Albumin Is a Component of the Esterase Status of Human Blood Plasma.

The esterase status of blood plasma can claim to be one of the universal markers of various diseases; therefore, it deserves attention when searching for markers of the severity of COVID-19 and other infectious and non-infectious pathologies. When analyzing the esterase status of blood plasma, the esterase activity of serum albumin, which is the major protein in the blood of mammals, should not be ignored. The purpose of this study is to expand understanding of the esterase status of blood plasma and to evaluate the relationship of the esterase status, which includes information on the amount and enzymatic activity of human serum albumin (HSA), with other biochemical parameters of human blood, using the example of surviving and deceased patients with confirmed COVID-19. In experiments in vitro and in silico, the activity of human plasma and pure HSA towards various substrates was studied, and the effect of various inhibitors on this activity was tested. Then, a comparative analysis of the esterase status and a number of basic biochemical parameters of the blood plasma of healthy subjects and patients with confirmed COVID-19 was performed. Statistically significant differences have been found in esterase status and biochemical indices (including albumin levels) between healthy subjects and patients with COVID-19, as well as between surviving and deceased patients. Additional evidence has been obtained for the importance of albumin as a diagnostic marker. Of particular interest is a new index, [Urea] × [MDA] × 1000/(BChEb × [ALB]), which in the group of deceased patients was 10 times higher than in the group of survivors and 26 times higher than the value in the group of apparently healthy elderly subjects.

Leveraging Human Plasma-Derived Small Extracellular Vesicles as Liquid Biopsy to Study the Induction of Cytochrome P450 3A4 by Modafinil.

Preparations of plasma-derived small extracellular vesicles (sEVs) were deployed as liquid biopsy to study cytochrome P450 (CYP) 3A4 (CYP3A4) induction following modafinil 400 mg once daily × 14 days (young healthy volunteers, N = 10 subjects). Induction was confirmed using the 4ß-hydroxycholesterol-to-cholesterol (4ßHC/C) ratio, a plasma CYP3A4/5 biomarker, with a mean 2.1-fold increase (Day 15 vs. Day 1; 90% confidence interval (CI) = 1.8-2.3; P value = 0.0004). Proteomic analysis revealed the induction (mean Day 15 vs. Day 1 fold-increase (90% CI)) of both liver (1.3 (1.1-1.5), P value = 0.014) and nonliver (1.9 (1.6-2.2), P value = 0.04) sEV CYP3A4 protein expression. In CYP3A5 nonexpresser subjects, the baseline (pre-dose) 4ßHC/C plasma ratio was more highly correlated with liver sEVs (r = 0.937, P value = 0.001) than nonliver sEVs (r = 0.619, P value = 0.101) CYP3A4 protein expression. When CYP3A5 expressers (CYP3A5*1/*3) were included, the correlation with liver sEVs (r = 0.761, P value = 0.011) and nonliver sEVs (r = 0.391, P value = 0.264) CYP3A4 protein was weaker. Although modafinil-induced changes in plasma 4ßHC/C ratio did not correlate with sEVs CYP3A4 protein expression, the individual subject sEVs proteomic data were used successfully to predict victim drug (midazolam, triazolam, dextromethorphan, 17α-ethinylestradiol, and abemaciclib) area under the plasma concentration-time curve (AUC) ratios (AUCRs) following modafinil. Based on the AUCR values, modafinil was classified as a weak to moderate CYP3A4 inducer (vs. rifampicin). For the first time, it was possible to deploy plasma-derived sEVs to study CYP3A4 induction beyond rifampicin, a more potent CYP3A4 inducer.

Influence of exogenous phytase supplementation on phytate degradation, plasma inositol, alkaline phosphatase, and glucose concentrations of broilers at 28 days of age.

