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Citation: Kang, Y.; Dillon, K.N.;
Martinez, M.A.; Maharaj, A.; Fischer,
S.M.; Figueroa, A. L-Citrulline
Supplementation Improves Arterial
Blood Flow and Muscle Oxygenation
during Handgrip Exercise in
Hypertensive Postmenopausal
Women. Nutrients 2024,16, 1935.
https://doi.org/10.3390/nu16121935
Academic Editor: Roberto Iacone
Received: 29 April 2024
Revised: 7 June 2024
Accepted: 16 June 2024
Published: 19 June 2024
Copyright: © 2024 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/).
nutrients
Article
L-Citrulline Supplementation Improves Arterial Blood Flow and
Muscle Oxygenation during Handgrip Exercise in Hypertensive
Postmenopausal Women
Yejin Kang 1, Katherine N. Dillon 1, Mauricio A. Martinez 1, Arun Maharaj 2, Stephen M. Fischer 3
and Arturo Figueroa 1, *
1Department of Kinesiology and Sport Management, Texas Tech University, Lubbock, TX 79409, USA;
yejin.kang@ttu.edu (Y.K.); katherine.dillon@ttu.edu (K.N.D.); mauricio.martinez@ttu.edu (M.A.M.)
2Department of Epidemiology and Cancer Control, St. Jude Children’s Research Hospital,
Memphis, TN 38105, USA; arun.maharaj@stjude.org
3
Department of Family and Community Medicine, The University of Texas Health Science Center at Houston,
Houston, TX 77030, USA; stephen.fischer@uth.tmc.edu
*Correspondence: arturo.figueroa@ttu.edu; Tel.: +1-(806)-834-5587; Fax: +1-(806)-742-1688
Abstract: Endothelial dysfunction decreases exercise limb blood flow (BF) and muscle oxygenation.
Acute L-Citrulline supplementation (CIT) improves muscle tissue oxygen saturation index (TSI) and
deoxygenated hemoglobin (HHb) during exercise. Although CIT improves endothelial function
(flow-mediated dilation [FMD]) in hypertensive women, the impact of CIT on exercise BF and
muscle oxygenation (TSI) and extraction (HHb) are unknown. We examined the effects of CIT
(10 g/day) and a placebo for 4 weeks on blood pressure (BP), arterial vasodilation (FMD, BF, and
vascular conductance [VC]), and forearm muscle oxygenation (TSI and HHb) at rest and during
exercise in
22 hypertensive
postmenopausal women. Compared to the placebo, CIT significantly
(
p< 0.05
) increased FMD (
∆−
0.7
±
0.6% vs.
∆
1.6
±
0.7%) and reduced aortic systolic BP (
∆
3
±
5 vs.
∆−4±6 mmHg
) at rest and improved exercise BF (
∆
17
±
12 vs.
∆
48
±
16 mL/min), VC (
∆−
21
±
9
vs.
∆
41
±
14 mL/mmHg/min), TSI (
∆−
0.84
±
0.58% vs.
∆
1.61
±
0.46%), and HHb (
∆
1.03
±
0.69
vs.
∆−
2.76
±
0.77
µ
M). Exercise BF and VC were positively correlated with improved FMD and TSI
during exercise (all p< 0.05). CIT improved exercise artery vasodilation and muscle oxygenation via
increased endothelial function in hypertensive postmenopausal women.
Keywords: L-citrulline; endothelial function; muscle oxygenation; blood flow; vascular conductance;
handgrip exercise; postmenopausal women
1. Introduction
Endothelial function is characterized by adequate nitric oxide (NO) bioavailability [
1
],
an essential vasodilator for normal vascular tone and blood flow (BF) regulation [
2
]. En-
dothelial cells are stimulated via an increase in BF induced by shear stress [
3
], leading to
the catabolism of L-arginine (ARG) into NO by endothelial NO synthase (eNOS) [
4
]. NO
increases BF in conduit arteries (macrovascular) [
5
] and resistance arterioles (microvascular)
at rest [
6
] via the relaxation of vascular smooth muscle cells [
2
]. Conduit arteries are respon-
sible for maintaining a steady BF to resistance arterioles, while arterioles regulate oxygen
and nutrient delivery to capillaries [
7
]. Menopause and hypertension are associated with
macro- and microvascular endothelial dysfunction at rest due to ARG and NO deficiency,
leading to reduced vasodilatory capacity and arterial stiffness [
8
–
11
]. Microvascular dys-
function in hypertension leads to impaired muscle BF and end organ damage, contributing
to increased cardiovascular event risk [11].
During exercise, active skeletal muscles require sufficient oxygen delivery to match
metabolic demands; thus, increasing BF to the contracting muscles is essential [
12
,
13
].
Nutrients 2024,16, 1935. https://doi.org/10.3390/nu16121935 https://www.mdpi.com/journal/nutrients
Nutrients 2024,16, 1935 2 of 16
Muscle BF during exercise is regulated by the balance between local vasodilators, including
NO and muscle metabolites, and vasoconstriction via sympathetic activity [
14
]. Aging
and hypertension lower NO availability and impair the ability of contracting muscles to
blunt sympathetic vasoconstriction during exercise (functional sympatholysis), reducing
BF and oxygen delivery to skeletal muscles [15]. The tissue oxygen saturation index (TSI),
an indicator of perfusion and oxygen delivery to skeletal muscles [
16
,
17
], decreases during
exercise in older women, indicating microvascular dysfunction and impaired vasodilatory
capacity [
17
]. NIRS-derived deoxygenated hemoglobin (HHb) content reflects local muscle
oxygen extraction as the ratio of oxygen utilization to the oxygen supply within the mi-
crovasculature [
18
,
19
]. Diminished BF in older adults [
19
] and NOS inhibition [
20
] cause
increased muscle oxygen extraction (measured as HHb) during exercise to meet the oxygen
demand for mitochondrial oxidative energy production. Taken together, impaired NO may
contribute to reductions in arterial BF and muscle oxygenation during exercise [
11
,
15
] in
hypertensive postmenopausal women.
NO, a potent vasodilator, can be synthesized through the conversion of ARG via
eNOS [
21
], which plays a major role in regulating muscle BF and oxygen delivery during
exercise [
22
]. While ARG supplementation can increase plasma ARG concentration and
macrovascular endothelial function at rest [
23
], it is not optimal to produce NO due
to stimulation of arginase activity [
24
], leading to no effects on oxygen delivery and
consumption during exercise [
25
]. Unlike oral ARG, the majority of ingested L-citrulline
(CIT) bypasses arginase catabolism in the intestines and the liver, and is converted to ARG
in the kidneys [
26
]. Thus, CIT supplementation is more efficient to enhance ARG and
NO availability than oral ARG supplementation [
27
]. Comparing increasing doses of CIT
(2–15 g), a 10 g dose was well tolerated and resulted in greater plasma ARG availability
in the elderly [
28
], suggesting this could be the most appropriate dose for clinical use [
29
].
Improvements in macro- and microvascular endothelial function at rest following CIT
supplementation were observed in hypertensive postmenopausal women [
30
] and patients
with heart failure with preserved ejection fraction [
31
] by enhancing ARG availability.
CIT supplementation for 7 days showed higher TSI and lower HHb in the leg muscle
during cycling exercise in young active men, suggesting improved microvascular perfusion
and reduced oxygen consumption [
25
]. Although CIT may enhance arterial vasodilatory
capacity at rest and muscle oxygenation responses to exercise in young men, the efficacy of
CIT supplementation to improve macrovascular function and muscle oxygenation during
exercise in postmenopausal women with hypertension is unknown. Thus, the purpose
of this study was to examine the impact of 4 weeks of CIT supplementation on brachial
artery vasodilatory capacity and muscle oxygenation at rest and during handgrip exercise
in hypertensive postmenopausal women. We hypothesized that 4 weeks of CIT would
improve arterial vasodilation, muscle oxygenation, and muscle extraction during exercise
via improved brachial artery endothelial-dependent vasodilation.
