# Milk polar lipids favorably alter circulating and intestinal ceramide and sphingomyelin species in postmenopausal women

Mélanie Le Barz, Cécile Vors, Emmanuel Combe, Laurie Joumard-Cubizolles, Manon Lecomte, Florent Joffre, Michèle Trauchessec, Sandra Pesenti, Emmanuelle Loizon, Anne-Esther Breyton, et al.

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## To cite this version:

Mélanie Le Barz, Cécile Vors, Emmanuel Combe, Laurie Joumard-Cubizolles, Manon Lecomte, et al.. Milk polar lipids favorably alter circulating and intestinal ceramide and sphingomyelin species in postmenopausal women. JCI Insight, 2021, 10.1172/jci.insight.146161. [https://doi.org/10.1172/jci.insight.146168](https://doi.org/10.1172/jci.insight.146168)

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# Milk polar lipids favorably alter circulating and intestinal ceramide and sphingomyelin species in postmenopausal women

Mélanie Le Barz, ..., David Cheillan, Marie-Caroline Michalski

JCI Insight. 2021. https://doi.org/10.1172/jci.insight.146161.

Clinical Medicine

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Clinical trials

Metabolism

## Graphical abstract

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1

Milk polar lipids favorably alter circulating and intestinal

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ceramide and sphingomyelin species in postmenopausal women

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4

Mélanie Le Barz¹,#, Cécile Vors¹,²,#, Emmanuel Combe¹, Laurie Joumard-Cubizolles³, Manon

5

Lecomte¹,², Florent Joffre⁴, Michèle Trauchessec⁵,⁶, Sandra Pesenti¹, Emmanuelle Loizon¹, Anne-

6

Esther Breyton¹,², Emmanuelle Meugnier¹, Karène Bertrand⁴, Jocelyne Drai¹,⁵,⁷, Chloé Robert¹,²,

7

Annie Durand¹, Charlotte Cuerq¹,⁵,⁷, Patrice Gaborit⁸,⁹, Nadine Leconte¹⁰, Annick Bernalier-

8

Donadille¹¹, Eddy Cotte⁵,¹²,¹³, Martine Laville¹,²,⁵,¹³, Stéphanie Lambert-Porcheron²,⁵, Lemlih

9

Ouchchane¹⁴,¹⁵, Hubert Vidal¹, Corinne Malpuech-Brugère³, David Cheillan¹,⁵,⁶, Marie-Caroline

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Michalski*¹,²

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¹ Univ Lyon, CarMeN laboratory, INSERM, INRAE, INSA Lyon, Université Claude Bernard Lyon 1,
Charles Mérieux Medical School, 69310, Pierre-Bénite, France.

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² Centre de Recherche en Nutrition Humaine Rhône-Alpes, Univ-Lyon, CarMeN Laboratory,
Université Claude Bernard Lyon1, Hospices Civils de Lyon, CENS, FCRIN/FORCE Network, 69310,
Pierre-Bénite, France.

15

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³ Université Clermont Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH Auvergne,
63000, Clermont-Ferrand, France.

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⁴ ITERG, ZA Pessac-Canéjan, 11 Rue Gaspard Monge, 33610, Canéjan, France.

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⁵ Hospices Civils de Lyon, 69000, Lyon, France.

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⁶ Unité Maladies Héréditaires du Métabolisme, Service de Biochimie et Biologie Moléculaire Grand
Est, Centre de Biologie et de Pathologie Est, Hospices Civils de Lyon, 69677, Bron, France.

22
23 ⁷ Unité de Nutrition Endocrinologie Métabolisme, Service de Biochimie, Centre de Biologie et de Pathologie Sud, Hospices Civils de Lyon, 69495, Pierre-Bénite, France.

25 ⁸ ACTALIA Dairy Products and Technologies, Avenue François Mitterrand, BP49, 17700, Surgères, France.

27 ⁹ ENILIA ENSMIC, Avenue François Mitterrand, 17700, Surgères, France.

28 ¹⁰ INRAE, Institut Agro, STLO (Science et Technologie du Lait et de l'Œuf), F-35042, Rennes, France.

30 ¹¹ Université Clermont Auvergne, INRAE, UMR 454, MEDIS, 63000, Clermont-Ferrand, France.

31 ¹² Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Service de chirurgie digestive, 69310, Pierre-Bénite, France.

33 ¹³ Université Claude Bernard Lyon 1, Faculté de médecine Lyon-Sud-Charles Mérieux, EMR 3738, 69600, Oullins, France.

35 ¹⁴ Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut Pascal, 63000, Clermont-Ferrand, France.

37 ¹⁵ CHU Clermont-Ferrand, Unité de Biostatistique-Informatique Médicale, 63000, Clermont-Ferrand, France.

39 # MLB and CV contributed equally and are co-first authors.

40

41 * Corresponding author:

42 Michalski Marie-Caroline

43 INRAE UMR1397 - INSERM U1060 - CarMeN laboratory (Laboratoire de Recherche en Cardiovasculaire Métabolisme Diabétologie et Nutrition)

45 Bâtiment CENS-ELI 2D, Hôpitaux Lyon Sud, Secteur 2,

46 165, chemin du Grand Revoyet,
47 69310 Pierre-Bénite, France

48 E-mail: [marie-caroline.michalski@inrae.fr](mailto:marie-caroline.michalski@inrae.fr),

49 Phone: +33 4 26 23 61 71; Twitter: @Michalski MC

50

51 **Conflict of interest:** This work was supported in part by a grant from the French Dairy Interbranch

52 Organisation (CNIEL). M-CM has received research funding for other research projects from CNIEL,

53 Danone-Nutricia Research, Sodiaal-Candia R&D. M-CM has consultancy activities for food, fats and
54 oils and dairy companies. M-CM is a member of the scientific advisory board of ITERG, the Industrial

55 Technical Centre for the oils and fats business sector. These activities had no link with the present

56 study. FJ was, and KB is, an employee of ITERG. PG is an employee of ACTALIA Produits Laitiers,

57 an Agri-Food Technical Institute, with a strong specialization in dairy research and development, and

58 food safety. MLa had research collaborations with Mondelez and Bridor without link with the present
59 study. HV has research collaborations with PiLeJe and Roquette without link with the present study.

