Background: Obesity is a chronic endocrine-metabolic disorder. The risk of comorbidities increases with a higher body mass index (BMI), particularly when BMI ≥ 35.0 kg/m2. Common complications include insulin resistance, type 2 diabetes, dyslipidemia, and chronic low-grade inflammation, which collectively impair DNA stability by promoting the formation of genotoxic species. Methods: This non-randomised, quasi-experimental clinical intervention study included 53 participants (both sexes) with a BMI ≥ 35.0 kg/m2, who were assigned to parallel experimental or control streams based on clinical needs and institutional eligibility. During a three-week intervention, the experimental group received a hospital-supervised very-low-calorie diet (VLCD; ~600 kcal/day) under continuous medical monitoring. Conversely, the control group followed a standard reduced-calorie diet (SRD) of 1500 kcal/day in a free-living home environment. Before and after the intervention, primary, oxidative, and permanent DNA damage were measured using alkaline, FPG-modified comet (peripheral blood mononuclear cells), and cytokinesis-block micronucleus cytome assays (phytohaemagglutinin-stimulated binucleated lymphocytes), alongside anthropometric and biochemical tracking. Results: Within-group evaluations revealed that both dietary regimens improved several metabolic health indicators, notably modulating insulin resistance, lipid profiles, and leukocyte counts. However, participants in the VLCD stream experienced significantly greater downward changes in body weight, BMI, and absolute lipid values. Crucially, the VLCD intervention was associated with a highly significant within-group reduction in parameters of permanent chromosomal damage, effectively halving the frequencies of micronuclei and nuclear buds, independent of baseline variations, in adjusted multivariate regression models. Conversely, the home-based SRD regimen demonstrated no measurable impact on permanent genomic damage. Neither diet induced a significant change in repairable primary or oxidative DNA lesions over this short timeframe. Conclusions: These exploratory findings suggest that strict calorie restriction can rapidly stabilise genome stability in advanced clinical settings, warranting future randomised controlled trials with long-term longitudinal follow-up to assess permanent risk reductions. Due to structural baseline variations in age, chronic comorbidities, and compliance environments between the cohorts, direct comparative superiority cannot be definitively established.

Although obesity with its comorbidities is linked with higher cancer risk, the data on genome stability in the obese/severely obese are scarce. This is the first study with three DNA damage assessment assays (Fpg-modified and alkaline comet assays and micronucleus cytome assay) performed on a severely obese population (n = 53) where the results were compared with daily intake of food groups, nutrient intake, dietary inflammatory index (DII), and anthropometric and biochemical parameters usually measured in obese individuals. Results demonstrated the association between DNA damage levels and a decrease in cell proliferation with anthropometric measurements and the severity of obese status, together with elevated levels of urates, inorganic phosphates, chlorides, and hs troponin I levels. DII was connected with oxidative DNA damage, while BMI and basal metabolic rate (BMR) were associated with a decrease in cell proliferation and DNA damage creation. Measured daily BMR and calculated daily energy intake from the food frequency questionnaire (FFQ) demonstrated no significant difference (1792.80 vs. 1869.86 kcal day−1 mean values). Groups with higher DNA damage than expected (tail intensity in comet assay >9% and >12.4%, micronucleus frequency >13), consumed daily, weekly, and monthly more often some type of food groups, but differences did not show a clear influence on the elevated DNA damage levels. Combination of all three DNA damage assays demonstrated that some type of damage can start earlier in the obese individual lifespan, such as nuclear buds and nucleoplasmic bridges, then comes decrease in cell proliferation and then elevated micronucleus frequencies, and that primary DNA damage is not maybe crucial in the overweight, but in severely obese. Biochemically changed parameters pointed out that obesity can have an impact on changes in blood cell counts and division and also on genomic instability. Assays were able to demonstrate groups of sensitive individuals that should be further monitored for genomic instability and cancer prevention, especially when obesity is already connected with comorbidities, 13 different cancers, and a higher mortality risk with 7–10 disease-free years loss. In the future, both DNA damage and biochemical parameters should be combined with anthropometric ones for further obese monitoring, better insight into biological changes in the severely obese, and a more individual approach in therapy and treatment. Patients should also get a proper education about the foodstuff with pro- and anti-inflammatory effect.

