Physiological and Cellular Functions of Vitamin K on Cardiovascular Function

*Meneerah A. Aljafary, Hussah Alshwyeh, Nada Alahmadi, Adeeb Shehzad, Huseyin Tombuloglu, Zagit Gaymalov, Abdelqader Homieda and Ebtesam Al-Suhaimi*

## **Abstract**

This chapter reviews the physiological and cellular functions of vitamin K in the cardiovascular system based on the latest pre-clinical and clinical evidence. Vitamin K belongs to a family of structurally similar fat-soluble vitamins, actively required by the body for the synthesis of essential proteins as well as regulate blood clotting, bone metabolism and calcium level. The authors emphasize the quintessential association between dietary vitamin K2 and cardiovascular diseases shown in various studies. The association, through the vitamin K - dependent hormones, plays a primary role in regulating calcification of different cell types, especially their role in calcification of the vascular endothelial cells. The consequences of vitamin K deficiency in the vascular system are unfavorable, shown in various clinical studies on statins - well-known inhibitors of vitamin K production in the body. New clinical insights suggest that vitamin K levels in the body and its dietary supplementation play a crucial role in cardiovascular disease prevention. There is negative influence of these antagonist's pate in vascular composition and functions. Therefore, there is a need for prospective studies to make more in-depth exploration and increase the current understanding of this critical relationship to confidently apply such knowledge to prevent cardiovascular diseases and improve their outcomes.

**Keywords:** Vitamin K, hormone, heart, cardiac disease, vascular system, gene expression, statin

## **1. Introduction**

Vitamin K applies to fat-soluble vitamins, which are similar in structure and essential in the blood coagulation process and to control the calcium mineral binding in bones and other tissues. The discovery of vitamin K can be attributed to the observations of a high incidence of bleeding in chickens on a low-lipid diet during the 1930s [1]. Until the 1970s, it was believed that vitamin K was essential exclusively for homeostasis, maintaining an adequate blood supply, and preserving vascular

integrity in animals and humans. Today, we know that vitamin K is involved in gamma-carboxylation as a co-factor in several essential proteins located within the bone, heart, and blood vessels. Moreover, only in the presence of vitamin K, specific essential proteins called vitamin K-dependent proteins (VKDPs) are able to switch from inactive uncarboxylated forms to active carboxylated forms. Vitamin K allows switching VKDPs to their active states by the carboxylation of VKDPs glutamic acid (Glu) residues in specific tissues and organs [2]. VKDPs include seven proteins involved in blood coagulation (coagulation factor II, VII, IX, and X, and anticoagulant proteins C, S, and Z); proteins responsible for bone mineralization (osteocalcin (OC) and matrix gamma-carboxyglutamic acid (Gla)-protein (MGP)); and recently discovered proteins, including growth arrest-specific gene 6 (Gas-6), the transmembrane Gla proteins (TMG3 and TMG4), the proline-rich Gla proteins (PRGP1 and PRGP2), the Gla-rich protein (GRP), periostin and transthyretin [3–7]. In nature, there are two main variants of vitamin K: phylloquinone (or vitamin K1) can be found in some green vegetables, and menaquinone (or vitamin K2) can be found in some meat, fermented milk, and fermented soybean products. Structurally, vitamers differ in their degree of saturation and side-chain lengths; vitamin K2 is more biologically active than vitamin K1 and circulates longer in the body [8]. Cardiovascular diseases (CVDs) comprise a group of disorders affecting the heart and blood vessels that cause conditions such as coronary heart disease (CHD) and cerebrovascular disease, which affect the blood vessels that supply the brain [9]. In recent years, numerous physiological studies have pointed out the critical role of vitamin K as an anti-vascular calcification (VC) factor. VC is recognized as an autonomous and prominent risk factor for CVDs, and several human observational studies have shown a positive correlation between low vitamin K supplementation and VC [10, 11]. Meta-analysis studies showed that patients with regular dietary of vitamin K showed significantly less VC while maintaining vascular stiffness compared with patients with no vitamin K supplementation [12]. In this chapter, we will cover the following topics: the importance of dietary vitamin K; physiological functions of vitamin K beyond blood coagulation; vitamin K-dependent hormones; physiological and protective roles of vitamin K on cardiovascular (CV) processes; cellular and molecular mechanisms of vitamin K in the vascular cells and whether vitamin K promotes or encounters statins.

## **2. The importance of dietary vitamin K in CVD**

Vitamin K is vital for healthy bones and the heart and increases blood clotting. Although a deficiency of vitamin K is not common, its deficiency may affect the body over time. Bleeding and weak bones, as well as higher CV risks, are some consequences of vitamin K deficiency [13, 14]. Hence, vitamin K intake must not be ignored. Usually, the daily value (DV) of 120 mcg of vitamin K is sufficient in adult males and less than this in females and children (**Table 1**) [17, 18]. Vitamin K can be in both forms in our diets, where phylloquinone can be sourced from leafy green vegetables, and menaquinone can be sourced from animal-based food that includes meats, fermented dairy, fermented soybeans, and dietary supplements [19]. Vitamin K2 is also available on the market in the form of synthetic menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in natural or synthesized form. Naturally, in animals, MK-4 is more commonly produced, while MK-7 (as well as MK-5 to MK-14) is made by bacteria. Many animals can convert vitamin K1 to vitamin K2 (MK-4).


*Physiological and Cellular Functions of Vitamin K on Cardiovascular Function DOI: http://dx.doi.org/10.5772/intechopen.99344*

#### **Table 1.**

*Amount (% of daily value) of vitamin K2 in 100 g of animal products that prevent insufficiency in man.*

Vascular health can be improved by ensuring the consumption of sufficient amounts of vitamin K2. Vitamin K2 stimulates MGP, which prevents calcium from depositing inside the vessel walls. When calcium is not deposited in the arteries, it offers dual benefits of clear arteries as well as the availability of calcium for various functions in the human body [20]. Presently, MGP is found to be highly effective for the modulation of arterial calcification. Although MGP binds calcium to protect calcification within blood vessels, it needs to be first activated via an adequate dose of vitamin K2 [20]. A total of 4807 healthy individuals from both genders with ages above 55 were involved in a population-based study conducted in Rotterdam. The study aimed to investigate the impact of dietary intake of vitamin K on calcification within the aorta, CVDs, and all-cause mortality [21]. It was found that the risk of calcification within the arteries and CVDs were reduced by half, while the all-cause mortality risk was reduced by one-quarter as a result of a higher dietary intake of vitamin K2 (minimum daily intake of 32 μg) instead of vitamin K1 [22]. Another populationbased study that involved 16,000 healthy females with ages between 49 and 70 was conducted; this study showed corresponding results. The study participants were selected from the cohort population of the European Prospective Investigation into Cancer and Nutrition (EPIC) study [23]. The data obtained from the study depicted that vitamin K2 instead of vitamin K1 had to be consumed in high quantities to prevent CV disorders. The data revealed that there was a 9% reduction in the risk of CHDs with every dose of 10 μg of vitamin K2 (taken as MK-7, MK-8, and MK-9). In the Netherlands, ultrasound and pulse wave velocity methods were utilized by the researchers working at the research and development (R&D) Group Vita K of Maastricht University to study 244 healthy postmenopausal females [23]. The subjects were observed for three years. Some of the subjects were administered a dose of vitamin K2 (180 μg) in the form of MK-7 (as MenaQ7 from NattoPharma), while some of the participants were given a placebo capsule every day for three years [22].

This was conducted on a random basis. At the end of the treatment, the group given vitamin K2 supplementation demonstrated a steady decline in stiffness index than the placebo group that showed a slight rise in the index. The study outcomes showed the positive impact of MenaQ7 on vascular health by enhancing vascular elasticity in females with stiff arteries and by suppressing age-related artery-wall stiffening. The researchers also found that the CV conditions improved as the subjects were administered a nutritional dose of vitamin K2 in the form of MK-7 (as MenaQ7). Moreover, if vitamin K2 is taken on a daily basis, there is a high chance of preventing the hardening of arteries [23, 24].