2. Materials and Methods
2.1. Participants
Postmenopausal women (at least 1 year without menstruation), aged 50 to 71 years,
were enrolled in this study. All participants were hypertensive (resting systolic blood
pressure (SBP)
≥
130 mmHg or
≥
120 mmHg if they were on anti-hypertensive medication)
and sedentary (<120 min/week of exercise). Hypertension was defined according to
the 2017 American Heart Association guidelines [
32
], which differ from international
guidelines (SBP > 140 mmHg). Exclusion criteria included a body mass index > 40 kg/m
2
,
SBP ≥160 mmHg,
current use of tobacco, or >7 alcoholic drinks consumed per week.
Participants were excluded if they were diagnosed with cardiovascular diseases, type 1 or
2 diabetes,
or any metabolic/chronic diseases or if they were taking beta-blockers, more
than one vasoactive drug, or dietary supplements with vasodilatory and/or antioxidant
effects. No participants changed their medications at least 3 months prior to the study
participation and during the study period. Participants signed a written informed consent
Nutrients 2024,16, 1935 3 of 16
and completed a health questionnaire. All study protocols were explained by a researcher
and participants were familiarized with the protocols prior to the experimental visits.
All procedures were approved by the Texas Tech University Institutional Review Board
(IRB2018-463; approved on 31 May 2019) and registered in ClinicalTrials.gov https://
clinicaltrials.gov/study/NCT05227781 (accessed on 7 February 2022). under NCT05227781.
2.2. Experimental Protocol
This study was of a double-blind, randomized, placebo-controlled, and parallel design.
Measurements were performed in the morning after an overnight fast of at least 8 h. Partici-
pants refrained from caffeine and prescription medications (~12 h) before each visit, and from
alcohol and physical activity for at least 24 h. Following at least 20 min of rest in the supine
position, vascular measurements were performed in a quiet, temperature-controlled, dimly lit
(~23
◦
C) room. All measurements were collected 4 weeks after the supplementations at the
same time of the day (±1 h) following the sequence utilized at baseline.
The principal investigator, who was not involved in data collection, performed the
group randomization stratified by age and SBP using an online program. Participants
were randomized to consume either CIT (10 g/day) (n= 11) or the placebo (maltodextrin)
(n= 11) for 4 weeks (NOW
®
Foods). Participants consumed 4.5 g (6 capsules of 750 mg)
and 5. 25 g (7 capsules of 750 mg) in the morning and evening, respectively, for 4 weeks.
Participants were asked to not consume foods containing high levels of ARG and CIT
(e.g., nuts, almonds, watermelon) or supplements containing antioxidants during the study
duration. Adherence to the supplements was calculated by counting capsules from the
returned bottles. Participants were asked to keep their habitual diet and physical activity
until the completion of the study.
2.3. Measurements
2.3.1. Anthropometrics
Height (m) was measured using a stadiometer (Free-Standing Portable Height Rod,
Detecto, Webb City, MO, USA) to the nearest 0.01 m. Weight (kg) was obtained using
a beam scale (Weigh Beam, Detecto, Webb City, MO, USA) to the nearest 0.1 kg. The
body mass index was calculated by dividing weight (kg) by height squared (m
2
). Waist
circumference was measured with a non-elastic tape measure at the point between the last
rib and the upper border of the iliac crest [33].
2.3.2. Forearm Muscle Strength and Dynamic Handgrip Exercise
Muscle strength of the dominant hand was determined through maximal voluntary
contraction (MVC) using a digital handgrip dynamometer (Lafayette Instrument Co.,
Lafayette, IN, USA). Participants performed three MVCs with a minute break between
trials, and the highest value was considered as the MVC. Following the collection of resting
measurements, participants performed a rhythmic handgrip exercise at 30% of MVC for
3 min with a metronome-controlled rate (3-s concentric/3-s eccentric). Participants had
continuous visual guidance of the target force on the screen and verbal feedback from the
researchers to keep on the target MVC during exercise.
2.3.3. Brachial and Aortic Blood Pressure and Arterial Stiffness
Following 20 min of rest in the supine position, brachial BP was measured at least
twice using an automated oscillometric device (HEM-907XL; Omron Healthcare, Vernon
Hill, IL, USA) and an average of two measures with a difference of less than 5 mmHg was
used in the analysis. An arterial tonometer (SphygmoCor CPV, AtCor Medical, Sydney,
Australia) was placed on the radial artery to collect pressure waveforms. Radial waveforms
were calibrated with brachial diastolic BP (DBP) and mean arterial pressure (MAP) to
generate aortic pressure waveforms. Brachial and aortic SBP, DBP, MAP, and pulse pressure
(PP) were determined at rest. Brachial BP was measured once at minute 3 of handgrip
exercise. Changes (
∆
) in SBP, DBP, MAP, and PP were calculated from rest to minute 3
Nutrients 2024,16, 1935 4 of 16
of exercise. Carotid–femoral pulse wave velocity (cfPWV), a measure of aortic stiffness,
was assessed using wave sensors (Complior Analyse, Alam Medical, Vincennes, France)
positioned over the common carotid and femoral arteries. The distance between the two
arterial points was measured above the body surface using a non-elastic segmometer. The
value of cfPWV was determined by dividing the distance of the carotid-femoral segment by
the transit time between the two pulse waves. At least two cfPWV readings were obtained
and averaged if there was a ≤0.3 m/s difference between two values.
2.3.4. Brachial Artery Flow-Mediated Dilation (FMD) and Hemodynamics
A 12 MHz linear array Doppler ultrasound probe (Logiq S7, General Electric, Milwau-
kee, WI, USA) was placed on the brachial artery (2–3 cm proximal to the antecubital fossa)
and held with a probe holder at an insonation angle < 60
◦
. The baseline diameter was
recorded for 2 min, and the cuff was inflated to 250 mmHg for 5 min using a rapid-inflating
cuff (Hokanson E20, Bellevue, WA, USA) placed on the right forearm distal to the brachial
artery. Arterial occlusion was followed by rapid cuff deflation and 3 min of reactive hyper-
emia. The brachial artery diameter and mean blood velocity were continuously recorded
during the 10 min protocol. Images were recorded through an online video software (OBS
Studio version 29.1.3). The images were analyzed using automated edge detection software
(Quipu Cardiovascular Suite version 3.6.0, Pisa, Italy), and brachial artery flow-mediated
dilation (FMD) was calculated by using the following formula.
FMD (%) = (peak diameter −baseline diameter)/baseline diameter ×100
During handgrip exercise protocol, the ultrasound probe was placed on the upper arm,
and the brachial artery diameter and mean blood velocity were continuously recorded at
rest and during 3 min of exercise. Forearm muscle BF (FBF) and VC (FVC) were calculated
as described in the previous study [
34
] and averaged by every 1 min for data analysis.
Changes (
∆
) in FBF and FVC were calculated from rest (0 min) to the 1st, 2nd, and 3rd
minute of exercise. ∆FBF and ∆FVC were averaged over the 3 min (∆3-min) of exercise.
FBF (mL/min) = mean blood velocity (cm/sec) ×π(brachial artery diameter (cm)/2)2 ×60
FVC (mL/min/mmHg) = (FBF (mL/min)/MAP (mmHg)) ×100
2.3.5. Muscle Oxygenation
Forearm muscle oxygenation was measured using a frequency domain, non-invasive, near-
infrared spectroscopy (NIRS) system (PortaMon, Artinis Medical System BV, Elst, Gelderland,
The Netherlands) positioned on the skin over the flexor digitorum profundus of the right hand.