Inositol is the final product of phytate degradation, which has the potential to serve as an indicator of phytase efficacy. An experiment was conducted to evaluate effects of supplementing broiler diets with phytase on phytate degradation and plasma inositol concentrations at 28 d of age. Twenty-four Ross × Ross 708 male chicks were placed in battery cages (4 birds per cage) from 1 to 21 d of age and individually from 22 to 28 d of age. At 27 d of age, a catheter was placed in the brachial vein of broilers to avoid repeated puncture of the vein during blood collection. At 28 d of age, broilers received 1 of 3 experimental diets formulated to contain 0, 400, or 1,200 phytase units (FTU)/kg, respectively, in diet 1, 2, and 3. Blood was collected 1 h before feeding experimental diets and from 20 to 240 min after feeding experimental diets at 20-min intervals with a final blood collection at 480 min to determine plasma inositol concentrations. Inositol phosphate (IP) ester degradation was determined in gizzard contents and ileal digesta. Broilers provided the 1,200 FTU/kg phytase diet had 60% less (P < 0.01) IP6 concentration in gizzard content (1,264 vs. 4,176 nmol/g) and ileal digesta (13,472 vs. 33,244 nmol/g) than birds fed the 400 FTU/kg diet. Adding phytase at 1,200 FTU/kg increased (P < 0.01) inositol concentrations in gizzard content and ileal digesta of broilers by 2.5 (2,703 vs. 1,071 nmol/g) and 3.5 (16,485 vs. 4,667 nmol/g) fold, respectively, compared with adding 400 FTU/kg. Plasma inositol concentration of broilers was not different (P = 0.94) among the dietary treatments at each collection time. Inositol liberation in the digesta of broilers fed diets with 1,200 FTU/kg phytase did not translate to increased plasma inositol concentrations, which warrants further investigation.

Effects of supplementation of DL-methionine on tissue and plasma antioxidant status during heat-induced oxidative stress in broilers.

Exposure to high ambient temperature has been shown to impair growth performance and to cause oxidative stress in broilers. This study investigated the hypothesis that supplementation with methionine (Met) as DL-Met (DLM) more than the National Research Council recommendations improves growth performance and alleviates oxidative stress in broilers exposed to high ambient temperature. One-day-old male Cobb-500 broilers (n = 68) were allotted to 4 groups and phase-fed 3 basal diets during days 1 to 10, 11 to 21, and 22 to 35. One group was kept under thermoneutral temperature conditions and received the basal diets with Met + cysteine (Cys) concentrations according to recommendations of NRC. The other 3 groups were kept in a room with an increased ambient temperature from week 3 to 5 and were fed either the basal diet or the basal diets supplemented with 2 levels of DLM in which Met + Cys concentrations exceeded NRC recommendations by around 20% (group DLM1) and 40% (group DLM2), respectively. As expected, the broilers exposed to high ambient temperature showed a lower feed intake, lower body weight gains, a higher feed:gain ratio, and biochemical indications of oxidative stress in comparison to broilers kept under thermoneutral temperature conditions. Supplementation of DLM did not improve the growth performance in broilers exposed to high ambient temperature. However, the broilers supplemented with DLM had increased concentrations of glutathione in liver and breast muscle (groups DLM1 and DLM2), increased concentrations of tocopherols in the liver (group DLM2), and reduced concentrations of 7α-hydroxycholesterol and 7-ketocholesterol in heat-processed thigh muscle (groups DLM1 and DLM2) in comparison to the control group exposed to high ambient temperature. Concentrations of thiobarbituric acid-reactive substances and vitamin C in plasma, liver, and muscle were not different between the 3 groups exposed to heat stress. Nevertheless, the study shows that supplementation of DLM in slight excess of the Met concentration required for maximum growth performance improved the antioxidant status in tissues and reduced the susceptibility of muscle toward oxidation in heat-stressed broilers.

Investigation of optimal conditions for production of antioxidant peptides from duck blood plasma: response surface methodology.

Duck blood is rich in protein. It is one of the main by-products in the slaughter industry. The objective of this research was to optimize and establish a method for producing duck plasma antioxidant peptides. The composition of duck plasma powder was analyzed. Protease selection experiment (Alcalase, Protamex, and Flavourzyme) and single-factor experiment were performed, and response surface methodology was used to determine the optimal hydrolysis conditions for duck plasma. Among the proteases, Alcalase hydrolysate exhibited the strongest 1,1-diphenyl-2-picrylhydrazyl scavenging rate. The optimum enzymatic hydrolysis conditions were hydrolysis time of 6 h, temperature of 65.5°C, pH 10.0, and enzyme-to-substrate ratio of 0.3%. The 1,1-diphenyl-2-picrylhydrazyl scavenging rate reached 64.84%, and the ratio of essential amino acids was 38.76%. Briefly, the duck plasma hydrolysate exhibited strong antioxidant properties and reasonable composition of amino acids. Thus, it may be used as a nutritional or functional ingredient in foods or medicines. This research provides a theoretical basis for comprehensive processing and high value utilization of duck plasma.

