**1. Introduction**

The endothelium has a major role in the regulation of vascular function and structure by releasing vasodilatory molecules [1]. Nitric oxide (NO), the main vasodilator, is produced from the amino acid L-arginine (ARG) by endothelial NO synthase (eNOS) [2] in response to increased shear stress induced by blood flow. NO is the primary endothelial factor for relaxing vascular smooth muscle cells and preventing pathological structure remodeling that leads to arterial stiffness [1,3]. Reduced NO availability, a main cause of endothelial dysfunction [1,3], is associated with ARG deficiency driven by increased levels of endogenous inhibitors of eNOS [4] and ARG catabolism to ornithine (ORN) by the enzyme arginase [5,6]. Oxidative stress is a main mechanism of endothelial dysfunction via increasing asymmetric dimethylarginine (ADMA, an eNOS inhibitor) levels [7–9] and arginase activity [10]. Since ARG and ADMA compete for binding to eNOS, a low ARG/ADMA ratio is a biomarker of reduced ARG availability and NO production [7].

Impaired endothelial function, assessed as low brachial artery flow-mediated dilation (FMD), begins before and progresses after menopause in healthy women [4,11]. Endothelial

**Citation:** Figueroa, A.; Maharaj, A.; Kang, Y.; Dillon, K.N.; Martinez, M.A.; Morita, M.; Nogimura, D.; Fischer, S.M. Combined Citrulline and Glutathione Supplementation Improves Endothelial Function and Blood Pressure Reactivity in Postmenopausal Women. *Nutrients* **2023**, *15*, 1557. https://doi.org/ 10.3390/nu15071557

Academic Editors: Shane Phillips and Abeer M. Mahmoud

Received: 16 February 2023 Revised: 7 March 2023 Accepted: 21 March 2023 Published: 23 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

dysfunction precedes the development of arterial stiffness [12] and hypertension in healthy postmenopausal women [13]. Another mechanism for hypertension in postmenopausal women is elevated sympathetic activity [14]. Systolic hypertension and consequent heart failure with preserved ejection fraction are highly prevalent in older women due to increased aortic stiffness [15] and sympathetic-mediated vasoconstriction [16,17]. Evidence indicates that augmented systolic blood pressure (BP) reactivity to a sympathetic stimulus (cold pressor test, CPT) predicts future hypertension [18] and heart failure [19]. Exaggerated BP reactivity to sympathetic stimuli seen in postmenopausal women [16], may be due to the inability of the endothelium to counteract sympathetic-mediated vasoconstriction [17,20].

Dietary strategies to increase circulating ARG may improve vascular function. The effectiveness of short-term oral ARG supplementation for improving FMD was observed in healthy older adults [21]. However, oral ARG becomes ineffective for vascular benefits due to stimulation of arginase activity [22]. Contrary to ARG, citrulline (CIT) is not catabolized by arginase and inhibits arginase activity [23], and thus, is more efficient than oral ARG in increasing circulating ARG levels [24]. Short-term CIT supplementation has been shown to improve plasma ARG, ARG/ADMA, arterial stiffness, and resting BP in middle-aged and older adults [23,25–29]. Evidence supports the benefit of CIT supplementation on the attenuation of BP reactivity to CPT in healthy adults [30–32]. A placebo-controlled study examined the effect of CIT supplementation on FMD but failed to detect improvements in healthy adults [24]. Since oxidative stress is implicated in endothelial dysfunction, the addition of the antioxidant glutathione (GSH) may synergize the vascular benefits of CIT. Recent evidence supports the combined use of CIT and GSH (CIT+GSH) to improve eNOS activity in a mouse ischemia model [33] and the long-lasting increase of plasma NO levels in young men [34]. Although combined CIT+GSH supplementation increased post-exercise plasma NO metabolites (NOx), an indicator of improved endothelial function [34], vascular function measures were not evaluated. Therefore, it is unknown whether CIT+GSH supplementation can improve vascular function and BP reactivity in postmenopausal women. Given that CIT supplementation increased circulating ARG levels and the ARG/ADMA ratio, an indicator of increased NO availability [27], improvements in brachial FMD are expected in postmenopausal women.

The purpose of this study was to investigate the effects of supplementing with CIT alone and CIT+GSH on vascular function, assessed by brachial FMD, arterial stiffness (pulse wave velocity, PWV), and BP reactivity to CPT in postmenopausal women. Moreover, we assessed fasting blood glucose (FBG), serum levels of ARG and its metabolites, and markers of oxidative stress. Our hypothesis was that CIT alone and CIT+GSH supplementations would increase brachial FMD and attenuate BP responses to sympathetic activation induced by CPT compared to a placebo in postmenopausal women.
