Search Results (68)

Lithium is used as a medication in the treatment of bipolar disorder. Lithium has a narrow therapeutic index, and fatal intoxications have been described. The therapeutic drug monitoring of lithium is routinely performed in serum. Serum is commonly unavailable in forensic postmortem analysis, where whole blood is the matrix of choice. In this study, an inductively coupled plasma mass spectrometry (ICP-MS) method was developed and validated for the quantification of lithium in postmortem whole blood. Sample preparation consisted of a 100-fold dilution with acid and required only 40 µL of blood. Carry-over was deemed appropriately reduced with a rinse solution containing 5% hydrochloric acid. A nebulizer gas flow rate of 1.15 L/min showed a sufficient improvement of lithium sensitivity while simultaneously minimizing the background. Germanium was determined to be the most optimal internal standard. The method was validated in terms of linearity, accuracy, precision, and lower limit of quantification. Linearity was demonstrated within the analytical measurement range of 0.10–1.5 mmol/L. The method showed acceptable precision and accuracy, with a total coefficient of a variation ≤2.3% and accuracies ranging from 105 to 108% at all concentrations in the quality control samples. The final method was applied to postmortem blood from 103 consecutive autopsy cases and demonstrated robustness by low intermediate precision and high and consistent recovery of the internal standard. Full article

Volatile organic compounds (VOCs) in biological samples originate both from exogenous and endogenous sources. Recent studies have highlighted their potential as cancer biomarkers, emphasizing the need for accurate detection methods in clinical settings. However, analysis of VOCs in whole blood (WB) samples remains challenging due to the complex matrix effects caused by the protein−VOC binding phenomenon and lack of standardized sample preparation protocols. Therefore, this study suggests a standardized method for advanced VOC analysis in WB samples specifically for veterinary applications. We compared 12 combinations of reagents composed of protein denaturing reagents and salts, particularly urea mixtures, to enhance VOC decoupling from proteins and improve matrix effect uniformity in gas chromatography−mass spectrometry (GC-MS) analysis. Among all combinations, urea with NaCl showed an optimal performance, demonstrating an advancement in the detection sensitivity of up to 151.3% and a significantly reduced matrix effect variation (−35.5% to 25%) compared with the water-only control. This novel approach eliminates complex procedures while maintaining accuracy, making it particularly suitable for veterinary uses. The method’s standardization and improved performance characteristics offer a practical solution for efficient VOC detection in veterinary diagnostics, potentially advancing tumor biomarker research. Full article

Equine Piroplasmosis (EP) is a significant tick-borne disease affecting horses, and one of the causative protozoan parasites is Theileria equi, hence the need to understand the prevalence and associated factors influencing it. Considering the population of horses in the study areas, a sample size of 272 horses comprising 171 and 101 horses from Taif and Jeddah was estimated. Thin and thick blood smears were made from the animals’ whole blood for microscopic examination. At the same time, serum samples were prepared and examined for antibodies to antigens using commercial Theileria equi antibody test kit ELISA. The relationships of gender and age with the presence or absence of T. equi parasite infection were determined using the chi-square test. The results revealed no significant association between gender and T. equi prevalence using both microscopic (χ² = 2.748, p = 0.07) and ELISA (χ² = 2.412, p = 0.096) diagnostic methods. In Taif, the microscopic results revealed that 86% of female horses tested negative, while 14% tested positive. In contrast, 75% of male horses tested negative, with 25% testing positive for T. equi. In terms of age groups of horses, a significant association (χ² = 31.966, p = 0.032) between age groups and the prevalence of T. equi in samples from Jeddah using the ELISA method was recorded. Understanding the relationship between the prevalence of T. equi and factors such as gender and age is crucial for developing effective control measures and improving equine health management, especially in Saudi Arabia. Full article

Effective blood glucose management is essential for individuals with type 1 diabetes, particularly when dietary adjustments involve staple foods like rice. As a primary carbohydrate worldwide, rice significantly influences the glycemic index (GI) based on its type and cooking method. This study investigated the impact of rice type and boiling duration on the GI in healthy adults using an in vivo approach aligned with ISO 2010 standards. The glycemic response to four rice types (white round-grain, parboiled medium-grain, white long-grain, and whole-grain long-grain) was measured through postprandial blood glucose levels under both standard and extended boiling conditions to assess their implications for dietary glycemic control. Ten healthy participants (mean age 25 years, body mass index (BMI) 23.0 ± 1.6 kg/m²) consumed rice samples containing 50 g of available carbohydrates, prepared under controlled boiling conditions. Postprandial glycemic response was measured at regular intervals over 2 h following ingestion, with glucose solution as a reference food. The GI was calculated based on the incremental area under the glycemic response curve for each rice sample. Extended boiling significantly increased the GI across all rice types. White round-grain rice exhibited the highest relative increase (+15.8%) in the GI, while whole-grain long-grain rice, despite showing a greater percentage increase (+25.4%), maintained the lowest overall GI due to its high amylose and fiber content. Rice types with higher amylopectin content demonstrated faster glycemic responses and higher GI compared to high-amylose types. This study highlights rice type and cooking time as critical factors influencing postprandial glycemic response. Shorter boiling durations may benefit individuals requiring strict glycemic control, particularly those with diabetes, underscoring the importance of personalized dietary guidance for managing glycemic outcomes effectively. Full article

