Blood Plasma Concentrations Research Articles

Embryonic Exposure to the Benzotriazole Ultraviolet Stabilizer 2-(2H-benzotriazol-2-yl)-4-methylphenol Decreases Fertility of Adult Zebrafish (Danio rerio).

Benzotriazole ultraviolet stabilizers (BUVSs) are emerging contaminants of concern. They are added to a variety of products, including building materials, personal care products, paints, and plastics, to prevent degradation caused by ultraviolet (UV) light. Despite widespread occurrence in aquatic environments, little is known regarding the effects of BUVSs on aquatic organisms. The aim of the present study was to characterize the effects of exposure to 2-(2H-benzotriazol-2-yl)-4-methylphenol (UV-P) on the reproductive success of zebrafish (Danio rerio) following embryonic exposure. Embryos were exposed, by use of microinjection, to UV-P at <1.5 (control), 2.77, and 24.25 ng/g egg, and reared until sexual maturity, when reproductive performance was assessed, following which molecular and biochemical endpoints were analyzed. Exposure to UV-P did not have a significant effect on fecundity. However, there was a significant effect on fertilization success. Using UV-P-exposed males and females, fertility was decreased by 8.75% in the low treatment group and by 15.02% in the high treatment group relative to control. In a reproduction assay with UV-P-exposed males and control females, fertility was decreased by 11.47% in the high treatment group relative to the control. Embryonic exposure to UV-P might have perturbed male sex steroid synthesis as indicated by small changes in blood plasma concentrations of 17β-estradiol and 11-ketotestosterone, and small statistically nonsignificant decreases in mRNA abundances of cyp19a1a, cyp11c1, and hsd17b3. In addition, decreased transcript abundances of genes involved in spermatogenesis, such as nanos2 and dazl, were observed. Decreases in later stages of sperm development were observed, suggesting that embryonic exposure to UV-P impaired spematogenesis, resulting in decreased sperm quantity. The present study is the first to demonstrate latent effects of BUVSs, specifically on fish reproduction. Environ Toxicol Chem 2024;43:385-397. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Dose-response relationship between toothpaste soluble fluoride absorbed in the gastrointestinal tract and saliva fluoride secretion

Aim: This study aimed to evaluate if there is a dose-response relationship between toothpaste chemically soluble fluoride absorbed in the gastrointestinal tract and fluoride secreted by saliva, giving support to the use of saliva as surrogate for plasma fluoride. Methods: A 4-phase single blind study was conducted, in which 10 participants were subjected in each phase to one of the assigned treatment groups: group I: fresh sample of a Na2FPO3/CaCO3-based toothpaste with 1,334 μg F/g of total soluble fluoride (TSF) and groups II–IV: aged samples of this toothpaste presenting TSF concentrations of 1,128, 808, and 687 μg F/g, respectively. In all phases, the participants ingested an amount of toothpaste equivalent to 70.0 μg F/Kg body weight, as total fluoride (TF). Saliva and blood samples were collected before (baseline) and up to 180 min after toothpaste ingestion as indicator of fluoride bioavailability. F concentration in saliva and blood plasma was determined with a fluoride ion-specific electrode. The areas under the curve (AUC) of F concentration versus time (AUC = ng F/mL × min) and the peaks of fluoride concentration (Cmax) in saliva and plasma were calculated. Results: A significant correlation between mg of TSF ingested and the AUC (r=0.47; p&lt;0.01), and Cmax (r=0.59; p&lt;0.01) in saliva was found; for TF, the correlation was not significant (p&gt;0.05). In addition, the correlations between plasma and saliva fluoride concentrations were statistically significant for AUC (r=0.55; p&lt;0.01) as for Cmax (r=0.68; p&lt;0.01). Conclusion: The findings support that saliva can be used as a systemic biomarker of bioavailable fluoride present in Na2FPO3/ CaCO3-based toothpaste.

The probiotic Lacticaseibacillus rhamnosus HN001 influences the architecture and gene expression of small intestine tissue in a piglet model.

