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Prolonged monitoring of postprandial lipid metabolism after a western meal rich in linoleic acid (LA) and carbohydrates

Journal: Applied Physiology, Nutrition, and Metabolism

Manuscript ID apnm-2018-0798.R2

Manuscript Type: Article

Date Submitted by the Author: 20-Feb-2019

Complete List of Authors: Shokry, Engy; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreRaab, Roxana; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreKirchberg, Franca; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreHellmuth, Christian; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreKlingler, Mario; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreDemmelmair, Hans; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreKoletzko, Berthold; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical CentreUhl, Olaf; Ludwig-Maximilians-Universitat Munchen, Division of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical Centre

Keyword: meal challenge, metabolism, linoleic acid, postprandial, very long-chain saturated fatty acids (VLCSFA), polyunsaturated fatty acids (PUFA)

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Applied Physiology, Nutrition, and Metabolism

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Prolonged monitoring of postprandial lipid metabolism after a western meal rich in

linoleic acid (LA) and carbohydrates

Engy Shokry, Roxana Raab, Franca F. Kirchberg, Christian Hellmuth, Mario Klingler,

Hans Demmelmair, Berthold Koletzko*, Olaf Uhl

LMU - Ludwig-Maximilians-Universität München, Division of Metabolic and

Nutritional Medicine, Dr. von Hauner Children’s Hospital, University of Munich Medical

Centre, Munich, Germany

Co-authors’ email addresses:

Engy Shokry: [email protected]

Roxana Raab: [email protected]

Franca F. Kirchberg: [email protected]

Christian Hellmuth: [email protected]

Mario Klingler: [email protected]

Hans Demmelmair: [email protected]

Berthold Koletzko: [email protected]

Olaf Uhl: [email protected]

* Correspondence: Dr. Berthold Koletzko, Prof. Of Paediatrics, LMU - Ludwig-

Maximilians-Universität München, Dr. von Hauner Children’s Hospital, LMU Medical

Center, Campus Innenstadt, Lindwurmstr. 4, D-80337 Munich, Germany.

Tel: +49 89 44005 2826, Fax: +49 89 44005 7742, E-mail: [email protected]; [email protected]

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mailto:[email protected]





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Abstract: Today, increased awareness has been raised regarding high consumption of

n-6 polyunsaturated fatty acids (n-6 PUFA) in western diets. A comprehensive analysis

of total and individual postprandial fatty acid (FA) profiles would provide insights into

metabolic turnover and related health effects. After an overnight fast, nine healthy adults

consumed a mixed meal composed of 97 g carbohydrate and 45 g fat of which 26.4 g was

linoleic acid (LA). Non-esterified fatty acids (NEFA), phospholipid fatty acids (PL-FA)

and triacylglycerol fatty acids (TG-FA) were monitored in plasma samples, at baseline

and hourly over 7 h postprandial. Total TG-FA concentration peaked at 2h postprandial

followed by a constant decline. LA from TG18:2n-6 and behenic acid from TG22:0

showed the highest response among TG-FA, with a biphasic response detected for the

former. PL-FA exhibited no change. Total NEFA initially decreased reaching nadir at 1h,

then increased reaching its maximum value at 7h. The individual NEFA showed the same

response curve except LA and some very long chain saturated fatty acids (VLCSFA, ≥

20 carbon chain length) that markedly increased shortly after the meal intake. The

similarities and dissimilarities in lipid profiles between study subjects at different time

points were visualized using non-metric multidimensional scaling (NMDS). Overall, the

results indicate that postprandial levels of LA and VLCSFA either as NEFA or TG were

most affected by the test meal, which might provide an explanation for the health effects

of this dietary lifestyle characterized by high intake of mixed meals rich in n-6 PUFA.

Key words: meal challenge, metabolism, linoleic acid, postprandial, very long-chain

saturated fatty acids (VLCSFA), polyunsaturated fatty acids (PUFA).

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Introduction

Fatty acid (FA) trafficking across the tissues is highly dependent on the nutritional state

(Hodson and Fielding 2010). In the fasted state, activated lipolysis leads to a net flow of

non-esterified FAs (NEFA) from the adipose tissue to peripheral tissues to serve as an

energy source (Hodson and Fielding 2010). On the other hand, after a meal, especially

following a high-fat meal, major changes occur in FA trafficking as a response to the

large influx of meal fat. Dietary fat is mainly composed of triacylglycerols (TG), which

after digestion and absorption appear in the circulation as chylomicron-TG (CM-TG)

(Snook et al. 1996). The adipose tissue plays a key role in buffering postprandial flux of

dietary fat into the circulation as it suppresses the release of NEFA into the circulation,

increases plasma TG clearance, and increases fat reuptake (Frayn 2002). This clearance

is largely mediated by the action of lipoprotein lipase (LPL), which hydrolyzes CM-TG,

releasing FA which are subsequently taken-up primarily by the adipose tissue. Therefore,

the net trafficking of FA in the postprandial state is directed towards storage rather than

release. This is one of the metabolic pathways used to limit the increase in plasma lipids,

thus protecting other tissues from exposure to excessive lipid levels (Hodson and Fielding

2010). The FA composition of the meal is an important factor affecting the absorption,

transport, and uptake by adipose tissue of dietary fat, thus it can influence the duration,

magnitude, and composition of postprandial lipemia, both qualitatively and quantitatively

(Tumova et al. 2016). This is important to note, as sequential meal intake (< 5 h) is

common among western communities, and means that most individuals spend most of

the day in the postprandial state (Leech et al. 2015).

