Effects of Dietary Nitrate in Women With Secondary Amenorrhea

Due to excessive exercise, disordered attitudes toward eating, physical and psychological stress, and/or hormonal imbalances, young women may be predisposed to developing secondary amenorrhea. Secondary amenorrhea is defined as the absence of menses for at least 3 months in regularly menstruating women or at least 6 months in irregularly menstruating women [1]. This disorder of the hypothalamic-pituitary axis (HPO) is associated with a short-term lack of endogenous estradiol (E2), which may have negative health consequences [2-7]. For example, E2 plays an important role in regulating skeletal muscle mass and contractile function, including both strength and power [4]. In part, this is because E2 increases phosphorylation of the regulatory light chain of myosin, thereby enhancing muscle contractility [6, 8]. Lack of E2 also alters lipid and carbohydrate metabolism [9, 10], which may be due to the important role E2 plays in regulating mitochondrial function, more specifically respiration and efficiency. This impairment in metabolism and mitochondrial respiration could increase CVD risk and alter plasma levels of lipids, cortisol, and insulin [2, 9, 10]. Indeed, Ronkainen et al. [11] found that postmenopausal women not on hormone replacement therapy (HRT) have reduced skeletal muscle strength and power compared to those on HRT, whereas Kleis-Olsen et al. [12] reported similar findings with respect to fat mass and mitochondrial respiratory capacity. Younger women with secondary amenorrhea may also suffer from similar impairments due to E2 deficiency.

It has also been shown that short term lack of E2 compromises nitric oxide synthase (NOS) activity, which plays a critical role in cardiovascular health, exercise capacity, and vasomodulation and mitochondrial function within skeletal muscle [7, 13, 14]. Conversely, dietary nitrate supplementation can increase nitric oxide bioavailailbity, via an enterosalivary pathway in which nitrate is reduced to nitrite that in turn is further reduced to nitric oxide [15]. It has been demonstrated that dietary nitrate improves muscle contractility [16], exercise capacity and performance [15, 17], vascular function [18], and mitochondrial efficiency during exercise [5, 19, 20]. Indeed, we have found nitrate supplementation to be particularly effective in increasing muscular speed and power in postmenopausal women [21]. Increasing NO bioavailability via ingestion of nitrate therefore may have beneficial effects on both muscle function and mitochondrial function in women with secondary amenorrhea. To date, however, this hypothesis has not been tested.

Although the aforementioned research has highlighted the beneficial effects of dietary nitrate supplementation on muscle contractile function and mitochondrial respiration in various populations, no studies have examined the effects of this supplementation on these parameters in women with secondary amenorrhea. The proposed study will therefore provide a better understanding of the effects of dietary nitrate supplementation on muscle function and mitochondrial respiration in women with secondary amenorrhea, potentially improving health, recovery, and performance outcomes within this vulnerable population.