Hepatic progenitor cell activation is induced by the depletion of the gut microbiome in mice.

The homeostasis of the gut microbiome is crucial for human health and for liver function. However, it has not been established whether the gut microbiome influence hepatic progenitor cells (HPCs). HPCs are capable of self-renewal and differentiate into hepatocytes and cholangiocytes; however, HPCs are normally quiescent and are rare in adults. After sustained liver damage, a ductular reaction occurs, and the number of HPCs is substantially increased. Here, we administered five broad-spectrum antibiotics for 14 days to deplete the gut microbiomes of male C57BL/6 mice, and we measured the plasma aminotransferases and other biochemical indices. The expression levels of two HPC markers, SRY-related high mobility group-box gene 9 (Sox9) and cytokeratin (CK), were also measured. The plasma aminotransferase activities were not affected, but the triglyceride, lactate dehydrogenase, low-density lipoprotein, and high-density lipoprotein concentrations were significantly altered; this suggests that liver function is affected by the composition of the gut microbiome. The mRNA expression of Sox9 was significantly higher in the treated mice than it was in the control mice (p < 0.0001), and a substantial expression of Sox9 and CK was observed around the bile ducts. The mRNA expression levels of proinflammatory factors (interleukin [IL]-1ß, IL-6, tumor necrosis factor [TNF]-α, and TNF-like weak inducer of apoptosis [Tweak]) were also significantly higher in the antibiotic-treated mice than the levels in the control mice. These data imply that the depletion of the gut microbiome leads to liver damage, negatively impacts the hepatic metabolism and function, and activates HPCs. However, the underlying mechanisms remain to be determined.

The Plethora of Angiotensin-Converting Enzyme-Processed Peptides in Mouse Plasma.

Angiotensin-converting enzyme (ACE) converts angiotensin I into the potent vasoconstrictor angiotensin II, which regulates blood pressure. However, ACE activity is also essential for other physiological functions, presumably through processing of peptides unrelated to angiotensin. The goal of this study was to identify novel natural substrates and products of ACE through a series of mass-spectrometric experiments. This included comparing the ACE-treated and untreated plasma peptidomes of ACE-knockout (KO) mice, validation with select synthetic peptides, and a quantitative in vivo study of ACE substrates in mice with distinct genetic ACE backgrounds. In total, 244 natural peptides were identified ex vivo as possible substrates or products of ACE, demonstrating high promiscuity of the enzyme. ACE prefers to cleave substrates with Phe or Leu at the C-terminal P2' position and Gly in the P6 position. Pro in P1' and Iso in P1 are typical residues in peptides that ACE does not cleave. Several of the novel ACE substrates are known to have biological activities, including a fragment of complement C3, the spasmogenic C3f, which was processed by ACE ex vivo and in vitro. Analyses with N-domain-inactive (NKO) ACE allowed clarification of domain selectivity toward substrates. The in vivo ACE-substrate concentrations in WT, transgenic ACE-KO, NKO, and CKO mice correspond well with the in vitro observations in that higher levels of the ACE substrates were observed when the processing domain was knocked out. This study highlights the vast extent of ACE promiscuity and provides a valuable platform for further investigations of ACE functionality.

In vitro assessment of the long-term stability of the inhibitory effects of canine and feline plasma on MMP 2 and 9.