In the majority of the previously conducted postprandial studies, large, calorie dense and

high-fat test meals (≈1300 kcal and 60% fat) were used for monitoring postprandial lipid

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levels (Brandauer et al. 2013; Harrison et al. 2009; Katsanos et al. 2004; Koutsari et al.

2001a, 2001b; Peddie et al. 2012; Peluso et al. 2012; Schwander et al. 2014; Tsetsonis et

al. 1997). The problem with large high-fat test meals is, that at very high doses of dietary

lipids, there is no clear dose dependence of postprandial hypertriglyceridemia on the fat

load. However, a dose-dependent increase has been reported for mixed meals (650-950

kcal) with moderate intake of fat (30-60g, 50-65% of total energy content) and a

significant proportion of carbohydrates (50-100g, 25-40% of total energy content) to

ensure the effective release of insulin (Zhang et al. 1998). These parameters were used to

design the test diet in the current study. The composition of macronutrients in the meal

used is typical for a meal consumed by many individuals in western communities on a

daily basis, according to reports released by UN Food and Agriculture Organization

(FAO) (Roser and Ritchie 2018). Specifically, the test diet was rich in linoleic acid (LA,

NEFA18:2n-6), which is the most frequently consumed polyunsaturated fatty acid

(PUFA) in the western diet and is found in virtually all commonly consumed foods. LA

is an essential fatty acid (EFA), which cannot be synthesized de novo by humans.

Therefore, it can be used as a reliable biomarker of dietary intake by tracking its

movement within the endogenous plasma pool. According to the existing literature,

adipose tissue and plasma PUFA, specifically LA and alpha-linolenic acids (ALA) were

found to be the best indicators of dietary intake (Baylin et al. 2002; Hedrick et al. 2012).

On these bases, the objective of the present work was to introduce a hypothesis-free

explorative study addressing the question of real-life postprandial individual FA response

following a typical western meal, containing moderately high fat (45g, ≈45% of total

energy content) and energy contents (≈905kcal). The present study also monitored other

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PUFA including arachidonic acid, the precursor of leukotrienes and prostaglandins which

play a role in inflammation.

Materials and methods

Participants and sample collection

Briefly, nine adults (3 males and 6 females) initially participated in the study. The study

participants did not suffer from any endocrine, gastroenterological or glucose metabolic

disorders, renal or liver failure, and had not undergone any bariatric surgeries or

procedures. Medical history of the study participants was obtained based on self-reported

data before inclusion in the study. The study was approved by the Ethics Committee of

the Medical Faculty, Ludwig-Maximilians-Universität München (LMU) (251-10) and

written informed consent was obtained from all participants. Later, one female participant

was excluded from further investigation because of missing blood sampling at three time

points of the study. Six of the remaining 8 study participants were normal-weight (NW)

(18.50≥BMI≤24.99) and 2 were overweight (OW) or preobese (25.00≥BMI≤29.99)

(WHO 2000) with an overall mean body mass index (BMI; in kg/m2) of 21.0 ± 3.0. All

participants showed normal baseline concentrations of HDL-cholesterol (HDL-c) with a

median of 1.8 mol/m3. Three of the 8 participants showed borderline high baseline LDL-

cholesterol (LDL-c) but normal LDL-c/HDL-c ratios with overall medians of 3.1 mol/m3

and 1.7, respectively. Only one participant (#1) presented borderline high values of TG,

LDL-c and LDL-c/HDL-c ratios with values of 1.8 mol/m3, 3.5 mol/m3, and 3.9,

respectively.

The subjects were asked to avoid alcohol consumption and strenuous physical activity for

24 h preceding the study, to minimize environmental influences on metabolism. Weight

was assessed in a standardized manner wearing light clothes and no shoes. After an

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overnight fast of 12 h, participants consumed a standardized breakfast meal consisting of

a muffin, a chicken sandwich and a glass of orange juice. The energy content of the meal

was 3785 KJ (≈905kcal), provided as 26 g, 45 g, and 97 g of protein, fat, and

carbohydrates, respectively. A detailed overview of the meal composition is given in

Table 1. Most of the total fat content in the meal (42 g, 93.3%) was provided by sunflower

oil. The total FA composition of sunflower oil was determined as described by Drzymała-

Czyż et al. (2017) using a 25 μl oil sample and the results are presented in Table 2. Both

the chicken sandwich and the muffin were rich in PUFA and MUFA, represented

principally by LA and OA. The meal was consumed within 20 minutes. Fasting blood

samples were collected right before the ingestion of the meal and blood samples were

taken hourly over 7 h postprandial. Blood was collected via venipuncture from antecubital

vein in 7.7 ml ethylenediamine tetraacetic acid (EDTA) monovettes (Sarstedt,

Nümbrecht, Germany). Collected blood samples were immediately centrifuged (1000xg,

10 min, 4°C), or kept on ice and centrifuged within 2 h. Samples were stored at -80 °C

until analysis.

