Ion-exchange Chromatographic Method Research Articles

A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin–Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol−1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL−1, 273.10 U mL−1, and 168.52 s−1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

Efficient separation of radium from natural thorium using a mesoporous silica-supported composite resin with sulfonic acid groups for the acquisition of targeted α-nuclides 212Pb

212Pb serves as one of the most important nuclides for targeted alpha therapy (TAT), with good potential in the treatment of malignant tumors. However, medical and clinical applications are seriously limited by its extremely short supply, which strongly depends on the acquisition of its parent nuclides such as 228Ra or 224Ra. To address this issue, we conducted a systematic investigation on the separation of trace radium from natural thorium using the ion exchange chromatography method, and a novel porous silica-based composite resin functionalized with sulfonic acid groups was prepared, characterized, and employed. Experimental results showed that the silica-supported resin was successfully prepared with the sulfonic acid groups, and possessed small particle size, mesoporous pore structure and large specific surface area. The resin exhibited high selectivity toward thorium compared with barium, and the separation factor reached 123 at low acidity and at low temperature. The resin demonstrated a fast adsorption speed and an excellent separation performance, and the adsorption mechanism was confirmed based on the ion exchange between the sodium carried and the target cations. On the basis, column experiments were further conducted and finally the separation of trace radium from thorium was realized using a small column filled with about 1.0 g resin. The chemical recovery of radium reached 97 %, and the decontamination factor for thorium exceeded 40,000. This work verifies the feasibility of directly extracting 228Ra and 224Ra from natural thorium using the sulfonic acid group functionalized materials in the dilute nitric acid solution, and presents an excellent material and systematic experimental data on the separation activities, rendering this study particularly significant.

Platelet amino acid spectrum and gut microbiota, their links in patients with coronary artery disease and atrial fibrillation

Background. The aim of our work was to identify the links between platelet amino acid (AA) spectrum and gut microbiota composition in patients with coronary artery disease (CAD) and atrial fibrillation (AF) and to evaluate them. Materials and methods. Three hundred patients were enrolled in the study. They were divided into 3 groups: I (CAD) — 149 patients with CAD but without arrhythmias, II (CAD + AF) — 124 people with CAD and AF paroxysm, and control group (CG) — 27 individuals without CAD and arrhythmias. Platelet AA level was assessed by method of ion-exchange liquid column chromatography. Gut microbiota composition was studied by 16-S rRNA sequencing. Results. A significant increase in isoleucine (10.73 %), leucine (12.63 %) and a decrease in threonine (23.05 %), serine (5.06 %), glycine (32.21 %) and valine (30.83 %) platelets levels was found in patients with CAD and AF (P < 0.05). In addition, they had a significant increase in Bacteroides spp., Faecalibacterium prausnitzii, Actinobacter spp., Streptococcus spp., Ruminococcus spp. and a decrease in Lactobacillus spp., Bifidobacterium spp., Eubacterium rectale (P < 0.05). Platelet glutamine acid, valine, glycine, asparagine acid, threonine had the highest number of significant correlations with gut microbiota species (P < 0.05). Actinobacter spp., Blautia spp., Streptococcus spp., Akkermansia muciniphila and Roseburia inulinivorans had the highest number of significant correlations with platelet amino acids (P < 0.05). Conclusions. Platelet amino acid spectrum and gut microbiota composition in patients with coronary artery disease and atrial fibrillation are closely linked.

A-326 Argininosuccinic Acid - is it Routinely Detectable in the Urine of Healthy Individuals?

