Plasma Total Levels Research Articles

Plasma levels of polyunsaturated fatty acids and multiple sclerosis susceptibility in a US case-control study

BackgroundThere are plausible mechanisms, yet mixed evidence, that higher polyunsaturated fatty acids (PUFAs) levels reduces the risk of multiple sclerosis (MS). Prior studies relied on dietary surveys to estimate levels. ObjectiveWe tested associations between plasma levels of n-3 and n-6 PUFAs and likelihood of MS onset or clinically isolated syndrome (CIS) using data from the MS Sunshine Study, a case-control study conducted in the United States. MethodsCase participants (n = 589) aged ≥ 18 years and matched control participants (n = 630) were recruited between 2011 and 2015. Plasma phospholipid fatty acid profiling was conducted by gas-liquid chromatography. We used logistic regression to report odds ratios, testing for interactions, adjusting for covariates and correcting for multiple comparisons. ResultsThere was a 6 % lower probability of MS/CIS per unit increase in total n-6 PUFA level, expressed as a percentage of total plasma phospholipid fatty acids (odds ratio = 0.94; 95 % confidence interval = 0.90,0.98; p = 0.012). We found no statistically significant association between individual or total plasma levels of n-3 PUFAs and probability of MS/CIS; however, plasma levels of n-3 PUFAs were low across the cohort. No other individual or aggregate PUFA levels were significantly associated with MS/CIS. ConclusionA higher total n-6 PUFA level may be beneficial in terms of MS susceptibility.Further research is needed to determine whether n-3 PUFAs may be beneficial only above a threshold that is achievable by supplementation.

Evaluation of In Vitro Metabolism- and Transporter-Based Drug Interactions with Sunscreen Active Ingredients.

The aim of this study was to examine the ability of sunscreen active ingredients to inhibit in vitro drug metabolism via cytochrome P450 (CYP) enzymes and drug uptake transporters. Metabolism assays with human liver microsomes were conducted for CYP2C9, CYP2D6 and CYP3A4 using probe substrates warfarin, bufuralol and midazolam, respectively. Uptake transporter assays with transfected cell lines were conducted for OAT3, OCT2 and OATP1B1 with probe substrates estrone-3-sulfate, metformin and rosuvastatin, respectively. Six sunscreen active ingredients, avobenzone, enzacamene, oxybenzone, octinoxate, trolamine, and homosalate, were evaluated up to their aqueous solubility limits in the assays. None of the sunscreen active ingredients inhibited CYP2D6 or CYP3A4 activities in the microsomes at concentration ranges up to tenfold higher than their known clinical total plasma levels. Only enzacamene, oxybenzone and trolamine were found to be inhibitory to CYP2C9 activity with IC50 values of 14.76, 22.46 and 154.7µM, respectively. Avobenzone, enzacamene, homosalate and octinoxate were not inhibitory to the uptake transporters at the evaluated concentrations. Oxybenzone was inhibitory to OAT3 and OCT2 with IC50 values of 39.93 and 42.77µM, respectively. Trolamine also inhibited uptake in OAT3 and OCT2 transfected cells with IC50 values of 448.1 and 1376μM, respectively. Although enzacamene, oxybenzone and trolamine inhibited CYP2C9 and the renal transporters OAT3 and OCT2 in vitro, their IC50 values exceeded total plasma levels found in clinical studies. Therefore, it is unlikely that these sunscreen active ingredients in sunscreen products will inhibit the metabolism or transport of co-administered drugs in consumers.

PON1 rs662, rs854560 and TRIB1 rs17321515, rs2954029 Gene Polymorphisms Are Associated with Lipid Parameters in Patients with Unstable Angina.

Acute coronary heart disease (CHD) is mainly caused by the rupture of an unstable atherosclerotic plaque. Many different factors can cause stenosis or even occlusion of the coronary artery lumen, such as vasculitis and platelet aggregation. Our study was performed to assess the association between PON1 rs662, rs854560 and TRIB1 rs17321515, rs2954029 polymorphisms and the risk of CHD, as well as the association between studied polymorphisms and selected clinical parameters affecting the risk of developing ischemic heart disease. A total of 232 patients with unstable angina were enrolled in this study. There were no statistically significant differences in the PON1 rs662, rs854560 and TRIB1 rs17321515, rs2954029 polymorphism distributions between the total study and control groups. Total cholesterol plasma levels were significantly higher in patients with the PON1 rs662 TT genotype compared to those with the CC+TC genotypes, as well as in patients with the PON1 rs854560 TT genotype compared to those with the AA+AT genotypes. LDL plasma levels were significantly increased in patients with the PON1 rs854560 TT genotype compared to those with the AA+AT genotypes. Plasma levels of HDL were significantly decreased in patients with the TRIB1 rs17321515 AA+AG genotypes compared to those with the GG genotype, as well as in patients with the TRIB1 rs2954029 AA+AT genotypes compared to those with the TT genotype. Our results suggest that the analysed polymorphisms are not risk factors for unstable angina in the Polish population. However, the results of this study indicate an association between the PON1 rs662, rs854560 and TRIB1 rs17321515, rs2954029 polymorphisms with lipid parameters in patients with coronary artery disease.