The optodes were covered and stabilized by wrapping with a black elastic bandage around the
forearm. The NIRS-derived data were acquired by transmitting to a computer via Bluetooth at
10 Hz, and recorded using software (Oxysoft version 3.0, Artinis Medical Systems BV, Elst, The
Netherlands). The NIRS device continuously monitored the relative changes in oxygenated
hemoglobin (O
2
Hb) and HHb from rest to exercise. The HHb reflects the balance between the
local oxygen supply and utilization and provides an estimate of changes in fractional oxygen
extraction [
18
]. TSI is an absolute measure of muscle oxygenation [
35
] and was calculated as
follows: TSI (%) = (O
2
Hb/(O
2
Hb + HHb))
×
100. Changes (
∆
) in TSI, HHb, and O
2
Hb were
calculated from rest to minutes 1, 2, and 3 of exercise.
∆
TSI,
∆
HHb, and
∆
O
2
Hb were averaged
over the 3 min (∆3-min) of exercise.
2.4. Statistical Analysis
Based on previous studies that showed increased FBF [
36
] and higher O
2
Hb concen-
tration [
37
] during exercise after the acute ingestion of an NO precursor in young adults,
10 participants per group were estimated to detect a significant difference in muscle oxy-
genation with
≥
80% power at the
α
= 0.05 level. All statistical analyses were performed
with SPSS 29.0 (IBM SPSS Statistics, Chicago, IL, USA). The normality of the data was
Nutrients 2024,16, 1935 5 of 16
tested using the Shapiro–Wilk test. Between-group differences at 0 week were compared
using an independent t-test. Two-way repeated measures analysis of variance (ANOVA)
with Bonferroni adjustments were used to detect significant changes in brachial and aortic
BP, FMD, and cfPWV between groups (placebo and CIT) over time (0 week and 4 weeks).
Two-way repeated measures ANOVA with Bonferroni adjustments were used to determine
significant differences in arterial vasodilation (
∆
FBF and
∆
FVC) and NIRS-derived (
∆
TSI,
∆
HHb, and
∆
O
2
Hb) responses to exercise (0, 1, 2, 3 min) from 0 to 4 weeks between groups.
When a significant group-by-time interaction was detected, pairwise comparisons were
performed through the Bonferroni adjustment that corrects the error of multiple compar-
isons. An independent t-test was performed to determine significant differences of average
changes over the 3 min of exercise on arterial vasodilation (
∆
3-min_FBF and
∆
3-min_FVC)
and NIRS-derived measures (
∆
3-min_TSI,
∆
3-min_HHb, and
∆
3-min_O
2
Hb) from 0 to
4 weeks between groups. Pearson’s correlation was performed to identify relationships
between the changes in FMD, arterial vasodilation (
∆
3-min_FBF and
∆
3-min_FVC), and
muscle oxygenation measures (
∆
3-min_TSI,
∆
3-min_HHb, and
∆
3-min_O
2
Hb) during
exercise from 0 to 4 week. Data were presented as mean
±
standard deviation (SD) in tables
and standard error (SE) in figures. Statistical significance was set at p< 0.05.
3. Results
Twenty-two participants finished the study (Figure 1). Compliance with the supple-
ments were 94.2
±
2.0% (placebo) and 93.7
±
2.2% (CIT). No adverse effects were reported
during the study. Participant characteristics and medications are shown in Table 1. There
were no significant baseline differences between the groups (all p> 0.05, Table 1). Partici-
pants in both groups were on antihypertensive medications (PL = 3 and CIT = 4) and statins
(PL = 1 and CIT = 1).
Nutrients 2024, 16, x FOR PEER REVIEW 5 of 17
1, 2, and 3 of exercise. ΔTSI, ΔHHb, and ΔO
2
Hb were averaged over the 3 min (Δ3-min) of
exercise.
2.4. Statistical Analysis
Based on previous studies that showed increased FBF [36] and higher O
2
Hb concen-
tration [37] during exercise after the acute ingestion of an NO precursor in young adults,
10 participants per group were estimated to detect a significant difference in muscle oxy-
genation with ≥80% power at the α = 0.05 level. All statistical analyses were performed
with SPSS 29.0 (IBM SPSS Statistics, Chicago, IL, USA). The normality of the data was
tested using the Shapiro–Wilk test. Between-group differences at 0 week were compared
using an independent t-test. Two-way repeated measures analysis of variance (ANOVA)
with Bonferroni adjustments were used to detect significant changes in brachial and aortic
BP, FMD, and cfPWV between groups (placebo and CIT) over time (0 week and 4 weeks).
Two-way repeated measures ANOVA with Bonferroni adjustments were used to deter-
mine significant differences in arterial vasodilation (ΔFBF and ΔFVC) and NIRS-derived
(ΔTSI, ΔHHb, and ΔO
2
Hb) responses to exercise (0, 1, 2, 3 min) from 0 to 4 weeks between
groups. When a significant group-by-time interaction was detected, pairwise comparisons
were performed through the Bonferroni adjustment that corrects the error of multiple
comparisons. An independent t-test was performed to determine significant differences
of average changes over the 3 min of exercise on arterial vasodilation (Δ3-min_FBF and
Δ3min_FVC) and NIRS-derived measures (Δ3-min_TSI, Δ3-min_HHb, and Δ3-
min_O
2
Hb) from 0 to 4 weeks between groups. Pearson’s correlation was performed to
identify relationships between the changes in FMD, arterial vasodilation (Δ3-min_FBF
and Δ3-min_FVC), and muscle oxygenation measures (Δ3-min_TSI, Δ3-min_HHb, and
Δ3-min_O
2
Hb) during exercise from 0 to 4 week. Data were presented as mean ± standard
deviation (SD) in tables and standard error (SE) in figures. Statistical significance was set
at p < 0.05.
3. Results
Twenty-two participants finished the study (Figure 1). Compliance with the supple-
ments were 94.2 ± 2.0% (placebo) and 93.7 ± 2.2% (CIT). No adverse effects were reported
during the study. Participant characteristics and medications are shown in Table 1. There
were no significant baseline differences between the groups (all p > 0.05, Table 1). Partici-
pants in both groups were on antihypertensive medications (PL = 3 and CIT = 4) and
statins (PL = 1 and CIT = 1).
Figure 1. CONSORT flow chart of participants through the study. CIT, L-citrulline; NIRS, near-
infrared spectroscopy.
Nutrients 2024,16, 1935 6 of 16
Table 1. Participant characteristics and medications.
Characteristics Placebo (n= 11) CIT (n= 11) p
Age (years) 64 ±5 61 ±7 0.35
Height (m) 1.58 ±0.09 1.59 ±0.05 0.45
Weight (kg) 74.4 ±15.7 75.0 ±10.8 0.92
Body mass index (kg/m2)30.2 ±5.9 29.8 ±4.0 0.85
Waist circumference (cm) 97.5 ±18.3 90.8 ±10.4 0.30
MVC (kg) 30 ±7 30 ±6 0.92
Medication (n)
ARB 1 2
ACE inhibitors 0 1
Diuretics 1 1
Calcium channel blockers 1 0
Statin 1 1
Values are the mean
±
SD or number (n). Abbreviations: CIT, L-citrulline; MVC, maximal voluntary contraction;
ARB, angiotensin receptor blocker; ACE, angiotensin converting enzyme. p-values are the between-group
differences from the t-test.
3.1. Effects of Supplementations on Blood Pressure, Endothelial Function, and Arterial Stiffness
There were no group differences in cfPWV, FMD, resting brachial and aortic BP, and
brachial BP responses to exercise at 0 week (all p> 0.05, Table 2). No significant group-
by-time interaction was observed for cfPWV (p> 0.05, Table 2). There was a significant
group-by-time interaction for brachial FMD (p< 0.05, Figure 2A). Compared to the placebo,
FMD significantly increased after 4 weeks of CIT supplementation (placebo:
∆−
0.7
±
0.6%
vs. CIT: ∆1.6 ±0.7%, p< 0.05) (Figure 2B).
Table 2. Vascular function and blood pressure at rest, and blood pressure responses to exercise at 0
and 4 weeks.