This study investigated the effects of Lacticaseibacillus rhamnosus HN001 supplementation on the architecture and gene expression in small intestinal tissues of piglets used as an animal model for infant humans. Twenty-four 10-d-old entire male piglets (4·3 (sd 0·59) kg body weight) were fed an infant formula (IF) (control) or IF supplemented with 1·3 × 105 (low dose) or 7·9 × 106 (high dose) colony-forming units HN001 per ml of reconstituted formula (n 8 piglets/treatment). After 24 d, piglets were euthanised. Samples were collected to analyse the histology and gene expression (RNAseq and qPCR) in the jejunal and ileal tissues, blood cytokine concentrations, and blood and faecal calprotectin concentrations. HN001 consumption altered (false discovery rate < 0·05) gene expression (RNAseq) in jejunal tissues but not in ileal tissues. The number of ileal goblet cells and crypt surface area increased quadratically (P < 0·05) as dietary HN001 levels increased, but no increase was observed in the jejunal tissues. Similarly, blood plasma concentrations of IL-10 and calprotectin increased linearly (P < 0·05) as dietary HN001 levels increased. In conclusion, supplementation of IF with HN001 affected the architecture and gene expression of small intestine tissue, blood cytokine concentration and frequencies, and blood calprotectin concentrations, indicating that HN001 modulated small intestinal tissue maturation and immunity in the piglet model.

Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called “protein corona” as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

Pharmacokinetics of a New Dimethylamineethanol Derivative Administered Orally to Laboratory Animals

A new derivative of dimethylaminoethanol, butanedioic and trans-butenedioic acids (laboratory code ADK-17) was synthesized at the Organic Chemistry Department of the Saint Petersburg State Chemical and Pharmaceutical University (SPSPU). This promising nootropic and anti-asthmatic agent is planned for use in an oral dosage form. The aim was to calculate the pharmacokinetic parameters of the synthesized compound and to determine its possible biotransformation pathways. Rabbits of the Soviet Chinchilla breed were used as a test system in the preclinical study of the compound pharmacokinetics. In accordance with the designed schedule, blood was taken from the marginal vein of the ear, and urine samples were taken. Quantitative determination in biological media was carried out using an Ultimate 3000 liquid chromatograph with a Q-Exactive mass-selective detector with electrospray ionization in accordance with OFS 1.2.1.100015 (Chromatography) of the State Pharmacopoeia by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS). To study the pharmacokinetics of the ADK-17 preparation, a measurement procedure was developed with a detection limit in biological media ranging from 10-6 to 10-3 mg/ml. The determined harmacokinetic parameters show that the absorption of ADK-17 into the systemic circulation from the gastrointestinal tract occurs at an average rate, with its maximum concentration in blood plasma during the oral route of administration being observed between 60–90 min. The elimination half-life from the systemic circulation comprises 4.3 h. Urinary excretion in the first 4 h proceeds at a high rate; however, the compound is detected in the urine within 3 days and in the blood within 2 days from the moment of administration.

Parametric and non-parametric Poisson regression for modelling of the arterial input function in positron emission tomography

Full quantification of Positron Emission Tomography (PET) requires an arterial input function (AIF) for measurement of certain targets, or using particular radiotracers, or for the quantification of specific outcome measures. The AIF represents the measurement of radiotracer concentrations in the arterial blood plasma over the course of the PET examination. Measurement of the AIF is prone to error as it is a composite measure created from the combination of multiple measurements of different samples with different equipment, each of which can be sources of measurement error. Moreover, its measurement requires a high degree of temporal granularity for early time points, which necessitates a compromise between quality and quantity of recorded samples. For these reasons, it is often desirable to fit models to this data in order to improve its quality before using it for quantification of radiotracer binding in the tissue. The raw observations of radioactivity in arterial blood and plasma samples are derived from radioactive decay, which is measured as a number of recorded counts. Count data have several specific properties, including the fact that they cannot be negative as well as a particular mean-variance relationship. Poisson regression is the most principled modelling strategy for working with count data, as it both incorporates and exploits these properties. However, no previous studies to our knowledge have taken this approach, despite the advantages of greater efficiency and accuracy which result from using the appropriate distributional assumptions. Here, we implement a Poisson regression modelling approach for the AIF as proof-of-concept of its application. We applied both parametric and non-parametric models for the input function curve. We show that a negative binomial distribution is a more appropriate error distribution for handling overdispersion. Furthermore, we extend this approach to a hierarchical non-parametric model which is shown to be highly resilient to missing data. We thus demonstrate that Poisson regression is both feasible and effective when applied to AIF data, and propose that this is a promising strategy for modelling blood count data for PET in future.