## **3. Vitamin K-dependent hormones**

The growing clinical evidence suggests that regular vitamin K supplementation may improve bone structure, prevent VC, improve the body's sensitivity to the insulin hormone, which increases the life expectancy and treatment outcome in patients [25]. In the past ten years, more evidence has been published supporting the hypothesis that vitamin K2 should be considered a hormone. Vitamin K2 was found to activate many genes directly and indirectly by binding to the intranuclear receptor SXR, activating sirtuins and/or histone deacetylases (HDACs) responsible for cell-type determination and specific cell functions [26]. A study by Lanham et al. on rats and their offspring explored the effect of a high-fat diet on bone development and vascular development, particularly the role of VKDPs, including Gas-6, MGP) and OC [27]. The study also shows the importance of proper nutrition during pregnancy. During the study, the team observed increased levels of Gas-6 proteins, increased expression of the gene responsible for vitamin K-dependent gamma-glutamyl carboxylase (GGCX) in the cardiovascular tissues, while decreased levels of MGP in the femoral bones of female offsprings of high-fat dietary fed mothers [27]. The osteoblastic synthesis gives rise to OC production, deposited into bone or released into circulation, giving the histological measures of bone formation. OC's structure is greatly affected by vitamin K-dependent Gla residues, resulting in bone mineral maturation. The circulating uncarboxylated OC (unOC) levels have been applied as biomarkers for vitamin K deficiency and correlated with age-related bone loss. In animals, in-vivo and in-vitro tests have revealed unOC as an active hormone affecting glucose metabolism; however, the results are inconclusive on human levels and need to be investigated further [28]. Post-translational GGCX enzymes detected both hepatically and extrahepatically are critical for the functionality of Gla residues in VKDPs. OC (bone-derived protein) has been associated with energy metabolism as the skeleton system has been considered an endocrine organ [29]. Via molecular mechanisms, OC mediates vitamin K positive effects, improves insulin resistance, lipid, and glucose profiles. OC is also detected by insulin to regulate bone mineralization. It has been hypothesized that normal VKDP carboxylation is an essential step in the prevention of vascular endothelial calcification [30]. Vitamin K2 has been found to affect bone and CV health. A study of the vitamin K2 homolog MK-7 found that serum levels increased, as evidenced by healthy Japanese women, who supplemented their diet with MK-7, which can be particularly important for extrahepatic tissue health [31]. In a study of the murine model, the importance of the vitamin K-dependent MGP on the inhibition of extraskeletal calcification was suggested. With a high dose dietary supplementation of MK-7, the induced VC was inhibited, and the aortic alkaline phosphatase tissue concentration was reduced [32].

## **4. Molecular mechanism of vitamin K-dependent calcification on vascular system**

Carboxylation is one of the post-translational modifications on proteins and is essential for the activity of VKDPs. The activation of VKDPs, which includes coagulation factors, OC, MGP, Gas-6, GRP, and periostin, is achieved by carboxylation of the proteins' Glu residues [33, 34]. The carboxylation of VKDPs happens in the case of an abundance of vitamin K, which is required for the activation of the GGCX enzyme (**Figure 1**) [35]. This enzyme adds carboxyl groups on Glu residues of VKDPs and converts them to Gla (**Figure 2**) [35]. This conversion enables the Gla residues to capture free Ca2+ ions that are circulating in the vascular system [36, 37]. For instance, there are five Glu residues on MGP, which is the only protein known as an inhibitor of arterial calcification. The Glu residues on the protein are carboxylated and converted to Gla, the active form of the protein. Without being activated, MGP is unable to hold free Ca2+ ions, which are eventually deposited in the vascular system and cause VC, such as calcium deposits and atherosclerotic plaques (**Figure 3**) [38, 39]. After the

#### **Figure 1.**

*Vitamin K-dependent post-translational carboxylation of MGP protein.*

### **Figure 2.**

*General overview of vitamin-K induced activation of VKPDs.*

inflammation and hyperlipidemia, the soft-tissue mineralization phenomena occur in vascular smooth muscle cells (VSMCs), leading to their hardening and differentiation into osteoblast-type cells [40]. In addition to the mineralization phenomena, it has been hypothesized that active forms of MGP proteins may attach to the calcified crystals in the vasculature resulting in apoptotic bodies and vesicles. Another assumption is their potential to hinder VSMCs' trans-differentiation into an osteogenic phenotype [34, 41, 42]. A three-year clinical study by Shea et al. with 229 patients who were routinely given dietary vitamin K versus 223 patients in the placebo group showed that the addition of dietary vitamin K significantly correlated with decreased levels of calcium in coronary arteries [43]. In addition to carboxylation, posttranslational phosphorylation of serine residues occurs on MGP. The enzyme casein kinase adds phosphate groups on three serine residues, regulating the secretion of protein into the extracellular matrix [44]. The unique relationship among circulating MGP forms, aortic stiffness, and arterial calcification was proposed in a recent article by Roumeliotis et al. [37]. The study has shown that more than one form of the MGP protein can be detected in the circulation and extracellular matrix governed by the degree of carboxylation and phosphorylation of the protein (**Figure 1**) [37].

## **5. Statins effect on vitamin K2 function**

Statins, or more precisely, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase-inhibiting molecules, are a family of molecules that interfere with cholesterol synthesis and induce the uptake of the low-density lipoproteins (LDLs) in the body [45]. Available as a prescription since 1987 [46], statins today are one of the most commonly prescribed medications worldwide [47]. However, some researchers are starting to suggest that physicians may be overprescribing statins to their patients [48–50]. A recent study published in the Annals of Internal Medicine journal found that the statins' potential side effects seem to outweigh the benefits for people whose 10-year CVD risk is approximately 7.5–10% [51]. The United States Food and Drug Administration's consumer update from 2017, named "Controlling Cholesterol with Statins," states that statins have been linked to associated muscle

## *Physiological and Cellular Functions of Vitamin K on Cardiovascular Function DOI: http://dx.doi.org/10.5772/intechopen.99344*

symptoms and a high chance of developing type 2 diabetes in patients [52]. It seems crucial to understand the exact phenomena behind statins' mechanism of action and clarify the medical community's created bias [53]. One potential explanation is given by the Kinjo Gakuin University group led by Harumi Okuyama, who suggests that statins may have an essential role in the increased probability of developing diabetes and arteriosclerosis [54] via the inhibition of vitamin K2 synthesis [55]. Researchers have indicated that statins may inhibit vitamin K2 production via the inhibition of geranylgeranyl diphosphate (GGPP) synthesis by HMG-CoA reductases (**Figure 4**) [56]. Some authors hypothesize that prolonged HMG-CoA reductase suppression by chronic statins treatment could adversely affect patients by diminishing the vitamin K2 supply to their bodies [56]. This phenomenon may be an essential factor in diabetes, atherosclerosis, and osteoporosis causation. The recently aggregated data from available randomized controlled trials and observational studies suggest a 10 to 45 percent higher risk of new-onset development of diabetes mellitus type 2 in statin patients than non-users [57]. Studies have shown that postmenopausal women taking statins are 150% more likely to develop type 2 diabetes [58]. In a large clinical study (n = 2,142), Cederberg et al. show a 46% increase in the risk of developing diabetes alongside decreased insulin secretion and overall body sensitivity to insulin [59]. Furthermore, particular varieties of statins—simvastatin and atorvastatin—showed a dose-dependent effect on insulin sensitivity and its secretion in patients [60]. These clinical observations may collectively suggest a relationship between statins' inhibition of Vitamin K2 and GGPP production and statins' influence on insulin

#### **Figure 4.**

*Statins and vitamin K2 related biochemical pathways. Statins inhibit HMG-CoA reductase in the mevalonate pathway. Geranylgeranyl-PP is essential for the synthesis of vitamin K2 from vitamin K1.*

synthesis, secretion, and sensitivity. Insufficient levels of vitamin K2 may be linked to atherosclerosis and other CVDs [61]. In the large population-based Rotterdam study (n = 7,983 men and women, age > 55 years), Geleijnse et al. report a positive correlation between the reduced risk of CHD with regular dietary consumption of vitamin K2 [59]. The authors hypothesize the link between the depletion of vitamin K2 levels and severe coronary artery calcification in patients.

