Effects of dietary sodium on metabolites: the Dietary Approaches to
Stop Hypertension (DASH)–Sodium Feeding Study
Andriy Derkach,1 Joshua Sampson,1 Justin Joseph,1,2 Mary C Playdon,1 and Rachael Z Stolzenberg-Solomon1
1
Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Rockville, MD; and 2Department of Chemistry, Virginia Commonwealth
University, Richmond, VA
ABSTRACT
Background: High sodium intake is known to increase blood pres-
sure and is difficult to measure in epidemiologic studies.
Objective: We examined the effect of sodium intake on metabolites
within the DASH (Dietary Approaches to Stop Hypertension Trial)–
Sodium Trial to further our understanding of the biological effects
of sodium intake beyond blood pressure.
Design: The DASH-Sodium Trial randomly assigned individuals to
either the DASH diet (low in fat and high in protein, low-fat dairy,
and fruits and vegetables) or a control diet for 12 wk. Participants
within each diet arm received, in random order, diets containing high
(150 nmol or 3450 mg), medium (100 nmol or 2300 mg), and low
(50 nmol or 1150 mg) amounts of sodium for 30 d (crossover design).
Fasting blood samples were collected at the end of each sodium inter-
vention. We measured 531 identified plasma metabolites in 73 partic-
ipants at the end of their high- and low-sodium interventions and in 46
participants at the end of their high- and medium-sodium interventions
(N = 119). We used linear mixed-effects regression to model the re-
lation between each log-transformed metabolite and sodium intake. We
also combined the resulting P values with Fisher’s method to estimate
the association between sodium intake and 38 metabolic pathways or
groups.
Results: Six pathways were associated with sodium intake at a
Bonferroni-corrected threshold of 0.0013 (e.g., fatty acid, food com-
ponent or plant, benzoate, g-glutamyl amino acid, methionine, and
tryptophan). Although 82 metabolites were associated with sodium
intake at a false discovery rate #0.10, only 4-ethylphenylsufate, a
xenobiotic related to benzoate metabolism, was significant at a
Bonferroni-corrected threshold (P , 1025). Adjustment for co-
inciding change in blood pressure did not substantively alter the
association for the top-ranked metabolites.
Conclusion: Sodium intake is associated with changes in circulating
metabolites, including gut microbial, tryptophan, plant component, and
g-glutamyl amino acid–related metabolites. This trial was registered at
clinicaltrials.gov as NCT00000608. Am J Clin Nutr 2017;106:1131–
41.
Keywords: metabolomics, epidemiology, sodium intake, trial,
high and low sodium intake, feeding trial, African American
INTRODUCTION
Most adults in the United States, including those with hy-
pertension, consume sodium in excess of the recommended
Am J Clin Nutr 2017;106:1131–41. Printed in USA.
2017 American Society for Nutrition
Downloaded from https://academic.oup.com/ajcn/article-abstract/106/4/1131/4651949 by ASN Member Access user on 09 September 2019
2300 mg/d, with the mean daily sodium intake being w3500 mg
(1). High sodium intake is known to be associated with high
blood pressure. In 2013, the Institute of Medicine concluded that
there was evidence to support an association between sodium
intake and an increased risk of cardiovascular disease, stroke,
and all-cause mortality in accordance with the known effects of
sodium on blood pressure (2). However, associations between
sodium intake and other health outcomes have been difficult to
identify (2). To our knowledge, sodium intake is poorly measured
in epidemiologic studies and this measurement error leads to in-
accurate risk estimates (2). In addition, the specific biological
effects of high sodium intake, aside from increased blood pressure,
are not well understood (2).
We studied the effect of sodium intake on plasma metabolites
among participants in the DASH (Dietary Approaches to Stop
Hypertension Trial)–Sodium Trial (3), a landmark clinical feeding
study. The DASH-Sodium Trial was designed to evaluate the ef-
fects of the DASH diet (high in fruits, vegetables, and low-fat
dairy products) and 3 levels of sodium intake on blood pressure
(3). As part of the study, each subject received, in random order
(i.e., a crossover design), diets containing low (1150 mg/d), medium
(2300 mg/d), and high (3450 mg/d) sodium (3). The trial demon-
strated that lowering sodium intake significantly reduced blood
pressure, independent of the dietary patterns (3). Our current study
targets a different question and evaluates the effect of sodium intake
on metabolomic profiles. This study offers an important step toward
determining whether there are associations between sodium intake
and health outcomes. First, strongly associated metabolites, if
discovered, may eventually be used as surrogate measures of so-
dium consumption in large epidemiologic studies. Second, these
associations might provide further insight into potential biological
Supported by the NIH Intramural Research Program and the Division of
Cancer Epidemiology and Genetics, National Cancer Institute, NIH, US
Department of Health and Human Services.
Supplemental Figures 1 and 2 and Supplemental Tables 1–7 are available
from the “Online Supporting Material” link in the online posting of the article
and from the same link in the online table of contents at http://ajcn.nutrition.org.
Address correspondence to RZS-S (e-mail: rs221z@nih.gov).
Abbreviations used: DASH, Dietary Approaches to Stop Hypertension
Trial; MS, mass spectrometry; PC, principal component; SS, salt sensitive.
Received November 25, 2016. Accepted for publication August 1, 2017.
First published online August 30, 2017; doi: https://doi.org/10.3945/ajcn.
116.150136.
1131
1132 DERKACH ET AL.
mechanisms linking dietary sodium intake and disease. We
hypothesized that unique metabolic profiles were associated
with sodium intake.
METHODS
Study population
The DASH-Sodium Trial (clinicaltrials.gov NCT00000608)
was a multicenter, randomized feeding trial. The trial compared
the effects of 2 dietary patterns and 3 sodium intake interventions
on blood pressure. Details of the methods for the DASH-Sodium
Trial were published previously (3–5). Briefly, the original study
(conducted between 1997 and 1999) included 412 participants
$22 y of age who had systolic and diastolic blood pressures
between 120 and 159 and 80 and 95 mm Hg, respectively (3). Ex-
clusion criteria included renal insufficiency, diabetes requiring in-
sulin, poorly controlled hyperlipidemia, special dietary requirements,
alcohol consumption of .14 drinks/wk, and use of antihypertension
drugs or other medicines that would affect blood pressure. Four
clinical centers and a coordinating center participated in the trial. All
participants signed an informed consent form and the study was
approved by the human subject committees of each center (3).
DASH-Sodium Trial participants were randomly assigned to
consume either a control diet (i.e., similar to the typical diet in the
United States) or the DASH diet (high in fruits, vegetables, and
low-fat dairy products) for 12 wk (3, 4). Within their designated
dietary pattern, each participant received, in random order, low-,
medium-, and high-sodium versions of their diet for 30 d (crossover
design, Supplemental Figure 1) (3, 4). The sodium levels desig-
nated as low, medium, and high were 50 nmol/d (1150 mg),
100 nmol/d (2300 mg), and 150 nmol/d (3450 mg), respectively.
The highest amount of sodium was comparable to typical US
intake, the intermediate amount corresponded to the upper range
of public health recommendations, and the low amount was lower
than current recommendations. Participants’ energy intake was
adjusted to ensure that their weight remained constant during the
study (3, 4). Per the study protocol, trained staff measured par-
ticipants’ blood pressure using a random-zero sphygmomanometer
while participants were seated (3). Fasting EDTA plasma samples
were collected from participants at the end of each sodium in-
tervention and were stored at 2808C at the National Heart, Lung,
and Blood Institute repository.
Our metabolomic study includes a subset of 119 participants
from the DASH trial. For 73 participants, we measured the
metabolomic profiles in plasma collected after the high- and low-
sodium interventions. These 73 participants included all individuals
who had a low-sodium plasma sample that had not undergone a
freeze-thaw cycle, a requirement for all samples used in our study.
For the remaining 46 participants, we measured the profiles of
plasma collected after the high- and medium-sodium interventions.
Approximately half of participants were in each dietary pattern
intervention arm (DASH diet, n = 60; typical American control
diet, n = 59; Supplemental Figure 2).
Laboratory analysis
All blood samples were thawed, aliquoted, and processed in a
controlled and consistent manner. Samples from the same par-
ticipant were analyzed consecutively within batches, and we
included 24 blinded replicate samples for quality control.
The samples were sent to Metabolon Inc. on dry ice. Plasma
samples were assayed with untargeted ultra-HPLC coupled to
tandem mass spectrometry (MS) and gas chromatography–MS
(6). The liquid chromatography–MS portion of the platform was
based on a Waters ACQUITY ultra-performance liquid chroma-
tographer and a Thermo Scientific Q-Exactive high-resolution
accurate mass spectrometer interfaced with a heated electrospray
ionization source and an Orbitrap mass analyzer operated at
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35,000 mass resolution. The gas chromatography–MS portion was
analyzed on a Thermo Finnigan Trace DSQ fast-scanning single-
quadrupole mass spectrometer using electron impact ionization
and operated at unit mass resolving power (6). Peaks were iden-
tified via linkage to Metabolon’s known chemical reference li-
brary. Metabolon grouped the metabolites into 9 chemical classes
(amino acids, carbohydrates, cofactors and vitamins, energy me-
tabolites, lipids, nucleotide metabolites, peptides, and xenobiotics)
and metabolic subpathways based on Kyoto Encyclopedia of Genes
and Genomes classifications (7). Unknown chemical identities were
tagged beginning with “X” followed by numbers.
In total, 1027 plasma metabolites were detected in the samples;
613 were chemically identified and 414 were unnamed. We ex-
cluded metabolites for which $50% of participants had metabolite
values below the limit of detection. We focused on the 531
identified metabolites that exceeded the limit of detection in $50%
of participants. However, in supplementary analyses, we report
the statistically significant results for the unidentified metabolites
(n = 340) that were present above this limit of detection. Metabolite
peak intensity was normalized according to run day by dividing
each metabolite observation by the median for that metabolite on
that run day. Multiple approaches can validly be used to account
for biomarkers below the limit of detection without compromising
the associations observed (1–3). We chose to assign the minimum
observed value. Metabolites below detection were assigned the
minimum observed value for that metabolite. Metabolite mea-
surements were highly reproducible, with a median (IQR) intra-
class correlation coefficient of 0.84 (0.62–0.91).
Statistical analysis
We calculated means 6 SDs and proportions for the char-
acteristics of the study population shown in Table 1.
In our evaluation of the sodium intervention, we first considered
2 groups of participants separately: those with plasma collected
after the high- and low-sodium interventions and those with plasma
collected after the high- and medium-sodium interventions. We
evaluated the effect of sodium intervention with mixed-effect models.
We designated Yji as the level of metabolite j in sample i, Xi as a
binary indicator equaling 1 if sample i was collected after the lower-
sodium diet (medium or low) or 0 if the sample was collected after
the high-sodium diet, and Ci = [Ci1.Ci5]T as a vector of covariates
that included categorical age (#55 or .55 y), sex, race (African
American, non-Hispanic white, or other), dietary pattern (DASH
or control), and sodium intervention order (1, 2, or 3). For each
metabolite, we then fit a separate model as follows using mixed-
effects linear regression with a subject-specific random intercept:
X
log10 ðYji Þ ¼ bj0 þ bj1 Xi þ bjk Cik ð1Þ
k
We reported an estimate of the relative change (Rj = 10bj1) in
metabolite level when switching from a high- to lower-sodium
 SODIUM FEEDING ASSOCIATION WITH METABOLITES 1133
TABLE 1
Baseline characteristics of DASH-Sodium participants (N = 119)1
Sodium intervention
All subjects
Characteristic (N = 119) High and medium (n = 46) High and low (n = 73)