60 Other authors declared no conflict of interest.
61 **ABSTRACT**

62 **BACKGROUND.** High circulating levels of ceramides (Cer) and sphingomyelins (SM) have been
63 associated with cardiometabolic diseases. The consumption of whole-fat dairy products, which
64 naturally contain such polar lipids (PL), is associated with health benefits, but the impact on
65 sphingolipidome remains unknown. We investigated how milk PL supplementation impacts
66 circulating and intestinal SM and Cer composition in association with improvement of cardiovascular
67 markers.

68 **METHODS.** In a 4 week-randomized double-blind controlled study, 58 postmenopausal women 69 consumed daily a cream cheese containing 0, 3 or 5 g of milk PL. Postprandial metabolic explorations 70 were performed before and after the supplementation. SM and Cer species were analyzed in serum, 71 intestine-derived chylomicrons and feces. The ileal content of 4 ileostomy patients was also explored 72 after milk PL intake in a crossover double-blind study.

73 **RESULTS.** Milk PL consumption decreased serum atherogenic C24:1 Cer (Pgroup = 0.033), C16:1
74 (Pgroup = 0.007) and C18:1 (Pgroup = 0.003) SM species. Changes in serum C16+18 SM species were
75 positively correlated with the reduction of total cholesterol (r = 0.706, P < 0.001), LDL-C (r = 0.666,
76 P < 0.001) and ApoB (r = 0.705, P < 0.001). Milk PL decreased the concentration in chylomicrons of
77 total SM (Pgroup < 0.0001) and of C24:1 Cer (Pgroup = 0.001). Saturated SM and Cer species, which are
78 also the major species found in milk PL-enriched cheeses, increased in ileal efflux and feces. There
79 was a marked increase in total fecal Cer after milk PL supplementation (Pgroup = 0.0002). Milk PL also
80 modulated the abundance of some specific SM and Cer species in ileal efflux and feces, suggesting
81 differential absorption and metabolization processes in the gut.

82 **CONCLUSION.** These data demonstrate that milk PL supplementation decreases atherogenic SM
83 and Cer species associated with an improvement of cardiovascular risk markers. Our findings bring
84 new insights on sphingolipid metabolism in the gastrointestinal tract, especially Cer as such signaling
molecules potentially participating in the beneficial effect of milk PL. [ClinicalTrials.gov](https://ClinicalTrials.gov),
NCT02099032, NCT02146339.

**FUNDINGS.** Agence Nationale de la Recherche, ANR-11-ALID-007-01; Regional Hospital Clinical Research Program (PHRCI-2014: VALOBAB, n°14-007); French Dairy Interbranch Organization (CNIEL); Groupe Lipides et Nutrition (GLN 2018-11-07), Hospices Civils de Lyon as sponsor.
90 INTRODUCTION

91 Sphingolipids (SP) represent a large class of bioactive polar lipids (PL) that play a pivotal role in
92 structural and metabolic functions in the regulation of cardiometabolic homeostasis, intestinal health
93 and inflammatory signaling pathways (1–3). SP metabolism represents a vast and complex network
94 (4), including a plethora of metabolically relevant species regulated by several key enzymes and
95 metabolite fluxes in mammals (5). Dysregulated SP metabolism is related to negative health outcomes
96 (6–8), and an increase in circulating ceramide (Cer) species (particularly C24:1 Cer), which are key
97 precursors of the biosynthesis of several other SP molecules, correlates with markers of
98 cardiometabolic complications (9–11). Sphingomyelins (SM) represent one of the most abundant SP
99 families. High serum SM concentrations correlate with coronary heart diseases in obese patients (13)
100 and high-fat diet increases specifically C16+C18 SM species (i.e., C16:0, C16:1, C18:0 and C18:1
101 species) in rodents (14).

102 There is growing evidence showing that several factors may affect circulating SM and Cer
103 concentrations such as drugs and lifestyle modifications, including exercise and dietary changes (15,
104 16). SP being found in plant and animal cell membranes, the daily SP intake represents approximately
105 0.3-0.4 g / day in humans (17), but the impact of such consumption on the endogenous
106 sphingolipidome remains largely unknown. Cow milk has recently attracted more attention because it
107 naturally contains SM and Cer (~25% of PL in the milk fat globule membrane, MFGM), including
108 C20+22 species (i.e., C20:0, C20:1, C22:0 and C22:1 species) that are found in higher amounts in milk
109 fat than in human blood. Recent meta-analysis and research papers highlighted the beneficial
110 cardiometabolic effects of the consumption of whole fat dairy product, which contain sizeable amounts
111 of milk PL (18, 19). Given that milk PL also contain C16+18 SM and C24:1 Cer species with potential
112 deleterious effects, understanding the impact of milk SP on circulating levels and endogenous SP
113 metabolism is of uttermost importance.
Preclinical studies revealed that milk SM supplementation prevents hyperlipemia, hypercholesterolemia, low-grade inflammation, and improves intestinal health related to gut microbiota modulations (2, 20–26). Buttermilk, a by-product of butter industry, represents a natural source of SM-rich MFGM (1-10 g of milk PL / L) (27, 28). In the VALOBAB-C trial, we demonstrated recently that the 4-week consumption of milk PL-enriched test cheese decreases circulating total cholesterol (C) (primary outcome), triacylglycerols (TAG), LDL-C and ApoB, at both fasting and postprandial states, (Supplemental Figure 1) in postmenopausal women at risk of cardiovascular disease (29). However, there are still open questions regarding the potential involvement of the endogenous metabolism and intestinal fate of milk SP species in these benefits. We thus explored the prespecified secondary outcomes of the VALOBAB clinical trial by analyzing SM and Cer molecular species of particular interest in several biological compartments. We aimed to determine how the 4-week supplementation with milk PL impacts the circulating and fecal SM and Cer species at fasting state, and their amount in intestine-derived chylomicrons during the postprandial period, in postmenopausal women at cardiovascular risk. We then verified whether this contributes to the beneficial effects of milk PL on lipid cardiovascular markers. In a complementary study conducted in ileostomy patients, we further aimed to identify the digestive fate of milk SP in the upper gastrointestinal tract after acute consumption of milk PL-rich meals.
131 RESULTS

