PKB/AKT has also been shown to affect the proteasomal degradation of HIF-1α through glycogen synthase kinase 3β (GSK3β) [75]. GSK3β-mediated phosphorylation of HIF-1α facilitates its binding to FBW7, an E3 ubiquitin ligase, which recognizes GSK3β-phosphorylated proteins and targets them for proteasomal degradation [76]. Since inactivating phosphorylation of GSK3β is primarily mediated by PKB/AKT, activation of the PKB/AKT pathway not only influences the translational rate of HIF-1α via mTOR but also mimics the effect of hypoxia via

To make the picture even more colorful, mTOR also phosphorylates MINT3, a membrane-type matrix metalloproteinase (MT-MMPs) regulator, at its threonine 5/serine 7 residues [77]. This modification promotes binding of MINT3 to FIH-1 leading to the inactivation of the latter [78]. By sequestering the HIF-1 suppressor FIH-1 to the Golgi membrane in cooperation with the MT1-MMP, the mTOR/MINT3/MT1-MMP axis can also support transcriptional activity of HIF-1 independently of its translational rate. Interestingly, in renal cell carcinoma, MT1-MMP has been found to be a target gene for HIF-2 raising the question if the mTOR-regulated MINT3/MT1- MMP/FIH-1-mediated positive feedback loop is a general mechanism in the regulation of HIFs [79]. Although its biological relevance is yet to be determined, it is noteworthy that mTOR has also been reported to associate with HIF-1α via the mTOR complex 1 member RAPTOR and a putative TOR motif within the HIF-1α polypeptide [66]. Since mTOR is a serine/threonine kinase and one of the known posttranslational modifications that favors HIF-1α transcriptional activity is phosphorylation, the possibility that this co-localization also supports the effect of mTOR on

HIF-1 via direct phosphorylation cannot be excluded but is yet to be confirmed [66, 80].

Eventually, rapamycin-sensitive upregulation of HIF-1 supports the induction of a wide range of HIF-1 targets, of which many have been found to form feedback loops via regulation of the hypoxia-related activity of mTOR. These include REDD1 that activates the tuberous sclerosis complex 1/2 (TSC1/2) [81]. The TSC1/2 possesses GTPase-activating function that renders the mTOR activator RHEB inactive [82]. BNIP3, another known HIF-1 target, also facilitates the accumulation of the GDP-bound form of RHEB and the consequent downregulation of mTOR

). Current data indicate that, following its

activates the PKB/AKT-mTOR

O2

matrix while producing hydrogen peroxide (H2

54 Cell Signalling - Thermodynamics and Molecular Control

HIF-dependent upregulation in hypoxia, LOX-generated H2

inhibition of proteasomal degradation of the HIF-1α polypeptide.

Besides its role in the mTOR-mediated HIF regulation discussed above, hypoxia-inducible miR-155 is one of the identified measures directly targeting HIF-1α mRNA, indicating its pivotal role in the regulation of the HIF pathway. Sequence analyses revealed that, besides the HIF responsive element, NF-κB consensus sequences are also present in the miR-155 promoter indicating the capacity of NF-κB-mediated stimuli to influence the HIF pathway via miR-155 [47]. Studies on the proposed link between hypoxia and inflammation revealed that NF-κB can also induce *HIF1A* via evolutionary conserved consensus binding sites identified in the *HIF1A* promoter [72, 85–87]. Since this induction is not sufficient for the accumulation of HIF-1α in the absence of hypoxia, current data suggest that the canonical NF-κB pathway is rather for pre-setting the HIF-1α mRNA level according to the redox state and inflammatory cytokine composition of the extracellular milieu [88]. This concept is further supported by the observation that NF-κB activity can be both up- and downregulated upon inhibition of prolyl-4-hydroxylases depending on the NF-κB stimulus received [89].

Intriguingly, besides *HIF1A*, NF-κB transactivates *ARNT* as well, leading to enhanced formation of HIF-1β:HIF-2α that attenuates the proteasomal degradation of the latter. Considering the celltype specific expression pattern of HIF-2α, the NF-κB-mediated induction of *ARNT* may represent a tissue-specific arm of the NF-κB-governed regulation of hypoxia signaling [90]. The interplay, however, is apparently bidirectional and the hypoxic signal can also be conveyed to the inflammatory pathway. Under normoxic conditions, PHDs inhibit the I kappa B kinase (IKK) complex attenuating the dissociation of inhibitory kappa B (IκB) from NF-κB [85, 88, 91]. In the absence of oxygen, however, the PHD-mediated blockade of IKK is resolved, the IKK complex becomes active leading to the phosphorylation of IκB and release of sequestered NF-κB subunits. The consequent formation of active NF-κB heterodimers culminates in moderate upregulation of the basal NF-κB activity that is believed to potentiate NF-κB responsiveness to cytokines like the tumor necrosis factor-alpha (TNF-α) or reactive oxygen species, stimuli typically accompanying inflammatory conditions [72, 92–95]. Thus, the interaction between the NF-κB and HIF pathways well illustrates the close pathophysiologic connection between hypoxia and inflammation and allows the cell to integrate inflammatory stimuli in the adaptive response under hypoxic conditions.

Anabolic extracellular signals that activate the mTOR pathway often diverge and activate the ERK signaling cascade as well, raising the question if ERK and hypoxia signaling interplay. Experimental data indicate that the HIF-1α polypeptide can be phosphorylated by p42/44 MAP kinases both under hypoxic conditions and in response to receptor-mediated ERKactivating stimuli [96–98]. The ERK-mediated phosphorylation was found to enhance the transcriptional activity of HIF-1 in various model systems, although the exact mechanism is still not clear [99, 100]. On one hand, it was proposed that phosphorylation of HIF-1α at positions 641 and 643 supports the transcriptional activity by attenuating its nuclear export [101, 102]. On the other, it was also demonstrated that ERK activity fundamentally alters the predicted composition of HIF-1-containing nuclear complexes suggesting multiple effects of ERK activity on hypoxia signaling [103]. Independently of the mechanistic details, current data suggest that ERK-mediated upregulation of the HIF pathway differs from the mTORmediated effect and, primarily, acts on the transactivation function of HIFs, possibly, complementing the mTOR-mediated effects (**Figure 4**).

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## **5. Conclusion**

Extensive experimental work over the past three decades deciphered the molecular background of the cellular response to oxygen depletion, one of the fundamental physiologic processes. To date, these efforts depictured an intricate molecular network that bridges, apparently, every aspect of cellular physiology. Within this network, the PHD-HIF axis plays an integrative role of various signals that allows the hypoxic cell to shape dynamics of the adaptive response according to the actual endogenous metabolic state and surrounding microenvironment alike. Deeper understanding of these molecular machineries gives the opportunity to develop more efficient medical modalities for pathologies like chronic inflammation, ischemia or neoplasms.

## **Acknowledgements**

The Open Access fee of this chapter was funded by the Munchausen fund provided by the Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Faculty of Medicine, Semmelweis University.

## **Conflict of interest**

The author declares no conflict of interest.