Age, y
18–30 1 (0.8) 0 1 (1.4)
31–55 76 (63.8) 28 (60.9) 48 (65.8)

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56–65 32 (27.0) 14 (30.4) 18 (24.7)
.65 10 (8.4) 4 (8.7) 6 (8.2)
Women 65 (54.6) 25 (54.4) 40 (54.8)
Race/ethnicity
Black or African American 59 (49.6) 4 (8.7) 55 (75.3)
Non-Hispanic white or other 60 (50.4) 42 (91.3) 18 (24.7)
BMI, kg/m2 29.3 6 4.1 28.7 6 3.6 29.8 6 4.4
Waist circumference, cm 100.8 6 12.6 97.5 6 11.1 102.9 6 13.1
Blood pressure,2 mm Hg
Systolic 136.0 6 9.1 136.9 6 9.9 135.4 6 8.7
Diastolic 85.5 6 4.0 85.7 6 4.7 85.4 6 3.6
Hypertension3 47 (39.5) 23 (50.0) 24 (32.9)
Education
High school graduate or less 19 (16.0) 6 (13.0) 13 (17.8)
Some college 48 (40.3) 14 (30.4) 34 (46.6)
College degree 23 (19.3) 8 (17.4) 15 (20.6)
Postgraduate work/degree 29 (24.4) 18 (39.1) 11 (15.1)
Household income, $/y
,30,000 34 (28.6) 17 (37.0) 17 (23.3)
30,000–60,000 46 (38.7) 15 (32.6) 31 (42.5)
.60,000 35 (29.4) 12 (26.1) 23 (31.5)
Missing 4 (3.3) 2 (4.3) 2 (2.7)
Smoker
Never 67 (56.3) 28 (60.9) 39 (53.4)
Former 41 (34.5) 15 (32.6) 26 (35.6)
Current 11 (9.2) 3 (6.5) 8 (11.0)
1
Values are means 6 SDs or n (%). DASH, Dietary Approaches to Stop Hypertension Trial.
2
Blood pressure was the average of 3 screening measurements and 2 measurements during the run-in period.
3
Hypertension was defined as an average systolic blood pressure of 140–159 mm Hg or an average diastolic blood
pressure of 90–95 mm Hg during the 3 screening visits.