The study aimed to investigate the protective effects of chokeberry fruit products and by-products against cisplatin-induced acute nephrotoxicity in rats. Potential mechanisms involving oxidative stress and inflammatory responses were examined through biochemical and histopathological analyses of kidney tissue. Chokeberry waste, along with the whole fruit extract and juice, was evaluated as a potential raw material for pharmaceutical use. The chemical composition of chokeberry juice and extracts was analyzed using spectrophotometry and HPLC. Rats were treated with chokeberry preparations via intragastric tube for ten days, with a single intraperitoneal dose of cisplatin (8 mg/kg BW) administered on the third day. Post-sacrifice, plasma samples were analyzed for biochemical nephrotoxicity markers, oxidative stress, and inflammatory markers. Kidneys were removed for histopathological and biochemical analysis. Cisplatin-induced acute nephrotoxicity was confirmed by elevated plasma creatinine and blood urea nitrogen levels. Additionally, lipid peroxidation was significantly elevated, while reduced glutathione and catalase activity were significantly reduced. Pro-inflammatory mediators IL-1β, TNF-α, and IL-6 levels were significantly increased in the cisplatin group. Treatment with chokeberry extracts and juice significantly mitigated these nephrotoxic effects, as confirmed by histopathological examination and biochemical marker analysis. Notably, the waste extract demonstrated greater efficacy than the whole fruit extract, likely due to its higher concentration of polyphenolic compounds, especially anthocyanins. These results highlight the potential of chokeberry as a therapeutic and preventive agent for kidney protection, emphasizing the value of by-products rich in biologically active compounds. Full article

African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR. Full article

Diabetes mellitus is considered to be the most common health issue affecting almost 1 in 11 adults globally. Oral health complications including xerostomia, periodontal disease, dental caries, and soft tissue lesions are prevalent among individuals with diabetes, and therefore an understanding of the potential association between salivary metabolites and dental caries progression would enable the early detection and prevention of this non-communicable disease. Therefore, the aim of this study was to compare salivary biomarkers between individuals with type 2 diabetes (T2DM) with those without this disorder (ND) using ¹H NMR-based metabolomics strategies. The objectives were to identify T2DM-associated biomarker signatures and their potential impact on dental caries. In addition, HbA1c and vitamin D levels were also analysed for this purpose. Methods: Stimulated whole-mouth saliva (SWS) samples were collected from T2DM and ND (n = 30 in each case) participants randomly selected from a group of 128 participants recruited for this case–control study. All participants were advised to refrain from eating, drinking, and smoking for at least 1–2 h prior to sample collection. Following preparation, SWS supernatants underwent ¹H NMR analysis at an operating frequency of 800 MHz, and the dataset acquired was analysed using a range of multivariate metabolomics techniques. Results: Metabolomics analysis of data acquired demonstrated that, together with up- and downregulated blood HbA1c and vitamin D levels, key salivary discriminators between these two classifications included lactate, taurine, creatinine, α-glucose, and formate to a lesser extent. The bacterial catabolites lactate and formate were both significantly upregulated in the T2DM group, and these have previously been implicated in the pathogenesis of dental caries. Significance analysis of metabolites (SAM)-facilitated AUROC analysis yielded an 83% accuracy for this distinction. Conclusion: In conclusion, this study highlights the significant differences in salivary metabolites between individuals with T2DM and healthy controls. Such differences appear to be related to the development and progression of dental caries in T2DM patients. Full article

The present study describes the enhancement of the nutritional values of gluten-free rice crackers by adding whole black rice grain flour. The crackers were prepared by combining whole brown rice flour (WRF) and whole black rice flour (BRF) in ratios of 0% (WRC), 25% (25-BRC), 50% (50-BRC), 75% (75-BRC), and 100% (BRC). The resulting samples underwent in-vivo effects on postprandial blood glucose levels as well as physicochemical and sensory analysis. In comparison to WRC, the samples containing 100% added black rice flour presented higher nutritional qualities in terms of protein, by 16.61%, 8.64% for lipids, 5.61% for ash, 36.94% for crude fiber, 58.04% for total polyphenols, 95.49% for proanthocyanidins, and 88.07% for flavonoids. The addition of BRF had a suppressing effect on lightness (L*) and yellowness (b*), while redness (a*) increased. The results of the glycemic measurements confirmed that consumption of crackers made from brown or black whole-grain rice grain flour does not generate glycemic peaks above the limit of 30 mg/dL in baseline blood glucose levels. The results of developing rice crackers from black and brown flour blends showed promising physicochemical and nutritional properties and could provide a good alternative to wheat flour as a gluten-free product. Full article