Immunotherapy targeting isoDGR‐protein damage extends lifespan in a mouse model of protein deamidation

Aging results from the accumulation of molecular damage that impairs normal biochemical processes. We previously reported that age‐linked damage to amino acid sequence NGR (Asn‐Gly‐Arg) results in “gain‐of‐function” conformational switching to isoDGR (isoAsp‐Gly‐Arg). This integrin‐binding motif activates leukocytes and promotes chronic inflammation, which are characteristic features of age‐linked cardiovascular disorders. We now report that anti‐isoDGR immunotherapy mitigates lifespan reduction of Pcmt1−/− mouse. We observed extensive accumulation of isoDGR and inflammatory cytokine expression in multiple tissues from Pcmt1−/− and naturally aged WT animals, which could also be induced via injection of isoDGR‐modified plasma proteins or synthetic peptides into young WT animals. However, weekly injection of anti‐isoDGR mAb (1 mg/kg) was sufficient to significantly reduce isoDGR‐protein levels in body tissues, decreased pro‐inflammatory cytokine concentrations in blood plasma, improved cognition/coordination metrics, and extended the average lifespan of Pcmt1−/− mice. Mechanistically, isoDGR‐mAb mediated immune clearance of damaged isoDGR‐proteins via antibody‐dependent cellular phagocytosis (ADCP). These results indicate that immunotherapy targeting age‐linked protein damage may represent an effective intervention strategy in a range of human degenerative disorders.

PSIV-13 Influence of Differing Levels of Concentrate on Circulating Cytokine Concentrations in Beef Heifers

Abstract Components of the immune system, such as cytokines and chemokines, can influence female reproductive efficiency. Recently, studies have begun to characterize the influence of nutrition on cytokines and reproductive function. Characterizing the influence of nutritional components on shifting circulating cytokine concentrations will allow for a better understanding of reproductive efficiency in cattle. The objective of this study was to determine the effect of diet composition on cytokine concentrations in blood plasma in beef heifers. Using a 3 x 3 Latin square design, fifteen rumen-cannulated, pubertal Bos taurus-influenced heifers were fed a diet based on ranging concentrate percentages. Their treatment period consisted of 28-day feeding periods with a washout interval of 21 days. The treatment groups were fed 100% grass hay (high forage; HF), 60% grass hay with 40% corn-based concentrate (intermediate; INT), and 25% grass hay with 75% corn-based concentrate (high grain; HG) within their respective treatment group. Heifers were offered 2% of their body weight daily. Blood was collected on days 0 and 28 of the treatment period for cytokine analysis. Plasma cytokine concentrations were quantified using RayBiotech Quantibody Bovine Cytokine Array Q1 kit according to manufacturer instructions. Data analysis was conducted using orthogonal contrasts (linear and quadratic) with day 0 as independent covariates and heifers as the experimental unit. There were no significant quadratic contrasts. Concentrations of interferon gamma-induced protein 10 (IP-10) linearly decreased with an increased concentrate diet (P = 0.04). Concentrations of IP10 significantly differed for heifers consuming the HF diet versus the HG diet (3,069.5 ± 672.2 vs. 1,001.8 ± 669.3 pg/mL, respectively) and heifers consuming the INT diet versus the HG diet (2,886.8 ± 665.6 vs. 1,001.8 ± 669.3 pg/mL, respectively); however, there were no significant differences in IP10 concentrations between the HF and INT heifers. Interferon-gamma (IFNg) concentrations significantly differed between feeding periods with period 2 having the least circulating concentrations when compared with periods 1 and 3 (5.2 ± 66.2 vs. 183.4 ± 68.4 and 221.3 ± 67.8 pg/mL, respectively; P &amp;lt; 0.05). Additionally, monokine induced by gamma interferon (MIG) significantly differed by period with period 2 having the least circulating concentrations compared with periods 1 and 3 (862.2 ± 285.4 vs. 1675.8 ± 283.4 and 1,170.8 ± 286.9 pg/mL, respectively; P &amp;lt; 0.05). Interestingly, both IP10 and MIG are chemokines, or small cytokines with chemotactic functions, that are secreted by immune cells in response to IFNg. Results suggest that heifers consuming a high concentrate diet have decreased concentrations of circulating cytokines, specifically IP10. Additionally, in the current study, cytokines that were influenced were associated with IFNg. Further research is necessary to better comprehend the dietary influence on the immune system in developing heifers and how this is associated with reproductive efficiency.

