Polyethylene glycols (PEGs) are amphiphilic polymers that are used extensively in consumer products and PEGylated biotherapeutics. Although PEGs are considered biologically inert with a low toxicity, anti-PEG antibodies have been detected in patients receiving treatment with PEGylated biotherapeutics as well as in healthy individuals. Despite continual exposure in daily life, the prevalence of PEGs within the general population remains unclear. This study aimed to evaluate a healthy population for the presence of PEGs. A validated enzyme-linked immunosorbent assay (ELISA) platform was used to analyze 200 plasma samples from healthy adults for the presence of PEGs. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was also used to analyze the samples for the presence of four PEG analytes: PEG3350; PEG4000; PEG10000; and PEG2ru20K COOH, a 20 kDa PEG used to modify therapeutic proteins. The detection limit for PEG3350 calculated by extrapolation with linear regression was assumed to be 3.60 ng/mL. PEG levels above the lower limit of quantification (LLOQ) (21.6 ng/mL) were detected in 41 plasma samples, range 22.1-43.9 ng/mL, using the ELISA platform. None of the samples were found to contain detectable levels of PEG4000, PEG10000, or PEG2ru20K COOH. Following extrapolation, PEG3350 levels above the LLOQ (3.60 ng/mL) were detected in 154 plasma samples, range 3.69-46.0 ng/mL. Using an ELISA platform and HPLC-MS/MS analysis, PEGs were detected in the plasma of 77.4% of healthy adults. The findings are suggestive that expanding worldwide exposure to PEGs may have resulted in a much higher incidence of detectable plasma burdens of PEG than was previously realized.

Background Diagnosis of sepsis at the ICU admission is burdened by uncertainty. There is a need for an accurate identification of patients with sepsis from those with non-infectious, e.g. post-surgical and random trauma. We tested diagnostic utility of a novel biomarker, nucleoside t6A (N6-threonylcarbamoyladenosine) in animal sepsis models and in adult patients with sepsis compared to two different patient cohorts with elective coronary artery bypass graft surgery (CABG) and polytrauma. Methods We performed a multicenter retrospective comparative observational study in adult intensive care units (ICUs) at three university hospitals: 81 patients presenting to the ICU with sepsis; 81 patients after CABG (center 1), 64 patients with polytrauma (center 2; Injury Severity Score >15) and 49 patients with COVID-19 (center 3). All animal modeling was performed by a research institution (center 4). Results Circulating t6A measured by tandem mass spectrometry accurately identified patients with sepsis at the ICU admission when compared to CABG (AUC 95%) and polytrauma (AUC 97%) patients was superior to procalcitonin (PCT) with an AUC 88% (p<0.05) by ROC test. In identification of 49 COVID-19 patients at the ICU admission, t6A reached AUC of 88%. t6A was released to circulation approximately 6–8 h post-infection onset as demonstrated in the baboon E. coli septic shock and non-lethal abdominal sepsis mouse models. While PCT declined, t6A median concentration was constantly above the optimal ROC diagnostic threshold until day 10 post-admission. Both t6A (AUC 62%) and PCT (AUC 72%) poorly predicted sepsis outcome at-admission. There was no increase of t6A concentration in pig trauma. Compared to a normal human value, circulating t6A was of similar magnitude in the healthy baboon, pig, dog, rabbit, rat and mouse. Conclusion t6A was highly accurate in detecting sepsis at the ICU admission compared to surgical CABG and severe polytrauma patients. t6A rapid rise at the sepsis onset and its prolonged elevation makes t6A an apt marker for diagnosis of sepsis. Overall, t6A shows potential for a precise and early differentiation between septic and non-septic patients in the ICU.

Within the bioanalytical community, the use of blank matrix from preclinical animals for bioanalytical method validation and sample analysis is common practice and required in the context of guidelines for bioanalytical method validation. At the same time, its use has been challenged by the scientific community for decades, since there is ample scientific evidence to allow the use surrogate matrices for this purpose. Nevertheless, legacy and current regulatory thinking continues to be reluctant to allow the use of surrogate matrices in bioanalytical testing except for so-called rare matrices. As part of ongoing discussions in relation to the ICH M10 Guideline, the European Bioanalysis Forum re-challenges the unnecessary use of blank matrices from preclinical animals and believes that, as part of community responsibility and ethical standards and when supported by data, the use of surrogate matrices should become widely accepted. It is in this context that targeted experiments were conducted within the European Bioanalysis Forum to gather additional data and re-open the discussions with all involved and that it should become acceptable to use surrogate matrices wherever possible.

