Beyond the Coagulation Cascade: Vitamin K and Its Multifaceted Impact on Human and Domesticated Animal Health.

Vitamin K (VK) is an essential micronutrient impacting many systems in the body. This lipid-soluble vitamin is found in various plant and animal products and is absorbed via the lymphatic system. This biomolecule's importance to human health includes but is not limited to its promotion of brain, cardiovascular, bone, and immune functions. These biological properties are also necessary for maintaining domesticated animal health. The synergistic impact of both VK and vitamin D (VD) maximizes these health benefits, specifically for the circulatory and skeletal systems. This manuscript reviews VK's properties, molecular structures, nutrikinetics, mechanisms of action, daily requirements, safety in supplemental form, biomarkers used for its detection, and impacts on various organs. The purpose of synthesizing this information is to evaluate the potential uses of VK for the treatment or prevention of diseases.

Sustainable menaquinone-7 production through continuous fermentation in biofilm bioreactors.

Menaquinone-7 (MK-7), a vital vitamin with numerous health benefits, is synthesized and secreted extracellularly by the formation of biofilm, dominantly in Bacillus strains. Our team developed an innovative biofilm reactor utilizing Bacillus subtilis natto cells to foster biofilm growth on plastic composite supports to produce MK-7. Continuous fermentation in biofilm reactors offers a promising strategy for achieving sustainable and efficient production of Menaquinone-7 (MK-7). Unlike conventional batch fermentation, continuous biofilm reactors maintain a steady state of operation, which reduces resource consumption and waste generation, contributing to sustainability. By optimizing fermentation conditions, MK-7 production was significantly enhanced in this study, demonstrating the potential for sustainable industrial-scale production. To determine the optimal operational parameters, various dilution rates were tested. These rates were selected based on their potential to enhance nutrient supply and biofilm stability, thereby improving MK-7 production. By carefully considering the fermentation conditions and systematically varying the dilution rates, MK-7 production was significantly enhanced during continuous fermentation. The MK-7 productivity was found to increase from 0.12 mg/L/h to 0.33 mg/L/h with a dilution rate increment from 0.007 to 0.042 h-1). This range was chosen to explore the impact of various nutrient supply rates on MK-7 production and to identify the optimal conditions for maximizing productivity. However, a further increase in the dilution rate to 0.084 h-1 led to reduced productivity at approximately 0.16 mg/L/h, likely due to insufficient retention time for effective biofilm formation. Consequently, a dilution rate of 0.042 h-1 exhibited the highest productivity of 0.33 mg/L/h, outperforming all investigated dilution rates and demonstrating the critical balance between nutrient supply and retention time in continuous fermentation. These findings validate the feasibility of operating continuous fermentation at a 0.084 h-1 dilution rate, corresponding to a 48 h retention time, to achieve the highest MK-7 productivity compared to conventional batch fermentation. The significant advancements achieved in enhancing Menaquinone-7 (MK-7) productivity through continuous fermentation at optimal dilution rates in the present work indicate promising prospects for even greater efficiency and sustainability in MK-7 production through future developments.

The impact of gut bacteria producing long chain homologs of vitamin K2 on colorectal carcinogenesis.

Colorectal cancer (CRC) is one of the foremost causes of cancer-related deaths. Lately, a close connection between the course of CRC and the intestinal microbiota has been revealed. Vitamin K2 (VK2) is a bacterially derived compound that plays a crucial role in the human body. Its significant anti-cancer properties may result, inter alia, from a quinone ring possessing a specific chemical structure found in many chemotherapeutics. VK2 can be supplied to our body exogenously, i.e., through dietary supplements or fermented food (e.g., yellow cheese, fermented soybeans -Natto), and endogenously, i.e., through the production of bacteria that constantly colonize the human microbiome of the large intestine.This paper focuses on endogenous K2 synthesized by the most active members of the human gut microbiome. This analysis tested 86 intestinally derived bacterial strains, among which the largest VK2 producers (Lactobacillus, Bifidobacterium, Bacillus) were selected. Moreover, based on the chosen VK2-MK4 homolog, the potential of VK2 penetration into Caco-2 cells in an aqueous environment without the coexistence of fats, pancreatic enzymes, or bile salts has been displayed. The influence of three VK2 homologs: VK2-MK4, VK2-MK7 and VK2-MK9 on apoptosis and necrosis of Caco-2 cells was tested proving the lack of their harmful effects on the tested cells. Moreover, the unique role of long-chain homologs (VK2-MK9 and VK2-MK7) in inhibiting the secretion of pro-inflammatory cytokines such as IL-8 (for Caco-2 tissue) and IL-6 and TNFα (for RAW 264.7) has been documented.