Non-esterified fatty acid (NEFA) analysis

NEFA in plasma were analyzed by liquid chromatography (1200 Agilent, Santa Clara,

USA), coupled to triple quadrupole mass spectrometry (4000 QTRAP, Sciex,

Framingham, USA) as described previously (Hellmuth et al. 2012). In short, proteins

were precipitated by adding isopropanol (200 μl) to 20 μl of serum in a 96-deep-well

plate. After centrifugation, 10 μl of the supernatant were injected with an eluent flow rate

of 700 μl/min. Gradient elution was performed with eluent A (5 mM ammonium acetate

and 2.1 mM acetic acid) and eluent B (acetonitrile with 20% isopropanol) on Pursuit UPS

Diphenyl column (1.9 μm, 100 x 3.0 mm; Varian, Darmstadt, Germany) at 40°C. All

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samples were measured within one batch, and the quality of the measurements was

assessed with quality control (QC) samples. Three QC samples were analyzed with the

study samples. NEFA with a coefficient of variation (CV %) > 10% in the QC samples

were excluded from analysis. Absolute baseline and postprandial concentrations were

calculated in µmol/L then converted to mol/m3 (standard international (SI) unit).

Fatty acid (FA) analysis of phospholipids (PL) and triacylglycerols (TG)

PL and TG were extracted from plasma according to a modified Folch procedure with

chloroform/methanol (2:1, v/v). Lipid fractions were separated by thin layer

chromatography (TLC) and fatty acid methyl esters (FAME) were synthesized at room

temperature by adding 50 μl of sodium methoxide (25% weight in methanol, Sigma

Aldrich). The transesterification reaction was stopped after 3 minutes by adding 150 μl

of 3 M methanolic HCl. FAME were extracted twice in 600 μl of hexane. Then, the

extracts were combined, evaporated under nitrogen, and re-dissolved in 40 μl hexane.

Individual FAME were quantified by gas chromatography with flame ionization detection

(GC-FID) as µmol/L (Agilent 5890 series II, Waldbronn, Germany) using a BPX 70

column (25 m × 0.22 mm, 0.25 µm film, SGE, Weiterstadt, Germany).

Statistical analyses

Data were analyzed using Excel 2010 and R version 3.3.1. Concentration data for the

individual FA were provided as absolute postprandial concentrations. To calculate the

relative concentrations, postprandial concentrations were divided by the respective

baseline values. Areas under the curves (AUC) were calculated using the relative values.

Concentrations and AUC are provided in Supplementary Table 1 (Apnm-2018-

0798suppla). Non-metric multidimensional scaling (NMDS) using Bray–Curtis

dissimilarity index was performed in the R vegan package to visualize the clustering of

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samples, based on concentration data of the measured FA species at the different time

points (0-7 h postprandial) (Oksanen 2018). A NMDS plot was performed to show the

spatial distribution of samples collected from participants at different time points in the

study settings or “scores plot” using the measured species (both at baseline &

postprandial) (Fig. 1) and significant differences were tested by PERMANOVA, using

Adonis methodology. Adonis function in R vegan package implements a multivariate

analysis of variances using distance matrices. It partitions dissimilarities for the sources

of variation and uses permutation tests to inspect the significances of those partitions. In

Adonis, a R2 value (effect size) is computed showing the percentage of variation

explained by the supplied category, as well as a p-value indicating the statistical

significance (Oksanen 2018).

Time versus concentration data were plotted collectively for the three main lipid classes

(TG, NEFA, PL) (Fig. 2) as well as for their individual subclasses (saturated fatty acids

(SFA), very long chain saturated fatty acids (VLCSFA), PUFA, and MUFA) (Fig. 3-5).

Error bars were not included in Fig. 3-5 due to the high overlap between the curves.

Results

To examine the effect of a mixed meal rich in LA on a broad spectrum of FA, plasma

concentrations were assessed at baseline (before the meal intake) and hourly over a period

of 7 h in healthy volunteers. Detailed demographic characteristics and baseline metabolic

data of the study participants are provided in Supplementary Table 2 (Apnm-2018-

0798supplb). Each subject provided 8 blood samples (from 8 time points). In total, 44

NEFA, 27 TG-FA and 26 PL-FA were quantified.

Non-multidimensional scaling (NMDS) plot

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NMDS ordination plots of Bray-Curtis dissimilarities between samples taken from 8

participants at 8 time points demonstrated significant clustering (p=0.001, R2=0.285).

Eight Clusters of samples corresponding to the 8 time points of the experiment were

identified in the NMDS plot. Clusters corresponding to baseline, 5, 6, and to a lesser

extent 7 h postprandial were ordinated close to one another (Group A). The same applied

to those at 1, 3 and to a lesser extent 2 h postprandial (Group B). A single cluster

corresponding to 4 h postprandial showed almost a similar spatial distance to groups A

and B, thus ordinated midway between the 2 groups (Fig. 1).

Total lipid response

A concentration-time course plot for the medians of the total FA of the investigated lipid

species (TG, PL, and NEFA) is presented in Fig. 2. The plot shows similar profiles of the

lipid species at baseline and 5 h postprandial. The total PL-FA did not respond to the meal

challenge in contrast to the TG-FA which peaked at 2 h postprandial, then steadily

decreased reaching baseline levels by 6 h and nadir by 7 h after ingestion of the test meal.

Total NEFA followed the expected postprandial fall and rise pattern. The median total

NEFA concentration decreased to nadir by 1 h following the meal challenge, then steadily

increased reaching baseline and highest values at 5 and 7 h postprandial, respectively.