Abstract Background Septic encephalopathy is frequent but its pathophysiology is enigmatic. We studied expression of neurotransmitters, inflammation and integrity of the blood–brain barrier (BBB) in several brain regions during abdominal sepsis. We compared mice with either lethal or surviving phenotype in the first 4 sepsis days. Mature CD-1 females underwent cecal ligation and puncture (CLP). Body temperature (BT) was measured daily and predicted-to-die (within 24 h) mice (for P-DIE; BT < 28 °C) were sacrificed together (1:1 ratio) with mice predicted-to-survive (P-SUR; BT > 35 °C), and healthy controls (CON). Brains were dissected into neocortex, cerebellum, midbrain, medulla, striatum, hypothalamus and hippocampus. Results CLP mice showed an up to threefold rise of serotonin in the hippocampus, 5-hydroxyindoleacetic and homovanillic acid (HVA) in nearly all regions vs. CON. Compared to P-SUR, P-DIE mice showed a 1.7 to twofold rise of HVA (386 ng/g of tissue), dopamine (265 ng/g) and 3,4-Dihydroxyphenylacetic acid (DOPAC; 140 ng/g) in the hippocampus, hypothalamus and medulla (174, 156, 82 ng/g of tissue, respectively). CLP increased expression of TNFα, IL-1β and IL-6 mRNA by several folds in the midbrain, cerebellum and hippocampus versus CON. The same cytokines were further elevated in P-DIE vs P-SUR in the midbrain and cerebellum. Activation of astrocytes and microglia was robust across regions but remained typically phenotype independent. There was a similar influx of sodium fluorescein across the BBB in both P-DIE and P-SUR mice. Conclusions Compared to survivors, the lethal phenotype induced a stronger deregulation of amine metabolism and cytokine expression in selected brain regions, but the BBB permeability remained similar regardless of the predicted outcome.

Fabry disease (FD, OMIM 301500) is a rare X-linked inherited lysosomal storage disorder associated with reduced activities of α-galactosidase A (aGal, EC 3.2.1.22). The current standard of care for FD is based on enzyme replacement therapy (ERT), in which a recombinantly produced version of αGal is intravenously (iv) applied to Fabry patients in biweekly intervals. Though the iv application is clinically efficacious, periodical infusions are inconvenient, time- and resource-consuming and they negatively impact the patients' quality of life. Subcutaneous (sc) injection, in contrast, is an established route of administration for treatment of chronic conditions. It opens the beneficial option of self-administration, thereby improving patients' quality of life and at the same time reducing treatment costs. We have previously shown that Moss-α-Galactosidase (moss-aGal), recombinantly produced in the moss Physcomitrium patens, is efficient in degrading accumulated Gb3 in target organs of murine model of FD and in the phase I clinical study, we obtained first efficacy evidence in human patients following single iv infusion. Here, we tested the efficacy of subcutaneous administration of moss-aGal and compared it with the results observed following iv infusion in Fabry mice. The obtained findings demonstrate that subcutaneously applied moss-aGal is correctly transported to target organs and efficacious in degrading Gb3 deposits there and thus suggest the possibility of using this route of administration for therapy of Fabry disease.

OBJECTIVE:Lidocaine 5% patches are approved for the treatment of post-herpetic neuralgia in adults. Little information is available on the penetration of lidocaine into skin and skin-related soft tissue, which are thought to be closer to the site where lidocaine exerts its pharmacological action on neuronal structures. This pilot study investigated subcutaneous and systemic pharmacokinetics of lidocaine during topical application of two different lidocaine 5% patches.MATERIALS AND METHODS:This randomized two-way, two-period crossover study assessed lidocaine concentrations in subcutaneous tissue (by microdialysis) and plasma of n = 5 healthy subjects during 12-hour-long applications of a recently developed lidocaine 5% patch (Laboratorios Gebro Pharma, SA, Barcelona, Spain) and a marketed reference patch (Versatis 5% lidocaine patch, Grünenthal, Brunn am Gebirge, Austria), respectively.RESULTS:Lidocaine was detectable in subcutaneous tissue within 60 minutes from start of patch application, and in plasma only after a marked delay. The test formulation led to increased exposure to lidocaine in both subcutaneous tissue and plasma.CONCLUSION:This study has underscored the potential of microdialysis to comparatively assess the pharmacokinetics of two different drug formulations and encourages its further use in this area.

