimed to investigate the relationships between advanced glycation end-products, measured as skin autofluorescence, and vascular stiffness in various glycemic strata. Methods We performed a cross-sectional analysis within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort, comprising n = 3535 participants (median age 67 years, 60% women). Advanced glycation end-products were measured as skin autofluorescence with AGE-Reader (TM), vascular stiffness was measured as pulse wave velocity, augmentation index and ankle-brachial index with Vascular Explorer (TM). A subset of 1348 participants underwent an oral glucose tolerance test. Participants were sub-phenotyped into normoglycemic, prediabetes and diabetes groups. Associations between skin autofluorescence and various indices of vascular stiffness were assessed by multivariable regression analyses and were adjusted for age, sex, measures of adiposity and lifestyle, blood pressure, prevalent conditions, medication use and blood biomarkers. Results Skin autofluorescence associated with pulse wave velocity, augmentation index and ankle-brachial index, adjusted beta coefficients (95% CI) per unit skin autofluorescence increase: 0.38 (0.21; 0.55) for carotid-femoral pulse wave velocity, 0.25 (0.14; 0.37) for aortic pulse wave velocity, 1.00 (0.29; 1.70) for aortic augmentation index, 4.12 (2.24; 6.00) for brachial augmentation index and - 0.04 (- 0.05; - 0.02) for ankle-brachial index. The associations were strongest in men, younger individuals and were consistent across all glycemic strata: for carotid-femoral pulse wave velocity 0.36 (0.12; 0.60) in normoglycemic, 0.33 (- 0.01; 0.67) in prediabetes and 0.45 (0.09; 0.80) in diabetes groups; with similar estimates for aortic pulse wave velocity. Augmentation index was associated with skin autofluorescence only in normoglycemic and diabetes groups. Ankle-brachial index inversely associated with skin autofluorescence across all sex, age and glycemic strata. Conclusions Our findings indicate that advanced glycation end-products measured as skin autofluorescence might be involved in vascular stiffening independent of age and other cardiometabolic risk factors not only in individuals with diabetes but also in normoglycemic and prediabetic conditions. Skin autofluorescence might prove as a rapid and non-invasive method for assessment of macrovascular disease progression across all glycemic strata.
Plant cultivation and processing may impact nutrient and phytochemical content of vegetables. The present study aimed at determining the influence of cultivation and processing on the health promoting capacity of African nightshade (Solanum scabrum Mill.) leaves, an indigenous vegetable, rich in nutrients and phytochemicals. Anti-genotoxicity against the human liver carcinogen aflatoxin B1 (AFB(1)) as determined by the comet assay and radical oxygen species (ROS) scavenging capacity of ethanolic and aqueous extracts were investigated in human derived liver (HepG2) cells. ROS scavenging activity was assessed using electron paramagnetic spin resonance and quantification of ARE/Nrf2 mediated gene expression. The cultivation was done under different environmental conditions. The processing included fermentation and cooking; postharvest ultraviolet irradiation (UV-C) treatment was also investigated. Overall, S. scabrum extracts showed strong health promoting potential, the highest potential was observed with the fermented extract, which showed a 60% reduction of AFB(1) induced DNA damage and a 38% reduction in FeSO4 induced oxidative stress. The content of total polyphenols, carotenoids and chlorophylls was indeed affected by cultivation and processing. Based on the present in vitro findings consumption of S. scabrum leaves could be further encouraged, preferentially after cooking or fermentation of the plant.
Aggravation by prostaglandin e-2 of interleukin-6-dependent insulin resistance in hepatocytes
(2009)
Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E-2 (PGE(2)) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE(2) and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE(2) while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE(2) induced suppressors of cytokine signaling (SOCS3). PGE(2) but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE(2)-dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE(2) pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. Conclusion: PGE(2) might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulin-dependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE(2) may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3.
Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4- Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG- rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG- hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG- hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R- expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.
Background: Alcohol abuse is a major risk factor for somatic and neuropsychiatric diseases. Despite their potential clinical importance, little is known about the alterations of plasma glycerophospholipid (GPL) and sphingolipid (SPL) species associated with alcohol abuse.
Methods: Plasma GPL and SPL species were quantified using electrospray ionization tandem mass spectrometry in samples from 23 male alcohol-dependent patients before and after detoxification, as well as from 20 healthy male controls.
Results: A comparison of alcohol-dependent patients with controls revealed higher phosphatidylcholine (PC; P-value = 0.008) and phosphatidylinositol (PI; P-value = 0.001) concentrations in patients before detoxification, and higher PI (P-value = 0.001) and phosphatidylethanolamine (PE)-based plasmalogen (PEP; P-value = 0.003) concentrations after detoxification. Lysophosphatidylcholines (LPC) were increased by acute intoxication (P-value = 0.002). Sphingomyelin (SM) concentration increased during detoxification (P-value = 0.011). The concentration of SM 23:0 was lower in patients (P-value = 2.79 x 10(-5)), and the concentrations of ceramide Cer d18:1/16:0 and Cer d18:1/18:0 were higher in patients (P-value = 2.45 x 10(-5) and 3.73 x 10(-5)). Activity of lysosomal acid sphingomyelinase (ASM) in patients correlated positively with the concentrations of eight LPC species, while activity of secreted ASM was inversely correlated with several PE, PI and PC species, and positively correlated with the molar ratio of PC to SM (Pearson's r = 0.432; P-value = 0.039).
