Point of care testing in Croatia: a survey of the Working group for point of care testing of the Croatian society of medical biochemistry and laboratory medicine

Graphical abstract Highlights Majority of participants find point of care testing a valuable tool that can enhance patient care Communication between laboratory and clinical personnel should be improved National point of care testing regulations are available but not implemented Education and training are crucial in ensuring high-quality results and patient safety IntroductionThe aim of this study was to investigate attitudes and routine procedures in point of care testing (POCT) among non-laboratory and laboratory healthcare professionals in Croatia. Materials and methodsThe Working Group (WG) for POCT of the Croatian society of medical biochemistry and laboratory medicine has designed two anonymous surveys for laboratory staff and non-laboratory staff with a total of 44 questions/statements on POCT (27 questions for non-laboratory staff and 17 for laboratory staff). Surveys were sent to 184 medical biochemistry laboratory (MBL) managers, the Croatian medical chamber and the Croatian chamber of nurses. The survey was disseminated using the online survey platform SurveyMonkey. ResultsA total of 112 non-laboratory healthcare professionals and 50 laboratories participated in the survey, which represents a response rate of 0.25% for non-laboratory professionals and 27% for MBLs. The majority of non-laboratory staff stated that POCT enables better medical care for the patient (90/112) and that the implementation of new POCT devices should be the responsibility of a POCT team comprising laboratory and clinical healthcare professionals. The great majority of responding MBLs (42/50) acknowledge that POCT is necessary for better patient care, and also realize that validation of POCT devices and comparison to the central laboratory is necessary before implementation (49/50). ConclusionsThe majority of participants consider POCT as a medical tool that enables better patient care but there is still a lack of communication between laboratory and clinical staff. The study identified some critical spots that will help to create national guidelines to ensure high patient safety when using POCT devices

Paraoxonase and arylesterase activity of paraoxonase 1 and oxidative stress parameters in cervical intraepithelial neoplasia

Graphical abstract Highlights Reduced paraoxonase 1 arylesterase activity was observed in cervical intraepithelial neoplasia Malondialdehyde was lower in the cervical intraepithelial neoplasia group Reduced glutathione was higher in cervical intraepithelial neoplasia IntroductionParaoxonase 1 (PON1) is the enzyme that removes carcinogenic radicals from lipids. The aim of the study was to investigate the differences in PON1 activity and oxidation stress parameters between patients with cervical intraepithelial neoplasia (CIN) and healthy controls. Materials and methodsThe study included 65 women with CIN and 109 healthy women. Lipid parameters were determined on Cobas Integra 400 plus (Roche, Mannheim, Germany). Tiols and reduced glutathione (GSH) were determined spectrophotometric using Eliman reagent. Activity of PON1 was assessed with two substrates, paraoxon and phenylacetate by spectrophotometric method. Malondialdehyde (MDA) was determined by high performance liquid chromatography (Shimadzu Corporation, Kyoto, Japan). Mann-Whitney-test, t-test, χ2-test, correlation and logistic regression was used in statistical analysis. P < 0.05 was considered statistically significant. ResultsThe basal (P = 0.929) and NaCl-stimulated (P = 0.985) PON1 activity and activities standardised on the concentration of high-density lipoprotein (HDL; P = 0.076; P = 0.065, respectively) and apolipoprotein AI (apo AI; P = 0.444; P = 0.499, respectively) as well as PON1 phenotypes (P = 0.842) did not differ significantly between the groups. The PON1 arylesterase activity (53±19 kU/L vs. 77±17 kU/L; P < 0.001) and HDL-standardized activity (37 (28-44) kU/mmol vs. 43 (37-50) kU/mmol; P < 0.001) and apoAI (29±11 kU/g vs. 44±11 kU/g; P < 0.001) was significantly reduced in the CIN group. The concentration of the thiol groups was similar (P = 0.519), of MDA was lower (0.39 (0.27-0.55) µmol/L vs. 0.76 (0.57-1.15) µmol/L; P < 0.001) and of GSH was higher (112.0 (66.0-129.6) µg/mL vs. 53.4 (34.8-134.4) µg/mL; P < 0.001) in the CIN group. ConclusionReduced PON1 arylesterase activity, lower MDA and higher GSH concentration were observed in CIN patients

