Free Radical Biology and Medicine 176 (2021) 345–355

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Changes in the plasma lipidome of healthy subjects after coffee

consumption reveal potential cardiovascular benefits: A randomized
controlled trial
Oscar J. Lara-Guzmán a, Rafael Álvarez b, c, Katalina Muñoz-Durango a, *
a
Vidarium - Nutrition, Health and Wellness Research Center, Nutresa Business Group, Calle 8 Sur No. 50-67, Medellín, Colombia
b
Grupo de Investigación en Ciencias Farmacéuticas-ICIF-CES. Facultad de Ciencias y Biotecnología, Universidad CES, Calle 10A No. 22-04, Medellín, Colombia
c
Grupo de Investigación en Sustancias Bioactivas, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Calle 70 No. 52-21, Medellín, Colombia

A R T I C L E I N F O A B S T R A C T

Keywords: Lipid metabolism dysregulation is associated with cardiovascular disease (CVD) risk. Specific oxidized lipids are
Coffee beverage recognized CVD biomarkers involved in all stages of atherosclerosis, including foam cell formation. Moderate
Lipidome coffee intake is positively associated with cardiovascular health. A randomized, controlled (n = 25) clinical trial
Oxysterols
was conducted in healthy subjects to assess the changes in lipid species relevant to CVD (main inclusion criteria:
Chlorogenic acids
OxLDL
coffee drinkers, nonsmokers, with no history and/or diagnosis of chronic disease and not consuming any med­
Macrophage foam cells ications). Volunteers consumed a coffee beverage (400 mL/day) containing either 787 mg (coffee A; n = 24) or
407 mg (coffee B; n = 25) of chlorogenic acids for eight weeks. We measured the total plasma levels of 46 lipids,
including fatty acids, sterols, and oxysterols, at baseline and after eight weeks and assessed the effects of
chlorogenic and phenolic acids, the major coffee antioxidants, in an in vitro foam cell model via targeted lip­
idomics. At baseline (n = 74), all participants presented oxysterols and free fatty acids (FFAs) (CVD risk
markers), which are closely correlated to among them, but not with the classical clinical variables (lipid profile,
waist circumference, and BMI). After eight weeks, the control group lipidome showed an increase in oxysterols
(+7 ± 10%) and was strongly correlated with FFAs (e.g., arachidonic acid) and cholesteryl ester reduction (− 13
± 7%). Notably, the coffee group subjects (n = 49) had increased cholesteryl esters (+9 ± 11%), while oxysterols
(− 71 ± 30%) and FFAs (− 29 ± 26%) decreased. No differences were found between the consumption of coffees
A and B. Additionally, coffee antioxidants decreased oxysterols and regulated arachidonic acid in foam cells. Our
results suggest that coffee consumption modulates the generation of oxidized and inflammatory lipids in healthy
subjects, which are fundamental during CVD development. The clinical trial was registered on the International
Clinical Trials Registry Platform, WHO primary registry (RPCEC00000168).

Previously, clinical- and population-based metabolomic and lip­

1. Introduction
Coffee is a source of natural bioactive compounds such as chloro­
genic acids (CGAs), which are widely recognized for their beneficial
effects on cardiovascular health [1]. Some clinical trials have demon­
strated a reduction in biomarkers related to cardiovascular diseases
(CVDs), type 2 diabetes, and obesity in apparently healthy subjects with
moderate daily coffee intake [2,3]. Evidence has shown that CGAs may
play a key role in inhibiting lipid peroxidation and modulating oxidative
stress, thereby contributing to a lower risk of atherosclerosis [4]. Despite
the above, it is imperative to determine how bioactive coffee compounds
impact metabolism.
idomic studies on coffee consumption have reported a significant
enrichment in circulating metabolites from several metabolic pathways
(i.e., xanthine, benzoate, steroids, and endocannabinoids) [5]; a
reduction in fatty acids and lysophosphatidylcholine has been linked to
an increase in phospholipids [6]; and a strong positive association with
sphingomyelins and a negative association with long- and
medium-chain acylcarnitines has been noted [7]. Since many of these
lipids are closely related to CVD risk [8–11], evidence indicates that
coffee intake affects several biological pathways that are clearly distinct
from those traditionally used in clinical and epidemiological studies.
On the other hand, a promising group of biomarkers is oxysterols,
which are cholesterol derivatives with 27 carbons that contain

* Corresponding author. Vidarium, Nutrition, Health and Wellness Research Center, Calle 67 No. 52-20, Medellín, Colombia.
E-mail addresses: kmunoz@serviciosnutresa.com, kmunos@gmail.com (K. Muñoz-Durango).

https://doi.org/10.1016/j.freeradbiomed.2021.10.012
Received 30 August 2021; Received in revised form 7 October 2021; Accepted 10 October 2021
Available online 12 October 2021
0891-5849/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
O.J. Lara-Guzmán et al. Free Radical Biology and Medicine 176 (2021) 345–355

Abbreviations (PBS) phosphate-buffered saline

(PMA) phorbol 12-myristate-13-acetate
(AA) arachidonic acid (PCA) principal component analysis
(AI) atherogenic index (PG) prostaglandin
(CA) caffeic acid (PUFA) polyunsaturated fatty acid
(CQA) caffeoylquinic acid (ROS) reactive oxygen species
(CGA) chlorogenic acid (SFA) saturated fatty acid
(CVD) cardiovascular diseases (SPE) solid phase extraction
(ChoEs) Cholesteryl esters (UHPLC) ultra-high performance liquid chromatography
(DHFA) dihydroferulic acid (TFAs) total fatty acids
(diCQA) dicaffeoylquinic acid (TGs) triglycerides
(FAME) fatty acids methyl ester (CoA) 4-coumaric acid
(FA) ferulic acid (4α-OHC) 4α-hydroxycholesterol
(FFAs) free fatty acids (4β-OHC) 4β-hydroxycholesterol
(GC-MS) gas chromatography-mass spectrometry (7α-OHC) 7α-hydroxycholesterol
(iFA) isoferulic acid (7β-OHC) 7β-hydroxycholesterol
(IsoP) isoprostane (7-KC) 7-ketocholesterol
(LDL) low-density lipoprotein (24(S)–OHC) 24(S) hydroxycholesterol
(oxLDL) oxidized low-density lipoprotein (25-OHC) 25-hydroxycholesterol
(MUFA) monounsaturated fatty acid (27-OHC) 27-hydroxycholesterol