132 Sphingolipidome of serum SM and Cer molecular species is modified by milk PL

133 The dietary intervention with milk PL significantly modified the amount of several molecular SP
134 species in fasting serum. In the 5g-PL group, the serum concentrations of the following species
135 decreased between the first (V1) and second (V2) exploration visit: C16:1 SM (ΔV2-V1 CTL: +0.44
136 ± 0.97 µM; 3g-PL: -0.64 ± 0.84 µM; 5g-PL: -3.35 ± 0.50 µM; Pgroup = 0.007; post hoc analysis: PCTL-
137 5gPL = 0.006), C18:1 SM (ΔV2-V1 CTL: +1.02 ± 0.87 µM; 3g-PL: -2.21 ± 0.69 µM; 5g-PL: -2.99 ±
138 0.79 µM; Pgroup = 0.003; PCTL-5gPL = 0.003) and C20:1 SM (ΔV2-V1 CTL: +0.62 ± 0.61 µM; 3g-PL: -
139 0.71 ± 0.50 µM; 5g-PL: -1.57 ± 0.48 µM; Pgroup = 0.025; PCTL-5gPL = 0.019) (Table 1). A decrease in
140 serum C24:1 Cer species was also observed in milk PL groups regardless of dose (ΔV2-V1 CTL: +0.11
141 ± 0.08 µM; 3g-PL: -0.19 ± 0.08 µM; 5g-PL: -0.37 ± 0.18 µM; PPL=0.016), without any effect on the
142 other identified Cer species. No difference between groups was observed in the circulating fasting
143 concentrations of total SM and Cer and of phospholipids (Table 1). Parallel to their amount, changes
144 in the relative abundance of fasting SM and Cer species after intervention, i.e., the proportion of each
145 SM or Cer species in total serum SM or Cer respectively, revealed decreased proportions of C18:1 SM
146 species (Pgroup = 0.002) and C24:1 Cer species (ΔV2-V1 CTL: +0.65 ± 0.76%; 3g-PL: -2.67 ± 0.96%;
147 5g-PL: -2.65 ± 0.71%; Pgroup = 0.010, PPL = 0.002; PCTL-5g = 0.021, PCTL-3g = 0.020; Supplemental Table
148 2). These beneficial effects were associated with the increase of the relative proportions of specific
149 SM and Cer species usually poorly represented in human blood: C20:0 SM (ΔV2-V1 CTL: +0.14 ±
150 0.14%; 3g-PL: +0.62 ± 0.08%; 5g-PL: +0.95 ± 0.09%; Pgroup = 0.00005; PCTL-5g = 0.00003, PCTL-3g =
151 0.010), C22:1 SM (ΔV2-V1 CTL: -0.02 ± 0.10%; 3g-PL: +0.65 ± 0.27%; 5g-PL: +0.56 ± 0.23%; Pgroup
152 = 0.07; PPL = 0.021) and C20:0 Cer species (ΔV2-V1 CTL: -1.44 ± 0.65%; 3g-PL: +0.38 ± 0.56%; 5g-
153 PL: +0.47 ± 0.60%; Pgroup = 0.057, PPL = 0.016) (Supplemental Table 2).
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Milk PL-induced modulations of serum SM and Cer profiles are correlated with the decrease of
CVD risk markers

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Results demonstrated a significant correlation between change in serum SM (particularly C16+18 SM species) and ΔLDL-C, Δtotal C and ΔApoB (Figure 2A, *P* < 0.001). These correlations were mainly mediated by the dietary intervention regardless of milk PL dose (Figure 2B; *versus* no correlation in control group, Figure 2C), as also illustrated in Figure 2D-F by the specific correlations between ΔC16+18 SM species and ΔLDL-C (r = 0.666, *P* < 0.0001), Δtotal C (r = 0.706, *P* < 0.0001), and ΔApoB (r = 0.705, *P* < 0.0001). Fewer correlations were observed between changes in Cer concentrations and those of blood lipids. Because results revealed changes in the SP species proportions (Supplemental Table 2), we analyzed potential correlations with blood lipid concentrations (Figure 2G). Changes in C24+26 Cer species proportions positively correlated with ΔLDL-C (r = 0.418, *P* = 0.022), Δtotal C (r = 0.585, *P* < 0.001) (Figure 2, G and J-K) and ΔApoB (r = 0.492, *P* = 0.006). Conversely, variations in C20+22 Cer species proportions negatively correlated with ΔLDL-C (r = -0.424, *P* = 0.020) and Δtotal C (r = -0.476, *P* = 0.008) (Figure 2, H-I). In parallel, we determined the magnitude effect of ΔC16+18 SM, Δ%C20+22 and Δ%C24+26 Cer species on Total C, LDL-C and ApoB by estimating the regression coefficient associated to each variable in a general linear mixed model. This shows that each variation of 1 µM of ΔC16+18 SM species would result in a variation of 0.0074 mM of LDL-C (*P* = 0.0065), 0.0088 mM of total C (*P* = 0.0042), 0.0017 g / L of ApoB (*P* = 0.017). The magnitude effect of Δ%C20+22 species on cardiovascular lipid markers was not significant, while each variation of 1% of ΔC24+26 Cer species proportions would result in a variation of 0.039 mM of LDL-C (*P* = 0.040), 0.054 mM of total C (*P* = 0.010) and 0.010 g / L of ApoB (*P* = 0.041).
177

178 Milk PL decrease SM content in intestine-derived chylomicrons and impact their SM and Cer
179 molecular profiles

180 The variations of plasma concentrations of CMRF-bound SM (CMRF-SM) decreased in the 5g-PL
181 group during all the postprandial period ($P_{group}$ = 0.015; $P_{CTL-5g}$ = 0.013), and the variation of plasma
182 CMRF-Cer concentration also tended to decrease ($P_{group}$ = 0.053, $P_{PL}$ = 0.051) (Supplemental Table
183 3). To focus on potential modifications of chylomicron lipid composition regardless of their circulating
184 concentration, we also determined their enrichment in SP by analyzing the SM/TAG and Cer/TAG
185 ratios in CMRF particles. Milk PL reduced significantly CMRF-SM/TAG ratio (Figure 3A, $P_{group}$ =
186 0.00095, $P_{PL}$ = 0.001; $P_{CTL-3g}$ = 0.001; $P_{CTL-5g}$ = 0.009), notably after lunch that contained the test cream
187 cheese (240-480 min). CMRF-Cer/TAG ratio also significantly decreased in milk PL-treated groups,
188 regardless of dose ($P_{group}$ = 0.071, $P_{PL}$ = 0.024) (Figure 3B). Milk PL effects on SM molecular
189 composition in intestine-derived chylomicrons were mainly mediated by a significant decrease in
190 several CMRF-SM species content relative to CMRF-TAG including C16:0, C16:1, C18:0, C18:1,
191 C20:1, C24:0 and C24:1 SM species (Figure 3, C-E; Supplemental Figure 2). Changes in CMRF-Cer
192 molecular composition was mainly driven by a decrease of C22:0 and C24:1 Cer species content
193 relative to CMRF-TAG (Figure 3, F-H; Supplemental Figure 2).

