Enzymes and Lipids – a Healthy Vasculature Can Make

It was over 2,000 years ago that the Japanese population made natto a staple of their diet. Natto is a cheese-like food composed of soybeans fermented with a bacterium – Bacillus subtillis. Recognised for its beneficial cardiovascular effects, it has been extensively studied[1].

Relatively little was known regarding the mechanism by which natto intake led to an overall improvement in cardiovascular health until the 1980s. A potent fibrinolytic enzyme called nattokinase was discovered in natto in 1987[2], and since then, nattokinase has been the subject of a considerable amount of research conducted in both Asian countries (Japan, China, Korea) and the United States.

Kinases are used extensively to transmit signals and regulate complex processes in cells. The phosphorylation of molecules can enhance or inhibit their activity and modulate their ability to interact with other molecules.

Phosphorylation reaction is of particular importance in biology because several biological processes depend upon this reaction such as apoptosis, inflammation, regulation of metabolism, subcellular trafficking, and proliferation. In biology, phosphorylation is the transfer of phosphate molecules to a protein. This transfer prepares the proteins for specialised tasks in a living being. Phosphorylation is a reversible reaction; it means that a phosphate molecule can be added and removed. The enzymes that are responsible for adding phosphate groups to proteins are known as “kinases”.

Actions to Assist Vascular Health

Not only does nattokinase possess potent fibrinolytic/antithrombotic activity[3], but nattokinase has also been shown to exert antihypertensive, anti-atherosclerotic, lipid-lowering, antiplatelet/anticoagulant, and neuroprotective effects in both animal and human studies[4].

Supplementation with nattokinase, enhanced markers of fibrinolysis and anticoagulation in human subjects, along with decreasing blood pressure and atherosclerosis[5]. The most unique characteristic of nattokinase is that it is safe and has multiple cardiovascular disease-related preventative and alleviating pharmacologic effects. To the best of current knowledge, there are no other nutraceuticals with a similar range of pharmacologic properties[6].

Nattokinase has also been observed to have a beneficial effect on cardiovascular diseases (CVD) by lowering blood pressure (BP). A study by Jensen et al suggested that nattokinase consumption for 8 weeks was associated with beneficial changes to BP in patients having hypertension[7]. This is consistent with several laboratory studies demonstrating effective reduction of BP in spontaneous hypertensive rats by nattokinase administration.

Dosing and Timing

The recommended amount of nattokinase is more than 2,000FU/ day. The nattokinase activity level in natto commercially available varies from 1,400 FU/ pack (50g) to 2,000FU/ pack. The enzymatic activity of nattokinase is indicated by FU which stands for Fibrin Degradation Unit. Fibrin degradation method (using FU) is used to indicate a nattokinase activity level as it is reproducible and accurate to quantify. It is considered best to take nattokinase after dinner or before sleep since thrombus is more likely to be produced around midnight to the early morning.

It is recommended to take nattokinase on a regular basis for those who are over 40 years old, stressed-out, have relatively high blood pressure, and have high blood viscosity due to hyperlipidaemia or diabetes.

Membrane Lipid Replacement (MLR)

For almost 20 years regular publications have explored the potential role of providing membrane-related lipids via supplementation to improve cell-related function and mitochondrial quality. In essence, oral supplementation of selected phospholipids in a form that withstands digestive challenges and permits cell uptake can change numerous health-related functions.

Defects in cellular and intracellular membranes are characteristic of all chronic medical conditions, including cancer, and normal processes, such as aging[8]. Membrane glycerolphospholipids form the matrix for all cellular membranes and provide the separation of enzymatic and chemical reactions into discrete cellular compartments and organelles. They are also essential for the function of a variety of membrane-intercalated and membrane-bound enzymes, and they afford cells with an important energy storage system. Moreover, they provide precursors for bioactive molecules that function in signalling and recognition pathways.

MLR and Vascular Health

Unsaturated fatty acids (FAs) are particularly sensitive to reactive oxygen species and reactive nitrogen species (ROS/RNS) becoming oxidised, a recognised component of vascular and CVD dysfunction, resulting in the formation of lipid peroxides[9]. Lipid peroxides can be cytotoxic and lead to free-radical damage to other lipids, proteins and DNA[10].

In Metabolic Syndrome (MetSyn), Type 2 Diabetes (T2D), cardiovascular disease and renal diseases, free FAs accumulate inside cells and mitochondria, where they are prone to peroxidation. Also when mitochondrial inner membranes (MIM) are oxidatively damaged, this can result in MIM proton leakage and electron transport chain (ETC) and mitochondrial DNA (mtDNA) damage, and subsequent activation of the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome[11].

Inflammasomes – a Therapeutic Target

The NLRP3 inflammasome is a multiprotein complex that assembles to engage essential innate immune defences by processing the maturation of pro-inflammatory cytokines IL-1β and IL-18. Substantial evidence has positioned the NLRP3 inflammasome at the centre of vascular disease progression, with a particular significance in the context of aging and the low-grade chronic inflammation associated (inflammaging).

