Milka Marinova

The vitamin K cycle.

Vitamin K is a collective term for lipid-like naphthoquinone derivatives synthesized only in eubacteria and plants and functioning as electron carriers in energy transduction pathways and as free radical scavengers maintaining intracellular redox homeostasis. Paradoxically, vitamin K is a required micronutrient in animals for protein posttranslational modification of some glutamate side chains to gamma-carboxyglutamate. The majority of gamma-carboxylated proteins function in blood coagulation. Vitamin K shuttles reducing equivalents as electrons between two enzymes: VKORC1, which is itself reduced by an unknown ER lumenal reductant in order to reduce vitamin K epoxide (K>O) to the quinone form (KH2); and gamma-glutamyl carboxylase, which catalyzes posttranslational gamma-carboxylation and oxidizes KH2 to K>O. This article reviews vitamin K synthesis and the vitamin K cycle, outlines physiological roles of various vitamin K-dependent, gamma-carboxylated proteins, and summarizes the current understanding of clinical phenotypes caused by genetic mutations affecting both enzymes of the vitamin K cycle.

Human Vitamin K 2,3-Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Mediates Vitamin K-dependent Intracellular Antioxidant Function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: Km (μm) = 4.15 (vitamin K1 epoxide) and 11.24 (vitamin K2 epoxide); Vmax (nmol·mg−1·hr−1) = 2.57 (vitamin K1 epoxide) and 13.46 (vitamin K2 epoxide). Oxidative stress induced by H2O2 applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K1 and K2 but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K1. Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.

4beta-hydroxycholesterol as a marker of CYP3A4 inhibition in vivo - effects of itraconazole in man.

OBJECTIVE Itraconazole, a triazole antifungal agent, has been demonstrated to act as an inhibitor of the ligand induced pregnane X receptor-mediated transcriptional regulation of the CYP3A4 gene. Here, we study the potential endogenous serum marker of CYP3A4 activity, 4beta-hydroxycholesterol, during therapy with itraconazole. PATIENTS AND METHODS 8 male patients with onychomycosis received two 1-week cycles of treatment with 400 mg itraconazole once daily in an open, prospective exploratory trial. Fasting serum samples were taken at the beginning and at the end of each cycle. The levels of cholesterol were measured using gas chromatography-flame ionization detection, while cholesterol and bile acid precursors were quantified by gas chromatography-mass spectrometry. RESULTS Total cholesterol decreased by 10% (p < 0.0005) during the itraconazole treatment. Concentrations of the cholesterol precursor lanosterol and 24, 25-dihydrolanosterol increased 10- and 240-fold, respectively (p < 0.001 for both). Interestingly, the ratio of serum lathosterol to cholesterol, an indicator of endogenous cholesterol synthesis downstream from lanosterol, remained unchanged. Absolute and cholesterol-corrected concentrations of 4beta-hydroxycholesterol, formed by CYP3A4-mediated oxidation, decreased significantly during both cycles, on average by 29.1% (p = 0.0006) and 20.8% (p = 0.0062), respectively. The brain-specific cholesterol metabolite 24S-hydroxycholesterol as well as its ratio to cholesterol increased by 19.7% (p = 0.0422) and 34.9% (p = 0.0013), respectively, while the concentrations of the other bile acid precursors, 7alpha-hydroxycholesterol and 27-hydroxycholesterol, remained unchanged. CONCLUSIONS In conclusion, 4beta-hydroxycholesterol appears to be a sensitive endogenous surrogate marker in human serum for inhibition of CYP3A4 by itraconazole.

A systematic review on the role of vitamins, minerals, proteins, and other supplements for the treatment of cachexia in cancer: a European Palliative Care Research Centre cachexia project

We provide a systematic review to support the European Palliative Care Research Collaboration development of clinical guidelines for cancer patients suffering from cachexia. CENTRAL, MEDLINE, PsycINFO, ClinicalTrials.gov, and a selection of cancer journals have been searched up until 15 April 2016. The systematic literature research yielded 4214 publications with 21 of these included in the final evaluation. Regarding minerals, our search identified only one study examining the use of magnesium with no effect on weight loss. As far as vitamins are concerned, vitamin E in combination with omega‐3 fatty acids displayed an effect on survival in a single study, vitamin D showed improvement of muscle weakness in prostate cancer patients, and vitamin C supplementation led to an improvement of various quality of life aspects in a sample with a variety of cancer diagnoses. For proteins, a combination therapy of β‐hydroxy‐β‐methylbutyrate (HMB), arginine, and glutamine showed an increase in lean body mass after 4 weeks in a study of advanced solid tumour patients, whereas the same combination did not show a benefit on lean body mass in a large sample of advanced lung and other cancer patients after 8 weeks. L‐carnitine led to an increase of body mass index and an increase in overall survival in advanced pancreatic cancer patients. Adverse effects of food supplementation were rare and showed mild intensity. There is not enough solid evidence for the use of minerals, vitamins, proteins, or other supplements in cancer. No serious adverse effects have been reported with dietary supplementation.