Plasma Levels of Estradiol and Progesterone are Positively Associated with IL-1α and IL-1Ra in Young Women with PTSD

Introduction: Post-traumatic stress disorder (PTSD) is independently associated with greater cardiovascular disease (CVD) risk. Prior studies have reported higher inflammation as a possible underlying mechanism. Premenopausal females are typically thought to be protected from CVD due to estradiol (E2) and available data supports growing CVD risk in females before menopause by virtue of having PTSD. Given that E2 and progesterone (P4) have been shown to have a broad range of both pro- and anti-inflammatory effects, the purpose of our study was to investigate whether plasma levels of E2 and P4 are associated with subclinical inflammation in young women with PTSD. We hypothesized that E2 and P4 will be negatively associated with IL-1α, IL-1Ra, and IL-6. Methods: Seventy-five otherwise healthy young women (age, 28±7 years; BMI, 26.7±6 kg/m2) with a prior clinical diagnosis of PTSD (PTSD+, n=39) and without a PTSD diagnosis (PTSD-, n=36) were included in this study. We collected blood plasma samples to quantify E2 and P4 and inflammatory biomarkers Interleukin-1α (IL-1α), Interleukin-1 receptor antagonist (IL-1Ra) and Interleukin-6 (IL-6). Total plasma levels of E2 and P4 were measured using the quantitative sandwich enzyme immunoassay technique (ELISA). The inflammatory biomarkers were quantified using a Luminex assay. Participants were also asked to complete a self-administered PTSD checklist for DSM-5 (PCL5) to assess the severity of PTSD symptoms. We ran Pearson correlations between the sex hormones and the inflammatory biomarkers stratified by PTSD diagnosis. Results: We found a positive correlation between E2 levels and IL-1α (r=0.39, p=0.018), E2 and IL-1Ra (r=0.35, p=0.030), P4 and IL-1α (r=0.45, p=0.005), and P4 and IL-1Ra (r=0.27, p=0.101) in the PTSD+ group. In contrast, in the PTSD- group, there was no correlation between E2 and IL-1α (r=0.05, p=0.77), E2 and IL-1Ra (r=-0.06, p=0.73), P4 and IL-1α (r=-0.04, p=0.83) and P4 and IL-1Ra (r=0.07, p=0.68). Conclusion: Our analysis revealed that in young women with a clinical diagnosis of PTSD, E2 and P4 levels in the blood are positively associated with an increased IL-1α and IL-1Ra but not in young trauma-exposed women without PTSD. These findings suggest that in young women with PTSD, E2 and P4 might have a paradoxical effect on inflammation such that E2 and P4 promote the release of both pro-inflammatory (i.e. IL-1α) and anti-inflammatory (i.e. IL-1Ra) cytokines. Conversely, an increase in subclinical inflammation could lead to an increase in E2 and P4 levels, which in turn would promote the release of anti-inflammatory cytokines in order to inhibit the inflammatory response. K01HL161027 and UMN CTSI UL1TR002494 No Disclosure. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The intake of ultra-processed foods and homocysteine levels in women with(out) overweight and obesity: The Rotterdam Periconceptional Cohort