194

195 Ileostomy model reveals an important increase of saturated SM and Cer species in ileal efflux

196 We performed a complementary mechanistic study in ileostomy patients to determine whether the
197 digestive fate of milk PL in the upper gastrointestinal tract may contribute to the above results, notably
198 before absorption and enterocyte metabolism (29). Each milk PL-enriched meal resulted in higher 8h-
199 cumulative ileal efflux of total SM and Cer (CTL: 4.4 ± 1.3 µmol; 3g-PL: 143.2 ± 51.4 µmol; 5g-PL:
200 250.2 ± 117.3 µmol, $P_{meal}$ = 0.04; CTL: 3.5 ± 1.0 µmol; 3g-PL: 67.9 ± 21.5 µmol; 5g-PL: 109.1 ± 15.0
201 µmol, $P_{\text{meal}}$ = 0.005 respectively, Figure 4A). Detailed molecular composition analysis showed a
202 significant increase in C16:0 SM (CTL: 1.6 ± 0.4 µmol; 3g-PL: 32.2 ± 12.7 µmol; 5g-PL: 55.7 ± 29.9
203 µmol, $P_{\text{meal}}$ = 0.04), C20:0 SM (CTL: 0.3 ± 0.1 µmol; 3g-PL: 15.2 ± 4.8 µmol; 5g-PL: 26.7 ± 11.6
204 µmol, $P_{\text{meal}}$ = 0.04), C22:0 SM (CTL: 0.7 ± 0.2 µmol; 3g-PL: 46.9 ± 15.8 µmol; 5g-PL: 83.3 ± 36.3
205 µmol, $P_{\text{meal}}$ = 0.04) and C16:0 Cer (CTL: 1.4 ± 0.4 µmol; 3g-PL: 20.0 ± 4.4 µmol; 5g-PL: 31.1 ± 3.1
206 µmol, $P_{\text{meal}}$ = 0.01), C20:0 Cer (CTL: 0.1 ± 0.0 µmol; 3g-PL: 1.0 ± 0.3 µmol; 5g-PL: 1.5 ± 0.3 µmol,
207 $P_{\text{meal}}$ = 0.009), C22:0 Cer species (CTL: 0.4 ± 0.1 µmol; 3g-PL: 17.7 ± 7.2 µmol; 5g-PL: 26.4 ± 6.3
208 µmol, $P_{\text{meal}}$ = 0.02) (Figure 4, B and C). However, the analysis of SP species relative abundance
209 revealed a reduction in the proportions of atherogenic C16:0, C18:0 SM and C24:1 Cer species ($P_{\text{meal}}$
210 = 0.02, $P_{\text{meal}}$ = 0.04 and $P_{\text{meal}}$ = 0.04 respectively), and an increase in the proportions of C22:0 and
211 C24:0 SM species ($P_{\text{meal}}$ = 0.005 and $P_{\text{meal}}$ < 0.001) and Cer species ($P_{\text{meal}}$ = 0.02 and $P_{\text{meal}}$ = 0.04,
212 respectively; Supplemental Figure 3, A and B). Considering that such lipids cannot be absorbed
213 directly as such by enterocytes and that SM digestion is incomplete in the gastrointestinal tract (30),
214 we explored the molecular composition of SM and Cer species in fecal samples collected by the
215 postmenopausal women included in the VALOBAB-C trial.
216

216
217 Fecal sphingolipidome is largely enriched in Cer after milk PL supplementation

218 The 4-week nutritional intervention significantly increased total fecal SM and Cer in milk PL
219 supplemented groups compared to control (Figure 5, A and B) (ΔSM: CTL -0.09 ± 0.04 µmol; 3g-PL
220 +1.46 ± 0.95 µmol; 5g-PL +1.76 ± 0.83 µmol / g of dry feces, $P_{\text{group}}$ = 0.006, $P_{\text{PL}}$ = 0.001; ΔCer: CTL
221 -0.15 ± 0.08 µmol; 3g-PL +4.09 ± 1.38 µmol; 5g-PL +7.69 ± 2.95 µmol / g of dry feces, $P_{\text{group}}$ = 0.0002,
222 $P_{\text{PL}}$ = 0.00006). Altogether, this increase of total fecal Cer was higher than that of SM ($P$ = 0.015, ΔCer
223 versus ΔSM in milk PL groups). The detailed molecular analysis revealed a major impact of
224 intervention on saturated SP species, notably an increase of C22:0 SM ($P_{\text{group}}$ = 0.009, $P_{\text{PL}}$ = 0.003),
225 C24:0 SM ($P_{\text{group}}$ = 0.011, $P_{\text{PL}}$ = 0.002), C16:0 Cer ($P_{\text{group}}$ = 0.0005, $P_{\text{PL}}$ = 0.0001), C22:0 Cer ($P_{\text{group}}$ =
226 0.00001, $P_{\text{PL}}$ = 0.00001) and C24:0 Cer species ($P_{\text{group}}$ = 0.00002, $P_{\text{PL}}$ = 0.006) (Figure 5, C-E and I-
227 K, Supplemental Table 4). To a lower extent, the milk PL supplementation also increased the fecal
228 amount of some unsaturated SP species (Figure 5, F-H and L-N, Supplemental Table 4).
229 DISCUSSION

