Assessment of Plasma-Free Cortisol Concentrations by LC-MS/MS in Patients with Autonomous Cortisol Secretion

Autonomous cortisol secretion (ACS) of an adrenal incidentaloma (AI) is associated with mild cortisol excess that could result in poor metabolic and cardiovascular outcomes. The biological activity of glucocorticoids depends on the unbound, free fraction. We aimed to evaluate plasma free cortisol (FC) concentrations in patients with ACS in this cross-sectional study. One hundred and ten AI patients in 3 groups; non-functioning (NFA, n=33), possible ACS (n=65), ACS (n=12) were enrolled. Following measurements were conducted: Clinical data and total serum cortisol (TC), plasma corticotrophin (ACTH), serum dehydroepiandrosterone sulfate (DHEA-S), cortisol after 1 mg dexamethasone by both immunoassay and LC-MS/MS (DexF), serum corticosteroid binding globulin (CBG), plasma dexamethasone concentration [DEX] and plasma FC by LC-MS/MS. Patients with ACS featured an unfavorable metabolic profile. Plasma [DEX] and serum CBG levels were similar between groups. Plasma FC was significantly higher in ACS when compared to NFA and possible ACS groups p<0.05 and p<0.01, respectively. In multiple regression analysis DexF (beta=0.402, p<0.001) and CBG (beta=−0.257, p=0.03) remained as the independent predictors of plasma FC while age, sex, BMI, smoking habit, and existing cardiovascular disease did not make a significant contribution to the regression model. In conclusion, the magnitude of cortisol excess in ACS could lead to increased plasma FC concentrations. Further studies in AI patients are needed to demonstrate whether any alterations of cortisol affinity for CBG exist and to establish whether plasma FC concentrations predict the unfavorable metabolic profile in ACS.

Optimization of Diagnosis of Secondary Metabolic Disorders and Treatment Tactics in Patients with Malignant Neoplasm Anemia in Colorectal Cancer

The purpose of the study was to study the content of free fraction of heparin, histamine and serotonin in plasma of peripheral venous blood of patients with malignant neoplasm anemia in colorectal cancer, depending on the severity of anemia. Materials and methods. The material for the study was the blood plasma of 445 patients (228 men and 217 women). Among them, 53 patients (31 women and 22 men) with iron deficiency anemia were examined and included in the first observation group (І) and 392 patients (206 men (52.55 %) and 186 women (47.45 %)) with colorectal cancer whose course of the underlying disease was burdened with malignant neoplasm anemia (ICD-10 code: D63.0) were included in the second observation group (II). Among the patients in the second (II) observation group, there were 222 individuals (119 men and 103 women) with malignant neoplasms of the colon (ICD-10 code: С.18), 29 individuals (16 men and 13 women) with malignant neoplasms of the rectosigmoid junction (ICD-10 code: C.19), 138 individuals (82 men and 56 women) with malignant neoplasms of the rectum (code ICD-10 C.20) and 3 patients (2 men and 1 woman) with malignant neoplasms of the anus (ICD-10 code: C.21). The mean age of the patients was 63.3 ± 1.2 years old. The plasma level of free fraction of heparin of the examined patients was determined using the photocolorimetric method on photoelectric colorimeter 56-M after its preliminary isolation by electrophoretic method according to the appropriate procedure (B. V. Mykhailychenko, S. V. Vydyborets (2000)). The plasma level of free histamine and serotonin of the examined patients was studied using the method of fluorometric analysis on the analyzer “BIAN-130”-“BIAN-100” according to the procedure of B. V. Mykhailychenko, S. V. Vydyborets (1999). Results and discussion. It was found that prior to the initiation of treatment in patients with malignant neoplasm anemia, regardless of the course of colorectal cancer, there was a significant increase in the plasma level of free fraction of heparin, histamine, serotonin (p < 0.001); the ratio of free histamine to free serotonin was also changed in comparison with the values in the control group (p < 0.05), which indicated both an increased release of heparin, histamine and serotonin from the depot, and an impaired inactivation processes of these biologically active substances. Considering all of the above and the quite obvious reasons, namely, the secondary metabolic disorders of serotonin, histamine, heparin which manifested by a significant increase in their plasma level of patients with malignant neoplasm anemia in colorectal cancer, we suggested the need to use a medicinal product in a complex of therapeutic measures which can cause antihypoxic, membrane stabilizing and anti-edema action. Conclusion. Malignant neoplasm anemia in colorectal cancer is accompanied by significant changes in the metabolism of biologically active substances – free fraction of heparin, histamine, serotonin, and the ratio of free histamine to serotonin. It was correctly concluded that in addition to the baseline therapy the administration of arginine glutamate which causes both antihypoxic and membrane-stabilizing action, reliably contributes to the normalization of secondary metabolic disorders of histamine, serotonin and heparin metabolism in malignant neoplasm anemia in patients with colorectal cancer