Variables
Placebo (n= 11) CIT (n= 11)
p
0 Week 4 Weeks ∆0 to 4 Weeks 0 Week 4 Weeks ∆0 to 4 Weeks
Rest
cfPWV (m/s) 9.2 ±1.3 8.6 ±1.6 ∆−0.6 ±0.8 9.4 ±2.0 8.5 ±1.1 * ∆−0.9 ±1.3 0.46
Baseline Diameter (mm) 3.67 ±0.48 3.81 ±0.51 ∆0.14 ±0.21 3.64 ±0.43 3.60 ±0.32 ∆−0.04 ±0.34 0.14
Peak Diameter (mm) 3.82 ±0.32 3.98 ±0.53 ∆0.13 ±0.18 3.83 ±0.44 3.82 ±0.32 ∆−0.01 ±0.38 0.31
Brachial FMD (%) 4.84 ±1.75 4.18 ±2.19 ∆−0.7 ±2.1 5.03 ±2.34 6.62 ±2.22 *†∆1.6 ±2.2 0.02
Baseline Shear Rate (s−1)125 ±45 131 ±36 ∆6±32 125 ±46 169 ±61 ∆43 ±164 0.47
Peak Shear Rate (s−1)1028 ±264 1016 ±302 ∆−12 ±209 1055 ±245 1148 ±401 ∆92 ±428 0.78
FMD/Shear RateAUC
(u.a.) 2.18 ±1.23 1.82 ±1.29 ∆−0.36 ±1.55 1.52 ±0.83 2.40 ±1.06 ∆0.88 ±1.35 0.06
Brachial SBP (mmHg) 133 ±14 135 ±15 ∆2±5 132 ±12 128 ±8 *†∆−4±6 0.04
Brachial DBP (mmHg) 78 ±10 79 ±11 ∆2±3 82 ±8 81 ±7∆−1±4 0.09
Brachial MAP (mmHg) 96 ±10 98 ±11 ∆2±4 99 ±8 97 ±6∆−2±4 0.04
Brachial PP (mmHg) 55 ±13 55 ±12 ∆1±5 50 ±9 47 ±8∆−2±7 0.21
Aortic SBP (mmHg) 127 ±13 130 ±15 ∆3±5 126 ±10 123 ±6 *†∆−4±6 0.01
Aortic DBP (mmHg) 78 ±11 79 ±12 ∆1±4 83 ±8 80 ±7 * ∆−3±6 0.06
Aortic MAP (mmHg) 95 ±10 96 ±12 ∆1±4 95 ±894 ±6 *†∆−3±5 0.02
Aortic PP (mmHg) 49 ±12 51 ±11 ∆2±3 43 ±7 43 ±7∆0±6 0.28
Exercise
∆Brachial SBP (mmHg) 19 ±10 19 ±10 ∆0±10 20 ±9 22 ±7∆2±8 0.71
∆Brachial DBP (mmHg) 6 ±10 8 ±5∆2±9 8 ±7 9 ±8∆1±6 0.73
∆
Brachial MAP (mmHg)
10 ±10 11 ±6∆1±8 12 ±6 13 ±7∆1±4 0.96
∆Brachial PP (mmHg) 13 ±8 12 ±7∆−2±11 12 ±10 13 ±7∆1±11 0.58
Values are the mean
±
SD. Abbreviations: CIT, L-citrulline; cfPWV, carotid–femoral pulse wave velocity; FMD,
flow-mediated dilation; AUC, area under the curve; SBP, systolic blood pressure; DBP, diastolic blood pressure;
MAP, mean arterial pressure; PP, pulse pressure;
∆
, change from rest to the last minute of exercise. p-values are the
group-by-time interaction from two-way repeated measures ANOVA. * p< 0.05 vs. 0 week;
†
p< 0.05 vs. placebo.
Nutrients 2024,16, 1935 7 of 16
Nutrients 2024, 16, x FOR PEER REVIEW 7 of 17
Brachial MAP (mmHg) 96 ± 10 98 ± 11 Δ2 ± 4 99 ± 8 97 ± 6 Δ−2 ± 4 0.04
Brachial PP (mmHg) 55 ± 13 55 ± 12 Δ1 ± 5 50 ± 9 47 ± 8 Δ−2 ± 7 0.21
Aortic SBP (mmHg) 127 ± 13 130 ± 15 Δ3 ± 5 126 ± 10 123 ± 6 *
†
Δ−4 ± 6 0.01
Aortic DBP (mmHg) 78 ± 11 79 ± 12 Δ1 ± 4 83 ± 8 80 ± 7 * Δ−3 ± 6 0.06
Aortic MAP (mmHg) 95 ± 10 96 ± 12 Δ1 ± 4 95 ± 8 94 ± 6 *
†
Δ−3 ± 5 0.02
Aortic PP (mmHg) 49 ± 12 51 ± 11 Δ2 ± 3 43 ± 7 43 ± 7 Δ0 ± 6 0.28
Exercise
Δ Brachial SBP (mmHg) 19 ± 10 19 ± 10 Δ0 ± 10 20 ± 9 22 ± 7 Δ2 ± 8 0.71
Δ Brachial DBP (mmHg) 6 ± 10 8 ± 5 Δ2 ± 9 8 ± 7 9 ± 8 Δ1 ± 6 0.73
Δ Brachial MAP
(mmHg) 10 ± 10 11 ± 6 Δ1 ± 8 12 ± 6 13 ± 7 Δ1 ± 4 0.96
Δ Brachial PP (mmHg) 13 ± 8 12 ± 7 Δ−2 ± 11 12 ± 10 13 ± 7 Δ1 ± 11 0.58
Values are the mean ± SD. Abbreviations: CIT, L-citrulline; cfPWV, carotid–femoral pulse wave ve-
locity; FMD, flow-mediated dilation; AUC, area under the curve; SBP, systolic blood pressure; DBP,
diastolic blood pressure; MAP, mean arterial pressure; PP, pulse pressure; Δ, change from rest to
the last minute of exercise. p-values are the group-by-time interaction from two-way repeated
measures ANOVA. * p < 0.05 vs. 0 week;
†
p < 0.05 vs. placebo.
Figure 2. Brachial artery flow-mediated dilation (FMD) at 0 and 4 weeks (A) and change (Δ) in FMD
from 0 to 4 weeks (B). Abbreviations: CIT, L-citrulline. Data are means ± standard error (SE). * p <
0.05 vs. 0 week;
†
p < 0.05 vs. placebo.
3.2. Effects of Supplementations on Arterial Vasodilation during Exercise
There were no group differences in the absolute values of FBF and FVC at rest at 0
and 4 weeks (all p > 0.05, Table 3). Significant group-by-time interactions were found for
ΔFBF and ΔFVC (both p < 0.01). ΔFBF and ΔFVC were significantly enhanced during the
whole exercise period (all p < 0.05, Figure 3A,C). Compared to the placebo, Δ3-min_FBF
(placebo: Δ−17 ± 12 vs. CIT: Δ 48 ± 16 mL/min, p < 0.01, Figure 3B) and Δ3-min_FVC (pla-
cebo: Δ−21 ± 9 vs. CIT: Δ41 ± 14 mL/mmHg/min vs. p < 0.01, Figure 3D) during exercise
were significantly higher after CIT.
Tab l e 3. Brachial artery vasodilation capacity at rest and responses to handgrip exercise at 0 and 4
weeks.