PurposeToday’s diet consists of a substantial proportion of ultra-processed foods (UPF), especially in women with overweight and obesity in the reproductive period. High UPF intake results in an inadequate and unbalanced diet leading to derangements of several metabolic pathways detrimental to pregnancy and birth outcomes. Therefore, we aim to investigate whether UPF intake in the periconceptional period affects total homocysteine plasma levels (tHcy).Methods1532 participants were included from the prospective Rotterdam Periconceptional Cohort. UPF intake was calculated using Food Frequency Questionnaires including items classified as 4 in the Nova classification, and tHcy was measured by using liquid chromatography-tandem mass spectrometry system, with an interassay coefficient of variation of < 5.5%. Multivariable linear regression modeling was used and adjusted for covariates and significant interaction terms.ResultsWomen with overweight or obesity showed significantly higher percentage of UPF intake (respectively, 50.3 and 51.3%) and higher tHcy (respectively, 6.6 and 6.3 µmol/L, Kruskal–Wallis test; respectively, p < 0.001 and p = 0.04) compared to women with normal BMI (UPF intake: 46.8%, tHcy: 6.1 µmol/L). A 10% higher intake of UPF was associated with an increase in tHcy (adjusted: β = 1.31, 95% CI = 0.38–2.23). Analysis stratified for BMI classification showed comparable associations in normal weight participants (adjusted: β = 1.07, 95% CI = 0.06–2.07); however, no significant association in participants with overweight (adjusted: β = 0.06, 95% CI = − 0.95–1.07) and obesity (adjusted: β = 1.70, 95% CI = − 0.52–3.92) was shown.ConclusionThis study showed that a higher intake of UPF is associated with increased tHcy. Better knowledge and awareness of the nutritional quality of the diet in the periconceptional period may contribute to 1-CM and subsequently improve pregnancy course and outcome.Trial registration number and dateNTR4356, November 2010.

Plasma Transforming Growth Factor-β1 (TGF-β1) Levels Increase with Age and Aortic Stenosis Progression and Low-Dose Galunisertib Attenuates Disease Progression in Mice

Objective: Aortic stenosis (AS) is an age-related degenerative valvular disease that ultimately leads to heart failure and sudden death. Platelet-derived TGF-β1 is a major source of plasma TGF-β1 and contributes to AS progression in LDLR -/-ApoB 100/100(LA100), a mouse model that spontaneously develops AS that simulates human pathology, including fibrosis and high shear gradient. Although risk factors, such as hypercholesteremia and hyperglycemia, are like those for coronary artery disease, lowering cholesterol and glucose has not been successful in stopping progression, and there is no biomarker for early detection of AS. In this study, we measured plasma TGF-β1 levels as a function of age in LA100 mice and human AS patients and tested a new approach of partial inhibition of TGF-β signaling to inhibit AS progression in LA100 mice, while limiting cardiotoxicity. Methods: We measured plasma TGF-β1 levels in controls, severe AS patients, and LA100 mice before they developed AS (2-months of age), when they developed moderate AS (1-year), and when they developed severe AS (2-years). LA100 mice at different stages of AS were treated with low-dose galunisertib, an inhibitor of the TGF-β receptor, ALK5. Results: Plasma levels of total TGF-β1 were significantly higher in AS patients (n=34) compared to healthy controls (n=22) and patient controls (n=47) (p&amp;lt;0.001 for AS patients vs. healthy controls and p=0.049 for AS patients vs. patient controls). Age was positively correlated with total TGF-β1 levels in AS patients (r=0.475; p=0.005), but not patient or healthy controls (r=0.12, p=0.43). LA100mice developed AS with aging and plasma TGF-β1 levels in LA100mice (n=21-52; ages 2 months-2 years) were significantly higher than those in age-matched WT mice (n=23); (p&amp;lt;0.0001). Plasma TGF-β1 levels correlated with age in LA100 mice (R=0.6, p=&amp;lt;0.0001). Low-dose oral galunisertib was administered at a concentration of 1 µM in drinking water, giving an average daily dose of ~0.06 mg/kg, compared to a human cancer treatment dose of ~4.2 mg/kg daily. Galunisertib treatment: a) halted AS progression at all stages in LA100 mice compared to vehicle-treated control mice (p&amp;lt;0.05, n=9-13), b) did not change plasma TGF-β1 levels, c) inhibited TGF-β1 signaling as shown by decreased Smad2 phosphorylation in valve cells compared to vehicle-treated animals. An age-dependent increase in neutrophil to lymphocyte ratio (NY/LY), a marker of inflammation, was observed in LA100 mice compared to control mice (n=12-31; p&amp;lt;0.01), and the NY/LY ratio correlated with total TGF-β1 plasma levels (R=0.16; p=0.020). Galunisertib treatment reduced NY/LY ratios in LA100 mice (p=0.03). Conclusions: 1. Increases in plasma TGF-β1 levels may be a valuable surrogate marker of AS progression. 2. Low-dose galunisertib attenuates AS progression in the LA100 mouse model without producing the cardiotoxicity associated with higher doses and thus may warrant further study in human AS patients.