Several clinical studies have demonstrated that administering vitamin K2 (but not vitamin K1) was an effective method of osteoporosis fracture prevention in patients [62, 63]. Studies have shown that vitamin K2 up-regulates bone markers' expression and sustains lumbar bone mineral density in patients [63]. Pre-clinical and clinical evidence shows that statins may have an essential role in reducing the supply of vitamin K2 to the tissues through the body. Additional studies are required to explore the mechanisms for statin-associated diseases that were identified in pre-clinical and clinical studies, including diabetes, atherosclerosis, osteoporosis, chronic kidney disease, and cancer, and the effects of the various types of statins on the vitamin K2 synthesis, delivery, and accumulation in the body.

## **6. Physiological roles of vitamin K on CV functions**

There is growing preclinical and clinical evidence of the crucial role of vitamin K in VC prevention [64]. Several VKDPs are regulated by vitamin K share entirely Gla, which is the remarkable amino acid produced by the posttranslational modification of the vitamin K-mediated enzyme GGCX [65]. The prothrombin molecule is carboxylated at ten glutamyl residues to produce the active form of prothrombin. These Gla are deposited at the amino-terminal domain of all VKDP, which share a common amino acid sequence [66]. Studies have shown that Gla also regulates calcium due to the placement of Gla in calcium-binding sites of the protein [67].

Available literature has limitations regarding the average requirement of vitamin K for normal homeostasis. In 2001, the United States Pharmacopeia Health and Medicine Division established adequate intake (AI) values based on median intake values reported by the National Health and Nutrition Examination Survey (NHANES) III; the AI value for vitamin K1 was set to 90 μg/dl for adult females and 120 μg/dl for adult males [67, 68]. This study does not advocate that this concentration of vitamin K will be enough to maintain the carboxylation of VKDPs. Undercarboxylated, biologically inactive Gla proteins are caused by vitamin K deficiency, resulting in the synthesis of calcification, which is considered a risk factor for VC and CVD [67, 68].

CVD is a cluster of abnormal conditions, such as CHD, and influences the functions of the heart and blood vessels that supply blood to various parts of the body [69]. Heart diseases and stroke are the primary causes of death and disability worldwide. According to the American Heart Association 2020 report, the age-adjusted death rate of CVD is 219.4 per 100,000, which means that someone is dying of CVD every 37 seconds, with a total of 2,353 deaths from CVD each day in the U.S. Consistent with these data, there are approximately 795,000 new or recurrent strokes each year, as well as approximately 401 deaths from stroke each day, based on data for previous years [70]. CVD-related diseases, such as angina, carotid artery diseases, and peripheral artery diseases, are characterized by the formation of fatty deposits in the arteries, which is known as atherosclerosis. These deposits consist of calcium, cellular waste products, fatty substances, cholesterol, and fibrin (a clotting material in the blood), which ultimately leads to narrowing and blockage of the arteries and

### *Physiological and Cellular Functions of Vitamin K on Cardiovascular Function DOI: http://dx.doi.org/10.5772/intechopen.99344*

reduced blood flow to the heart muscle. Studies have shown that lifestyle, healthy diets, vitamins, and physical activity may have a potential role in preventing the development of CVD [71, 72].

There is compelling evidence that vitamin K is involved in various biological processes in the body and mediates anti-calcification, anti-cancer, bone-forming, and insulin-sensitization effects, and plays a vital role in the prevention management of CVD (**Figure 5**) [72]. It has been reported that vascular deficiency of vitamin K can lead to CVD by increasing calcium deposition and coronary artery calcification because vitamin K-synthesized osteocalcin and MGP strongly inhibit VC by regulating bone metabolism. Previous studies have shown a strong association between reduced intake of vitamin K and the development of coronary calcification, advocating that adequate vitamin K intakes can prevent CVD [73, 74]. The involvement of vitamin K in VC by the carboxylation of MGP has been confirmed in various animal studies that; MGP-knock out mice died within two months due to VC-induced rupturing of blood vessels followed by short stature, osteopenia, and fractures [75]. Sweatt et al. hypothesized that in rodents, a specific calcium-mediated and vitamin K-dependent Gla region in MGP protein is involved in binding bone morphogenetic protein-2 (BMP-2) that may link the age-related arterial calcification and low carboxylation of MGP [76]. Vitamin K antagonist warfarin has been shown to antagonize vitamin K-dependent carboxylation of MGP, leading to extensive VC [77]. However, vitamin K intake can suppress arterial calcification after treatment with warfarin in rats [78]. VKDPs, such as MGP and Gas-6, have the ability to protect the vasculature and have an essential role in blood coagulation by preventing tissue calcification and cell death in VSMCs and arterial vessel walls [79]. Several clinical observational studies have hypothesized that chronic dietary supplementation of both vitamins K1 and K2 may negatively correlate with risks of VC and CVD [79].

#### **Figure 5.**

*A model showing the therapeutic potential of vitamin K against various cardiovascular diseases (CVD). MGP and Gas6 are vitamin K dependent proteins, which are activated by carboxylation. Subsequently, Gas6 exerts inhibitory effects on the apoptosis of endothelial cells and VSMCs, thus preventing angiosteosis and protecting blood vessels and improved various heart diseases.*

Additionally, subtypes of vitamin K2 (MK4 to MK9) have been examined in the Prospect–EPIC cohort, which consists of 16,057 women, aged 49–70 years old with no history of CVD [79]. This study concluded that a high intake of menaquinones (MK7, MK8, and MK9) could protect against CVD. However, this kind of protection has not been observed with vitamin K1 (phylloquinone) against CVD in other cohort observations [79, 80]. Notably, a Nurses' Health Study conducted with 72,874 female nurses aged between 38 and 65 years old has confirmed that vitamin K1 and lower risk of CVD is not significant because vitamin K1 intake may be a substitute marker for a healthy diet rather than an independent risk factor for CHD [81]. Nevertheless, data from National Health and Nutrition Examination Surveys examined the data of 5296 individuals with a minimum age of 50 years and concluded that vitamin K1 shows an independent assessment of high arterial pulse pressure [69]. In another prospective cohort study, 7216 participants were assessed by different types of vitamin K intake and mortality [82]. This study concluded that a high vitamin K intake is linked to the reduced risk of CVD in a Mediterranean population [82]. Vitamin K has shown promising results against vascular calcification in vitamin K-deficient individuals. Further research is justified to explore a relationship between vitamin K supplementation and the prevention of CVD.

## **7. Therapeutic role of vitamin K**

CVD is a public health burden and a serious challenge to the health system throughout the world. CVD is a leading cause of death globally, with approximately 18 million deaths in 2015; the World Health Organization (WHO) forecasts that approximately 23.3 million deaths could occur from CVD by 2030 [83, 84]. The WHO has defined CVD as a "*group of illnesses that affect the heart and blood vessels*" [83]. These conditions include CHD and cerebrovascular disease. Scientific evidence has shown that factors related to nutrition have an important role in the development of CVDs and that these dietary factors may contribute to the differences in the morbidity and mortality from CVD seen in various regions of the world [83].

