Association of the trimethylamine N-oxide with cardiovascular risk and vascular alterations in middle-aged patients with risk factors for cardiovascular diseases.

BACKGROUND: Trimethylamine N-oxide (TMAO) is synthesized by the intestinal microbiota and is an independent predictor of cardiovascular disease (CVD). However, its underlying mechanisms remain unclear. We investigated TMAO levels across different CVD-risk patient groups, and evaluated associations between TMAO and vascular alterations (e.g., arterial stiffness, intima-media thickness [IMT], and the presence and grade of carotid artery plaques [CAPs]). METHODS: We examined 95 patients (58.5 ± 7.3 years): 40 with clinical atherosclerotic cardiovascular disease (ASCVD), 40 with atherosclerosis risk factors (RF), and 15 controls. Arterial stiffness was measured by Carotid-Femoral Pulse Wave Velocity (C-F PWV). B-mode ultrasound was used to evaluate the presence and grade of CAPs and carotid IMT (CIMT). TMAO was measured by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and results were presented as the median (interquartile range). RESULTS: TMAO levels were higher in patients with ASCVD (251.5 [164.5] µg/l) when compared with patients with RFs (194.0 [174] µg/l, P=0.04) and controls (122.0 (77) µg/l, P<0.001). A significant correlation was observed between TMAO and PWV (r = 0.31, P=0.003), which was not confirmed after adjustment for RFs. TMAO levels were significantly correlated with plaque score (r = 0.46, P<0.001) and plaque height (r=0.41, P=0.003), and were independent predictors for grade III plaques (odds ratio [OR] = 1.002, confidence interval (CI) 95%: 1.000047-1.003, P=0.044). CONCLUSIONS: TMAO levels are increased with expanded CVD risk. Across different types of vascular damage, TMAO is associated with atherosclerotic changes.

Serum and Urinary Biomarkers in COVID-19 Patients with or without Baseline Chronic Kidney Disease.

In a prospective, observational, non-interventional, single-center study, we assessed various plasma and urinary biomarkers of kidney injury (neutrophil gelatinase-associated Lipocain [NGAL], kidney-injury molecule-1 [KIM-1], and interleukin-18 [IL-18]); inflammation (IL-6, C-reactive protein [CRP]); plus angiotensin converting enzyme 2 (ACE2) in 120 COVID-19 patients (of whom 70 had chronic kidney disease (CKD) at emergency-department (ED) admission). Our aim was to correlate the biomarkers with the outcomes (death, acute kidney injury [AKI]). All patients had received a chest-CT scan at admission to calculate the severity score (0-5). Biomarkers were also assessed in healthy volunteers and non-COVID-19-CKD patients. These biomarkers statistically differed across subgroups, i.e., they were significantly increased in COVID-19 patients, except for urinary (u)KIM1 and uIL-18. Amongst the biomarkers, only IL-6 was independently associated with mortality, along with AKI and not using remdesivir. Regarding the prediction of AKI, only IL-6 and uKIM1 were significantly elevated in patients presenting with AKI. However, AKI could not be predicted. Having high baseline IL-6 levels was associated with subsequent ventilation requirement and death. The mortality rate was almost 90% when the chest CT-scan severity score was 3 or 4 vs. 6.8% when the severity score was 0-2 (p < 0.0001).

IL-6 and SAA-Strong Predictors for the Outcome in COVID-19 CKD Patients.

In this prospective study, we assessed biomarkers of inflammation (IL-6 and SAA) from the serum of 120 COVID-19 patients, of whom 70 had chronic kidney disease. All the samples were taken at emergency-department (ED) admission. Our goal was to relate the biomarkers to the results of death and acute kidney injury. All the patients underwent chest computer tomography to estimate the severity score (0-5), which was performed at hospital admission. Finally, biomarkers were also evaluated in a healthy control group and in non-COVID-19-CKD patients. IL-6 and SAA were statistically different between the subgroups, i.e., they were significantly increased in patients with COVID-19. Both of the biomarkers (IL-6 and SAA) were independently associated with mortality, AKI and a higher grade of pathological changes in the lung's parenchyma. Both high baseline levels of IL-6 and SAA on hospital admission were highly correlated with a later ventilatory requirement and mortality, independent of hospital stay. Mortality was found to be significantly higher when the chest CT severity score was 3-4, compared with a severity score of 0-2 (p < 0.0001). Conclusions: at the admission stage, IL-6 and SAA are useful markers for COVID-19 patients with CKD.