Background & aims:Severe obesity and its comorbidities relate to increased genomic instability/cancer risk. Obesity in Croatia is rapidly increasing, and long diets are sometimes the reason for obese to quit health improvement programs. A shorter diet with more strict calorie reduction could also lead to weight reduction and health improvements, but data are scarce. We tested for the first time if a very low-calorie diet (VLCD) can improve anthropometric, biochemical and genomic stability parameters in severely obese with BMI > 35 kg m-2. Methods:22 participants were chosen among those regularly attending the hospital for obesity control, with no other previous treatment for bodyweight reduction. Under 24 h medical surveillance, patients received 3-weeks-567-kcal-hospital-controlled-VLCD composed of 50-60% complex carbohydrates, 20 -25% proteins, and 25-30% fat, with the attention to food carbo-glycemic index, in 3 meals freshly prepared in hospital. We analyzed changes in body weight, BMI, basal metabolism rate, waist-hip ratio, visceral fat level, body fat mass, percent body fat, skeletal muscle mass, basal metabolism, energy intake, lipid profile, thyroid hormones, TSH, and genomic instability (alkaline and oxidative FPG comet assay) before and on the last VLCD day. Results:Diet caused BMI reduction (in average 3-4 BMI units' loss), excessive weight loss (between 10 and 35%), significant weight loss (average 9 kg, range 4.8-14.4 kg) and a significant decrease in glucose, insulin, urea, cholesterol, HDL-c, LDL-c, oxidative (FPG) and DNA damage (alkaline comet assay) levels. Conclusions:The diet can lead to >10% excessive weight loss, significant health, and genomic stability improvement, and keep severely obese interest in maintaining healthy habits. The study was registered at ClinicalTrials.gov as NCT05007171 (10.08.2021). (c) 2022 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

Although a very-low-calorie diet (VLCD) is considered safe and has demonstrated benefits among other types of diets, data are scarce concerning its effects on improving health and weight loss in severely obese patients. As part of the personalized weight loss program developed at the Duga Resa Special Hospital for Extended Treatment, Croatia, we evaluated anthropometric, biochemical, and permanent DNA damage parameters (assessed with the cytochalasin B-blocked micronucleus cytome assay—CBMN) in severely obese patients (BMI ≥ 35 kg m−2) after 3-weeks on a 567 kcal, hospital-controlled VLCD. This is the first study on the permanent genomic (in)stability in such VLCD patients. VLCDs caused significant decreases in weight (loss), parameters of the lipid profile, urea, insulin resistance, and reduced glutathione (GSH). Genomic instability parameters were lowered by half, reaching reference values usually found in the healthy population. A correlation was found between GSH decrease and reduced DNA damage. VLCDs revealed susceptible individuals with remaining higher DNA damage for further monitoring. In a highly heterogeneous group (class II and III in obesity, differences in weight, BMI, and other categories) consisting of 26 obese patients, the approach demonstrated its usefulness and benefits in health improvement, enabling an individual approach to further monitoring, diagnosis, treatment, and risk assessment based on changing anthropometric/biochemical VLCD parameters, and CBMN results.

Using alkaline comet assay, DNA damage tail length (TL) and tail intensity (TI) parameters were compared between fresh whole blood and 1-year frozen small volume whole blood in EDTA at -80 °C without cryo-preservation. The studied group consisted of 25 volunteers with different health conditions who served as their own controls for frozen blood results. Without the purification step after thawing, the results and the usefulness of this protocol for future/retrospective (including re-analysations of putative outliers) studies were analysed. Medical surveillance and blood sampling were done at Merkur University Hospital Zagreb. No significant differences between fresh and frozen blood samples in terms of the mean TL values (mean ± SD: 29.03 ± 12.26 vs. 25.36 ± 6.97, respectively) and the mean TI values (9.19 ± 10.37 vs. 10.17 ± 8.55, respectively), and highly damaged cell percentage were determined among 25 volunteers. Median TI frozen samples values of entire group were within the allowed 10-11% (8.24). At the individual levels, no correlation between fresh and frozen whole blood samples was observed in 11 volunteers who suffered from diabetes mellitus type 2. Strong correlation between fresh/frozen samples was seen for TL (r = 0.64, p < 0.015) and TI (r = 0.71, p < 0.005) in nondiabetic subgroup. Overall, the results demonstrated the usefulness of the 1-year frozen blood without induction of heavily damaged DNA. Due to the different DNA damage behaviour connected with different health conditions, future studies should involve more volunteers, oxidative DNA damage comet assay measurements, the inclusion of a washing step after thawing and inclusion of disease/antioxidant biomarkers.