Conclusion: Plasma concentrations of numerous GPL and SPL species were altered in alcohol-dependent patients. These molecules might serve as potential biomarkers to improve the diagnosis of patients and to indicate health risks associated with alcohol abuse. Our study further indicates that there are strong interactions between plasma GPL concentrations and SPL metabolism. (C) 2015 Elsevier B.V. All rights reserved.
Retinol-binding protein 4 (RBP4) is elevated in patients with chronic kidney disease (CKD) and has been discussed as marker of kidney function. In addition to an elevated concentration, the existence of truncated RBP4 species, RBP4-L (truncated at last C-terminal leucine) and RBP4-LL (truncated at both C-terminal leucines), has been reported in serum of hemodialysis patients. Since little is known about the occurrence of RBP4 species during the progression of CKD it was the aim of this study to analyse this possible association. The presence of RBP4, RBP4-L, RBP4- LL and transthyretin (TTR) was assessed in serum of 45 healthy controls and 52 patients with stage 2-5 of CKD using ELISA and RBP4 immunoprecipitation with subsequent MALDI-TOF-MS analysis. A reduction of glomerular filtration rate was accompanied by a gradual elevation of RBP4 serum levels and relative amounts of RBP4-LL. Correlation analysis revealed a strong association of the RBP4-TTR ratio with parameters of lipid metabolism and with diabetes-related factors. In conclusion, RBP4 serum concentration and the appearance of RBP4-LL seem to be influenced by kidney function. Furthermore, the RBP4-TTR ratio may provide diagnostic potential with regard to metabolic complications in CKD patients.
Vitamin A, vitamin E and retinol-binding protein 4 (RBP4) are a focus of current obesity research in humans. The impact of body weight (BW) gain on fat-soluble vitamins and its associated parameters in equines has not been previously reported. Ten Shetland ponies and 9 Warmblood horses, all adult geldings, non-obese and healthy, were fed an excessive energy diet for 20 months to induce BW gain. Serum alpha-tocopherol (vitamin E), retinol (vitamin A), retinol-binding protein 4 (RBP4) and retinol/RBP4 ratio were analysed before BW gain induction and at six timepoints during the BW gaining period. The mean (+/- SD) % BW gain achieved during two years of excess energy intake was 29.9 +/- 19.4% for ponies and 17 +/- 6.74% for horses. Serum alpha-tocopherol increased significantly in ponies and horses during excess energy intake and circulating alpha-tocopherol levels correlated positively with alpha-tocopherol intake (r = .6; p < .001). Serum retinol concentrations showed variations during the study but without relation to intake. Serum RBP4 decreased at the end of the study. The retinol/RBP4 ratio increased with BW gain without differences between ponies and horses. In comparison with human research, the increase in the retinol/RBP4 ratio was unexpected and needs further elucidation.
For centuries, Amaranthus sp. were used as food, ornamentals, and medication. Molecular mechanisms, explaining the health beneficial properties of amaranth, are not yet understood, but have been attributed to secondary metabolites, such as phenolic compounds. One of the most abundant phenolic compounds in amaranth leaves is 2-caffeoylisocitric acid (C-IA) and regarding food occurrence, C-IA is exclusively found in various amaranth species. In the present study, the anti-inflammatory activity of C-IA, chlorogenic acid, and caffeic acid in LPS-challenged macrophages (RAW 264.7) has been investigated and cellular contents of the caffeic acid derivatives (CADs) were quantified in the cells and media. The CADs were quantified in the cell lysates in nanomolar concentrations, indicating a cellular uptake. Treatment of LPS-challenged RAW 264.7 cells with 10 µM of CADs counteracted the LPS effects and led to significantly lower mRNA and protein levels of inducible nitric oxide synthase, tumor necrosis factor alpha, and interleukin 6, by directly decreasing the translocation of the nuclear factor κB/Rel-like containing protein 65 into the nucleus. This work provides new insights into the molecular mechanisms that attribute to amaranth’s anti-inflammatory properties and highlights C-IA’s potential as a health-beneficial compound for future research.