Frequency Distribution of CYP2C9 and VKORC1 Mutations among Bulgarian Patients and their Importance for Anticoagulant Therapy.

BACKGROUND: Genetic polymorphisms of CYP2C9 and VKORC1 play a major role in pharmacokinetics and pharmacodynamics of coumarin anticoagulants. The purpose of our study was to assess the relative frequency of the above mutations in Bulgarian population in order to predict bleeding tendencies and precisely manage the anticoagulant therapy during the postoperative period after cardiac surgery with extracorporeal circulation. METHODS: Genomic DNA samples from 200 Bulgarian patients subjected to cardiac surgery with extracorporeal circulation were analyzed for VKORC1 1639G>A and CYP2C9*2&*3 polymorphisms by real-time polymerase chain reaction (PCR), then allele frequencies of various genotypes were calculated by Hardy-Weinberg Equilibrium. RESULTS: Median patients' age was 63.9 ± 10.8 years; 66.5% were male. Median BMI was 28.6 ± 5.4 kg/m2. Genotype distribution for CYP2C9 was *1/*1 - 51%, *1/*2 - 21%, *1/*3 - 13.5%, *2/*3 - 4%, *3/*3 - 2%, and *2/*2 - 1.5%. The calculated frequency of CYP2C9*1 allele was 74.25%, CYP2C9*2 allele was 13%, and CYP2C9*3 allele was 12.75%, and all allelic frequencies were in Hardy-Weinberg equilibrium (p-value = 0.358). The major VKORC1 genotype was G/A - 47%, followed by G/G - 35.5% and A/A - 17.5%). Based on Hardy-Weinberg Equilibrium, there was no significant difference between observed and expected frequencies (X - -3.779), presumably as a result of the homogeneity in the population. CONCLUSIONS: Analysis of the data obtained in the course of the study suggested that identification of homozygous carriers of VKORC1-1639 G>A (rs9923231) in Bulgarians may be useful in developing recommendations for personalized therapy. On the contrary, homozygous carriers of CYP2C9*2 or *3, included only 4.5% of the studied patients, thus indicating that this group would benefit less from dosing algorithms. Our results demonstrated good agreement with the results obtained in other studies conducted in the Caucasian population.

Renin-angiotensin-aldosterone system blockers in Bulgarian COVID-19 patients with or without chronic kidney disease.

When angiotensin-converting enzyme inhibitor/angiotensin receptor blocker-treated patients present with SARS-CoV-2 infection there is a debate to know whether renin-angiotensin-aldosterone (RAAS) blockers should be stopped or not. We conducted a prospective observational study in Bulgarian COVID-19-infected patients with or without chronic kidney disease (CKD) to assess whether maintenance RAAS blocker therapy has an impact on SARS-CoV-2 infection and its complications. We included 120 in-patient COVID-19 subjects, of whom 70 had CKD and 50 had normal renal function. A total of 30% of the patients (total number of 36 patients, 21 females) were receiving RAAS therapy at admission and it was maintained throughout hospitalization. The overall mortality was 19.2% (23 patients); there was no significant difference across the 2 groups (P-value = .21), except in RAAS blockers-treated hypertensive patients who had a significantly lower mortality as compared to non-RAAS-blockers-treated hypertensive patients (P = .04). Regarding subsequent intensive-care unit admission, there were 50% less patients in the RAAS group (4 out of 36, i.e., 11%) as compared to 19 out of 84 from the non-RAAS group, that is, 22.6% (P = .29). Overall, 37 patients developed acute kidney injury (any stage by KDIGO); of them 14 (37.8%) were receiving RAAS blockers. Acute kidney injury was not significantly associated with the use of RAAS blockers (P-value = .28). Likewise, both in non-CKD and in CKD patients the use of RAAS blockers did not have an impact on renal function recovery after SARS-CoV-2 infection. Finally, regarding RAAS blockers and the biological parameters outcome only D-dimers were significantly lower at the follow-up as compared to that in non-RAAS blocker treated patients. RAAS blockers benefited patients with hypertension by lowering mortality rate. Other than that, RAAS blocker therapy continuation during SARS-CoV-2 infection in CKD and non-CKD patients had no significant impact upon major outcomes.