intervention. For example, an Rj of 1.2 suggests that switching
from the high- to lower-sodium intervention increased the me-
tabolite level by a factor of 1.2 or 20%. For metabolites detected
in ,50% of participants, we modeled the presence or absence of
the metabolite with mixed-effects logistic regression.
We then considered all subjects together and assumed that log-
metabolite levels changed linearly with sodium intake. We used a
similar model to the one above but designated Xi = 0, 1/2, or 1,
according to whether sample i was collected after the high-,
medium-, or low-sodium interventions and we included an ad-
ditional covariate for group (i.e., the covariate equaled 1 if the
participant’s samples were collected after the high- and low-
sodium interventions or 0 if the samples were collected after
the high- and medium-sodium interventions). We estimated the
relative change (Rj = 10bj1) in metabolite for a 100-nmol/d de-
crease in sodium (i.e., the difference between the high- and low-
sodium interventions). We denote the P and Q values for bj1
from this model with Plin and Qlin, respectively, where the
subscript “lin” indicates that the sodium level was assumed to
have a linear effect on log-metabolite levels. The Q value ef-
fectively estimates the proportion of associations with P # Plin
that are likely to be false positives (8, 9). We tested for a non-
linear effect of sodium intake with a likelihood ratio test
comparing 2 models, and we denote the resulting P and Q values
from this likelihood ratio test with Pnl and Qnl, respectively.
Here, Qnl is based only on metabolites with Qlin # 0.1. The
larger model allows each sodium diet to have its own effect by
including sodium as a categorical variable (i.e., the mean log-
metabolite for the high-, medium-, and low-sodium diets are bj0,
bj0 1 g1, and bj0 1 g 2, respectively). The smaller, nested model
forces sodium amount to have a linear effect by including sodium
as a continuous variable with Xi ˛f0; 1=2; 1g (i.e., the mean log-
metabolite for the high-, medium-, and low-sodium diets are bj0,
bj0 + 0.5 g2, and bj0 + g2).
We next determined the association between metabolic
pathways and sodium intake. The metabolites were divided into
38 defined pathways (Supplemental Table 1). For each pathway,
we combined the P values of the included metabolites by
Fisher’s method [Fisher’s statistic = + 2 2 lnðPj Þ]. We obtained
j
the distribution of Fisher’s statistic under the null hypothesis of
no association by permutation. We created 105 permuted data-
sets. For each permuted dataset, we randomly assigned the high-
and lower-sodium designations to the 2 measures for every
participant. We then calculated Fisher’s statistic for each per-
muted dataset. Finally, we reported the statistical significance of
1134 DERKACH ET AL.
the pathway association by a pathway P value (Ppath), defined as
the proportion of these 105 Fisher’s statistics that are below the
value observed for the actual data.
As a second multimetabolite test, we identified the first 10
principal components (PCs) of the metabolomic profile using PC
analysis and then tested whether each PC was associated with
sodium intervention at the Bonferroni-adjusted significance of
0.005 (0.05/10) with the same linear mixed-effects models used
for individual metabolites.
We also performed secondary or sensitivity analyses to test
whether covariates (e.g., race, sex, dietary pattern, and age)
modified the effect of sodium intake and whether change in blood
pressure mediated the effect. Building on the model that assumed a
linear trend (i.e., Xi ˛f0; 1=2; 1g), we considered the following:
X when participants switched from the high- to medium-sodium
log10 ðYji Þ ¼ bj0 þ bj1 Xi þ bjk Cik þ bjI Cil Xi ð2Þ
k dence to confidently reject the hypothesis that sodium intake was
We then tested whether each covariate modified the effect of in-
tervention (i.e., bjI ¼ 0). We reported the relative change in
effect size (Rj ¼ 10bjI ). We also fit the following model:
X nificantly altered after adjusting for blood pressure (Supplemental
log10 ðYji Þ ¼ b 
j0 þ bj1 Xi þ b  
jk Cik þ bjS Si þ bjD Di ð3Þ
k
where the covariates Si and Di are the measured systolic and
diastolic blood pressure at the end of each sodium intervention
for the corresponding individual when the ith sample was col-
lected, respectively. We also reported estimates of the direct
b
effect (RDj ¼ 10 ) for a 100-nmol/d decrease in sodium intake
j1
after adjusting for the mediating effects on blood pressure.
The Bonferroni significance method for adjusting P values
when multiple hypotheses are being tested is considered very
conservative and may lead to a high number of false negatives;
therefore, we used a false discovery rate of 0.10 for statistical
significance (Q value). The Bonferroni-adjusted a considering
all of the comparisons including the stratified analyses was 3.14 3
1025 (0.05/1593) for individual metabolites and 0.00044 (0.05/114)
for the pathways. However, we noted that the Bonferroni-adjusted
a for the combined analyses was 9.4 3 1025 (0.05/531) for in-
dividual metabolites and 0.0013 (0.05/38) for metabolic pathways
because this was the focus of our results and discussion.
All statistical analyses were performed using R programming
language (R Foundation for Statistical Computing) (10).
RESULTS
Table 1 shows the characteristics of the study participants.
Overall, the majority of participants were 31–55 y of age, were
women, were not hypertensive, were never smokers, attained some
college education, and had an income of $30,000–60,000/y. The
mean BMI (in kg/m2) was 29.3 (overweight) and the mean systolic
and diastolic blood pressure was 136.0 and 85.5 mm Hg, re-
spectively. Compared with participants in the high- to medium-
sodium group, participants in the high- to low-sodium groups
were more often African American and had less education but
were similar with respect to other characteristics. Compared
with the high- to low-sodium group, participants in the high- to
medium-sodium group had a larger proportion of clinically defined
high blood pressure; however, both groups had similar mean
systolic and diastolic blood pressure and the difference could be
attributable to chance because of the small number of partici-
pants in the medium- to high-sodium group (n = 43).
Six metabolic pathways were associated with sodium intake
at a Bonferroni-corrected threshold of 0.0013 (i.e., fatty acid,
food component or plant, benzoate, g-glutamyl amino acid,
methionine, and tryptophan; Table 2). Although 82 metabolites
were associated with sodium intake at a false discovery rate of
0.1 (Qlin # 0.1), only 4-ethylphenylsufate, a xenobiotic related
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to benzoate metabolism, was significant at a Bonferroni-corrected
threshold (Plin , 1025) (Table 3). The relative change in me-
tabolite levels was larger in participants switching from the high-
to low-sodium interventions than the high- to medium-sodium
interventions. For many metabolites (including our most strongly
associated metabolite, 4-ethylphenysulfate), there was little change
interventions. However, in general, there was not enough evi-
linearly related to log-metabolite levels (Q . 0.10; Supplemental
Table 2). In secondary analyses, the effect of reducing sodium
intake was not significantly modified by dietary pattern, sex, race,
or age (Q . 0.90; Supplemental Table 3). The effect was not sig-
Tables 4 and 5), with 68 of the initial 82 metabolites remaining
statistically significant (Qlin # 0.10) after adjustment. Despite these
strong and robust associations, the top 10 PCs were not signif-
icantly associated with sodium intake. Additional analyses on
unknown metabolites detected 2 statistically significant metabolites
(Plin , 1025) that were highly correlated to g-glutamyliosoleucine
and g-glutamylleucine (Supplemental Table 6); no metabolite
detected in ,50% of participants was significant at Q , 0.1.
The most strongly associated pathway was the fatty acid group,
in which the short-chain fatty acid isovalerate decreased and the
lipids butyrylcarnitine and valerylcarnitine increased with sodium
reduction. Seven xenobiotic metabolites in the food component
or plant group increased with sodium reduction, including 4-
allylphenol sulfate, methyl glucopyranoside (a plus b), N-
acetylalliin, methyl indole-3-acetate, gluconate, homostachydrine,
and erothioneine (Table 3). Three xenobiotic metabolites increased
with low sodium intake within the benzoate metabolism pathway,
including 4-ethylphenysulfate, which reached Bonferroni signifi-
cance (Plin , 5.85 3 10210); other significant metabolites in this
pathway included 4-methylcatechol sulfate and O-methylcatechol
sulfate. In the g-glutamyl amino acid group, 7 peptide metabolites
(g-glutamylvaline, g-glutamylisoleucine, g-glutamylleucine, g-
glutamylmethionine, g-glutamylglutamate, g-glutamylphenylalanine,
and g-glutamyltyrosine) decreased with lower sodium intake.
In the methionine metabolism pathway, 6 amino acid metabolites
(methionine sulfone, a-ketobutyrate, S-adenosylhomocysteine, N-
formylmethionine, N-acetylmethionine, and methionine) increased,
whereas amino acid methionine sulfoxide decreased with the low-
sodium intervention. Finally, in the tryptophan group, 6 amino acid
metabolites (namely, indoleacetate, indolebutyrate, methyl indole-
3-acetate, tryptophan betaine, indoleacetylglutamine, and C-glycosyl
tryptophan) increased with lower sodium intake. The results for all
531 known metabolites contributing to each pathway are shown
in Supplemental Table 7.
A few additional metabolites were among the most strongly
associated metabolites (P , 0.0005). 4-Hydroxyphenylpuruvate,
an amino acid related to phenlyanalanine and tyrosine me-
tabolism, increased with the low-sodium intervention, as did 2
 SODIUM FEEDING ASSOCIATION WITH METABOLITES 1135
TABLE 2
Metabolic pathways associated with sodium intervention1
Sodium intervention, P value2