Abstract Background Argininosuccinic aciduria is a genetic disorder that results in elevated levels of argininosuccinic acid (ASA) in urine and blood. While there is seemingly a consensus within the clinical literature that any detectable amount of ASA in blood/plasma is diagnostic for argininosuccinic aciduria, yet there is conflict as to whether ASA in urine can be found in unaffected individuals. Some literature states that ASA should not be found in the urine of a healthy individual, while other literature reports low levels of ASA in the urine of healthy individuals. Additionally, many laboratories do not have a normal range for ASA within urine, which implies that any level of ASA found within urine is indicative of argininosuccinic aciduria. This conflict may in part be due to differences in the methods used to determine ASA levels. Newer liquid chromatography tandem mass spectrometry (LCMSMS) methods have been shown to be more sensitive than ion-exchange chromatography (IEC) based amino acid methods for detection of ASA. Additionally, the detection of ASA via IEC requires the heating and acidification of the sample to convert ASA into a single peak for quantitation. Heating and acidification is not routinely done for urine amino acid analysis with IEC; with routine IEC urine amino acid analysis, ASA migrates as three small peaks that may go unnoticed. In our clinical laboratory, which utilizes the less sensitive IEC method, our procedure describes the presence of any ASA in urine as diagnostic for argininosuccinic aciduria. Given the conflicting literature, we set forth to determine whether low levels can be detected in unaffected individuals with an IEC method, and if so, establish a normal range of ASA levels in urine with IEC. Methods Twenty waste urine samples from patients with no prior diagnoses of argininosuccinic aciduria were evaluated. ASA levels within the samples were determined via high performance ion exchange chromatography for separation of compounds followed by derivatization with ninhydrin for spectrophotometric detection with a Hitachi Amino Acid analyzer. The samples were acidified and heated, which converts ASA into a single anhydride peak for accurate quantitation, and the results were normalized to urine creatinine. Results ASA was detected in all twenty urine samples, with an average ASA concentration of 27.2 μmol/g Cr and a range of 11.3–47.7 μmol/g Cr. Conclusion Given these findings, our clinical laboratory procedure has been updated to include the information that ASA can be present in the urine of healthy individuals up to a concentration of 50 μmol/g Cr. Other clinical laboratories that analyze urine to identify argininosuccinic aciduria should be aware of the misinformation in the literature that claims that ASA is not present in the urine of healthy individuals.

Ion exchange chromatography as a simple and scalable method to isolate biologically active small extracellular vesicles from conditioned media.

In the last few years, extracellular vesicles (EVs) have become of great interest due to their potential as biomarkers, drug delivery systems, and, in particular, therapeutic agents. However, there is no consensus on which is the best way to isolate these EVs. The choice of the isolation method depends on the starting material (i.e., conditioned culture media, urine, serum, etc.) and their downstream applications. Even though there are numerous methods to isolate EVs, few are compatible with clinical applications as they are not scalable. In the present work, we set up a protocol to isolate EVs from conditioned media by ion exchange chromatography, a simple, fast, and scalable method, suitable for clinical production. We performed the isolation using an anion exchange resin (Q sepharose) and eluted the EVs using 500 mM NaCl. We characterized the elution profile by measuring protein and lipid concentration, and CD63 by ELISA. Moreover, we immunophenotyped all the eluted fractions, assessed the presence of TSG101, calnexin, and cytochrome C by western blot, analyzed nanoparticle size and distribution by tRPS, and morphology by TEM. Finally, we evaluated the immunomodulatory activity in vitro. We found that most EVs are eluted and concentrated in a single peak fraction, with a mean particle size of <150nm and expression of CD9, CD63, CD81, and TSG101 markers. Moreover, sEVs in fraction 4 exerted an anti-inflammatory activity on LPS-stimulated macrophages. In summary, we set up a chromatographic, scalable, and clinically compatible method to isolate and concentrate small EVs from conditioned media, which preserves the EVs biological activity.