Variables Placebo (n = 11) CIT (n = 11)
0 Week 4 Weeks 0 Week 4 Weeks
FBF (mL/min)
Rest 68 ± 41 - 107 ± 105 - 62 ± 18 - 90 ± 90 -
1 min 150 ± 47 * Δ82 ± 43 * 172 ± 115 * Δ65 ± 39 * 127 ± 40 * Δ65 ± 30 * 189 ± 127 * Δ100 ± 52 *
†
2 min 176 ± 41 * Δ107 ± 35 * 204 ± 125 * Δ98 ± 35 * 149 ± 54 * Δ87 ± 46 * 225 ± 133 * Δ135 ± 65 *
†
3 min 200 ± 35 * Δ132 ± 21 * 212 ± 112 * Δ106 ± 22 * 177 ± 51 * Δ115 ± 38 * 265 ± 136 * Δ176 ± 77 *
†
Figure 2. Brachial artery flow-mediated dilation (FMD) at 0 and 4 weeks (A) and change (
∆
) in
FMD from 0 to 4 weeks (B). Abbreviations: CIT, L-citrulline. Data are means
±
standard error (SE).
*p< 0.05 vs. 0 week; †p< 0.05 vs. placebo.
There were significant group-by-time interactions in resting brachial and aortic SBP
and MAP (all p< 0.05, Table 2). Four weeks of CIT supplementation significantly reduced
brachial SBP (placebo:
∆
2
±
5 vs. CIT:
∆−
4
±
6 mmHg), brachial MAP (placebo:
∆2±4
vs.
CIT:
∆−
2
±
4 mmHg), aortic SBP (placebo:
∆
3
±
5 vs. CIT:
∆−
4
±
6 mmHg), and aortic
MAP (placebo:
∆
2
±
4 vs. CIT:
∆−
3
±
5 mmHg) compared to the placebo (all p< 0.05,
Table 2). No significant group-by-time interactions were observed for brachial BP responses
to exercise (all p> 0.05, Table 2).
3.2. Effects of Supplementations on Arterial Vasodilation during Exercise
There were no group differences in the absolute values of FBF and FVC at rest at 0 and
4 weeks (all p> 0.05, Table 3). Significant group-by-time interactions were found for
∆
FBF
and
∆
FVC (both p< 0.01).
∆
FBF and
∆
FVC were significantly enhanced during the whole
exercise period (all p< 0.05, Figure 3A,C). Compared to the placebo,
∆
3-min_FBF (placebo:
∆−
17
±
12 vs. CIT:
∆
48
±
16 mL/min, p< 0.01, Figure 3B) and
∆
3-min_FVC (placebo:
∆−
21
±
9 vs. CIT:
∆
41
±
14 mL/mmHg/min vs. p< 0.01, Figure 3D) during exercise were
significantly higher after CIT.
Table 3. Brachial artery vasodilation capacity at rest and responses to handgrip exercise at 0 and
4 weeks.
Variables
Placebo (n= 11) CIT (n= 11)
0 Week 4 Weeks 0 Week 4 Weeks
FBF (mL/min)
Rest 68 ±41 - 107 ±105 - 62 ±18 - 90 ±90 -
1 min 150 ±47 * ∆82 ±43 * 172 ±115 * ∆65 ±39 * 127 ±40 * ∆65 ±30 * 189 ±127 * ∆100 ±52 *†
2 min 176 ±41 * ∆107 ±35 * 204 ±125 * ∆98 ±35 * 149 ±54 * ∆87 ±46 * 225 ±133 * ∆135 ±65 *†
3 min 200 ±35 * ∆132 ±21 * 212 ±112 * ∆106 ±22 * 177 ±51 * ∆115 ±38 * 265 ±136 * ∆176 ±77 *†
Average over 3 min
175 ±38 * ∆107 ±30 * 191 ±116 * ∆90 ±26 * 151 ±44 * ∆89 ±33 * 226 ±126 * ∆137 ±53 *†
FVC
(mL/min/mmHg)
Rest 68 ±44 64 ±19 110 ±116 91 ±82
1 min 133 ±44 * ∆65 ±41 * 120 ±43 * ∆46 ±36 156 ±116 * ∆56 ±31 * 177 ±112 * ∆86 ±43 *†
2 min 155 ±39 * ∆87 ±33 * 140 ±53 * ∆74 ±19 * 184 ±118 * ∆76 ±43 * 206 ±117 * ∆115 ±54 *†
3 min 176 ±37 * ∆108 ±23 * 161 ±53 * ∆79 ±16 * 189 ±113 * ∆97 ±40 * 240 ±125 * ∆149 ±69 *†
Average over 3 min
155 ±38 * ∆87 ±30 * 176 ±115 * ∆92 ±42 * 140 ±47 * ∆76 ±35 * 208 ±113 * ∆117 ±44 *†
Values are the mean
±
SD. Abbreviations: CIT, L-citrulline; FBF, forearm blood flow; FVC, forearm vascular
conductance; ∆, change from rest to exercise. * p< 0.05 vs. rest; †p< 0.05 vs. placebo the same week.
Nutrients 2024,16, 1935 8 of 16
Nutrients 2024, 16, x FOR PEER REVIEW 8 of 17
Average over 3 min 175 ± 38 * Δ107 ± 30 * 191 ± 116 * Δ90 ± 26 * 151 ± 44 * Δ89 ± 33 * 226 ± 126 * Δ137 ± 53 *
†
FVC
(mL/min/mmHg)
Rest 68 ± 44 64 ± 19 110 ±116 91 ± 82
1 min 133 ± 44 * Δ65 ± 41 * 120 ± 43 * Δ46 ± 36 156 ± 116 * Δ56 ± 31 * 177 ± 112 * Δ86 ± 43 *
†
2 min 155 ± 39 * Δ87 ± 33 * 140 ± 53 * Δ74 ± 19 * 184 ± 118 * Δ76 ± 43 * 206 ± 117 * Δ115 ± 54 *
†
3 min 176 ± 37 * Δ108 ± 23 * 161 ± 53 * Δ79 ± 16 * 189 ± 113 * Δ97 ± 40 * 240 ± 125 * Δ149 ± 69 *
†
Average over 3 min 155 ± 38 * Δ87 ± 30 * 176 ± 115 * Δ92 ± 42 * 140 ± 47 * Δ76 ± 35 * 208 ± 113 * Δ117 ± 44 *
†
Values are the mean ± SD. Abbreviations: CIT, L-citrulline; FBF, forearm blood flow; FVC, forearm
vascular conductance; Δ, change from rest to exercise. * p < 0.05 vs. rest;
†
p < 0.05 vs. placebo the
same week.
Figure 3. Changes (Δ) in forearm blood flow (FBF) (A) and vascular conductance (FVC) (C) during
3 min exercise from 0 to 4 weeks, and average changes in FBF (Δ3-min_FBF) (B) and FVC (Δ3-
min_FVC) (D) over the 3 min of exercise from 0 to 4 weeks. Abbreviation: CIT, L-citrulline. Data are
means ± standard error (SE).
†
p < 0.05;
‡
p < 0.01 vs. placebo.
3.3. Effects of Supplementations on Muscle Oxygenation Responses to Exercise
There were no group differences in the absolute values of TSI, HHb, and O
2
Hb at rest
at 0 and 4 weeks (all p > 0.05, Table 4). There were significant group-by-time interactions
for ΔTSI, ΔHHb, and ΔO
2
Hb during exercise (all p < 0.05, Figure 4). ΔTSI significantly
increased during the whole exercise period after CIT compared to the placebo (all p < 0.05,
Figure 4A). Compared to the placebo, Δ3-min_TSI during exercise was significantly higher
after CIT (placebo: Δ−0.84 ± 0.58 vs. CIT: Δ1.61 ± 0.46%, p < 0.01) (Figure 4B). ΔHHb was
significantly lower throughout the exercise after CIT compared to the placebo (all p < 0.05,
Figure 4C). Compared to the placebo, Δ3-min_HHb during exercise was lower after CIT
(placebo: Δ1.03 ± 0.69 vs. CIT: Δ −2.76 ± 0.77 µM, p < 0.01) (Figure 4D). Despite no signifi-
cant between-group difference at minute 1, ΔO
2
Hb significantly increased during minutes
2 and 3 of exercise after CIT compared to the placebo (all p < 0.05, Figure 4E). Compared
Figure 3. Changes (
∆
) in forearm blood flow (FBF) (A) and vascular conductance (FVC) (C) dur-
ing
3 min
exercise from 0 to 4 weeks, and average changes in FBF (
∆
3-min_FBF) (B) and FVC
(
∆3-min_FVC
) (D) over the 3 min of exercise from 0 to 4 weeks. Abbreviation: CIT, L-citrulline. Data
are means ±standard error (SE). †p< 0.05; ‡p< 0.01 vs. placebo.