PURPOSE: To evaluate the effect of increasing storage time on the inhibitory effects of canine and feline plasma on matrix metalloproteinases (MMP) 2 and 9 in vitro. METHODS: Matrix metalloproteinases 2 and 9 activity in the presence of canine plasma stored for 57, 155, 222, 316, 367, and 438 days, and feline plasma stored for 17, 198, 565, and 954 days was assayed using a commercially available colorimetric assay kit. RESULTS: For canine plasma, the MMP 2 activity for older samples was not significantly different than the 57-day sample (P = 0.2025-0.9033). Two canine samples had significantly lower MMP 9 activity than the 57-day sample (367 days: P = 0.0099, 438 days: P = 0.0348, others P = 0.0778-0.9928). For feline plasma, storage time did not significantly affect inhibition of MMP 2 and MMP 9 activity (ANOVA, P = 0.2688 and P = 0.2404, respectively). CONCLUSIONS: Increasing storage time does not significantly decrease the inhibiting activity of plasma on MMP 2 and 9 for up to 14 months in dogs and 31 months in cats.

HPLC-UV assay for the evaluation of inhibitors of plasma amine oxidase using crude bovine plasma.

Recently, we have described a method for evaluation of plasma amine oxidase (PAO) inhibitors, which monitors the formation of 6-(5-phenyl-2H-tetrazol-2-yl)hexanal from the corresponding amine substrate by HPLC with UV-detection using purified bovine PAO. We now investigated, whether crude bovine plasma can be used as enzyme source in this assay instead of the purified enzyme. With the aid of specific inhibitors, it was ensured that there was no detectable activity of other important amine oxidases in the plasma, namely monoamine oxidase (MAO) A and B and diamine oxidase (DAO). For a series of ω-(5-phenyl-2H-tetrazol-2-yl)alkan-1-amine substrates similar conversion rates were measured for both the purified PAO and crude plasma. The inhibition values determined for the PAO inhibitor 2-(4-phenylphenyl)acetohydrazide (16) under different conditions also corresponded. Additionally, inhibition data of the known PAO inhibitor 2-amino-N-(3-phenylbenzyl)acetamide (17) and a newly synthesised meta-substituted derivative of 16 were determined, which together reflect the two-step inhibition mechanism of these covalent inhibitors.

Pharmacokinetics of plasma infusion in congenital thrombotic thrombocytopenic purpura.

Essentials Congenital thrombotic thrombocytopenic purpura (TTP) is primarily treated with plasma infusion. We present a pharmacokinetic analysis of ADAMTS-13 in six patients following plasma infusion. A median half-life of 130 h was demonstrated, ranging between 82.6 and 189.5 h. Investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment. SUMMARY: Background Congenital thrombotic thrombocytopenic purpura (TTP) is defined by persistent severe deficiency of ADAMTS-13 in the absence of anti-ADAMTS-13 inhibitory antibodies, confirmed by mutational analysis. Replacement of the missing protease prevents disease relapse, primarily using plasma infusion (PI). Objectives, patients and methods There is scant evidence regarding optimal dose and frequency of treatment, which tends to be empirically guided. We present a pharmacokinetic analysis of ADAMTS-13 in six patients with congenital TTP on established regimes following PI. Results We found a median clearance of 25.41 mL h-1 and half-life of 130 h, ranging between 82.6 and 189.5 h (3.4-7.9 days, respectively). All patients reached baseline ADAMTS-13 level within 7-10 days post-plasma. Median ADAMTS-13 activity peak post-PI was 24.05 IU dL-1 . Variation was related to elimination rate, which, in turn, was affected by weight and metabolism, but not to von Willebrand factor antigen or activity levels. Using the pharmacokinetic parameters, we simulated individualized protocols based on PI dose or frequency to target hypothetical optimal plasma levels of ADAMTS-13 of 10 and 50 IU dL-1 , respectively. Results suggest a target trough ADAMTS-13 of 10 IU dL-1 is feasible but 50 IU dL-1 would not be achievable taking into account volume required. Conclusions Further work is needed to compare treatment of congenital TTP with PI vs. recombinant ADAMTS-13. PI may provide longer duration of ADAMTS-13 effect, but is limited by plasma volume required, whereas recombinant therapy can provide a higher ADAMTS-13 peak. We propose that investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment and provide the rationale for dose and frequency of prophylaxis.

A solution to Nature's haemoglobin knockout: a plasma-accessible carbonic anhydrase catalyses CO2 excretion in Antarctic icefish gills.

