Mol. Nutr. Food Res. 61, 2, 2017, 1600552 DOI 10.1002/mnfr.

201600552 (1 of 12) 1600552

RESEARCH ARTICLE

Reduction in cardiometabolic risk factors by a

multifunctional diet is mediated via several branches of
metabolism as evidenced by nontargeted metabolite
profiling approach
Juscelino Tovar1 , Vanessa D. de Mello2 , Anne Nilsson1 , Maria Johansson1 , Jussi Paananen2 ,
Marko Lehtonen3 , Kati Hanhineva2 and Inger Bjorck
1

1
Food for Health Science Centre, Lund University, Lund, Sweden
2
Department of Clinical Nutrition, Institute of Public Health and Clinical Nutrition, University of Eastern Finland,
Kuopio Campus, Kuopio, Finland
3
School of Pharmacy, University of Eastern Finland, Kuopio, Finland

Scope: Multifunctional diet (MFD), a diet based on multiple functional concepts and ingredi- Received: July 1, 2016
ents with anti-inflammatory activity, was previously shown to improve different cardiometabolic Revised: September 5, 2016
risk-associated markers in healthy subjects. Here, we assessed the impact of MFD on plasma Accepted: September 11, 2016
metabolome and explored associations of the differential metabolites with clinical parameters,
searching for metabolic determinants related to the effects of MFD.
Methods and results: Forty-four overweight healthy volunteers completed a randomized
crossover intervention comparing MFD with a control diet devoid of the active components of
MFD. Fasting plasma samples were analyzed with nontargeted metabolite profiling at base-
line and at the end (4 wk) of each diet period by LC coupled to quadrupole-TOF-MS system,
revealing a vast impact of MFD on metabolic homeostasis. Main metabolite classes affected
included acylcarnitines, furan fatty acids, phospholipids (plasmalogens, phosphatidylcholines,
phosphatidylethanolamines), and various low-molecular weight products from the bioactivity
of gut microbiota. Circulating levels of several of these metabolites correlated with changes in
clinical blood lipid biomarkers.
Conclusions: The metabolomics approach revealed that consumption of MFD affected different
areas of metabolism, highlighting the impact of a healthy diet on plasma metabolome. This
seems linked to reduced cardiometabolic risk and provides mechanistic insight into the effects
of MFD.
Keywords:
Cardiometabolic diseases / Dietary prevention / Functional foods / Metabolic syn-
drome / Plasma metabolomics

 Additional supporting information may be found in the online version of this article at
the publishers web-site

1 Introduction

Although cardiometabolic diseases (CMDs) are the main

Correspondence: Dr. Juscelino Tovar
E-mail: juscelino.tovar@food-health-science.lu.se
Abbreviations: BP, blood pressure; CMPF, 3-carboxy-4-
methyl-5-propyl-2-furanpropanoic acid; CD, control diet; CMD,
cardiometabolic diseases; LPC, lysophosphatidylcholine; LPE,
lysophosphatidylethanolamine; MFD, multifunctional diet; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; TAGs, tria-
cylglycerols
cause of morbidity and mortality in contemporary societies
[1], efforts to prevent these illnesses are relatively scarce.
As modification of dietary habits can contribute to healthy
ageing [2], customary healthy dietary patterns, such as the
Mediterranean diet, may be promoters of lifelong health,
Current address: Dr. Juscelino Tovar, Food for Health Science Cen-
tre, Lund University, Medicon Village, SE-223 81, Lund, Sweden


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1600552 (2 of 12) J. Tovar et al.
reducing the risk for cardiovascular complications [3]. Sim-
ilarly, more defined, evidence-based regimens may also
contribute to facilitate the adoption of healthier eating
habits.
We have designed a mixed diet that encompasses mul-
tiple functional ingredients and foods that exert beneficial
effects related to cardiometabolic risk. The diet follows the
Nordic Nutrition Recommendations, but it specifically targets
subclinical inflammation, combining whole barley kernel-
and soy protein containing products, plant stanols, almonds,
oily fish, foods, and ingredients rich in natural antioxidants,
oat and barley products rich in viscous dietary fibers, low
glycemic index meals, and a probiotic strain [4]. The phys-
iological effects of this multifunctional diet (MFD) were
evaluated in a randomized controlled trial in healthy subjects
considered at risk of developing cardiometabolic complica-
tions, i.e. age 50 years old and overweight (BMI 25) [4].
The study compared MFD with a control diet (CD) also formu-
lated according to the general Nordic Nutrition Recommenda-
tions. Four week consumption of MFD resulted in markedly
reduced values of circulating CMD-related biomarkers, while
CD had no effect. The biomarkers affected by MFD were total
and LDL-cholesterol levels, triglycerides, CRP, HbA1c, and
systolic blood pressure (BP), with 30% decrease in estimated
coronary heart disease risk [4]. MFD may thus be a potential
tool for implementing innovative dietary strategies for the
prevention of CMDs.
Metabolomics analyses of human fluids and tissues can
reveal changes in endogenous metabolites that may lead to
the identification of novel, disease-staging biomarkers of par-
ticular conditions, such as CMDs [5,6]. In addition, metabolite
profiling approaches have applications in nutritional studies,
providing information at molecular level that can be used to
establish reliable markers of consumption of specific foods
groups [79]. They also allow to monitor diet-related changes
in endogenous metabolites, thus helping to understand the
effects of certain foods and dietary regimens on metabolic
pathways and illnesses [10, 11]. Moreover, when diet-related
metabolic differences are investigated for associations with
clinical parameters, further mechanistic understanding may
be achieved and novel hypotheses drawn [5].
Here, fasting plasma samples from the above-mentioned
intervention study [4] were analyzed with a nontargeted
LCMS based metabolite profiling approach. Evaluation of
the metabolite profiles against conventional clinical vari-
ables revealed that consumption of MFD promotes impor-
tant metabolome changes, providing additional insight into
the beneficial effects of this regimen.
2 Methods
2.1 Participants
Nonsmoking healthy volunteers, were recruited through ad-
vertisement in local newspapers and gave written acceptance