Substantial incorporation of isotopically heavy reduced U species into marine carbonate sediments

The uranium (U) isotope ratio (238U/235U, reported as δ238U) in marine carbonates is an important proxy for reconstructing changes in past oceanic redox conditions, based on the essential assumption that U in carbonates originates from dissolved U(VI) in seawater. However, diagenetic processes may induce the addition of isotopically heavy U(IV) into the carbonates, complicating the application of the U isotope proxy. So far, the valence states of trace amounts of U and its relation with U isotope composition in marine carbonate sediments have not been directly studied. In this study, we have calibrated an ion-exchange chromatographic method to separate U(IV) from U(VI) in geological carbonate samples (e.g., modern and fossil corals, stalagmites, cold seep carbonates) and quantified their contents by mass spectrometry. Our study confirms that modern coral carbonates are faithful archives of U(VI) sourced from seawater. Surprisingly, drill core samples from a modern coral carbonate platform, which bear no sign of alteration inferred from conventional diagenetic proxies (e.g., Mn/Sr), show significant positive correlation between U(IV) fraction and bulk carbonate δ238U, suggesting U(IV) is present in the marine carbonates and drives δ238U toward higher values than that of seawater. We therefore suggest that coupled valence and isotope analyses of U in marine carbonates could provide critical constraints toward reliable reconstruction of marine redox evolution in the Earth’s history.

Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of NANOBODYⓇ molecules

NANOBODYⓇ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODYⓇ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODYⓇ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODYⓇ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODYⓇ molecule retention. Furthermore, the protein charge plot also showed that NANOBODYⓇ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODYⓇ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.

Detection of bacteria causing mastitis in cows with immunoelectric tool.

This study has investigated designing, producing, and evaluating a newly developed immunoelectric device for the identification of the Staphylococcus aureus and Escherichia coli in experimentally or clinically polluted milk samples. The design of the immunoelectric tool parts was carried out and produced by using a 3D printer. The animals were immunized against S. aureus and E. coli; purification of the specific IgG from hyperimmune serum was carried out by ion exchange and affinity chromatography methods. Coupling of the specific rabbit antibodies to the polystyrene filters was performed by DTPA linker. The purified rat antibodies were coupled to Fe nanoparticles and used as detector elements. The experimentally polluted milk and PBS samples were prepared. After optimization, the minimum traceable number of the bacteria was determined using immunoelectric tool. Also, the cow's milk samples with clinical mastitis were tested for bacterial detection by bacterial culture and immunoelectric methods. Coupling of the antibody to the filters and capturing of the target bacteria to the filters were successfully confirmed using enzyme immune assay and electron microscopy. The detection limit of the developed immunoelectric tool was equal to 15 cells/mL in PBS or milk samples for 30 min. Also, in agreement with bacterial cell culture, the clinically infected milk samples by S. aureus and E. coli were successfully detected using the immunoelectric device. The developed immunoelectric device could be utilized for rapid and cost-benefit detection of the bacterial organism.

Characterization of antithrombin isoforms and latent forms by ion exchange chromatography coupled to mass spectrometry

Antithrombin is a key protein of the coagulation system belonging to the serine protease inhibitor family. Antithrombin preparations are used as a therapeutic treatment for patients with decreased antithrombin activity. Elucidating the structural features of this protein is an important part of the control strategy to assure a high quality. This study presents an ion exchange chromatographic method coupled to mass spectrometry capable of characterizing antithrombin post-translational modifications such as N-glycosylation, phosphorylation or deamidation. Furthermore, the method was successfully used to evidence irreversible/inactive conformers of antithrombin which are commonly observed for serine protease inhibitors and referred to as latent forms.

Role of overexpression mTORC1 in diabetes mellitus type 2: activation PI3K/Akt pathway

The objective of this study is investigating the role of excessive activation PI3K/Akt/mTORC1 in diabetes mellitus type 2. It’s known that the PI3K/Akt/mTORC1 pathway is involved in the pathogenesis of cancer. Study design was carried out in accordance with the guidelines of the Helsinki Declaration that included normal patients as control (n = 15), second group included patients with diabetes mellitus type 2 (n = 22) while, third group included patients with malignancy positive mTORC1 (n = 22). 81 women were examined and enrolled in this study with age from 40 to 75-year-old. Diabetes mellitus type 2 compensation was assessed by determining the level of HbA1c by the method of ion-exchange chromatography, using the BIO-RAD D-10 analyzer, PRAS40 was determined in units depending on the amount of protein in the blood cell lysates.