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This study is the first to report how the daily consumption of a significant amount of dietary SP present in milk PL impacts the endogenous sphingolipidome in the bloodstream and along the gastrointestinal tract in humans. Firstly, we reveal that the increased intake of milk SM and Cer did not increase their total amount in serum, but the molecular composition of SM and Cer species was markedly improved by the 4-week intervention with milk PL. Notably, the atherogenic C16+18 SM and C24:1 Cer species decreased significantly despite their increased intake from the provided supplementation. These variations even correlated with the beneficial impacts of milk PL on lipid cardiovascular markers reported previously (29). In addition, the Mayo Clinic published the reference values for circulating level of C24:1 Cer species (i.e., 0.65-1.65µM, https://www.mayocliniclabs.com). Here, we found that serum C24:1 Cer species concentration returned within the normal range after the intervention in the 5g-PL group only (V1: 1.96 ± 0.17 µM, V2: 1.59 ± 0.16 µM, $P_{group}$ = 0.033). Our results demonstrate that milk PL supplementation positively impacts the endogenous sphingolipidome, with the specific decrease of serum SM and Cer species known for being associated with inflammation and metabolic disorders (31, 32). Previous studies reported that high concentrations of serum C18:0, C20:0, and C24:1 Cer species are associated with type 2 diabetes, while high serum levels of C16:0 Cer and C18:0 SM species correlate with insulin resistance (33). Regardless of milk PL dose, the analysis of the relative abundance of each SM and Cer species in the bloodstream revealed a significant increase in the proportions of C20:0 SM, C22:1 SM and C20:0 Cer species that are normally poorly detected in human blood but found in non-negligible amount in MFGM. We also estimated to what extent the changes in major SM or Cer species could explain the relationships between milk PL consumption and enhanced lipid cardiometabolic risk factors. According to the estimated regression coefficients, assuming a mean variation of C16+18 SM species of about -22 µM as observed in the 5g-PL group,
253 its mean effect is expected to be (i) -0.16 mM on LDL-C (with a global effect of -0.34 mM observed in this group) (29), and (ii) -0.19 mM on total C (with an observed effect of -0.4 mM). Moreover, assuming a mean variation of the relative proportions of C24+26 Cer of about -0.83% as observed in the 5g-PL group, its mean effect is expected to be (i) -0.032 mM on LDL-C and (ii) -0.045 mM on total C. These results show that changes in serum total C and LDL-C are significantly associated with changes in serum C16+18 SM species, and to a lower extent with the modulation of the relative proportions of C24+26 Cer species (here mainly driven by the variation of C24:1 Cer species).

260

261 To investigate underlying mechanisms involved in the effects of milk PL consumption on circulating SP species, we first estimated the contribution of intestine-derived chylomicrons, which are the dietary lipid carriers secreted by the small intestine during the postprandial phase. Chylomicrons represent a major source of circulating SM, although the mechanisms by which SM is inserted into these lipoproteins have not been established (34). Dietary SM and Cer are not absorbed as such; their lipolysis products released in the small intestine can be absorbed and a small proportion of their sphingoid bases contribute to the newly formed SP ultimately found in chylomicrons (34). Herein, the 4-week milk PL supplementation decreased chylomicron total SM and Cer, especially during the second part of the postprandial period (after test cheese consumption), without change in particle size (i.e., no change in the surface-to-TAG core ratio) (29). These modifications were also observed at species level for almost all SM and Cer species, including those whose concentrations in total serum decreased, namely C16:1 SM, C20:1 SM and C24:1 Cer. Because SP are located at the surface of lipoproteins, this reveals a lower SP amount in the chylomicron composition. Whether this is due to decreased SM synthesis in enterocytes after intervention with dietary SP remains to be elucidated. Milk PL also modified SM molecular profile in chylomicrons with an increase in the proportions of C20 SM species and a decrease in the proportions of C24:1 SM species, possible precursor of C24:1
277 Cer *via* acid SMase (35). These results suggest that these modifications may originate from the gut or
278 from enterocyte metabolism during the intestinal digestion and absorption processes.

279

280 To determine the contribution of SM and Cer in the intestine, we analyzed their molecular profiles in
281 the gut lumen of ileostomy patients after the acute intake of milk PL, as well as in feces of the
282 postmenopausal women after 4-week milk PL supplementation. These analyses revealed an increase
283 of both total SM and Cer in gut contents in milk PL supplemented groups. At a molecular level, the
284 amounts of most detected SP species of interest were significantly increased by milk PL consumption,
285 especially C16:0, C22:0, C24:0 SM and Cer, and also C20:0 SM species. Altogether, SM and Cer
286 species whose amounts increased in gut contents reflect species that are present in milk PL-enriched
287 cheeses. These results are consistent with the fact that SM digestion is incomplete, as only 75-80% of
288 milk SM was reported to be digested and absorbed in humans (36). It has been previously reported that
289 ileal efflux of C16:0 SM was only ~10% of ingested dose versus ~20% for C24:0 SM after intake of
290 lower doses of SM, i.e., 50-200 mg (36), suggesting that longer-chain saturated species of SM and Cer
291 are less efficiently digested and absorbed. In addition, the digestion of SM being slow and incomplete,
292 it may induce an important increase of non-digested SM and non-absorbed Cer in the lumen content
293 (3, 30), which may explain the present results. Moreover, fecal metabolites, including the various lipid
294 species normally found in feces, may originate directly from food, but also from host cells, bacterial
295 cell components or indirectly from the molecular conversion of SP by gut microorganisms or host
296 enzymes (37). In a recent study performed in healthy patients, plasma and fecal lipidomic analyses
297 demonstrated that the lipid fraction of fecal samples contains significant amounts of Cer species with
298 only two SM species detected, while plasma samples commonly contain significant amounts of several
299 SM species and lower quantities of Cer (37, 38). Herein, we chose to determine the concentration of
300 12 SM and Cer species of interest in serum and we were also able to quantify all these species in feces.
301 In ileostomy patients, we report higher total SM amount in ileal content compared to total Cer, while 302 total Cer was largely more abundant in the fecal samples compared to total SM. The latter could be 303 the result of several metabolic pathways such as the conversion of dietary SM species in Cer species 304 by host enzymes present in the lumen and in enterocytes. At a molecular level, monounsaturated SM 305 and Cer species increased in both ileal efflux and fecal samples, despite being found in minority in test 306 cheeses compared to saturated species. A potential differential absorption process between 307 monounsaturated and saturated species would thus deserve to be investigated. Interestingly, the major 308 changes reported in the serum after the dietary intervention with milk PL mainly concern 309 monounsaturated SM and Cer species. It may suggest that some modifications of serum and 310 chylomicron SP profiles occur in response to changes in the SP fate in the small intestine.