Prediction of phenprocoumon maintenance dose and phenprocoumon plasma concentration by genetic and non-genetic parameters

PurposeThe anticoagulation response to vitamin K antagonists is characterised by high inter-individual variability. The impact of single nucleotide polymorphisms (SNPs) in several genes of enzymes involved in the vitamin K cycle on phenprocoumon dose variability and phenprocoumon plasma concentrations is still under investigation.MethodsWe assessed the influence of VKORC1 c.-1639G>A, CYP2C9*2, CYP2C9*3, CYP4F2 c.1297G>A, CALU c.*4A>G, EPHX1 c.337T>C, GGCX c.214+597G>A, F7 c.-402G>A, F7 c.-401G>T, PROC c.-228C>T and PROC c.-215G>A along with clinical and demographic parameters on steady-state phenprocoumon therapy in 75 patients. A prediction model was developed for total phenprocoumon plasma concentrations and daily phenprocoumon doses required for therapeutic anticoagulation.ResultsThe VKORC1 c.-1639 genotype was the main predictor of the phenprocoumon daily dose (adjusted R2 = 37.6%) and the total phenprocoumon concentration (adjusted R2 = 38.3%). CYP2C9 affected the phenprocoumon concentration, but not the dose requirements. SNPs in the other genes of the vitamin K cycle, concomitant medication, nicotine use and alcohol consumption did not predict phenprocoumon concentrations and phenprocoumon dose requirements in a multiple linear regression model. Phenprocoumon concentrations were predicted by VKORC1 c.-1639, CYP2C9 genotype, age and BMI. The final prediction model for the daily phenprocoumon dose requirements comprised VKORC1 c.-1639 genotype, age and height accounting for 48.6% of the inter-individual variability.ConclusionsA rough prediction of phenprocoumon maintenance doses can be achieved by a limited set of parameters (VKORC1, age, height). The investigated SNPs in CYP4F2, CALU, EPHX1, GGCX, F7, and PROC did not improve the predictive value of a pharmacogenetic-based dosing equation for phenprocoumon.

Use of routine thoracic and abdominal computed tomography scans for assessing bone mineral density and detecting osteoporosis

Abstract Objective: Evaluation of computed tomography (CT) attenuation measurements for assessing bone mineral density (BMD) and predicting osteoporosis in thoracic and abdominal CT scans for various clinical indications using dual-energy X-ray absorptiometry (DXA) as reference standard. Research design and methods: A total of 234 patients (147 women, 87 men) undergoing DXA and CT were examined retrospectively. Mean time between both studies was 0.5 years. CT-attenuation values in Hounsfield units (HU) were measured at the thoracic and lumbar spine (T1, T6, T12, L1–L5), at the femoral neck, and then assigned to their corresponding DXA scores. Results: Patients with DXA-defined osteoporosis or osteopenia showed significantly lower HU values of trabecular bone at all measured levels compared to healthy subjects (p < 0.001). HU values were highest at T1 and T6, lowest at L1–L3 and the femoral neck. There were no significant intraindividual differences between HU values in the sagittal, coronal or transversal plane. Significant differences between normal and abnormal BMD categories were verified for three CT scanners. More than half of all fractures were detected in patients with non-osteoporotic DXA T-scores. Conclusions: Abdominal and particularly thoracic CT scans obtained for other clinical indications can sensibly be applied toward determining low BMD, detecting osteoporosis and identifying persons at increased fracture risk. Osteoporotic morbidity and mortality might be minimized. Superiorly to DXA, fragility fractures can be found without additional imaging or radiation exposure which can initiate early adequate treatment. Limitations: Key limitations of the study were as following: a retrospective, single-center study; small patient cohort – larger cohorts are needed to evaluate the sensitivity and specificity of diagnostic performance measurements; more complex CT evaluation of the hip for BMD assessment; DXA measurements were used as a reference standard, however, patients with unsuspected compression fractures but showing osteopenic or even normal BMD outline the limitations of DXA.