## **Author details**

Zsolt Fabian

Address all correspondence to<EMAIL_ADDRESS>
Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of Medicine, Semmelweis University, Budapest, Hungary

## **References**

[101, 102]. On the other, it was also demonstrated that ERK activity fundamentally alters the predicted composition of HIF-1-containing nuclear complexes suggesting multiple effects of ERK activity on hypoxia signaling [103]. Independently of the mechanistic details, current data suggest that ERK-mediated upregulation of the HIF pathway differs from the mTORmediated effect and, primarily, acts on the transactivation function of HIFs, possibly, comple-

Extensive experimental work over the past three decades deciphered the molecular background of the cellular response to oxygen depletion, one of the fundamental physiologic processes. To date, these efforts depictured an intricate molecular network that bridges, apparently, every aspect of cellular physiology. Within this network, the PHD-HIF axis plays an integrative role of various signals that allows the hypoxic cell to shape dynamics of the adaptive response according to the actual endogenous metabolic state and surrounding microenvironment alike. Deeper understanding of these molecular machineries gives the opportunity to develop more efficient medical modalities for pathologies like chronic inflam-

The Open Access fee of this chapter was funded by the Munchausen fund provided by the Department of Medical Chemistry, Molecular Biology, and Pathobiochemistry, Faculty of

menting the mTOR-mediated effects (**Figure 4**).

56 Cell Signalling - Thermodynamics and Molecular Control

**5. Conclusion**

mation, ischemia or neoplasms.

Medicine, Semmelweis University.

The author declares no conflict of interest.

Address all correspondence to<EMAIL_ADDRESS>
Medicine, Semmelweis University, Budapest, Hungary

Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Faculty of

**Acknowledgements**

**Conflict of interest**

**Author details**

Zsolt Fabian

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**Chapter 5**

**Provisional chapter**

**Vitamin K2: A Vitamin that Works like a Hormone,**

**Vitamin K2: A Vitamin that Works like a Hormone,**

DOI: 10.5772/intechopen.80388

Vitamin K2 binds to the intranuclear receptor SXR and results in the activation of a plethora of genes, both directly and indirectly. Among these genes are important biological markers of cellular characteristics or features (also known as cell phenotypes), as well as a set of molecules known to be involved in both hormone-induced, G-protein-mediated cell signalling, either directly or indirectly activating so-called sirtuins and/or histone deacetylaces (HDACs), known as determinants of cell types and their specific functions in a given tissue. Hence, vitamin K2 may be closely involved in or serving as a traditional molecular 'link' between hormonal receptors and intracellular signalling pathways. It has been stated that a true hormone is a product of living cells, which circulates in body fluids (such as blood) and elicits a specific and often stimulatory effect on the activity of cells situated remotely from its point of origin. A large bulk of evidence published over the past 10 years establishes vitamin K2 in this category of substances. Hence, vitamin K2

should be considered and consequently classified as a hormone.

**Keywords:** vitamin K2, SXR, G-proteins, vitamin A-D-K2 cascade, bioinformatics,

© 2016 The Author(s). Licensee InTech. 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,

© 2019 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.

and reproduction in any medium, provided the original work is properly cited.

Vitamin K has since more than 25 years been known to serve as a powerful nutrient factor in the preservation of homeostatic bone turnover, along with blood clotting biochemistry. In addition, vitamin K has been used as a therapeutic remedy in the clinic to treat and prevent bone brittleness (osteoporosis) in Japan and many other countries around the world. Moreover, beyond its enzymatic character as a cofactor for the vitamin K-dependent GGCX (gammaglutamyl carboxylase), Professor Inoue and his co-workers have shown that the K2 variant is,

**Impinging on Gene Expression**

**Impinging on Gene Expression**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80388

Jan Oxholm Gordeladze

Jan Oxholm Gordeladze

**Abstract**

in vitro model systems

**1. Introduction**

#### **Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression**

DOI: 10.5772/intechopen.80388

Jan Oxholm Gordeladze Jan Oxholm Gordeladze

[101] Mylonis I et al. Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1 alpha. The Journal of Biological

[102] Mylonis I et al. Atypical CRM1-dependent nuclear export signal mediates regulation of hypoxia-inducible factor-1 alpha by MAPK. The Journal of Biological Chemistry.

[103] Fabian Z et al. Basic fibroblast growth factor modifies the hypoxic response of human bone marrow stromal cells by ERK-mediated enhancement of HIF-1 alpha activity.

Chemistry. 2006;**281**(44):33095-33106

Stem Cell Research. 2014;**12**(3):646-658

2008;**283**(41):27620-27627

64 Cell Signalling - Thermodynamics and Molecular Control

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.80388

#### **Abstract**

Vitamin K2 binds to the intranuclear receptor SXR and results in the activation of a plethora of genes, both directly and indirectly. Among these genes are important biological markers of cellular characteristics or features (also known as cell phenotypes), as well as a set of molecules known to be involved in both hormone-induced, G-protein-mediated cell signalling, either directly or indirectly activating so-called sirtuins and/or histone deacetylaces (HDACs), known as determinants of cell types and their specific functions in a given tissue. Hence, vitamin K2 may be closely involved in or serving as a traditional molecular 'link' between hormonal receptors and intracellular signalling pathways. It has been stated that a true hormone is a product of living cells, which circulates in body fluids (such as blood) and elicits a specific and often stimulatory effect on the activity of cells situated remotely from its point of origin. A large bulk of evidence published over the past 10 years establishes vitamin K2 in this category of substances. Hence, vitamin K2 should be considered and consequently classified as a hormone.

**Keywords:** vitamin K2, SXR, G-proteins, vitamin A-D-K2 cascade, bioinformatics, in vitro model systems

## **1. Introduction**

Vitamin K has since more than 25 years been known to serve as a powerful nutrient factor in the preservation of homeostatic bone turnover, along with blood clotting biochemistry. In addition, vitamin K has been used as a therapeutic remedy in the clinic to treat and prevent bone brittleness (osteoporosis) in Japan and many other countries around the world. Moreover, beyond its enzymatic character as a cofactor for the vitamin K-dependent GGCX (gammaglutamyl carboxylase), Professor Inoue and his co-workers have shown that the K2 variant is,

© 2016 The Author(s). Licensee InTech. 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. © 2019 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.

in fact, a transcriptional modulator of osseous marker genes, also serving as an extracellular matrix-related molecule, via the stimulation of the 'steroid and xenobiotic receptor' SXR [1]. A microarray-based revelation of the present action of vitamin K2 in bone-derived osteoblastic cell genes corroborated the notion that the K2 variant menaquinone-4 (MK-4), in fact, is a hormone. Among the significantly upregulated genes, both growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were concluded to serve as novel target genes, which both circumvented the traditional GGCX- and SXR-mediated pathways found in osteoblast-like cells.

selected populations. The chief mechanisms pertain to anti-atherogenic potential, substantial augmentation of endothelial functionality (pertaining to vitamins A, C, D and E, respectively) and general metabolic profiling (pertaining to vitamins A, B12, C, D and K, respectively), suppression of the renin-angiotensin-aldosterone (R-R-A) system (vitamin D), anti-inflammatory (vitamins A-D-E-K) and antioxidant effects (vitamins A-C-E), diminished homocysteine levels (vitamin B12) and a reversal of the calcification of arteries (vitamin K). Vitamins A, B12, C, D and E, as well as vitamin K status, are important in evaluating the risk of cardiovascular events, and, finally, supplementation with vitamins may serve as an efficient, individually

Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression

http://dx.doi.org/10.5772/intechopen.80388

67

Vitamin K is renowned for being an important (vital) nutrient, sustaining both bone homeostasis and blood coagulation. Therefore, it is both clinically and ubiquitously used as a therapeutic agent or treatment for osteoporosis in Japan and western countries. Besides its powerful enzymatic, cofactor role of the vitamin K-dependent g-glutamyl carboxylase (GGCX), it has since long been known that vitamin K2 may serve as a transcriptional regulator of marker osteoblastic genes, as well as in matrix-related, extracellular genes. In this context the activa-

Hence, genes known to be upregulated by vitamin K2 isoforms like menaquinone-4 (MK-4) were applied using oligonucleotide-based microarray analyses. Among the MK-4 upregulated gene species, the growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were discovered as new MK-4 target genes, being independent of the GGCX and SXR pathways in osteoblastic cells. The observed induction of GDF15 and STC2 was construed as specific to MK-4, since it was not seen with exposure to MK-7 or vitamin K1. Surprisingly, a scrutiny of the main signalling pathways showed that MK-4 stimulated PKA (protein kinase A) phosphorylation. Furthermore, the MK-4-dependent induction of both GDF15 and STC2 genes was obliterated subsequent to the treatment with PKA inhibitors or siRNAs against PKA. Therefore, it was postulated that vitamin K2 (MK-7) could modulate target gene expression in osteoblastic cells via PKA-dependent mechanisms, which were distinct from any pre-

The paper found that vitamin K2 is recognised, along with calcium, vitamin D and magnesium, as essential in supporting strong bones and healthy arteries. In the paper, *Nutritional strategies for skeletal and cardiovascular health: hard bones, soft arteries, rather than vice versa*, the authors cite a US Surgeon General's Report that states that one in two Americans over 50 is expected to have or to be at risk of developing osteoporosis, which causes 8.9 million fractures annually, with an estimated cumulative cost of incident fractures predicted at \$474 billion

Furthermore, a study conducted by the Mayo Clinic [4, 5] reported that 'compared with 30 years ago, forearm fractures have risen more than 32% in boys and 56% in girls'. Meanwhile, strong epidemiological associations exist between decreased bone mineral density (BMD) and increased risk of cardiovascular (CV) disease. For example, individuals with osteoporosis have a higher risk of coronary artery disease and vice versa. This problem will be magnified, according to the paper, if the therapies for osteoporosis (calcium supplements) independently increase risk of myocardial infarction. To that end, the authors conducted a comprehensive and

tion of the so-called steroid and xenobiotic receptor (known as SXR) is mandatory.

based and less costly 'destiffening' therapeutic mode.

viously known vitamin K-mediated signalling pathway.

during the next 20 years in the USA.

## **2. Vitamin K2: vitamin and hormone**

The induction of both GDF15 and STC2 genes is construed as specific to MK-4, since it was shown not to be brought about by another vitamin K(2) isoform MK-7, vitamin K(1) or the MK-4 side chain containing the geranylgeraniol group. A survey into the signalling pathways in question indicated that MK-4 sustained phosphorylation of protein kinase A (PKA) and that MK-4 mediated upregulation of genes, such as GDF15 and STC2, was diminished by the exposure to a PKA inhibitor or by siRNA constructs against PKA. These observations were in line with the concept that vitamin K(2) was capable of modulating its own target gene expression in bone-derived (osteoblastic) cell entities through a PKA-driven pathway, which in essence was different from the traditional vitamin K-dependent signalling pathways [2].

Vitamin K2 has been included as a member in a group of molecules, constituting the 'requirement' for blood to coagulate; however, it has been demonstrated to function or serve as a key in the homeostasis of osseous tissue, thus showing effectiveness as one therapeutic agent, among others, in the curative treatment portfolio of bone ailments and diseases, i.e. like bone loss or brittleness (osteomalacia and osteoporosis). Furthermore, it has, since several decades, been acknowledged that vitamin K2 mediates transcriptional modulation of marker genes in osteoblastic cells, as well as reinforcing bone formation via the nuclear steroid and xenobiotic receptor, SXR. In this context, Dr. Satoshi Inoue and his research team identified several genes, which were upregulated by vitamin K2 (and a prototypical SXR like ligand, rifampicin) in osteoblastic cells, through microarray analysis and PCR.A plethora of genes was upregulated, among which collagen synthesis and accumulation in osteoblast-like MG63 cells were enhanced several times over by vitamin K2 treatment. Therefore, the results of Dr. Inoue and his research group more than suggested a novel function for vitamin K2 in the formation of osseous tissues, i.e. that K2 was a true transcriptional regulator of extracellular matrix-related genes, being involved in the assembly of collagen. At present, we know that vitamin K2 (or menaquinone-7 = MK-7, among other vitamin K2 metabolites) works through this nuclear receptor and consequently should be classified as a hormone and not solely be construed as 'a vitamin'.

Arterial stiffness is always associated with an enhanced cardiovascular risk, morbidity and mortality [3]. The present article reviews the main vitamins being involved in arterial stiffness and enabling destiffening; their mechanism of action, providing a brief description of the latest studies in the area; and their implications for primary cardiovascular prevention, clinical practice and therapy. Despite inconsistent proof for 'softening' brought about by vitamin supplementation in a plethora of clinical trials, promising results were observed in selected populations. The chief mechanisms pertain to anti-atherogenic potential, substantial augmentation of endothelial functionality (pertaining to vitamins A, C, D and E, respectively) and general metabolic profiling (pertaining to vitamins A, B12, C, D and K, respectively), suppression of the renin-angiotensin-aldosterone (R-R-A) system (vitamin D), anti-inflammatory (vitamins A-D-E-K) and antioxidant effects (vitamins A-C-E), diminished homocysteine levels (vitamin B12) and a reversal of the calcification of arteries (vitamin K). Vitamins A, B12, C, D and E, as well as vitamin K status, are important in evaluating the risk of cardiovascular events, and, finally, supplementation with vitamins may serve as an efficient, individually based and less costly 'destiffening' therapeutic mode.

in fact, a transcriptional modulator of osseous marker genes, also serving as an extracellular matrix-related molecule, via the stimulation of the 'steroid and xenobiotic receptor' SXR [1]. A microarray-based revelation of the present action of vitamin K2 in bone-derived osteoblastic cell genes corroborated the notion that the K2 variant menaquinone-4 (MK-4), in fact, is a hormone. Among the significantly upregulated genes, both growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were concluded to serve as novel target genes, which both circumvented