In all vertebrates studied to date, CO2 excretion depends on the enzyme carbonic anhydrase (CA) that catalyses the rapid conversion of HCO3- to CO2 at the gas-exchange organs. The largest pool of CA is present within red blood cells (RBCs) and, in some vertebrates, plasma-accessible CA (paCA) isoforms participate in CO2 excretion. However, teleost fishes typically do not have paCA at the gills and CO2 excretion is reliant entirely on RBC CA - a strategy that is not possible in icefishes. As the result of a natural knockout, Antarctic icefishes (Channichthyidae) are the only known vertebrates that do not express haemoglobin (Hb) as adults, and largely lack RBCs in the circulation (haematocrit <1%). Previous work has indicated the presence of high levels of membrane-bound CA activity in the gills of icefishes, but without determining its cellular orientation. Thus, we hypothesised that icefishes express a membrane-bound CA isoform at the gill that is accessible to the blood plasma. The CA distribution was compared in the gills of two closely related notothenioid species, one with Hb and RBCs (Notothenia rossii) and one without (Champsocephalus gunnari). Molecular, biochemical and immunohistochemical markers indicate high levels of a Ca4 isoform in the gills of the icefish (but not the red-blooded N. rossii), in a plasma-accessible location that is consistent with a role in CO2 excretion. Thus, in the absence of RBC CA, the icefish gill could exclusively provide the catalytic activity necessary for CO2 excretion - a pathway that is unlike that of any other vertebrate.

Identification of human, rat and mouse hydrolyzing enzymes bioconverting amino acid ester prodrug of ketoprofen.

Alkyl ester prodrugs are well known to be bioconverted by carboxylesterases, particularly in rodents' by first-pass metabolism in the systemic circulation and liver. However, the bioconversion of structurally more complex esters with polar functional groups is less well understood, especially in humans. Therefore, it is not clear if ester prodrugs can be utilized for targeted drug delivery. In the present study a brain-targeted ester prodrug (1) of ketoprofen, utilizing the l-type amino acid transporter 1 (LAT1) was prepared and the enzymes involved in its metabolism in human plasma and liver S9 subcellular fraction as well as rat brain S9 fraction were identified. Furthermore, species differences among mouse, rat and human plasma and liver S9 fraction were compared. The results showed that bioconversion of the ester prodrug was much faster in mouse plasma compared to human, while it's half-life in rat plasma was closer to the one of human. Moreover, both rodent species showed more efficient bioconversion in the liver S9 fractions compared to human and relatively efficient bioconversion in the brain S9 fractions. More specifically, butyrylcholinesterase (BChE) and paraoxygenase 1 (PON1) were the main hydrolyzing enzymes of the prodrug 1 in human plasma, while carboxylesterases 1 and 2 (CES1 and CES2) as well as PONs were the main bioconverting enzymes in human liver S9 fractions. In rat brain S9 fraction, acetylcholinesterase (AChE) was hydrolyzing the prodrug 1, although also other unidentified metal-and pH-dependent enzyme(s) were recognized to be participating to the total bioconversion of the compound 1 in the brain.

Assessment of the Dual Role of Clumping Factor A in S. Aureus Adhesion to Endothelium in Absence and Presence of Plasma.

Adhesion of Staphylococcus aureus to endothelial cells (ECs) is paramount in infective endocarditis. Bacterial proteins such as clumping factor A (ClfA) and fibronectin binding protein A (FnbpA) mediate adhesion to EC surface molecules and (sub)endothelial matrix proteins including fibrinogen (Fg), fibrin, fibronectin (Fn) and von Willebrand factor (vWF). We studied the influence of shear flow and plasma on the binding of ClfA and FnbpA (including its sub-domains A, A16+, ABC, CD) to coverslip-coated vWF, Fg/fibrin, Fn or confluent ECs, making use of Lactococcus lactis, expressing these adhesins heterologously. Global adherence profiles were similar in static and flow conditions. In the absence of plasma, L. lactis-clfA binding to Fg increased with shear forces, whereas binding to fibrin did not. The degree of adhesion of L. lactis-fnbpA to EC-bound Fn and of L. lactis-clfA to EC-bound Fg, furthermore, was similar to that of L. lactis-clfA to coated vWF domain A1, in the presence of vWF-binding protein (vWbp). Yet, in plasma, L. lactis-clfA adherence to activated EC-vWF/vWbp dropped over 10 minutes by 80% due to vWF-hydrolysis by a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 and that of L. lactis-fnbpA likewise by > 70% compared to the adhesion in absence of plasma. In contrast, plasma Fg supported high L. lactis-clfA binding to resting and activated ECs. Or, in plasma S. aureus adhesion to active endothelium occurs mainly via two complementary pathways: a rapid but short-lived vWF/vWbp pathway and a stable integrin-coupled Fg-pathway. Hence, the pharmacological inhibition of ClfA-Fg interactions may constitute a valuable additive treatment in infective endocarditis.