The NLRP3 receptor is unique among innate immunity elements as it can recognise a vast variety of pathogenic and non-pathogenic endogenous stressors, unlike the majority of pattern recognition receptors (PRRs), which have limited specificity to one or few unrelated stimuli[12]. As the sources of NLRP3 inflammasome activation are diverse, this complex can also mediate a heightened state of sterile inflammation when over-activated.

Mitochondria at the Nexus of Inflammation

Sterile inflammation occurs in the absence of microorganisms and is typically associated with the pattern recognition of intracellular contents released from damaged and necrotic cells (also known as damage-associated molecular patterns) by inflammatory signalling receptors. These triggers include oxidised cellular components and oxidised mtDNA[13].

Atherosclerosis is the main cause underlying vascular disease[14] and there are several pieces of evidence pointing out that inflammation and NLRP3 activation play an important role in this disease[15]. Oxidised LDL (oxLDL), which is known to accumulate in the vessel wall during atherosclerosis and is phagocyted by macrophages, activates NLRP3 by processes related to the influx of Ca2+, reactive oxygen species, and mitochondrial dysfunction, among others. Improving the cell wall and the inner membrane of mitochondria are two of the proposed and evidenced mechanisms that MLR uses to contain and reverse vascular disease.

A narrative review in the journal Membranes in late 2021, looked at a range of clinical possibilities for the use of MLR. The authors explain that the activation of inflammation complexes such as the NLRP3 inflammasome complex also contributes to the development of visceral adiposity, endothelial dysfunction and insulin resistance, precursors to type 2 diabetes (T2D)[16].

Specific Supplements

Food supplements, however, can be taken orally to prevent some of the damage to cellular and mitochondrial membranes seen during MetSyn development and potentially slow the process of progression to T2D and the co-development of vascular diseases. Such supplements are also important in preventing loss of ETC function seen in MetSyn and T2D[17]. Dietary use of various types of antioxidants or other supplements that increase free-radical scavenging systems have been employed to reduce or prevent the process of development of MetSyn, CVD and T2D[18].

In MetSyn, T2D, CVD, and diseases caused or promoted by continuing excess ROS/RNS, food supplementation with low molecular weight antioxidants, plus some replacements of accessory MLR molecules have been employed. By adding enzyme cofactors such as zinc, manganese, copper, vanadium, chromium, and selenium ions necessary for antioxidant and some enzyme functions plus certain vitamins with antioxidant properties and coenzymes (examples: C, E, A, CoQ10) such combinations can be used to maintain antioxidant levels and free-radical scavenging systems.

It is worth noting that controlled clinical trials, for the most part containing single oral antioxidants, failed to show significant prevention benefits[19]. This should not be unexpected, as these complex phenomena involve multiple, complex mechanisms and pathways, and it is unlikely that a simple, singular approach could be effective.

Chronic inflammatory damage to blood vessels due to lipid accumulation, inflammatory responses, endothelial cell death, and thrombosis, can eventually result in atherosclerosis and CVD. That MLR alone can modify or reverse the conditions described above and result in the reversal of T2D, atherosclerosis, CVD, and other diseases remain extremely unlikely, if not completely impossible.

However, the use of MLR phospholipids can change the composition and oxidation state of circulating lipoproteins and lipids[20]. Thus, this has the potential to modify pathologic processes and limit the progression of more lethal forms of metabolic diseases and also over time reverse disease states.

Conclusion

Whilst there are many approaches to improving the health of the cardiovascular system, they require some core mechanistic interventions to be combined with lifestyle changes to achieve a clinically relevant outcome. This author’s experience with the combined use of nattokinase and MLR intervention is that the two operate in a synergistic manner impacting multiple beneficial mechanistic outcomes gently and effectively.

To quote Thomas Sydenham, an English physician (1624-1689), “A man is only as old as his arteries.” His belief was certainly ahead of his time since we now know that arterial aging is one of the primary mechanisms for chronic illnesses, such as CVD, including heart attacks, strokes, dementia, erectile dysfunction and more.

Additional lifestyle interventions utilised in conjunction with the suggestions and other allied nutrient combinations provides a strategic intervention of modest cost, minimal risk and outcome potential that may add years to the life of and enhance functionality of the client/patient. Remembering you are only as old as your vascular system suggests that now may be the time to undertake some antiaging vascular therapy[21]!

References

[1] Nagata C, Wada K, Tamura T, Konishi K, Goto Y, Koda S, et al. Dietary Soy and Natto Intake and Cardiovascular Disease Mortality in Japanese Adults: The Takayama Study. Am J Clin Nutr (2017) 105:426–31.