Total fasting NEFA levels ranged from 0.17 to 0.79 mol/m3 with a median of 0.33 mol/m3.

Triacylglycerols (TG) response

The majority of individual TG-FA followed the total TG concentration-time course

pattern with different magnitudes. LA from TG18:2n-6 and behenic acid from TG22:0

showed the highest response to the meal challenge with 0-7 h AUC values of 13.22 and

21.27, respectively. Additionally, a biphasic response was detected for LA from

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TG18:2n-6 at 2 and 6 h (Fig. 3). AUCs (0-7 h) of all other TG-FA ranged between -0.76

(TG16:1n-7) and 4.78 (TG24:0).

Non-esterified fatty acids (NEFA) response

By examining the concentrations of individual NEFA, the highest baseline levels were

detected for NEFA18:1 with a median of 0.14 mol/m3. NEFA18:2, NEFA16:0, and

NEFA18:0 also showed high baseline concentrations. After meal ingestion, the majority

of individual NEFA showed the same concentration-time curve observed for total NEFA

with initial suppression followed by a continuous rise to highest levels, reached by the

end of the study. Characteristically, LA did not show the initial decrease suffered by the

majority of other NEFA (≤ 1 h). It directly started to increase till 4 h, decreased at 5 h

concomitant with the increase in the other NEFA, then continuously increased reaching

the highest value at 7 h after the meal intake. Other NEFA, including NEFA22:0,

NEFA24:0, and NEFA26:0 also did not decrease shortly after meal ingestion, but

markedly increased reaching the highest values at 2-3 h. This was followed by a slow

decreasing trend and a kink by 5 h, after which the concentrations rather stagnated (Fig.

4). The observations seen from the curves are also reflected in the 0-7 h AUCs with values

of 30.31, 19.72, 10.04, and 6.83 obtained for NEFA22:0, NEFA24:0, NEFA26:0, and

NEFA18:2, respectively.

Phospholipids (PL) response

No substantial changes from baseline levels were seen for individual PL-FA

concentrations in response to the test meal over a period of 7 h (Fig. 5). AUC were in the

range of -0.87 (PL18:3n-3) to 0.65 (PL22:2n-6).

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Discussion

The present study investigated the effect of the composition of a typical western meal on

levels of different circulating FA in TG, PL, and NEFA fractions, unlike the majority of

the previously conducted postprandial studies, in which often only the total NEFA

concentration has been determined (Jackson et al. 2005). Using a test meal rich in LA

helped in interpreting the findings since it is a reliable biomarker useful to track the

ingested lipid within the endogenous plasma pool. This was reflected in the study results

which demonstrated that LA in addition to VLCSFA either as NEFA or TG were the most

affected by the test meal, where they not only showed the highest response but also a

different behaviour from the other individual species. The study also allowed monitoring

of the residual lipid profile usually subjected to the second meal effect by using a time

course of 7h postprandial in the study design. The residual lipid profile is the amount of

fat left-over after the assimilation of nutrients of the previous meal usually taking place

within 5-6 h after the meal consumption, thus produces an additive effect with fat from

the next meal consumed (Woerle et al. 2003). Elevation of the residual lipid profile might

indicate potential health risks, since it is associated with accumulation of lipids in plasma

and extended hyperlipidemia, triggering coronary artery diseases (CAD) and

cardiovascular incidents (Bell et al. 2008; Tushuizen et al. 2005; Garber 2012). At 7h

postprandial, LA and behenic acid as TG18:2n-6 and TG22:0, respectively were the only

TG-FA that did not return to their baseline values and the same applied to the total NEFA,

especially LA which reached their highest response at 7h. Interestingly, the study was

also able to detect the interindividual differences in postprandial lipid responses which

were related to the corresponding baseline levels according to results of NMDS analysis.

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In detail, the NMDS plot showed a similarity in the lipid responses at baseline with the

late postprandial period (5-7 h postprandial), where samples at these time points were

ordinated close to each other (Fig. 1). Additionally, it was observed, that samples obtained

from participant #5, especially at time point 4, 5, 6, 7 h, and to a lesser extent at 1 h, 3 h,

and baseline, were ordinated further away from the rest of the study participants’ samples,

indicating a different profile. The same applied to participant #1 and #7, especially among

samples collected in the late postprandial period (6 h and 7 h). Inspection of the raw data

available in Supplementary Table 2 (Apnm-2018-0798supplb) showed that participant #5

and #1 have the lowest and highest baseline TG concentrations, respectively (especially

TG18:2n-6, TG18:1n-7, TG18:1n-9, and total TG concentrations). They also maintained

similar behaviour of these TG species postprandially at 1, 4, 6 and 7 h in comparison to

corresponding samples from other study participants. Samples from participant #7 at time

points 0, 4, 6 and 7 h were also relatively distant from the samples of the rest of the study

participants. This participant showed higher baseline and postprandial TG concentrations

relative to participant #5, however lower than the rest of the study participants. These

observations provide information on the correlations between baseline and postprandial

TG concentrations, especially at the lowest and highest ranges. Previous studies have

demonstrated that fasting hypertriglyceridemia displayed exaggerated and prolonged

triglyceride responses and vice versa, which indicates a close correlation between

postprandial and baseline blood TG concentrations (Tiihonen et al. 2015). This elevated

TG response, especially in the late postprandial phase (5–8 h) was linked to increased risk

of CVD (Patsch et al. 1992). Interestingly, according to the present findings, participants

#5 & 7 with the lowest TG values (baseline & postprandial) showed the lowest LDL-

c/HDL-c ratios while participant #1 with the highest TG values showed the highest LDL-

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c/HDL-c ratios. LDL-c/ HDL-c ratio was reported as a risk indicator for CVD (Kunutsor

et al. 2017).