Different forms of vitamin K exist in the diet sourced from plants and animals [83, 85–88]. Vitamin K2 is usually a product of bacterial synthesis; however, meat, dairy, and fermented food products provide a minimal amount of vitamin K-2 [86, 88, 89]. Discovered in 1936, vitamin K has been known as an enzyme co-factor for the carboxylation of VKDPs [85, 86, 89]. Its key function in the synthesis of clotting factors in the liver has made the relationship between vitamin K and coagulation of blood a well-known phenomenon; however, recent studies are offering more insight into the diversity of functions associated with vitamin K [85, 86, 89, 90]. Many disease conditions related to the activities of vitamin K are now being described [89]. The carboxylation or activation of VKDPs requires vitamin K as a cofactor to the GGCX enzyme and occurs in the liver [83, 85, 86, 89]. The process converts specific Glu into calcium-binding Gla residues [86, 89, 90]. The uncarboxylated forms of the VKDPs are inactive, and carboxylation turns them into active and functioning proteins [86]. Some of the VKDPs that are carboxylated in the liver include clotting factors, such as factor II (prothrombin) and factor X [86]. Studies have confirmed that the process of activation of VKDPs also occurs outside the liver in smooth muscle cells. The extracellular matrix MGP protein is produced by smooth muscle cells and inhibits soft-tissue mineralization by binding to ca2+ ions to the vascular walls [44, 83, 85, 86]. MGP is a VKDP with Gla and serine residues. MGP is activated via carboxylation of the Gla residues, followed by phosphorylation of the serine residues

*Physiological and Cellular Functions of Vitamin K on Cardiovascular Function DOI: http://dx.doi.org/10.5772/intechopen.99344*

**Figure 6.** *Key functions of vitamin K.*

[44, 86]. Vitamin K is essential to the carboxylation and phosphorylation of MGP as the enzyme co-factor [44, 86]. Carboxylation of MGP leads to its structural changes, which are very important for its ability to bind to calcium crystals [44]. Although MGP becomes inactivated when there is vitamin K deficiency and leads to VC, a high intake of vitamin K can reverse conditions [44, 83, 85, 86]. MGP secreted by chondrocytes and VSMCs has been shown to inhibit VC and was described as the most potent natural inhibitor of calcification in the human body [44]. Apart from inhibition of calcification, MGP has also been recognized as having the ability to reverse the calcification process [44]. The protection of MGP from VC occurs via its high binding affinity to new crystals of hydroxyapatite, which prevents their increase within the vascular wall [44]. MGP also stimulates arterial macrophages, leading to phagocytosis and apoptosis of the MGP-hydroxyapatite complex (**Figure 6**).

A sub-optimal level of vitamin K in the body is associated with an increased risk of adverse health outcomes, especially in adult and elderly populations. Studies have linked vitamin K deficiency with CVD, insulin resistance, and inflammation, as well as cognitive impairment [13, 83, 85, 87]. A lack of vitamin K has been shown to lead to an increased risk of calcification of blood vessels and CVD due to the presence of nonfunctioning Gla proteins [44, 83, 85, 86]. Vitamin K deficiency may cause increased calcium deposition in the walls of the blood vessels, leading to calcification of the coronary artery, and ultimately, CVD [13, 83]. Earlier observational studies have also established a relationship between low vitamin K intake with calcification of blood vessels; other observations have suggested that high vitamin K supplementation in the diet may reduce long-term CVD risks [13, 83, 85]. An increased intake of dietary vitamin K is also associated with a decrease in the risk of all-cause mortality, as concluded by a study of a Mediterranean population with a high risk of CVD [83].

## **8. Vitamin K and inflammation**

Inflammation is a recognized contributor to the progression and onset of diseases related to aging, such as osteoarthritis, CVD, and other similar diseases [85, 89]. The production of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFɑ), C-reactive protein (CRP), and interleukin-6 (IL-6), has been found to be interfered by vitamin K. These findings were demonstrated in a cross-sectional study that showed a relationship among the high levels of vitamin K supplementation, the low levels of pro-inflammatory cytokines, and diminished inflammation in the body [85, 89]. Leptin hormone has a proinflammatory effect, menadione (VK3) has an apoptotic effect in Hepatocellular carcinoma through inhibiting leptin and through ROS generation which made VK3 a potential vitamin in preventing hepatocyte survival [91].

## **9. Conclusion**

New insights about the activities of vitamin K and its crucial protective role in CVD development have emerged. Good association between dietary vitamin K2 and CVD is now clinically established. The role of inhibitory effect of statins in synthesis of vitamin K2 should be emphasized. However, there is still a need for prospective in-depth studies to improve the current understanding of this critical relationship and apply such knowledge to prevent CVD and improve its outcomes. Research should focus on understanding the function and regulation of new proteins that enhance or inhibit vascular calcification as well as the combination of vitamin D with other therapeutic drugs. Prospective studies may assess the vitamin K status using multiple biomarkers to provide insight on the relationship of vitamin K to vascular calcification and CVD.

## **Conflict of interest**

All authors declare that there is no conflict of interest.

## **Author details**

Meneerah A. Aljafary1 , Hussah Alshwyeh1 , Nada Alahmadi1 , Adeeb Shehzad2 , Huseyin Tombuloglu3 , Zagit Gaymalov4 , Abdelqader Homieda1 and Ebtesam Al-Suhaimi1 \*

1 Biology Department, College of Sciences, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia

2 Department of Clinical Pharmacy Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia

3 Genetics Research Department, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, Dammam, Saudi Arabia

4 Earlystage OÜ, Tallinn, Estonia

\*Address all correspondence to: ealsuhaimi@iau.edu.sa

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 4**

## Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health

*Dilip Mehta, Anselm de Souza and Shashank S. Jadhav*

## **Abstract**

Menaquinone-7 plays a significant role in cardiovascular and bone health. In recent times there is a growing interest in understanding the role of Menaquinone-7 in health and diseases. Several population-based studies have reported specific health effects of the long-chain menaquinones, notably MK-7, MK-8, and MK-9. There are several epidemiological studies, clinical trials, along with *in vivo* and *in vitro* studies confirming the role of Menaquinone-7 in health and diseases. More recently, research group at Synergia Life Sciences has discovered a wider role for Menaquinone-7 in energy homeostasis (VO2max), peripheral neuropathy, muscle cramps and mitochondrial respiration not only through improvement of the electron transport but also the perfusion improving oxygen availability. In the current chapter, the authors have discussed the wider physiological role of Menaquinone-7 highlighting the recent research with Menaquinone-7 in the areas of Muscle and Nerve Health.

**Keywords:** Menaquinone-7, muscle cramps, peripheral neuropathy, bone health, cardiovascular, insulin resistance, deficiency, catabolism, SXR, energy homeostasis

## **1. Introduction**

Menaquinone-7 belongs to Vitamin K group. The two general categories of vitamin K are Phylloquinone (Vitamin K1) and Menaquinones, also referred as MK-n (clinical nomenclature Vitamin K2-n) having side chains with 4–12 prenyl units (MK-n where n stands for the number of isoprenoid units, MK-4 to MK-12) 9 (**Figure 1**). Their physiological and patho- physiological roles are specific.