New synthetic monophenolic antioxidant TS-13 penetrates the blood-brain barrier

TS-13 (sodium 3-(3’-tert-butyl-4’-hydroxyphenyl)propylthiosulfonate) is a synthetic antioxidant that has demonstrated biological effectiveness in numerous studies in modeling pathological conditions in vivo, in particular, in the model of Parkinson’s disease. In order to establish whether these effects are mediated or associated, among other things, with the direct effect of TS-13 on the organs and tissues of animals, in this work, the concentration of TS-13 in rat blood plasma and brain after intragastric administration was determined. Material and methods. After a single intragastric administration of a solution of TS-13 at a dose of 100 mg/kg, biomaterial (blood, brain) was taken for 24 h in male Sprague Dawley stock rats (n = 57). To measure the concentration of a substance in samples, a bioanalytical method was developed and validated using high-performance liquid chromatography with UV detection. Results and discussion. The method of quantitative determination was developed by us for the first time and validated before the study. It has been established that the calculated values of the calibration samples meet the acceptance criteria (have the required accuracy and precision) in the concentration range from 0.05 to 6 µg/ml, R=0.9998. The results of determining TS-13 concentration in rat blood plasma and brain showed that after a single administration per os, the compound enters the blood, where it is detected for 15 h (mean retention time 7.94 h, half-life 7.59 h, elimination constant 0.13 h-1, total clearance 40.1 l/(kg × h)), and also penetrates the blood-brain barrier, quickly entering the brain (maximum concentration is reached after 1 h). The compound has a low affinity for brain tissue (tissue availability 0.32), and therefore its concentration does not reach high values, however, a slow excretion of the substance is observed - the average retention time is 6.56 h, the half-life is 6.43 h, the elimination constant 0.11 h-1. Conclusions. After a single intragastric administration to rats, TS-13 enters the blood, where at least part of it is detected unchanged after 30 minutes, reaching maximum values after 1 hour. Similar kinetics of the substance is characteristic of the brain, where it is found in smaller amounts. Thus, as a result of the study, it was shown that TS-13 penetrates the blood-brain barrier and is able to directly affect brain structures, which, however, does not negate the possibility of an indirect effect mediated by the ability to change the activity of intra- and intercellular signaling systems.

Features of the biochemical composition of blood in calves with different dynamics of live weight gain

The biochemical markers of blood associated with the rate of body weight gain were studied in the work. For experimental work, 30 calves on the 1st and 14th day after birth received venous blood and conducted laboratory tests. At the time of birth, as well as after 6 months of cultivation, the live weight of young an-imals was determined and the average daily increase was calculated. It was found that on the first day after birth in intensively growing animals, the activity of AlAT was lower by 56.6% (P?0.01), and AsAT by 45.4% (P?0.05), in relation to the indicators of young animals with a low rate of weight gain. Similarly, calves with intensive growth had a higher concentration of VSNMM in plasma by 30.4...48.4% (P?0.001) and 37.1...48.6% (P?0.01) in blood. On day 14, the biochemical profile of calves with low growth rates was characterized by a higher activity of alkaline phosphatase (by 27.4...46.6%; P&lt;0.05), whereas in intensively growing young calves a higher level of calcium was observed (by 5.9%; P&lt;0.05). The analysis of correlations showed that on the first day after birth, there was a pronounced relationship between the growth rate in relation to the activity of AsAT (-0.510) and AlAT (-0.497), iron concentration (-0.650) and VSNMM in blood plasma (0.626). Subsequently, the connection of these markers weakened, and the growth rate of calves was more dependent on the activity of alkaline phosphatase. So on the 14th day of postnatal ontogenesis, the correlation between the increase in live weight and enzyme activity was -0.744. Thus, it can be concluded that the activity of alkaline phosphatase is the most promising biochemical marker associated with the dynamics of body weight gain.

Pharmacokinetics of a New Neuroprotector - Indenoquinoxalinone Derivative after Intravenous Administration in Rabbits and Rats.

The specific JNK inhibitor and NO donor 11H-indeno[1,2-b]quinoxalin-11-one oxime (IQ-1) demonstrated pronounced neuroprotective properties in an in vivo model of ischemic stroke in rats. The pharmacokinetic behavior of IQ-1 was studied in two animal species (rats, rabbits) after intravenous administration in a dose of 1 mg/kg. IQ-1 concentrations in venous blood plasma were measured by the liquid chromatography-tandem mass spectrometry method. The pharmacokinetics of IQ-1 was adequately described by the two-compartmental model. The calculated C0 for IQ-1 in rabbit and rat plasma were 2239.83±1229.55 and 1552.50±182.23 ng/ml, respectively. Two animal species are characterized by extensive tissue distribution of IQ-1 (Vss exceeded the total body water in rabbits and rats by 3.6 and 5.6 times, respectively) and high clearance values (88-94% of hepatic blood flow).