3.3. Effects of Supplementations on Muscle Oxygenation Responses to Exercise
There were no group differences in the absolute values of TSI, HHb, and O
2
Hb at rest
at 0 and 4 weeks (all p> 0.05, Table 4). There were significant group-by-time interactions
for
∆
TSI,
∆
HHb, and
∆
O
2
Hb during exercise (all p< 0.05, Figure 4).
∆
TSI significantly
increased during the whole exercise period after CIT compared to the placebo (all p< 0.05,
Figure 4A). Compared to the placebo,
∆
3-min_TSI during exercise was significantly higher
after CIT (placebo:
∆−
0.84
±
0.58 vs. CIT:
∆
1.61
±
0.46%, p< 0.01) (Figure 4B).
∆
HHb
was significantly lower throughout the exercise after CIT compared to the placebo (all
p< 0.05
, Figure 4C). Compared to the placebo,
∆
3-min_HHb during exercise was lower
after CIT (placebo:
∆
1.03
±
0.69 vs. CIT:
∆−
2.76
±
0.77
µ
M, p< 0.01) (Figure 4D). Despite
no significant between-group difference at minute 1,
∆
O
2
Hb significantly increased during
minutes 2 and 3 of exercise after CIT compared to the placebo (all p< 0.05, Figure 4E).
Compared to the placebo,
∆
3-min_O
2
Hb during exercise was higher after CIT (placebo:
∆−2.02 ±0.84 vs. CIT: ∆2.46 ±1.42 µM, p< 0.05) (Figure 4F).
Table 4. Forearm muscle oxygenation during handgrip exercise at 0 and 4 weeks.
Variables Placebo (n= 11) CIT (n= 11)
0 Week 4 Weeks 0 Week 4 Weeks
TSI (%)
Rest 61.4 ±2.3 - 59.6 ±2.7 - 61.8 ±4.1 - 60.6 ±3.3 -
1 min 61.5 ±2.5 ∆0.18 ±1.24 58.8 ±3.8 ∆−0.86 ±2.27 61.2 ±5.6 ∆−0.65 ±2.12 61.6 ±3.2 ∆0.98 ±1.21 †
2 min 60.6 ±2.2 ∆−0.71 ±1.46 58.0 ±4.2 ∆−1.64 ±2.75 60.2 ±5.7 ∆−1.60 ±2.66 60.6 ±3.2 ∆0.01 ±1.96 †
3 min 60.5 ±4.2 ∆−0.82 ±1.37 58.3 ±4.1 ∆−1.36 ±2.39 60.4 ±5.6 ∆−1.46 ±2.51 60.7 ±3.2 ∆0.13 ±1.73 †
Average
over 3 min 60.9 ±2.2 ∆−0.45 ±1.07 58.4 ±4.0 ∆−1.29 ±2.42 60.6 ±5.6 ∆−1.24 ±2.38 61.0 ±3.2 ∆0.37 ±1.62 †
Nutrients 2024,16, 1935 9 of 16
Table 4. Cont.
Variables Placebo (n= 11) CIT (n= 11)
0 Week 4 Weeks 0 Week 4 Weeks
HHb (µM)
Rest −0.60 ±0.62 - −0.95 ±0.94 - −0.38 ±0.49 - −0.51 ±0.56 -
1 min 0.96 ±1.48 ∆1.56 ±1.70 1.48 ±2.99 * ∆2.43 ±2.63 * 2.01 ±2.07 * ∆2.39 ±2.10 * −0.69 ±1.96 ∆−0.18 ±1.57 †
2 min 1.64 ±1.45 * ∆2.24 ±1.63 * 2.80 ±3.73 * ∆3.76 ±3.68 * 2.97 ±2.92 * ∆3.35 ±2.97 * −0.01 ±2.48 †∆0.49 ±2.13 †
3 min 2.45 ±2.82 * ∆3.04 ±2.54 * 2.80 ±3.98 * ∆3.75 ±3.90 * 2.93 ±3.10 * ∆3.30 ±3.19 * −0.06 ±2.23 †∆0.45 ±1.87 †
Average
over 3 min 1.68 ±1.57 * ∆2.28 ±1.66 * 2.36 ±3.43 * ∆3.31 ±3.27 * 2.64 ±2.65 * ∆3.02 ±2.71 * −0.25 ±2.20 †∆0.25 ±1.83 †
O2Hb (µM)
Rest 0.23 ±0.63 - 0.35 ±0.76 - 0.45 ±0.47 - 0.70 ±0.47 -
1 min 1.08 ±3.11 ∆0.85 ±3.07 0.17 ±2.60 ∆−0.18 ±2.30 1.43 ±4.06 ∆0.97 ±4.00 3.22 ±2.42 *†∆2.52 ±2.34 *†
2 min 1.06 ±3.34 ∆0.83 ±3.44 −0.85 ±3.99 ∆−1.20 ±3.81 0.21 ±5.77 ∆−0.24 ±5.73 3.45 ±3.15 †∆2.75 ±3.11 †
3 min 1.55 ±3.77 ∆2.32 ±3.79 −0.32 ±3.75 ∆−0.67 ±3.51 0.62 ±5.91 ∆0.17 ±5.93 3.71 ±2.88 *†∆3.01 ±2.86 *†
Average
over 3 min 1.56 ±3.33 ∆1.34 ±3.36 −0.34 ±3.32 ∆−0.69 ±3.08 0.75 ±5.17 ∆0.30 ±5.14 3.26 ±2.79 *†∆2.76 ±2.75 *†
Values are the mean
±
SD. Abbreviations: CIT, L-citrulline; TSI, tissue oxygen saturation index; HHb, deoxy-
genated hemoglobin; O
2
Hb, oxygenated hemoglobin;
∆
, change from rest to exercise. * p< 0.05 vs. rest;
†
p< 0.05
vs. placebo the same week.
Nutrients 2024, 16, x FOR PEER REVIEW 10 of 17
Figure 4. Changes (Δ) in the tissue oxygen saturation index (TSI) (A), deoxygenated hemoglobin
(HHb) (C), and oxygenated hemoglobin (O
2
Hb) (E) during handgrip exercise from 0 to 4 weeks, and
average changes in TSI (Δ3-min_TSI) (B), HHb (Δ3-min_HHb) (D), and O
2
Hb (Δ3-min_O
2
Hb) (F)
over the 3 min of exercise from 0 to 4 weeks. Abbreviation: CIT, L-citrulline. Data are means ± stand-
ard error (SE).
†
p < 0.05 vs. placebo;
‡
p < 0.01 vs. placebo.
3.4. Correlations between FMD with Arterial Vasodilation and Muscle Oxygenation after CIT
Supplementation
Enhanced Δ3-min_FBF (r = 0.53, p < 0.05, Figure 5A) and Δ3-min_FVC (r = 0.52, p <
0.05, Figure 5B) during exercise from 0 to 4 weeks were correlated with ΔFMD from 0 to
4 weeks. Δ3-min_NIRS-derived measures were not significantly correlated with ΔFMD
from 0 to 4 weeks. Moreover, improved Δ3-min_FBF was correlated with Δ3-min_TSI (r =
0.53, p < 0.05, Figure 5C), Δ3-min_O
2
Hb (r = 0.54, p < 0.05), but not with Δ3-min_HHb
during exercise (p > 0.05). Improved Δ3-min_FVC was correlated with Δ3-min_TSI (r =
0.44, p < 0.05, Figure 5D) Δ3-min_O
2
Hb (r = 0.62, p < 0.01), and Δ3-min_HHb during exer-
cise (r = −0.44, p < 0.05).