[2] Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A Novel Fibrinolytic Enzyme (Nattokinase) in the Vegetable Cheese Natto; a Typical and Popular Soybean Food in the Japanese Diet. Experientia (1987) 43:1110–1.

[3] Sumi H, Hamada H, Nakanishi K, Hiratani H. Enhancement of the Fibrinolytic Activity in Plasma by Oral Administration of Nattokinases. Acta Haematol (1990) 84:139–43.

[4] Kim JY, Gum SN, Paik JK, Lim HH, Kim KC, Ogasawara K, et al. Effects of Nattokinase on Blood Pressure: A Randomized, Controlled Trial. Hypertens Res (2008) 31:1583–8.

[5] Kurosawa Y, Nirengi S, Homma T, Esaki K, Ohta M, Clark JF, et al. A Single-Dose of Oral Nattokinase Potentiates Thrombolysis and Anti-Coagulation Profiles. Sci Rep (2015) 5:11601.

[6] Gallelli G, Di Mizio G, Palleria C, Siniscalchi A, Rubino P, Muraca L, Cione E, Salerno M, De Sarro G, Gallelli L. Data Recorded in Real Life Support the Safety of Nattokinase in Patients with Vascular Diseases. Nutrients. 2021 Jun 13;13(6):2031.

[7] Jensen GS, Lenninger M, Ero MP, Benson KF. Consumption of nattokinase is associated with reduced blood pressure and von Willebrand factor, a cardiovascular risk marker: results from a randomized, double-blind, placebo-controlled, multicenter North American clinical trial. Integr Blood Press Control. 2016;9:95–104.

[8] Nicolson GL, Ash ME. Membrane Lipid Replacement for chronic illnesses, aging and cancer using oral glycerolphospholipid formulations with fructooligosaccharides to restore phospholipid function in cellular membranes, organelles, cells and tissues. Biochim Biophys Acta Biomembr. 2017 Sep;1859(9 Pt B):1704-1724.

[9] A.W. Linnane, H. Eastwood. Cellular redox regulation and prooxidant signaling systems: a new perspective on the free radical theory of aging Ann. N. Y. Acad. Sci., 1067 (2006), pp. 47-55

[10] P. Schrauwen Skeletal muscle uncoupling protein 3 (UCP3): mitochondrial uncoupling protein in search of a function Curr. Opin. Clin. Nutr. Metab. Care, 5 (2002), pp. 265-270

[11] Schrauwen P, Schrauwen-Hinderling V, Hoeks J, Hesselink MK. Mitochondrial dysfunction and lipotoxicity. Biochim Biophys Acta. 2010 Mar;1801(3):266-71.

[12] Swanson K.V., Deng M., Ting J.P.-Y. The NLRP3 inflammasome: Molecular activation and regulation to therapeutics. Nat. Rev. Immunol. 2019;19:477–489.

[13] Nakayama H, Otsu K. Mitochondrial DNA as an inflammatory mediator in cardiovascular diseases. Biochem J. 2018;475(5):839-852. Published 2018 Mar 6.

[14] Frosteg?rd J. Immunity, atherosclerosis and cardiovascular disease. BMC Med. 2013 May 1;11:117.

[15] Zhou W, Chen C, Chen Z, Liu L, Jiang J, Wu Z, Zhao M, Chen Y. NLRP3: A Novel Mediator in Cardiovascular Disease. J Immunol Res. 2018 Apr 8;2018:5702103.

[16] Gora, I.M.; Ciechanowska, A.; Ladyzynski, P. NLRP3 Inflammasome at the Interface of inflammation, endothelial dysfunction, and type 2 diabetes. Cells 2021, 10, 314

[17] Butler, A.E.; Janson, J.; Bonner-Weir, S.; Ritzel, R.; Rizza, R.A.; Butler, P.C. Beta-cell deficit and increased beta-cell apoptosis in humans with in type type 2 diabetes. Diabetes 2003, 52, 102–110.

[18] Butler, A.E.; Janson, J.; Bonner-Weir, S.; Ritzel, R.; Rizza, R.A.; Butler, P.C. Beta-cell deficit and increased beta-cell apoptosis in humans with in type type 2 diabetes. Diabetes 2003, 52, 102–110.

[19] Ueda, S.; Yasunari, K. What we learned from randomized clinical trials and cohort studies of antioxidant vitamins. Focus on vitamin E and cardiovascular disease. Curr. Pharm. Biotechnol. 2006, 7, 69–72

[20] Blum, C.B.; Levy, R.I.; Eisenberg, S.; Hall, M.; Goebel, R.H.; Berman, M. High density lipoprotein metabolism in man. J. Clin. Investig. 1977, 60, 795–807.

[21] Seals DR, Kaplon RE, Gioscia-Ryan RA, LaRocca TJ. You're only as old as your arteries: translational strategies for preserving vascular endothelial function with aging. Physiology (Bethesda). 2014;29(4):250-264.