Development and validation of an LC-MS/MS method for determination of 8-iso-prostaglandin f2 Alpha in human saliva.

Background: Increased formation of reactive oxygen species may be caused by the ion release of the metal alloys used in prosthetic dental restorations due to the corrosion process. As products of lipid peroxidation, isoprostanes can be used as a marker for oxidative stress in the body. There are two significant advantages of using isoprostanes as an oxidative stress marker - presence in all fluids in the body and low reactivity. Saliva provides noninvasive, painless, and cost-effective sample collection and can be used as an alternative testing medium of blood and urine. Methods: This study presents the development and validation of a sample LC-MS/MS method to quantify 8-isoprostaglandin F2-a in human saliva using salt-out assisted liquid-liquid extraction (SALLE). Results: The selected sample preparation procedure optimized chromatographic separation and mass detection provided high recovery and sensitivity of the analysis. The calibration curve was obtained in the predefined range 25-329 ng/L with R2 larger than 0.995. Normalized matrix varied between 89.7 % and 113.5%. The method showed sufficient accuracy and precision - accuracy in the range 89.7 %-113.9 %, and precision between 2.3% and 5.4%. Conclusions: The proposed method is validated according to current EMA/FDA industrial guidance for bioanalysis and offers an appropriate level of sensitivity and sufficient accuracy and precision.

COVID-19 Infection in Chronic Kidney Disease Patients in Bulgaria: Risk Factors for Death and Acute Kidney Injury.

Regarding COVID-19 infection, Bulgaria has one of the lowest rates of vaccination in Europe, and its COVID-19-related mortality rate has been one of the highest in the European Union. Chronic kidney disease (CKD)-COVID-19 patients are at higher risk of developing acute kidney injury (AKI) and death after hospital admission. This single-center prospective cohort study from Bulgaria included 120 in-patient COVID-19 subjects of whom 70 had CKD and 50 normal renal function. Diabetes mellitus, hypertension, obesity, and cardiovascular disease were statistically more prevalent in the CKD group as compared to the non-CKD group. At admission, D-dimer, creatinine, and urea levels were significantly higher in the CKD group, whereas estimated glomerular-filtration rate was significantly lower as compared to the non-CKD patients. During hospitalization, 23 patients (19.1%) died, of which 19 were in the CKD group (p-value = 0.0096); in addition, 38 developed AKI (31.6%), of which 31 were in the CKD group (p-value = 0.0006). Using binary logistic regression, being male, having experienced AKI, and not having been treated with remdesivir were independent risk factors for COVID-19-induced mortality. Regarding risk of AKI, having had COVID-19-related symptoms for more than 6 days before admission, having CKD at baseline, and having not received remdesivir therapy were independent predictive factors for developing AKI after admission.

Evaluation of automated hematology analyzer DYMIND DH76 compared to SYSMEX XN 1000 system.