THE ROLE OF ENDOTOXICOSIS AND INFLAMMATION IN DEEPENING THE PANCREATIC FUNCTIONAL INSUFFICIENCY IN CHRONIC PANCREATITIS IN COMBINATION WITH TYPE 2 DIABETES.

Aim: To analyze the state of parameters of inflammation, endotoxicosis, and their influence on the functional capacity of the pancreas in the comorbid course of chronic pancreatitis and type 2 diabetes mellitus (DM2). Materials and methods: 115 patients with CP in the phase of mild therapeutic exacerbation in combination with DM2 in the stage of subcompensation were examined. To assess the impact of comorbid DM2 on the clinical condition of patients with CP, a comparison group of 25 patients with CP in the exacerbation phase was included in the study. The assessment of the presence and depth of pancreatic exocrine insufficiency (PEI) was carried out according to the "gold standard" - determination of the content of fecal α-elastase-1, which was determined by the method of enzyme immunoassay using standard kits. As the main criterion for diagnosis and monitoring of DM, the measurement of HbA1c was used, which was determined by the method of ion exchange chromatography. C-reactive protein (CRP) was determined by the immunoturbidometry method by photometric measurement of the antigen-antibody reaction to human CRP antibodies; reference values of CRP in blood serum are up to 3 mg/l. Endogenous intoxication (EI) was assessed based on the levels of medium-mass molecules (MMM) - MMM1 and MMM2 at wavelengths 254 and 280 nm. The level of circulating immune complexes (CIC) was determined by the method of selective precipitation in 3.75% ethylene glycol followed by photometry. Results: Moderate and moderate inverse correlations were established between CRP and fecal α-elastase in CP and CP-DM2 comorbidity (r=-0.423 and r=-0.565, p<0.05). This proved a reliable influence of the depth of inflammation according to the content of CRP on the increase in PEI according to the level of fecal α-elastase, which was higher in the CP-DM2 comorbidity compared to CP. A deeper level of secretory insufficiency of the pancreas was established in CP with concomitant DM2, which deepened when the CRP level increased, compared to that in isolated CP: an increase in the strength of reliable direct moderate HbA1c-CRP correlations in patients with CP in combination with DM2 was proved in relation to such cases isolated CP (respectively r=0.313 and r=0.410, p<0.05). Conclusions: We proved a reliable influence of the index of endogenous intoxication on the level of PEI according to the level of fecal α-elastase, which was higher in the CP-DM2 comorbidity compared to isolated CP: moderate and medium-strength inverse correlations were established IEI-fecal α-elastase in patients with CP and CP-DM2 comorbidity (r=-0.471 and r=-0.517, p<0.05). An increase in the strength of reliable direct, moderate, and moderate correlations between the levels of HbA1c and the index of endogenous intoxication in patients with isolated CP and CP-DM2 comorbidity (r=0.337 and r=0.552, p<0.05), which proved a deeper level of secretory pancreas insufficiency with concomitant DM2, which worsened with increasing endotoxicosis according to the value of the index of endogenous intoxication.

Association between Serum Calcium Concentration and Glycated Haemoglobin in Type II Diabetic Nigerian Patients