Determination of Vitamin K1, MK-4, MK-7, and D Levels in Human Serum of Postmenopausal Osteoporosis Women Based on High Stability LC-MS/MS: MK-7 May Be a New Marker of Bone Metabolism.

INTRODUCTION: Vitamin K (VK) as well as vitamin D (VD) plays an important role in osteoporosis. Vitamin K1 (VK1), vitamin K2 (VK2, menaquinone-4 (MK-4), and menaquinone-7 (MK-7)) are significant for the metabolism of skeletal muscle. 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 (25(OH)D2 and 25(OH)D3) reflect circulating VD levels. More sensitive measurements remain to be developed. In the present study, a new high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of VK1, VK2 (MK-4 and MK-7), as well as 25(OH)D2 and 25(OH)D3 levels in human serum. METHODS: We developed a simple LC-MS/MS method for the determination of VK1, MK-4, MK-7, 25(OH)D2, and 25(OH)D3 levels in human serum and validated the method in a study cohort of 200 patients divided into the premenopausal women group and postmenopausal osteoporosis patient group. RESULTS: The overall precision (coefficient of variation) ranged from 2.66 to 10.11% in the specified working range (0.05-5 ng/mL) for VK1, MK-4, and MK-7. Serum VK1, MK-4, and MK-7 levels in postmenopausal women with osteoporosis were 1.187 ± 0.094 ng/mL, 0.058 ± 0.009 ng/mL, and 0.885 ± 0.064 ng/mL, respectively (mean ± standard deviation). Serum VK1, MK-4, and MK-7 levels in premenopausal women were 1.143 ± 0.103 ng/mL, 0.028 ± 0.003 ng/mL, and 1.553 ± 0.226 ng/mL, respectively. Serum 25(OH)D2 and 25(OH)D3 levels in postmenopausal women with osteoporosis were 0.757 ± 0.056 ng/mL and 11.72 ± 0.632 ng/mL, respectively. Serum 25(OH)D2 and 25(OH)D3 levels in premenopausal were 1.793 ± 0.575 ng/mL and 12.42 ± 1.069 ng/mL, respectively. CONCLUSION: A new LC-MS/MS method for determination of serum VK and VD levels was evaluated and validated. MK-7 in plasma decreased earlier than VD in postmenopausal osteoporosis patients. MK-7 status is significantly associated with osteoporosis and could be considered a predictable biomarker in the diagnosis of osteoporosis in postmenopausal women.

Cis and trans isomers of the vitamin menaquinone-7: which one is biologically significant?

Recently, several studies have indicated that an adequate intake of menaquinone-7 (MK-7) offers numerous health benefits. However, the low availability of MK-7 in the diet necessitates the development of dietary supplements or functional food products to complement natural food sources and meet the daily intake requirements. Like most biological molecules, MK-7 can exist as geometric isomers that can occur in the cis, trans, and cis/trans forms; however, only the all-trans form is biologically significant. MK-7 is traditionally produced through bacterial fermentation, but various synthetic preparations have lately become available. The isomer composition in the final product is influenced by numerous factors, including the methods of production and purification, as well as particular environmental and storage conditions. The MK-7 profile obtained from the various production methods has not yet been elucidated, and the ideal method for the synthesis of the all-trans form of the vitamin is also debatable. Consequently, the quantification of the MK-7 profile of various products is necessary to develop an understanding of the factors that influence the proportion of isomers that are obtained in different preparations. Several possible methods exist for the quantification of MK-7 isomers, and of these, liquid chromatography in conjunction with mass spectrometry techniques appears to be the most promising. Evaluation of the isomer composition is an important consideration, as only the all-trans form sustains biological activity. Furthermore, knowledge of the prominent factors that influence the MK-7 composition may also enable their manipulation to obtain a more favorable MK-7 profile in the final product.

Biofilm reactors as a promising method for vitamin K (menaquinone-7) production.