Pathway group Metabolites, n High and medium High and low All subjects

Fatty acid 15 0.10 0.00041 0.00007

Food component or plant group 27 0.57 0.00005 0.00010
Benzoate metabolism 15 0.01 0.00061 0.00013
g-Glutamyl amino acid

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11 0.55 0.00106 0.00017
Methionine metabolism 14 0.11 0.00051 0.00020
Tryptophan metabolism 18 0.23 0.00010 0.00020
Phenylalanine and tyrosine metabolism 28 0.24 0.017 0.002
Others3 53 0.17 0.022 0.006
Purine metabolism 17 0.16 0.026 0.007
Valine, leucine, and isoleucine metabolism 30 0.54 0.029 0.009
Sterol or steroid 36 0.75 0.008 0.010
Pentose metabolism 9 0.67 0.023 0.010
Alanine and aspartate metabolism 8 0.58 0.020 0.013
Glutamate metabolism 6 0.25 0.052 0.021
Glycolysis, gluconeogenesis, and 7 0.48 0.13 0.04
pyruvate metabolism
Chemical group 15 0.97 0.08 0.04
Histidine metabolism 7 0.76 0.11 0.05
Fatty acid, dicarboxylate 13 0.10 0.21 0.06
Sphingolipid metabolism 10 0.90 0.07 0.06
Fatty acid, monohydroxy 13 0.37 0.25 0.07
Urea cycle 13 0.60 0.13 0.10
Long-chain fatty acid; PUFA [n–3 (v-3) and 14 0.04 0.27 0.10
n–6 (v-6)]
Fructose, mannose, galactose, starch, and 7 0.22 0.17 0.10
sucrose metabolism
Tocopherol metabolism 6 0.36 0.02 0.12
Lysolipid 31 0.92 0.23 0.12
Dipeptide group 9 0.26 0.40 0.12
Pyrimidine metabolism 12 0.77 0.12 0.12
Carnitine metabolism 12 0.56 0.40 0.18
Drug 5 0.03 0.50 0.20
Krebs or tricarboxylic acid cycle 6 0.48 0.22 0.23
Medium-chain fatty acid group 9 0.31 0.40 0.24
Long-chain fatty acid 15 0.14 0.81 0.36
Lysine metabolism 10 0.21 0.52 0.39
Monoacylglycerol 10 0.92 0.53 0.41
Glycine, serine, and threonine metabolism 10 0.65 0.73 0.62
Secondary bile acid metabolism 12 0.32 0.90 0.62
Primary bile acid metabolism 6 0.26 0.60 0.77
Xanthine metabolism 12 0.91 0.88 0.95
1
Pathways are based on the Kyoto Encyclopedia of Genes and Genomes and are described in Supplemental Table 1.
2
P values describe the significance of the association between sodium intake and metabolic pathway among in-
dividuals receiving the high- and medium-sodium interventions, high- and low-sodium interventions, and all subjects.
The reported pathway-level P values combine metabolite-level P values obtained from mixed models adjusted for age,
sex, race (African American, or non-Hispanic white or other), dietary pattern (Dietary Approaches to Stop Hypertension
Trial or control), sodium intervention order (1, 2, or 3), and group (when using all subjects; e.g., high and low or high and
medium) covariate. P values are not adjusted for multiple comparisons. Bonferroni-corrected significance for the 38
pathways was 0.05/114 = 0.00044 for all of the comparisons considering the stratified analyses and 0.05/38 = 0.0013
for the combined analyses alone.
3
“Others” includes categories with ,5 metabolites that could not otherwise be categorized.

nucleotides in the purine metabolism pathway (urate and N6-
carbamoylthreonyladenosine).
DISCUSSION
In this controlled feeding study, we observed that changes in
sodium intake resulted in numerous changes in plasma metabolite
concentrations. The strongest associations were observed in the
fatty acid, food plant component, benzoate, methionine, and
tryptophan pathways with metabolites that mostly increased with
the low-sodium intervention and metabolites in the g-glutamyl
amino acid pathway that decreased with sodium restriction. The
significant relative changes in metabolites were primarily driven
by the high- to low-sodium interventions. 4-Ethylphenysulfate
TABLE 3
Metabolites associated with sodium intervention1
1136

Sodium intervention
All subjects w,
High to medium High to low 100-nm/d decrease

Pathway or metabolite Class R (95% CI) P value R (95% CI) P value R (95% CI) P value Q value Rank2