Platelets are metabolically active, anucleated and small circulating cells mainly responsible for the prevention of bleeding and maintenance of hemostasis. Previous studies showed that platelets mitochondrial content, function, and energy supply change during several diseases such as HIV/AIDS, COVID-19, pulmonary arterial hypertension, and in preeclampsia during pregnancy. These changes in platelets contributed to the severity of diseases and mortality. In our previous studies, we have shown that the seahorse-based cellular stress assay (CSA) parameters are crucial to the understanding of the mitochondrial performance in peripheral blood mononuclear cells (PBMCS). Moreover, the results of CSA parameters were significantly influenced by the PBMC preparation methods. In this study, we assessed the correlation of CSA parameters and intracellular ATP content in platelets and evaluated the effects of platelet preparation methods on the results of CSA parameters and intracellular ATP content. We compared the results of CSA parameters and intracellular ATP content in platelets isolated by density centrifugation with Optiprep and simple centrifugation of blood samples without Optiprep. Platelets isolated by centrifugation with Optiprep showed a higher spare capacity, basal respiration, and maximal respiration than those isolated without Optiprep. There was a clear correlation between basal respiration and maximal respiration, and the whole-ATP content in both isolation methods. Moreover, a positive correlation was observed between the relative spare capacity and whole-cell ATP content. In conclusion, the results of seahorse-based CSA parameters and intracellular ATP content in platelets are markedly influenced by the platelet isolation methods employed. The results of basal respiration and maximal respiration are hallmarks of cellular activity in platelets, and whole-cell ATP content is a potential hint for basic platelet viability. We recommend further studies to evaluate the role of CSA parameters and intracellular ATP content in platelets as biomarkers for the diagnosis and prediction of disease states. Full article

For the treatment of human immunodeficiency virus (HIV)-infected patients, the regular assessment of the immune status is indispensable. The quantification of CD4⁺ T lymphocytes in blood by gold standard optical flow cytometry is not point-of-care testing (POCT) compatible. This incompatibility is due to unavoidable pre-analytics, expensive and bulky optics with limited portability, and complex workflow integration. Here, we propose a non-optical, magnetic flow cytometry (MFC) workflow that offers effortless integration opportunities, including minimal user interaction, integrated sample preparation and up-concentration, and miniaturization. Furthermore, we demonstrate immunomagnetic CD4⁺ T lymphocyte labeling in whole blood with subsequent quantification using sheath-less MFC. Showing linearity over two log scales and being largely unimpaired by hematocrit, evidence is provided for POCT capabilities of HIV patients. Full article

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation—control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7–23.1 ng/µL; persistent AF: 4.9–66.8 ng/µL; controls: 6.3–10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation. Full article

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC–MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH₃THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH⁺-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease–amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins’ isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC–MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH₃THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH⁺THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH₃THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC–MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples. Full article

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium. A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae, respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates. Full article

The radiolabeled iron oxide nanoparticles constitute an attractive choice to be used as dual-modality contrast agents (DMCAs) in nuclear medical diagnosis, due to their ability to combine the benefits of two imaging modalities, for instance single photon emission computed tomography (SPECT) with magnetic resonance imaging (MRI). Before the use of any DMCA, the investigation of its plasma extra- and on/intra cellular distribution in peripheral human blood is of paramount importance. Here, we focus on the in vitro investigation of the distribution of ^99mTc-DPD-Fe₃O₄ DMCA in donated peripheral human blood (the ligand 2-3-dicarboxypropane-1-1-diphosphonic-acid is denoted as DPD). Initially, we described the experimental methods we performed for the radiosynthesis of the ^99mTc-DPD-Fe₃O₄, the preparation of whole blood and blood plasma samples, and their incubation conditions with ^99mTc-DPD-Fe₃O₄. More importantly, we employed a gamma-camera apparatus for the direct imaging of the ^99mTc-DPD-Fe₃O₄-loaded whole blood and blood plasma samples when subjected to specialized centrifugation protocols. The direct comparison of the gamma-camera data obtained at the exact same samples before and after their centrifugation enabled us to clearly identify the distribution of the ^99mTc-DPD-Fe₃O₄ in the two components, plasma and cells, of peripheral human blood. Full article

Sample preparation for mass spectroscopy typically involves several liquid and solid phase clean-ups, extractions, and other unit operations, which are labour-intensive and error-prone. We demonstrate a centrifugal microfluidic platform that automates the whole blood sample’s preparation and clean-up by combining traditional liquid-phase and multiple solid-phase extractions for applications in mass spectroscopy (MS)-based small molecule detection. Liquid phase extraction was performed using methanol to precipitate proteins in plasma separated from a blood sample under centrifugal force. The preloaded solid phase composed of C18 beads then removed lipids with a combination of silica particles, which further cleaned up any remaining proteins. We further integrated the application of this sample prep disc with matrix-assisted laser desorption/ionization (MALDI) MS by using glancing angle deposition films, which further cleaned up the processed sample by segregating the electrolyte background from the sample salts. Additionally, hydrophilic interaction liquid chromatography (HILIC) MS was employed for detecting targeted free amino acids. Therefore, several representative ionic metabolites, including several amino acids and organic acids from blood samples, were analysed by both MALDI-MS and HILIC-MS to demonstrate the performance of this sample preparation disc. The fully automated blood sample preparation procedure only took 35 mins, with a throughput of three parallel units. Full article