Menaquinone-7 (MK-7) is the most potent subtype of vitamin K with extraordinarily high half-life in the circulatory system. Therefore, MK-7 plays a critical role in promoting human wellbeing today. Studies on MK-7 every year show more and more magnificent benefits of it in preventing cardiovascular diseases and osteoporosis to battling cancer cells, Alzheimer's and Parkinson's diseases. Thus, it needs to be supplemented to daily diet for accumulative and long-term benefits. Chemical synthesis of MK-7 produces a significant cis-isomer form of it, which has no biological activity. Fortunately, due to its key role in electron transfer in bacteria, trans-MK-7 is biosynthesized by especially Gram-positive strains mainly Bacillus genus. Concordantly, MK-7 could be produced via solid or liquid state fermentation strategies. In either regime, when static fermentation is applied in the absence of agitation and aeration, operational issues arise such as heat and mass transfer inefficiencies. Thus, scaling up the process becomes a challenge. On the other hand, studies have indicated that biofilm and pellicle formation that occur in static fermentations are key characteristics for extracellular MK-7 secretion. Therefore, this review covers the most recent discoveries of the therapeutic properties of MK-7 and optimization attempts at increasing its biosynthesis in different media compositions and effective growth parameters as well as the cutting-edge use of biofilm reactors where B. subtilis cells have the infrastructures to form mature biofilm formations on plastic composite supports. Biofilm reactors therefore can provide robust extracellular MK-7 secretion while simultaneously enduring high agitation and aeration rates, which then address the scale-up and operational issues associated with static fermentation strategies.

Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.

Menaquinone 7 Stability of Formulations and Its Relationship with Purity Profile.

Menaquinone-7 (MK7) is a member of the vitamin K family in which interest has considerably increased over the last decade, mainly due to its beneficial role in human health. MK7 can be produced by synthesis or fermentation, and its purity profile can differ depending on methodologies and extraction procedures. Finished formulations show a high heterogeneity of purity profiles, as well as frequent discrepancies in the nominal content, compared to the actual title. The present study compared purity profiles of different raw material and related them to their stability in normal (12 months/25 °C/60%RH) and accelerated conditions (6 months/40 °C/75% RH) in order to test their performance in the presence of different common excipients. Results showed higher purity profile results in enhanced stability, and this could explain title discrepancies found in finished products, which are present on the market worldwide.

Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.

Determination of vitamins K1 , MK-4, and MK-7 in human serum of postmenopausal women by HPLC with fluorescence detection.

BACKGROUND: New high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K1 and two forms of vitamin K2 (MK-4 and MK-7) in human serum, and the levels of vitamin K were determined in 350 samples of postmenopausal women. METHODS: Vitamin K was determined by HPLC with fluorescence detection after postcolumn zinc reduction. The detection was performed at 246 nm (excitation) and 430 nm (emission). The internal standard and 2 mL of ethanol were added to 500 µL of serum. The mixture was extracted with 4 mL of hexane, and solid phase extraction was then used. RESULTS: The HLPC method was fully validated. The intra- and interday accuracy and precision were evaluated on two QC samples by multiple analysis, and CV were less than 10%. The limit of quantification for MK-4 was found at 0.04 ng/mL, for K1 0.03 ng/mL, and for MK-7 0.03 ng/mL. The mean recoveries of the corresponding compounds were 98%-110%. Serum levels of MK-4, K1 , and MK-7 in postmenopausal women with osteoporosis were 0.890 ± 0.291 ng/mL, 0.433 ± 0.394 ng/mL, and 1.002 ± 1.020 ng/mL, respectively (mean ± SD). Serum levels of MK-4, K1 , and MK-7 in postmenopausal women without osteoporosis were 0.825 ± 0.266 ng/mL, 0.493 ± 0.399 ng/mL, and 1.186 ± 1.076 ng/mL, respectively (mean ± SD). CONCLUSION: New HPLC method for the determination of vitamins K1 , MK-4, and MK-7 in serum was evaluated and validated. This method is highly specific and sensitive with the low limit of quantification.

Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (Menaquinone-7) production in biofilm reactors.