The induction of both GDF15 and STC2 genes is construed as specific to MK-4, since it was shown not to be brought about by another vitamin K(2) isoform MK-7, vitamin K(1) or the MK-4 side chain containing the geranylgeraniol group. A survey into the signalling pathways in question indicated that MK-4 sustained phosphorylation of protein kinase A (PKA) and that MK-4 mediated upregulation of genes, such as GDF15 and STC2, was diminished by the exposure to a PKA inhibitor or by siRNA constructs against PKA. These observations were in line with the concept that vitamin K(2) was capable of modulating its own target gene expression in bone-derived (osteoblastic) cell entities through a PKA-driven pathway, which in essence was different from the traditional vitamin K-dependent signalling pathways [2]. Vitamin K2 has been included as a member in a group of molecules, constituting the 'requirement' for blood to coagulate; however, it has been demonstrated to function or serve as a key in the homeostasis of osseous tissue, thus showing effectiveness as one therapeutic agent, among others, in the curative treatment portfolio of bone ailments and diseases, i.e. like bone loss or brittleness (osteomalacia and osteoporosis). Furthermore, it has, since several decades, been acknowledged that vitamin K2 mediates transcriptional modulation of marker genes in osteoblastic cells, as well as reinforcing bone formation via the nuclear steroid and xenobiotic receptor, SXR. In this context, Dr. Satoshi Inoue and his research team identified several genes, which were upregulated by vitamin K2 (and a prototypical SXR like ligand, rifampicin) in osteoblastic cells, through microarray analysis and PCR.A plethora of genes was upregulated, among which collagen synthesis and accumulation in osteoblast-like MG63 cells were enhanced several times over by vitamin K2 treatment. Therefore, the results of Dr. Inoue and his research group more than suggested a novel function for vitamin K2 in the formation of osseous tissues, i.e. that K2 was a true transcriptional regulator of extracellular matrix-related genes, being involved in the assembly of collagen. At present, we know that vitamin K2 (or menaquinone-7 = MK-7, among other vitamin K2 metabolites) works through this nuclear receptor and consequently should be

the traditional GGCX- and SXR-mediated pathways found in osteoblast-like cells.

**2. Vitamin K2: vitamin and hormone**

66 Cell Signalling - Thermodynamics and Molecular Control

classified as a hormone and not solely be construed as 'a vitamin'.

Arterial stiffness is always associated with an enhanced cardiovascular risk, morbidity and mortality [3]. The present article reviews the main vitamins being involved in arterial stiffness and enabling destiffening; their mechanism of action, providing a brief description of the latest studies in the area; and their implications for primary cardiovascular prevention, clinical practice and therapy. Despite inconsistent proof for 'softening' brought about by vitamin supplementation in a plethora of clinical trials, promising results were observed in Vitamin K is renowned for being an important (vital) nutrient, sustaining both bone homeostasis and blood coagulation. Therefore, it is both clinically and ubiquitously used as a therapeutic agent or treatment for osteoporosis in Japan and western countries. Besides its powerful enzymatic, cofactor role of the vitamin K-dependent g-glutamyl carboxylase (GGCX), it has since long been known that vitamin K2 may serve as a transcriptional regulator of marker osteoblastic genes, as well as in matrix-related, extracellular genes. In this context the activation of the so-called steroid and xenobiotic receptor (known as SXR) is mandatory.

Hence, genes known to be upregulated by vitamin K2 isoforms like menaquinone-4 (MK-4) were applied using oligonucleotide-based microarray analyses. Among the MK-4 upregulated gene species, the growth differentiation factor 15 (GDF15) and stanniocalcin 2 (STC2) were discovered as new MK-4 target genes, being independent of the GGCX and SXR pathways in osteoblastic cells. The observed induction of GDF15 and STC2 was construed as specific to MK-4, since it was not seen with exposure to MK-7 or vitamin K1. Surprisingly, a scrutiny of the main signalling pathways showed that MK-4 stimulated PKA (protein kinase A) phosphorylation. Furthermore, the MK-4-dependent induction of both GDF15 and STC2 genes was obliterated subsequent to the treatment with PKA inhibitors or siRNAs against PKA. Therefore, it was postulated that vitamin K2 (MK-7) could modulate target gene expression in osteoblastic cells via PKA-dependent mechanisms, which were distinct from any previously known vitamin K-mediated signalling pathway.

The paper found that vitamin K2 is recognised, along with calcium, vitamin D and magnesium, as essential in supporting strong bones and healthy arteries. In the paper, *Nutritional strategies for skeletal and cardiovascular health: hard bones, soft arteries, rather than vice versa*, the authors cite a US Surgeon General's Report that states that one in two Americans over 50 is expected to have or to be at risk of developing osteoporosis, which causes 8.9 million fractures annually, with an estimated cumulative cost of incident fractures predicted at \$474 billion during the next 20 years in the USA.

Furthermore, a study conducted by the Mayo Clinic [4, 5] reported that 'compared with 30 years ago, forearm fractures have risen more than 32% in boys and 56% in girls'. Meanwhile, strong epidemiological associations exist between decreased bone mineral density (BMD) and increased risk of cardiovascular (CV) disease. For example, individuals with osteoporosis have a higher risk of coronary artery disease and vice versa. This problem will be magnified, according to the paper, if the therapies for osteoporosis (calcium supplements) independently increase risk of myocardial infarction. To that end, the authors conducted a comprehensive and systematic review of the scientific literature to determine the optimal dietary strategies and nutritional supplements for long-term skeletal health and cardiovascular health. They summarised what is helpful for building strong bones while maintaining soft and supple arteries:

**3. What features characterise a true hormone?**

**Figure 5**).

To make the picture clearer, let us start all over again. According to **Figure 1**, the impact of a hormone is one signal among differentiation signals (also called epigenator factors), which may be temperature variations, oxygen tension, mechano-stimulation or humoral factor/ hormones. The 'hormone' eventually activates a transcription factor or microRNA synthesis, which may impinge upon DNA in terms of a certain or given spectrum of mRNAs appearing in the cytosol of the cell. Of major interest here are eventually two classes of molecules utmost important for the acquisition of the final cell phenotype—the histones and the sirtuins (see

Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression

http://dx.doi.org/10.5772/intechopen.80388

69

The response elicited by a single hormone (epigenator) may look like the network of interacting factors (mostly transcription factors), such as the network representing the closely cooperating network (mostly represented by transcription factor), as seen in **Figure 2a** and **b**.

The reader is recommended to look up the remainder of the genes shown above and will be amazed as to the plethora of biological effects being modulated (directly and/or indirectly) by the nuclear receptor NR1/NR2 = SXR, to which vitamin K2 binds, exerting its multitude of biological effects. The crucial question is then: what may be the immediate effect of vitamin K2 (e.g. MK-7) on cell phenotype, for example, on bone chips harbouring live osteoblastic cells? In **Figure 3**, bovine chips from young calves were incubated for 14 days in growth medium, which were analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (osteoblast and

**Figure 1.** The epigenator-initiator-maintainer model of hormonal impact on the phenotype of a given cell. The hormone (binding to a given receptor—here represented by two different transmembrane proteins) will eventually elicit a response determined by the joint effect of transcription factors and microRNAs determining the end-point effect of the

epigenator, such as cell differentiation and/or efflux of secretory products.

osteoclast markers), respectively. For detailed summary of results, see **Figure 3**.