BLOOD PLASMA 25-HYDROXYCALCIPHEROL (25(OH)D) CONTENT IN PATIENTS WITH TYPE 2 DIABETES MELLITUS AT DIFFERENT STAGES OF DIABETIC RETINOPATHY

Background. To date, it has become necessary to study the consequences of vitamin D-deficiency conditions on the course of severe complications of diabetes mellitus (DM), in particular diabetic retinopathy (DR), when the pleiotropic metabolic effect of vitamin D is significantly reduced.&#x0D; Aim: to investigate the content of 25-hydroxycalciferol (25(OH)D) in the blood plasma of patients with type 2 diabetes at various stages of diabetic retinopathy.&#x0D; Materials and methods. An open observational single-center one-stage selective study was conducted. The study was approved by the Local Ethics Committee. 90 patients (173 eyes) with T2D and DR (men and women; mean age - 59,4±3,4 years; mean HbA1c - 7,82±0,51 %) were assigned to 3 groups, based on the stage of DR (nonproliferative, preproliferative and proliferative). The inclusion of patients in groups with DR was carried out in accordance with the ETDRS protocol (2019). Statistical analysis was included ANOVA and regression analysis.&#x0D; Results. It was shown that the blood plasma concentration of 25(OH)D in patients with T2D probably decreases at each stage of DR (I stage - 19.8 [18.8-22.6] ng\ml, II stage - 16.9 ng\ml [15.3- 18.8], stage III - 14.3 [12.5-15.8] ng/ml, p&lt;0.001). At the same time, as the stage of DR increases, the frequency of detection of 25(OH)D deficiency (concentration less than 20 ng/ml) probably increases (50.0% - stage I, 86.2% - stage II, 94.3% - stage III, p &lt;0.001). It was shown that the risk of a high degree of DR probably decreases (p&lt;0.001) with an increase in the concentration of 25(OH)D in blood plasma for each ng/ml (OR = 0.64 (0.53 – 0.78)).&#x0D; Conclusion. With an increassng in the stage of DR, the content of 25(OH)D in blood plasma decreases (p&lt;0.001). At the same time, the proportion of patients with 25(OH)D deficiency increases, especially at stage III DR (p&lt;0.001). An analysis of the relationship between the stage of DR and the concentration of 25(OH)D showed a decreasing in the risk of a high degree of DR with an increassng of the 25(OH)D.

Epstein-Barr virus (Herpesviridae: Lymphocryptovirus) types 1 and 2 and other viral markers in patients with nasopharyngeal carcinoma in two geographically and ethnically distinct regions of Russia

The discovery of two types of Epstein-Barr virus (EBV) (EBV-1 and EBV-2) that have different biological properties stimulated the search for neoplasms associated with each type of the virus. The aim of the work is to study the nature of the association of nasopharyngeal cancer (NPC) with EBV-1 and EBV-2, serological activity for each viral type and the concentration of EBV DNA in the blood plasma of two gender, age and ethnic groups of NPC patients that represent geographically and climatically different regions of Russia,. In the blood plasma of patients with NPC and other non- EBV associated tumors of oral cavity (OTOCEBV-) from the North Caucasian (NCFD) and Central (CFD) Federal Districts of Russia, the types of EBV and the concentration of viral DNA were determined using respectively «nested» and real time PCR; titers of IgG and IgA antibodies to viral capsid antigen (VCA) were measured in indirect immunofluorescence assay. The blood plasma samples testing showed that NPC and OTOCEBV- patients were infected with both types of EBV in approximately equal proportions. In two groups of NPC patients infected with one of the virus types only, EBV-1 or EBV-2, respectively, no statistically significant differences were found between the geometric mean values of IgG and IgA anti-EBV antibody titers and viral DNA concentrations in blood plasma. The distribution of virus types was not affected by either patient gender or ethnogeographic origin. The difference was found only between age groups: EBV-2 dominated in NPC patients up to 60 years, and EBV-1 was prevalent in patients over 60 years. The lack of the predominance of one of EBV types in NPC patients that are the representatives of different ethnic groups from geographically and climatically different regions, suggests that none of these factors play an important role in the NPC carcinogenesis. Evidently, each type of EBV, EBV-1 or EBV-2, if the necessary conditions arise, are able to exhibit its oncogenic potential to initiate tumor development.

Short communication: Predicting blood plasma non-esterified fatty acid and beta-hydroxybutyrate concentrations from cow milk-addressing systematic issues in modelling.