Regarding the individual concentration-time course curves of the three lipid classes

involved, they showed a decrease in NEFA in the immediate postprandial period (≤ 1h),

with a corresponding increase in TG, which typically reached its peak at 2 h and no

significant change in PL (Fig. 2). This is considered a normal response, since during

fasting; adipose tissue tends to release FA into the systemic circulation in order to supply

tissues with a high requirement for FA, thus causing high fasting plasma NEFA

concentrations. Shortly after meals, the adipose tissue metabolism starts to deal with the

increased concentration of the plasma TG resulting from entry of chylomicron-TG (CM-

TG) into the circulation (Hunter et al. 2001). However, it should not be excluded that the

hepatic very low-density lipoproteins (VLDL) might also partially explain the increase in

TG in the postprandial period, although CM are better substrates for LPL than VLDL

(Xiang et al. 1999, Heller et al. 1993). The total plasma TG typically increased to reach

a broad peak at 2 h before decreasing gradually due to clearance from the circulation (Fig.

2). This was found in agreement with reports on the monophasic response of the mean

concentrations of plasma TG postprandially, but in contrast with other studies

demonstrating biphasic response in the first 6 h period after a high fat meal (Cohn et al.

1988; Heller et al. 1993; Kashyap et al. 1983; Olefsky et al. 1976; Peel et al. 1993;

Williams et al. 1992). Interestingly, in this study, a biphasic response was detected in

TG18:2n-6 comprising LA, the most abundant FA in the test meal (Fig. 3b). TG18:2n-6

showed 2 peaks at 2 h and 6 h postprandial. The early sharp peak at 2 h corresponded to

the early rise in plasma TG concentrations due to the first entry of CM-TG containing LA

into the circulation (Bysted et al. 2005; Hodson et al. 2009; Yli-Jokipii et al. 2003).

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Ockner et al. (1969) demonstrated that the increase in exogenous postprandial TG-LA

levels is entirely a result of the increase in CM-TG with no increase in VLDL-TG. In

other reports, the TG fatty acid composition of CM in the systemic circulation was

consistently found to resemble that of the dietary fat consumed (Hodson and Fielding

2010). The delayed peak at 6 h might be attributed to the carry-over effect, suggesting the

release of preformed CM retained either in enterocytes (primary site of synthesis) or

lymph. (Zhu et al. 2009). This hypothesis is supported by the fact that LA was discovered

to form large sized bulky CM, which partially reside in the enterocytes before being

released by lymphatic drainage. In the literature, the size of CM produced by LA was

compared to palmitic acid (NEFA16:0), a SFA. It was found that both FA formed CM of

significantly different sizes corresponding to >800 angstroms (Å) in diameter for TG18:

2n-6 versus 300 to 800 Å for TG16:0, thus showing that the latter resulted in particles

similar to the smaller VLDL (Ockner et al. 1971, 1972). Carlier et al. (1991) reported that

LA produced an increase in both the size and proportion of CM, which may cause a

stepwise release. Thus, this could possibly provide an explanation for the delayed peak

detected for TG18:2n-6 at 6 h. Negative health impacts were related to the formation of

large CM particles (800-3600 Å) because of their role as the primary cause of formation

of atherosclerotic lesions (Kocmur 2017). It could also be that the second peak was no

longer confined to CM but might represent newly synthesized VLDL-TG.

This biphasic response of TG18:2n-6 could be also attributed to the consumption of a

mixed meal rich in carbohydrates where a high carbohydrate content might have led to a

rapid rate of gastric emptying and fat digestion which was absorbed in two waves

(Zampelas et al. 1998). This hypothesis is further supported by a similar finding

previously reported on another exogenous fat (alpha-linolenic acid, ALA), which showed

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a biphasic pattern in both its TG and NEFA fractions after consuming a meal rich in

carbohydrates (136g) with a fat intake of 0.5 g fat/kg body weight comprising ALA in

rapeseed oil (12 g/100 g) (Shishehbor et al. 1998). Overall, these findings provide

evidence that consuming a mixed meal of high carbohydrates with a fat meal could cause

increased residence time and reduced clearance of postprandial TG in the late stage of

postprandial phase, especially CM-TG which increase its atherogenicity (Bravo et al.

2010).

In the present study, TG18:2n-6 along with TG22:0 were the only two TG-FA that that

did not return to the baseline values at 7 h postprandial, unlike the other measured

individual TG-FA, that returned back to their baseline values or below baseline at the end

of the study (Fig. 3b & 3d). It is also worth mentioning, that at 2 h, the TG comprising

VLCSFA (≥ 20 carbon chain length) showed a relatively high increase from their baseline

values compared to the other individual TG species (Fig. 3d).