Total and free 25-hydroxyvitamin D concentrations and risk of urinary incontinence in women participating in Nurses Health Studies I & II

BackgroundUrgency urinary incontinence (UUI) occurs in >40% of older women. Our objective was to examine the relationship of total and free plasma 25-hydroxyvitamin D (25(OH)D) and UUI to evaluate vitamin D status as a novel target for prevention of UUI. MethodsWe conducted a nested case control study using the Nurses’ Health Study (NHS) and NHS II. Using stored plasma samples from 2000, we measured total 25(OH)D, free 25(OH)D, and intact parathyroid hormone (PTH) levels and examined their relationship to incident UUI from 2000 to 2013. Plasma biomarker levels were categorized as quartiles. Multivariable-adjusted odds ratios of UUI were estimated by conditional logistic regression models (with matching by age) across categories of each biomarker and covariates. ResultsThe analytic sample included 398 cases of incident UUI and 398 matched controls with a mean age of 50 years. We found a strong correlation of plasma levels of total 25(OH)D with free 25(OH)D (r=0.5). Plasma total 25(OH)D and free 25(OH)D concentrations were negatively correlated with PTH (r=-0.08 AND -0.09, respectively). Overall, we found no evidence that levels of total plasma 25(OH)D, free 25(OH)D, or PTH were related to incident UUI after adjustment for obesity, physical activity, cigarette smoking, menopausal status, hypertension, and type 2 diabetes. ConclusionsFree plasma 25(OH)D by quartile, as well as total plasma 25(OH)D, was not associated with incident UUI in women. We found that plasma total and free 25(OH)D were highly correlated with each other and inversely correlated with PTH. Plasma free 25(OH)D did not provide additional predictive value in determining risk of UUI.

The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.

Oral L-arginine supplements are popular mainly for their nitric oxide mediated vasodilation, but their physiological impact is not fully known. L-arginine is a substrate of several enzymes including arginase, nitric oxide synthase, arginine decarboxylase, and arginine: glycine amidinotransferase (AGAT). We have published a study on the physiological impact of oral L- and D-arginine at 500 mg/kg/day for 4 wks in male Sprague-Dawley rats. We investigated the effects of oral L-arginine and D-arginine at a higher dose of 1000 mg/kg/d for a longer treatment duration of 16 wks in 9-week-old male Sprague-Dawley rats. We measured the expression and activity of L-arginine metabolizing enzymes, and levels of their metabolites in the plasma and various organs. L-arginine did not affect the levels of L-arginine and L-lysine in the plasma and various organs. L-arginine decreased arginase protein expression in the upper small intestine, and arginase activity in the plasma. It also decreased AGAT protein expression in the liver, and creatinine levels in the urine. L-arginine altered arginine decarboxylase protein expression in the upper small intestine and liver, with increased total polyamines plasma levels. Endothelial nitric oxide synthase protein was increased with D-arginine, the presumed metabolically inert isomer, but not L-arginine. In conclusion, oral L-arginine and D-arginine at a higher dose and longer treatment duration significantly altered various enzymes and metabolites in the arginine metabolic pathways, which differed from alterations produced by a lower dose shorter duration treatment published earlier. Further studies with differing doses and duration would allow for a better understanding of oral L-arginine uses, and evidence based safe and effective dose range and duration.

High Prevalence of Polyclonal Plasmodium falciparum Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = -0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

DIETARY SODIUM RESTRICTION REDUCES NOCTURNAL BLOOD PRESSURE IN PATIENTS WITH TREATMENT RESISTANT HYPERTENSION

Objective: Patients with treatment resistant hypertension (TRH) are characterised by lack of blood pressure (BP) control despite antihypertensive treatment. They are at particular risk of cardiovascular disease and more likely to experience BP increase as a response to sodium loading than other individuals. In this study, we aimed to analyse if self-performed dietary sodium restriction could be implemented in patients with TRH. Moreover, we aimed to analyse the effect of this on nocturnal and 24h BP, and how BP changes were correlated to changes in plasma levels of BNP, nitric oxide and body water content. Design and method: The study was conducted as an interventional cross-over study including patients with TRH from 20-70 years. Patients were screened with 24h ambulatory BP monitoring initiated after observed intake of usual antihypertensive medication. They were included if 24h systolic BP&gt;130/80 mmHg. Study participation included two periods of 14 days; a standard period with usual diet and an interventional period with instructed dietary sodium restriction and handed-out sodium-free bread. At the end of each period, patients were examined with 24h BP monitoring, 24h urine collection (sodium excretion), blood samples (nitric oxide, BNP), and bioimpedance measurement (body water content). Results: Participation was completed by 15 patients with mean age 59 years and mean 24h BP 144/82 mmHg. Mean baseline urinary sodium excretion was 192(86) mmol/24h (11.2 grams of salt). As seen in Table 1, sodium excretion was reduced to 91 mmol/24h equalling 5.3 grams of salt. BP levels were reduced significantly. Plasma levels of nitric oxide increased significantly, and this was correlated to the change in 24h BP. Total body water content and plasma levels of BNP was reduced significantly. The changes in BP were not correlated to changes in sodium excretion, body water or BNP. Conclusions: Self-performed dietary sodium restriction could be implemented resulting in a significant reduction in BP levels. BP change was not related to changes in body water or sodium excretion. Increased levels of nitric oxide may be evidence of improved endothelial function and may be one of the involved mechanisms in BP decrease following sodium restriction

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.