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Mol. Nutr. Food Res. 61, 2, 2017, 1600552
to join the study. Inclusion criteria were age between 50 and
73 years old, body mass index in the 2533 kg/m2 range, and
fasting plasma glucose value 6.1 mmol/L. Forty-four (36
women, 8 men) completed the study and yielded usable data.
Baseline characteristics of the participants are presented in
Supporting Information 1.
2.2 Study protocol
The dietary intervention is described in detail elsewhere [4].
The study was a randomized, controlled, crossover trial of
the effect of MFD on biomarkers related to cardiometabolic
risk [4]. MFD was compared with a CD (see below). Each
diet phase lasted 4 wk separated by a 4 wk washout interval,
consisting of the subjects habitual diet.
Clinical outcomes measured were: body weight, BP and
fasting circulating concentrations of glucose, insulin, glycated
hemoglobin (HbA1c), cholesterol (total, LDL and HDL), tria-
cylglycerols (TAG), C-reactive protein (hs-CRP), apoA1, apoB,
IL-6, TNF-, free fatty acids, and plasminogen activator in-
hibitor 1 levels.
The study aimed to evaluate diet-related metabolic
changes under conditions favouring weight maintenance.
Accordingly, participants did not alter their physical activ-
ity habits during the intervention and recorded their body
weight weekly. Variations larger than 1 kg led to nutritionist-
guided compensatory dietary modifications, without altering
the intake of key components of MFD. The study was ap-
proved by the Regional Ethical Review Board, Lund, Sweden
(Dnr 5932008). Details about the study protocol are provided
in [4] and Supporting information 1.
2.3 Diets
The nutritional profiles of CD and MFD were described
previously [4] and are summarized in Table 1. Both diets
conformed to the Nordic Nutrition Recommendations [12],
except for dietary fiber contents which were higher in MFD
(49 and 62 g/day for women and men, respectively) and lower
in CD (22 and 26 g/day for women and men, respectively).
The diets supplied 25002600 Kcal/day for men and 2000
2100 Kcal/day for women, combining foods from plant and
animal origin. For a detailed list of products included in MFD
see ref. 4.
MFD combined several functional concepts capable of in-
fluencing different biomarkers related to the inflammatory
tonus and cardiometabolic risk, including: (i) foods rich in
natural anti-inflammatory antioxidants, with BP and blood
lipid improving features; (ii) omega-3 fatty acids in oily fish
and rapeseed oil, with anti-inflammatory and triglyceride-
lowering properties; (iii) ingredients with prebiotic activity,
i.e. beta-glucans and resistant starch in intact barley ker-
nels, whole kernel rye flour, oats, and isolated barley fiber.
An experimental guar gum containing bread was another
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Table 1. Nutritional profiles of CD and MFD Table 2. Main functional action and average content of active
components in MFDa)
CD MFD
Main functional Content in MFD
Women Men Women Men role (g/day)

Energy (kcal/day) 2045 2570 2100 2615 Women Men

Protein (E%) 15 14 19 18
Carbohydrate (E%) 56 55 51 50 Soybean/soy Cholesterol- 21 25
Fat (E%) 29 30 31 31 protein lowering,
Saturated fat (E%) 12.8 13.2 5.9 5.9 anti-
Monounsaturated fat (E%) 10.5 11.1 13.0 13.6 inflammatory
Polyunsaturated fat (E%) 3.6 3.7 8.2 8.4 Viscous fibers Cholesterol-
-6 fatty acids (E%) 2.9 3.1 4.2 4.3 lowering,
-3 fatty acids (E%) 0.8 0.8 2.2 2.3 prebiotic,
-6/-3 ratio 3.8 3.8 1.9 1.9 GI-reducing
Dietary fiber (g/day) 22 26 49 61 -glucans 5.8 6.2
Cholesterol (mg/day) 200 240 140 160 Guar gum 5.6 6.7
Total 11.4 12.9
Mean daily values from 14 days (2-week rotating menu plan) [4] Long chain 3 Triglyceride- 2.4 3.0
fatty acids (20:5 lowering,
+ 22:6) anti-
source of viscous fermentable fiber. A probiotic Lactobacillus inflammatory
plantarum strain was also included; (iv) low glycemic impact Almonds Cholesterol- 28 28
foods/meals, which are associated with reduced risk for car- lowering
Plant stanols Cholesterol- 2.0 2.7
diometabolic problems and inflammatory tonus. Besides low
lowering
glycemic index foods, MFD included ingredients that lower Cinnamon Antioxidant 3.0 3.0
the postprandial glycemic response (whey protein and vine- Blueberries Antioxidant, 74.5 94.5
gar); (v) blood cholesterol normalizing ingredients: soybean prebiotic
products, a margarine enriched in stanol esters and dry al- Vinegar GI -reducing 22.5 22.5
monds. Whey proteinb) GI-reducing 4.3 4.3
The mean daily quantities and functional properties con- a) For further details see ref. [4] and Supporting Information 2.
sidered for the selection of key components of MFD are b) Whey protein (10 g) was only consumed simultaneously with
summarized in Table 2. None of the active ingredients were high glycemic index meals (potatoes, parsnip), three times per
week.
present in the CD, except for minor amounts of -3 fatty
acids. Further details on the functional concepts and the ba-
sis for their inclusion in MFD are provided in Supporting mass qTOF spectrometry. The samples were analyzed using
Information 2. RP and hydrophilic interaction chromatography (HILIC) in
positive (+) and negative (-) polarity. For technical details see
Supporting Information 3.
2.4 Clinical analyses