Prolyl carboxypeptidase activity in the circulation and its correlation with body weight and adipose tissue in lean and obese subjects.

BACKGROUND: Prolyl carboxypeptidase (PRCP) is involved in the regulation of body weight, likely by hydrolysing alpha-melanocyte-stimulating hormone and apelin in the hypothalamus and in the periphery. A link between PRCP protein concentrations in plasma and metabolic disorders has been reported. In this study, we investigated the distribution of circulating PRCP activity and assessed its relation with body weight and adipose tissue in obese patients and patients who significantly lost weight. METHODS: PRCP activity was measured using reversed-phase high-performance liquid chromatography in different isolated blood fractions and primary human cells to investigate the distribution of circulating PRCP. PRCP activity was measured in serum of individuals (n = 75) categorized based on their body mass index (BMI < 25.0; 25.0-29.9; 30.0-39.9; ≥ 40.0 kg/m2) and the diagnosis of metabolic syndrome. Differences in serum PRCP activity were determined before and six months after weight loss, either by diet (n = 45) or by bariatric surgery (n = 24). Potential correlations between serum PRCP activity and several metabolic and biochemical parameters were assessed. Additionally, plasma PRCP concentrations were quantified using a sensitive ELISA in the bariatric surgery group. RESULTS: White blood cells and plasma contributed the most to circulating PRCP activity. Serum PRCP activity in lean subjects was 0.83 ± 0.04 U/L and increased significantly with a rising BMI (p<0.001) and decreased upon weight loss (diet, p<0.05; bariatric surgery, p<0.001). The serum PRCP activity alteration reflected body weight changes and was found to be positively correlated with several metabolic parameters, including: total, abdominal and visceral adipose tissue. Plasma PRCP concentration was found to be significantly correlated to serum PRCP activity (0.865; p<0.001). Additionally, a significant decrease (p<0.001) in plasma PRCP protein concentration (mean ± SD) before (18.2 ± 3.7 ng/mL) and 6 months after bariatric surgery (15.7 ± 2.7 ng/mL) was found. CONCLUSION: Our novel findings demonstrate that white blood cells and plasma contributed the most to circulating PRCP activity. Additionally, we have shown that there were significant correlations between serum PRCP activity and various metabolic parameters, and that plasma PRCP concentration was significantly correlated to serum PRCP activity. These novel findings on PRCP activity in serum support further investigation of its in vivo role and involvement in several metabolic diseases.

Controlling Plasma Stability of Hydroxamic Acids: A MedChem Toolbox.

Hydroxamic acids are outstanding zinc chelating groups that can be used to design potent and selective metalloenzyme inhibitors in various therapeutic areas. Some hydroxamic acids display a high plasma clearance resulting in poor in vivo activity, though they may be very potent compounds in vitro. We designed a 57-member library of hydroxamic acids to explore the structure-plasma stability relationships in these series and to identify which enzyme(s) and which pharmacophores are critical for plasma stability. Arylesterases and carboxylesterases were identified as the main metabolic enzymes for hydroxamic acids. Finally, we suggest structural features to be introduced or removed to improve stability. This work thus provides the first medicinal chemistry toolbox (experimental procedures and structural guidance) to assess and control the plasma stability of hydroxamic acids and realize their full potential as in vivo pharmacological probes and therapeutic agents. This study is particularly relevant to preclinical development as it allows obtaining compounds equally stable in human and rodent models.

The Impact of the Hepatocyte-to-Plasma pH Gradient on the Prediction of Hepatic Clearance and Drug-Drug Interactions for CYP2C9 and CYP3A4 Substrates.