Obesity-induced chronic low-grade inflammation originates from adipose tissue and is crucial for obesity-driven metabolic deterioration, including insulin resistance and type 2 diabetes. Chronic inflammation may be a consequence of a failure to actively resolve inflammation and could result from a lack of local specialized proresolving lipid mediators (SPMs), such as resolvins and protectins, which derive from the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We assessed obesity-induced changes of n-3–derived SPMs in adipose tissue and the effects of dietary EPA/DHA thereon. Moreover, we treated obese mice with SPM precursors and investigated the effects on inflammation and metabolic dysregulation. Obesity significantly decreased DHA-derived 17-hydroxydocosahexaenoic acid (17-HDHA, resolvin D1 precursor) and protectin D1 (PD1) levels in murine adipose tissue. Dietary EPA/DHA treatment restored endogenous biosynthesis of n-3–derived lipid mediators in obesity while attenuating adipose tissue inflammation and improving insulin sensitivity. Notably, 17-HDHA treatment reduced adipose tissue expression of inflammatory cytokines, increased adiponectin expression, and improved glucose tolerance parallel to insulin sensitivity in obese mice. These findings indicate that impaired biosynthesis of certain SPM and SPM precursors, including 17-HDHA and PD1, contributes to adipose tissue inflammation in obesity and suggest 17-HDHA as a novel treatment option for obesity-associated complications.

Recently, lyso-globotriaosylsphingosine (lyso-Gb3) was found to be elevated in plasma of treatment naive male patients and some female patients with Fabry Disease (FD). This study tested whether lyso-Gb3 could be analyzed in dried blood spots (DBS) from filter cards and whether concentrations are elevated in newborn infants with FD. Lyso-Gb3 concentrations were analyzed in DBS following extraction using a novel HPLC-mass spectrometry (MS)/MS method. Lyso-Gb3 levels in DBS were above the lower limit of quantitation (0.28 ng/mL) in 5/17 newborn FD infants (16 males; range: 1.02-8.81 ng/mL), but in none of the newborn controls, in all 13 patients (4 males) with classic FD (range: 2.06-54.1 ng/mL), in 125/159 Taiwanese individuals with symptomatic or asymptomatic FD who carry the late onset α-galactosidase A (GLA) mutation c.936+919G>A (IVS4+919G>A) (3.75±0.69 ng/mL; range: 0.418-3.97 ng/mL) and in 20/29 healthy controls (0.77±0.24 ng/mL; range: 0.507-1.4 ng/mL). The HPLC-MS/MS method for analysis of lyso-Gb3 is robust and yields reproducible results in DBS in patients with FD. However, concentrations of lyso-Gb3 were below the limit of quantitation in most newborn infants with FD rendering this approach not suitable for newborn screening. In addition, most females with the late onset mutation have undetectable lyso-Gb3 concentrations.

Background Gaucher disease (GD) is the most common lysosomal storage disorder (LSD). Based on a deficient β-glucocerebrosidase it leads to an accumulation of glucosylceramide. Standard diagnostic procedures include measurement of enzyme activity, genetic testing as well as analysis of chitotriosidase and CCL18/PARC as biomarkers. Even though chitotriosidase is the most well-established biomarker in GD, it is not specific for GD. Furthermore, it may be false negative in a significant percentage of GD patients due to mutation. Additionally, chitotriosidase reflects the changes in the course of the disease belatedly. This further enhances the need for a reliable biomarker, especially for the monitoring of the disease and the impact of potential treatments. Methodology Here, we evaluated the sensitivity and specificity of the previously reported biomarker Glucosylsphingosine with regard to different control groups (healthy control vs. GD carriers vs. other LSDs). Findings Only GD patients displayed elevated levels of Glucosylsphingosine higher than 12 ng/ml whereas the comparison controls groups revealed concentrations below the pathological cut-off, verifying the specificity of Glucosylsphingosine as a biomarker for GD. In addition, we evaluated the biomarker before and during enzyme replacement therapy (ERT) in 19 patients, demonstrating a decrease in Glucosylsphingosine over time with the most pronounced reduction within the first 6 months of ERT. Furthermore, our data reveals a correlation between the medical consequence of specific mutations and Glucosylsphingosine. Interpretation In summary, Glucosylsphingosine is a very promising, reliable and specific biomarker for GD.