The Clinical Chemistry Laboratory/Skane

University Hospi-
tal Malmo (Sweden) performed routine blood analyses on
fasting plasma (total and HDL cholesterol, TAG, apo A-1,
apo B, hs-CRP), serum (insulin), or on total blood samples
(HbA1c). LDL cholesterol concentrations were calculated [13].
All other analyses were performed as described in ref. 4.
EDTA plasma samples were kept frozen at 40C for the
LCMS metabolite profiling analysis.
2.5 Nontargeted LCMS metabolite profiling
analysis
Plasma samples were analyzed by the UHPLC-qTOF-MS
system (Agilent Technologies, Waldbronn, Karlsruhe, Ger-
many) that consisted of a 1290 LC system, a Jetstream elec-
trospray ionization (ESI) source, and a 6540 UHD accurate-
2.5.1 Data collection and alignment
Data were collected with Find by Molecular Feature al-
gorithm (MassHunter Qualitative Analysis B.05.00, Agilent
Technologies, USA). The peak collection threshold was 200
counts, and the allowed ion species were limited to [M+H]+
and [M+Na]+ in ESI(+), and [M-H] and [M+Cl] in ESI().
Data files (.cef-format) were exported to Mass Profiler Profes-
sional (Agilent Technologies) for peak alignment. After the
first initial alignment, the data were combined in one .cef file,
against which the original raw data were reanalyzed. For this
recursive analysis, compound mass tolerance was 15 ppm,
retention time 0.2 min, and symmetric expansion value
for chromatograms 10 ppm. Resulting compounds were
reexported to Mass Profiler Professional software for peak
alignment and data cleanup resulting in 3532, 3511, 1347,
1441 metabolite features from RP(+), RP(), hilic(+), and


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1600552 (4 of 12) J. Tovar et al.
hilic() respectively, which were subjected for chemometri-
cal and statistical evaluation.
2.6 Statistical analysis
To account for nonnormal distributions, metabolite data were
transformed using rank-based normal transformation. Each
metabolite was analyzed using linear mixed-effect model with
the metabolite as a dependent variable. Included independent
fixed-effect terms were diet (MFD, CD), time point, treatment
order, and interaction between study group and time point,
while subject identifier was included as a random term. Inter-
action between diet and time point was the main outcome. All
p-values were adjusted for multiple testing using Benjamini
Hochberg false discovery rate (p-FDR). p-FDR < 0.05 was
considered as statistically significant. Statistical analyses were
performed using R (version 3.1.2) and nlme package (version
3.1-118). Additionally, multivariate assessment of the data
was performed with supervised clustering algorithm par-
tial least-squares discriminant analysis (PLS-DA; Simca-13,
Umetrics, Sweden) for the samples collected at the end of the
interventions. The data were log10-transformed, paretoscaled
and the model was validated by the Simca-13 internal cross
validation.
All the data with combined results from the statistical
and chemometrical analysis were exported in excel (Mi-
crosoft Office 2013) for further processing. The most sig-
nificantly altered metabolite signals were filtered out from
the data matrix in each of the four analytical conditions by
matching with the following criteria: signal was present in
at least 80% of samples in one replicate group; p-FDR <
0.05; fold change < 0.8 or >1.2; and variable importance
for projection (VIP) > 1 in partial least-squares discriminant
analysis.
The differential metabolites were identified based on
the MS/MS spectral comparison of pure standard com-
pounds and on the search of the candidate compounds
in databases, including the Human Metabolome database,
METLIN, ChemSpider, and SciFinder, and the results ver-
ified with the MS/MS spectral features included in the
databases or reported in earlier publications.
Spearman correlation coefficients (rs ) were used to test
the relationship between the changes in the cardiometabolic-
related biomarkers and in the significantly differentiated
metabolites. A p < 0.05 was considered statistically signifi-
cant.
3 Results
3.1 CMD-related outcomes
The results of the intervention in terms of cardiometabolic
risk-associated markers have been reported earlier [4] and
can be summarized as follows: compared to baseline, there