The plausible explanation is the relatively high concentration of these TG in the meal,

which are absorbed and transported as CM to the lymphatic circulation. Some reports do

not consider diet as a major source of these FA inside the body (USDA 2014). However,

hydrogenated fats or partially hydrogenated vegetable oils, commonly found in processed

food, could represent an unusual dietary source of VLCSFA (Ajmi 2005). These

VLCSFA were suggested to be products of desaturation and elongation of trans fatty

acids (TFA) from hydrogenated oils in the human body (Ajmi 2005). Hydrogenated fat

has also been previously recognized as a source of VLCSFA based on studies performed

on rats fed a diet with partially and totally hydrogenated fish oils (Granlund et al. 2000).

Additionally, increased levels of serum TG of arachidic (20:0) and behenic (22:0) acids

were found in rats fed a diet rich in VLCSFA (Ajmi 2005). Some human studies found

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that the proportions of behenic acid (22:0) and lignoceric acid (24:0) in PL and cholesterol

esters (CE) increased by n-6 PUFA consumption compared with SFA, while arachidic

acid (20:0) exhibited no change (Lauritzen and Hellgren 2015).

In agreement with previous literature, the total NEFA concentration was initially

suppressed after meal ingestion followed by a steady increase to values above baseline

levels (Bickerton et al. 2007; Dubois et al. 1998; Griffiths et al. 1994; Jackson et al. 2005).

The initial decrease was attributed to insulin driven inhibition of lipolysis in adipose

tissue and subsequent suppression of NEFA release into plasma (Fielding 2011). The

following rising part of the NEFA curve likely represented an interplay between (1) spill-

over of FA into plasma caused by the ineffective uptake of FA released from circulating

TG (especially after a fat-rich meal) by the adipose tissue (Evans et al. 2002; Fielding et

al. 1996), (2) FA release from adipose tissue by lipolysis, and (3) uptake of NEFAs into

peripheral tissues (Fielding 2011).

In contrast to the general behaviour observed in the majority of NEFA, LA showed a

characteristic profile, since its concentration did not decrease initially unlike the majority

of NEFA (≤ 1 h). Several hypotheses could be suggested to provide an explanation for

this finding. One of which is the spill-over effect, which accounts for 40-50% of the total

plasma NEFA pool in the postprandial period. NEFA18:2n-6 might be affected to a large

extent by this effect, as it represented the most abundant FA in the test meal (64.59% in

sunflower oil) (Fielding 1996). It could also be that the high baseline concentration of

this NEFA dampened the effect on its plasma levels. Another hypothesis is, that LA has

the ability to bypass the lymphatic pathway and went directly to the portal circulation.

Previous reports demonstrated that an important proportion of PUFA, especially LA and

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ALA might pass through the enterocyte cytosol and go directly to the portal venous flow

(Mc Donald and Weidman 1987; Ramı´rez et al. 2001; Thomson et al. 1989).

LA is assumed to be preferentially taken-up by the adipose tissue because it is the

principal FA in the test meal and postprandial TG. Therefore, the unexpected increase in

its plasma levels in the early postprandial period would imply a decreased capacity of its

entrapment within the adipose tissue. This hypothesis was generated based on earlier

reports on the close correlation between FA composition of the adipose tissue TG and

diet in the early postprandial period (2-4 h) (Hynes et al. 2003; Summers et al. 2000).

This means that FA derived from LPL-mediated CM-TG hydrolysis are more likely to be

taken-up by the adipose tissue than FA from the circulating NEFA pool. This assumption

is further supported by the sudden decrease of LA at 5 h concomitant with the increase in

the other NEFA because, at this time point, lipolysis reached its maximum and the uptake

load of the adipose tissue was spared. It should not be disregarded that this pattern was

also obtained in a previous study where participants consumed a meal high in

carbohydrates and fat in the form of rapeseed oil, as mentioned earlier. ALA, similarly,

showed a characteristic “surge-null-surge” response for both plasma TG and NEFA

fractions with no clear explanation (Shishehbor et al. 1998).

Another class of FA that stood out regarding their postprandial response were VLCSFA,

namely: NEFA22:0, NEFA24:0, and NEFA26:0), one very long chain monounsaturated

fatty acid (VLCMUFA, NEFA26:1), and two very long chain polyunsaturated fatty acids

VLCPUFA (NEFA26:2 and NEFA26:3) (Fig. 4). Even though baseline concentrations of

these FA were very low, the extent to which these FA responded was not expected and

did not seem to be proportional to their initial concentrations.VLCSFA (NEFA22:0 and

NEFA24:0) showed a biphasic pattern where they showed two maxima and two minima

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at 3 h and 5 h and similar to NEFA18:2, they showed initial increase in their levels shortly

after meal intake and did not return to the baseline at any time point during the experiment

(Fig. 4b & 4d). Moreover, the magnitude of their response was very high relative to

NEFA18:2n-6, in spite that the latter is the major component of the meal. This could

suggest different magnitudes of spill-over effect, resulting from selective uptake of PUFA

relative to VLCSFA, either by the liver or the adipose tissue or both. In fact, this finding

is difficult to be directly interpreted since the plasma levels of NEFA are governed by

different factors, including passive diffusion along a concentration gradient and

facilitated diffusion by transfer proteins. These factors may be affected by concentration,

chain length, saturation/unsaturation, as well as competition between different FA,

particularly in case of saturation. Previous studies have demonstrated that in case of the

competition of chain lengths, the shorter chain length is preferred and in case of equal

chain length, the more unsaturated FA are preferred. Based on that, in the current

situation of a high PUFA meal especially LA, when saturation of adipose tissue occurs,

spill-over will show much stronger influence on the less polar VLCSFA (longer chain

length and with no double bonds) than the more polar PUFA (Abumrad et al. 1998).