The study notes: 'A meta-analysis concluded that while supplementation with phytonadione (vitamin K1) improved bone health, vitamin K2 was even more effective in this regard. This large and statistically rigorous meta-analysis concluded that high vitamin K2 levels were associated with reduced vertebral fractures by approximately 60%, hip fractures by 77% and all non-vertebral fractures by approximately 81%. Supplementation with vitamin K2 as MK-7 increased bone strength in postmenopausal women in 3-year clinical study'.

'Additionally, increased vitamin K2 intake has been associated with decreased arterial calcium deposition and the ability to reverse vascular calcification in animal models. Moreover clinical trial proved that vitamin K2 supplementation increases elasticity of the arteries (in 3 years)', the paper stated. The authors recommend increasing the intake of foods rich in vitamins K1 and K2 to secure skeletal and cardiovascular health. 'The positive health potential of vitamin K2 is more effective than for vitamin K1, the paper reads. Yet, Dr. Hogne Vik, Chief Medical Officer with NattoPharma, world leader in vitamin K2 R&D, exclusive global supplier of MenaQ7 vitamin K2 as MK-7, and sponsor of the 3-year studies cited in the paper, explains that it is not possible to get sufficient amounts of vitamin K2 through a European or US diet'.

The only food that contains enough vitamin K2 is the Japanese dish Natto. 'This means that if you want to get enough vitamin K2 into your body, then you have to take dietary supplements or functional foods containing vitamin K2', he said. 'We are gratified, but not surprised, that our 3-year clinical studies were cited in this paper', Dr. Vik continues. 'NattoPharma has driven the clinical research that has demonstrated vitamin K2's benefits for human health, and our breakthrough studies provided the first intervention data confirming the associations that observational studies made previously: that vitamin K2 as MK-7 is available beyond the liver to support bone and cardiovascular health. And it does this by activating proteins that help the body to properly utilise calcium – there by simultaneously supporting both skeletal and cardiovascular health'.

How are we going to interpret the above-described information about the mechanism of action of vitamin K2? Well, according to the definition given in dictionaries and scientific papers, vitamin K2 (as in the form or MK-4 or MK-7) fits this definition and should be classified as one.

## **3. What features characterise a true hormone?**

systematic review of the scientific literature to determine the optimal dietary strategies and nutritional supplements for long-term skeletal health and cardiovascular health. They summarised what is helpful for building strong bones while maintaining soft and supple arteries:

• Obtain calcium from dietary sources (the best choice is a calcium hydroxyapatite) with the

• Concomitantly increase potassium consumption, while reducing sodium intake should be

The study notes: 'A meta-analysis concluded that while supplementation with phytonadione (vitamin K1) improved bone health, vitamin K2 was even more effective in this regard. This large and statistically rigorous meta-analysis concluded that high vitamin K2 levels were associated with reduced vertebral fractures by approximately 60%, hip fractures by 77% and all non-vertebral fractures by approximately 81%. Supplementation with vitamin K2 as MK-7

'Additionally, increased vitamin K2 intake has been associated with decreased arterial calcium deposition and the ability to reverse vascular calcification in animal models. Moreover clinical trial proved that vitamin K2 supplementation increases elasticity of the arteries (in 3 years)', the paper stated. The authors recommend increasing the intake of foods rich in vitamins K1 and K2 to secure skeletal and cardiovascular health. 'The positive health potential of vitamin K2 is more effective than for vitamin K1, the paper reads. Yet, Dr. Hogne Vik, Chief Medical Officer with NattoPharma, world leader in vitamin K2 R&D, exclusive global supplier of MenaQ7 vitamin K2 as MK-7, and sponsor of the 3-year studies cited in the paper, explains that it is not possible to get sufficient amounts of vitamin K2 through a

The only food that contains enough vitamin K2 is the Japanese dish Natto. 'This means that if you want to get enough vitamin K2 into your body, then you have to take dietary supplements or functional foods containing vitamin K2', he said. 'We are gratified, but not surprised, that our 3-year clinical studies were cited in this paper', Dr. Vik continues. 'NattoPharma has driven the clinical research that has demonstrated vitamin K2's benefits for human health, and our breakthrough studies provided the first intervention data confirming the associations that observational studies made previously: that vitamin K2 as MK-7 is available beyond the liver to support bone and cardiovascular health. And it does this by activating proteins that help the body to properly utilise calcium – there by simultaneously supporting both skeletal

How are we going to interpret the above-described information about the mechanism of action of vitamin K2? Well, according to the definition given in dictionaries and scientific papers, vitamin K2 (as in the form or MK-4 or MK-7) fits this definition and should be classi-

increased bone strength in postmenopausal women in 3-year clinical study'.

adequate animal protein, fruit and vegetable intake.

• Increase the intake of foods rich in vitamins K1 and K2.

• Maintain vitamin D levels in the normal range.

68 Cell Signalling - Thermodynamics and Molecular Control

taken into account.

European or US diet'.

and cardiovascular health'.

fied as one.

To make the picture clearer, let us start all over again. According to **Figure 1**, the impact of a hormone is one signal among differentiation signals (also called epigenator factors), which may be temperature variations, oxygen tension, mechano-stimulation or humoral factor/ hormones. The 'hormone' eventually activates a transcription factor or microRNA synthesis, which may impinge upon DNA in terms of a certain or given spectrum of mRNAs appearing in the cytosol of the cell. Of major interest here are eventually two classes of molecules utmost important for the acquisition of the final cell phenotype—the histones and the sirtuins (see **Figure 5**).

The response elicited by a single hormone (epigenator) may look like the network of interacting factors (mostly transcription factors), such as the network representing the closely cooperating network (mostly represented by transcription factor), as seen in **Figure 2a** and **b**.

The reader is recommended to look up the remainder of the genes shown above and will be amazed as to the plethora of biological effects being modulated (directly and/or indirectly) by the nuclear receptor NR1/NR2 = SXR, to which vitamin K2 binds, exerting its multitude of biological effects. The crucial question is then: what may be the immediate effect of vitamin K2 (e.g. MK-7) on cell phenotype, for example, on bone chips harbouring live osteoblastic cells? In **Figure 3**, bovine chips from young calves were incubated for 14 days in growth medium, which were analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (osteoblast and osteoclast markers), respectively. For detailed summary of results, see **Figure 3**.

**Figure 1.** The epigenator-initiator-maintainer model of hormonal impact on the phenotype of a given cell. The hormone (binding to a given receptor—here represented by two different transmembrane proteins) will eventually elicit a response determined by the joint effect of transcription factors and microRNAs determining the end-point effect of the epigenator, such as cell differentiation and/or efflux of secretory products.

**Figure 2.** (a) and (b) GeneCards-based emulation of molecules interacting with NR1/NR2 (which is identical to SXR, identified/described by the Japanese researcher, Professor Satoshi Inoue). Interestingly, NR1/NR2 is integrated in a large network of interacting molecules (genes), representing various classes of transcription factors (e.g. PPARG, RORC, RARA, RXR) and not to forget the thyroid receptors A and B!