Negative energy status in early lactation is linked to a variety of metabolic disorders, reduced fertility, and decreased milk production. To improve the energy status of cows by breeding and management, the identification of negative energy status is crucial. While biomarkers such as non-esterified fatty acid (NEFA) concentration and beta-hydroxybutyrate (BHB) in blood plasma could be used to identify a negative energy state, measuring them directly from blood is both invasive and expensive. In this work, we developed prediction equations for blood plasma NEFA and BHB levels based on mid-IR spectral measurements of milk. The models were fitted using partial least squares regression and evaluated using both cross-validation and independent-herd validation. A total of 3183 spectral records from 606 lactations originating from three different herds were utilised. R2 values of 0.53 (RMSE=0.206mmol/l, RMSE of cross-validation (RMSECV) 0.217mmol/l) for NEFA and 0.63 (RMSE=0.326mmol/l, RMSECV=0.353mmol/l) for BHB were obtained. Furthermore, relatively similar prediction accuracies were found for BHB (RMSE of prediction (RMSEP) 0.411mmol/l and 0.422mmol/l) and NEFA (RMSEP=0.186mmol/l and 0.221mmol/l) when model training was done using two herds and validated on the third herd. The results from the model fits confirm that it is possible to build blood plasma BHB and NEFA models based on mid-IR spectra that are sufficiently accurate for practical use.

Spitting for Science: Non-Invasive Drug Monitoring Through a Modified Liquid Micro Junction-Surface Sampling Probe

The use of saliva as a convenient, non-invasive alternative for therapeutic drug monitoring has become increasingly favorable with the emergence of pharmacokinetic and bioequivalence research providing a link between blood plasma and saliva concentrations. Therapeutic drug monitoring is a practice through which specific drugs are monitored at given time intervals to maintain a constant concentration in the blood. It is only necessary for patients taking drugs with known pharmacokinetic variability, narrow therapeutic indices and potential adverse effects.&#x0D; Although the drug concentration in saliva is lower than in plasma, saliva has a complex matrix, consisting of salts, immunoglobins, proteins and more. The polar matrix in the saliva introduces potential interference with the sensitivity of the drug detection through salt suppression.&#x0D; In this project, 6 drugs used as anticonvulsants and antidepressants were analysed in artificial saliva using a modified version of the Liquid Micro Junction-Surface Sampling Probe (LMJ-SSP) to maximize the sensitivity of observed signals and minimize the detection limits.&#x0D; The main parameter which was targeted for optimizing the extraction and ionization of the drug in the saliva samples was the polarity of the carrier liquid, which continuously flows in the coaxial tube of the LMJ-SSP. Upon analyzing four different carrier liquids, the mixture containing methanol, water and chloroform was determined to provide the best sensitivity. Additionally, the enrichment factors of the peak areas using the chloroform mixture in the modified LMJ-SSP were enhanced by a factor ranging between 2.9 to 31.4, depending on the analyte.&#x0D; Overall, the data obtained from this project thus far displays that modifying the polarity by modifying the carrier liquid composition to balance the extraction and ionization during analysis using the modified LMJ-SSP optimizes the sensitivity of the detection.

Epinephrine Affects Ribosomes, Cell Division, and Catabolic Processes in Micrococcus luteus Skin Strain C01: Revelation of the Conditionally Extensive Hormone Effect Using Orbitrap Mass Spectrometry and Proteomic Analysis.

In the current study, extensive Orbitrap mass spectrometry analysis was conducted for skin strain Micrococcus luteus C01 planktonic cultures and biofilms after 24 h and 72 h of incubation either in the presence of epinephrine or without any implementations. The investigation revealed the complex and conditionally extensive effect of epinephrine at concentrations closer to normal blood plasma concentrations on both planktonic cultures and biofilms of skin strain M. luteus C01. The concentrations of hundreds of proteins changed during the shift from planktonic growth mode to biofilm and hundreds of proteins were downregulated or upregulated in the presence of epinephrine. Ribosomal, TCA, and cell division proteins appear to be the most altered in their amounts in the presence of the hormone. Potentially, the regulatory mechanism of this process is connected with c-di-GMP and histidine kinases, which were affected by epinephrine in different samples. The phenomenon of epinephrine-based biofilm regulation in M. luteus C01 has wide implications for microbial endocrinology and other research areas.