Background: Diabetes is a metabolic disease that affects key biochemical parameters in the body. The study was carried out to evaluate the serum calcium concentration and the relationship between serum calcium and glycosylated haemoglobin in type 2 diabetics. Methods: A total of 126 subjects consisting of 63 Diabetic patients and 63 controls participated in this study. Serum calcium and glycosylated haemoglobin concentrations were measured. Fasting blood sugar was also measured. Total calcium was measured using colorimetric determination of plasma total calcium by the o-cresolphthalein o-complexone (CPC) assay principle. Plasma albumin was determined using Bromocresol green (BCG) method, and thus corrected calcium was deduced. Glycosylated haemoglobin (HbA1C) was estimated using an ion-exchange chromatographic method. A semi-structured questionnaire was used for data collection. Results: The mean serum calcium levels of diabetics compared to non-diabetics did not differ statistically even though that of diabetics was in the lower range of normal. There was no statistically significant difference (p=0.099) between the mean serum calcium levels of diabetic patients who had glycosylated haemoglobin values below 6.5 (2.26 mmol/l±0.07) and diabetic patients who had glycosylated haemoglobin values above 6.5 (2.17mmol/l±0.09). Surprisingly, there was a weak positive correlation between glycosylated haemoglobin levels above 6.5 and serum calcium levels (p=0.087) and HbA1C below 6.5 correlated negatively albeit weakly with serum calcium levels though they were both insignificant. Socio-demographic data show that more females were diabetic. Conclusion: Overall, the study reveals that mean serum calcium concentrations in diabetic and non-diabetic patients are not different and this may be due to good blood glucose control in these diabetic patients

Environmental iodine speciation quantification in seawater and snow using ion exchange chromatography and UV spectrophotometric detection

The behaviour and distribution of iodine in the environment are of significant interest in a range of scientific disciplines, from health, as iodine is an essential element for humans and animals, to climate and air quality, to geochemistry. Aquatic environments are the reservoir for iodine, where it exists in low concentrations as iodide, iodate and dissolved organic iodine and in which it undergoes redox reactions. The current measurement techniques for iodine species are typically time-consuming, subject to relatively poor precision and require specialist instrumentation including those that require mercury as an electrode. We present a new method for measuring iodine species, that is tailored towards lower dissolved organic carbon waters, such as seawater, rainwater and snow, using ion exchange chromatography (IC) with direct ultra-violet spectrophotometric detection of iodide and without the need for sample pre-concentration. Simple chemical amendments to the sample allow for the quantification of both iodate and dissolved organic iodine in addition to iodide. The developed IC method, which takes 16 min, was applied to contrasting samples that encompass a wide range of aqueous environments, from Arctic sea-ice snow (low concentrations) to coastal seawater (complex sample matrix). Linear calibrations are demonstrated for all matrices, using gravimetrically prepared potassium iodide standards. The detection limit for the iodide ion is 0.12 nM based on the standard deviation of the blank, while sample reproducibility is typically <2% at >8 nM and ∼4% at <8 nM. Since there is no environmental certified reference material for iodine species, the measurements made on seawater samples using this IC method were compared to those obtained using established analytical techniques; iodide voltammetry and iodate spectrophotometry. We calculated recoveries of 102 ± 16% (n = 107) for iodide and 116 ± 9% (n = 103) for iodate, the latter difference may be due to an underestimation of iodate by the spectrophotometric method. We further compared a chemical oxidation and reduction of the sample to an ultra-violet digestion to establish the total dissolved iodine content, the average recovery following chemical amendments was 98 ± 4% (n = 92). The new method represents a simple, efficient, green, precise and sensitive method for measuring dissolved speciated iodine in complex matrices.

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors.

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.

Total plasma homocysteine measurement: Evaluation of the Abbott immunoassay, comparison with the JEOL ion exchange chromatography and investigation of its clinical utility