Fatty acid
Isovalerate3 Lipid or amino acid 1.04 (0.98, 1.12) 0.20 0.55 (0.39, 0.78) 1.49 3 1023 0.61 (0.47, 0.79) 2.82 3 1024 0.01 7
Butyrylcarnitine Lipid 1.07 (0.97, 1.18) 0.19 1.26 (1.02, 1.55) 7.08 3 1023 1.27 (1.11, 1.46) 7.45 3 1024 0.01 20
Valerylcarnitine Lipid 1.40 (0.81, 2.42) 0.23 1.66 (1.13, 2.45) 1.28 3 1022 1.69 (1.16, 2.47) 7.16 3 1023 0.06 49
Food component or plant group
4-Allylphenol sulfate Xenobiotic 1.02 (0.88, 1.18) 0.78 1.30 (1.12, 1.51) 7.35 3 1024 1.26 (1.11, 1.44) 5.05 3 1024 0.01 10
Methyl glucopyranoside (a plus b) Xenobiotic 1.10 (0.93, 1.29) 0.27 1.29 (1.09, 1.53) 5.37 3 1023 1.28 (1.11, 1.48) 9.02 3 1024 0.01 24
N-Acetylalliin Xenobiotic 0.84 (0.49, 1.44) 0.54 2.18 (1.48, 3.20) 9.33 3 1025 1.91 (1.31, 2.78) 1.06 3 1023 0.02 26
Methyl indole-3-acetate4 Xenobiotic 1.13 (0.98, 1.3) 0.10 1.25 (1.07, 1.47) 6.40 3 1023 1.25 (1.09, 1.43) 1.77 3 1023 0.02 33
Gluconate Xenobiotic 1.04 (0.96, 1.13) 0.32 1.14 (1.03, 1.25) 1.24 3 1022 1.13 (1.05, 1.22) 2.55 3 1023 0.03 34
Homostachydrine Xenobiotic 1.08 (0.75, 1.56) 0.70 1.50 (1.15, 1.95) 4.60 3 1023 1.47 (1.13, 1.91) 4.95 3 1023 0.04 44
Ergothioneine Xenobiotic 1.01 (0.81, 1.27) 0.92 1.45 (1.06, 1.98) 1.74 3 1022 1.39 (1.08, 1.77) 1.02 3 1022 0.07 55
Benzoate metabolism
4-Ethylphenysulfate Xenobiotic 0.96 (0.82, 1.13) 0.61 2.03 (1.67, 2.47) 2.03 3 1029 1.78 (1.51, 2.11) 5.85 3 10210 2.24 3 1027 1
4-Methycatechol sulfate Xenobiotic 1.09 (0.90, 1.32) 0.36 1.28 (1.06, 1.54) 1.20 3 1022 1.23 (1.06, 1.43) 7.87 3 1023 0.06 51
O-Methylcatechol sulfate Xenobiotic 1.26 (1.00, 1.58) 0.06 1.26 (0.99, 1.60) 5.39 3 1022 1.29 (1.05, 1.58) 1.45 3 1022 0.08 66
g-Glutamyl amino acid
g-Glutamylvaline Peptide 0.99 (0.94, 1.03) 0.55 0.59 (0.43, 0.80) 1.45 3 1023 0.63 (0.51, 0.79) 1.08 3 1024 0.01 2
g-Glutamylisoleucine Peptide 0.98 (0.93, 1.03) 0.35 0.59 (0.43, 0.80) 1.57 3 1023 0.63 (0.51, 0.79) 1.09 3 1024 0.01 3
g-Glutamylleucine Peptide 0.98 (0.93, 1.03) 0.48 0.65 (0.51, 0.84) 1.95 3 1023 0.69 (0.58, 0.83) 1.46 3 1024 0.01 5
g-Glutamylmethionine Peptide 0.94 (0.85, 1.04) 0.22 0.78 (0.66, 0.91) 3.22 3 1023 0.79 (0.70, 0.90) 4.40 3 1024 0.01 9
DERKACH ET AL.

g-Glutamylglutamate Peptide 0.98 (0.86, 1.13) 0.81 0.40 (0.22, 0.73) 4.10 3 1023 0.45 (0.29, 0.7) 5.43 3 1024 0.01 12
g-Glutamylphenylalanine Peptide 0.97 (0.94, 1.01) 0.17 0.82 (0.71, 0.95) 9.07 3 1023 0.84 (0.76, 0.94) 1.75 3 1023 0.02 32
g-Glutamyltyrosine Peptide 0.97 (0.89, 1.05) 0.46 0.84 (0.74, 0.95) 7.43 3 1023 0.86 (0.78, 0.95) 3.19 3 1023 0.03 37
Methionine metabolism
Methionine sulfone Amino acid 1.09 (0.98, 1.21) 0.10 1.13 (1.05, 1.22) 1.63 3 1023 1.14 (1.06, 1.22) 5.20 3 1024 0.01 11
a-Ketobutyrate Amino acid 1.08 (0.87, 1.34) 0.48 1.86 (1.18, 2.93) 1.17 3 1022 1.75 (1.25, 2.46) 1.63 3 1023 0.02 31
S-Adenosylhomocysteine Amino acid 0.91 (0.78, 1.07) 0.28 1.35 (1.11, 1.63) 3.91 3 1023 1.26 (1.08, 1.47) 4.48 3 1023 0.04 40
Methionine sulfoxide Amino acid 1.02 (0.95, 1.09) 0.63 0.67 (0.48, 0.92) 2.03 3 1022 0.71 (0.56, 0.90) 6.19 3 1023 0.05 48
N-Formylmethionine Amino acid 0.96 (0.90, 1.03) 0.25 1.15 (1.04, 1.28) 1.79 3 1022 1.12 (1.03, 1.22) 8.06 3 1023 0.06 52
N-Acetylmethionine Amino acid 0.97 (0.90, 1.05) 0.49 1.13 (1.03, 1.24) 2.09 3 1022 1.10 (1.02, 1.19) 1.37 3 1022 0.08 63
Methionine Amino acid 0.94 (0.87, 1.02) 0.13 1.18 (1.04, 1.34) 2.64 3 1022 1.13 (1.03, 1.25) 1.58 3 1022 0.09 67
Tryptophan metabolism
Indoleacetate Amino acid 1.07 (0.97, 1.18) 0.21 1.15 (1.05, 1.26) 2.93 3 1023 1.15 (1.06, 1.24) 6.49 3 1024 0.01 17
Indolebutyrate Amino acid 1.05 (0.85, 1.30) 0.65 1.27 (1.13, 1.44) 1.95 3 1024 1.25 (1.10, 1.43) 8.96 3 1024 0.01 23
Methyl indole-3-acetate4 Amino acid 1.13 (0.98, 1.30) 0.10 1.25 (1.07, 1.47) 6.40 3 1023 1.25 (1.09, 1.43) 1.77 3 1023 0.02 33
Tryptophan betaine Amino acid 1.11 (0.98, 1.25) 0.09 1.15 (1.04, 1.28) 9.85 3 1023 1.15 (1.05, 1.25) 3.05 3 1023 0.03 36
Indoleacetylglutamine Amino acid 0.96 (0.80, 1.15) 0.64 1.27 (1.08, 1.49) 4.82 3 1023 1.22 (1.06, 1.41) 7.30 3 1023 0.06 50
C-Glycosyl tryptophan Amino acid 1.01 (0.93, 1.08) 0.88 1.08 (1.02, 1.14) 1.05 3 1022 1.07 (1.02, 1.13) 9.59 3 1023 0.07 54
(Continued)

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TABLE 3 (Continued )

Sodium intervention
All subjects w,
High to medium High to low 100-nm/d decrease

Pathway or metabolite Class R (95% CI) P value R (95% CI) P value R (95% CI) P value Q value Rank2