Quality and safety of hemp meal as a protein supplement for nonlactating dairy cows

Hemp seed meal may be a suitable protein supplement for dairy cows, but its quality and safety as a dairy cow feed has not yet been fully investigated. As a result, dry matter intake (DMI), rumen fermentation, blood metabolites, total-tract digestibility, and concentrations of cannabinoids in blood plasma, urine, muscle, and adipose tissues were compared among nonlactating Holstein dairy cows receiving a basal partial mixed ration that was supplemented with either 10.2% dry matter (DM) hemp meal (HM treatment), 13.5% DM canola meal (CM treatment), or 6.25% DM hemp meal and 6.16% DM canola meal (HC treatment). Diets were formulated to be isoenergetic and isonitrogenous. Six nonlactating, nonpregnant Holstein cows were used in a repeated 3 × 3 Latin square design trial with three 3-wk experimental periods. The first 2 weeks of each served as adaptation. Sample and data collection occurred during the third week of each period. Neither the partial mixed ration nor canola meal contain cannabidiol (CBD), cannabidiolic acid (CBDA), d9-tetrahydrocannabinol (THC), or tetrahydrocannabinolic acid A (THCA-A). However, the hemp meal contained 3.0, 4.4, 0, and 0.1 μg/g DM of CBD, CBDA, THC, and THCA-A, respectively. Treatment did not affect DMI, pH, concentrations of volatile fatty acids or ammonia in the rumen, total-tract digestibilities of DM and crude protein, or blood plasma concentrations of glucose, urea, β-hydroxybutyrate, and nonesterified fatty acids. Hence, based on these metabolites, treatment did not affect the nutritional status of the cows. However, the total-tract neutral detergent fiber digestibility of the CM treatment (43%) was higher than that of the HM treatment (38%). No cannabinoids were detected in blood plasma, rumen fluid, and urine. Cannabinoids were also not detected in kidney, liver, urine, muscle, or adipose tissues at the end of the experiment when cows had undergone all treatments. Feces from all treatments did not contain detectable concentrations of THC or THCA-A, but feces of cows on the HC treatment contained 0.42 and 0.40 μg/g DM of CBD and CBDA, respectively. Feces of cows on the HM treatment contained 0.68 and 0.67 μg/g DM of CBD and CBDA, respectively. This indicated that most ingested CBD and CBDA were not absorbed but instead were excreted in the feces. Our data show dietary inclusion rates of up to 10.2% of DM. We find that hemp meal is a high-quality and safe protein supplement for nonlactating dairy cows.

Comparing dehulled hemp meal and canola meal as a protein supplement for lactating dairy cows

Effects of replacing canola meal with dehulled hemp meal in the diet of lactating dairy cows on the dry matter intake (DMI), milk production, milk fatty acid profile, blood metabolites, total-tract nutrient digestibility, and transfer of cannabinoids were determined in 12 lactating, nonpregnant Holstein cows. These cows were used in a 3 × 3 Latin square design with three 3-wk experimental periods consisting of 2 wk of adaptation and 1 wk of sampling. Cows received basal partial mixed rations supplemented with either 15% dry matter (DM) canola meal (CM15), 15% DM dehulled hemp meal (HM15), or 7.5% DM dehulled hemp meal and 7.5% DM canola meal (CM7.5HM7.5). Diets were formulated to be isoenergetic and isonitrogenous, but the HM15 and CM7.5HM7.5 diets contained, on average 1.2 percentage units more crude protein (CP) that the CM15 diet. The CP of the dehulled hemp meal contained less soluble protein than that of canola meal. Hence, the intake of soluble protein did not differ among diets. Canola meal contained less crude fat than hemp seed meal (3.46% vs. 8.25% DM). The lipid fraction of canola meal fat contained more oleic acid (C18:1 cis-9; 47.3 vs. 14.9 g/100 g of fatty acids, FA) and vaccenic acid (18:1 cis-11; 13.7 vs. 1.2 g/100 g of FA) and less linoleic acid (C18:2n-6; 21.9 vs. 55.7 g/100 g of FA) and α linolenic acid (C18:3n-3; 3.2 vs. 8.9 g/100 g of FA) than the lipid fraction of hemp seed meal. The hemp seed meal contained 4.9 µg/g cannabidiol, 5.1 µg/g cannabidiolic acid, and 0.1 µg/g tertahydroxycannabinolic acid A. Treatments did not differ in DMI, yields of milk, milk protein and milk fat, total-tract neutral detergent fiber digestibility, and blood plasma concentrations of β-hydroxybutyrate and nonesterified FA. Apparent total-tract DM digestibility was lowest in the HM15 treatment, whereas the CP digestibility and the concentrations of urea in blood, urine, and milk were lowest in the CM15 treatment. Cannabinoids were not detected in urine, milk, and blood plasma. Replacing canola meal with hemp seed meal increased milk fat contents of polyunsaturated fatty acids (PUFA), which were 3.42, 3.90, and 4.25 g/100 g of FA for the CM15, CM7.5HM7.5, and HM15 treatments, respectively. Especially, the milk fat contents of 18:2n-6 (1.99 vs. 1.56 g/100 g FA) and 18:3n-3 (0.31 vs. 0.43 g/100 g FA) were increased by hemp meal feeding. Especially, the milk fat contents of 18:2n-6 (1.99 vs. 1.56 g/100 g FA) and 18:3n-3 (0.31 vs. 0.43 g/100 g FA) were increased by hemp meal feeding. Our data show that hemp seed meal is a suitable and safe replacement for canola meal as a feed for lactating dairy cows and that this replacement increases CP digestibility and urea in urine, milk, and blood plasma, as well the PUFA content of milk fat.