oxygenated groups such as hydroxyl, carbonyl, or epoxide groups.
Increased levels of oxysterols are associated with macrophage foam cells
and atherosclerotic plaque formation [12,13]. These molecules can be
produced enzymatically by mitochondrial or microsomal cytochrome
enzymes or nonenzymatically by autoxidation processes [14]. The main
enzymatically formed oxysterols in human circulation are 27-, 24-, and
7α− hydroxycholesterol, while the major nonenzymatic forms are
7-ketocholesterol, 7β-hydroxycholesterol, and 5β,6β-epoxycholesterol,
which have prominent cytotoxic properties and have been implicated in
various pathological states [12,13,15]. Among the naturally occurring
oxysterols, 7β-hydroxycholesterol and 7-ketocholesterol have been re­
ported to be highly toxic to a number of tumor and normal cells,
including those of the vascular wall [16]. Additionally, it has been
observed that oxysterols generated by autoxidation are elevated by up to
45-fold in men with hypercholesteremia, which can then be reduced
after simvastatin treatment [17].
Previously, we determined that coffee consumption increases the
antioxidant capacity of plasma in healthy subjects without altering
clinical parameters such as the lipid profile and vascular function [18].
Therefore, we analyzed the impact of coffee consumption on urinary
oxylipins, which are well-recognized markers of inflammation and
oxidative stress in vivo, and found a significant reduction [19]. Our
hypothesis in the current study is that coffee consumption regulates the
production and generation of oxysterols and specific lipid species asso­
ciated with cardiovascular risk and atherogenesis. Thus, we examined
the changes in the concentrations of fatty acids (free (FFAs) and total
(TFAs)), sterols (cholesterol, cholesteryl esters, and desmosterol), and
oxysterols by using gas chromatography-mass spectrometry (GC-MS) on
plasma samples collected from a previous randomized controlled trial in
which subjects consumed coffee for eight weeks [18]. Since several
lipids that have been studied in human plasma samples are implicated in
inflammation and atherogenesis [20–23], we studied a subset of those
lipids in macrophages exposed to oxidized LDL (oxLDL) and evaluated
the effects of 11 typical antioxidants derived from coffee consumption.
2. Materials and methods
2.1. Design of the study
The plasma samples used in the current study were obtained from
healthy subjects who consumed coffee in a controlled, parallel clinical
trial [18]. The purpose of the original investigation was to determine the
effects of coffee consumption on the lipid profile, vascular function, and
plasma antioxidant capacity. Therefore, the sample size was calculated
based on differences in plasma antioxidant capacity (22 ± 12 μmol
Fe2+/L using ferric reducing antioxidant power (FRAP)) [24] and on
changes in LDL (17.1 ± 18.8 mg/dL). A total of 25 subjects were needed,
for a power of 0.8 and a significance level of 0.05. The design of the
study is presented in Fig. S1. Before randomization, all potential vol­
unteers were classified according to age (category 1: 20–40 or category
2: 41–60 years old), BMI (adequate: 18,5–24,9 or overweight: 25,0–29,
9 kg/m2) and sex (female or male). Then volunteers were randomly
assigned to three study groups matched by age, sex, and body mass index
(BMI). Two intervention groups drank one of 2 types of coffee (400
mL/day) (coffee A: 787 mg/day CGAs and coffee B: 407 mg/day CGAs)
for 8 wk while the control group did not consume coffee.
Inclusion criteria: men or women between 20 and 60 years old; BMI
between 18.8 and 30 kg/m2; regular coffee drinker, at least 300 mL/d;
nonsmoker; physical activity of less than 10 h/week; no history and/or
diagnosis of chronic disease; not currently consuming any medications
(lipid-lowering, antioxidant dietary supplements, anticonvulsants,
anti–inflammatory steroids, hypnotics, or caffeine); and a maximum
alcohol consumption of 10 g/day for women and 15 g/day for men.
Vegetarians, high-performance athletes, and pregnant and nursing
women were excluded. The clinical trial was registered in the Interna­
tional Clinical Trials Registry Platform, WHO primary registry
(RPCEC00000168) and was approved by the CES University Ethics
Committee (Act 47; project 142; May 16th, 2012).
One week before beginning the study, all participants had a washout
period. During the washout period and the eight weeks of intervention,
all participants avoided tea, dark chocolate, red wine, cocoa-derived
products, herbal infusions, berries, soy, antioxidant supplements, and
foods and beverages with caffeine and naturally high antioxidant levels.
The consumption of caffeine-containing drugs was forbidden. The coffee
beverages consumed during the study were provided at the workplace
and brewed at home on the weekend.
Anthropometric assessment and biochemical tests: Weight, height, and
waist circumference were measured at the beginning and end of the
intervention. Specialized personnel were standardized by using inter­
nationally accepted equipment and techniques [25]. BMI and waist
circumference values were calculated and classified according to WHO
criteria [26]. Plasma TC, HDL cholesterol, and triglycerides (TGs) were
measured with cholesterol oxidase/peroxidase, cholesterol HDL direct,
and TG glycerol phosphate oxidase/peroxidase commercial kits