Figure 4. Changes (
∆
) in the tissue oxygen saturation index (TSI) (A), deoxygenated hemoglobin
(HHb) (C), and oxygenated hemoglobin (O
2
Hb) (E) during handgrip exercise from 0 to 4 weeks, and
average changes in TSI (
∆
3-min_TSI) (B), HHb (
∆
3-min_HHb) (D), and O
2
Hb (
∆
3-min_O
2
Hb)
(F) over the 3 min of exercise from 0 to 4 weeks. Abbreviation: CIT, L-citrulline. Data are
means ±standard error (SE). †p< 0.05 vs. placebo; ‡p< 0.01 vs. placebo.
Nutrients 2024,16, 1935 10 of 16
3.4. Correlations between FMD with Arterial Vasodilation and Muscle Oxygenation after
CIT Supplementation
Enhanced
∆
3-min_FBF (r = 0.53, p< 0.05, Figure 5A) and
∆
3-min_FVC (r = 0.52,
p< 0.05,
Figure 5B) during exercise from 0 to 4 weeks were correlated with
∆
FMD from 0
to 4 weeks.
∆
3-min_NIRS-derived measures were not significantly correlated with
∆
FMD
from 0 to 4 weeks. Moreover, improved
∆
3-min_FBF was correlated with
∆
3-min_TSI
(
r = 0.53,
p< 0.05, Figure 5C),
∆
3-min_O
2
Hb (r = 0.54, p< 0.05), but not with
∆
3-min_HHb
during exercise (p> 0.05). Improved
∆
3-min_FVC was correlated with
∆
3-min_TSI (r = 0.44,
p< 0.05, Figure 5D)
∆
3-min_O
2
Hb (r = 0.62, p< 0.01), and
∆
3-min_HHb during exercise
(r = −0.44, p< 0.05).
Nutrients 2024, 16, x FOR PEER REVIEW 11 of 17
Figure 5. Correlations between average changes over the 3 min of exercise (Δ3-min) in forearm blood
flow (Δ3-min_FBF) and vascular conductance (Δ3-min_FVC) from 0 to 4 weeks with changes in bra-
chial artery flow-mediated dilation (ΔFMD) from 0 to 4 weeks (A,B) and average changes in the
tissue oxygen saturation index (Δ3-min_TSI) from 0 to 4 weeks (C,D).
4. Discussion
Our findings indicate that CIT supplementation increased brachial FMD and reduced
brachial and aortic SBP and MAP at rest. The novel findings of this study are that 4 weeks
of CIT supplementation increased FBF, FVC, TSI, and O
2
Hb and aenuated HHb during
dynamic handgrip exercise. Furthermore, enhanced arterial vasodilation (FBF and FVC)
was correlated with improvements in FMD and muscle oxygenation. Our findings indi-
cate that CIT supplementation improves muscle oxygenation and oxygen extraction re-
sponses to exercise by increasing local artery endothelial-dependent vasodilation in hy-
pertensive postmenopausal women.
Aging and hypertension cause endothelial dysfunction due to the structural and
functional changes in limb arteries and arterioles [10]. Specifically, brachial artery endo-
thelial function progressively declines in women across the menopause transition due to
ARG deficiency [8]. Reduced NO production and increased vasoconstrictors (e.g., cate-
cholamines and endothelin) in postmenopausal women contribute to the development of
hypertension [38]. Postmenopausal women have a higher prevalence of systolic hyperten-
sion than men due to the proximal aortic stiffness [39,40], leading to greater risk of heart
failure [40]. In this study, we observed that CIT supplementation reduced resting brachial
and aortic SBP by 4 mmHg and 3 mmHg, respectively. Moreover, although there was no
change in aortic stiffness, we found that 4 weeks of CIT supplementation improved FMD
by 1.6%, showing the clinical significance of a potential reduction in the risk of cardiovas-
cular disease by approximately 19% [41]. Similarly, previous studies have reported the
efficacy of CIT supplementation on endothelial function in postmenopausal women
Figure 5. Correlations between average changes over the 3 min of exercise (
∆
3-min) in forearm blood
flow (
∆
3-min_FBF) and vascular conductance (
∆
3-min_FVC) from 0 to 4 weeks with changes in
brachial artery flow-mediated dilation (
∆
FMD) from 0 to 4 weeks (A,B) and average changes in the
tissue oxygen saturation index (∆3-min_TSI) from 0 to 4 weeks (C,D).
4. Discussion
Our findings indicate that CIT supplementation increased brachial FMD and reduced
brachial and aortic SBP and MAP at rest. The novel findings of this study are that 4 weeks
of CIT supplementation increased FBF, FVC, TSI, and O
2
Hb and attenuated HHb during
dynamic handgrip exercise. Furthermore, enhanced arterial vasodilation (FBF and FVC)
was correlated with improvements in FMD and muscle oxygenation. Our findings indicate
that CIT supplementation improves muscle oxygenation and oxygen extraction responses
to exercise by increasing local artery endothelial-dependent vasodilation in hypertensive
postmenopausal women.
Aging and hypertension cause endothelial dysfunction due to the structural and func-
tional changes in limb arteries and arterioles [
10
]. Specifically, brachial artery endothelial
function progressively declines in women across the menopause transition due to ARG de-
ficiency [
8
]. Reduced NO production and increased vasoconstrictors (e.g., catecholamines
and endothelin) in postmenopausal women contribute to the development of hyperten-
sion [
38
]. Postmenopausal women have a higher prevalence of systolic hypertension than
men due to the proximal aortic stiffness [
39
,
40
], leading to greater risk of heart failure [
40
].
In this study, we observed that CIT supplementation reduced resting brachial and aortic
SBP by 4 mmHg and 3 mmHg, respectively. Moreover, although there was no change in
aortic stiffness, we found that 4 weeks of CIT supplementation improved FMD by 1.6%,
Nutrients 2024,16, 1935 11 of 16
showing the clinical significance of a potential reduction in the risk of cardiovascular dis-
ease by approximately 19% [
41
]. Similarly, previous studies have reported the efficacy of
CIT supplementation on endothelial function in postmenopausal women [
30
,
42
] but not
on aortic stiffness [
43
,
44
]. Our findings suggest that oral CIT supplementation may lead
to functional improvement in limb arteries by enhancing the ARG-NO pathway [
30
,
31
],
despite no change in the structural property of the conduit arteries.
Muscle BF is regulated by a balance between vasodilation and vasoconstriction [
45
].
During exercise, contracting muscles require greater BF and oxygen delivery to capillar-
ies in order to meet an elevated metabolic demand [
15
,
46
], which primarily occurs via
local vasodilation in limb arteries and arterioles [
12
,
47
]. However, arterial BF during ex-
ercise is blunted in older adults [
34
,
48
] due, in part, to reduced NO-mediated endothelial
vasodilation [
34
,
49
,
50
]. In the current study, we found that 4 weeks of CIT (10 g/day)
supplementation increased FBF and FVC during handgrip exercise by 47% and 49%, respec-
tively, in hypertensive postmenopausal women. These findings are in line with a previous
study showing that acute dietary nitrate, an NO donor, enhanced FBF and FVC during
handgrip exercise in young healthy adults [
36
]. On the other hand, CIT supplementation
(6 g/day) for 7 days failed to increase FVC during handgrip exercise performed at 10%
of MVC in young, healthy women [
51
], suggesting that CIT does not improve arterial
vasodilation at a low workload in women with apparently normal endothelial function [
13
].
In addition, 2 weeks of CIT (6 g/day) supplementation increased leg BF and VC during
calf exercise in older men, but not in apparently healthy older women [
52
]. Despite the
use of CIT, the disparity between the previous [
52
] and present findings in older women
may be attributed to the short duration of the intervention (2 and 4 weeks), lower CIT dose
(6 and 10 g), and non-hypertensive status (normotensives and hypertensives), and lower
NO-dependent vasodilator response in leg compared to arm arteries [53].