Phenylalanine and tyrosine metabolism

4-Hydroxyphenylpyruvate Amino acid 1.34 (1.02, 1.76) 0.04 1.72 (1.22, 2.44) 2.10 3 1023 1.76 (1.33, 2.34) 1.39 3 1024 0.01 4
N-Formylphenylalanine Amino acid 0.92 (0.77, 1.11) 0.39 0.76 (0.59, 0.97) 4.32 3 1022 0.76 (0.63, 0.92) 5.58 3 1023 0.05 47
Phenyllactate Amino acid 1.01 (0.97, 1.06) 0.65 1.06 (1.01, 1.12) 2.74 3 1022 1.06 (1.01, 1.11) 1.08 3 1022 0.07 57
Phenylpyruvate Amino acid 0.93 (0.58, 1.48) 0.75 1.55 (1.16, 2.07) 4.46 3 1023 1.44 (1.06, 1.96) 1.97 3 1022 0.098 77
Others (pathway)
Acisoga (polyamine metabolism) Amino acid 1.04 (0.97, 1.11) 0.25 1.10 (1.04, 1.17) 2.15 3 1023 1.10 (1.04, 1.16) 6.10 3 1024 0.01 16
HWESASXX (polypeptide) Peptide 1.11 (0.97, 1.26) 0.13 0.58 (0.42, 0.8) 1.65 3 1023 0.64 (0.50, 0.82) 6.64 3 1024 0.01 18
Prostaglandin E2 (eicosanoid) Lipid 1.00 (0.91, 1.09) 0.92 0.39 (0.20, 0.77) 7.65 3 1023 0.43 (0.27, 0.7) 7.96 3 1024 0.01 22
Leukotriene B4 Lipid 1.06 (0.97, 1.14) 0.19 0.26 (0.10, 0.68) 8.00 3 1023 0.30 (0.15, 0.6) 9.55 3 1024 0.01 25
4-Guanidinobutanoate (guanidino and acetamido metabolism) Amino acid 0.82 (0.58, 1.18) 0.29 0.51 (0.32, 0.83) 7.61 3 1023 0.55 (0.38, 0.81) 2.86 3 1023 0.03 35
Erythrulose (advanced glycation end product) Carbohydrate 1.28 (1.07, 1.55) 0.01 1.19 (0.99, 1.43) 6.14 3 1022 1.27 (1.08, 1.49) 3.60 3 1023 0.04 39
L-Urobilin (hemoglobin and porphyrin metabolism) Cofactors and vitamins 1.09 (0.72, 1.64) 0.69 0.61 (0.45, 0.83) 2.24 3 1023 0.66 (0.49, 0.87) 4.74 3 1023 0.04 41
Bradykinin, hydroxy-pro(3) (polypeptide) Peptide 0.85 (0.61, 1.18) 0.34 0.54 (0.30, 0.95) 3.62 3 1022 0.55 (0.35, 0.85) 8.13 3 1023 0.06 53
Bilirubin (Z,Z) (hemoglobin and porphyrin metabolism) Cofactors and vitamins 1.09 (0.67, 1.77) 0.74 1.84 (1.08, 3.12) 2.74 3 1022 1.77 (1.14, 2.75) 1.21 3 1022 0.08 59
Creatinine (creatine metabolism) Amino acid 0.97 (0.86, 1.10) 0.68 1.15 (1.04, 1.28) 9.24 3 1023 0.46 (0.24, 0.88) 1.40 3 1022 0.08 64
Myo-inositol (inositol metabolism) Lipid 1.03 (0.98, 1.09) 0.24 1.08 (0.99, 1.17) 8.90 3 1022 1.08 (1.02, 1.15) 1.43 3 1022 0.08 65
4-Acetamidobutanoate (polyamine metabolism) Amino acid 1.03 (0.92, 1.16) 0.62 1.15 (1.01, 1.31) 3.29 3 1022 1.12 (1.02, 1.23) 1.92 3 1022 0.10 75
HWESASLLR (polypeptide) Peptide 0.96 (0.88, 1.04) 0.33 0.46 (0.20, 1.10) 8.42 3 1022 0.46 (0.24, 0.88) 1.95 3 1022 0.10 76
Erythronate (aminosugar metabolism) Carbohydrate 1.01 (0.97, 1.04) 0.75 0.93 (0.87, 1.00) 4.22 3 1022 0.94 (0.89, 0.99) 2.14 3 1022 0.10 80
Purine metabolism
Urate Nucleotide 1.03 (0.99, 1.07) 0.17 1.07 (1.03, 1.11) 1.62 3 1023 1.06 (1.03, 1.1) 2.35 3 1024 0.01 6
N6-Carbamoylthreonyladenosine Nucleotide 1.05 (0.94, 1.16) 0.39 1.17 (1.07, 1.28) 9.08 3 1024 1.15 (1.07, 1.24) 4.36 3 1024 0.01 8
Valine, leucine, and isoleucine metabolism
Isovalerate3 Amino acid 1.04 (0.98, 1.12) 0.20 0.55 (0.39, 0.78) 1.49 3 1023 0.61 (0.47, 0.79) 2.82 3 1024 0.01 7
2-Methylbutyrylcarnitine Amino acid 0.97 (0.89, 1.06) 0.48 1.18 (1.05, 1.34) 7.71 3 1023 1.11 (1.04, 1.19) 3.39 3 1023 0.03 38
b-hydroxyisovaleroylcarnitine5 Amino acid 1.00 (0.95, 1.06) 0.93 1.07 (1.01, 1.14) 2.08 3 1022 1.07 (1.01, 1.12) 1.36 3 1022 0.08 61
Ethylmalonate Amino acid 1.04 (0.99, 1.09) 0.15 1.06 (0.98, 1.15) 1.60 3 1021 1.07 (1.01, 1.13) 2.25 3 1022 0.10 82
SODIUM FEEDING ASSOCIATION WITH METABOLITES

Isobutyrylcarnitine Amino acid 1.08 (0.98, 1.18) 0.13 1.11 (0.99, 1.24) 8.64 3 1022 1.11 (1.02, 1.22) 2.13 3 1022 0.10 79
Sterol or steroid
Etiocholanolone glucuronide Lipid 1.05 (0.95, 1.16) 0.34 1.19 (1.07, 1.32) 2.15 3 1023 1.17 (1.07, 1.28) 6.03 3 1024 0.01 14
4-Androsten-3b,17b-diol monosulfate(1) Lipid 1.00 (0.96, 1.04) 0.91 1.10 (1.04, 1.17) 1.53 3 1023 1.08 (1.04, 1.14) 7.96 3 1024 0.01 21
5a-Androstane-3b, 17b-diol monosulfate Lipid 1.00 (0.93, 1.08) 0.95 1.11 (1.05, 1.18) 5.38 3 1024 1.1 (1.04, 1.16) 1.06 3 1023 0.02 27
Andro steroid monosulfate(1) Lipid 0.93 (0.78, 1.10) 0.39 1.34 (1.16, 1.55) 1.51 3 1024 1.26 (1.10, 1.44) 1.26 3 1023 0.02 28
Epiandrosterone sulfate Lipid 1.01 (0.95, 1.06) 0.81 1.09 (1.02, 1.17) 9.60 3 1023 1.08 (1.02, 1.14) 5.51 3 1023 0.05 46
Androsterone sulfate Lipid 1.02 (0.98, 1.07) 0.32 1.07 (1.01, 1.15) 3.79 3 1022 1.07 (1.02, 1.13) 1.16 3 1022 0.08 58
5a-Androstane-3-a,17b-diol disulfate Lipid 1.07 (0.88, 1.29) 0.51 1.21 (1.03, 1.41) 2.10 3 1022 1.19 (1.03, 1.37) 1.92 3 1022 0.10 74
Pentose metabolism
Arabitol Carbohydrate 0.97 (0.90, 1.04) 0.39 1.16 (1.07, 1.25) 3.97 3 1024 1.11 (1.04, 1.18) 1.32 3 1023 0.02 29
(Continued)
1137