BACKGROUND: DYMIND DH76 (DYMIND BIOTECH, China) is a new automated hematology system designed to provide CBC count, including a 5-part WBC differential count, and its analytical performance should be assessed before adoption for clinical use. METHODS: The analyzer was evaluated according to the International Council for Standardization in Haematology guideline. The purposes of this study were to assess its analytical performance in comparison to SYSMEX XN 1000 hematology analyzer currently used in our laboratory, as well as to compare the automated and manual WBC differential. RESULTS: Within-run precision in all concentration ranges was very good with coefficients of variation (CVs) between 0.02% and 2.5% except for platelets over 500×109/L (CV 9.5%). Within-batch imprecision showed CVs lower the declared deviation ranges. Accuracy (defined as trueness) was excellent for all CBC and white cell differential parameters, compared with the state of the art%. Linearity was confirmed with excellent regression coefficients (0.999-1.000), even in the lowest values, and carryover was ≤ 1%. Comparison between DYMIND DH76 and SYSMEX XN 1000 was also very good with correlation coefficients (R2) for WBC (1.000), RBC (0.999), hemoglobin (0.999) and PLT over 50×109/L (0.994) and R2 was lower but still acceptable (0.910) for PLT<50×109/L. R2 for neutrophils, lymphocytes, eosinophils, basophils, and monocytes were 0.974, 0.982, 0.957, 0.625, and 0.836, respectively, in the comparison between the manual and DYMIND DH76 automated differential WBC counts. CONCLUSIONS: With excellent analytical performance and acceptable comparative analysis, DYMIND DH76 hematology analyser covered the predefined international standards and requirements and is fully appropriate for clinical application.

A proposed Common Training Framework for Specialists in Laboratory Medicine under EU Directive 2013/55/EC (The Recognition of Professional Qualifications).

European Union (EU) Directive 2013/55/EC (The Recognition of Professional Qualifications) allows Member States to decide on a common set of minimum knowledge, skills and competences that are needed to pursue a given profession through a Common Training Framework. To be adopted the framework must combine the knowledge, skills and competences of at least one third of the Member States. Professionals who have gained their qualifications under a Common Training Framework will be able to have these recognised automatically within the Union. The backbone of the European Federation of Clinical Chemistry and Laboratory Medicine's (EFLM) proposed Common Training Framework for non-medical Specialists in Laboratory Medicine is outlined here. It is based on an Equivalence of Standards in education, training, qualifications, knowledge, skills, competences and the professional conduct associated with specialist practice. In proposing the recognition of specialist practice EFLM has identified 15 EU Member States able to meet Equivalence and in whom the profession and/or its training is regulated (an additional EU Commission requirement). The framework supports and contributes to the Directive's enabling goals for increasing professional mobility, safeguarding consumers and ensuring a more equitable distribution of skills and expertise across the Member States. It represents EFLM's position statement and provides a template for professional societies and/or competent authorities to engage with the EU Commission.

Phase separation liquid-liquid extraction for the quantification of 8-iso-Prostaglandin F2 Alpha in human plasma by LC-MS/MS.

BACKGROUND: Reactive oxygen species (ROS) are produced in the body during normal metabolism by means of enzymes and non-enzymatic chemical reduction of molecular oxygen. In case of the prevalence of ROS formation over their elimination, highly reactive free radicals can be accumulated and can cause multiple damages to the biomolecules and cells. Determination of isoprostanes in biological matrices is most often used to register free radical damage and requires selective, sensitive and specific techniques. METHODS: This study presents the development and validation of the LC-MS/MS method for the determination of 8-iso-Prostaglandin F2α in human plasma utilising a modified liquid-liquid extraction procedure with phase separation. RESULTS: Modified sample preparation procedure assured higher extraction yield, clear separation of organic layer from the plasma water phase and protein precipitates, and better-purified product for instrumental analysis. Linearity was validated in the range 0.1-5.0 µg/L with R2 > 0.996; normalised matrix varied between 86.0% and 108.3%, accuracy ranged from 90.4 % to 113.9% and precision both within runs and between runs was less than 7%. With a run time of 10 min, a throughput of over 50 samples per working day could be performed. CONCLUSIONS: The method meets all the current industrial validation criteria and allows the accurate and precise determination of 8-iso-PGF2α in human plasma at diagnostically significant concentration range.

Blood-Brain Barrier and Intrathecal Immune Response in patients with neuroinfections.