Aging and hypertension augment muscle sympathetic nerve activity and local vaso-
constriction during exercise, reducing BF to active skeletal muscles [
48
,
50
]. SBP responses
to exercise are augmented by menopause and hypertension [
54
,
55
]. A greater increase
in SBP during exercise in older women, compared to young women and men, and older
men is due to an inability to attenuate vasoconstriction [
55
]. Postmenopausal women
have exaggerated SBP responses to exercise due, in part, to endothelial dysfunction and
increased sympathetic-mediated vasoconstriction [
33
,
56
,
57
]. Although several endothelial-
derived and skeletal muscle-derived (ATP, adenosine, potassium) factors are involved in
augmenting local BF, NO and prostaglandin are the main vasodilators during exercise. In
humans, the infusion of inhibitors of NO and prostaglandin synthesis resulted in reduced
BF during exercise [
58
,
59
]. However, NO caused a consistent contribution to forearm hy-
peremia while prostaglandin had a modest and transient vasodilator effect [
58
]. Moreover,
the inhibition of endothelial-derived hyperpolarizing factors did not affect exercise BF [
59
].
Therefore, the imbalance between local vasodilation and sympathetic vasoconstriction
during exercise can be explained, in part, by impaired NO availability [
15
]. Improved leg
BF and VC responses to exercise after CIT supplementation in older men may be influenced
by a reduced MAP during exercise due to attenuated sympathetic vasomotor activity [
52
].
In the present study, CIT supplementation increased FBF and FVC during exercise without
a significant change in MAP; thus, improved ARG and NO bioavailability to produce
vascular smooth muscle relaxation may be the most likely mechanism. Our group recently
demonstrated that increases in leg BF and VC during exercise were strongly associated with
endothelial-mediated vasodilation in non-obese postmenopausal women [
34
]. In contrast,
obesity attenuated BF and VC responses to exercise. Moreover, we previously reported that
CIT supplementation improved brachial artery endothelial function and ARG availability
in hypertensive postmenopausal women [
30
]. Taken together, our findings suggest that
CIT supplementation can enhance arm artery vasodilation during low-intensity exercise in
hypertensive postmenopausal women, which may be due to improved endothelial func-
tion [
30
,
34
] and related sympathetic-mediated vasoconstriction attenuation [
48
] commonly
known as functional sympatholysis.
Nutrients 2024,16, 1935 12 of 16
Microvascular function is important for the delivery of oxygen and nutrients to skeletal
muscles [
60
,
61
]. In individuals with hypertension, structural and functional microvascular
abnormalities contribute to reduced oxygen delivery during exercise due to increased
sympathetic-mediated vasoconstriction and reduced vasodilatory capacity [
62
,
63
]. In
the present study, CIT supplementation improved forearm muscle TSI, O
2
Hb, and HHb
levels during low-intensity handgrip exercise. Consistent with our findings, 7 days of
CIT supplementation (6 g daily) raised TSI and attenuated the increase in HHb during
moderate-intensity cycling exercise in young healthy males, suggesting improved oxy-
gen availability within the muscle microvasculature by increasing muscle perfusion and
reducing oxygen extraction [
25
]. Similarly, an improvement in vastus lateralis TSI dur-
ing moderate-intensity cycling exercise was observed after 2 weeks of watermelon juice,
a naturally rich source of CIT, in active young men [
64
]. These improvements may be
explained by increased oxygen delivery coupled with reduced oxygen extraction during
exercise [
18
,
65
,
66
], suggesting improved oxygen efficiency and oxygen cost [
18
,
67
]. At the
same relative intensity, a greater BP response is required for achieving similar muscle TSI
during exercise in hypertensive compared to normotensive adults due to less capillary den-
sity, attenuated vasodilation, and exaggerated sympathetic-mediated vasoconstriction [
35
].
In the present study, there was a significant improvement in muscle oxygenation during
exercise without an excessive BP response in postmenopausal women with hypertension
after CIT supplementation compared to placebo. This could be explained by greater local
artery vasodilation providing improved perfusion to contracting skeletal muscles [
14
],
thereby increased oxygen delivery [
25
]. Taken together, 4 weeks of CIT supplementation
was effective to improve muscle oxygenation and extraction during exercise in hyperten-
sive postmenopausal women. Improved microvascular function and muscle oxygenation
may enhance exercise capacity and performance [
18
] in hypertensive postmenopausal
women. This is the first study to demonstrate the effects of a longer and higher dose of
CIT supplementation on muscle oxygenation during low-intensity exercise in hypertensive
postmenopausal women, whereas most of the previous studies examined shorter supple-
mentation in young healthy men [
25
,
64
]. Endothelial dysfunction and hypertension are
independent precursors to the increased risk of cardiovascular disease in postmenopausal
women [
68
,
69
]. Our findings are clinically important considering that 4 weeks of CIT
improved resting BP, endothelial-mediated vasodilation, and vasodilatory capacity during
exercise, suggesting a reduction in future cardiovascular risk in this vulnerable population.
There are some limitations in the present study. First, we did not report ARG and
NO bioavailability in this study. However, the present data were part of a previous study
from our group that demonstrated improved endothelial function via enhanced ARG
bioavailability after CIT supplementation in hypertensive postmenopausal women [
30
].
Further, we used NIRS as a non-invasive method to evaluate augmented skeletal muscle
oxygenation during dynamic exercise [
66
], which may have been influenced by NO and
muscle metabolites (e.g., ATP, adenosine) [
70
]. Accordingly, the improvements in arterial
vasodilation and muscle oxygenation during exercise found after CIT may be associated
with improved macro-and microvascular endothelial function at rest [
30
,
31
]. Second, we
did not measure the subcutaneous adipose tissue thickness of the forearm using skinfolds,
which may reduce absolute NIRS signals [
71
]. However, the forearm (around flexor
digitorum profundus) has a relatively lower subcutaneous fat than other sites [
72
]. In
addition, since NIRS-derived measures in forearm muscles were not affected in obese
individuals with higher BMI (32.9
±
1.9 kg/m
2
) than our participants [
73
], the influence
of adipose tissue thickness on NIRS signal in our study can be neglected. Third, we did
not measure serum Hb levels, which may affect NIRS-derived measures. Lastly, this study
was conducted in hypertensive postmenopausal women and the present findings cannot
be extrapolated to other populations. Future studies are needed to investigate the chronic
longer effects of CIT supplementation on arm and leg macro- and microvascular function
at rest and during exercise in various clinical populations with cardiometabolic risk factors
or diseases.
Nutrients 2024,16, 1935 13 of 16
5. Conclusions
Four weeks of CIT supplementation increased brachial artery endothelial function and
vasodilatory capacity during low-intensity handgrip exercise in hypertensive postmenopausal
women. Our findings suggest that CIT supplementation can enhance peripheral artery
vasodilation, leading to improvements in skeletal muscle oxygenation during exercise.
Author Contributions: Conceptualization, A.F.; Data curation, Y.K. and A.M.; Formal analysis, Y.K.
and A.F.; Investigation, Y.K., K.N.D., M.A.M., A.M. and S.M.F.; Methodology, A.M., S.M.F. and A.F.;
Project administration, A.F.; Resources, A.F.; Supervision, A.F.; Visualization, Y.K.; Writing—original
draft, Y.K.; Writing—review and editing, K.N.D., M.A.M., A.M., S.M.F. and A.F. All authors have
read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: The experimental protocol and informed consent was approved
by Texas tech University Institutional Review Board (IRB2018-463; approved on 31 May 2019).
Informed Consent Statement: Written informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding author.
Acknowledgments: We appreciate all participants who volunteered their time to make this project a
success. We also thank NowFoods for providing the CIT and placebo capsules.
Conflicts of Interest: The authors declare no conflicts of interest.
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