In silico and multi-spectroscopic analyses on the interaction of 5-amino-8-hydroxyquinoline and bovine serum albumin as a potential anticancer agent

Abstract5-Amino-8-hydroxyquinoline (5A8HQ), an amino derivative of 8-hydroxyquinoline, has become a potential anticancer candidate because of its promising proteasome inhibitory activity to overcome and yet synergize bortezomib for fighting cancers. Therefore, in this study, its physicochemical properties and interaction activities with serum protein have extensively been elucidated by both in vitro and in silico approaches to fulfill the pharmacokinetic and pharmacodynamic gaps. 5A8HQ exhibited the drug-likeness properties, where oral administration seems to be a route of choice owing to its high-water solubility and intestinal absorptivity. Multi-spectroscopic investigations suggested that 5A8HQ tended to associate with bovine serum albumin (BSA), a representative of serum protein, via the ground-state complexation. It apparently bound in a protein cleft between subdomains IIA and IIIA of BSA as suggested by the molecular docking and molecular dynamics simulations. The binding was mainly driven by hydrogen bonding and electrostatic interactions with a moderate binding constant at 104 M−1, conforming with the predicted free fraction in serum at 0.484. Therefore, 5A8HQ seems to display a good bioavailability in plasma to reach target sites and exerts its potent pharmacological activity. Likewise, serum albumin is a good candidate to be reservoir and transporter of 5A8HQ in the circulatory system.

Removal of Protein-Bound Uremic Toxins Using Binding Competitors in Hemodialysis: A Narrative Review

Removal of protein-bound uremic toxins (PBUTs) during conventional dialysis is insufficient. PBUTs are associated with comorbidities and mortality in dialysis patients. Albumin is the primary carrier for PBUTs and only a small free fraction of PBUTs are dialyzable. In the past, we proposed a novel method where a binding competitor is infused upstream of a dialyzer into an extracorporeal circuit. The competitor competes with PBUTs for their binding sites on albumin and increases the free PBUT fraction. Essentially, binding competitor-augmented hemodialysis is a reactive membrane separation technique and is a paradigm shift from conventional dialysis therapies. The proposed method has been tested in silico, ex vivo, and in vivo, and has proven to be very effective in all scenarios. In an ex vivo study and a proof-of-concept clinical study with 18 patients, ibuprofen was used as a binding competitor; however, chronic ibuprofen infusion may affect residual kidney function. Binding competition with free fatty acids significantly improved PBUT removal in pre-clinical rat models. Based on in silico analysis, tryptophan can also be used as a binding competitor; importantly, fatty acids or tryptophan may have salutary effects in HD patients. More chemoinformatics research, pre-clinical, and clinical studies are required to identify ideal binding competitors before routine clinical use.

Valproic acid autoinduction: a case-based review

Although valproic acid (VPA) induces the metabolism of multiple other drugs, the clinical reports of VPA autoinduction are rare. A comprehensive literature search yielded only one published case series, which provided the rationale to conduct a review of the published cases along with a new case of VPA autoinduction. Although there may be myriad of reasons for lack of published cases of VPA autoinduction, potential underreporting may be one of the core reasons. Lack of understanding into the highly complex metabolism of VPA may also make it difficult to recognize and report VPA autoinduction. However, it is important to mention that in addition to autoinduction increased elimination of VPA may be mediated by several pharmacokinetic (PK) factors, such as drug interactions, genetic polymorphisms of metabolic enzymes, and protein displacement reactions. As VPA is metabolized by multiple metabolic pathways, the risk for drug interactions is relatively high. There is also a growing evidence for high genetic inducibility of some enzymes involved in VPA metabolism. Protein displacement reactions with VPA increase the biologically active and readily metabolizable free fraction and pose a diagnostic challenge as they are usually not requested by most clinicians. Thus, monitoring of free fraction with total VPA levels may prevent clinically serious outcomes and optimize VPA treatment in clinically challenging patients. This case-based review compares the clinical data from three published cases and a new case of VPA autoinduction to enhance clinicians' awareness of this relatively rare but clinically relevant phenomenon along with a discussion of potential underlying mechanisms.