311 The increased amount of total Cer reported in the fecal samples of milk PL supplemented volunteers 312 could also be the result of gut bacteria metabolism, because several bacteria, including those belonging 313 to the *Bacteroides* genus, were reported to be able to produce SP (39, 40). Very recently, Lee *et al.* 314 demonstrated in female mice that sphinganine, which is the main sphingoid base of SM and Cer present 315 in MFGM, is assimilated by gut bacteria (41). In this study, 99% of gavaged fluorescent sphinganine 316 was assimilated by *Bacteroides* spp.; the remaining 1% by *Prevotella* spp., *Lactobacillus* spp. and 317 *Bifidobacterium* genus (41). Also, *Bifidobacterium* spp., which are known to be increased after milk 318 SM consumption in rodents (42, 43), can release free milk Cer by hydrolyzing milk gangliosides (44). 319 In this context, the contribution of the gut microbiota SP metabolism in the effects of milk PL 320 consumption on the intestinal and circulating SM and Cer profiles cannot be ruled out.

321

322 To further explore potential endogenous mechanisms, we analyzed whole blood cells gene expression 323 of some key enzymes involved in SM synthesis (SM synthase 1 and 2, SGMS1 and SGMS2) and 324 hydrolysis (an acid sphingomyelinase – SMase, also called SM phosphodiesterase 1, SMPD1). We
found only slight effects on SGMS1 and SGMS2 expression (Supplemental Figure 4), while SMPD1 expression decreased in milk PL-treated groups compared to control ($P_{group}$ = 0.052, $P_{PL}$ = 0.030). As previously described, acid SMase activity in plasma is increased in acute coronary syndromes (45). However, these results were not likely to explain the changes observed in the circulating sphingolipidome. It would be also relevant to consider the possible contribution of intestinal enzymes given that the small intestine is rich in enzymes known to contribute to SP metabolism, such as alkaline SMase that converts SM in Cer (35). Unfortunately, we could not collect intestinal biopsies from the healthy postmenopausal women in the present study. However, in an 8-week milk PL supplementation performed (0.9 wt%) in high-fat diet fed mice (22), we observed a significant increase of jejunal expression of Enpp7, coding for the alkaline SMase, compared to the high-fat control group (1.7-fold change). Interestingly, previous preclinical studies reported opposite impacts of Cer production depending on SMase activity: Cer generated from neutral or acid intestinal SMases are more prompted to exert pro-inflammatory effects while Cer generated from alkaline SMase promote anti-inflammatory pathways (46, 47). The conversion of exogenous SM in Cer by the alkaline SMase could also play a role in the inhibition of cholesterol absorption (47, 48), which is concordant with the present findings and supports the role of SM metabolism in cholesterol absorption. Previous preclinical studies demonstrated that dietary SM are able to play a beneficial role on cholesterol levels and more largely in the prevention of cardiometabolic disorders (25, 26, 49, 50). In mice fed high-fat diets, supplementation with egg SM lowered intestinal absorption of cholesterol and lipids with a reduction of hepatic cholesterol (51). In vitro, both SM and Cer inhibit cholesterol absorption in Caco-2 intestinal epithelial cells (47). However, it has been suggested that small SM catabolites, such as Cer and sphingosine, might be the effectors of the beneficial impact of milk SM (2). The present findings consolidate our previous clinical results given that the observed reductions in circulating total cholesterol, LDL-C and ApoB (29) significantly correlate with the reduction of serum pro-
349 inflammatory C16+18 SM in the milk PL-treated groups. In accordance with above mentioned studies, 350 our findings also bring new information and insights on Cer in the gastrointestinal tract as signaling 351 molecules potentially participating in the beneficial effect of milk PL consumption on cholesterol 352 metabolism. 353

354 The present study has several strengths but also some limitations that need to be outlined. The clinical 355 trials were performed in real-life in a targeted population (i.e., overweight postmenopausal women) 356 known to present an important risk of cardiovascular diseases, but results cannot be extrapolated to 357 other populations. We took care to include 4-day dietary records before and after the nutritional 358 intervention to show that volunteers of the three groups did not differentially modify their energy and 359 dietary intakes (Supplemental Table 5). Many parameters of the present study were measured in a 360 limited number of subjects. Nevertheless, we performed for the first time a broad sphingolipidomic 361 analysis, including a large scale of measurements of SM and Cer species at both fasting and 362 postprandial states in various biological compartments: serum, chylomicron fractions, and feces, but 363 also in 8 h-cumulative ileal efflux from ileostomy patients. The sphingolipidome is a complex and 364 dynamic system that encompasses several important SP families, including dihydroceramides, 365 gangliosides or cerebrosides (17). Considering the variations in the sphingoid bases, FA and 366 headgroups of SP molecules, the number of species exceeds thousands. SP are localized in cellular 367 membranes (lipid rafts) and are carried by albumin, lipoprotein particles, blood cells and platelets in 368 the bloodstream (52, 53). Based on the present findings, future studies should thus explore the 369 sphingolipidome in other blood compartments and potentially epithelial cells to better understand the 370 fate of milk SM and Cer species. Furthermore, we cannot exclude the potential contribution of other 371 components of the PL fraction/MFGM from buttermilk concentrate and/or the lower milk TAG content 372 in the PL-enriched cheeses in the reported metabolic effects in both trials. Putting aside these
limitations, this is to the best of our knowledge the first time that such a wide sphingolipidomic analysis is performed in humans in response to a controlled dietary intervention in the context of cardiometabolic disorders. The present study clearly responds to the need to identify relevant dietary strategies to improve the endogenous SP metabolism, which was highlighted in recent reviews (2, 54).

