Measurement of vitamin K and its derivatives in human plasma through enzyme-assisted liquid chromatography-tandem mass spectrometry assay.

Vitamin K is an essential lipophilic vitamin that acts as a coenzyme in several metabolic pathways. Accurate measurement of apolar metabolites transported by lipoproteins in serum matrices requires high-recovery extractions of vitamin K and its derivatives following standardized protocols. Conventionally developed methods in this field have predominantly employed solid-phase extraction for the measurement of vitamin K and its derivatives. In this study, our objective was to develop an enzyme-assisted extraction method for the precise measurement of vitamin K and its derivatives. Our methodology involved mixing 450 µL of serum samples with 50 µL of an internal standard and 50 µL of a lipase enzyme solution. Following vortexing, the mixture was incubated at 37°C for 15 min to activate the enzymes. The enzyme reaction was subsequently quenched with a mixture of 250 µL of methanol and 1 mL of hexane, followed by centrifugation at 12,000 g for 5 min. The upper phase was collected, concentrated using a concentrator device, and dissolved in a 100 µL solution of methanol/acetone/isopropanol (7:1:1, v/v/v) for analysis. Spectrum analysis was performed using the open-source MZmine 3 software, and a reference interval was established using the Python programming language on the Google Colab platform. The developed method for measuring vitamin K and its derivatives exhibited limit of detection and limit of quantitation values of 0.005 and 0.01 ng/mL, respectively. In conclusion, our study presents a precise and reliable method for the measurement of vitamin K and its derivatives using enzyme-assisted extraction.

Mass spectrometry-based untargeted metabolomics study of non-obese individuals with non-alcoholic fatty liver disease.

OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is a disease characterized by the accumulation of excessive fat in the liver, which can lead to fibrosis and has an increasing prevalence. NAFLD requires non-invasive diagnostic biomarkers. While typically observed in overweight individuals, it can also occur in non-obese/non-overweight individuals. Comparative studies on non-obese NAFLD patients are scarce. This study aimed to conduct a using liquid chromatography-high resolution mass spectrometry (LC-MS/MS)-based metabolic profiling of non-obese NAFLD patients and healthy controls. MATERIALS AND METHODS: The patient group consisted of 27 individuals with NAFLD, while the healthy control group included 39 individuals. Both groups were between 18 and 40 years old, had a BMI of less than 25 and had alcohol consumption less than 20 g/week for men and 10 g/week for women. Serum samples were collected and analyzed using LC-MS/MS. The data were analyzed using the TidyMass and MetaboAnalyst. RESULTS: The LC-MS/MS analyses detected significant changes in D-amino acid metabolism, vitamin B6 metabolism, apoptosis, mTOR signaling pathway, lysine degradation, and phenylalanine metabolism pathways in non-obese NAFLD patients. Significant changes were also observed in the metabolites D-pantothenic acid, hypoxanthine, citric acid, citramalic acid, L-phenylalanine, glutamine, and histamine-trifluoromethyl-toluidide, ß-hydroxymyristic acid, DL-Lactic acid, and 3-methyl-2-oxopentanoic. Overall, the study provides valuable insights into the metabolic changes associated with non-obese NAFLD patients and can contribute to the development of non-invasive diagnostic biomarkers for NAFLD. CONCLUSIONS: This study sheds light on the metabolic changes in non-obese NAFLD patients. Further research is needed to better understand the metabolic changes associated with NAFLD and to develop effective treatment options.

Investigation of serum enzyme activities and oxidative stress markers in preeclampsia: a multiparameter analysis.

OBJECTIVES: Preeclampsia, a high cause of fetomaternal morbidity-mortality, remains a significant burden affecting 8% of all pregnancies. Environmental conditions induce disease development leading to endothelial dysfunction in genetically predisposed women. Our aim is to discuss oxidative stress as a well-established contributing factor to disease progression with being the first study to show new evidence about serum dehydrogenase enzyme levels (isocitrate, malate, glutamate dehydrogenase) with oxidative markers (myeloperoxidase, total antioxidant-oxidant status, oxidative stress index). MATERIAL AND METHODS: Serum parameters were analyzed with photometric method (Abbott ARCHITECT c8000). RESULTS: The enzyme levels and oxidative markers were significantly higher in patients, supporting the redox imbalance in preeclampsia. According to ROC analysis, malate dehydrogenase showed an outstanding diagnostic ability with the highest AUC value of 0.9 and the cut-off value of 51.2 IU/L. Discriminant analysis including malate, isocitrate and glutamate dehydrogenase had predicted preeclampsia with an overall 87.9% accuracy. CONCLUSIONS: Considering the above results, we propose that the enzyme levels increase with oxidative stress functioning as antioxidant defense factors. The unique finding of the study is that the serum levels of malate, isocitrate and glutamate dehydrogenase can be used both separately and combined in the early prediction of preeclampsia. As a novel approach, we also offer combining serum isocitrate and glutamate dehydrogenase levels with ALT, AST tests to state liver functions more reliably in patients. Still, larger sample-sized studies investigating enzyme expression levels are required to confirm the recent findings and to reveal underlying mechanisms.