The interpretation of the experiment with 'live' bovine bone chips is the following: since MK-7 (a vitamin K2 analogue) is stimulated, while SXR siRNA and pre-mir-760 markedly reduced the secretory profile of the bovine osteoblast, it was concluded that the effect of vitamin K2 (MK-7) on the osteoblast profile of secretory molecules was stimulatory and that the effect of

**Figure 4.** The impact of insulin and growth factors (GFs) on members of the FoxA and FoxO families of insulin and

Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression

http://dx.doi.org/10.5772/intechopen.80388

71

Furthermore, it was shown that one well-known and well-represented vitamin K2 'analogue' (MK-7) was able to activate (upregulate) some of the members of the FoxA and FoxO family of transcription factors. In our hands, vitamin K2 analogue MK-7 (and to a lesser extent MK-4) was able to substitute for insulin, as well as some growth factors (like growth hormone and a

**Figure 5** shows a strong relationship (network between a human osteoblast microRNA profile, respectively, and a well-known plethora of marker genes) which has previously been listed according to their sequential appearance as the osteoblast develops from a stem cell into a 'full-blown', mature mineralizing osteoblast. By adding a plethora of transcription factors, sirtuins (SIRTs), histone deacetylases (HDACs) and transcription factors (with special references to the osteoblastic phenotype), a distinct pattern of interaction appeared. By adding vitamin K2, i.e. MK-7 and/or SXR, to the system, a similar pattern appeared (not shown1

This was also the case for other tissues as well, e.g. lung, heart, brain, muscle, white/beige fat

).

).

vitamin K2 was mediated solely through the action of the nuclear receptor SXR.

few interleukins (not shown)) (**Figure 4**).

tissues and many others (not mentioned here2

Details not included—patents pending.

Details not included—patents pending.

1

growth factors.

2

**Figure 3.** Bovine chips from young calves incubated as stated above. The chips were (a) incubated for 7 or 21 days and analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (all parameters' characteristics for the osteoblast phenotype); (b) incubated for 7 days and thereafter for 14 days in the presence of either vitamin K2 = MK-7; siRNA against the vitamin K2 receptor SXR or with pre-mir-760. The final measurements of secretory products were as indicated above (osteocalcin, IL-10, TGFβ, OPG and RANKL).

Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression http://dx.doi.org/10.5772/intechopen.80388 71

**Figure 4.** The impact of insulin and growth factors (GFs) on members of the FoxA and FoxO families of insulin and growth factors.

The interpretation of the experiment with 'live' bovine bone chips is the following: since MK-7 (a vitamin K2 analogue) is stimulated, while SXR siRNA and pre-mir-760 markedly reduced the secretory profile of the bovine osteoblast, it was concluded that the effect of vitamin K2 (MK-7) on the osteoblast profile of secretory molecules was stimulatory and that the effect of vitamin K2 was mediated solely through the action of the nuclear receptor SXR.

Furthermore, it was shown that one well-known and well-represented vitamin K2 'analogue' (MK-7) was able to activate (upregulate) some of the members of the FoxA and FoxO family of transcription factors. In our hands, vitamin K2 analogue MK-7 (and to a lesser extent MK-4) was able to substitute for insulin, as well as some growth factors (like growth hormone and a few interleukins (not shown)) (**Figure 4**).

**Figure 5** shows a strong relationship (network between a human osteoblast microRNA profile, respectively, and a well-known plethora of marker genes) which has previously been listed according to their sequential appearance as the osteoblast develops from a stem cell into a 'full-blown', mature mineralizing osteoblast. By adding a plethora of transcription factors, sirtuins (SIRTs), histone deacetylases (HDACs) and transcription factors (with special references to the osteoblastic phenotype), a distinct pattern of interaction appeared. By adding vitamin K2, i.e. MK-7 and/or SXR, to the system, a similar pattern appeared (not shown1 ). This was also the case for other tissues as well, e.g. lung, heart, brain, muscle, white/beige fat tissues and many others (not mentioned here2 ).

**Figure 3.** Bovine chips from young calves incubated as stated above. The chips were (a) incubated for 7 or 21 days and analysed for osteocalcin, IL-10, TGFβ, OPG and RANKL (all parameters' characteristics for the osteoblast phenotype); (b) incubated for 7 days and thereafter for 14 days in the presence of either vitamin K2 = MK-7; siRNA against the vitamin K2 receptor SXR or with pre-mir-760. The final measurements of secretory products were as indicated above

**Figure 2.** (a) and (b) GeneCards-based emulation of molecules interacting with NR1/NR2 (which is identical to SXR, identified/described by the Japanese researcher, Professor Satoshi Inoue). Interestingly, NR1/NR2 is integrated in a large network of interacting molecules (genes), representing various classes of transcription factors (e.g. PPARG, RORC,

RARA, RXR) and not to forget the thyroid receptors A and B!

70 Cell Signalling - Thermodynamics and Molecular Control

(osteocalcin, IL-10, TGFβ, OPG and RANKL).

1 Details not included—patents pending.

2 Details not included—patents pending.

[3] Mosos I. Review article—Crosstalk between vitamins A, B12, D, K, C, and E status and arterial stiffness. Disease Markers. 2017;**2017**:14. Article ID: 8784971. DOI: 10.1155/2017/

Vitamin K2: A Vitamin that Works like a Hormone, Impinging on Gene Expression

http://dx.doi.org/10.5772/intechopen.80388

73

[5] Gordeladze JO. General Reading on Vitamin K2: Vitamin K2—Vital for Health and Well-

[4] https://www.bodyscience.com.au/blog/dr-hogne-vik-vitamin-k2-interview/

Being. IntechOpen. 2017. DOI: 10.5772/61430

8784971

**Figure 5.** Network (based on the computerprogram Mir@nt@n) postulating the interaction between key microRNAs, transcription factors, sirtuins, and histone deacetylases (HDACs) in the developing osteoblast from stem cells via preosteoblasts. The compilation of genes was based on various searches on PubMed for genes appearing during the course of preosteoblast to mature (mineralizing) osteoblasts from mammalian species, including humans.

## **Author details**

#### Jan Oxholm Gordeladze

Address all correspondence to<EMAIL_ADDRESS>
Department of Molecular Medicine, Section for Biochemistry, Institute of Basic Medical Science, Blindern, Norway

## **References**

**Author details**

**References**

Jan Oxholm Gordeladze

72 Cell Signalling - Thermodynamics and Molecular Control

Science, Blindern, Norway

Address all correspondence to<EMAIL_ADDRESS>
of Agricultural Science, Tohoku University. 2018

Journal of Molecular Endocrinology. 2007;**39**(4):239-247

Department of Molecular Medicine, Section for Biochemistry, Institute of Basic Medical

**Figure 5.** Network (based on the computerprogram Mir@nt@n) postulating the interaction between key microRNAs, transcription factors, sirtuins, and histone deacetylases (HDACs) in the developing osteoblast from stem cells via preosteoblasts. The compilation of genes was based on various searches on PubMed for genes appearing during the

course of preosteoblast to mature (mineralizing) osteoblasts from mammalian species, including humans.