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TABLE 3 (Continued ) 1138

Sodium intervention
All subjects w,
High to medium High to low 100-nm/d decrease

Pathway or metabolite Class R (95% CI) P value R (95% CI) P value R (95% CI) P value Q value Rank2

Alanine and aspartate metabolism

Asparagine Amino acid 1.03 (0.96, 1.11) 0.47 1.19 (1.06, 1.34) 4.33 3 1023 1.17 (1.07, 1.28) 6.07 3 1024 0.01 15
Glutamate metabolism
N-Acetylglutamate Amino acid 1.34 (1.03, 1.74) 0.03 1.25 (0.99, 1.57) 6.25 3 1022 1.30 (1.06, 1.60) 1.34 3 1022 0.08 60
Glutamine Amino acid 1.03 (0.97, 1.10) 0.34 1.12 (0.96, 1.30) 1.52 3 1021 1.13 (1.02, 1.25) 1.86 3 1022 0.10 72
Glycolysis, gluconeogenesis, and pyruvate metabolism
Glucose Carbohydrate 1.01 (0.99, 1.04) 0.24 1.03 (1.00, 1.06) 2.35 3 1022 1.03 (1.01, 1.06) 4.91 3 1023 0.04 43
Chemical group
O-Sulfo-L-tyrosine Xenobiotics 1.00 (0.95, 1.05) 1.00 1.09 (1.03, 1.15) 2.04 3 1023 1.08 (1.03, 1.13) 6.76 3 1024 0.01 19
Phenylcarnitine Xenobiotics 1.13 (0.97, 1.31) 0.12 1.14 (0.99, 1.30) 6.55 3 1022 1.15 (1.03, 1.3) 1.90 3 1022 0.10 73
Histidine metabolism
trans-Urocanate Amino acid 1.04 (0.93, 1.16) 0.48 1.14 (1.02, 1.28) 2.50 3 1022 1.13 (1.03, 1.23) 1.37 3 1022 0.08 62
Imidazole lactate Amino acid 1.04 (0.92, 1.18) 0.53 1.14 (1.02, 1.28) 2.48 3 1022 1.13 (1.02, 1.26) 1.78 3 1022 0.10 70
Fatty acid, dicarboxylate
3-Carboxy-4-methyl-5-propyl-2-furanpropanoate Lipid 1.05 (0.96, 1.14) 0.29 1.19 (1.06, 1.32) 2.86 3 1023 1.17 (1.07, 1.27) 5.46 3 1024 0.01 13
Sphingolipid metabolism
Myristoyl sphingomyelin Lipid 1.02 (0.92, 1.14) 0.68 1.13 (1.03, 1.24) 8.79 3 1023 1.13 (1.04, 1.22) 4.88 3 1023 0.04 42
Fatty acid, monohydroxy
13-hode, 9-hode Lipid 1.01 (0.93, 1.10) 0.78 0.56 (0.36, 0.85) 9.07 3 1023 0.60 (0.44, 0.82) 1.53 3 1023 0.02 30
5-Hydroxyhexanoate Lipid 1.16 (0.98, 1.38) 0.09 1.15 (0.97, 1.36) 1.05 3 1021 1.17 (1.03, 1.33) 1.83 3 1022 0.10 71
Urea cycle
DERKACH ET AL.

Citrulline Amino acid 1.00 (0.92, 1.1) 0.95 1.08 (1.02, 1.15) 1.57 3 1022 1.08 (1.01, 1.14) 1.77 3 1022 0.10 69
Fructose, mannose, galactose, starch, and sucrose metabolism
Maltotriose Carbohydrate 1.24 (0.79, 1.94) 0.36 1.87 (1.17, 3.00) 1.14 3 1022 1.78 (1.20, 2.64) 5.16 3 1023 0.04 45
Lysolipid
1-Linoleoylglycerophosphoinositol Lipid 1.06 (0.97, 1.15) 0.20 1.12 (1.00, 1.26) 4.77 3 1022 1.13 (1.03, 1.23) 1.05 3 1022 0.07 56
Dipeptide group
cys-Gly, oxidized (glutathione metabolism) Amino acid 1.10 (0.82, 1.46) 0.52 1.59 (1.00, 2.52) 5.43 3 1022 1.54 (1.07, 2.21) 2.03 3 1022 0.10 78
Lysine metabolism
Glutarylcarnitine (C5) Amino acid 0.96 (0.93, 1.00) 0.05 1.06 (1.02, 1.10) 4.47 3 1023 1.04 (1.01, 1.08) 2.18 3 1022 0.10 81
Secondary bile acid metabolism
Ursodeoxycholate Lipid 0.66 (0.37, 1.19) 0.17 0.52 (0.27, 0.99) 5.19 3 1022 0.51 (0.30, 0.88) 1.62 3 1022 0.09 68
1
The relative change (R) in metabolite level is reported when switching from the high- to medium-sodium intervention and the high- to low-sodium intervention and when decreasing sodium intake by
100 nm/d (e.g., assuming a linear relation and combining data from all participants). R and its associated 95% CI, P value, and Q value were calculated using mixed-effect models adjusted for age, sex, race
(African American, or non-Hispanic white or other), dietary pattern (Dietary Approaches to Stop Hypertension Trial or control), sodium intervention order (1, 2, or 3) and group (when using all subjects; e.g.,
high-low or high-medium) covariate. Metabolites with Qlin , 0.1 are included. The P values were not adjusted for multiple comparisons. The Bonferroni-adjusted a level considering all the comparisons
including the stratified analyses was 3.14 3 10–5 (0.05/1593) and is 9.4 3 10–5 (0.05/531) for the combined analyses alone.
2
Relative rank of metabolites based on Plin.
3
Isovalerate is in the fatty acid and the valine, leucine, and isoleucine metabolism group pathway.
4
Methyl indole-3-acetate is in the tryptophan metabolism and the food component pathways.
5
b-Hydroxyisovaleroylcarnitine is also in carnitine metabolism group.