## **2. Biological activity of Menaquinone-7**

Vitamin K was discovered by the Danish scientist, Henrik Dam, in the 1930s. Dam's discovery was during his quest to understand chicken's cholesterol metabolism by feeding them a diet free of sterols and low in fat [1]. This reduced their intake of fat-soluble vitamin K, resulting in chickens developing large subcutaneous and intramuscular hemorrhages. This initial finding led to isolating, identifying, and

#### **Figure 1.**

*Chemical structures of various isoforms of Vitamin K. chemical structures of some K vitamins and metabolites. Nomenclature: Chemical name and IUPAC name and abbreviation in brackets: (I) 2-methyl1,4-naphthoquinone (Menadione; K3), (II) 2-methyl3-phytyl-1,4-naphthoquinone (Phylloquinone; K1), (III) 2-methyl-3-phytyl-1,4-naphthoquinone-2,3-epoxide (Phylloquinone epoxide; K1O), (IV) 2-methyl-3-geranyl-geranyl1,4 naphthoquinone (Menaquinone-4; MK-4), (V) 2-methyl-3-farnesylgeranylgeranyl-1,4-naphthoquinone (Menaquinone-7; MK-7).*

characterizing the structure of vitamin K and its importance as an anti-haemorrhagic agent. Of the many metabolic processes related to vitamin K deficiency, bleeding remains the potentially most serious generally known consequence. However, the role of vitamin K's impact on osteoporosis and its inhibitory role in arterial calcification and vascular biology is now recognized in general populations. It is axiomatic that these metabolisms require vitamin K for γ-carboxylation and that this step is essential to their proper functioning. However, there are many other functions of vitamin K recently discovered that seem to be independent of its classical co-factor function. Vitamin K's metabolic effects, e.g., ameliorating effect on peripheral neuropathy, cramps, autonomic nervous system, improving perfusion, etc., remain unexplained. Additionally, vitamin K also acts as a ligand for the receptor SXR, the steroid and xenobiotic sensing nuclear receptor (SXR), which is a transcriptional regulator of the cytochrome P450 gene CYP3A4.

Over the years, the understanding of the vitamin K family has evolved, with the recognition of two primary forms of vitamin K- vitamin K1 (phylloquinone) and vitamin K2 (menaquinones). All K-vitamins have same function, but they exhibit differences in bioavailability and bioactivity. Vitamin K2, the main storage form in animals, has several subtypes, which differ in isoprenoid sidechain length. These vitamin K2 homologs are called menaquinones and are characterized by the number of isoprenoid residues in their side chains. Menaquinones are abbreviated as MK-n, where M stands for menaquinone, the K stands for vitamin K, and the n signifies the number of isoprenoid side chain residues. MK-4 and MK-7 are the two prominent menaquinones in human nutrition. MK-7 and other long-chain menaquinones are different from MK-4 in that they are not produced by human tissue but are generated *Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

by bacteria in the gut. The available information suggests that a range of vitamin K2 analogues are present as a mixture in several foods, e.g., in sauerkraut, hard cheese, soft cheese and curd cheese [2]. These foods have a long history of consumption by humans as basic foods.

Diet (Natto and cheeses) rich in menaquinones (primarily MK-7) is safe and requires no systemic validation for toxicity. However, because of the role of vitamin K (K1 and K2) in blood coagulation and potential health benefits, there has been considerable effort to elucidate the mechanism of action of menaquinones, primarily MK-7. There is no known toxicity associated with high doses (dietary or supplemental) of the phylloquinone (vitamin K1) or menaquinones (vitamin K2) forms of vitamin K. In several human studies, Natto food, known to contain MK-7, has been investigated for its health benefits.

## **3. Menaquinone-7 safety**

The adverse effects of menaquinones, including Menaquinone-7 has been investigated in several animal and *in vitro* toxicity studies. Findings from animal studies for acute, chronic, and genotoxicity and *in vitro* studies for mutagenicity and carcinogenicity showed no significant risks associated with exposure to menaquinones [3]. The absence of adverse effects or death suggest that the minimum lethal dose of Menaquinone-7 is greater than 2000 mg/kg bw [4].

The European Union has permitted the use of Menaquinone-7 as a source of vitamin K for nutritional purposes in foodstuffs. The European Food Safety Authority (EFSA, 2008) [5] examined the safety of Menaquinone-7. The chemistry, nomenclature, dietary sources, intake levels, and pharmacokinetics of menaquinones, and data of nonclinical toxicity and on clinical outcomes related to safety (adverse events) was extensively reviewed by US Pharmacopeia Convention [6] and by the Institute of Medicine (IOM, 2000). The report considers menaquinone as an active form of vitamin K [7].

## **4. Menaquinone-7 in diet**

Schurgers et al. [2] have studied levels of Menaquinone-7 in many food products globally and found that the Menaquinone-7 levels are quite negligible in all the food products except Natto, a staple food in Eastern Japan which contains almost 998 mcg of Menaquinone-7 per 100 gm of Natto. The investigators also found small amounts of Menaquinone-7 in natural cheese. Researchers at Synergia Life Sciences have undertaken a study where a number of Indian food products were examined for the levels of Menaquinone-7. The foods tested had particularly included fermented foods consumed by Indians at large. It was observed that the regularly consumed food including fermented foods lack in Menaquinone-7. So, it can be said that Menaquinone-7 is negligible in Indian diet.

The only rich source of Menaquinone-7 is Natto which contains early 900 mcg of Menaquinone-7 in 100 gm's breakfast [2, 8] and different types of cheese [9] though in small amounts. The various common 18 varieties of Dutch cheeses and 13 varieties of European cheeses contain approximately on an average 1.14 and 1.36 mcg Menaquinone-7 per 100 gm of cheese respectively [9]. The hard cheese, soft cheese and curd cheese from Netherlands contains approximately 1.3, 0.5 and 0.3

mcg Menaquinone-7 per 100 gm cheese respectively [2]. Processed cheese from Japan reported 0.3 mcg Menaquinone-7 per 100 gm cheese [10].

Serum concentrations of Menaquinone-7 are higher in frequent natto eaters. Natto is a popular breakfast item used more widely in win Eastern Japan, as compared to Western Japan. The study by Kaneki *et al*. reports an average serum Menaquinone-7 concentration of 5.26 ng/ml in Eastern Japanese women (Tokyo), 1.22 ng/ml in the Western Japanese women (Hiroshima) and 0.37 ng/ml in British women (London) [11]. The serum concentrations in British women are negligible since they do not consume Natto, but their diet may include cheese which contributes to the small amounts of Menaquinone-7 in their serum.

Globally speaking Menaquinone-7 is negligible in diet. It is true that many bacteria that populate microbiota of the human intestine synthesize Menaquinones. However, it is realized that in the small intestine bacterial growth availing Menaquinone-7 is limited by the rapid transit times. Most synthesis of Menaquinones occur in the large intestine. Shearer *et al*. [12] and Suttie *et al*. [13] have examined the evidence of the contribution of gut menaquinones and concluded that while they do contribute to the human nutrition but not significantly. Karl JP *et al*. have shown that total Menaquinone (Menaquinone-4 to Menaquinone-13) concentration in human gut is highly variable. They measured total daily excretion of menaquinones in feces. The median total daily excretion of menaquinones in feces was 850 nmol/d but was highly variable (Range: 64–5358 nmol/day) [14].

## **5. Role of Menaquinone-7 in various diseases**

#### **5.1 Cardiovascular diseases**

Geleijnse et al. [15] studied 4807 men and women of aged 55 yrs. for 10 years to assess the association of dietary intake of K1 and K2 with aortic calcification, CVD, and total mortality. They concluded that "When consuming daily 45 mcg dietary K2, you have: 50% reduction of arterial calcification, 50% reduction of cardiovascular death, 25 % reduction of all-cause mortality as compared to low intake of dietary K2!"

Gast et al. [16] studied 16,057 women, aged 49–70 years and free of cardiovascular diseases (at baseline) for 8.1 ± 1.6 years. The intake of vitamin K1 was 211.7 ± 100.3 mcg/d and of vitamin K2 intake was 29.1 ± 12.8 mcg/d. They concluded that there is inverse association of vitamin K2 with CHD with reduction of 9.1% per 10 mcg/day. They also found out that vitamin K1 is not related to CHD.

The publication of the above two epidemiological studies, viz. Geleijnse *et al*. and Gast *et al*. Study, has expanded interest the investigations of various beneficial health effects of Menaquinone-7.