Cerebrospinal fluid/serum albumin ratio is one of the most informative parameters for blood-brain barrier (BBB) integrity in cases of central nervous system (CNS) infectious diseases. Normally, CNS albumin concentration is a function of diffusion processes along with CSF drainage and resorption. In pathological processes CSF albumin levels are dependent only on the rate of CSF drainage resulting in non-linear reciprocal changes of albumin quotient (Qalb). IgG, IgA and IgM concentrations both in CSF and serum can be compared to Qalb, thus determining the intrathecal immune response. The aim of the study was to detect BBB permeability impairment and the intrathecal immune response in patients with CNS infections with various etiologies. CSF/serum ratios were calculated and related to IgG IgA and IgM concentrations in CSF and blood serum. The results were integrated and presented by Reibergrams. The results demonstrated typical patterns which prove albumin to be the main modulator of protein dynamics and at the same time explicates the complex pathophysiological mechanisms involved in BBB disruption and intrathecal immune response in CNS infections. The diagnostic model presented in our study seeks to explain the observations of meningitis and meningoencephalitis pathophysiology and points out the mandatory cooperation between clinicians and laboratory for accurate diagnosis and proper treatment.

High-Throughput HPLC-MS/MS Method for Quantification of Ibuprofen Enantiomers in Human Plasma: Focus on Investigation of Metabolite Interference.

In this research, as a part of the development of fast and reliable HPLC-MS/MS method for quantification of ibuprofen (IBP) enantiomers in human plasma, the possibility of IBP acylglucoronide (IBP-Glu) back-conversion was assessed. This involved investigation of in source and in vitro back-conversion. The separation of IBP enantiomers, its metabolite and rac-IBP-d3 (internal standard), was achieved within 6 min using Chiracel OJ-RH chromatographic column (150 × 2.1 mm, 5 µm). The followed selected reaction monitoring transitions for IBP-Glu (m/z 381.4 → 205.4, m/z 381.4 → 161.4 and m/z 205.4 → 161.4) implied that under the optimized electrospray ionization parameters, in source back-conversion of IBP-Glu was insignificant. The results obtained after liquid-liquid extraction of plasma samples spiked with IBP-Glu revealed that the amount of IBP enantiomers generated by IBP-Glu back-conversion was far <20% of lower limit of quantification sample. These results indicate that the presence of IBP-Glu in real samples will not affect the quantification of the IBP enantiomers; thereby reliability of the method was improved. Additional advantage of the method is the short analysis time making it suitable for the large number of samples. The method was fully validated according to the EMA guideline and was shown to meet all requirements to be applied in a pharmacokinetic study.

Higher 25-Hydroxyvitamin D Levels Are Associated With Lower Proteinuria in Kidney Transplant Recipients.

OBJECTIVES: Proteinuria is associated with decreased graft and patient survival after kidney transplantation. Increasing evidence shows that vitamin D has antiproteinuric and renoprotective effects. The aim of our study was to assess the influence of 25-hydroxyvitamin D levels on proteinuria after kidney transplantation. MATERIAL AND METHODS: Between May 1, 2012, and November 30, 2012, we tested 395 kidney transplant recipients for 25-hydroxyvitamin D levels during their regular visits to our transplant center together with routine blood sampling and proteinuria testing. Patients within 12 months of transplant, who had undergone parathyroidectomy, had unstable graft function, had concomitant intake of calcineurin inhibitors and mammalian target of rapamycin inhibitors were not included in the study. Subjects with advanced liver disease, or receiving vitamin D supplementation were also excluded. All laboratory, clinical, and therapeutic factors for proteinuria were taken into consideration. Statistical analyses included descriptive statistics and univariate and multivariate log-log regression with backward selection (SPSS version 22.0; SPSS Inc., Chicago, IL, USA), with significance at P < .05. Determination of total 25-hydroxyvitamin D levels was performed by a validated liquid chromatography-tandem mass spectrometry method. RESULTS: Our study group included 230 patients (148 men, 82 women). Positive association was established between proteinuria and history of diabetes mellitus, rejection episode 12 months within testing for 25-hydroxyvitamin D levels, and use of mammalian target of rapamycin inhibitors (P < .05). Significant negative relations were detected for patient age, graft function, and 25-hydroxyvitamin D concentrations (P < .05). CONCLUSIONS: Our study established that better vitamin D status is associated with lower proteinuria. However, further research is needed to clarify the possible renoprotective properties of vitamin D.