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Mol. Nutr. Food Res. 61, 2, 2017, 1600552
was a minor weight reduction with both diets (0.9 %, CD;
1.8%; MFD, p < 0.05). CD did not modify the metabolic
variables measured but MFD promoted changes in total
serum cholesterol (26 1% versus baseline; p < 0.0001),
LDL-cholesterol (34 1%; p < 0.0001), TAG (19 3%;
p = 0.0056), LDL/HDL (27 2%; p < 0.0001), apoB/apoA1
(10 2%; p < 0.0001), HbA1c (2 0.4%; p = 0.0013),
hs-CRP (29 9%; p = 0.0497), and systolic BP (8 1%;
p = 0.0123). The differences remained significant after
adjustment for weight change [4]. After consumption of
MFD, both the Framingham cardiovascular risk estimate
and the Reynolds cardiovascular risk score were reduced
by one-third (30 4%; p < 0.0001 and 35 3%; p < 0.0001,
respectively).
3.2 Metabolomics analysis of fasting plasma
Nontargeted metabolite profiling of plasma samples revealed
marked changes in numerous metabolites after the MFD
period of the intervention while only modest variations
were recorded with CD. After strict filtering of the data,
the number of metabolic features considered as differen-
tial was 242, 181, 69, 49 in RP(+), RP(), hilic(+), and
hilic() modes, respectively, therefore comprising altogether
541 metabolite features. The examination of the MS/MS
spectral and chromatographic features showed that major-
ity of the differential signals were belonging to different
classes of compounds in lipid metabolism, namely phos-
phatidylcholines (PCs), lysophosphatidylcholines (LPCs),
phosphatidylethanolamines (PEs), plasmalogens, carnitines,
furan fatty acids, and other compounds resulting from the
bioactivity of the colonic microflora, including indolepropi-
onic acid (Table 3, Fig. 1).
3.2.1 Furan fatty acids
Plasma 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid
(CMPF) was clearly increased after ingestion of MFD,
whereas a significant decrease was recorded for CD.
In addition to CMPF, four other compounds that were el-
evated by the MFD intervention were tentatively identified as
furan fatty acids. These included 3-carboxy-4-methyl-5-pentyl-
2-furanpropionic acid and three other signals (Table 3). The
compound with molecular weight of 238.158 has elemental
composition of C14H22O3, which refers to the furan fatty
acid 3,4-dimethyl-5-pentyl-2-furanpropanoic acid identified
from crayfish [14], whereas the compound with molecular
weight of 226.084 refers to molecular formula C11H14O5.
This formula returns several furan fatty acid hits from the
SciFinder database, among which, e.g. 3-carboxy-5-propyl-2-
furanpropanoic acid exhibits a chemical structure that would
match the neutral loss of 44 amu (COO) twice from the molec-
ular structure as visible in the MS/MS spectrum in analogous
manner with CMPF. Likewise, the compound C15H24O3
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Table 3. Main differential metabolites analyzed in the nontargeted LC-qTOF-MS metabolite profiling analysisa)

Compound Compound Mass Retention Column ESI p-FDR av FC MFD av FC CD PLS-DA

class Time mode VIP

Hydroxybutyrylcarnitine Carnitines 247.1422 4.16 hilic pos 0.00 1.58 1.05 1.37
2797
373
Carnitine (8:2) Carnitines 283.178 1.04 hilic pos 0.01 1.49 1.03 1.74
0295
614
Carnitine (8:1) Carnitines 285.1939 0.93 hilic pos 0.04 1.45 1.03 1.50
0401
113
Carnitine (10:2) Carnitines 311.209 0.77 hilic pos 0.00 1.44 1.01 1.70
0786
075
Carnitine 345.26@10.4 Carnitines 345.2683 10.45 RP pos 0.00 1.74 1.19 1.56
1942
763
Carnitine (13:1) Carnitines 355.2721 8.05 RP pos 7.81 109 2.33 1.01 2.48
Carnitine (13:0) Carnitines 357.2879 8.33 RP pos 9.42 1011 4.32 1.10 2.77
Carnitine (14:2) Carnitines 367.2722 0.65 hilic pos 0.01 1.92 1.39 1.36
8943
623
Carnitine (14:1) Carnitines 369.2876 0.65 hilic pos 0.01 1.47 1.22 1.08
8743
12
Carnitine 391.27@8.1 Carnitines 391.2722 8.06 RP pos 2.11 106 2.07 1.01 2.34
Palmitoylcarnitine (16:0) Carnitines 399.3346 9.25 RP pos 0.01 0.86 1.03 1.65
9707
722
Stearoylcarnitine (18:0) Carnitines 427.366 9.64 RP pos 0.00 0.73 0.93 2.25
3799
828
Furan fatty acid Furan fatty 226.0842 6.80 RP neg 5.38 1019 3.09 0.30 3.31
226.08@6.8 acids
3-4-dimethyl-5-pentyl-2- Furan fatty 238.1576 9.35 RP neg 0.00 1.28 0.91 2.00
furanpropanoic acids
acid
7751
553
CMPF Furan fatty 240.0998 7.45 RP neg 2.18 1016 2.77 0.69 3.56
(3-carboxy-4-methyl-5- acids
propyl-2-furanpropanoic
acid)
Furan fatty acid Furan fatty 252.1722 9.54 RP neg 0.00 1.42 0.91 2.63
252.17@9.5 acids
1267
115
3-carboxy-4-methyl-5- Furan fatty 268.1311 8.54 RP neg 1.86 108 1.49 0.82 2.34
pentyl-2-furanpropionic acids
acid
EPA (20:5) Fatty acids 302.2246 10.26 RP pos 1.17 107 1.98 0.56 2.03
DHA (22:6) Fatty acids 328.2399 10.48 RP neg 7.60 105 1.52 1.06 2.17
FA (22:1) Fatty acids 338.3188 11.46 RP neg 0.00 2.16 0.83 1.93
2864
61
LPC (14:0) PCs 527.3234 9.67 RP neg 2.07 105 0.66 1.01 2.41
LPC (20:5) PCs 541.317 9.67 RP pos 4.83 1012 2.57 0.89 3.02
LPC (20:3) PCs 545.3486 10.13 RP pos 9.88 108 0.64 1.07 3.13
LPC (22:6) PCs 567.3333 9.91 RP pos 1.62 105 1.98 1.27 2.25
LPC (15:0) PCs 481.3169 9.73 RP pos 0.03 3.81 1.85 1.37
6888