The protective effect of endogenous melatonin on gilthead seabream sperm during cryopreservation

Cryopreservation is widely used for artificial reproduction techniques (ART) but requires good gamete quality to succeed. Considering the endogenously produced melatonin by the fish and its protective role in the organism, the objective of this experiment was to search for the best moment of the day to collect gilthead seabream (Sparus aurata) sperm with better quality and, at the same time, to test the potential protective effect of the exogenous melatonin during sperm freezing and thawing processes. Fish were first sampled every 6 h, corresponding to the beginning of the light period (6 h), mid-light (12h), beginning of the dark period (18 h), and mid-dark (24 h) to determine melatonin concentration in blood plasma by radioimmunoassay (RIA). The analysis showed higher values at 24 h (244 pg/mL) compared with 12 h (99 pg/mL), which allowed the selection of those moments for the next experiment. After, fish were sampled for sperm collection at mid-light (ML, 12 h) and mid-dark (MD, 24 h), and fresh sperm was used to assess gamete quality and for the cryopreservation assay. Sperm samples were cryopreserved to test supplementation of different melatonin concentrations (0.001 mM, 0.01 mM and 0.1 mM) together with a control group without added melatonin. Gamete quality was assessed through spermatozoa concentration and motility (CASA system), cell viability (PI/SYBR-green) and DNA fragmentation (Comet assay). Despite cell viability that was higher at ML, most of the fresh sperm motility parameters did not differ between ML and MD, only linearity (LIN) was enhanced at MD. Nevertheless, in cryopreserved samples, total motility (TM) was significantly higher at MD in all melatonin treatments, control, and fresh samples, revealing an endogenous night-effect. Moreover, spermatozoa concentration was also higher at MD (28.9 × 109/mL) than at ML (20.7 × 109/mL). Supplemented melatonin did not confer extra protection to gilthead seabream sperm during cryopreservation since the tested concentrations did not differ between the control in any sperm quality test. It is here suggested that endogenously produced melatonin may contribute to the improvement of some gamete quality parameters at mid-dark, allowing the aquaculture sector to select better sperm quality in a non-invasive way by choosing it as the best moment of the day for sperm collection.

The stent thrombosis in Belgium (STIB) scoring system reliability in Indonesia patients and the modified STIB scoring (M-STIB)

IntroductionClopidogrel is recommended as an alternative to Acetyl Salicylic Acid (ASA), the first-line drug for secondary stroke prevention. Clopidogrel resistance in Indonesia is reportedly 15.8%. The Stent Thrombosis in Belgium (STIB) is a scoring system proposed to assess clopidogrel resistance which has not been tested for the reliability in Indonesian population.ObjectivesThe objective of the study is to test the reliability of the STIB scoring and modified-STIB scoring system in Indonesian population.MethodsThis study was conducted cross-sectionally in Dr. Cipto Mangunkusumo Hospital and Universitas Indonesia Hospital from January 2020 to December 2021. Laboratory examinations of human CYP450 concentration in blood plasma from 112 subjects were carried out using the ELISA method. The clopidogrel resistance test was carried out using the VerifyNow method.ResultsOut of 112 ischemic stroke patients in this study, 14.3% of them did not respond to clopidogrel. Cross-tabulation between the STIB score and clopidogrel resistance showed significant results (p < 0.05) on the Hb variable and a combination of two or three factors involving Hb levels. Both Hb and CYP450 levels can be independent factors predicting clopidogrel resistance. Once combined, the Hb and CYP450 cutoff levels of 458.9 pg/mL had a sensitivity of 100% with a specificity of 30.2%.ConclusionsSTIB scoring system did not prove to be reliable in screening for Clopidogrel resistance in Indonesian population. The modified STIB scoring system proposed in this study showed a promising result. Further research with larger population should be conducted.