ObjectivesHomocysteine is an intermediary amino acid formed in methionine metabolism, with elevated total homocysteine (tHCY) being a biomarker of cardiovascular and cerebrovascular diseases. We evaluated the Abbott ARCHITECT tHCY immunoassay, compared it with the current established JEOL ion exchange chromatography (IEC) method and evaluated its clinical utility. Design and methodsFollowing immunoassay method verification, plasma samples of 91 patients were analysed for tHCY using immunoassay and IEC. ResultsFor the Abbott immunoassay, accuracy was assessed, with UK NEQAS EQA specimens, by the correlation of our Abbott immunoassay measurements to the Abbott ARCHITECT immunoassay mean (bias = 1.6%), and to the overall immunoassay mean (bias = 2.0%). The total imprecision was 2.7% (11.00 μmol/L), 2.4% (16.80 μmol/L) and 2.8% (24.30 μmol/L) respectively. Bias in linearity assessment was 0.12%–2.58%. The inter-method correlation was strong in Passing-Bablok regression: immunoassay = IEC x0.857 + 2.445 (95% CI: slope = [0.742,0.947], intercept = [1.340,3.582]), with Spearman correlation = 0.803 (p < 0.001). The Bland-Altman plot showed an average difference of −0.284 μmol/L (95% CI: [-1.043,0.474]) with limits of agreement (mean ± 1.96SD) from −7.425 μmol/L to 6.857 μmol/L.No significant difference in tHCY was found using both methods in patients with cerebrovascular diseases and cardiovascular diseases. Most tHCY measurements were within the reference ranges of both methods. All homocystinuria patients had tHCY values above the reference ranges of both methods. ConclusionsThe immunoassay demonstrated robust performance in its verification and showed good comparability with the IEC but with some biases so caution is needed if both are used interchangeably. The immunoassay offers an automated alternative to IEC in the assessment of hyperhomocysteinaemia.

Seed Protein from Artocarpus hirsutus Lam. with Trypsin Inhibitory, Micro-bicidal and Antioxidant Activities Induces Depletion of Human Skin Cancer (A431) and Colon Cancer (HT29) Cells

: Natural trypsin inhibitors are increasingly recognised as putative anticancer or antimicrobial therapeutics. In the present study, we isolated a proteinaceous trypsin inhibitor from the seeds of Artocarpus hirsutus to assess its anticancer, antioxidant and microbicidal effects. A trypsin inhibitory protein (AhTI) was isolated from the seeds of A. hirsutus using ion exchange and gel filtration chromatographic methods. The trypsin inhibitory activity of AhTI was confirmed by activity staining using Reverse Zymography. The effect of AhTI on human cancer cell lines (A431 and HT29) was studied by microscopic examination, MTT assay and LDH leakage analysis. Antioxidant activity of AhTI was analysed by ferric reducing power and DPPH radical scavenging assays. An agar well diffusion method was used to check the antimicrobial activity of AhTI. The purification protocols used in the study significantly increased the specific activity of the target protein from 72.65 ± 0.86 to 2048 ± 27.3. AhTI offers significant activity against the proliferation of the A431 and HT29 cancer cell lines. In vitro AhTI shows potent antioxidant activity and exhibits antimicrobial activity against all the bacteria and fungi isolates used in this study. Of particular note, AhTI was particularly effective against Staphylococcus aureus and Escherichia coli. This in vitro study shows that AhTIis a promising candidate for further development novel therapeutics targeting cancer, microbial infections and oxidative stress.

Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography

We describe a quenching-free, ‘online’ ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SubjectEnzymatic assays and analysis;