Increased HSP70 and TLR2 mRNA expression and association of HSP70 polymorphism rs6457452 with the risk of chronic obstructive pulmonary disease in the Croatian population

Heat shock protein 70 (Hsp70) engages Toll-like receptors (TLR) 2 and 4 when found in the extracellular compartment and contributes to inflammation in chronic obstructive pulmonary disease (COPD). Since there is growing evidence for the genetic risk factors for COPD, the gene expression of HSP70, TLR2 and TLR4 was determined, as well as the association between HSP70, TLR2 and TLR4 single nucleotide polymorphisms, (SNPs) and COPD. The gene expression was assessed in peripheral blood cells of 137 COPD patients and 95 controls by a quantitative polymerase chain reaction (qPCR), while a total of nine SNPs were genotyped by TaqMan allelic discrimination real-time PCR. HSP70 and TLR2 gene expression was increased in COPD patients compared to the controls, regardless of the disease severity and smoking status of participants. The rs6457452 SNP of HSP70 was associated with COPD, indicating the protective role of the T allele (OR = 0.46, 95% CI = 0.24-0.89, p = 0.022). Furthermore, COPD C/T heterozygotes showed a decreased HSP70 mRNA level compared to COPD C/C homozygotes. In conclusion, HSP70 and TLR2 may have a role in the pathogenesis of COPD, and the HSP70 rs6457452 variant might influence the genetic susceptibility to COPD in the Croatian population

Impact of anticoagulants on assessment of zinc in plasma

The aim of this study was to compare zinc (Zn) level in EDTA, heparin, citrate plasma and serum. The blood samples from the same healthy volunteers (n = 10) were sampled by use of different anticoagulants (EDTA, heparin and citrate) or with no anticoagulant. Zn level was determined by flame atomic-absorption spectrometer (AAnalyst 800, PerkinElmer, USA). Zn level in EDTA, heparin, citrate plasma and serum was in reference range (0.7 to 1.5 μg mL–1), and the difference between sample groups was not observed (P = 0.079). However, correlation was observed only between Zn level in heparin plasma and citrate plasma (rs = 0.72; P = 0.016) and citrate plasma and serum (rs = 0.76; P = 0.012). In conclusion, in Zn blood analysis, sample collection step is important and should be observed when planning study and when comparing the results of Zn level from various studies. This work is licensed under a Creative Commons Attribution 4.0 International License

Association of Plasma Heat Shock Protein 70 with Disease Severity, Smoking and Lung Function of Patients with Chronic Obstructive Pulmonary Disease

Extracellular heat shock protein 70 (eHsp70) might modulate immune responses in chronic obstructive pulmonary disease (COPD). The aim of the study was to explore eHsp70 concentration in stable COPD, its association with disease severity and smoking status as well as its diagnostic performance in COPD assessment. Plasma samples were collected from 137 COPD patients and 95 healthy individuals, and concentration of eHsp70 was assessed by commercially available enzyme-linked immunosorbent assay (ELISA) kit (Enzo Life Science, Farmingdale, NY, USA). COPD patients were subdivided regarding airflow obstruction severity and symptoms severity according to the Global Initiative for COPD (GOLD) guidelines. eHsp70 concentration increased in COPD patients when compared to controls and increased with the severity of airflow limitation as well as symptoms burden and exacerbation history. eHsp70 concentration did not differ among COPD patients based on smoking status, yet it increased in healthy smokers compared to healthy nonsmokers. In addition, eHsp70 negatively correlated with lung function parameters forced expiratory volume in one second (FEV1) and FEV1/ forced vital capacity (FVC), and positively with COPD multicomponent indices BODCAT (BMI, airflow obstruction, dyspnea, CAT score), BODEx (BMI, airflow obstruction, dyspnea, previous exacerbations), CODEx (Charlson&rsquo;s comorbidity index, airflow obstruction, dyspnea, previous exacerbations) and DOSE (dyspnea, airflow obstruction, smoking status, previous exacerbations) With great predictive value (OR = 7.63) obtained from univariate logistic regression, eHsp70 correctly classified 76% of cases. eHsp70 is associated with COPD prediction and disease severity and might have the potential for becoming an additional biomarker in COPD assessment

Valorization of Olive Pomace-Based Nutraceuticals as Antioxidants in Chemical, Food, and Biological Models