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 SODIUM FEEDING ASSOCIATION WITH METABOLITES
was associated with changes in sodium intake after correction
for multiple comparisons. The most significant metabolite changes
were not mediated by coinciding changes in blood pressure and
adjustment for blood pressure did not substantially change their
associations.
Our study provides evidence that suggests that sodium intake
may affect the gut microbiome. 4-Ethylphenylsulfate increased
with sodium restriction and was the most strongly associated with
change in sodium intake. This metabolite is produced by the gut
microflora (11), and higher serum concentrations are associated
with various health outcomes such as periodontal disease and lean
body mass in animals or humans (12–16). 4-Ethylphenylsulfate is
correlated with the consumption of soy products (17, 18) and is
elevated in the plasma of vegans compared with omnivores; a
vegan diet increases concentrations of several products pro-
duced by the gut microbiota (19). Salt has known antibacterial
properties (20) and has been used for medicinal purposes for
centuries (21, 22). Others have suggested that both diet and the
microbiome mediate associations between metabolites and
blood pressure (23). Although speculative, the effect of sodium
restriction on the gut microbiome may contribute to the in-
creases in 4-ethylphenylsulfate and possibly to changes in other
metabolites (e.g., 4-hydroxyphenylpyruvate, indole-related metab-
olites, and isovalerate) produced by gut bacterial metabolism (11).
Metabolites in the g-glutamyl amino acid metabolite group
decreased with lower sodium intake. These metabolites are formed
using the enzyme g-glutamyl transferase, which transfers a
g-glutamyl moiety from glutathione to amino acids and peptides
(24). g-Glutamyltransferase is a cell-surface protein that is im-
portant for maintaining glutathione homeostasis and its activity
increases in response to oxidative stress (25). In an in vitro ex-
periment, the sodium chloride concentration was directly pro-
portional to g-glutamyl transferase activity (26). To our knowledge,
most epidemiologic studies have shown blood concentrations
of g-glutamyltransferase to be positively associated with pre-
hypertension (27), hypertension (28), and cardiovascular disease
(29, 30). There is evidence that excess salt intake induces oxi-
dative stress in salt-sensitive (SS) hypertension (31). Although
speculative, it is possible that compared with high sodium intake,
sodium restriction decreases g-glutamyl transferase activity via
reduction of oxidative stress and accounts for the significant
decreases in g-glutamyl amino acid metabolites we observed.
Metabolites within the tryptophan metabolism pathway, par-
ticularly indole-related metabolites, significantly increased with
sodium restriction. Although some endogenous production oc-
curs in mammals, commensal enteric bacteria catabolize tryp-
tophan to indole compounds and derivatives, which are absorbed
passively through the colonic epithelia into the portal circulation
(32). Indole and its metabolites are metabolized through several
different pathways that have distinct biological activity, including
maintaining mucosal reactivity and homeostasis, expression of
pro- and anti-inflammatory genes in intestinal epithelial cells, and
modulating the release of intestinal glucagon-like peptide-1 that
acts to signal satiety, reducing food intake and obesity (32). 4-
Hydroxyphenylpyruvate was also significantly increased with
sodium restriction in our study. 4-Hydroxyphenylpyruvate is a
keto acid that is an intermediate in the metabolism of phenyl-
alanine and synthesized from tyrosine. Studies of patients with
disorders of tyrosine metabolism who are treated with the drug
nitisinone suggest that 4-hydrophenylpyruvate can stimulate the
1139
production of indole metabolites from tryptophan via tryptophan
aminotransferase (33). More research is needed to understand
how sodium intake impacts tryptophan-related metabolites, 4-
hydroxyophenylpyruvate, and the biochemical relation between
these metabolites.
A few additional metabolites that changed with sodium re-
striction deserve mention. The increase in food component or plant
group metabolites could be related to the low-sodium diet food
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preparation. For example, 4-allylphenol sulfate (chavicol) and
homostachydrine are found in herbs and are flavorings that may
have been added to enhance the palatability of the low-sodium diet.
It is uncertain how sodium restriction contributes to the observed
reduction in the short-chain fatty acid isovalerate. Isovalerate is a
degradation product of the branched-chain amino acid valine and is
modulated by both mammalian and bacteria metabolism (11).
Alterations in the gut microbiota owing to changes in salt intake
might alter carbohydrate fermentation and contribute to increased
production of short-chain fatty acids such as isovalerate (34). Urate
(a purine derivative) increased with sodium restriction, as observed
previously (35). Humans produce only small quantities of uric
acid, with excess accumulation leading to gout (36). Uric acid is
also a natural antioxidant produced endogenously that may protect
against oxygen radicals (37). HWESASXX (a peptide linked to
inflammation and positively associated with blood pressure) (38),
and 2 eicosanoids (prostaglandin E2 and leukotriene B4) decreased
with sodium restriction. Eicosanoids are known to play a role in
the regulation of blood pressure (39).
Three previous studies (40–42), 2 in humans and 1 in mice,
examined the association between sodium intake or salt sensi-
tivity and metabolites. First, in a 10-wk double-blind random-
ized crossover study of 17 middle-aged adults with elevated
blood pressure, participants were instructed to consume a low-
sodium diet and were given both a placebo and a slow-release
NaCl (150 nmol/d) tablet. Metabolites were measured in 24-h
urine samples collected at the end of each intervention. Sodium
restriction was associated with increases in succinate, methio-
nine sulfoxide, S-adenosylhomocysteine, D-gluconate, and as-
paragine (40). Our study of plasma metabolites replicated the
increases in succinate, gluconate, and asparagine, but we did not
observe the same direction of associations for succinate or
methionine sulfoxide. Another study of 13 treated patients with
hypertensive heart failure compared 152 metabolites measured
in 24-h urine samples at baseline and after participants consumed a
DASH sodium-restricted diet (50 nmol/d) for 21 d (41). Similar to
our study, this study showed decreases in isovalerate with the
sodium-restricted diet (41). Finally, an experimental study that
explored differences in metabolites owing to genetic variation in
Dahl SS rats compared with normotensive consomic SS.13 rats
showed that 23 metabolites differed (P , 0.05) between the 2
animal groups (42). Metabolites that were intermediates or linked
(glycolysis) to the tricarboxylic acid cycle (cis-aconitate, iso-
citrate, citrate, succinate, fumarate, and pyruvate) were upregu-
lated in SS rats (42). Valine, isoleucine, methionine, and urea
were increased in SS rats, whereas glutamate, asparagine, glycine,
and tyrosine were decreased (42). We observed significant asso-
ciations for some of these metabolites or molecules in related
pathways (e.g., succinate, methionine, glutamate, urea, aspara-
gine, branched-chain amino acids, glucose, and tyrosine) (42).
This study has several strengths. First, this was a carefully
conducted feeding study. Dietary intake was controlled, the
1140 DERKACH ET AL.
participants maintained a constant weight, and nonadherence was
minimized to facilitate measuring only the true biological effects
of sodium intake (3). The sodium intervention was a crossover
design: each participant served as his or her own control,
diminishing the effects of between-person genetic variation and
confounding by race, sex, age, and other exposures, thereby in-
creasing study power. Blood pressure was measured in a standard
manner, allowing us to attempt to untangle the effect of sodium
intake from that of changes in blood pressure. Given the original
design of the DASH-Sodium Trial, our results are likely gener-
alizable to many people in the United States (3). The absence of the
PC analysis not being associated with sodium intake suggests that
sodium is not the strongest factor driving the variability of the
metabolites. Correctly identifying metabolites is important for
biochemical interpretation. Although they were not subsequently
validated with pure compounds, the methods used to identify
metabolites in our study are based on a multiple orthogonal criteria
linkage to a unit mass spectral library built from authentic stan-
dards, which has been shown to accurately identify metabolites (6,
43). Limitations of our study include its relatively modest sample
size, the lack of an independent replication sample, and the in-
ability to directly examine metabolites for medium to low sodium
intake in the same individuals.
In conclusion, metabolites and associated metabolic pathways
differed by sodium intake. Some of our observed associations
may be related to salt’s effect on the gut microbiome; however,
more research is needed to understand the biochemical and
molecular basis of our observations.
The authors’ responsibilities were as follows—RZS-S: conceived the
study and acquired biomarker data for the research; RZS-S and JS: designed
the study; AD: analyzed the data and performed the statistical analysis; JS:
supervised the statistical analysis; AD, JS, and RZS-S: wrote the paper; JS,
JJ, and MCP: provided intellectual content to revise the manuscript; JJ:
conducted literature reviews for understanding metabolites; and all authors:
read and approved the final manuscript and assumed full responsibility for
the analyses, interpretation of these data, and final content of the manuscript.
None of the authors reported a conflict of interest related to the study.
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