Surrogate assays for drug metabolism and inhibition are traditionally performed in buffer systems at pH 7.4, despite evidence that hepatocyte intracellular pH is 7.0. This pH gradient can result in a pKa-dependent change in intracellular/extracellular concentrations for ionizable drugs that could affect predictions of clearance and P450 inhibition. The effect of microsomal incubation pH on in vitro enzyme kinetic parameters for CYP2C9 (diclofenac, (S)-warfarin) and CYP3A4 (midazolam, dextromethorphan, testosterone) substrates, enzyme specific reversible inhibitors (amiodarone, desethylamiodarone, clozapine, nicardipine, fluconazole, fluvoxamine, itraconazole) and a mechanism-based inhibitor (amiodarone) was investigated. Intrinsic clearance through CYP2C9 significantly increased (25% and 50% for diclofenac and (S)-warfarin respectively) at intracellular pH 7.0 compared with traditional pH 7.4. The CYP3A4 substrate dextromethorphan intrinsic clearance was decreased by 320% at pH 7.0, while midazolam and testosterone remained unchanged. Reversible inhibition of CYP2C9 was less potent at pH 7.0 compared with 7.4, while CYP3A4 inhibition potency was variably affected. Maximum enzyme inactivation rate of amiodarone toward CYP2C9 and CYP3A4 decreased at pH 7.0, while the irreversible inhibition constant remained unchanged for CYP2C9, but decreased for CYP3A4 at pH 7.0. Predictions of clearance and drug-drug interactions made through physiologically based pharmacokinetic models were improved with the inclusion of predicted intracellular concentrations based at pH 7.0 and in vitro parameters determined at pH 7.0. No general conclusion on the impact of pH could be made and therefore a recommendation to change buffer pH to 7.0 cannot be made at this time. It is recommended that the appropriate hepatocyte intracellular pH 7.0 be used for in vitro determinations when in vivo predictions are made.

Metabolic Syndrome Modulates Association between Endothelial Lipase and Lipid/Lipoprotein Plasma Levels in Acute Heart Failure Patients.

We hypothesised that the established association of endothelial lipase (EL) plasma levels with atherogenic lipid profile is altered in acute heart failure (AHF) and additionally affected by overlapping metabolic syndrome (MetS). We examined the association of EL plasma levels and lipid/lipoprotein plasma levels in AHF patients without and with overlapping MetS. The study was performed as a single-centre, observational study on 152 AHF patients, out of which 85 had overlapping MetS. In the no-MetS group, EL plasma levels were significantly positively correlated with plasma levels of atherogenic lipids/lipoproteins, including total cholesterol, low-density lipoprotein (LDL)-cholesterol, total LDL particles and triglycerides, but also with plasma levels of antiatherogenic high-density lipoprotein (HDL)-cholesterol, total HDL particles and small HDL particles. In the MetS group, EL plasma levels were positively correlated with triglyceride and small LDL-particle levels, and significantly negatively correlated with plasma levels of large HDL particles as well as with LDL- and HDL-particle size, respectively. EL- and lipid/lipoprotein- plasma levels were different in the no-MetS patients, compared to MetS patients. The association of EL with atherogenic lipid profile is altered in AHF and additionally modified by MetS, which strongly modulates EL- and lipid/lipoprotein-plasma levels in AHF.

N-Acetylcysteine supplementation alleviates intestinal injury in piglets infected by porcine epidemic diarrhea virus.

Porcine epidemic diarrhea virus (PEDV) infects the intestine of young pigs, but effective measures for prevention and treatment are lacking. N-Acetylcysteine (NAC) has been shown to reduce endotoxin-induced intestinal dysfunction. This study was conducted with the PEDV-infected neonatal piglet model to determine the effect of NAC supplementation on intestinal function. Thirty-two 7-day-old piglets were randomly allocated to one of four treatments in a 2 × 2 factorial design consisting of two liquid diets (0 or 50 mg/kg BW NAC supplementation) and oral administration of 0 or 104.5 TCID50 (50% tissue culture infectious dose) PEDV. On day 7 of the trial, half of the pigs (n = 8) in each dietary treatment received either sterile saline or PEDV (Yunnan province strain) solution at 104.5 TCID50 per pig. On day 10 of the trial, D-xylose (0.1 g/kg BW) was orally administrated to all pigs. One hour later, jugular vein blood samples were collected, and then all pigs were killed to obtain the small intestine. PEDV infection increased diarrhea incidence, while reducing ADG. PEDV infection also decreased plasma D-xylose concentration, small intestinal villus height, mucosal I-FABP and villin mRNA levels but increased mucosal MX1 and GCNT3 mRNA levels (P < 0.05). Dietary NAC supplementation ameliorated the PEDV-induced abnormal changes in all the measured variables. Moreover, NAC reduced oxidative stress, as indicated by decreases in plasma and mucosal H2O2 levels. Collectively, these novel results indicate that dietary supplementation with NAC alleviates intestinal mucosal damage and improves the absorptive function of the small intestine in PEDV-infected piglets.