[1] Sultana H, Watanabe K, Md MR, Takashima R, Ai O, Komai M, et al. Effects of Vitamin K2 on the Expression of Genes Involved in Bile Acid Synthesis and Glucose Homeostasis in Mice with Humanized PXR. Sendai, Japan: Laboratory of Nutrition, Graduate School

[2] Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S. Vitamin K2 induces phosphorylation of protein kinase A and expression of novel target genes in osteoblastic cells. [5] Gordeladze JO. General Reading on Vitamin K2: Vitamin K2—Vital for Health and Well-Being. IntechOpen. 2017. DOI: 10.5772/61430

## *Edited by Sajal Ray*

Intracellular signal transduction is an important topic of research in cell and molecular biology. It has a wide range of implications in growth, differentiation, development, animal polarity, cellular response monitoring, and many disease processes, including cancer. The functional homeostasis of a cell is assumed to be maintained by an intricate network of signaling that evolved through natural selection. This book includes chapters written in the frontier areas of research on cell signaling. Issues such as information thermodynamics, master regulation of immune tolerance, genetically encoded reporter circuits and their applications, hypoxia, and vitamin K2 mediated signaling processes are addressed and discussed by eminent researchers of the field.

Published in London, UK © 2019 IntechOpen © vchal / iStock

Cell Signalling - Thermodynamics and Molecular Control

Cell Signalling

Thermodynamics and Molecular Control

*Edited by Sajal Ray*


1.1.2. Operation

A 3.5 mm LCP PHILOS (Synthes, Solothurn, Switzerland) was inverted so that the locking screws could be inserted into both the talus and calcaneus and compression screws into the tibia. The plate was fixed to the lateral portion of the calcaneus and tibia using four 3.5 mm cortical screws proximally in the tibia and seven 3.5 mm locking screws distally. A 6.5 cannulated screw (Zimmer, Warsaw, IN, USA) was placed from the tibia across the talus and into the calcaneus to provide compression across the tibiotalar and subtalar joints. Three additional 3.5 mm screws with washers were placed from the navicular into the talus in order to fuse the talonavicular joint.

An additional 3.5 mm screw and washer were inserted from the anterior process of the calcaneus to the cuboid to achieve fusion at this joint. The patient was to remain non-weight bearing on her surgical extremity for three months.

#### 1.1.3. Post-Operative Follow-Up

Clinically, in the first year of follow up appointments, the patient was doing very well. Weight bearing as tolerated began after three months. She returned to the surgeon's office once yearly for follow up. During every follow up, new imaging (by X-ray) of the ankle was obtained. Imaging over this time showed that her ankle did not fuse and formed a non-union. Her hardware failed as seen with radiographic evidence of breakage. The hardware breakage progressed from a single screw to involving multiple screws and the plate over the post-operative course. At six years post-operative she complained of a new onset pain in her operative ankle. With the new onset symptoms and evidence of failed fusion and hardware failure, a removal of hardware and revision ankle fusion was planned.

#### 1.1.4. Post-Operative Imaging

X-rays from the 18-month post-op visit showed evidence of breakage of a distal 3.5 mm talonavicular screw (Figure A2). The fracture line was located in the thread near the screw head. At two years post-op, the screw breakage was more displaced and there was an additional fracture in the most proximal 3.5 mm cortical screw in the tibia (Figure A3). The fracture line of this screw was again in the thread near the screw head. At three years post-op, a new fracture of the most proximal 3.5 mm locking screw in the talus was present (Figure A4). There was also evidence of a newly forming valgus deformity. At five years post-op, an additional 3.5 mm cortical screw in the tibia, a second 3.5 mm talonavicular screw, the 6.5 mm cannulated screw, and the 3.5 mm LCP plate all exhibited fracture on imaging (Figure A5). At six years post-op, an additional 3.5 mm cortical screw in the tibia broke and the fractured plate became more widely displaced (Figure A6). The valgus deformity seen earlier had now progressed to nearly 30 degrees.

#### **2. Materials and Methods**

The PHILOS plate was made of Stainless Steel 316L, originally manufactured by Synthes, now Depuy Synthes Joint Reconstruction (Warsaw, IN, USA). The plate is anatomically shaped to the complex contours of the proximal humerus. The construct contains both locking and combination of locking and compression holes using different types of screws [24,25]. There are 10 locking holes in the distal end of the PHILOS permitting multiple points of fixation for support. The PHILOS has 5 combination holes, one elongated hole to aid in-plate positioning with a 3.5 mm locking screws hole in the threaded portion in the proximal shaft. There are also 3.5 and 4.0 mm cortical screw holes and 4.0 mm cancellous bone screws in the compression portion. All 18 pieces of the implant submitted for investigation are shown in Figure 1. Among the submitted pieces, only 4 screws were intact. The LCP, which was fractured into three pieces, was measured to be 114 mm in length and had a thickness of 3.5 mm.

In order to assess the mechanical and manufacturing integrity of the failed device, we preformed Rockwell hardness B-scale test and X-ray energy dispersive spectroscopy (EDS) using a Quanta 600 scanning electron microscope (SEM, Thermo Fisher Scientific, Hillsboro, OR, USA) with a 15 kV voltage and a spot size of 5. We compared the results from our testing to ASTM (American Society for Testing and Materials) standards reported for stainless steel 316L [26]. The Rockwell hardness test was performed in three different locations of the fractured plate with each test location increasing in distance away from the fracture surface. The EDS was performed using a 10 mm square detector and genesis software package. EDS was performed on both the fracture plate. EDS samples were taken both at the plate surface and plate interior to ensure homogeneity throughout both pieces.

One of the plate fragments was cleaned in ethanol followed by water sonication for 10 min to study the fracture surface. Fractography was performed using the SEM with a voltage of 15 kV and a spot size of 3. Electron backscatter diffraction (EBSD) was performed on an FEIXL-30 using an EDAX EBSD detector to determine the grain size and orientation.

#### **3. Results and Discussion**

#### *3.1. Chemical Composition Characterization*

The results of the X-ray EDS for the fracture plate is shown in Table 1 and compared with reported ASTM standard for SS 316L stainless steel (F138-03). An additional requirement set forth by the ASTM standard is shown in Equation (1), which yielded 24.77 for the plate [26]. One factor to keep in mind when comparing EDS results to the ASTM standard is that EDS is known to be a semi-quantitative technique. In addition, adventitious carbon may have accumulated on the surface preventing C from being quantified. There is also an overlap between the X-ray peaks of Mo and S, precluding deconvolution of their concentrations. The EDS system used in this case was not sensitive enough to detect the trace amounts of P, S, and N expected according to the standard. The EDS spectrum is shown in Figure 2.

$$\% \text{ Cr} + \text{3.3} \times \% \text{Mo} \overset{>}{\geq} \text{26.0} \tag{1}$$

**Table 1.** X-Ray energy dispersive spectroscopy (EDS) measured composition for plate (wt %).

**Figure 2.** Energy dispersive X-ray analysis shows qualitatively the peaks of different element weight percent present in the PHILOS plate.

The plate conformed to ASTM standards according to the tolerated ranges of element composition but failed equation 1 in this instance shown in Table 1. However, in another instance, the results of the composition analysis gave a result of 28.7 for Equation (1), thus passing the requirements.

#### *3.2. Microstructural Characterization*

EBSD and backscatter imaging methods were used to characterize the structure of the material. The microstructure is shown in Figure 3. The range in the average grain size of the plate was four to six micrometers shown in Figure 4.