Menaquinone-7 (MK-7) is the key form of vitamin K used as a dietary supplement and its production revolves around Bacillus subtilis natto. Current fermentation strategies, which suggest static fermentations without aeration and agitation, can be problematic for large scale MK-7 production due to biofilm formation. The use of biofilm reactors, therefore, is proposed in the present study, which could utilize both agitation and aeration without interrupting MK-7 secretion. In this study, biofilm reactors were constructed using the selected plastic composite support (PCS) and B. subtilis natto strain for MK-7 production. Using response surface methodology (RSM), optimum growth parameters including temperature, pH, and agitation were determined in a glycerol-based medium. Results were presented in a statistical model (R 2 = 0.90), leading to optimum growth conditions of temperature (35 °C), agitation (200 rpm) and pH (6.58). Model-predicted MK-7 concentration was validated and MK-7 concentration of 12.09 mg/L was produced in the biofilm reactor. The obtained concentration was 58% higher as compared to the suspended-cell culture (7.67 mg/L). The results of this study will provide a critical step towards improved industrial scale production of MK-7.

Roles of Vitamins D and K, Nutrition, and Lifestyle in Low-Energy Bone Fractures in Children and Young Adults.

The research on skeletal system health in children and young adults, while recognizing the important role of calcium and vitamin D, goes beyond these nutritional standards. This review focuses on the role of vitamin K in combination with vitamin D and other factors in bone health. The current understanding is that maintaining bone health and prevention of low-energy fractures in any pediatric population includes nutritional factors combined with an active lifestyle. Calcium, vitamin D, and vitamin K supplementation contribute independently and collectively to bone health. The beneficial role of vitamin K, particularly vitamin K2 as menaquinone-7 (MK-7), in bone and cardiovascular health is reasonably well supported scientifically, with several preclinical, epidemiological, and clinical studies published over the last decade. Osteocalcin and matrix-Gla (glutamate-containing) protein (MGP) exemplify vitamin K-dependent proteins involved in building bone matrix and keeping calcium from accumulating in the arterial walls, respectively. An important part of the mechanism of vitamin K involves carboxylation and posttranslational activation of the family of vitamin K-dependent proteins, which prevent expression of pro-inflammatory factors and support improvement in bone mineral concentration, bone mineral density, and the quality of bone matrix. Understanding the combined approach to a healthy skeletal system in children and young adults, including the roles of vitamins D and K, calcium, healthy diet, and exercise, is particularly important in view of reports of subclinical insufficiency of vitamins D and K in otherwise healthy pediatric populations with low-energy bone fractures.

Bioavailability and Chemical/Functional Aspects of Synthetic MK-7 vs Fermentation-Derived MK-7 in Randomised Controlled Trials.

We investigated the bioavailability of a synthetic form of the vitamin K2 molecule menaquinone-7 (MK-7) in a randomised single-blinded two-way cross-over study. Healthy subjects (20 - 66 years of age) took a single 180 µg dose of synthetic MK-7 or fermentation-derived MK-7, and serum MK-7 concentrations were monitored for 72 hours to calculate AUC(0 - 72 h) and Cmax. We also compared the biological effects of placebo, fermentation-derived MK-7 (90 µg) and 3 doses of synthetic MK-7(45, 90 and 180 µg) in a randomised double-blinded parallel study. Healthy subjects (20 - 60 years of age) took one of the supplements daily for 43 days, and the fraction of carboxylated osteocalcin (OC) was compared between day 1 and day 43 as a marker for vitamin K activity. In the bioavailability study, the 90 % confidence interval for the ratio of the AUC(0-72 h) values for synthetic and fermentationderived MK-7 was 83 - 111, indicating bioequivalence. The 90 % confidence interval for the Cmax ratio was 83 - 131. The serum concentrations of carboxylated OC and undercarboxylated OC were increased (p = 0.01) and reduced (p = 0.02), respectively, after daily intake of 180 µg of synthetic MK-7 for 43 days, indicating increased vitamin K activity. Across both studies, only 1 participant reported an adverse event (dry mouth; 180 µg synthetic MK-7 group, functional study) that was considered possibly related to synthetic MK-7 supplementation. Our findings provide evidence that the tested synthetic form of MK-7 is bioequivalent to fermentation-derived MK-7, exhibits vitamin K activity and is well tolerated in healthy subjects.

Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors.