Differences in the prevalence of vitamin D deficiency and hip fractures in nursing home residents and independently living elderly

ABSTRACT Objective To compare the prevalence of vitamin D deficiency and fracture history in nursing home residents and community-dwelling elderly subjects and to explore the association of vitamin D levels with various characteristics. Materials and methods Sixty-six nursing home residents and 139 community-dwelling elderly subjects participated. Marital status, medical history, medication including vitamin D supplements, smoking, past fractures were assessed. Weight and height were measured and body mass index calculated. Serum 25-hydroxyvitamin D (25-OHD), PTH, Ca, phosphate, creatinine and eGFR were determined. Results In the nursing home residents 25-OHD was lower (17.8 nmol/l, [9.4-28.6] vs. 36.7 nmol/l, [26.9-50], p < 0.001), PTH was higher (5.6 pmol/l, [3.9-8.9] vs. 4.7 pmol/l [3.6-5.8], P = 0.003) and 25-OHD deficiency was more prevalent (65.2% [53.7-76.7] vs. 22.3% [15.4-29.2], p < 0.001) as was elevated PTH (23% [12.8-33] vs. 5.8% [2-10], p = 0.001). 25-OHD correlated negatively with PTH (institutionalized r = -0.28, p = 0.025 and community-dwelling r = -0.36, p < 0.001). Hip fractures were reported by 8% of the residents and 2% of the independent elderly. The only predictor for hip fracture was elevated PTH (OR = 7.6 (1.5-36.9), p = 0.013). Conclusion The prevalence of vitamin D deficiency and secondary hyperparathyroidism was high in the institutionalized subjects. Hip fracture risk was associated with elevated PTH and not directly with vitamin D levels or the residency status.

Differences in the prevalence of vitamin D deficiency and hip fractures in nursing home residents and independently living elderly.

OBJECTIVE: To compare the prevalence of vitamin D deficiency and fracture history in nursing home residents and community-dwelling elderly subjects and to explore the association of vitamin D levels with various characteristics. MATERIALS AND METHODS: Sixty-six nursing home residents and 139 community-dwelling elderly subjects participated. Marital status, medical history, medication including vitamin D supplements, smoking, past fractures were assessed. Weight and height were measured and body mass index calculated. Serum 25-hydroxyvitamin D (25-OHD), PTH, Ca, phosphate, creatinine and eGFR were determined. RESULTS: In the nursing home residents 25-OHD was lower (17.8 nmol/l, [9.4-28.6] vs. 36.7 nmol/l, [26.9-50], p < 0.001), PTH was higher (5.6 pmol/l, [3.9-8.9] vs. 4.7 pmol/l [3.6-5.8], P = 0.003) and 25-OHD deficiency was more prevalent (65.2% [53.7-76.7] vs. 22.3% [15.4-29.2], p < 0.001) as was elevated PTH (23% [12.8-33] vs. 5.8% [2-10], p = 0.001). 25-OHD correlated negatively with PTH (institutionalized r = -0.28, p = 0.025 and community-dwelling r = -0.36, p < 0.001). Hip fractures were reported by 8% of the residents and 2% of the independent elderly. The only predictor for hip fracture was elevated PTH (OR = 7.6 (1.5-36.9), p = 0.013). CONCLUSION: The prevalence of vitamin D deficiency and secondary hyperparathyroidism was high in the institutionalized subjects. Hip fracture risk was associated with elevated PTH and not directly with vitamin D levels or the residency status.

Relationship between vitamin D status and immunosuppressive therapy in kidney transplant recipients.