The present findings uncover that milk PL supplementation providing particular SP species markedly improved the endogenous sphingolipidome by reducing serum atherogenic C16+18 SM and C24:1 Cer species in overweight postmenopausal women at risk of cardiovascular disease. These reductions in SP were (i) correlated with, and (ii) significantly involved in the decrease of lipid cardiovascular risk markers induced by milk PL intervention. We further demonstrate that despite a significant ingestion of SP provided by milk PL, SM and Cer concentrations decreased in intestine-derived chylomicrons while their concentration increased in gut contents. The related differences in SM and Cer profiles between gut contents and circulating compartments suggest that small intestinal mechanisms occurred during digestion and absorption processes of milk SM and Cer, and that a contribution of the gut microbiota may be possible. Considering that milk PL are naturally found in large amounts in buttermilk, which is still poorly valued in human food, such bioactive lipids could be envisioned as promising ingredients for the development of new functional foods providing health effects in the frame of chronic diseases.
391 METHODS

392

393 VALOBAB-C trial. Details of the VALOBAB-C study have been published previously (29). Briefly, the multicenter study used a double-blind randomized placebo-controlled parallel design and was conducted in 58 overweight postmenopausal women, without metabolic syndrome but at risk of CVD.

396 The eligibility criteria and sample size calculation have been described previously (29). Volunteers were randomly divided into 3 groups. Randomization was performed electronically using random number generator and supervised by the biostatistician (29). Both volunteers and investigators were kept blind regarding group allocation. Volunteers were subjected to the daily consumption of either control or milk PL-enriched cream cheese (100 g of cream cheese containing 13 g of total fat including 0 (control), 3 or 5g-milk PL during 28 days (n = 19; 19 and 20, respectively). The strategic approach was to formulate cheeses with identical total lipid content with partial substitution of TAG by milk PL to avoid increased energy intake. The 3g- and 5g-PL cream cheeses were based on a butterserum concentrate rich in milk PL prepared according to Gassi et al. (55) representing a 3- to 5-fold increased daily consumption of milk SM and Cer compared to an estimated intake of dairy SP in Western countries) (Supplemental Table 1) (17). After the “run-in” period, volunteers were subjected to a first exploratory visit (V1), followed by 28 days of intervention and ended by a second exploratory visit (V2). During each visit, overnight fasted-participants received a breakfast meal rich in fat and carbohydrates and 4 h later, they consumed a standardized lunch containing the corresponding test cream cheese, thus dividing the exploratory visit in two specific postprandial periods (0-240 min and 240-480 min, as detailed previously) (29). Volunteers were asked to continue their usual diet and physical activity all along the study. Participants were told to avoid the consumption of cheeses other than the test cream cheese, and listed foods that may influence the gut microbiota composition. Particular attention was drawn to standardize the meal consumed the evening before each postprandial
exploratory visit. Subjects recorded their food consumption for 4 days before and after the nutritional intervention. No difference in changes in energy and macronutrient intakes, fibers, alcohol, cholesterol and FA intakes was observed between groups (29). The primary outcome was the impact of the 4-week milk PL consumption on fasting serum concentration of total C (29). The predefined secondary outcomes tested in the present study were related to the impact of the dietary intervention on serum, chylomicron and fecal SM and Cer profiles. Considering available samples and practical/technical aspects, some analyses were performed on a subgroup of individuals only (Figure 1).

**VALOBAB-D trial.** The double-blind RCT was performed in 4 ileostomy patients following a crossover design, as previously described (29) (Supplemental Figure 5). An ileostomy is a surgical opening in the abdomen in which a piece of the ileum is brought outside the abdominal wall to create a stoma through which digestive contents leave the body and are collected in a pouch (ileal efflux). Selected patients according to eligibility criteria were invited to participate to 3 distinct exploratory visits separated by a 4 to 6-week washout period (29). During each visit, overnight fasted patients consumed one of the test cream cheese containing 0, 3- or 5g-milk PL and their ileal efflux was collected over 8 h. Sequences of meal allocation were based on random number generator (29). Both patients and investigators were kept blind regarding meal allocation.

**Isolation of chylomicron-rich fractions (CMRF).** Isolation of intestine-derived CMRF was performed by ultracentrifugation from plasma collected at different time points as previously described (29, 56).

**Analysis of serum phospholipids.** Total lipids were extracted from 300 µL of serum with chloroform:methanol (2:1, v/v) according to the method of Folch (57). After drying under nitrogen,
total lipids were determined gravimetrically and were dissolved precisely with 1 mL of chloroform:methanol (2:1, v/v). This stock solution of total lipids was stored at -20 °C. Phospholipid classes were then separated by high-performance liquid-chromatography coupled to an evaporative light-scattering detector (SEDEX LT-ELSD SOLT, HPLC DDL SEDERE, ThermoFisher) (58, 59), using a silica normal-phase column (Lichrospher Si 60, 3 µm, 100 x 4.6 mm, Waters). The chromatographic separation was carried out using a linear binary gradient according to the following scheme: t0 min: 90%A, 10%B 0%C, t20 min 42%A 52%B 6%C, t30 min 32%A 52%B 16%C, t55 min 30%A 70%B 0%C, t60 min 90%A 10%B 0%C. Total chromatographic run time was 75 min per sample, which consisted of a 60 min analysis and 15 min to restore initial conditions and re-equilibration. Eluent A consisted of hexane:tetrahydrofuran (99:1, v/v), eluent B of isopropanol:chloroform (80:20, v/v/v) and eluent C of isopropanol:water (50:50, v/v/v). The flow rate of the eluent was 1mL/min. Identification of phospholipids and lysophospholipids was carried out by comparison with the retention time of pure standards (Avanti polar Lipids, USA). Calibration curves for each compound were calculated from the area values of stock solution of pure standards between 0.1 to 1 mg / mL. Results were analyzed using Chromeleon software (Thermofisher) and expressed as µg / 100 µL of serum.