Furthermore, it should not be neglected that the higher magnitude of response in VLCSFA

relative to LA, could be due to their very low baseline values in comparison to high

baseline values for LA, as shown in Supplementary Table 1 (Apnm-2018-0798suppla).

In spite of the novelty of these results, it is difficult to relate them to potential health

effects. In the literature, VLCSFA in plasma were associated with a favourable metabolic

pattern and a reduced risk for diabetes and coronary heart diseases (Forouhi et al. 2014;

Lemaitre et al. 2015; Malik et al. 2015). Conversely, excess serum VLCSFA were

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identified as hepatotoxic substances which induce lipoapoptosis and liver damage (Malhi

et al. 2006).

In general, it was monitored that the plasma NEFA concentrations were higher at the end

of the study period than at baseline which could also be explained by the so-called

“rebound” effect of spill-over FA, which were less easily up-taken by the adipose tissue

against a concentration gradient, i.e. when the adipose tissue lipolysis is higher at the end

of the postprandial state (Evans et al. 2008; Miles and Nelson 2007). It could also be

speculated, that physical activity during the study period resulted in these elevated NEFA

levels since the majority of energy used by muscles is derived by NEFA from the adipose

tissue during moderate physical activity (Frayn 2010). The subjects were advised to stay

in the examination room, but they were allowed to move and work in a sitting position

(e.g. on computers). This activity consumes obviously more energy than overnight

sleeping, thus the higher NEFA levels might have resulted from the increased energy

demand. Overall, both prolonged postprandial hypertriglyceridemia and elevated NEFA

concentrations (caused by less stimulated tissue uptake leading to augmented spill over)

have been previously linked to obesity and diabetes (Lairon 1996).

Limitations

The study presented limitations that deserve to be mentioned. The study design did not

involve measurement of the partitioning or contribution of the FA within the individual

lipoprotein subclasses (CM- or VLDL-TG) using tracer technology. This makes

interpretation of the findings somewhat speculative and provides limited information

about the associated health risks of the ingested lipids, which is highly dependent on the

dynamic metabolic processes of the different major lipoprotein fractions. However, tracer

technology might not be appropriate in studies involving highly non-steady metabolic

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states such as the postprandial state (i.e. kinetic studies) which requires an ‘instantaneous’

replacement of carbon source with the labeled nutrient, followed by rapid quenching and

extraction of metabolites at defined time points (Magkos and Mittendorfer 2009). This

goes along with relatively high costs and a substantial burden added to subjects as they

have to avoid special foods naturally enriched in 13C if 13C-tracer method is used

(Lambert and Parks 2012).

Conclusion

In this work, the behaviour of a broad spectrum of plasma FA species was investigated

over an extended postprandial period of 7 h, in response to a typical western meal rich in

LA. The study results demonstrated that, postprandially, LA and VLCSFA either as

NEFA or TG were most affected by the test meal. Among the TG-FA, LA from TG18:2n-

6 and behenic acid from TG22:0 showed the highest response and were the only two TG-

FA that did not return to their baseline values at the end of the study in addition to a

characteristic postprandial biphasic response observed for the former. As NEFA, LA and

some VLCSFA showed a different behaviour from the other individual NEFA, since they

markedly increased shortly after the meal intake. The study also demonstrated that

postprandial TG responses were elevated in subjects with the highest baseline TG

concentrations indicating a high correlation between postprandial and baseline TG levels.

Concomitantly, the results pose new questions to be further investigated, especially

regarding potential health implications of western dietary lifestyle characterized by high

intake of mixed meals rich in n-6 PUFA, especially in high metabolic risk subjects (obese

and diabetic).

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Abbreviations:

Å (angstrom), ALA (alpha linolenic acid), AUC (Areas under the curves), BMI (body

mass index), CV% (coefficient of variation), FA (fatty acid), FABP (fatty acid binding

protein), FAME (fatty acid methyl esters), GC-FID (gas chromatography with flame

ionization detection), HDL-c (high-density lipoprotein cholesterol), IQR (interquartile

ranges), LA (linoleic acid), LC-MUFA (long chain monounsaturated fatty acid), LC-

PUFA (long chain polyunsaturated fatty acid), LC-SFA (long chain saturated fatty acid),

LDL-c (low-density lipoprotein cholesterol), LPL (lipoprotein lipase), MUFA

(monounsaturated fatty acid), NEFA (non-esterified fatty acid), OA (oleic acid), PL

(phospholipids), PUFA (polyunsaturated fatty acid), QC (quality control), SFA (saturated

fatty acid), TFA (trans fatty acids), triacylglycerols (TG), TLC (thin layer

chromatography), VLDL (very low-density lipoprotein), VLCMUFA (very long chain

monounsaturated fatty acid), VLCPUFA (very long chain polyunsaturated fatty acids),

VLCSFA (very long chain saturated fatty acid).