**Figure 3.** Microstructure of PHILOS plate at 1000×.

**Figure 4.** Electron backscatter diffraction (EBSD) grain map of the PHILOS plate.

An attempt was made to identify and characterize the inclusions. Two regions of interest identified in Figure 1 as area 2 and 3, were investigated for inclusions and presented in Figure 5a,b. The composition of one inclusion was mapped in Figure 6 using EDS. The maps in Figure 6 show that inclusions are likely Mo rich (though this could be S), these inclusions may be fine, localized chi and sigma intermetallic phases which may have started the crack. There is a need to characterize inclusions in surgical grade SS 316L as a dedicated basic research focus since this paper deals with failure analysis and the importance that the inclusions have on failure initiation.

**Figure 5.** Backscattered electron images of likely inclusions (**a**) SEM EBSD image of area 2 (**b**) Topographical SEM image of area 3.

**Figure 6.** Characterization of inclusions using EDS. (**a**) The inclusions, (**b**) Gray scale of inclusions. Overlapping spectra of S and Mo visible in (**c**) Sulfur and/or (**d**) Nickel, (**e**) Molybdenum in the microstructure of the PHILOS plate. While the rest (**f**) Chromium, (**g**) Silicon, (**h**) Iron and (**i**) Manganese are not visible.

#### *3.3. Material Property Analysis*

The results of the Rockwell hardness B-scale test on the fractured plate are shown in Table 2. The ASTM standard upper limit for SS 316L is 95 HRB, and a 95% confidence interval was calculated for each trial. The hardness was then converted to Vickers hardness and tensile strength, and a 95% confidence interval was also calculated and used to compare against the ASTM standard for SS316L [26]. Hardness of the plate varied across the plate. It is evident that as the fatigue related deformation accrued, the material hardened across or near the fracture plane. It is likely that the material may have been supplied at higher strength conditions via cold working and may also have had reduced elongation.

#### *3.4. Optical Microscopic Analysis*

Visual observation indicated a large number of scratches on the surface of the plate. Two broken screw heads (with StarDrive recess) remained in the plate, while one partially intact locking screw was not removed from the distal end. Figure 1 shows that the crack initiated from under one of the locking screw holes near the middle of the plate. This fracture initiation has progressed in two different directions, one diagonally 45◦ in the directions of maximum shear and one perpendicularly (Figure 1), which caused the plate to fail into three pieces. The observed features are summarized in Table 3. Since the plate and screw construct was removed after it had failed, we do not know whether the material dissolution or contact between two mating surfaces during removal removed the pitting and or other localized damage sites prior to crack formation.

> **Table 3.** Various damage features on different parts of implant.


**6. Design of experiments**

66 Multivariate Analysis in Management, Engineering and the Sciences

(Gabardo Filho, 2005; Domènech et al., 2001):

Full Mineralization of pollutants;

(Domenech et al., 2001; Moral, 2005):

**5. Heterogeneous photocatalysis**

such as the biologic;

biological process; Allow *in situ* treatment;

mineralized;

or color.

**4.1. Advantages of advanced oxidation processes**

Able to assimilate large variety of organic compounds;

Improve organoleptic properties of treated water;

Present high power with high oxidizing reaction kinetics.

**4.2. Disadvantages of advanced oxidation processes**

Some processes are not available at appropriate scales;

the chemical structure of the semiconductor (Custo et al., 2006).

According to Nogueira and Jardim (1998), a semiconductor is characterized by:

Costs can be high due to energy consumption;

 Valence bands, where vacancies are generated; Driving Bands that are generating electrons; A region between bands called bandgap;

from the valence band to the conduction band;

schematically the behavior described above.

Can be integrated with other processes such as pre or post treatment;

AOPs have a number of advantages when compared to conventional oxidation processes

Employed in the destruction of refractory compounds resistant to other treatments,

Used in high toxicity wastewater that can cause some difficulty in the treatment of

Develop byproducts reaction intermediates that submitted to a post treatment may be

AOPs can be applied to certain types of waste under some restrictions, as follows

Some types do not apply to wastewater with high organic capability, turbidity, optical

The heterogeneous photocatalysis is a process based on the absorption of UV-visible by a solid semiconductor. In the interface area between the solution and the electrically excited solid, a degradation reaction or transformation of pollutants may occur, without changing

Semiconductor particles, when irradiated, absorb photons that may excite electrons

This generates vacancy electrons. The electron / vacancy pair migrates to the surface of the particle resulting in oxidation and reduction sites (Carp et al. 2004). Figure 1 illustrates Design of Experiments (DOE) has been widely used in the optimization of process parameters and improving of quality products by the application of engineering concepts and Statistics (Wang, Huang, 2007).

Design of Experiments is defined as a set of statistical techniques applied to planning, conduction, analysis and interpretation of controlled tests, in order to find define factors that influence the values of a parameter or group of parameters (Bruns; Neto; Scarmínio, 2010).

According to Franceschini and Macchietto (2008), DOE is a statistical tool used to maximize the value of variable responses obtained on each experiment and also to minimize cost and time by reducing the number of experimental conditions.

Interactions between variables are considered in the experimental design and can be used for optimizing the operating parameters in multivariable systems (Ay; Catalkaya; Kargi, 2009).

According to Salazar (2009), the experimental has been studied as an important mathematical tool in the area of Advanced Oxidation Processes (Heterogeneous Photocatalysis). In this study, fractionated schedules for the degradation of organic matter and COD percentage of dairy effluent were used to obtain 93.70% of the treatment.


*Article* **Corrosion Behavior of High-Strength C71500 Copper-Nickel Alloy in Simulated Seawater with High Concentration of Sulfide**

**Xin Gao 1,2,\* ,† and Ming Liu 3,\* ,†**

**Abstract:** The corrosion behavior of high-strength C71500 copper-nickel alloy in high concentrations of sulfide-polluted seawater was studied by potentiodynamic polarization measurements, electrochemical impedance spectroscopy (EIS), immersion testing, and combined with SEM, EDS, XPS, and XRD surface analysis methods. The results showed that the C71500 alloy shows activation polarization during the entire corrosion process, the corrosion rate is much higher (0.15 mm/a) at the initial stage of immersion, and the appearance of diffusion limitation by corrosion product formation was in line with the appearance of a Warburg element in the EIS fitting after 24 h of immersion. As the corrosion process progressed, the formed dark-brown corrosion product film had a certain protective effect preventing the alloy from corrosion, and the corrosion rate gradually decreased. After 168 h of immersion, the corrosion rate stabilized at about 0.09 mm/a. The alloy was uniformly corroded, and the corrosion products were mainly composed of Cu2S, CuS, Cu2 (OH)3Cl, Mn2O3 , Mn2O, MnS2 , FeO(OH), etc. The content of Cu2S gradually increased with the extension of immersion time. The addition of S2− caused a large amount of dissolution of Fe and Ni, and prevented the simultaneous formation of a more protective Cu2O film, which promoted the corrosion process to some extent.

**Keywords:** C71500 copper-nickel alloy; sulfide; immersion test; corrosion behavior; seawater environment