#### **5.2 Bone health**

Knapen *et al*. [17] investigated the effects of low-dose Menaquinone-7 on bone health in healthy postmenopausal women. Menaquinone-7 intake significantly improved vitamin K status and decreased the age-related decline in Bone Mineral Content (BMC) and Bone Mineral Density (BMD) at the lumbar spine and femoral neck. In another placebo-controlled study, the authors investigated the effect of Menaquinone-7 on BMD and found out that Menaquinone-7 preserves trabecular bone structure at the tibia along with decrease in undercarboxylated osteocalcin

### *Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

(ucOC) [18]. In another clinical study Kanellakis *et al*. [19] assessed the effect of dairy products enriched with calcium, vitamin D3, and Menaquinone-7 on parameters of bone metabolism in postmenopausal women following a 12-month intervention. The study revealed more favorable changes in bone metabolism and bone mass indices for the Vitamin K2 supplemented groups. Van Summeran *et al*. [20] studied the effect of 45 mcg Menaquinone-7 on the circulating levels of undercarboxylated osteocalcin (ucOC) and carboxylated osteocalcin (cOC) along with Menaquinone-7 levels in healthy prepubertal children. They showed that the levels of Menaquinone-7 increased with the supplementation of Menaquinone-7 as compared to baseline levels.

Spronk HMH *et al*. in 2003 conducted an *in vivo* study for assessing tissue specific utilization of Vitamin K2 which resulted in prevention of arterial calcification in warfarin-treated rats. It was shown that the utilization of Vitamin K2 was more efficient in the aorta as compared to other tissues [21].

In a study by Yamaguchi *et al*., the authors have shown the anabolic effect of Menaquinone-7 on bone tissue and osteoblastic MC3T3-E1 cells *in vitro* [22]. Min Zhu *et al*. have shown that Menaquinone-7 has a stimulatory effect on bone tissue and osteoblastic SAOS-2 cells *in vitro* [23]. These studies suggest the role of Menaquinone-7 in osteoblastic bone formation. Recently, it has also been shown that Menaquinone-7 protects osteoblasts from oxidative stress and has beneficial effects on proliferation, differentiation, and mineralization of osteoblasts [24].

#### **5.3 Insulin resistance**

A decade-long study of 38,094 Dutch males and females aged 20–70 found that the quartile of participants who consumed the most dietary Vitamin K were 20% less likely to develop type 2 diabetes than the quartile with the lowest intake of Vitamin K [25]. Vitamin K2 is linked to lowered risk of developing type 2 diabetes and also a stronger relationship exists for Vitamin K2 intake. The risk of developing type 2 diabetes drops for every 10 mcg (0.01 mg) increase in Vitamin K2 intake. In this study participants with the highest intake of K2 consumed 250–360 mcg (0.25–0.36 mg)/ day. Thus, higher intake of Vitamin K2 is linked to lower diabetes risk.

In an attempt to better understand how Vitamin K2 improves insulin sensitivity, researchers from S. Korea studied 42 healthy male volunteers. Participants were either given 30 mg (30,000 mcg) of Vitamin K2 or a placebo each day for 4 weeks. Vitamin K2 supplementation significantly increased insulin sensitivity and seemed to be related to increased carboxylation (activation) of osteocalcin. Researchers concluded that Vitamin K2 can help regulate glucose metabolism by activating osteocalcin, an endocrine hormone that increases insulin sensitivity in humans [26].

Research has shown that for elderly men Vitamin K slows the development of insulin resistance [27]. The researchers concluded that Vitamin K2 plays a potentially beneficial role in reducing the progression of insulin resistance amongst elderly men.

## **6. Recent research**

## **6.1 Energy homeostasis (VO2max)**

In a recent randomized controlled trial, McFarlin et al. investigated the effects of dietary supplementation of Menaquinone-7 on cardiovascular responses to a graded cycle ergometer test. Menaquinone-7 supplementation was associated with a 12%

increase in maximal cardiac output, with a trend toward an increase in heart-rate AUC. No significant changes occurred in stroke volume [28]. **Figure 2** demonstrates that Menaquinone-7 treatment was associated with increased cardiac output, stroke volume, heart rate, and decreased blood lactate. Overall, these changes are consistent with increase maximal cardiovascular performance with oral Menaquinone-7 supplementation.

### **6.2 Mitochondrial respiration**

Synergia research group has identified Menaquinone-7's pivotal role in mitochondrial ATP generation by acting as a mitochondrial electron transport carrier, thus participating in the energy cycle of the cell. In human cell experiments, it has been shown that the cells' maximum capacity to generate energy, defined as the reserve energy, increases by 30–40% with Menaquinone-7, thus, identifying the role of Menaquinone-7 in redox cycle by transporting electrons in electron transport chain and also mitochondrial generation of ATP (**Figure 3**). This dual role of Menaquinone-7 is especially important to the aging geriatric population and athletes in their need of a greater oxygen supply for the oxidative phosphorylation.

In another *in vitro* study, Menaquinone-7 rescued mitochondrial defects in numerous conditions that affect mitochondrial function. Menaquinone-7 was also effective at improving systemic locomotion defects in fully developed adult pink1 and parkin mutant flies. Menaquinone-7 did not affect mitochondrial remodeling directly, but by increasing Electron Transfer Chain efficiency, it contributed to the proton motif force that facilitates ATP production. Menaquinone-7 may thus constitute a promising compound to treat mitochondrial pathology, also in Parkinson's disease (PD) patients suffering from Pink1 or Parkin deficiency [29]. A clinical study has been proposed to investigate the potential effects of Menaquinone-7 in genetically determined PD with mitochondrial dysfunction [30].

#### **Figure 2.**

*To visualize the 5 outcome variables (heart rate, stroke volume, cardiac output, oxygen consumption, and blood lactate) on the same scale all data maximal response data after 8 weeks of treatment with either a vitamin K2 (red) or control (rice flour; blue) were normalized using a Log10 adjustment. Plotted values represent increments of a Log10 scale consuming a specific supplement.*

*Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

**Figure 3.** *Mitochondrial respiration: Test sequence in sea horse XF-96 platform.*

### **6.3 Anti-inflammatory**

Chronic inflammation is considered an underlying pathology of many diseases that remain poorly understood and treated. Several important chronic diseases with an inflammatory background have been associated with vitamin K deficiency. These include cystic fibrosis, inflammatory bowel disease, pancreatitis, chronic kidney disease and osteoporosis [31, 32]. Circulatory markers of low-grade inflammation such as tumor necrosis factor-alpha (TNF-α), interleukin-1 alpha (IL-1α), and interleukin-1 beta (IL-1β) positively correlate with endothelial damage, atheroma formation, cardiovascular disease, and aging. Menaquinone-7 can modulate immune and inflammatory reactions in the dose–response inhibition of TNF-α, IL-1α, and IL-1β gene expression and protein production [33]. These findings highlight the anti-inflammatory properties of Menaquinone-7, elucidating the anti-inflammatory mechanism of Menaquinone-7 and in establishing the potential biomarker targets in clinical testing of the role of Menaquinone-7 in cardiovascular health as well as other chronic degenerative conditions.

### **6.4 Muscle health**

Vitamin K deficiency impacts neuromuscular and vascular function, thus affecting the physical functioning. Vitamin K has a function in promoting vascular smooth muscle differentiation [34]. As disabilities in patients are directly related to muscle strength and physical performance, therefore it is crucial to focus on muscle strength and performance rather than muscle mass [35].

Handgrip indicates muscle strength and is directly related to lower-extremity strength. Calf circumference indicates skeletal muscle mass and is associated with higher strength [36, 37]. A longitudinal cohort study conducted in communitydwelling adults (n: 633, aged: 55–65 years) analyzed the association between vitamin K status and physical functioning over 13 years. An association of low vitamin K status with lower handgrip strength, smaller calf circumference was observed. Low vitamin status in women indicated an existence of association of low vitamin status with poorer functional performance score [38].

Some observational studies conducted in sarcopenia patients showed an association of high vitamin K status in plasma with muscle strength, large muscle mass, and high physical performance.