346
O.J. Lara-Guzmán et al.
(BioSystems S.A.). The LDL value was calculated [27]. The concentra­
tion of oxLDL in plasma was determined by ELISA (Mercodia kit;
Uppsala, Sweden) at 450 nm using a Synergy HT Multi-Mode microplate
reader (BioTek Instruments Inc., Winooski, USA). The oxLDL results are
reported as U/L.
Sample collection: Venous blood samples were taken after a 12 h
overnight fast and centrifuged for plasma separation. Plasma samples
were obtained using EDTA as an anticoagulant by centrifugation at
1200×g for 15 min at 4 ◦ C and were stored at − 80 ◦ C until analysis.
2.2. Chemicals
Analytical grade solvents were purchased from J.T. Baker (Phillips­
burg, New Jersey, USA). The fatty acids methyl ester (FAME) mix con­
taining 37 components was provided by Supelco (Bellefonte, PA, USA).
The internal and external standards nonadecanoic acid (C19:0), meth­
ylnonadecanoic acid, coprostanol, desmosterol, cholesterol, cholesteryl
esters and BSTFA + TMCS 1% reagent for derivatization were supplied
by Sigma–Aldrich (St. Louis, Missouri, USA); cholesterol nonadecanoate
(Cho-C19:0), 4β-hydroxycholesterol (4β-OHC), 7α-hydroxycholesterol
(7α-OHC), 7β-hydroxycholesterol (7β-OHC), 7-ketocholesterol (7-KC),
24(S)-hydroxycholesterol (24(S)–OHC), 25-hydroxycholesterol (25-
OHC), and 27-hydroxycholesterol (27-OHC) were purchased from
Avanti Polar Lipid (Alabaster, Alabama, USA); and 4α-hydrox­
ycholesterol was provided by Bertin Pharma (Montigny le Bretonneux,
Yvelines, FR). A Zebron ZB-5MSi 5 m guardian GC column (30 m × 0.25
mm × 0.25 μm) was purchased from Phenomenex (Torrance, California,
USA); an HP-5MS GC column (30 m; 0.25 mm id; 0.25 mm) was pur­
chased from Agilent (Santa Clara, CA, USA); and a CPTAB triglyceride
analysis GC column (25 m, 0.25 mm i.d., film thickness 0.1 μm) was
purchased from J&W Scientific (Santa Clara, CA, USA).
Chlorogenic and phenolic acids: Ferulic acid (FA; 98.5%; CAS number
1135-24-6), 4-caffeoylquinic acid (4-CQA; 99.8%; CAS number 905-99-
7), 5-caffeoylquinic acid (5-CQA; 99.3%; CAS number 906-33-2), 3,4-
dicaffeoylquinic acid (3,4-diCQA; 98.9%; CAS number 14534-61-3),
3,5-dicaffeoylquinic acid (3,5-diCQA; 99.2%; CAS number 2450-53-5),
and 4,5-dicaffeoylquinic acid (4,5-diCQA; 98.2%; CAS number 57378-
72-0) were obtained from Biopurify Phytochemicals., Ltd. (Chengdu,
Sichuan, CN). Caffeic acid (CA; 98.5%; CAS number 331-39-5), iso­
ferulic acid (iFA; 90% CAS number 537-73-5), 4-coumaric acid (CoA;
90%; CAS number 7400-08-0) and 3-caffeoylquinic acid (3-CQA; 99%;
CAS number 327-97-9) were obtained from Extrasynthese S.A. (Lyon,
Auvernia-Ródano-Alpes, FR). Dihydroferulic acid (DHFA; 98.6%; CAS
number 1135-23-5) was purchased from TCI America, Ltd. (Tokyo,
Kanto, JP). Compound stock solutions were prepared at a concentration
of 20 mM in DMSO and stored at − 20 ◦ C.
2.3. Coffee beverages
The coffee beverages used in this study were prepared by dripping
followed by filtration through filter paper (6 g/100 mL of hot water).
The coffee used was Colombian Arabica variety, produced under light
(coffee A) and dark (coffee B) roasting processes (Colombian industry
Colcafé S.A.S.). Ground coffee was stored in laminate packaging with a
nitrogen–modified atmosphere until preparation, and the chemical
characterizations are presented in Table S1.
2.4. Macrophage foam cell model
LDL isolation and oxidation: The detailed procedure has already been
published [28]. Briefly, LDL was purified from plasma by a discontin­
uous density gradient using a Beckman XL-100 ultracentrifuge (Brea,
California, USA) and desalted by ultrafiltration with an Amicon Ultra
0.5 mL Ultracel 3 K device (Tullagreen, Cork, IRL). Concentrated LDL
was diluted in PBS, filtered (0.22 μm, sterile), and stored at 4 ◦ C until
use. The protein concentration was measured using a Pierce BCA Protein
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Free Radical Biology and Medicine 176 (2021) 345–355
Assay Kit (Pierce, Rockford, Illinois, USA). The oxLDL used in this
experiment was obtained after LDL oxidation with 5 μM CuSO4 at 37 ◦ C
for 6 h.
Macrophage culture and treatment: THP-1 monocyte (ATCC TIB-
202™) culture and macrophage differentiation were described by Lara-
Guzmán et al. [28]. To assess changes in the lipid profile, macrophages
were seeded and treated in 12-well plates at a density of 1 × 106
cells/well and exposed to three different concentrations of oxLDL or LDL
(12.5, 25, and 50 μg/mL). To assess the effects of phenolic compounds
on the cell lipid profile, the cells were pretreated with 1 μM of each
compound (CA, FA, iFA, 4-CoA, DHFA, 3-CQA, 4-CQA, 5-CQA, 3,
4-diCQA, 3,5-diCQA, and 4,5 diCQA) for 12 h. Then, the cells were
exposed for an additional 12 h to 25 μg/mL oxLDL. The negative control
was vehicle (<0.05% DMSO), and the positive control was oxLDL in
vehicle.
2.5. Sample preparation
Plasma extraction and derivatization: Samples were handled according
to the method proposed by Griffiths and Wang [29] and modified ac­
cording to our laboratory conditions. Briefly, 200 μL plasma aliquots
were pipetted into 2 mL vials, and then 50 μL of internal standards were
added for sample normalization as follows: nonadecanoic acid (10
μg/mL in hexane) for FFAs; methyl nonadecanoic acid (10 μg/mL in
hexane) for TFAs; coprostanol (10 μg/mL in chloroform) for cholesterol,
and C19:0 cholesteryl ester (10 μg/mL in hexane) for CEs [29]. The
mixtures were vortexed for 30 s the total lipids were subsequently
extracted using degassed organic solvents (chloroform and methanol,
stored at − 20 ◦ C) to prevent lipid oxidation. Then, 100 μL of chloroform
was added, and the samples were vortexed for 30 s for cold extraction
(6 ◦ C). An additional 100 μL of methanol was added, and the samples
were vortexed for 60 s. The samples were centrifuged at 9464×g for 10
min at 4 ◦ C. Then, the supernatant of each sample was recovered and
stored in a microcentrifuge tube. The pellets were washed by two suc­
cessive extractions with 100 μL of chloroform/methanol (2:1) and
centrifuged at 9464×g for 10 min at 4 ◦ C. Finally, the three supernatants
were combined in glass vials for chromatography, the solvent was
evaporated to dryness under a nitrogen stream (UAP 5.0), and the res­
idues were dissolved in 500 μL of conditioned chloroform using a
Hamilton precision microsyringe (lipid extract, LEx), homogenizing the
sample and taking aliquots as described.
Total fatty acids: FAMEs were quantified by transesterification ac­
cording to the method included in Supelco Bulletin 909 A [30]. Briefly,
200 μL of each lipid extract was evaporated to dryness under a nitrogen
stream, 600 μL of 2.0% H2SO4 (v/v) was added to methanol (HPLC
grade) followed by heating at 70 ◦ C for 2 h and cooling at room tem­
perature. Next, 50 μL of saturated NaCl was added to each solution and
vortexed. Subsequently, the samples were extracted with three portions
of hexane, first with 500 μL and then twice with 400 μL (2 × 400 μL).
The hexane supernatants were combined and evaporated under a ni­
trogen stream. Finally, the residues were dissolved in 100 μL of hexane
and transferred to vials with glass inserts for analysis by GC/MS.
Free fatty acids: Samples were silylated according to the Supelco
procedure, with some modifications before GC analysis [30]. Briefly,
100 μL of each lipid extract was dried under nitrogen prior to derivati­
zation. The residues were first dissolved in 100 μL of pyridine/acetoni­
trile (50:50), then added to 50 μL of BSTFA + TMCS 1% reagent and
heated for 2 h at 70 ◦ C. Finally, the derivatized samples were pipetted
into chromatography vials with glass inserts for GC/MS analysis.
Cholesterol: Fifty microliters of each lipid extract was diluted to 150
μL with chloroform and used directly for analysis. The samples were
transferred to chromatography vials with glass inserts for and injected
directly into the gas chromatography-mass spectrometer according to a
previously reported method [31].
Cholesteryl esters (ChoEs): Sample mining and instrumental parame­
ters were based on the method proposed by Son HH et al. and adapted to
O.J. Lara-Guzmán et al.
our laboratory conditions [32]. Briefly, 50 μL of lipid extracts diluted to