There is a growing body of evidence for the protective role of vitamin D in diabetes mellitus (DM), infection, cancer, cardiovascular disease, immune disorders and kidney function. Considering the reported high prevalence of vitamin D insufficiency among kidney transplant recipients (KTRs), the aim of this study was to assess the influence of immunosuppressive therapy and other factors on vitamin D status in such patients. The study included 289 KTRs (189 males and 100 females) who consented to participate. The first test for 25-hydrohyvitamin D [25(OH)D] was performed by a validated liquid chromatography-tandem mass spectrometry method. Influence of immunosuppressive drugs and previously reported predictors on vitamin D status was assessed by descriptive statistics, univariate and multivariate regression. Our results showed that only 53 patients (18.34%) of the studied KTRs were vitamin D sufficient. In addition to a well expected positive association between serum 25(OH)D and summer blood sampling (p < 0.05) and inverse relationship between vitamin D status and DM, gender (female) and body mass index, serum 25(OH)D was found to be inversely associated with calcineurin inhibitors (CNI) (p < 0.05) and unaffected by other immunosuppressive agents. Our study demonstrated high prevalence of vitamin D insufficiency after kidney transplantation in the studied cohort of patients. Apart from female gender, winter months, DM and overweight, the use of CNI could be considered an additional significant predictor of lower 25(OH)D in Bulgarian KTRs.

Critical development by design of a rugged HPLC-MS/MS method for direct determination of ibuprofen enantiomers in human plasma.

Development and validation of a HPLC-MS/MS method for direct determination of R- and S-ibuprofen (Ibu) in human plasma without a need of derivatization or other complexities such as postcolumn infusion of solvents or reagents was performed. Critical steps were investigated during method development using experimental design to achieve a reliable and rugged assay. The LC-MS/MS separation of R-Ibu and S-Ibu was obtained on Lux Cellulose chiral column utilizing 0.1% (v/v) acetic acid in mixture of methanol and water (90:10%, v/v) as a mobile phase. Two types of extraction procedure for Ibu and Ketoprofen (internal standard, IS) were optimized using Full factorial 3(2) design (LLE) and D-Optimal Experimental Design (SPE). Excellent recovery values, 80% (mean) and 95% (mean) for LLE and SPE respectively, were obtained using 50µL plasma. The matrix effect was assessed for both of the extraction procedures, including hyperlipidaemic and haemolyzed plasma. The extensive investigation of matrix effect showed that LLE yields cleaner extracts than the SPE. The result of the investigation of in vitro interconversion of R-Ibu and S-Ibu showed that it does not occur under the influence of pH, temperature, and in the overall analytical procedure. The validation data, adhered to EMA guideline for validation of bioanalytical methods, showed that the proposed method provides accurate and reproducible results in range of 0.1-50mg/L with a lower limit of detection of 0.02mg/L. The applicability of the method was demonstrated through determination of R-Ibu and S-Ibu in human plasma after oral administration of 400mg rac-Ibu.

Prevalence of vitamin D deficiency and insufficiency in Bulgarian patients with chronic hepatitis C viral infection.

AIMS: The present pilot study aimed to determine vitamin D status in Bulgarian patients with chronic HCV infection in respect to the severity of liver disease and response to interferon-ribavirin therapy. METHODS: The study encompassed 296 patients: 161 males (54.4%) aged 42.08 ± 14.87 years, 135 females (45.6%) aged 45.72 ± 14.34 years, 86.5% of them infected with HCV genotype 1. Total 25-hydroxyvitamin-D (25OHD) was determined by liquid chromatography/tandem-mass spectrometric detection. RESULTS: The median 25OHD level of the studied cohort was 50.40 nmol/L (range: 29.6-71.05). 25OHD deficient (< 25 nmol/L) were 16% of patients, 33% showed profound insufficiency (25-50 nmol/L), another 33% were in the range 50-80 nmol/L (mild insufficiency), the rest 18% were 25OHD sufficient. Significantly lower 25OHD levels were registered in cases with advanced fibrosis compared to those with mild or absent fibrosis (37.10 nmol/L vs. 53.00 nmol/L, respectively, p < 0.05). This association remained unchanged by seasonal variations in 25OHD levels. Inverse relationship was found between 25OHD and HCV-RNA (p < 0.01). Patients with sustained virological response to therapy had significantly higher 25OHD levels, compared to patients who failed to achieve viral eradication (56.90 nmol/L vs. 45.00 nmol/L, p = 0.012). CONCLUSION: More than 80% of HCV-infected patients were vitamin D-deficient and -insufficient. The inverse relationship between 25OHD levels and viral load, liver fibrosis and treatment outcomes supports the hypothesis that improvement of vitamin D status may have considerable potential to amend the host defense against HCV infection and response to therapy.