Thus, it was concluded that physical performance scores rather than muscle mass indicated the beneficial effect of vitamin K on muscle quality [35].

Systremma or leg cramps is a common and distressing problem characterized by involuntary, painful, sudden contractions of the skeletal muscles. It has affecting 30% of people who are over sixty-year-of age and 50% of people over eighty years of age [39, 40]. Muscle cramps may occur in normal subjects during a strong voluntary contraction, sleep, sports or pregnancy but it can also occur due to several pathological conditions such as myopathies, neuropathies, motoneuron diseases, metabolic disorders, hydroelectrolyte imbalances or endocrine pathologies, cirrhosis of liver, in patients on dialysis or may be triggered by intake of certain drugs such as diuretics, laxatives, beta2-agonists, cimetidine, and phenothiazines. Treatment of the underlying cause could successfully relieve this symptom [41, 42].

Diverse causes of muscle cramps has led to varied treatment modalities in clinical practice with varying degree of success in relieving the symptoms [43, 44]. These modalities include quinine Sulphate [45], calcium channel blockers [46], magnesium [47], gabapentin [48], botulinum toxin [49], phenytoin [50], Vit E [51], carisoprodol and orphenadrine [52]. Although quinine is the most used treatment modality in this condition [41], it is associated with several side effects like arrhythmia, tinnitus, headache, nausea, tremor, hypotension, and gastrointestinal upset, and occasionally, potentially fatal hypersensitivity reactions and thrombocytopenia [40, 41]. Due to severe toxicity encountered, US FDA has banned over-the-counter quinine-based products used for leg cramps [41, 53, 54]. This has generated a need for alternative therapeutic agents.

A preliminary open labeled observational study conducted by Vaidya *et al*. showed that daily administration of 100 mcg of Menaquinone-7 for 3 months was associated with a reduction in the frequency, intensity, and duration of idiopathic muscle cramps [55]. Menaquinone-7 at a dose of 100 mcg /day for 3 months was found to be well tolerated and safe and resulted in therapeutic relief of muscle cramps (**Figure 4**).

#### **Figure 4.**

*Decrease in mean severity of cramps as noted on VAS score in both the groups which were divided depending upon the frequency of cramps viz., cramps every day in group A and 2–3 cramps every week in group B.*

*Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

A research done by Mehta and Vaidya showed that daily administration of vitamin K relieves muscle cramps and prevents its recurrence. Vitamin MK-7 has longer halflife which facilitates its further utilization as it stays in the body for a longer duration. Vitamin K is a safe prophylactic for muscle cramps. Vitamin K also improves the muscle strength evident by relief of fatigue. The inventors have discovered relief from cramps when sufficient dose of vitamin K was administered systematically daily once or more. The preferred range was 10 μg to 1000 μg per day, and the preferred vitamin K was vitamin MK-7 [56].

#### **6.5 Nerve health**

Peripheral neuropathy (PN) also known as distal symmetric neuropathy or sensorimotor neuropathy, is a common problem with multifactorial aetiologies. Diabetes mellitus is the most common etiology of PN. Neural signals from sensory receptors in the cellular pathway of the peripheral nervous system are damaged in PN. It is a neurological complication where the nerves carrying sensory neurons from different parts of the body to the central nervous system are denervated hence causing numbness, tingling, motor paralysis and gland or organ dysfunction. PN is a disease with vast spectrum, found in a variety of groups of populations, commonly observed in geriatrics, obese and diabetic population [57].

An epidemiological study conducted by Martyn *et al*. has stated the worldwide prevalence of PN to be around 2.4% which is considerably increasing to 8% in patients older than 55 years [58].

Indian population is susceptible to PN due to large population density, exposed to different adverse environments for a living [59]. Amongst diabetic Indian population, prevalence of neuropathy has been 26–31% [60–62]. In an Indian epidemiological survey conducted amongst about 40 million diabetics in India, at least 10.4 million diabetics showed the symptoms of PN [63].

Some patients with neuropathy may experience extremely painful symptoms, whereas others may have objectively marked neurological deficit without significant painful neurological symptoms [64].

A systematic review analyzing the data of several studies stated that painful diabetic PN occurs in about one in six people with diabetes, impairing the quality of life of people and increasing healthcare cost. Although guidelines have suggested several treatment related recommendations, but they are associated with adjuvant side effects [65]. The risk of developing PN increases with the duration of diabetes and deteriorating glycemic control [64].

Neuropathy is a devastating event in patients with myeloma. Prolonged treatment of Multiple Myeloma (MM) related drugs leads to development of PN in 70% of patients [66]. Neuropathic events in such patients leads to dose reduction of the primary agents (Bortezomib, Thalidomide and Lenalidomide) or reduction in frequency of the therapy. This could further lead to discontinuation of therapy by some of the patients. Therefore, neuropathy in MM needs to be addressed.

The etiopathology of PN is poorly understood. Many factors, including dietary deficiencies, may contribute to the clinical manifestation of the condition [67].

The neurologic manifestations of folate deficiency overlap with those of vitamin B12 deficiency and include cognitive impairment, dementia, depression and commonly PN [64].

Myelopathy with or without an associated neuropathy is the commonly recognized neurological manifestations of vitamin B12 deficiency [68]. Methyl cobalamin is a

vitamin B12 analogue, necessary for the maintenance of the nervous system [69]. The diagnosis of neuropathy due to B12 vitamin deficiency remains a real challenge for the clinician [70].

The etiology of diabetic neuropathy has been a debatable topic however, neuropathy due to an inflammatory autoimmune condition that damages the myelin sheet of peripheral nerves and role of menaquinone-7 deficiency in alleviating this condition are considered as the evolving possibilities for diabetic neuropathy.

Vitamin K is considered to have a role in myelin synthesis and repair in central and peripheral nervous systems. Myelin is a sphingolipid, a group of complex lipids which are found in all mammalian cells as a major components of cell membranes, present particularly in high concentrations in cells of the central and peripheral nervous systems [71]. Certain sphingolipids found in the central and peripheral nervous systems have shown a high correlation with the tissue levels of vitamin K. Initially recognized for their structural role, sphingolipids are now considered as the key players in major cellular events such as proliferation, differentiation, senescence, cell–cell interaction, and transformation [72]. Furthermore, several recent research have shown a corelation of alterations in sphingolipids metabolism with aging process [73] and neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease [52, 74].

Scientific legacy of Meir Lev's group depicted the role for vitamin K in sphingolipid metabolism in a report published in Nature in 1958 [75]. The report showed that Vitamin K serves as a growth factor for the rumen strain *Bacteroides melaninogenicus* (also known as *Fusiformis nigrescens*), which was later found to be linked to cell membrane homeostasis. When *Bacteroides melaninogenicus* was cultured in a medium without vitamin K, cells grew as filaments (i.e., elongated cells), were more fragile when subjected to shaking with glass beads and tended to auto-agglutinate when placed in buffer. Growth of bacteria under such condition was also greatly affected. Vitamin K deficient cultures yielding around 80% lower bacteria weight than those grown from vitamin K replete conditions [76]. Two other reports also explained the role of vitamin K at the membrane level. The reports showed the essential requirement of vitamin K for sphingolipid synthesis. Recent published studies that confirm the modulation of brain sphingolipids by vitamin K nutritional status, underscore the potentially far-reaching effect of vitamin K in brain function given the key role of these lipids in cell-signaling functions.

In the nervous system, vitamin K activates the carboxylation and activation of Gla residues on GAS6 protein (growth arrest-specific gene 6 protein) which is structurally related to another vitamin K-dependent protein (VKDP), anticoagulation factor protein S [77]. GAS6 and related S protein bind and activate the receptor tyrosine kinases of the Tyro3, Axl, and Mer (TAM) family. They are responsible for cell signaling which stimulates the generation of central nervous system repair cells (oligodendrocytes) and increased myelin production including repair after myelin injury (demyelinating injury) [78]. Vitamin K may also act in the central nervous system independent to its role in the carboxylation reaction [79]. Vitamin K independent of VKDP, activates enzyme 3-ketodihydrosphingosine (3-KDS), involved in sphingolipid synthesis which is critical for healthy myelin [80].