Menaquinone-7 (MK-7), a subtype of vitamin K, has received a significant attention due to its effect on improving bone and cardiovascular health. Current fermentation strategies, which involve static fermentation without aeration or agitation, are associated with low productivity and scale-up issues and hardly justify the commercial production needs of this vitamin. Previous studies indicate that static fermentation is associated with pellicle and biofilm formations, which are critical for MK-7 secretion while posing significant operational issues. Therefore, the present study is undertaken to evaluate the possibility of using a biofilm reactor as a new strategy for MK-7 fermentation. Bacillus species, namely, Bacillus subtilis natto, Bacillus licheniformis, and Bacillus amyloliquifaciens as well as plastic composite, supports (PCS) were investigated in terms of MK-7 production and biofilm formation. Results show the possibility of using a biofilm reactor for MK-7 biosynthesis. Bacillus subtilis natto and soybean flour yeast extract PCS in glucose medium were found as the most potent combination for production of MK-7 as high as 35.5 mg/L, which includes both intracellular and extracellular MK-7.

Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation.

Production of menaquinone-7 (MK-7) by Bacillus subtilis natto is associated with major drawbacks. To address the current challenges in MK-7 fermentation, studying the effect of magnetic nanoparticles on the bacterial cells can open up a new domain for intensified bioprocesses. This article introduces the new concept of application of iron oxide nanoparticles (IONs) as a pioneer tool for MK-7 process intensification. In this order, IONs with the average size of 11 nm were successfully fabricated and characterized for possible in situ removal of target substances from the fermentation media. The prepared particles were used for decoration and immobilization of B. subtilis natto cells. Presence of iron oxide nanoparticles significantly enhanced the MK-7 specific yield (15 %) as compared to the control samples. In addition, fabricated IONs showed a promising ability for in situ recovery of bacterial cells from the fermentation media with more than 95 % capture efficiency. Based on the results, IONs can be implemented successfully as a novel tool for MK-7 production. This study provides a considerable interest for industrial application of magnetic nanoparticles and their future role in designing an intensified biological process.

Comparative genomics analysis and transposon mutagenesis provides new insights into high menaquinone-7 biosynthetic potential of Bacillus subtilis natto.

This research combined Whole-Genome sequencing, intraspecific comparative genomics and transposon mutagenesis to investigate the menaquinone-7 (MK-7) synthesis potential in Bacillus subtilis natto. First, Whole-Genome sequencing showed that Bacillus subtilis natto BN-P15-11-1 contains one single circular chromosome in size of 3,982,436 bp with a GC content of 43.85 %, harboring 4,053 predicted coding genes. Next, the comparative genomics analysis among strain BN-P15-11-1 with model Bacillus subtilis 168 and four typical Bacillus subtilis natto strains proves that the closer evolutionary relationship Bacillus subtilis natto BN-P15-11-1 and Bacillus subtilis 168 both exhibit strong biosynthetic potential. To further dig for MK-7 biosynthesis latent capacity of BN-P15-11-1, we constructed a mutant library using transposons and a high throughput screening method using microplates. We obtained a YqgQ deficient high MK-7 yield strain F4 with a yield 3.02 times that of the parent strain. Experiments also showed that the high yield mutants had defects in different transcription and translation regulatory factor genes, indicating that regulatory factor defects may affect the biosynthesis and accumulation of MK-7 by altering the overall metabolic level. The findings of this study will provide more novel insights on the precise identification and rational utilization of the Bacillus subtilis subspecies for biosynthesis latent capacity.

Simultaneous Production of MK-7 and Iturin A by Bacillus velezensis ND.

Bacillus velezensis can produce various secondary metabolites, such as the antibacterial compound iturin A and the coagulation-promoting menaquinone-7 (MK-7). To enhance the economic feasibility of the fermentation process, a co-production strategy, involving the simultaneous production of MK-7 and iturin A by Bacillus velezensis ND, was investigated in this study. Firstly, the effects of cultivation temperature and initial pH on the synthesis of MK-7 and iturin A were investigated. Considering the co-production of iturin A and MK-7, the optimal temperature and pH were determined as 32 °C and 7, respectively. Subsequently, important nutrients for the co-production process were investigated. It was observed that glycerol, soybean meal, yeast extract, and L-glutamate had a significant effect on the co-produce process. An optimal medium composed of glycerol (72.19 mL L-1), L-glutamate (1.4 g L-1), yeast extract (16.88 g L-1), and soybean meal (130.95 g L-1) was obtained by response surface methodology (RSM). This co-produce process was further scaled up in a biofilm reactor, and the maximum concentration of MK-7 and iturin A reached 46.88 mg L-1 and 5.58 g L-1, respectively. Finally, we established an effective method for separately extracting the two metabolites from the fermentation broth. The superiority of this co-production fermentation strategy demonstrates its significant potential for industrial production.