The Effect of Plasma Protein Binding on the Therapeutic Monitoring of Antiseizure Medications

Epilepsy is a widely diffused neurological disorder including a heterogeneous range of syndromes with different aetiology, severity and prognosis. Pharmacological treatments are based on the use, either in mono- or in polytherapy, of antiseizure medications (ASMs), which act at different synaptic levels, generally modifying the excitatory and/or inhibitory response through different action mechanisms. To reduce the risk of adverse effects and drug interactions, ASMs levels should be closely evaluated in biological fluids performing an appropriate Therapeutic Drug Monitoring (TDM). However, many decisions in TDM are based on the determination of the total drug concentration although measurement of the free fraction, which is not bound to plasma proteins, is becoming of ever-increasing importance since it correlates better with pharmacological and toxicological effects. Aim of this work has been to review methodological aspects concerning the evaluation of the free plasmatic fraction of some ASMs, focusing on the effect and the clinical significance that drug-protein binding has in the case of widely used drugs such as valproic acid, phenytoin, perampanel and carbamazepine. Although several validated methodologies are currently available which are effective in separating and quantifying the different forms of a drug, prospective validation studies are undoubtedly needed to better correlate, in real-world clinical contexts, pharmacokinetic monitoring to clinical outcomes.

Changes in organic matter composition caused by EDTA washing of two soils contaminated with toxic metals

Two soils contaminated with potentially toxic metals (PTMs) contrasting in pH and mineralogy were remediated with CaEDTA, and changes in soil organic matter (SOM) composition were investigated. Previous studies showed no significant loss of SOM from CaEDTA-treated soils, but the results of our study reflected significant decreases (from 46 to 49%) in the free fraction of humic acids (HAs). Remediation affected the composition of the free HA fraction via disturbance of intermolecular bonds — an increase in phenolic and aromatic groups with a simultaneous decrease in carbohydrates — which was confirmed by FTIR spectroscopy in both soils. Because non-radical molecules such as carbohydrates were selectively removed, the concentration of free radicals in the free HA fraction increased in acidic soil. The bound fraction of HAs and fulvic acids (FAs) in SOM, which are important due to their stability and the permanent effects they have on the soil’s physical properties, remained unchanged in both remediated soils. The effect of soil recultivation was observed only in the excitation emission matrix (EEM) fluorescence spectra of HAs. In terms of SOM, CaEDTA soil washing can be considered moderately conservative; however, the restoration of free humic fractions is likely to be a long-term process.

Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC–MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

Changes in Organic Matter Composition Caused by EDTA Washing of two Soils Contaminated with Toxic Metals

Two soils contaminated with potentially toxic metals (PTMs) contrasting in pH and mineralogy were remediated with CaEDTA, and changes in soil organic matter (SOM) composition were investigated. Previous studies showed no significant loss of SOM from CaEDTA-treated soils, but the results of our study reflected significant decreases (from 46 to 49%) in the free fraction of humic acids (HAs). Remediation affected the composition of the free HA fraction via disturbance of intermolecular bonds - an increase in phenolic and aromatic groups with a simultaneous decrease in carbohydrates - which was confirmed by FTIR spectroscopy in both soils. Because non-radical molecules such as carbohydrates were selectively removed, the concentration of free radicals in the free HA fraction increased in acidic soil. The bound fraction of HAs and fulvic acids (FAs) in SOM, which are important due to their stability and the permanent effects they have on the soil’s physical properties, remained unchanged in both remediated soils. The effect of soil recultivation was observed only in the EEM fluorescence spectra of HAs. In terms of SOM, CaEDTA soil washing can be considered moderately conservative, however, the restoration of free humic fractions is likely to be a long-term process.

Higher Dosing of Rifamycins Does Not Increase Activity against Mycobacterium tuberculosis in the Hollow-Fiber Infection Model

ABSTRACT Improvements in the translational value of preclinical models can allow more-successful and more-focused research on shortening the duration of tuberculosis treatment. Although the hollow-fiber infection model (HFIM) is considered a valuable addition to the drug development pipeline, its exact role has not been fully determined yet. Since the strategy of increasing the dose of rifamycins is being evaluated for its treatment-shortening potential, additional in vitro modeling is important. Therefore, we assessed increased dosing of rifampin and rifapentine in our HFIM in order to gain more insight into the place of the HFIM in the drug development pipeline. Total and free-fraction concentrations corresponding to daily dosing of 2.7, 10, and 50 mg of rifampin/kg of body weight, as well as 600 mg and 1,500 mg rifapentine, were assessed in our HFIM using the Mycobacterium tuberculosis H37Rv strain. Drug activity and the emergence of drug resistance were assessed by CFU counting and subsequent mathematical modeling over 14 days, and pharmacokinetic exposures were checked. We found that increasing rifampin exposure above what is expected with the standard dose did not result in higher antimycobacterial activity. For rifapentine, only the highest concentration showed increased activity, but the clinical relevance of this observation is questionable. Moreover, for both drugs, the emergence of resistance was unrelated to exposure. In conclusion, in the simplest experimental setup, the results of the HFIM did not fully correspond to preexisting clinical data. The inclusion of additional parameters and readouts in this preclinical model could be of interest for proper assessment of the translational value of the HFIM.