A Candidate Gliotransmitter, L-β-Aminoisobutyrate, Contributes to Weight Gain and Metabolic Complication Induced by Atypical Antipsychotics.

Lurasidone and quetiapine are effective atypical mood-stabilizing antipsychotics, but lurasidone and quetiapine are listed as lower-risk and high-risk for weight gain/metabolic complications, respectively. The pathophysiology of the discrepancy of metabolic adverse reactions between these antipsychotics remains to be clarified. The GABA isomer, β-aminoisobutyric acid (BAIBA) enantiomer, was recently re-discovered as myokine via an AMP-activated protein kinase activator (AMPK) enhancer and inhibitory gliotransmitter. Notably, activation of AMPK in peripheral organs improves, but in the hypothalamus, it aggravates metabolic disturbances. Therefore, we determined effects of chronic administration of lurasidone and quetiapine on intracellular and extracellular levels of the BAIBA enantiomer. L-BAIBA is a major BAIBA enantiomer in the hypothalamus and astrocytes, whereas L-BAIBA only accounted for about 5% of total plasma BAIBA enantiomers. Chronic lurasidone administration did not affect body weight but decreased the L-BAIBA level in hypothalamus and cultured astrocytes, whereas chronic quetiapine administration increased body weight and the L-BAIBA level in hypothalamus and astrocytes. Contrary, neither lurasidone nor quetiapine affected total plasma levels of the BAIBA enantiomer since D-BAIBA levels were not affected by these antipsychotics. These results suggest that activation of intracellular L-BAIBA signaling is, at least partially, involved in the pathophysiology of metabolic adverse reaction of quetiapine. Furthermore, this study also demonstrated that lurasidone and quetiapine suppressed and enhanced astroglial L-BAIBA release induced by ripple-burst stimulation (which physiologically contributes to cognitive memory integration during sleep), respectively. Therefore, L-BAIBA probably contributes to the pathophysiology of not only metabolic adverse reactions, but also a part of clinical action of lurasidone or quetiapine.

Role of Hepatocyte Transporters in Drug-Induced Liver Injury (DILI)-In Vitro Testing.

Bile acids and bile salts (BA/BS) are substrates of both influx and efflux transporters on hepatocytes. Canalicular efflux transporters, such as BSEP and MRP2, are crucial for the removal of BA/BS to the bile. Basolateral influx transporters, such as NTCP, OATP1B1/1B3, and OSTα/β, cooperate with canalicular transporters in the transcellular vectorial flux of BA/BS from the sinusoids to the bile. The blockage of canalicular transporters not only impairs the bile flow but also causes the intracellular accumulation of BA/BS in hepatocytes that contributes to, or even triggers, liver injury. In the case of BA/BS overload, the efflux of these toxic substances back to the blood via MRP3, MRP4, and OST α/β is considered a relief function. FXR, a key regulator of defense against BA/BS toxicity suppresses de novo bile acid synthesis and bile acid uptake, and promotes bile acid removal via increased efflux. In drug development, the early testing of the inhibition of these transporters, BSEP in particular, is important to flag compounds that could potentially inflict drug-induced liver injury (DILI). In vitro test systems for efflux transporters employ membrane vesicles, whereas those for influx transporters employ whole cells. Additional in vitro pharmaceutical testing panels usually include cellular toxicity tests using hepatocytes, as well as assessments of the mitochondrial toxicity and accumulation of reactive oxygen species (ROS). Primary hepatocytes are the cells of choice for toxicity testing, with HepaRG cells emerging as an alternative. Inhibition of the FXR function is also included in some testing panels. The molecular weight and hydrophobicity of the drug, as well as the steady-state total plasma levels, may positively correlate with the DILI potential. Depending on the phase of drug development, the physicochemical properties, dosing, and cut-off values of BSEP IC50 ≤ 25-50 µM or total Css,plasma/BSEP IC50 ≥ 0.1 may be an indication for further testing to minimize the risk of DILI liability.