100 μL with chloroform were used for analysis. The samples were
transferred to chromatography vials with glass inserts for direct injec­
tion and analysis by GC/MS.
Desmosterol and total oxysterols: The silylation procedure and
instrumental parameters for chromatography and mass spectrometry
were adapted from previous references [31,33]. Briefly, 200 μL plasma
aliquots were added to 1 mL of 0.7 M potassium hydroxide in methanol,
and alkaline hydrolysis was performed at room temperature for 2 h with
sporadic vortexing. Finally, the pH of the samples was adjusted using
100 μL of 3 M phosphoric acid and 100 μL of saturated NaCl was added,
followed by vortexing and centrifugation at 9464×g for 10 min. Samples
were then extracted with three 200 μL portions of conditioned hexane
(centrifugation at 9464×g for 5 min at 4 ◦ C was used to improve the
formation of the interface after each extraction step). The three hexane
supernatants were pipetted into chromatography vials and evaporated
to dryness at room temperature in a rotary evaporator (Centrivap,
Labconco; Kansas City, MO, USA). The residues were dissolved in 100 μL
of dichloromethane/pyridine (50:50). Finally, derivatization was per­
formed for 3 h at 60 ◦ C by adding 50 μL of BSTFA + TMCS 1%, and the
samples were transferred to chromatography vials with glass inserts for
GC/MS injection and analysis.
Macrophages: After exposure to oxLDL and phenolic acids, macro­
phages were washed twice with 500 μL of PBS. The cell monolayers were
detached from the wells using 300 μL of 0.2% EDTA for 15 min at 37 ◦ C,
and cells were collected and transferred into a new vial. Then, 100 μL of
0.05% BHT in PBS was added to each well to recover all of the cells,
which were added to their corresponding vial for storage at − 80 ◦ C until
analysis. For lipid extraction, the Folch method was used with some
modifications [34]. Briefly, each sample was mixed with 320 μL of BHT
(5% in MeOH) and 640 μL of CHCl3 for 20 min with constant agitation in
a vortexer. Then, 50 μL of the internal standards were added and mixed
as previously described for plasma sample preparation. After that, 150
μL of type II water was added for incubation under constant agitation for
10 min. The samples were then centrifuged at 2000×g for 5 min, and the
aqueous phases were collected for extraction with an additional portion
of 250 μl of CHCl3:MeOH (2:1). The organic phases were combined in
glass vials for chromatography and evaporated to dryness. The dried
residues were dissolved in 500 μL of chloroform and processed for
GC/MS analysis as described above for FAMEs, cholesterol, desmosterol,
and oxysterols in plasma.
2.6. Gas chromatography/mass spectrometry analysis
One microliter of the prepared samples was injected into an Agilent
7890 gas chromatography (GC) System (Wilmington, Delaware, USA)
equipped with a 5975C mass spectrometer (MS) (Wilmington, Delaware,
USA) and a CTC Combipal 3 autosampler in splitless mode. The pa­
rameters of the chromatographic system for each group of metabolites
are shown in Table S2. The mass spectrometer was tuned during all
experiments; signal acquisition for identification was performed in full
scan mode, and quantification was performed in single-ion monitoring
(SIM) mode. Dwell times during data acquisition for the quantifier and
identifier ions were optimized for better sensitivity (Table S3). The
temperatures of the ionization source and quadrupole were 230 ◦ C and
150 ◦ C, respectively. The electron impact ionization energy was 70 eV.
The chromatographic peaks were examined for homogeneity using the
extracted ions of the characteristic fragments to optimize resolution and
peak symmetry. The signals of the standard compounds and analytes in
each matrix were evaluated in terms of their identity. In addition, the
Kovats retention indices (KIs) were estimated for the analytes with an
acceptable chromatographic resolution, as in the case of esterified and
free C18 fatty acids, applying a standard mixture of alkanes (C6–C40)
using NIST AMDIS 2.68 software. Data analysis for quantification was
performed using a MassHunter WorkStation (Agilent; Santa Clara, CA).
The concentrations of metabolites are expressed according to the nature
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Free Radical Biology and Medicine 176 (2021) 345–355
of the matrix (plasma: ng/mL and macrophages: ng/106 cells).
2.7. Quantitation
The signal of each analyte was deconvoluted according to the ions
acquired in SIM mode. The chromatographic areas of the calibrators and
samples were normalized to the area response of their internal stan­
dards, as shown in Table S2. Calibration curves were constructed from
six concentrations each in triplicate on three different days to control
variability and establish a tolerable level for precision according to our
methods and laboratory conditions. Each curve was fitted by linear
regression, and the Pearson correlation coefficients were evaluated. The
homogeneity of the response factors was also assessed. In all cases, the
quantitative performance in the selected range was acceptable. Free
fatty acids were semiquantified using the response factor of the internal
standard to compare the experimental variations and the response of the
metabolites in the samples, so their values correspond to equivalents of
C19:0.
2.8. Statistical analysis
The quantitative variables age, sex, waist circumference, and BMI of
the volunteers are described with measures of central tendency and
dispersion (means ± SD). The normality of the continuous variables in
each group at baseline was evaluated with the Shapiro–Wilk test for one-
way analysis of variance (ANOVA). P values ≤ 0.05 were considered
statistically significant. To evaluate the effects of coffee consumption, a
dataset with clinical and lipidomic data at 2 time points (baseline and 8
weeks) from the 3 treatment groups (control, coffee A, and coffee B) was
constructed, as was the lipidomic data for the foam cell model. All
variables in the datasets were scaled and normalized (z-score) prior to
multivariate analyses. Lipidomic analysis was performed by principal
component analysis (PCA) using the Factoextra and FactoMineR pack­
ages in R to extract and visualize the output of exploratory multivariate
data analyses [35,36]. Additionally, the linear Pearson correlations
among variables were calculated and visualized as a correlation matrix
for each pair of variables using the corrplot package in R. Analyses were
performed with R-statistical open source software (Rx64 version 4.0.5, R
Foundation for Statistical Computing, Vienna, Austria; URL http://
www.R-project.org/). To evaluate the differences in blood chemistry
and lipid variables between the treatment groups, nonparametric tests
were applied. The Mann–Whitney U test was applied for unpaired
analysis of the change (Δ = 8 weeks-baseline) among the intervention
groups, while the Wilcoxon test was used for paired analysis. Each entry
labeled with * differs significantly (*p < 0.05, **p < 0.01, ***p < 0.001
and ****p < 0.0001). Finally, heatmaps were generated for comparison
between intervention groups at baseline and at 8 weeks, and the changes
were visualized using GraphPad Prism version 8.3.1 for Windows
(GraphPad Software, Inc., San Diego, California, USA). To evaluate the
effects of the treatments on macrophages, ANOVA was performed using
GraphPad Prism. P values ≤ 0.05 were considered statistically signifi­
cant. When differences were observed, Dunnett’s and Tukey’s multiple
comparison tests were used. The employed code and the database for
statistical lipidomic analyses are available on GitHub (https://github.
com/Vidarium/Lipidomic_Coffee_Antioxidant).
3. Results
Except for one individual who had alterations in their lipid profile at
baseline, all volunteers completed the study. The characteristics of the
subjects in each group at baseline are described in Table 1. No caffeine
was detected in the plasma samples in the control group at baseline or
eight weeks after intervention [18].
O.J. Lara-Guzmán et al. Free Radical Biology and Medicine 176 (2021) 345–355