Maternal Serum Levels of Zinc, Copper, and Thiols in Preeclampsia Patients: a Case-Control Study.

Preeclampsia is one of the leading causes of maternal mortality-morbidity, and environmental factors act as the main driving force for the development of disease in genetically lean women. Trace element levels (zinc, copper) and thiol state (total, native thiol) may affect involved risk factors and play a role in the pathogenesis. The objective of our study is to assess trace element and thiol levels in patient and control groups. A total number of 88 pregnant women (in their third trimester) included 43 preeclampsia patients and 45 normotensive pregnant women as controls. The main findings of this study were the significantly elevated copper levels and decreased thiol levels (native and total thiols) in the patient group compared to controls (p < 0.05). Disulfide levels were not statistically different between the groups (p > 0.05). In patients, the predictive cutoff value of copper was 224 µg/dL and was 1.19 for the copper/native thiol ratio. Zinc levels were not statistically different between the two groups. Correlation analysis revealed no relationship between zinc-copper and zinc-total thiol levels in patients, while a positive correlation was evident in controls (zinc-copper, p < 0.05, r = 0.425, and zinc-total thiol levels, p < 0.05, r = 0.642). Patients had marginally high ALT and AST values in the normal range, and a significant difference was found between the two groups (p < 0.05). According to these results, elevated copper levels and decreased thiol levels may have a value for early prediction. The mechanisms that may be responsible for the altered element and thiol status have been discussed here in the context of oxidative stress.

Effect of Camellia sinensis, Hypericum perforatum and Urtica dioica on kidney and liver injury induced by carbon tetrachloride in rats.

This study is aimed to investigate the effects of Camellia sinensis (CS), Hypericum perforatum (HP) and Urtica dioica (UD) in kidney and liver injury induced by carbon tetrachloride (CCl4) in rats. Highly toxic CCl4 which is used as a solvent in industry comprises experimental toxicity in rats and is widely used in hepatotoxicity and other tissue injury models. The purpose of this investigation is to monitor blood and various tissues by biochemical and histopathological analysis for preventive effects of CS, HP and UD on oxidative stress induced by administration of CCl4 and to enlighten the probable mechanism. Fifty eight rats were divided into five groups; sham group (Group 1, untreated animals), control CCl4 treated group (Group 2), HP extract-treated group (Group 3), UD extract-treated group (Group 4), CS extract-treated group (Group 5). All rats were anaesthetized at the end of the experiment and the blood was collected from each rat. Afterwards, tissue specimens were obtained. The tissue specimens were immersed in 10% formaldehyde for 24 hours. After routine tissue processing, the liver, kidney and stomach were sectioned in 5µm thickness, stained in hematoxylin and eosin. The histological study was performed by using light microscope. The serum marker enzymes were found to be significantly increased in CCl4-induced liver and kidney damage when compared with the sham group (p<0.05). However, treatment with CS, HP, and UD extracts resulted in decreased activity of serum enzymes. Malondialdehyde (MDA) levels were decreased by 20.51±0.95, 27.98±1.58, and 32.39±3.1 nmol/g wet weight protein in kidney homogenates and 16.65±1.75, 17.22±0.71 and 18.92±71 nmol/g wet weight protein in liver homogenates in CS, HP and UD treated groups, respectively. Our results have shown that additive antioxidants like CS, HP and UD will aid in diminishing these deviations in cases of liver and kidney dysfunction.

Effects of intra-articular administration of autologous bone marrow aspirate on healing of full-thickness meniscal tear: an experimental study on sheep.

OBJECTIVE: The aim of this study was to evaluate the effects of bone marrow-derived mesenchymal stem cell and bone marrow elements on the healing of meniscal tears. METHODS: This study was performed on twelve, 2-year-old male Tahirova sheep. In each subject, one knee was used for experiment purposes and the other knee was used as a control. After creating a longitudinal full-thickness tear in the red-white zone of the medial meniscus, aspirated autologous bone marrow material was injected into the tear site in the experiment group. The control group received no intervention for secondary healing. RESULTS: In the macroscopic evaluation of meniscus, a bridging reparation tissue and adhesion were observed between the rims of the tear in the experiment group. There was no statistical difference in collagen fibril formation between the groups (p=0.16). There was significantly more neovascularization in the experiment group than the control group (p=0.003). The cell count was also a significantly higher in the experiment group (p=0.004) and formation of cartilage plaques was more frequent in the experiment group (p=0.016). There was no evidence suggesting intrinsic repair in the meniscus of control group by light and electron microscopy. CONCLUSION: An injection of bone marrow into the meniscus tear site improves healing in a meniscal tear model as demonstrated by both light and electron microscopic findings.