Amyloidogenic and Neuroinflammatory Molecular Pathways Are Contrasted Using Menaquinone 4 (MK4) and Reduced Menaquinone 7 (MK7R) in Association with Increased DNA Methylation in SK-N-BE Neuroblastoma Cell Line.

Besides its role in coagulation, vitamin K seems to be involved in various other mechanisms, including inflammation and age-related diseases, also at the level of gene expression. This work examined the roles of two vitamin K2 (menaquinones) vitamers, namely, menaquinone-4 (MK4) and reduced menaquinone-7 (MK7R), as gene modulator compounds, as well as their potential role in the epigenetic regulation of genes involved in amyloidogenesis and neuroinflammation. The SK-N-BE human neuroblastoma cells provided a "first-line" model for screening the neuroinflammatory and neurodegenerative molecular pathways. MK7R, being a new vitamin K form, was first tested in terms of solubilization, uptake and cell viability, together with MK4 as an endogenous control. We assessed the expression of key factors in amyloidogenesis and neuroinflammation, observing that the MK7R treatment was associated with the downregulation of neurodegeneration- (PSEN1 and BACE1) and neuroinflammation- (IL-1ß and IL-6) associated genes, whereas genes retaining protective roles toward amiloidogenesis were upregulated (ADAM10 and ADAM17). By profiling the DNA methylation patterns of genes known to be epigenetically regulated, we observed a correlation between hypermethylation and the downregulation of PSEN1, IL-1ß and IL-6. These results suggest a possible role of MK7R in the treatment of cognitive impairment, giving a possible base for further preclinical experiments in animal models of neurodegenerative disease.

Characterization of K-binding factor involved in water-soluble complex of menaquinone-7 produced by Bacillus subtilis natto.

Vitamin Ks are expected to contribute bone and cardiovascular health. Especially, menaquinone-7 has a higher bioavailability and a longer half-life than other vitamin Ks in the human body. However, their low water-solubility limits their application. On the other hand, Bacillus subtilis natto produces a water-soluble complex, which comprises menaquinone-7 and peptides. The peptide named K-binding factor (KBF) has been reported as the main component of the complex. In the present, the structural characteristics of KBF were studied. Mass spectrometry showed significant peaks at m/z = 1050, while the previous PAGE suggested that molecular weight of KBF was ~ 3k. Amino acid analysis revealed that the 1k peptides were the various combinations of nine amino acids, among which Asx, Glx, Val, Leu and Met were found to be the most abundant. The peptides could serve as detergent properties. The 1k peptides could be isolated by reverse-phase high performance liquid chromatography. The bundle of three 1k detergent-like peptides would participate to the micelle structure containing menqauinone-7 inside. In conclusion, a basic unit of KBF would be the ~ 1k peptides, and the three basic unit assemble to the ~ 3k bundle, then the bundle form a water-soluble micelle including menqauinone-7 inside.

Does Vitamin K2 Influence the Interplay between Diabetes Mellitus and Intervertebral Disc Degeneration in a Rat Model?

Intervertebral disc (IVD) degeneration is a common cause of low back pain in diabetes mellitus type 2 (T2DM) patients. Its pathogenesis and the vitamin (vit.) K2 influence on this disease remain unclear. Lumbar motion segments of male Zucker Diabetes Fatty (ZDF) rats (non-diabetic [control] and diabetic; fed without or with vit. K2) were used. Femur lengths and vertebral epiphyseal cross-section areas were measured. IVDs were histopathologically examined. Protein synthesis and gene expression of isolated IVD fibrochondrocytes were analyzed. T2DM rats showed histopathological IVD degeneration. Femur lengths and epiphyseal areas were smaller in T2DM rats regardless of vit. K2 feeding. Fibrochondrocytes synthesized interleukin (IL)-24 and IL-10 with no major differences between groups. Alpha smooth muscle actin (αSMA) was strongly expressed, especially in cells of vit. K2-treated animals. Gene expression of aggrecan was low, and that of collagen type 2 was high in IVD cells of diabetic animals, whether treated with vit. K2 or not. Suppressor of cytokine signaling (Socs)3 and heme oxygenase (Hmox)1 gene expression was highest in the cells of diabetic animals treated with vit. K2. Vit. K2 influenced the expression of some stress-associated markers in IVD cells of diabetic rats, but not that of IL-10 and IL-24.