In patients with resistant hypertension (RH) we investigated the importance of urinary neutrophil gelatinase-associated lipocalin (uNGAL- a chemiluminescent microparticle immunoassay (CMIA) method became using (Abbott Diagnostics) for the measurement of NGAL in urine samples) and incidence of chronic kidney disease using the Modification of Diet in Renal Disease Study (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations in estimating glomerular filtration rate (eGFR) based on standardised serum creatinine method traceable to isotope dilution mass spectrometry (IDMS) method. It would have been difficult to predict that levels of these biomarker would perform better organ damage than traditional measurements of kidney function such as standardised serum creatinine, MDRD, or CKD-EPI equations in special population such as RH. Serum creatinine concentrations were measured in 50 patients (24M:26F from RH Registar in Clinical Hospital Merkur) by the kinetic Jaffe method. There were no significant differences between the GFR values derived by MDRD and CKD-EPI equations in the group of patients with RH. 62% of patients have eGFR > 60 mL/minl/1.73 m2, while a 38% of patients have eGFR < 60 mL/min/1.73 m2. The measurement of NGAL in urine samples of 40 patients with RH showed no difference and seems to be of no use in further determination of renal impairement. Higher value of uNGAL in some resistant hypertension patients could have link in the repair stage after AKI and would reveal pathways that could link AKI and CKD.

Introduction The aim of the study was to present a protocol for laboratory information system (LIS) and hospital information system (HIS) validation at the Institute of Clinical Chemistry and Laboratory Medicine of the Merkur University Hospital, Zagreb, Croatia. Materials and methods: Validity of data traceability was checked by entering all test requests for virtual patient into HIS/LIS and printing corresponding barcoded labels that provided laboratory analyzers with the information on requested tests. The original printouts of the test results from laboratory analyzer(s) were compared with the data obtained from LIS and entered into the provided template. Transfer of data from LIS to HIS was examined by requesting all tests in HIS and creating real data in a finding generated in LIS. Data obtained from LIS and HIS were entered into a corresponding template. The main outcome measure was the accuracy of transfer obtained from laboratory analyzers and results transferred from LIS and HIS expressed as percentage (%). Results: The accuracy of data transfer from laboratory analyzers to LIS was 99.5% and of that from LIS to HIS 100%. Conclusion: We presented our established validation protocol for laboratory information system and demonstrated that a system meets its intended purpose.

Background. Liver transplantation is accepted treatment of choice for many liver diseases. Long-term survival is limited by toxicity of immunosuppresive agents, sub-clinical as well as cronic rejection. Newer classes of immunosuppresive agents, including calcineurin inhibitors CNI (cyclosporine and tacrolimus) and mammalian target of rapamycin (mTOR) inhibitors (sirolimus and everolimus) have potential to improve long-term outcomes. Many long-term survivors face a considerable risk of renal dysfunction due to CNI. The aim of the study was to detrmine benefits of sirolimus compared to CNI towards kidneys toxicity. Materials and methods. We have monitored nine orthotopic liver transplantation patients (OLT) who underwent conversion of immunosuppresive regiment from CNIs to sirolimus. Creatinine concentrations were measured with the creatinine enzymatic assay. Measured concentrations of serum creatinine were used to estimate renal function, after dosing CNIs and converting to sirolimus, each one, three and six months after the dose. Results. Reffering to the long-term outcomes of OLT patients following results could be seen: among all patients switched to sirolimus, the levels of serum creatinine (80, 3 umol/L+/-12, 8 vs. 76, 0 umol/L+/-12, 7) remained stable in three of them, whereas the levels of serum creatinine started to decrease (119, 4 umol/L+/- 29, 8 vs. 83, 2 umol/L+/- 26, 3) after administration of sirolimus in six of the patients. Conclusion. These preliminary results have shown that sirolimus is effective in preventing rejection in OLT recipients and it is associated with improved renal function. mTOR inhibitors might have a role as an early alternative to CNIs in patients with CNI nephrotoxicity.