A Validated HPLC Method for the Determination of Vitamin K in Human Serum – First Application in a Pharmacological Study

Dietary phylloquinone (vitamin K1) is considered to be a major determinant of human vitamin K status. For this reason, measurements of plasma vitamin K concentrations provide a useful tool of vitamin K status in man. There is a growing interest in the role, biochemical function, and metabolism of vitamin K in vivo. A modified reversed phase-HPLC method with fluorescence detection after post-column zinc reduction in serum samples was validated for vitamin K analysis. Two human volunteers were investigated after p.o. and i.v. administration of 2 mg Konakion ® MM. Blood samples were collected periodically after administration and subjected to pharmacokinetic evaluation. High sensitivity, analytical recoveries, accuracy and calibration curves linearities could be reached. Our results showed an overall coefficient of variation of less than 10% for the intraand interday reproducibility, while the recovery ranged from 91% to 114 %. The limit of detection and quantification were 0.015 ng mL -1 and 0.15 ng mL -1 , respectively. Long-term stability was verified over a period of six months. The accuracy was proven by good results from external quality assurance. The observed pharmacokinetic differences indicate significant intraand inter-individual variance of vitamin K fate in the human body. This highly accurate, robust and reliable method is appropriate for the evaluation of vitamin K status in human serum. The ability to determine vitamin K has the potential to improve pharmacokinetic studies.

Polymorphisms in VKORC1 and GGCX are not major genetic determinants of vitamin K-dependent coagulation factor activity in Western Germans

Polymorphisms in VKORC1 and GGCX are not major genetic determinants of vitamin K-dependent coagulation factor activity in Western Germans -

Clinical Use of High-Intensity Focused Ultrasound (HIFU) for Tumor and Pain Reduction in Advanced Pancreatic Cancer.

PURPOSE Evaluation of ultrasound-guided high-intensity focused ultrasound (HIFU) used for the first time in Germany in patients with inoperable pancreatic cancer for reduction of tumor volume and relief of tumor-associated pain. MATERIALS AND METHODS 15 patients with locally advanced inoperable pancreatic cancer and tumor-related pain symptoms were treated by HIFU (n = 6 UICC stage III, n = 9 UICC stage IV). 13 patients underwent simultaneous standard chemotherapy. Ablation was performed using the JC HIFU system (Chongqing, China HAIFU Company) with an ultrasonic device for real-time imaging. Imaging follow-up (US, CT, MRI) and clinical assessment using validated questionnaires (NRS, BPI) was performed before and up to 15 months after HIFU. RESULTS Despite biliary or duodenal stents (4/15) and encasement of visceral vessels (15/15), HIFU treatment was performed successfully in all patients. Treatment time and sonication time were 111 min and 1103 s, respectively. The applied total energy was 386 768 J. After HIFU ablation, contrast-enhanced imaging showed devascularization of treated tumor regions with a significant average volume reduction of 63.8 % after 3 months. Considerable pain relief was achieved in 12 patients after HIFU (complete or partial pain reduction in 6 patients). CONCLUSION US-guided HIFU with a suitable acoustic pathway can be used for local tumor control and relief of tumor-associated pain in patients with locally advanced pancreatic cancer. KEY POINTS • US-guided HIFU allows an additive treatment of unresectable pancreatic cancer.• HIFU can be used for tumor volume reduction.• Using HIFU, a significant reduction of cancer-related pain was achieved.• HIFU provides clinical benefit in patients with pancreatic cancer. Citation Format: • Strunk HM, Henseler J, Rauch M et al. Clinical Use of High-Intensity Focused Ultrasound (HIFU) for Tumor and Pain Reduction in Advanced Pancreatic Cancer. Fortschr Röntgenstr 2016; 188: 662 - 670.

Clinical Effectiveness and Potential Survival Benefit of US-Guided High-Intensity Focused Ultrasound Therapy in Patients with Advanced-Stage Pancreatic Cancer

PURPOSE  Pancreatic cancer (PaC) is a life-limiting tumor with a wide range of incapacitating symptoms such as cancer pain in more than 80 % of patients. This prospective interventional study addresses the clinical effectiveness of ultrasound-guided high-intensity focused ultrasound (HIFU) treatment for patients with advanced-stage PaC, including pain perception, tumor size and survival benefit. MATERIALS AND METHODS  50 patients with late-stage PaC underwent HIFU. Clinical assessment included evaluation of tumor volume by imaging and pain burden (pain severity, pain sensation, interference with daily activities) using the Brief Pain Inventory at baseline and follow-up. Median overall survival, progression-free survival and time to local progression were estimated using Kaplan-Meier analysis. RESULTS  In 84 % of patients, significant early relief of cancer-induced abdominal pain was achieved by HIFU independent of metastatic status; it persisted during follow-up. Tumor volume reduction was 37.8 ± 18.1 % after 6 weeks and 57.9 ± 25.9 % after 6 months. 21 % of HIFU-treated patients had local tumor progression with a median time of 14.4 months from intervention. The median overall survival and progression-free survival were 16.2 and 16.9 months from diagnosis and 8.3 and 6.8 months from intervention. CONCLUSION  In patients with advanced pancreatic cancer and otherwise limited treatment options, HIFU resulted in significant early and long-lasting pain relief and tumor size reduction over time independent of metastatic status. Clinical data suggest an additional potential survival benefit.