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Table 3. Continued

Compound Compound Mass Retention Column ESI p-FDR av FC MFD av FC CD PLS-DA

class Time mode VIP

465
LPC (18:3) PCs 517.3167 9.68 RP pos 0.02 0.75 1.03 1.57
1800
842
PC (32:2) PCs 729.5308 11.73 RP pos 0.00 0.77 1.04 2.01
1611
913
PC (32:1) PCs 731.5473 12.05 RP pos 2.31 108 0.60 1.11 2.46
PC (18:2/16:1) PCs 755.5472 11.88 RP pos 5.69 105 0.67 1.03 2.64
PC (18:2/16:1) isomer PCs 755.5472 11.81 RP pos 0.04 0.77 1.32 1.81
1846
266
PC (36:4) PCs 765.566 12.14 RP pos 1.72 1012 2.39 0.80 2.90
PC 779.54@11.8 PCs 779.549 11.80 RP pos 2.93 108 12.31 3.01 2.80
PC (38:6) PCs 789.5662 12.24 RP pos 3.27 1017 1.96 0.91 3.38
PC (20:5/18:2) PCs 803.5461 11.62 RP pos 0.00 2.24 0.93 1.77
0328
809
PC (20:5/18:2) isomer PCs 803.5463 11.65 RP pos 0.00 1.86 0.79 2.10
0708
53
PC (18:2/20:4) isomer PCs 805.5613 12.33 RP pos 6.44 1011 1.94 1.01 2.97
PC (18:2/20:4) PCs 805.5623 12.02 RP pos 1.04 108 1.36 1.02 2.37
PC (38:5) PCs 807.5786 12.29 RP pos 3.73 1013 1.96 0.99 2.93
PC (38:5) isomer PCs 807.579 12.32 RP pos 6.44 1011 2.02 1.02 3.08
PC (18:1/20:3) PCs 809.5925 12.48 RP pos 5.28 105 0.80 1.37 2.27
PC (18:0/20:3) isomer PCs 811.6061 12.92 RP pos 0.00 2.41 1.03 2.93
1230
731
PC (18:0/20:3) PCs 811.6084 13.07 RP pos 1.01 1014 0.43 1.34 2.83
PC (40:6) PCs 817.5961 12.48 RP pos 3.07 1016 1.68 0.99 3.23
PC 831.57@11.9 PCs 831.5754 11.99 RP pos 1.16 1011 0.49 1.29 1.84
PC (18:1/22:6) PCs 831.5779 12.11 RP pos 1.95 1011 1.76 1.07 2.65
PC (22:6/18:0) PCs 833.5934 12.58 RP pos 3.36 1010 1.62 1.06 2.44
PC 859.60@12.7 PCs 859.6055 12.68 RP pos 7.96 1014 3.15 1.09 3.07
PC 877.55@11.6 PCs 877.5578 11.64 RP pos 1.82 108 2.70 0.97 2.54
LPE (16:1) PEs 451.2708 9.78 RP neg 8.05 107 0.59 1.16 2.12
LPE (20:5) isomer PEs 499.2704 9.63 RP neg 0.00 6.27 2.21 1.63
0924
909
LPE (20:5) PEs 499.2709 9.70 RP neg 2.87 108 1.84 0.94 3.00
LPE (20:4) PEs 501.2869 9.95 RP neg 2.84 106 0.78 1.11 2.47
LPE (20:3) PEs 503.302 10.13 RP neg 7.92 1014 0.50 1.17 3.33
LPC (18:3) PCs 517.3167 9.68 RP pos 0.02 0.75 1.03 1.57
1800
842
LPE (22:6) PEs 525.2866 9.91 RP neg 0.00 3.66 1.69 1.48
6464
993
LPE (22:5) PEs 527.3021 10.07 RP neg 3.61 107 0.67 1.08 2.48
PE (20:4/16:0) PEs 739.5161 12.13 RP neg 0.01 0.80 1.18 1.95
3879
152
PE (20:5/P-16:0) Plasmalogens 721.5044 11.93 RP pos 1.24 109 1.92 0.85 2.79
PE (20:4/P-16:0) Plasmalogens 723.5192 12.23 RP pos 0.00 0.72 0.95 2.39
7986
974
PE (20:4/P-18:1) Plasmalogens 749.5353 12.33 RP pos 0.00 0.76 0.96 2.34
8522