Although transplantation of solid organs has become a more standardized method of treatment, liver transplantation represents an exceptional multidisciplinary clinical procedure requiring understanding of specific pathophysiological changes that occur in the end stage of liver disease. Liver transplantation has been performed at Merkur University Hospital since 1998, with 360 transplantations performed to date. The most common indications are alcohol liver disease, cirrhosis caused by hepatitis B and C virus, hepatocellular carcinoma and cryptogenetic liver cirrhosis. Laboratory tests required for liver transplantation are performed at Department of Clinical Chemistry, Merkur University Hospital, accredited according to ISO 15189 in 2007 for the areas of clinical chemistry, laboratory hematology and coagulation, laboratory immunology-cell immunophenotyping, and molecular diagnosis. The complexity of liver transplant patients requires constant interaction between the anesthesiologist team and clinical laboratory, which has to ensure fast and efficient intraoperative monitoring of biochemical and liver profile: electrolytes and acid-base status, complete blood count, coagulation profile and monitoring of graft function according to the individual patient's health status. Dynamics of intraoperative changes is measured in whole arterial blood samples on a Nova Biomedical Stat Profile Critical Care Xpress mobile acid-base analyzer. Frequent monitoring of ionized calcium and magnesium levels is very important because of citrated blood transfusion and for appropriate therapeutic procedure. During anhepatic stage, there is a progressive increase in lactate level concentration. After reperfusion, a rapid increase in lactate clearance is an excellent indicator of stable graft initial function and its adequate size. During the transplantation procedure, there is usually a biphasic acid-base disturbance characterized by metabolic acidosis and then by metabolic alkalosis. The loss of base equivalents starts during the dissection stage and accelerates during the anhepatic stage. Fast and efficient intraoperative monitoring of hematological tests and coagulation status is of great help in detecting the cause of possible hemorrhage and consequential complications during transplantation procedure. The possibility of organ and tissue transplantation mostly depends on well regulated international cooperation in the areas of donating, transplanting and exchange of required organs and tissues, while laboratory test results must be comparable regardless of their geographical area, methodology employed or analytical equipment used, which is mainly warranted through accreditation according to the international ISO 15189 standard.

Background. The introduction of accreditation according to ISO 15189 standard in medical biochemistry laboratories in Croatia is of high priority in order to guarantee continuous quality improvement as well as the harmonization and standardization of laboratory diagnostics. The Institute of Clinical Chemistry, Merkur University Hospital, Zagreb is one of the first two laboratories accredited in Croatia. Methods. Evaluation of the impact of an electronic information system in providing safe and effective patient identification as well as in optimizing the efficiency of the entire laboratory process through the implementation of the new Laboratory Information System-LIS (BioNET application-LabNet, Josipovac, Croatia). Results. Our data showed that the application of LIS greatly contributed to the optimization of the efficiency of the whole laboratory process and significantly decrease identification errors through: application of the automatic system for patient identification and specimen labelling (bar coding) using two or more patient identifiers (name, birth date, identification number), increasing the automation of the analytical phase - work in „query-host“ with automated data entry including patient result verification, introducing computer- based medical records thus enabling critical results to reach physicians rapidly for improved clinical outcomes, applying Delta Check technology to evaluate laboratory results, improving test turnaround time, enabling health care planning and development of comprehensive electronic patient records. Conclusions. Application of electronic information system improve the overall quality and efficiency, prevent patient indentification errors and enables clinical laboratory to meet requirements according ISO 15189 standard for assessing the highest quality goals throughout the total testing process.