Waste remaining after the production of olive oil (olive pomace) is known to contain significant amounts of phenolic compounds that exert different types of biological activities, primarily acting as antioxidants. In this work, a sustainable approach that combines ultrasound-assisted extraction with food-grade solvents and encapsulation with different types of cyclodextrins was used to prepare olive pomace-based polyphenol rich extracts that were tested as antioxidants in various chemical, food, and biological model systems. Encapsulation with cyclodextrins had a significant positive impact on the chemical composition of obtained extracts and it positively affected their antioxidant activity. Observed effects can be explained by an increased content of polyphenols in the formulations, specific physical properties of encapsulated compounds improving their antioxidant activity in complex food/physiological environment, and enhanced interaction with natural substrates. Depending on the applied model, the tested samples showed significant antioxidant protection in the concentration range 0.1–3%. Among the investigated cyclodextrins, hydroxypropyl-β-cyclodextrin and randomly methylated-β-cyclodextrin encapsulated extracts showed particularly good antioxidant activity and were especially potent in oil-in-water emulsion systems (1242 mg/g and 1422 mg/g of Trolox equivalents, respectively), showing significantly higher antioxidant activity than Trolox (reference antioxidant). In other models, they provided antioxidant protection comparable to commonly used synthetic antioxidants at concentration levels of 2–3%

Extracellular Hsp70 modulates 16HBE cells' inflammatory responses to cigarette smoke and bacterial components lipopolysaccharide and lipoteichoic acid

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), leading to chronic inflammation, while bacterial components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are often present in airways of COPD patients, especially during exacerbations. We hypothesised that extracellular heat shock protein 70 (eHsp70), a damage-associated molecular pattern elevated in serum of COPD patients, induces inflammation and alters cigarette smoke and LPS/LTA-induced inflammatory effects in the airway epithelium. We used 16HBE cells exposed to recombinant human (rh)Hsp70 and its combinations with cigarette smoke extract (CSE), LPS or LTA to investigate those assumptions, and we determined pro-inflammatory cytokines’ secretion as well as TLR2 and TLR4 gene expression. rhHsp70 and CSE alone stimulated IL-6, IL-8 and TNF-α secretion. CSE and rhHsp70 had antagonistic effect on IL-6 secretion, while combinations of LPS or LTA with rhHsp70 showed antagonistic effect on TNF-α release. By using specific inhibitors, we demonstrated that effects of rhHsp70 on cytokines’ secretion were mediated via NF-κB and/or MAPK signalling pathways. rhHsp70 increased, and CSE decreased TLR2 gene expression compared to untreated cells, but their combinations increased it compared to CSE alone. LPS and rhHsp70 combinations decreased TLR2 gene expression compared to untreated cells. TLR4 expression was not induced by any of the treatments. In conclusion, we demonstrated that extracellular Hsp70 modulates pro-inflammatory responses of human airway epithelial cells to cigarette smoke and bacterial components LPS and LTA. Simultaneous presence of those compounds and their interactions might lead to inappropriate immune responses and adverse consequences in COPD

Effect of Alpha Lipoic Acid Supplementation on Oxidative Stress and Lipid Parameters in Women Diagnosed with Low-Grade Squamous Intraepithelial Lesions (LSILs): A Double-Blind, Randomized, Placebo-Controlled Trial

Limited scientific evidence shows that alpha lipoic acid (ALA) can induce regression rates of low-grade squamous intraepithelial lesions (LSILs), but the mechanisms of these effects have not been elucidated. To gain a broader insight into its therapeutic potential and mechanisms of action, the effects of 3 months of supplementation with 600 mg of ALA on antioxidant and lipid status parameters in 100 patients with LSILs were investigated in a randomized, placebo-controlled study. The obtained results are discussed in terms of patients’ initial metabolic status and diet quality (particularly nutritional intake of antioxidants). The obtained results showed that oxidative status biomarkers were not significantly affected by ALA supplementation. However, serum superoxide dismutase (SOD) activity was positively affected in the subgroup of patients with higher dietary antioxidant intake. Surprisingly, ALA supplementation resulted in a small but statistically significant increase in serum low density lipoprotein (LDL), and the observed effect was significantly affected by the initial lipid status of the participants. Larger studies are necessary to gain additional insights on the clinical significance of ALA as an antioxidant and hypolipemic agent and to optimize its potential application in LSIL treatment