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Table 3. Continued

Compound Compound Mass Retention Column ESI p-FDR av FC MFD av FC CD PLS-DA

class Time mode VIP

794
PE(16:0/22:6) PEs 763.5179 11.93 RP pos 0.03 1.32 1.07 1.16
4705
965
PE (22:6/P-18:1) Plasmalogens 773.5353 12.24 RP pos 1.43 105 1.43 1.03 2.37
Pipecolic acid Other 129.0789 3.81 hilic pos 0.02 1.30 0.99 0.75
8088
974
Dihydroxybenzoic acid Other 154.0263 0.97 hilic neg 4.46 106 1.74 0.90 1.84
Aminophenol sulfate Other 189.0097 1.82 RP neg 7.63 105 3.09 0.61 3.37
isomer
Aminophenol sulfate Other 189.0099 1.94 RP neg 3.34 109 3.52 0.67 2.63
Indolepropionic acid Other 189.0792 5.54 RP neg 0.00 2.15 1.32 1.89
4557
171
Pyrocatechol sulfate Other 189.9943 0.48 hilic neg 0.01 1.77 1.08 1.15
1469
54
Unknown 210.12@8.4 Other 210.1257 8.48 RP neg 6.72 107 3.20 1.20 1.48
Unknown 238.08@5.1 Other 238.085 5.14 RP pos 0.02 1.31 1.07 1.19
2007
283
Unknown 250.12@8.4 Other 250.1207 8.48 RP pos 4.29 1010 1.48 0.80 2.23
Unknown 392.29@9.5 Other 392.2933 9.53 RP neg 0.01 3.38 2.10 1.91
3510
914
Bile acid 449.31@9.4 Other 449.3142 9.41 RP neg 0.01 2.23 1.41 1.54
0612
737
Unknown bile acid Other 492.3304 9.14 RP neg 6.90 1012 6.45 0.48 3.31
492.33@9.1
Unknown (steroid Other 657.351 8.62 RP pos 1.33 1013 4.10 0.30 2.48
glucuronide) 657.35@8.6
Unknown (steroid Other 659.3664 9.25 RP pos 6.18 1015 5.13 0.32 2.23
glucuronide) 659.36@9.2
a) Included are the neutral molecular mass of the compound (mass), retention time either in HILIC or RP chromatography, ESI, tentative
identification based on the evaluation of msms spectral data (detailed msm spectra included as Supporting Information 4), FDR corrected
P values from the mixed model analysis, average fold changes (Av FC) between the baseline and end of the diet, variable importance for
projection (VIP) value from the partial least-squares discriminant analysis (PLS-DA).
DHA (22:6), docosahexaenoic acid (C22:6), EPA (20:5), eicosapentenoic acid (C20:5), FA, erucic acid (C22:1).

eluting in the same chromatographic region as the other fu-
ran fatty acids (M 252.172) refers to furan fatty acid structures,
e.g. 3-methyl-5-pentyl-2-furanpentanoic acid. This structure,
however, was not confirmed with MS/MS.
Notably, modified fatty acid composition of lipids
was observed also in the case of plasmalogens, as
the plasmenylethalomines PE(20:4/P-16:0) and PE(20:4/P-
18:1), which were decreased after the MFD interven-
tion, whereas PE(20:5/P-16:0) and PE(22:6/P-18:1) were
increased.

3.2.2 Lipids and fatty acids

The levels of numerous PCs, LPCs, PEs, and lysophos-
phatidylethanolamines (LPEs) decreased after the MFD diet.
The most significant ones were PC (18:0/20:3), LPC (20:3),
LPE (20:3), and LPE (16:1), with fold changes <0.5 and
p < 6 107 . Likewise, a large number of lipid species were
increased after the MFD intervention, including LPC (20:5),
LPC (22:6), PC (20:5/18:2).
3.2.3 Carnitines
Several compounds affected by the dietary scheme showed
characteristic MS/MS fragmentation patterns of acylcar-
nitines. These included carnitines C13:0 and C13:1, which
were both clearly increased after MFD (p-values 9.4 1011
and 7.8 109 , and average fold change for MFD of 4.3


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1600552 (8 of 12) J. Tovar et al. Mol. Nutr. Food Res. 61, 2, 2017, 1600552

Figure 1. Correlation heatmap

of the changes in metabolites
and changes in clinical biomark-
ers and anthropometric mea-
surements. Correlations were
calculated using Spearman rank
correlation and red and blue col-
ors indicate positive and neg-
ative correlation, respectively.
Size of the circles describe statis-
tical significance (log10 trans-
formed p-value) where a larger
circle indicates a higher statisti-
cal significance.

and 2.3, respectively). Other acylcarnitine species that in-
creased after MFD included carnitines with fatty acid con-
figuration C14:2, C14:1, C8:2, C8:1, all exhibiting increased
number of unsaturated bonds, and hydroxybutyrylcarnitine
which exhibited a 1.6-fold change. In contrast, two long-
chain acylcarnitine species, palmitoylcarnitine C16:0, and
stearoylcarnitine C18:0, decreased after the MFD diet. No-
tably, the most common short-chain acylcarnitines (acetyl-,
butyryl-, propionyl-, and valerylcarnitine) and medium chain-
length acylcarnitines (hexanoyl-, octanoyl-, nonaoylcarnitine)
were not altered by the different diets when examined
from the hilic- (short-chain) and RP (medium-chain) LCMS
data.
3.2.4 Other compounds
There was a marked increase in the concentration of several
small compounds resulting from the bioactivity of colonic-
microbiota, such as indolepropionic acid, pyrocatechol sul-