Pegcetacoplan Corrects Hyperbilirubinemia in Both Untreated Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) and in Patients with PNH Who Previously Received Eculizumab

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease characterized by complement-mediated hemolysis and thrombosis. It results from loss of the glycosylphosphatidylinositol-linked complement-inhibitor proteins CD55 and CD59 that inhibit early and late complement proteins, respectively, on red blood cell (RBC) membranes. In untreated patients, formation of the C5b-9 membrane attack complex results in chronic intravascular hemolysis (IVH) and acute hemolytic attacks. Treatment with the C5 inhibitors eculizumab (ECU) and ravulizumab inhibits IVH and unmasks extravascular hemolysis (EVH), which is due to C3b-mediated opsonization of RBCs and their clearance by the reticuloendothelial system. This can result in unconjugated (indirect) hyperbilirubinemia, leading to jaundice and scleral icterus when plasma bilirubin levels reach 2.0-3.0 mg/dL. As in other conditions with EVH (eg, sickle cell disease), bilirubin gallstones have been reported in patients with PNH treated with C5 inhibitors. Pegcetacoplan (PEG), a C3 inhibitor that controls IVH and EVH, is approved in the US for the treatment of adults with PNH. We hypothesized that PEG could control hyperbilirubinemia in PNH patients, reducing jaundice and the risk of gallstone formation. Methods This post hoc analysis evaluated 16- and 26-week data from 2 Phase 3 randomized controlled trials. PEGASUS (NCT03500549) evaluated patients with hemoglobin <10.5 g/dL despite stable ECU treatment for 48 weeks; patients received PEG or ECU for 16 weeks. PRINCE (NCT04085601) evaluated PEG therapy for 26 weeks in patients who were untreated with complement inhibitors. Mean and median total and indirect bilirubin values were calculated at baseline and at the end of the treatment period for patients treated with PEG and ECU in PEGASUS and with PEG in PRINCE. Total bilirubin plasma levels were categorized as normal (<1.1 mg/dL), intermediate (1.1-3.0 mg/dL), and high (>3.0 mg/dL). The main endpoint was the distribution of bilirubin categories at baseline compared with the end of the treatment period and probability of normalization as analyzed by Bowker's test of symmetry. Patients were excluded from the analysis if they did not have both a baseline and end of treatment bilirubin value for analysis. Results In PEGASUS, 74 of 80 patients (PEG, n=36; ECU, n=38) had evaluable bilirubin data; 30 of 35 PEG-treated patients were evaluable from PRINCE. At baseline, 85% (n=63) of patients in PEGASUS had abnormal bilirubin values and 26% (n=20) had high bilirubin values. In PRINCE at baseline, 87% had abnormal bilirubin values, and 23% had high bilirubin values. The hyperbilirubinemia was primarily of the indirect form (TABLE). At 16 weeks, 72% of patients treated with PEG in PEGASUS had normal bilirubin values compared with 16% of patients treated with ECU. In PRINCE, 83% of patients treated with PEG had normal bilirubin at 26 weeks; none had high bilirubin values. The shifts toward normalization in bilirubin categorization (FIGURE) were statistically significant (p<0.0001) for patients on PEG therapy in both PEGASUS and PRINCE. Continued treatment with ECU was not associated with a significant change in bilirubin (p=0.39). In PEGASUS, the normalization rate was 71% (n=22/31; exact 95% CI: 52%, 86%) for PEG and 9% (n=3/32; exact CI: 2%, 25%) for ECU. In PRINCE, the normalization rate was 81% (n=21/26 patients; exact CI: 61%, 93%) after PEG treatment. Conclusions This post hoc analysis found a high prevalence of abnormal bilirubin values at baseline in patients with PNH who were treated with a C5 inhibitor or were untreated. Treatment with PEG significantly improved bilirubin levels, with the majority of patients shifting to normal values. Conversely, bilirubin levels did not improve with continued ECU treatment. These findings suggest that long-term treatment with PEG reduces the signs and symptoms of hyperbilirubinemia, improving jaundice, and decreasing the risk of bilirubin stones and associated complications (eg, biliary sepsis and pancreatitis) in patients with PNH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Vasoactive intestinal peptide promotes hypophagia and metabolic changes: Role of paraventricular hypothalamic nucleus and nitric oxide