Ways of optimization of lead-polluted black earth soils in the soil-plant system

the most urgent problems of today. Ensuring the environmental safety of food raw materials and food products is one of the main tasks that determine the health of the human population and the preservation of its gene pool. Analysis of recent research and publications. Toxic effects of Pb on human bodies have been confirmed by numerous clinical studies that have shown the negative effects of heavy metals on the nervous, cardiovascular, immune systems and oncology. The works is devoted to the development of detoxification techniques, in which scientists proposed to reduce the content of heavy metals in chernozems by detoxifying soils by growing battery plants on them, which will «extract» heavy metals from soils disinfecting them. The disadvantage of the proposed technologies is the economic costs borne by farmers, producers and consumers. Farmers will suffer financial losses due to idle sown areas and the cost of seed. Producers will be forced to raise prices for the final food product as a result of reduced consumer purchasing power. Formulation of the purpose of the article. The aim of the article is to determine ways to optimize lead-contaminated chernozem soils in the system «soil – plant» (on the example of chernozem soils within the test sites on the territory of the collection nursery «Agrotek» in Kyiv region). Presentation of the main research material. The article presents the results of a study of ways to optimize lead-contaminated chernozem soils in the system «soil – plant». The objects of the study were soybean and chickpea varieties of different vegetation varieties, namely: ultra-early varieties, maturation period 95… 105 days. Мedium, maturation period 100…115 days. Мedium-ripe varieties maturation period115…125 days. Early ripening varieties of chickpeas growing period 95…115 days, and medium-ripe sotu chickpeas, growing period 115…125 days. Experimental studies were conducted during 2019 – 2021 within the test sites on the territory of the collection nursery «Agrotek» in Kyiv region. The method of ion exchange and liquid chromatography on the liquid chromatograph Shimadzu LC-20 (Japan). Studied the variability of nutrients (protein, fat, carbohydrates) in soybeans and chickpeas of different vegetation varieties. With the help of the MATLAB program, mathematical optimization was carried out and the five-year dynamics of protein, fat, and carbohydrate content in legumes grown on chernozem soils of the «Agrotek» collection nursery in the Kyiv region was determined. By the method of inversion-voltammetry with the help of voltammetric analyzer «ABA-3» (Russia) which is equipped with an indicator electrode for the determination of lead, the concentration of Pb in chernozems and soybeans and chickpeas with different protein content was studied. Concentrations of Pb in soybean and chickpea grains depending on the chemical composition of soils for cultivation were grown and studied at the test sites of the «Agrotek» collection nursery. Conclusions. As a result of experimental studies, it was found that the contamination of chernozem soils Pb and subsequent processes of its translocation in the system «soil – plant» have negative consequences and are manifested in the accumulation of toxicants in plants. It is significant that the largest metal-accumulating properties have cultivated early-ripening legumes, and the smallest – medium-ripe. It was determined that the indicators of Pb concentration in the storage organs of legume assimilants are influenced to a greater extent by the protein content in them than by mobile forms of Pb, which come due to translocation from soils contaminated with Pb. It is established that with the increase of protein in the organs of plant assimilators, the indicators of accumulation of toxic concentrations of Pb increase. Given that the genetic characteristics of chernozems allow us to consider them the most environmentally friendly soils in terms of anthropogenesis, however, they deposit contaminants and, accordingly, require optimization and development of detoxification methods. It has been experimentally established that when growing legumes of different vegetation varieties on chernozems contaminated with Pb, the intensity of their detoxification to obtain environmentally friendly plant products can be distributed as follows: N180P180K180 + Pb + lime + manure &gt; N360P360K360+ Pb &gt; N180P180K180 + Pb + lime &gt; N180P180K180 + Pb + manure. The obtained laws provide an opportunity to develop recommendations and propose ways to detoxify contaminated Pb soils. Which will provide an opportunity to significantly reduce budget expenditures, which is socially necessary and cost-effective.

INTERFERENCES OF HbA1c ANALYSIS IN HOSPITAL UNIVERSITI SAINS MALAYSIA – 3 YEARS STUDY

Haemoglobin A1c (HbA1c) is used to monitor glycaemic control and predict diabetic complications. Measurement of HbA1c can be interfered by haemoglobin (Hb) variant and other Hb derivatives include carbamylated Hb and elevated labile A1c. This study is to determine the percentages and type of interferences during HbA1c analysis and the percentages of non- reportable HbA1c results. This is a cross-sectional study using retrospective data of HbA1c. The HbA1c is measured on Biorad D10 using the ion-exchange high-performance liquid chromatography method. The data were analyzed using descriptive statistics. A total of 26,560 patients were included. The result showed the presence of interferences of 2269 (8.56%). The most common causes of the interferences were the Hb variant (8.48%) followed by carbamylated Hb and labile A1c (0.03% each). The non-reportable HbA1c results were 0.46% with the Hb variant contributed most of the causes. By knowing the presence of interferences particularly the Hb variant, the HbA1c results hopefully are interpreted with caution and correct management can be given to the patients.