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Mol. Nutr. Food Res. 61, 2, 2017, 1600552
fate, and aminophenol sulfate, which showed fold changes in
the 1.73.5 range after MFD. Additionally, clear increase was
observed for several compounds not amenable for identifica-
tion but suggesting most likely steroid structure, such as bile
acids or derivatives of plant stanols.
3.3 Correlation between changes in metabolite
concentration and CMD-related outcomes
After finding the metabolites that were most significantly
changed during the intervention, we performed correlation
analyses to investigate how the metabolic changes are related
to the measured clinical biomarkers (Fig. 1, Supporting Infor-
mation 5). Overall, changes in most of the differential plasma
metabolites were mainly associated with changes in the clas-
sic circulating lipid biomarkers, such as cholesterol fractions,
Apo B, and triglycerides. The strongest correlations were
observed for changes in total and LDL cholesterol and the
LDL to HDL cholesterol ratio with particular metabolites, as
strong inverse correlation was observed with changes in furan
fatty acids, including CMPF and 3-carboxy-4-methyl-5-pentyl-
2-furanpropionic acid, and other lipid compounds such as
PC(38:6), PC(34:6), PC(40:6) as well as two compounds tenta-
tively identified as steroid glucuronides. In contrast, marked
positive correlation with the changes in cholesterol fractions
was observed for various lipids, the strongest one for the 20:3
fatty acid-containing phospholipids: LPC(20:3), LPE(20:3),
and PC(20:3/18:0). Other lipid species and fatty acids, such
as eicosapentenoic acid (C20:5) (EPA), also showed strong
inverse association with the changes in both LDL cholesterol
and the LDL to HDL ratio as well as with the cardiovascular
risk predictors.
Opposite associations with clinical lipid markers were also
observed for other metabolites within the same metabolic
classes. Long-chain acylcarnitines, for instance, showed posi-
tive correlation with changes in cholesterol fractions whereas
the short-chained ones were inversely associated. Likewise,
two plasmalogens, PE(22:6/P-18:1) and PE(20:5/P-16:0), were
inversely correlated with total and LDL cholesterol, while two
other species, PE(20:4/P-18:1 and PE(20:4/P-16:0), showed
positive association.
The correlation with changes in body weight, glucose and
insulin levels were less consistent and weaker, but neverthe-
less many were statistically significant (Fig. 1, Supporting
Information 5). There was no important correlation trend be-
tween the changes in BP or inflammatory markers and the
changes in metabolome.
4 Discussion
The present study shows that the beneficial effect of MFD, a
diet based on a combination of functional food concepts, on
cardiometabolic risk factors is mediated via several branches
of metabolism, as evidenced by the notable changes in the

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(9 of 12) 1600552
plasma metabolome of healthy overweight subjects. Main
changes were recorded for fatty acid-containing furans and
different lipidic compounds, namely acyl-carnitines, phos-
pholipids, and fatty acids, as well as for metabolites related to
the activity of the gut microbiota.
Furan fatty acids levels, particularly CMPF and 3-carboxy-
4-methyl-5-pentyl-2-furanpropionic acid, increased during
the MFD intervention. This observation is explained mainly
on the basis of the significant supply of oily fish that char-
acterizes MFD. Elevated plasma levels of CMPF have been
recently shown in an intervention study where they strongly
correlated with the total intake of fish [7]. Additionally, in-
creased circulating levels of 3-carboxy-4-methyl-5-pentyl-2-
furanpropionic acid were reported also after intervention
with a Mediterranean-type diet [11]. These observations, to-
gether with the higher post-MFD levels of three other com-
pounds classified as furan fatty acid species (Table 3), in-
dicate that the variety of diet-derived furan fatty acids is
far more diverse than known so far. Moreover, the inverse
correlation observed between the levels of circulating fatty
acid containing furan metabolites and LDL cholesterol and
LDL/HDL cholesterol ratio suggests that furan-derived com-
pounds could be involved in the beneficial action of MFD.
The notable radical-scavenging ability of this chemical group
has been previously claimed relevant for the cardiometabolic-
protective effects of the intake of fish and seafood in
general [1517].
The PUFAs EPA and docosahexaenoic acid (C22:6)
(DHA), and various PUFA-containing phosphatidylcolines,
phosphatidyletanolamines, and their lyso- derivatives in-
creased after the MFD intervention. Again, these raised lev-
els of PUFA-containing compounds most likely reflect the
relatively high fish intake associated with this diet. Cor-
relation analyses confirmed that the change in concentra-
tion of several PUFA-containing phospholipid species is
inversely associated with the variation in circulating choles-
terol fractions. On the other hand, the positive correlation
between three C20:3-containing phospholipid species (PC
(18:0/20:3), LPC(20:3) and LPE(20:3)) and LDL-cholesterol,
total cholesterol, and the cardiovascular risk estimates, seems
in line with the proposed association between blood lipid
profiles high in this fatty acid and cardiometabolic risk
[18, 19]. Additionally, we have recently identified LPC(20:3)
as a compound related to higher risk of developing type
2 diabetes, thus further highlighting the potential harm-
ful effect of 20:3 fatty acid-containing lipids on health
(de Mello et al, submitted).
Reduced plasma concentrations of LPC(18:2) have been
recently proposed as a prediabetes-specific marker [20]. Our
study, however, did not reveal differences between consump-
tion of CD and MFD in relation with this metabolite. In fact,
the level of LPC(18:2) was increased after consuming either
diet, and no clear correlation pattern was observed between
circulating glucose or insulin and the various LPC species.
An interesting observation relates to the elevated concen-
tration of LPC (15:0) after the MFD period, since circulat-
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1600552 (10 of 12) J. Tovar et al.
ing levels of C(15:0)-containing phospholipids have been in-
versely associated with the incidence of type 2 diabetes [21,22].
It is generally accepted that human cells do not produce odd-
chain fatty acids and their occurrence in adipose tissue and
blood is determined by the diet. Plasma pentadecanoic acid
(C(15:0)) and heptadecanoic acid (C(17:0)) are thus regarded
as markers of the intake of dairy products [23] and fish [24,25].
Since MFD does not include particularly important levels of
milk and derived products, the fourfold elevation recorded
for LPC (15:0) may originate from the abundance of oily
fish in MFD. However, production of odd-chain fatty acids
by particular gut microorganisms cannot be ruled out [26].
The relevance of this putative activity of gut microbiota for
the host cardiometabolic health and its modulation by diet
should be further investigated. Odd-chain fatty acid contain-
ing lipids have also been related to lower type 2 diabetes risk
in the Finnish Diabetes Prevention Study (de Mello et al,
submitted).
Ethanolamine plasmalogens were another group of lipids
markedly affected by consumption of MFD. Levels of plas-
malogens containing fatty acid C(20:4) decreased, while those
with fatty acids C(20:5) and C(22:6) increased after MFD.
In addition, the changes in C(20:4)-containing plasmalogens
correlated positively with the clinical lipid biomarkers and
those with C(20:5) or C(22:6) showed negative association.
These observations clearly indicate the influence of diet on
the metabolism of this unique type of molecules. However,
their interpretation awaits further insights on the biology
of plasmalogens. Although plasmalogens are generally ac-
knowledged as natural protectants against oxidative stress
and reservoirs of second messengers [27] our knowledge
on the physiological meaning of circulating plasmalogens
is still limited [28]. While certain choline-based plasmalogens
have been suggested as potential antiatherogenic biomarkers,
decreased total serum levels of ethanolamine plasmalogens
may be associated with reduced cognitive performance [28].
Our results suggest that the fatty acid composition of plas-
malogens might be important in the maintenance of healthy
metabolic homeostasis, as evidenced by the clear shift caused
by the dietary regimens evaluated.
Various circulating medium- and long-chain acylcar-
nitines (C8-C13), including two species with unsaturated fatty
acids, increased after MFD consumption and were inversely
correlated with changes in cholesterol fractions and cardio-
vascular risk estimates. This probably reflects the overall im-
proved fat quality of MFD [4]. On the other hand, the levels
of two long-chain species, palmitoylcarnitine and stearoylcar-
nitine, decreased markedly after MFD and were positively
correlated with the clinical lipid markers. Besides provid-
ing a clear evidence of the effect of diet on acylcarnitine
metabolism, these changes might be considered of potential
benefit since, in general terms, plasma long-chain acylcar-
nitines are associated with insulin resistance and increased
short-chain species may reflect improved efflux of mithocon-
drial fatty acids, a process of protective action against the
inhibition of glucose catabolism [29]. Plasma hydroxybutyryl-