Vasoactive intestinal peptide (VIP), a neuromodulator present in the hypothalamus, plays an important role in the regulation of food intake. Paraventricular nucleus of the hypothalamus (PVN) is involved in ingestive responses and regulates the nitric oxide (NO) pathway. The main objectives of this study were to investigate metabolic changes established after different doses and times of VIP microinjection on the PVN, and the effect of VIP microinjection on the PVN on food intake and the role of NO in this control. In anesthetized rats, increased blood plasma glucose and insulin levels were observed following the doses of 40 and 80 ng/g of body weight. At the dose of 40 ng/g, VIP promoted hyperglycemia and hyperinsulinemia 5, 10, and 30 min after microinjection, and increased free fatty acids and total lipids plasma levels after 5 min, and triglycerides after 10 min. In awake animals, once again, VIP administration increased plasmatic levels of glucose, free fatty acids, corticosterone, and insulin 10 min after the microinjection. Moreover, VIP promoted hypophagia in the morning and night periods, and L-arginine (L-Arg) and monosodium glutamate (MSG) or a combination of both attenuated VIP-induced reduction on food intake. In addition, nitrate concentration in the PVN was decreased after VIP microinjection. Our data show that the PVN participates in the anorexigenic and metabolic effects of VIP, and that VIP-induced hypophagia is likely mediated by reduction of NO.

Hyperphosphatemia and its relationship with blood pressure, vasoconstriction, and endothelial cell dysfunction in hypertensive hemodialysis patients

BackgroundHyperphosphatemia occurs frequently in end-stage renal disease patients on hemodialysis and is associated with increased mortality. Hyperphosphatemia contributes to vascular calcification in these patients, but there is emerging evidence that it is also associated with endothelial cell dysfunction.MethodsWe conducted a cross-sectional study in hypertensive hemodialysis patients. We obtained pre-hemodialysis measurements of total peripheral resistance index (TPRI, non-invasive cardiac output monitor) and plasma levels of endothelin-1 (ET-1) and asymmetric dimethylarginine (ADMA). We ascertained the routine peridialytic blood pressure (BP) measurements from that treatment and the most recent pre-hemodialysis serum phosphate levels. We used generalized linear regression analyses to determine independent associations between serum phosphate with BP, TPRI, ET-1, and ADMA while controlling for demographic variables, parathyroid hormone (PTH), and interdialytic weight gain.ResultsThere were 54 patients analyzed. Mean pre-HD supine and seated systolic and diastolic BP were 164 (27), 158 (21), 91.5 (17), and 86.1 (16) mmHg. Mean serum phosphate was 5.89 (1.8) mg/dL. There were significant correlations between phosphate with all pre-hemodialysis BP measurements (r = 0.3, p = .04; r = 0.4, p = .002; r = 0.5, p < .0001; and r = 0.5, p = .0003.) The correlations with phosphate and TPRI, ET-1, and ADMA were 0.3 (p = .01), 0.4 (p = .007), and 0.3 (p = .04). In our final linear regression analyses controlling for baseline characteristics, PTH, and interdialytic weight gain, independent associations between phosphate with pre-hemodialysis diastolic BP, TPRI, and ET-1 were retained (β = 4.33, p = .0002; log transformed β = 0.05, p = .005; reciprocal transformed β = -0.03, p = .047).ConclusionsSerum phosphate concentration is independently associated with higher pre-HD BP, vasoconstriction, and markers of endothelial cell dysfunction. These findings demonstrate an additional negative impact of hyperphosphatemia on cardiovascular health beyond vascular calcification.Trial registrationThe study was part of a registered clinical trial, NCT01862497 (May 24, 2013).

G-CuP: the effect of a forced oral glucose intake on alcohol craving and mesolimbic cue reactivity in alcohol dependence—study protocol of a randomized, double-blind, placebo-controlled crossover study

BackgroundMultiple studies indicate that a lower plasma level of the acetylated form of the appetite-regulating hormone ghrelin and higher plasma levels of insulin lead to a reduction in subjective alcohol craving and a reduced mesolimbic cue reactivity in functional magnetic resonance imaging (fMRI) when being exposed to alcohol-associated stimuli. The ghrelin level can physiologically be reduced by the induction of stomach distension and the ingestion of glucose or lipids.MethodsA total of 108 alcohol-dependent patients aged between 18 and 65 years are examined in the randomized, double-blind, placebo-controlled crossover study. After collecting demographic and psychometric data, participants take part in an alcohol exposure session. Afterwards, the participants go through the intervention condition (oral glucose intake) and the control condition (placebo intake) in a randomized order on two examination days. Blood samples are taken repeatedly (every 10 min) during the study course on both measuring days to determine changes in acetylated and total ghrelin and insulin plasma levels. In parallel, subjective alcohol craving after the glucose or placebo intake as the primary outcome is assessed using the Alcohol Urge Questionnaire (AUQ) and a visual analog scale (VAS). To examine the mesolimbic cue reactivity as the secondary outcome, a fMRI measurement is conducted while being exposed to alcohol-related stimuli. Appropriate statistical analysis will be used for the evaluation of the outcomes.DiscussionIf successful, the results of this study could offer alcohol-dependent patients a new potential option for acute short-term reduction of alcohol craving and thus prevent relapses and prolong periods of abstinence in the long term.Trial registrationGerman Clinical Trials Register DRKS00022419 (UTN: U1111-1278-9428). Retrospectively registered on September 15, 2020.