Acknowledgment

Work reported herein is carried out with partial financial support from the Commission

of the European Communities, the 7th Framework Programme, contract FP7-289346-

EARLY NUTRITION and the European Research Council Advanced Grant ERC-2012-

AdG – no.322605 META-GROWTH. This manuscript does not necessarily reflect the

views of the Commission and in no way anticipates the future policy in this area.

Conflict of interest statement

The authors have no conflicts of interest to report.

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Table 1. Nutritional composition of the test meal

Muffin Sandwich Juice TotalEnergy (KJ) 1735 1764 286 3785Protein (g) 6 19 1 26Carbohydrates (g) 47 35 15 97Fat* (g) 22 23 - 45C 18:2 n-6 (g) 13.2 13.1 - 26.4C 18:1 n-9 (g) 5.7 5.4 - 11.1C 16:0 (g) 1.8 1.6 - 3.4C 18:0 (g) 0.7 0.7 - 1.3

* 42g of total fat in the meal is provided by sunflower oil

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Table 2. Fatty acid composition of the sunflower oil

FA %C18:2n-6 64.59C18:1n-9 23.73C16:0 6.49C18:0 2.99C18:1n-7 0.78C22:0 0.55C20:0 0.21C24:1n-9 0.19C20:1n-9 0.14C16:1n-7 0.11C14:0 0.07C18:3n-3 0.05C17:0 0.03C22:2n-6 0.03C16:1 t 0.01C20:2n-6 0.01C20:3n-9 0.01n-6 PUFA 64.63

Data are expressed as weight percentage of total fatty acids.

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Figure legends

Fig. 1. NMDS ordinations based on Bray-Curtis distances between all samples displaying

the distance between samples collected from the study participants (n = 8) at different

time points after the standardized meal intake. The numbers (0-7) refer to the time points

starting from baseline till 7h postprandial in 1h intervals. The samples are identified by a

combination of 2 numbers, the participant ID (number) and the timepoint at which the

sample was collected, separated by an underscore (_).

Fig. 2. Median of the sums of triacylglycerol, phospholipid, and non-esterified fatty acid

levels of study participants (n = 8) at baseline and after a standard breakfast meal. Values

are given in relation to baseline values.

Fig. 3. Curves depicting postprandial relative concentrations of plasma triglycerides (TG)

of study participants (n = 8), comprising: (a) saturated fatty acids (SFA) (b)

polyunsaturated fatty acids (PUFA) (c) monounsaturated fatty acids (d) Very long chain

saturated fatty acids (VLCSFA) versus time (h) after the standardized meal intake.

Results are given in relation to baseline values.

Fig. 4. Curves depicting postprandial relative concentrations of plasma non-esterified

fatty acids (NEFA) of study participants (n = 8), comprising: (a) saturated fatty acids

(SFA) (b) polyunsaturated fatty acids (PUFA) (c) monounsaturated fatty acids (d) Very

long chain saturated fatty acids (VLCSFA) versus time (h) after the standardized meal

intake. Results are given in relation to baseline values.

Fig. 5. Curves depicting postprandial relative concentrations of plasma phospholipids

(PL) of study participants (n = 8), comprising: (a) saturated fatty acids (SFA) (b)

polyunsaturated fatty acids (PUFA) (c) monounsaturated fatty acids (d) Very long chain

saturated fatty acids (VLCSFA)versus time (h) after the standardized meal intake. Results

are given in relation to baseline values.

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Fig. 1. NMDS ordinations based on Bray-Curtis distances between all samples displaying the distance between samples collected from the study participants (n = 8) at different time points after the standardized

meal intake. The numbers (0-7) refer to the time points starting from baseline till 7h postprandial in 1h intervals. The samples are identified by a combination of 2 numbers, the participant ID (number) and the

timepoint at which the sample was collected, separated by an underscore (_).

251x190mm (300 x 300 DPI)

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Fig. 2. Median of the sums of triacylglycerol, phospholipid, and non-esterified fatty acid levels of study participants (n = 8) at baseline and after a standard breakfast meal. Values are given in relation to baseline

values.

162x115mm (300 x 300 DPI)

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Fig. 3. Curves depicting postprandial relative concentrations of plasma triglycerides (TG) of study participants (n = 8), comprising: (a) saturated fatty acids (SFA) (b) polyunsaturated fatty acids (PUFA) (c) monounsaturated fatty acids (d) Very long chain saturated fatty acids (VLCSFA) versus time (h) after the

standardized meal intake. Results are given in relation to baseline values.

254x190mm (300 x 300 DPI)

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Fig. 4. Curves depicting postprandial relative concentrations of plasma non-esterified fatty acids (NEFA) of study participants (n = 8), comprising: (a) saturated fatty acids (SFA) (b) polyunsaturated fatty acids

(PUFA) (c) monounsaturated fatty acids (d) Very long chain saturated fatty acids (VLCSFA) versus time (h) after the standardized meal intake. Results are given in relation to baseline values.

254x190mm (300 x 300 DPI)

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Fig. 5. Curves depicting postprandial relative concentrations of plasma phospholipids (PL) of study participants (n = 8), comprising: (a) saturated fatty acids (SFA) (b) polyunsaturated fatty acids (PUFA) (c) monounsaturated fatty acids (d) Very long chain saturated fatty acids (VLCSFA)versus time (h) after the

standardized meal intake. Results are given in relation to baseline values.

254x190mm (300 x 300 DPI)

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