Background: Estimating dietary intake is important for both epidemiological and clinical studies, but often lacks accuracy. Objective: To investigate the accuracy and validity of energy intake estimated by an easy-to-use semiquantitative food record (EISQFR) compared to total energy expenditure ( TEE) estimated by doubly labelled water technique (EEDLW). Design: TEE was measured in 29 nonobese subjects using the doubly labelled water method over a period of 14 days. Within this period, subjects reported their food consumption by a newly developed semiquantitative food record for 4 consecutive days. Energy intake was calculated using the German Food Code and Nutrition Data Base BLS II.3. Results: A good correlation was observed between EISQFR and EEDLW (r = 0.685, P<0.001). The mean difference between EISQFR and EEDLW was - 1.7 +/- 2.6 MJ/ day ( - 14 +/- 21%, P = 0.002). An underestimation of EISQFR <10% was observed in nine subjects (31%), of 10 - 20% in six subjects (21%), and of >20% in nine subjects (31%). In five subjects (17%), an overestimation of EISQFR was observed. Conclusions: The easy-to-use semiquantitative food record provided good estimates of EI in free-living and nonobese adults without prior detailed verbal instructions. The presented food record has limitations regarding accuracy at the individual level
Analyses of the correlation between dermal and blood carotenoids in female cattle by optical methods
(2013)
Herd health programs for the maintenance of welfare and productivity in cattle need efficient tools for monitoring the health of individual animals. Recent reports demonstrate that the oxidative status is related to various stress conditions in dairy cows. Biomarkers, among other carotenoids, could serve as indicators of stress originating from the environment (e.g., heat stress or sun radiation) or from the animal itself (e.g., disease). To date, only invasive in vitro tests are available to assess the oxidative status in cattle. The present study compares the results of optical noninvasive in vivo measurements of dermal carotenoids in cattle udder skin using an LED-based miniaturized spectroscopic system (MSS) with those obtained by photometric analysis of beta carotene in whole blood samples using a portable device. Correlations between the concentrations of dermal and blood carotenoids were calculated under consideration of the nutritional status of the animals. Significant correlation (R = 0.86) was found for cattle with a moderate to obese body condition. Thus, the blood and skin concentrations of the marker substance beta carotene are comparable under stable stress conditions of the cattle. This demonstrates that the MSS is suitable for noninvasive assessment of dermal carotenoid concentrations in cattle.
Analysis of electrochemical and liver microsomal transformation products of lasalocid by LC/HRMS
(2022)
Rationale:
Lasalocid (LAS), an ionophore, is used in cattle and poultry farming as feed additive for its antibiotic and growth-promoting properties. Literature on transformation products (TP) resulting from LAS degradation is limited. So far, only hydroxylation is found to occur as the metabolic reaction during the LAS degradation. To investigate potential TPs of LAS, we used electrochemistry (EC) and liver microsome (LM) assays to synthesize TPs, which were identified using liquid chromatography high-resolution mass spectrometry (LC/HRMS).
Methods:
Electrochemically produced TPs were analyzed online by direct coupling of the electrochemical cell to the electrospray ionization (ESI) source of a Sciex Triple-TOF high resolution mass spectrometer. Then, EC-treated LAS solution was collected and analyzed offline using LC/HRMS to confirm stable TPs and improve their annotation with a chemical structure due to informative MS/MS spectra. In a complementary approach, TPs formed by rat and human microsomal incubation were investigated using LC/HRMS. The resulting data were used to investigate LAS modification reactions and elucidate the chemical structure of obtained TPs.
Results:
The online measurements identified a broad variety of TPs, resulting from modification reactions like (de-)hydrogenation, hydration, methylation, oxidation as well as adduct formation with methanol. We consistently observed different ion complexations of LAS and LAS-TPs (Na+; 2Na(+) K+; NaNH4+; KNH4+). Two stable methylated EC-TPs were found, structurally annotated, and assigned to a likely modification reaction. Using LM incubation, seven TPs were formed, mostly by oxidation/hydroxylation. After the identification of LM-TPs as Na+-complexes, we identified LM-TPs as K+-complexes.
Conclusion:
We identified and characterized TPs of LAS using EC- and LM-based methods. Moreover, we found different ion complexes of LAS-based TPs. This knowledge, especially the different ion complexes, may help elucidate the metabolic and environmental degradation pathways of LAS.
Background: DNA-methylation is a common epigenetic tool which plays a crucial role in gene regulation and is essential for cell differentiation and embryonic development. The placenta is an important organ where gene activity can be regulated by epigenetic DNA modifications, including DNA methylation. This is of interest as, the placenta is the interface between the fetus and its environment, the mother. Exposure to environmental toxins and nutrition during pregnancy may alter DNA methylation of the placenta and subsequently placental function and as a result the phenotype of the offspring. The aim of this study was to develop a reliable method to quantify DNA methylation in large clinical studies. This will be a tool to analyze the degree of DNA methylation in the human placenta in relationship to clinical readouts. Methods: Liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS) technique was used for the quantification of the 5dmC/dG ratio in placentas from 248 healthy pregnancies. We were able to demonstrate that this method is a reliable and stable way to determine global placental DNA methylation in large clinical trials. Results/Conclusion: The degree of placental DNA methylation seen in our pilot study varies substantially from 2% to 5%. The clinical implications of this variation need to be demonstrated in adequately powered large studies.