Early predictors of unfavourable outcome in progressive cholestasis of northwestern Quebec.

Progressive cholestasis of northwestern Quebec (PCNQ) is a rare and severe form of cirrhosis affecting children from Quebec's First Nations. First described by our group in 1981 and historically named North American Indian childhood cirrhosis, such a condition often requires liver transplantation during the pediatric age. This study aimed at suggesting a more culturally sensitive name for the disease and identifying early prognostic factors for an unfavourable outcome. We retrospectively collected data of all 14 consecutive patients diagnosed with PCNQ over the last 20 years and compared children listed for liver transplant before 18 years of age (LT, n = 7) to those with milder disease progression (no-LT, n = 7). Compared with the no-LT group, LT children developed serious complications with an unusually high incidence of gastrointestinal bleeding. Over the first 12 months from presentation, a greater increase of alanine aminotransferase plasma levels, decrease of total bilirubin, and increase of alanine aminotransferase-to-total bilirubin ratio was observed in the LT group. Bone mineral density was lower in LT children independently of vitamin D levels. Patients with PCNQ showed poorer bone health than age-matched children with other cholestatic disorders. In the name of cultural sensitivity, PCNQ should be the preferred name for this condition. Variation of alanine aminotransferase and total bilirubin plasma levels over the first 12 months from presentation might be used for the early identification of children with PCNQ who are at higher risk of unfavourable outcomes. This might help optimize clinical management to populations that are underserved by health care services.

Valproate in status epilepticus: Correlation between loading dose, serum levels, and clinical response.

Background and purposeIntravenous valproate (VPA) is an established treatment of status epilepticus (SE), but optimal loading dose was not fully assessed. We aimed at analyzing the correlation between VPA loading dose and subsequent plasma levels with clinical response in SE.MethodsThis was a retrospective study in one referral center of all consecutive VPA‐naïve SE episodes treated with VPA between January 2013 and June 2019, in which total VPA trough plasma levels after intravenous loading dose were available. Response to VPA, defined as last antiseizure medication introduced before SE resolution (without the need for additional treatment), was correlated with VPA loading dose and trough level. Correlations were adjusted for other SE characteristics.ResultsAmong 128 SE episodes, 53 (41%) responded to VPA. Median VPA loading dose was 25.2 mg/kg (range, 7–58 mg/kg). Loading doses and total plasma levels were not associated with the probability of response or mortality. Correcting for other possible confounders (number of previously tried treatment, demographics, SE severity) did not alter these findings. Only 3.8% of SE episodes that responded to VPA received >30 mg/kg.ConclusionsA high loading dose (>30 mg/kg) is not associated with a greater response rate in patients with SE. Therefore, it seems to bring little benefit. If confirmed in further studies, a dosage of 25–30 mg/kg appears adequate in SE.

Mass spectrometry, data re-analysis, and homology modelling predict posttranslational modifications of leucine-rich alpha-2-glycoprotein as a marker of myelodysplastic syndrome.

Leucine-rich alpha-2-glycoprotein (LRG) has been repeatedly proposed as a potential plasma biomarker for myelodysplastic syndrome (MDS). The goal of our work was to establish the total LRG plasma level and LRG posttranslational modifications (PTMs) as a suitable MDS biomarker. The total plasma LRG concentration was determined with ELISA, whilst the LRG-specific PTMs and their locations, were established using mass spectrometry and public mass spectrometry data re-analysis. Homology modelling and sequence analysis were used to establish the potential impact of PTMs on LRG functions via their impact on the LRG structure. While the results showed that the total LRG plasma concentration is not a suitable MDS marker, alterations within two LRG sites correlated with MDS diagnosis (p= 0.0011). Sequence analysis and the homology model suggest the influence of PTMs within the two LRG sites on the function of this protein. We report the presence of LRG proteoforms that correlate with diagnosis in the plasma of MDS patients. The combination of mass spectrometry, re-analysis of publicly available data, and homology modelling, represents an approach that can be used for any protein to predict clinically relevant protein sites for biomarker research despite the character of the PTMs being unknown.