BACKGROUND Chronic adipose tissue inflammation is a hallmark of obesity, triggering the development of associated pathologies, particularly type 2 diabetes. Long-chain n-3 PUFAs reduce cardiovascular events and exert well-established antiinflammatory effects, but their effects on human adipose tissue inflammation are unknown. OBJECTIVE We investigated whether n-3 PUFAs reduce adipose tissue inflammation in severely obese nondiabetic patients. DESIGN We treated 55 severely obese nondiabetic patients, scheduled to undergo elective bariatric surgery, with 3.36 g long-chain n-3 PUFAs/d (EPA, DHA) or an equivalent amount of butterfat as control, for 8 wk, in a randomized open-label controlled clinical trial. The primary efficacy measure was inflammatory gene expression in visceral and subcutaneous adipose tissue samples (subcutaneous adipose tissue and visceral adipose tissue), collected during surgery after the intervention. Secondary efficacy variables were adipose tissue production of antiinflammatory n-3 PUFA-derived eicosanoids, plasma concentrations of inflammatory markers, metabolic control, and the effect of the Pro12Ala PPARG polymorphism on the treatment response. RESULTS Treatment with n-3 PUFAs, which was well tolerated, decreased the gene expression of most analyzed inflammatory genes in subcutaneous adipose tissue (P < 0.05) and increased production of antiinflammatory eicosanoids in visceral adipose tissue and subcutaneous adipose tissue (P < 0.05). In comparison with control subjects who received butterfat, circulating interleukin-6 and triglyceride concentrations decreased significantly in the n-3 PUFA group (P = 0.04 and P = 0.03, respectively). The Pro12Ala polymorphism affected the serum cholesterol response to n-3 PUFA treatment. CONCLUSIONS Treatment with long-chain n-3 PUFAs favorably modulated adipose tissue and systemic inflammation in severely obese nondiabetic patients and improved lipid metabolism. These effects may be beneficial in the long-term treatment of obesity. This trial was registered at clinicaltrials.gov as NCT00760760.

The AP301 peptide mimics the lectin-like domain of TNF-α. The synthetic peptide AP301 (molecular weight 1923.1amu) is composed of 17 amino acids and contains an intramolecular disulfide bond between the N-terminal and the C-terminal cysteine. AP301 interacts with the endothelial sodium channel (ENaC) and activates pulmonary liquid clearance both in vitro and in animal studies. Currently, AP301 is subject to clinical investigations for the treatment of pulmonary oedema. With HPLC-MS/MS on reversed phase chromatography a determination limit of 1ng AP301/mL human plasma can be achieved. The MS-ionisation was done with ESI positive. 50μL of human plasma was mixed with the internal standard (a stable isotope labelled AP301, with a total of 6 carbon 13) in acetonitrile for protein precipitation. After centrifugation a part of the clear supernatant was injected into HPLC-MS/MS. Validation was performed according to FDA-guideline in three batches [U.S. Department of Health and Human Services, Food and Drug Administration (FDA): Guidance for Industry, Bioanalytical Method Validation, May 2001]. By using a 6xC13 isotopically labelled internal standard good precision, accuracy and linearity can be gained. The inter-batch precision (CV) of the quality control samples in human plasma (conc. 2.50/20/240ng/mL) ranged from 5.54 to 10.15%. The inter-batch accuracy (with reference to the mean value) of the quality control samples in plasma ranged from 96.1% to 99.9%. The analyte was stable in human plasma over three freeze/thaw cycles, or for 4h at room temperature, or for at least 20weeks when stored at below -20°C. This method was used for quantifying AP301 after inhalative application in a phase I-study.

Background: Hyperlipidemia and obesity are associated with metabolic syndrome and increased risk in developing diabetes and cardiovascular disease. Nutritional supplements, e.g. L-carnitine and polyunsaturated fatty acids (PUFAs), exert lipid-lowering effects. Hence, the hypothesis that dietetic intervention reduces plasma lipid levels and metabolic enzymes in overweight hyperlipidemic subjects was tested. Subjects and Methods: In a prospective placebo-controlled double-blind study in 22 moderately hyperlipidemic obese humans consuming low-fat yoghurt enriched with a combination of low-dose PUFAs, polyphenols and L-carnitine (PPC) twice a day for 12 weeks were compared to 20 matching participants ingesting low-fat yoghurt. The effects on plasma lipids and expression of enzymes involved in regulation of fatty acid oxidation in peripheral blood mononuclear cells (PBMCs) and HepG2 cells were evaluated. Results: PPC consumption led to significantly reduced plasma free fatty acid (–29%) and triglyceride (–24%) concentrations (each p < 0.05). PPC application increased significantly peroxisome proliferator-activated receptor α (PPARα) mRNA abundances and those of PPARα target genes (carnitine palmitoyltransferases-1, CPT1A and CPT1B, carnitine acetyltransferase and organic cation transporter 2; each p < 0.05) in PBMCs. In controls, plasma lipid levels and PBMC gene expression did not change. These findings were substantiated by the results of cell culture experiments in HepG2 cells. Conclusion: Supplementation of PPC had marked lipid-lowering effects and PBMC gene expression profiles seemed to reflect nutrition-related metabolic changes.