C 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Mol. Nutr. Food Res. 61, 2, 2017, 1600552
carnitine has been suggested to promote insulin resistance
[29]. Surprisingly, MFD increased the concentration of this
compound, which showed negative association with clinical
lipid markers (Fig. 1). It is also worthwhile mentioning that
no appreciable changes were registered in plasma levels of
short-chain acylcarnitines, including acetylcarnitine, a com-
pound that qualifies as an early biomarker of type 2 diabetes
[20]. Evidently, more research on physiopathological aspects
of acylcarnitines would be needed for comprehensive inter-
pretation of present observations.
There was an increase in the plasma concentration of dif-
ferent low molecular mass compounds that are product of
gut microbial metabolism of dietary phenolics (pyrocathecol
sulfate), aminoacids (indolepropionic acid), and benzoxazi-
noids (aminophenol sulfate) [30, 31] that are abundant in
nonrefined cereals and other plant foods [30]. Since MFD
is rich in ingredients with prebiotic activity and contains a
probiotic bacterial strain, the increase in microbiota-derived
compounds might be consequence of both enhanced bioac-
tivity of the colonic flora and the higher amount of potential
precursor compounds included in the diet. Although rela-
tively little is known about the possible metabolic role of
these molecules in humans, the importance of microbiota-
mediated metabolism in maintenance of good human health
is being increasingly stressed [32]. In this respect, the positive
correlation shown here between aminophenol sulfate and the
changes in total and LDL-cholesterol deserves further inves-
tigation. We have recently found that high plasma levels of
indolepropionic acid are related to lowered risk of develop-
ing type 2 diabetes (de Mello et al., submitted). Additionally,
there were appreciable elevations in the circulating levels of
a number of compounds exhibiting most likely steroid struc-
ture, such as bile acids. Alternatively, these partially iden-
tified compounds could represent derivatives of the plant
stanols included in MFD as part of the cholesterol-lowering
strategy.
In conclusion, the present study revealed a set of impor-
tant changes in the plasma metabolome of healthy overweight
subjects in a controlled intervention with a diet based on mul-
tiple functional properties, and thus demonstrates clearly the
utilizability of nontargeted metabolite profiling as discovery
approach for unforeseen metabolic alterations. Some of the
changes may be associated with the composition of the diet
(markers of exposure) but others seem to reflect actual diet-
dependent modifications of the metabolic status of the sub-
jects, particularly regarding lipid metabolism. Changes in cir-
culating concentrations of several metabolites, including gut
microbiota-produced compounds, were correlated with ben-
eficial variations in the conventional clinic lipid profile, thus
providing further insight to the metabolic control related to
clinically relevant lipid markers. Although the investigation
was performed in a predominantly female group, there are
reasons to infer that our conclusions may be valid for the
healthy segment of the general population [4]. The metabolic
impact of MFD in patients affected by CMD remains to be
studied.
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Mol. Nutr. Food Res. 61, 2, 2017, 1600552
JT, IB, AN, MJ, KH were responsible for the study concept and
design; JT conducted the clinical research; KH collected and an-
alyzed metabolomics data; JP and VdM performed the statistical
evaluation of data; ML was responsible for the chemical analyzes;
JT drafted the manuscript and had primary responsibility for fi-
nal content. All authors revised, discussed and approved the final
paper.
This study was funded by the Lund University Antidiabetic
Food Center-AFC, a VINNOVA VINN Excellence Center (I.B.,
J.T.).
The authors have declared no conflict of interests.
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