Table 1
General characteristics of the subjects at baseline.
Groups Control (n = Coffee A (n Coffee B (n P
25) = 24) = 25)

BMI, kg/m2 (x‾ ± SD) 23.88 ± 24.45 ± 23.85 ± 0.67a

2.67 2.89 2.47
Waist circumference, cm 77.85 ± 79.33 ± 78.02 ± 0.81a
(x‾ ± SD) 7.94 8.88 9.98
Age, y (x‾ ± SD) 38.04 ± 38.00 ± 38.04 ± 0.99a
8.74 8.87 9.99
Men 13 12 12 0.91b
Women 12 12 13
a
Mean comparisons test (ANOVA).
b
Chi square test.

3.1. Lipidomic analysis at baseline in healthy adults

A total of 46 lipids were quantified and grouped into two categories:

fatty acids, both FFAs and TFAs, and sterols, including cholesterol, des­
mosterol, cholesteryl esters (ChoEs), and oxysterols. Fig. 1A summarizes
the lipids evaluated, their classification, and origin (e.g., de novo or from
diet) at baseline; this figure also describes the enzymes and other me­
diators involved in fatty acid biosynthesis [37] as well as oxysterol
biosynthesis by specific cytochrome oxidases (cyps) or reactive oxygen
species (ROS) [38]. The analysis included five additional variables: the
total levels of saturated (SFAs), monounsaturated (MUFAs), and poly­
unsaturated (PUFAs) fatty acids, the total ChoEs, and the ratio of
ChoEs/cholesterol. The lipids evaluated by GC/MS were compared with
conventional clinical assays for total cholesterol (TC), LDL, VLDL, HDL,
and triglycerides (TGs), as well as different cardiovascular risk indices
(LDL/HDL and TC/HDL or atherogenic index (AI)) [39], BMI, WC, and
plasma oxLDL levels. The lipidomic dataset was first explored by prin­
cipal component analysis (PCA), in which the first and second PCs
accounted for 31.7% and 13.5% of the variance, respectively (Fig. 1B
and C). The PCA score plot (Fig. 1B) does not show any tendencies in the
projection of each participant, and the centroids reveal a similar dis­
tribution within the groups. The PCA loading plot (Fig. 1C) shows that
TFAs had the highest quality representation (or correlation) for PC1,
while most of the sterols, oxysterols, FFAs and clinical variables had the
highest quality for PC2. Moreover, analysis of the distance measure­
ments regarding the (dis)similarity in the lipidome among all partici­
pants (Fig. S2A) showed a homogeneous distribution of subjects within Fig. 1. Lipidomic and clinical variables at baseline. (A) A simplified scheme
the groups. These data indicate that at baseline, the participants had including all lipid species evaluated in this study. Total fatty acids (TFAs), free
good distribution in each group in terms of lipid species. At the begin­ fatty acids (FFAs), sterols, and oxysterols are described. Black circles represent
ning of the study, no significant differences were observed between the lipid species and classes from de novo biosynthesis in animals, while white
circles represent those from diet. Biosynthetic mediators: Delta 5, 6, and 9
control group and the intervention groups (Fig. S3).
desaturases (Δ5-d, Δ6-d, and Δ9-d) and elongase (E.) add two carbons (+2C) to
The correlation network at baseline, including all lipids and clinical
the fatty acid chain, whereas cytochrome oxidase (cyp) and reactive oxygen
variables, showed specific and significant correlations (Fig. 1D) (posi­
species (ROS) oxidize cholesterol. The green background and intensity reveal
tive correlations: r2 > 0.3, colored in red). The color intensity indicates the degree of the positive correlation between lipid variables captured by the
the degree of correlation. We observed that most of the clinical variables correlation network. For principal component analysis, the score (B) and
related to CVD risk had a high positive correlation (Fig. 1D). Negative loading plots (C) (PC-1 and PC-2) show the participants and variables,
correlations between HDL and several CVD risk variables (AI, BMI, LDL/ respectively. B shows the control group (gray, n = 25), coffee A (red, n = 24),
HDL, TGs and WC) were also observed (Fig. S4). TFAs and nonoxidized and coffee B (yellow, n = 25). (C) Variables with high quality of representation
sterols had a closer association with the clinical variables than oxy­ are labeled in dark brown. Significant variables are represented in PC1 and PC2,
sterols and FFAs. and their correlations are shown in Table S4 (D). The correlation network of
The oxysterols 7-KC, 7α-OHC, 7β-OHC, 24(S)–OHC, 25-OHC, and 27- variables from all participants (n = 74); red lines represent positive correlations
and blue lines represent negative correlations (r2 > 0.3), while the size of each
OHC, all associated with CVD risk factors [21,38], showed a high cor­
cluster reflects the number of variables with a significant correlation. (For
relation and were clustered together. Additionally, these oxysterols were
interpretation of the references to color in this figure legend, the reader is
strongly correlated with free arachidonic acid (AA), another risk in­ referred to the Web version of this article.)
flammatory mediator. In contrast, 4α-OHC and 4β-OHC, which have not
been associated with CVD risk [21,38], clustered independently and
3.2. Lipidomic analysis among the control and intervention groups after 8
were associated with free palmitic and stearic acids (Fig. 1D).
weeks of coffee consumption

Pronounced differences in the lipidomes of the control and inter­

vention groups were observed. Compared to the baseline, most of the
FFAs and oxysterols increased in the noncoffee drinker control group,