Acute HIV-1 infection is associated with increased plasma levels of heme oxygenase-1 and presence of heme oxygenase-1-specific regulatory T cells.

OBJECTIVE: Heme oxygenase-1 (HO-1) is an inducible stress response protein with potent anti-inflammatory activity and recent data suggest a potentially beneficial role in HIV pathogenesis. We investigated the impact of HO-1 and a novel subset of HO-1-specific CD8 regulatory T cells on virus-specific T-cell immunity in HIV-1-infected individuals. METHODS: HO-1 protein levels were quantified in plasma from individuals at different stages of HIV-1 disease and longitudinally following primary HIV infection. HO-1-specific CD8 T cells were investigated by flow cytometry using human leukocyte antigen (HLA) class I pentamers. Flow-sorted HO-1-specific CD8 T cells were cultured and tested for suppressive activity on HIV-1-specific cytotoxic T-cell clones clones. HO-1 gene expression was determined in sorted peripheral blood mononuclear cell (PBMC) subsets from individuals with acute HIV-1 infection. RESULTS: HO-1 plasma levels were significantly increased in HIV-1 infection, with the highest levels in individuals with acute HIV-1 infection, and gradually declined over time. The frequency of CD8 T cells specific for HO-1 was elevated in study participants with primary HIV-1 infection and flow-sorted HO-1-specific CD8 T cells were capable of suppressing HIV-1-specific lysis of cytotoxic T-cell clones clones. HO-1 gene expression was upregulated in multiple immune cell subsets during acute HIV-1 infection and HO-1 overexpression modulated anti-HIV immunity in vitro. CONCLUSION: Our data suggest that HO-1 is induced during acute HIV-1 infection, likely mediating anti-inflammatory effects and driving expansion of HO-1-specific CD8 regulatory T cells capable of suppressing HIV-1-specific immune responses in vitro. The investigation of HO-1 and the novel CD8 regulatory cell type described here provide further insight into immune regulation in HIV-1 infection and may hold potential for future immunotherapeutic intervention.

Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma.

OBJECTIVE: Plasma acts as a good indicator of oxidative stress in blood. L-Carnitine is an antioxidant that reduces metabolic stress in cells, thereby providing a protective effect against oxidative stress (OS). L-Carnitine as an additive in storage has not been explored. Thus, this study attempts to analyze the role of L-carnitine in blood storage solution, citrate phosphate dextrose adenine (CPDA)-1, through OS markers including antioxidant enzymes, lipid peroxidation, and protein oxidation. MATERIALS AND METHODS: Blood was collected from male Wistar rats and stored in CPDA-1 solution with L-carnitine (10 mM, 30 mM, and 60 mM: groups LC 10, LC 30, and LC 60, respectively) and without L-carnitine (control group). Plasma was isolated every 5th day and the OS markers were analyzed. RESULTS: Superoxide dismutase (SOD) and sulfhydryl (SH) increased over storage in controls, LC 30, and LC 60. Catalase increased in LC 30 and LC 60 during storage. Thiobarbituric acid reactive substances (TBARS) and protein carbonyl (PrC) levels in all groups increased initially and reduced towards the end of storage. SOD and SH levels were maintained while TBARS and PrC levels increased in LC 10. CONCLUSION: L-Carnitine was beneficial in terms of increased antioxidant capacity and SH and decreased lipid peroxidation. This forms the basis for further studies on L-carnitine as a constituent in storage solutions.