1. Rebar, R., Evaluation of Amenorrhea, Anovulation, and Abnormal Bleeding, in Endotext, K.R. Feingold, et al., Editors. 2000: South Dartmouth (MA).
2. Gupte, A.A., H.J. Pownall, and D.J. Hamilton, Estrogen: an emerging regulator of insulin action and mitochondrial function. J Diabetes Res, 2015. 2015: p. 916585.
3. Ihalainen, J.K., et al., Beyond Menstrual Dysfunction: Does Altered Endocrine Function Caused by Problematic Low Energy Availability Impair Health and Sports Performance in Female Athletes? Sports Med, 2024. 54(9): p. 2267-2289.
4. Chidi-Ogbolu, N. and K. Baar, Effect of Estrogen on Musculoskeletal Performance and Injury Risk. Front Physiol, 2018. 9: p. 1834.
5. Larsen, F.J., et al., Dietary inorganic nitrate improves mitochondrial efficiency in humans. Cell Metab, 2011. 13(2): p. 149-59.
6. Lai, S., et al., Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice. Am J Physiol Endocrinol Metab, 2016. 310(9): p. E724-33.
7. O'Donnell, E. and M.J. De Souza, The cardiovascular effects of chronic hypoestrogenism in amenorrhoeic athletes: a critical review. Sports Med, 2004. 34(9): p. 601-27.
8. Greenberg, M.J., et al., The molecular effects of skeletal muscle myosin regulatory light chain phosphorylation. Am J Physiol Regul Integr Comp Physiol, 2009. 297(2): p. R265-74.
9. An, J., et al., Sex- and endurance training-mediated cardiovascular protection through lipids during exercise. Trends Endocrinol Metab, 2024.
10. Senoz, S., et al., Estrogen deprivation, rather than age, is responsible for the poor lipid profile and carbohydrate metabolism in women. Maturitas, 1996. 25(2): p. 107-14.
11. Ronkainen, P.H., et al., Postmenopausal hormone replacement therapy modifies skeletal muscle composition and function: a study with monozygotic twin pairs. J Appl Physiol (1985), 2009. 107(1): p. 25-33.
12. Kleis-Olsen, A.S., et al., Metabolic flexibility in postmenopausal women: Hormone replacement therapy is associated with higher mitochondrial content, respiratory capacity, and lower total fat mass. Acta Physiol (Oxf), 2024. 240(6): p. e14117.
13. Fadel, P.J., W. Zhao, and G.D. Thomas, Impaired vasomodulation is associated with reduced neuronal nitric oxide synthase in skeletal muscle of ovariectomized rats. J Physiol, 2003. 549(Pt 1): p. 243-53.
14. Tengan, C.H., G.S. Rodrigues, and R.O. Godinho, Nitric oxide in skeletal muscle: role on mitochondrial biogenesis and function. Int J Mol Sci, 2012. 13(12): p. 17160-84.
15. Amdahl, M.B., A.W. DeMartino, and M.T. Gladwin, Inorganic nitrite bioactivation and role in physiological signaling and therapeutics. Biol Chem, 2019. 401(1): p. 201-211.
16. Coggan, A.R. and L.R. Peterson, Dietary Nitrate Enhances the Contractile Properties of Human Skeletal Muscle. Exerc Sport Sci Rev, 2018. 46(4): p. 254-261.
17. Flueck, J.L., et al., Is beetroot juice more effective than sodium nitrate? The effects of equimolar nitrate dosages of nitrate-rich beetroot juice and sodium nitrate on oxygen consumption during exercise. Appl Physiol Nutr Metab, 2016. 41(4): p. 421-9.
18. Craig, J.C., et al., Effect of dietary nitrate supplementation on conduit artery blood flow, muscle oxygenation, and metabolic rate during handgrip exercise. J Appl Physiol (1985), 2018. 125(2): p. 254-262.
19. Bailey, S.J., et al., Dietary nitrate supplementation enhances muscle contractile efficiency during knee-extensor exercise in humans. J Appl Physiol (1985), 2010. 109(1): p. 135-48.
20. Pappas, G., M.L. Wilkinson, and A.J. Gow, Nitric oxide regulation of cellular metabolism: Adaptive tuning of cellular energy. Nitric Oxide, 2023. 131: p. 8-17.
21. Coggan, A.R., et al., Dietary nitrate-induced increases in human muscle power: high versus low responders. Physiol Rep, 2018. 6(2).
22. Jungersten, L., et al., Plasma nitrate as an index of nitric oxide formation in man: analyses of kinetics and confounding factors. Clin Physiol, 1996. 16(4): p. 369-79.
23. Govoni, M., et al., The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash. Nitric Oxide, 2008. 19(4): p. 333-7.
24. Joint FAO/WHO Expert Committee on Food Additives. Meeting (61st: 2003, R., Italy) \& International Programme on Chemical Safety., Safety evaluation of certain food additives and contaminants/prepared by the sixty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JEFCA). 2004.
25. Goodman, J., S. Thomas, and J.F. Burr, Physical activity series: cardiovascular risks of physical activity in apparently healthy individuals: risk evaluation for exercise clearance and prescription. Can Fam Physician, 2013. 59(1): p. 46-9, e6-e10.
26. Sumner, M.D., et al., Near Infrared Spectroscopy Measurements of Mitochondrial Capacity Using Partial Recovery Curves. Front Physiol, 2020. 11: p. 111.
27. Gallardo EJ, Gray DA, Hoffman RL, Yates BA, Moorthi RN, Coggan AR. Dose-response effect of dietary nitrate on muscle contractility and blood pressure in older subjects: a pilot study. J Gerontol A Biol Sci Med Sci 2021; 76:591-598.
28. Zoughaib WS, Hoffman RL, Yates BA, Moorthi RN, Lim K, Coggan AR. Short-term beetroot juice supplementation improves muscle contractility but does not reduce blood pressure or oxidative stress in 65-79 y old men and women. Nitric Oxide 20