349
O.J. Lara-Guzmán et al.
while the total ChoEs and ChoEs/cholesterol ratio decreased; the
opposite behavior was found in the groups that drank coffee (Fig. 2A).
Similarly, at the end of the intervention, the plasma levels of 14 oxy­
sterols and FFAs were significantly downregulated, whereas ChoEs were
upregulated in coffee drinkers compared to noncoffee drinkers (Fig. 2B).
The oxysterols (μg/mL of plasma) for the three groups, at baseline and 8
weeks after the intervention, are also presented in Fig. S5.
Next, PCA was used to explore data patterns in the lipidome, as
shown in Fig. 2C. The two first principal components (PCs), accounting
for 22.3% and 16.5% of the variance, respectively, were included in a
loadings and scores biplot to summarize the data. TFAs had high quality
of representation in PC1, while oxysterols, ChoEs, PUFAs, MUFAs and
most of the FFAs were best summarized in PC2. In contrast to the oxy­
sterols and most FFAs that were positively associated with PC2, ChoEs,
PUFAs, and MUFAs showed a negative correlation with this component.
Clearly, the centroids of Groups A and B were clustered together and
were distinguished from the control group based on the lipids repre­
sented in PC2 (oxysterols and FFAs). Moreover, in the lipidome
dissimilarity matrix among all participants (Fig. S2B), the coffee drinker
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Free Radical Biology and Medicine 176 (2021) 345–355
groups (A and B) and the noncoffee drinker control group formed two
distinguishable clusters with opposite lipidomic profiles. In general,
these results show that coffee restriction leads to increased levels of
circulating oxysterols and FFAs, which are significantly reduced with
coffee consumption.
Fig. 3 shows the comparative correlation matrix that includes the
clinical variables and plasma lipid levels (Δ = 8 W-Baseline) for the
control group (lower triangle) and the groups consuming the coffee
beverages (upper triangle). Importantly, in the control noncoffee
drinker group, most of the SFAs had a positive significant correlation
with TGs, VLDL, and AI, while MUFAs and PUFAs had either a negative
correlation or no correlation with these clinical variables. Additionally,
oxysterols and several FFAs had a high correlation among each other in
the control group (Fig. 3; lower triangle), and AA was strongly corre­
lated with most oxysterols. This response was different in the groups that
consumed coffee (Fig. 3; upper triangle), in which a reduction in oxy­
sterols and FFAs was observed to be clustered in two highly correlated
groups: the first included 24(S)–OHC, 25-OHC, 27-OHC, 7β-OHC and
AA, and the second included 4α-OHC, 4β-OHC and 7α-OHC, 7-KC and
Fig. 2. Comparison of the lipid species between
the control and intervention groups. (A) Heatmap
of the plasma lipid levels in the participants. Each
row represents the mean of the Z-score for each var­
iable, and each column represents the baseline (BL)
and 8-week (8 W) values for each group. (B) Heatmap
displaying the changes in the lipidomes after 8 weeks.
The values of each feature were plotted on a red–blue
color scale; a red tile indicates an increase, and blue
indicates a decrease. The scale from 1 to − 1 repre­
sents the distance in standard deviation in which a
value departs from the general mean (in A) or median
(in B). Tiles labeled with * differ significantly from
baseline after Wilcoxon test analysis for A, while a
Mann–Whitney test was used for B (*p < 0.05, **p <
0.01, ***p < 0.001 and ****p < 0.0001) compared
with the control group. (C) Principal component (PC1
and PC2) score and loading biplot of the changes in
lipid levels (Δ = 8 weeks-baseline) for the different
lipidomes explaining 38.8% of the variance; control
group (gray, n = 25) and groups consuming coffee
beverages (coffee A: red, n = 24; and coffee B: yellow,
n = 25). The variables that correlated significantly (p
< 0.001) with PC1 or PC2 are described in Table S5.
Variables are represented as full black circles,
whereas the quality of the representation is labeled as
shades of brown. Total fatty acids (TFAs). Free fatty
acids (FFAs). Total cholesterol/HDL (or atherogenic
index (AI)). (For interpretation of the references to
color in this figure legend, the reader is referred to
the Web version of this article.)
O.J. Lara-Guzmán et al.
stearic acid.
Fig. S6 shows an additional simplified scheme that summarizes the
impact of coffee restriction and coffee consumption at the end of 8 weeks
of intervention on all 46 lipids evaluated in the plasma from healthy
subjects; lipid groups, lipid species and levels of correlation are also
illustrated.
3.3. Phenolic compounds from coffee regulate the accumulation of fatty
acids and oxysterols in foam cells
The degree of alteration in the levels of specific fatty acids and ste­
rols, including cholesterol, desmosterol and oxysterols, as well as their
regulation by antioxidants, was determined in macrophages treated
with 12.5, 25, and 50 μg/mL oxLDL for 12 h (Fig. 4A and Fig. S7). In the
PCA loading/score biplot, PC1 and PC2 accounted for 41.5 and 32.9% of
the variance, respectively (Fig. 4A). Regarding the loadings, most of the
oxysterols as well as the total contents of palmitic, stearic, and oleic
acids had the highest correlation and quality of representation in PC1,
while 25-OHC among the total content of arachidic acid had the highest
correlation and quality of representation in PC2. In contrast, the total
contents of arachidonic and myristic acids showed a negative correlation
with PC1. Comparison of the lipid compositions of LDL and oxLDL is
presented in the first two columns of the heatmap in Fig. S7A. OxLDL
had higher concentrations of 4α-OHC and 24(S)–OHC and lower con­
centrations of linoleic acid, cholesterol and 25-OHC than the LDL par­
ticles. The score projections (treatments) in Fig. 4A also show that
compared to the controls (basal and LDL), exposure of macrophages to
oxLDL induced higher levels of all oxysterols while SFAs (palmitic,
stearic and arachidic acids) and oleic acid increased only slightly; PUFAs
(linoleic and arachidonic acids) were reduced and displayed signifi­
cantly lower levels than those found in the macrophages exposed to LDL.
351
Free Radical Biology and Medicine 176 (2021) 345–355
Fig. 3. Correlation between clinical lipid vari­
ables and the change in lipid levels in the con­
trol and intervention groups (Δ ¼ 8W-Baseline).
Correlation matrix comparing the control group (n
= 25; lower triangle) and the groups consuming
coffee beverages (coffees A and B, n = 49; upper
triangle). Significant positive Pearson’s correlations
(p < 0.01) are colored red, whereas negative cor­
relations are presented in blue. Total fatty acids
(TFAs). Free fatty acids (FFAs). Total cholesterol/
HDL (or atherogenic index (AI)). (For interpretation
of the references to color in this figure legend, the
reader is referred to the Web version of this article.)
One aspect to highlight from this PCA is that the oxysterols showed a
high positive correlation with SFAs and oleic acid, while AA had a
negative correlation with most of the oxysterols.
The effects of phenolic acids from coffee on cellular lipid production
was evaluated using the oxLDL-macrophage interaction model (Fig. 4B
and Fig. S7B). The dataset was reduced into two principal components,
PC1 and PC2, which accounted for 43.5 and 27.9% of the variance,
respectively, as shown in the loading/score biplot (Fig. 4B). Regarding
the loadings, the samples formed two distinguishable clusters based on
PC2; the oxysterols 4α-OCH, 7-KC, 24(S)–OHC, 25-OCH and 27-OCH