Sakaue M et al. investigated the protective effects of different forms of Vitamin K (Vitamin K1 and Vitamin K2–4) in an *in vitro* experiment conducted in primary cultured neurons from cerebella of rat pups where methylmercury-induced the cell death. They also investigated its protective effect against GSH-depletion-induced cell death by employing two intracellular glutathione (GSH) reducers, L-buthionine

### *Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

sulfoximine (BSO) and diethyl maleate (DEM), in primary cultured neurons and human neuroblastoma IMR-32 cells. It was observed that all the forms of Vitamin K inhibited the death of the primary cultured neurons indicating that vitamin K forms have the potential to protect neurons against cytotoxic methylmercury and agents that deplete GSH, without increasing intracellular GSH levels [81]. Kenji Onodera et al. while examining the antinociceptive effects of Vitamin K2–4 in diabetic mice found that no significant difference exist between non-diabetic and diabetic mice in the Vitamin K2–4 induced changes in the nociceptive threshold. This indicated the therapeutic effectiveness of Vitamin K2–4 for treating painful diabetic neuropathy [82].

A serendipitous discovery by two researchers, Mehta and Vaidya is that Menaquinone-7 relieves idiopathic muscle cramps as well as symptoms of diabetic neuropathy. PCT/IN2008/000465, application further claims the safety of usage of Menaquinone-7 in the various novel conditions like neuropathy [56].

In an open labeled study conducted by Kulkarni et al., it was shown that Menaquinone-7 at a dose of 100 mcg twice a day for 8 weeks (**Figure 5**) was well tolerated and safe with a therapeutic activity for the symptoms of peripheral neuropathy [83].

Based on the results of these studies, the next study which is a follow-up study in a larger cohort (n = 100) was planned to address the peripheral neuropathy experienced by patients. Menaquinone-7 capsules (100 mcg / capsule, twice a day) were given orally for 8 weeks and were followed up to 12 weeks. By twelfth week, the score was reduced in megaloblastic anemia as well as in diabetes mellitus groups to 1–2 (**Figure 6**). The decrease was statistically significant (P < 0.0001). The tingling and numbness had reduced significantly. There was a significant decrease in the weakness and fatigue [84].

Recently a double-blind placebo-controlled efficacy and safety study of Menaquinone-7 was conducted in 60 patients presenting with peripheral neuropathy and suffering from either vitamin B12 deficiency and/ or type 2 diabetes mellitus.

#### **Figure 5.**

*Decrease in the intensity and severity of PN from baseline to 8th week as noted on VAS score in the groups a and B, where group A (severe) had a VAS score of 8–9 and group B (moderate) with a score of 6–8 at baseline.*

#### **Figure 6.**

*Decrease in the intensity and severity of PN from baseline to 8th week as noted on VAS score in the groups VBD (Vitamin B12 deficiency) and T2DM (type 2 diabetes mellitus).*

Patients from both the groups' i.e., Vitamin B12 deficiency and type 2 diabetes mellitus had overall VAS score of 9 at baseline. By the end of the twelfth week, patients who were receiving Menaquinone-7 showed statistically significant reduction in the VAS score in Vitamin B12 deficiency as well as in type 2 diabetes mellitus to 2; whereas the patients who were taking placebo in Vitamin B12 deficiency group had reduced to 8, and in type 2 diabetes mellitus group to 9. This study was again performed with same protocol along with estimation of serum Menaquinone-7 levels in serum in a small sample size. The VAS score showed an inverse relationship between Menaquinone-7 levels and peripheral neuropathy symptoms (**Figure 7**) [85, 86].

Antineoplastic agents are the chemotherapy drugs used for cancer. Chemotherapyinduced peripheral neuropathy (CIPN) is one of the most frequent side effects caused by antineoplastic agents having a prevalence ranging from 19% to over 85%. CIPN is a mostly sensory neuropathy and is associated with motor and autonomic changes of varying intensity and duration [87]. Chemotherapeutics induces toxicity in peripheral nervous system. Oxaliplatin, an antineoplastic agent damages the blood brain barrier (BBB). The possible mechanisms of BBB damage may include proinflammatory cytokines, ROS, or other neurotransmitters, all of which are involved in the peripheral nervous system toxicity induced by chemotherapeutics [88, 89].

#### **Figure 7.**

*Average VAS score and serum Vitamin K2–7 levels (ng/ml) in Vitamin B12 deficiency group (A) and type 2 diabetes mellitus group (B).*

*Menaquinone-7: Wide Ranging Physiological Relevance in Muscle and Nerve Health DOI: http://dx.doi.org/10.5772/intechopen.99809*

The study of Sanna et al. has shown a direct correlation between structural changes in the central nervous system and chemotherapy-induced neurotoxicity [90].

An open labeled observational study to evaluate the iatrogenic neuropathy and its amelioration using Menaquinone-7 in patients with Multiple Myeloma with drug induced PN suggests for the first time that Menaquinone-7 has an ameliorative potential for relief of iatrogenic PN in Multiple Myeloma patients. Menaquinone-7 reduces the symptoms of PN like tingling, numbness, burning sensation, pain, causalgia, wooly feeling, and cramps caused during treatment of MM, thus Menaquinone-7 is found to be useful in the treatment of PN caused due to the therapy of MM [91].

Multiple myeloma (MM) is a type of hematological cancer which is characterized by excessive production of malignant plasma cell clones in the bone marrow [92]. Incidence of iatrogenic PN has been observed in patients with MM who received chemotherapy. It is primarily of a sensory or sensorimotor nature, and the symptoms of tingling, numbness, burning sensation and pain are predominantly bilaterally symmetric [93]. Development of debilitating drug induced PN is one of the major challenges in the treatment of MM, affecting compliance leading to discontinuation of therapy or dose/drug modification [94]. Thus, there is a need of any modality that could reduce the severity and allows continuation of effective therapy in the clinical setting. This preliminary observational study is the first study revealing the potential of Menaquinone-7 in relieving the symptoms of iatrogenic PN in MM patients.

## **7. Conclusion**

Menaquinone-7 appears promising in the areas of chronic degenerative conditions such as bone health, cardiovascular, diabetes, energy metabolism, peripheral neuropathy, cramps etc. Newer research is ongoing to confirm its role in many other areas including immunity, cognition, cancer etc. With the recent discovery of its many biological functions, Menaquinone-7 is sometimes referred to as a multitasking vitamin. Globally speaking Menaquinone-7 is negligible in diet consumed by the population all around the world except in small pockets leading to Menaquinone-7 insufficiency. Either lack or deficiency of a given vitamin invites multiple pathologies, some mild some severe, some experiential some silent, some acute some chronic. Until this knowledge is available to an individual, he is likely to consider multiple healing effects of a vitamin as panacea. Researchers have now realized that most of the global population is facing multiple severe morbidities due to lack of or inadequate levels of Menaquinone-7 in diet and supplementation. The "Next Big Thing" in medicine is Menaquinone-7 with what the science has revealed already. This vitamin will advance on an exponential curve.

## **Conflict of interest**

Dr. Dilip S. Mehta, Dr. Anselm de Souza, and Dr. Shashank S. Jadhav are CEO, Managing Director and Medical Director of Synergia Life Sciences Pvt. Ltd.

*Vitamin K - Recent Topics on the Biology and Chemistry*

## **Author details**

Dilip Mehta, Anselm de Souza and Shashank S. Jadhav\* Synergia Life Sciences Pvt. Ltd., Mumbai, India

\*Address all correspondence to: shashank@viridisbiopharma.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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## **Chapter 5**