were positively correlated with this PC, in contrast to 7β-OHC and AA,
which were negatively associated. The score projections in the biplot
(treatments) revealed that exposure of the oxLDL-induced macrophages
to a subgroup of compounds represented by CA, FA, iFA, DHFA, 3-CQA
and 4-CQAs resulted in lower levels of most of the oxysterols compared
with the positive control (C+; oxLDL-induced macrophages). In
contrast, diCQAs, 5-CQA, and 4-CoA less effectively reduced oxysterol
production in oxLDL-activated macrophages (Fig. 4B). Complementa­
rily, the heatmap in Fig. S7B summarizes the changes (up or down)
induced by each phenolic compound evaluated.
4. Discussion
This is the first study to evaluate 46 lipid species, including plasmatic
fatty acids, sterols, and oxysterols, some of which are atherogenic risk
factor markers, after coffee consumption in a healthy population.
Additionally, the impact of the most representative phenolic compounds
from coffee on the regulation of these factors was evaluated using an
oxLDL-macrophage atherogenic cell model.
O.J. Lara-Guzmán et al.
Fig. 4. Regulation of lipid species in foam cells by the phenolic com­
pounds from coffee. (A) Principal component score/loading biplot (PC1 and
PC2) explaining 74.4% of the variance in the lipidomes dataset from macro­
phages treated with 12.5, 25, and 50 μg/mL LDL or oxLDL. The large circles
represent the centroid of each population (gray: basal; yellow: LDL; red:
oxLDL); lipid species with high representation are labeled with dark brown and
indicate the variables that contributed the most to the variance explained. (B)
PCA score/loading biplot (PC-1 and PC-2) explaining 71.4% of the variance for
the different lipidomes from macrophages exposed to oxLDL (25 μg/mL; posi­
tive control, C+) and treated with coffee antioxidants. Caffeic (CA), ferulic
(FA), isoferulic (iFA), dihydroferulic (DHFA), 4-coumaric (CoA), caffeoylquinic
(3-CQA, 4-CQA and 5-CQA) and dicaffeoylquinic (3,4-diCQA, 3,5-diCQA and
4,5-diCQA) acids. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
4.1. Lipidome status at baseline
As expected, most of the clinical variables associated with CVD risk
(TC, LDL, VLDL, TGs, WC, oxLDL, BMI, AI, and LDL/HDL) were posi­
tively intercorrelated, and some (TGs, AI, LDL/HDL, BMI and WC) dis­
played a negative correlation with HDL, as presented in the correlation-
based network in which four nodes were clearly identified (Fig. 1D).
Among the most interesting cluster nodes, that represented by 7-KC, 7α-
OHC, 7β-OHC, 24(S)–OHC, 25-OHC, and 27-OHC, which are oxysterols
that have been frequently associated with cardiovascular risk [21,38],
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Free Radical Biology and Medicine 176 (2021) 345–355
were highly correlated with free AA. Both oxysterols and free AA can
affect many cellular functions and influence various physiological pro­
cesses, such as inflammation and atherogenic events, including foam cell
formation and plaque establishment and rupture [21,40]. Oxysterols
activate cells, mainly macrophages into the arterial wall [41], while the
free AA released during inflammation can be oxidized by enzymes or
free radicals, leading to the formation of oxylipins, which are also
proinflammatory mediators [28,40,42]. Both oxysterols and oxylipins
are closely involved in atherogenesis and are considered biomarkers of
CVD risk [38,42]. The correlation node characterized by most of the
FFAs and 4α- and 4β-OHCs [21,38] was highly correlated with free
stearic acid (Fig. 1A and D). 4α-OHC and 4β-OHC have not been asso­
ciated with CVD risk [21,38], and stearic acid appears to have little
proinflammatory activity [43]. Last, the correlation nodes of TFAs and
ChoEs that were close to the clinical lipid profile (e.g., CT, VLDL, TGs)
could be explained by the fact that lipoproteins are enriched with these
lipid species [44]; however, the analysis of cholesterol molecules did not
reveal a correlation with the clinical variables (Fig. 1D). In summary, the
baseline results showed the presence of plasmatic oxysterols and free
AA, which have been associated with CVD risk. However, it is important
to highlight that the population in the study was healthy; therefore, their
clinical variables were within the reference intervals for the overall
population. The lipidomic profiles of the subjects at baseline confirmed
good randomization prior to allocation into intervention groups due to
the diversity and homogeneous distribution of the lipidomes in each
group (Fig. 1B and Fig. S2A).
4.2. Lipidomic response to coffee intake suggests potential cardiovascular
benefits
After coffee consumption, relevant changes and associations in the
lipidome occurred (Fig. 2C). On the one hand, the contents of free fatty
acids and oxysterols increased in the control group and decreased in the
coffee-consuming groups and were the main discriminating features in
the PCAs (Fig. 2C). Although little is known about the effects of coffee
intake on FFA plasma levels, a previous study showed that plasmatic
FFAs and TGs decreased after one month of coffee consumption (600
mL/day) [6]. Although FFAs serve as an energy source, elevated con­
centrations of circulating FFAs are related to inflammation, oxidative
stress, and CDV risk [45,46]. Moreover, elevated plasmatic concentra­
tions of FFAs has been associated with coronary artery disease [8] and
heart failure [47], and is considered a marker to predict the risk of
developing atrial fibrillation [48]. Free AA has an important role in
inflammation since it can be further oxidized, yielding a variety of ei­
cosanoids related to CVD risk [42]. Furthermore, some metabolomic-
and lipidomic-based studies have reported an inverse association be­
tween coffee consumption and the plasmatic concentration of other
lipids relevant to inflammation and oxidative stress, such as lysophos­
phatidylcholines (LPCs) (16:1, 18:1, 20:4, 22:1, and 22:2) [5,6,49],
showing the possible beneficial effects of coffee beverages in CVD by
reducing the circulating levels of potentially harmful lipids. A possible
explanation for the increment of the FFAs and oxysterol levels in the
control group, could be that the subjects included in this study were
habitual coffee consumers. The restriction of coffee consumption in the
control group, could have a negative impact on the redox balance status
and lead the induction of the pathways related to cholesterol oxidation:
free radicals and enzymatic (Cytochrome P450) [14]. We previously
observed that the restrictions in the control group increase oxylipins,
other oxidized lipids associated with CVD risk [19].
Coffee consumption has also been positively associated with an
improvement in the antioxidant capacity of plasma [18] and a reduction
in the circulating levels of oxylipins [3]. In fact, we recently provided
clinical and in vitro evidence that coffee consumption, as well as its
phenolic compounds, regulate oxylipin levels [19]. Importantly, the
studies focused on the positive effects on health, like ours, have used
filtered coffee, a preparation method that reduces the content of cafestol
O.J. Lara-Guzmán et al.
and kahweol, two diterpenes associated with the increment of TC [50].
In the present study, the high correlation (at baseline and the change
after 8 weeks of coffee drinking) between the plasma levels of free AA
and oxysterols related to CVD risk (Figs. 1D and 3A) indicates an
interesting biological association. The decrease in AA in response to
coffee consumption [6] and the evidence suggesting that oxysterols
induce the cellular release of AA and eicosanoids [41,51] could explain,
in part, the possible effects exerted by the phenolic compounds from
coffee that have been observed in different studies, including ours.
It is important to note that not all FFAs have the same inflammatory
potential and their effects depend, in part, on their chemical structure.
For instance, some saturated FAs are able to stimulate inflammatory
gene expression, but their potency varies with chain length; e.g., lauric
acid displays the greatest activity via TLR4, whereas myristic and stearic
acids have little proinflammatory activity [43]. Here, at baseline and
after intervention, free stearic acid was highly correlated with oxysterols
that have not been associated with CVD (Figs. 1D and 3A).
Finally, cholesteryl esters with palmitic, oleic, and linoleic acids and
the ChoEs/Chol ratios were elevated in the groups that consumed coffee
but decreased in the control group at the end of the intervention (Fig. 2B
and C). Previous reports indicate that the ChoEs/Chol ratio in plasma is
significantly lower in subjects with CVD [52]; therefore, increasing the
levels of ChoEs could be related to a favorable outcome. Until now, only
one study has shown that coffee intake decreased the concentration of a
specific cholesteryl ester (with a 20:4 acid) [6], which is in contrast with
our results, probably due to considerable differences in the study design,
including a three-stage clinical trial and the volume of coffee con­
sumption; moreover, they profiled the lipidome of serum from partici­
pants with an elevated risk of type 2 diabetes.
4.3. Phenolic compounds from coffee decrease oxysterols and regulate AA
oxLDL-treated macrophages
The presence of oxysterols in the vasculature is associated with all
phases of atherosclerosis development and is recognized as a biomarker
of CVD risk [21,42]. Furthermore, emerging evidence suggests that
particular oxysterols play a major role in the initiation and progression
of foam cells [53]. In our study, when THP-1 macrophages were acti­
vated with oxLDL, the levels of cellular oxysterols increased, and the
levels of AA and linoleic acid decreased (Fig. 4A and Fig. S7A). It is well
documented that oxLDL is a proinflammatory and oxidative inductor
that promotes the formation of oxylipins and oxysterols from AA and
cholesterol, respectively, leading to their accumulation in macrophages
[28,53]. This can also be observed in the PCA presented in Fig. 4A. The
total content of esterified AA was negatively correlated with most oxy­
sterols, which suggests that oxidation and inflammation induce oxy­
sterol production and oxidative AA modifications. On the other hand,
desmosterol, a lipid known to have regulatory properties on inflamma­
tion and lipid metabolism [54], was upregulated in macrophages after
exposure to oxLDL. This result is similar to that from a previous report
related to oxysterol induction in foam cells [54], in which cell survival
pathways were activated against a harmful signal, in this case, oxLDL.
Finally, when macrophages were treated with phenolic compounds
prior to the interaction with oxLDL, we observed that the biological
activity depends, in part, on the molecular structure of the polyphenol
(Fig. 4B, S7B and S8). Almost all phenolic compounds, except for 4-CoA,
3-CQA and 4-CQA, led to a decrease in most of the oxysterols but
increased 7β-OHC. In contrast, 7β-OHC was inhibited by the di-CQAs 5-
CQA and 4-CoA. Additionally, with the exception of caffeic acid, the
reduction of oxysterols by antioxidants was closely related to the in­
crease in esterified AA, which suggests its protection (e.g., deester­
ification and/or oxidation). Indeed, we previously observed that most of
these antioxidants decrease the contents of oxylipins in foam cells [19].
On the other hand, our results show that most antioxidants decreased
cholesterol accumulation in macrophages (Fig. S7B), which is in line
with a previous report showing that 3-CQA, caffeic acid and ferulic acid
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Free Radical Biology and Medicine 176 (2021) 345–355
decreased lipid accumulation and stimulated cholesterol efflux from
RAW264.7 macrophages exposed to oxLDL [55]. There is also evidence
that 3-CQA reduces the atherosclerotic lesion area and the plasma levels
of total cholesterol, triglycerides, LDL, and inflammatory markers in
ApoE-deficient mice [55]. Taken together, these results suggest that
phenolic compounds from coffee beverages have antiatherogenic ef­
fects, at least in part, through mechanisms that involve inhibition of
foam cells, cholesterol efflux, AA esterification, and oxysterol reduction.
The reduction of oxysterols and FFAs in plasma after coffee con­
sumption help to explain the potential role of coffee antioxidants
regulating lipid metabolism and markers of inflammation, which have
clinical relevance due to lower concentration of circulating oxysterols
and FFAs are inversely associated with atherogenesis and CVD risk
factors [12,13]. Importantly, our research contributes to explain why
moderate coffee intake is associate with positive cardiovascular benefits
found in observational studies [2,3]. The major strength of this study is
the analytical strategy, which was based on combined targeted lip­
idomic analysis with classical biomarkers to identify the effects of
phenolic compounds from coffee on a wide variety of lipids related to
CVD risk using both in vitro and in vivo approaches.
5. Conclusions
This study showed that the contents of oxidized sterols and FFAs
decreased as a result of exposure to phenolic compounds from coffee in
two different models, suggesting a potential favorable impact of coffee
consumption on the regulation of lipid metabolism and markers related
to oxidation and inflammation with respect to CVD risk. In addition, we
showed the lipid response after the consumption of two types of coffee
with different concentrations of chlorogenic acids for the first time; in
this case, the effect was not dose-dependent in terms of antioxidants.
Furthermore, we outlined additional evidence that improved the
comprehension of lipid changes during foam cell formation and
demonstrated that typical antioxidants from coffee may regulate the
production of oxysterols and FFAs. In summary, we illustrated the
complex way in which the production of FFAs (e.g., arachidonic acid)
and oxysterols are related and regulated and their possible interactions
with the phenolic compounds from coffee in the context of
atherogenesis.
Acknowledgments
The authors thank the volunteers who participated in the clinical
trial. The authors are grateful for the support of COLCIENCIAS (current
Colombian Ministry of Science, Technology and Innovation) through the
grant no. 528-2011 for doctoral students.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.freeradbiomed.2021.10.012.
Author contributions
KM-D designed the clinical trial and conducted the research; KM-D,
OJL-G and RA-Q designed the lipidomic study; RA-Q performed the
lipidomic analyses and quantification of lipid variables; and OJL-G
optimized the experimental conditions of the cell model and analyzed
the lipidomic datasets. All authors wrote and approved the final
manuscript.
Author disclosure statement
KM-D and OJL-G are researchers at the Vidarium, Nutrition, Health
and Wellness Research Center, Nutresa Business Group. RA-Q states that
he has no conflicts of interest.
O.J. Lara-Guzmán et al. Free Radical Biology and Medicine 176 (2021) 345–355

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