455

**Analysis of SM and Cer molecular profiles.** Concentrations of SM and Cer molecular species of interest were determined in serum, CMRF, ileal efflux, fecal samples and also in test cream cheese according to the method by Kyrklund (60) which was optimized as previously described (27). Ileal content from patients with ileostomy and fecal samples obtained from VALOBAB-C trial's volunteers were freeze-dried and approximately 15-40 mg of lyophilized matter, accurately weighted, were dissolved in 1mL of apyrogen water prior to lipid extraction. Briefly, for each sample, total lipids were extracted using 2.5 mL of chloroform:methanol (1:2 v/v) in the presence of two deuterium-labelled
463 internal standards (N-heptadecanoyl-D-erythro-sphingosine (C17:0-Ceramide); N-palmitoyl(d31)-D-erythro-sphingosylphosphorylcholine (C16:0D31SM) from Avanti Polar Lipids, Alabama, USA).
464 After 2 h of shaking and centrifugation (10 min, 1900 g), samples were evaporated with liquid nitrogen.
465 The dry samples were dissolved in 1.5mL of chloroform:methanol (1:2 v/v) and sonicated 30 sec on ice. SP were then isolated by saponification with potassium hydroxide during 2 h at 37 °C and then
466 fractionated and desalted using reverse-phase Bond Elut C18 columns. The final elutions were done with 2x1mL of chloroform:methanol (12:1 v/v) and 2x1mL of chloroform:methanol (1:2 v/v) prior to
467 the evaporation of samples with liquid nitrogen. The dry extracts were kept at -20 °C until tandem mass spectrometry analysis (MS/MS). Samples were homogenized in 1mL of chloroform:methanol (1:2 v/v) and analyzed by direct flow injection on a triple-quadrupole mass spectrometer (API 4500 QTRAP MS/MS; Sciex Applied Biosystems, Toronto, Canada) in the positive ionization mode using
468 the multiple reaction monitoring (MRM) method. Cer and SM species were measured separately, with two different methods with a flow rate of 200 µl / min (analysis time of 3 min). We quantified 12 SM and Cer species of particular interest regarding cardiovascular risk, being the most abundant in human
469 and also found in bovine milk in different proportions (Table 1). The concentration of each molecular species was calculated from the ratio of its signal to that of the corresponding internal standard. Total
470 Cer and SM concentrations were the sum of the concentrations of the various species. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined
471 SM and Cer species. These analyses were performed on a MS/MS platform accredited following EN
472 NF ISO 15189 requirements. The coefficient of variation (CV) for total SM and Cer was 4.4% and
473 5.4% respectively. For the most abundant isoforms (C16:0, C22:0, C24:0, C24:1 SM/Cer), the average CV was 7 ± 4%. The CV for the less abundant isoforms is slightly higher: 17 ± 5% for Cer and 9 ± 6%
474 for SM species. These elements are in agreement with the Methods and Protocols section of
475 LIPIDMAPS for the analysis of SP (61).
476
487

488

**Gene expression analysis in whole blood cells.** The PAXgene™ Fresh Whole Blood RNA samples were processed using the PAXgene™ Blood RNA Kit based on column purification of nucleic acids (PreAnalytiX, QIAGEN) as previously described (29). After reverse transcription, real-time PCR assays of *SGMS1* (F-CCTGGTATGCATTTCAACTG; R-TGGCCGCTGTACAGATAGTC), *SGMS2* (F-CAATAGTGGGACGCAGATTC and R-GGACAATCCACCACCAGAAA) *SMPD1* (F-CATCCTGCCAGGTTACATCG; R-CACACCTCCACCATGTCATC) were assessed using a Rotor-Gene 6000 (QIAGEN) and obtained values were normalized to the expression of the housekeeping gene *PGK1* (phosphoglycerate kinase 1, F-CCATGGTAGGAGTCAATCTG; R-AGCTGGATCTTGTCTGCAAC).

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494

495

496

497

498

**Statistics.** *VALOBAB-C:* Continuous variables are described as mean ± SEM. The 4-week intervention impact was determined by comparing the variation of each variable between exploratory visits (i.e., ΔV2-V1) between groups (*P*<sub>group</sub>) (i.e., control versus 3g-PL versus 5g-PL group; Figure 1). Single time point parameters were analyzed through a general linear model and a subsequent Tukey's *post hoc* test. *P*<sub>post hoc</sub> corresponds altogether to *P*<sub>CTL vs 3g-PL</sub>; *P*<sub>CTL vs 5g-PL</sub> and *P*<sub>3g vs 5g-PL</sub> as mentioned in the text and figures. For parameters analyzed along the postprandial period, a mixed linear modelling (MIXED procedure) was performed to account for within-subject repeated measures, seeking for main effects, at least “group” or time effect and interaction. *Post hoc* analyses were performed following Tukey-Kramer's test to both detail main effects and control for familywise type I error. In case of residual distribution departing from normality, the analyses were performed on ranks. Global “milk PL” effect was also considered as binary factor, and statistical analysis was performed by lumping together milk PL doses in one group versus control. Spearman's correlation analyses were also performed between blood lipid markers of cardiovascular risk and serum SM and Cer species grouped

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506

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510
in 3 subclasses (i.e., C16+C18; C20+C22 and C24+C26 SM or Cer species). In order to check for any confounding effect, these analyses were also carried-out adjusting for center, age and waist circumference quartiles. Analyses were performed on SAS v9.4 (SAS Institute Inc. Cary, NC, USA) with a two-sided type I error set at 0.05. In order to determine to what extent the changes in SM and Cer species could explain their relationship with lipid markers of cardiometabolic risk, we performed additional analyses. We aimed to adjust the analysis of ΔLDL-C, Δtotal C and ΔApoB variables with ΔC16+18 SM, Δ%C20+22 and Δ%C24+26 Cer species variables. We first transformed each covariate as a 4 classes ordinal variable, and then checked for a linear relationship between each covariate and each response variable, seeking for almost constant effect from an ordinal class to its neighbor. Since we found merely monotonic relationship, it allowed us to include these covariates in their original continuous form, associated with a unique and relevant regression coefficient, thereby simplifying interpretation. We then reported the magnitude of the effect of ΔC16+18 SM, Δ%C20+22 and Δ%C24+26 Cer species on Δtotal C, ΔLDL-C and ΔApoB by estimating the coefficient of regression associated to each variable in the mixed linear general model. VALOBAB-D: Data are presented as mean ± SEM and were analyzed with GraphPad Prism 8.3. For normally distributed data (Shapiro-Wilk's test), repeated measures one-way ANOVA were performed followed by Tukey's post hoc test. For non-normally distributed data, a Friedman's test was performed followed by Dunn's post hoc test. The variation between groups was reported using *P*<sub>meal</sub> values and *post hoc* analyses were added directly on corresponding figures using a, b letters. Graphs: All graphs and heat maps were created using GraphPad Prism 8.3 (San Diego, CA, USA).

531

532 **Study approval.** Both clinical trials were approved by the Scientific Ethics Committee of Lyon Sud-

533 Est-IV and ANSM (French Agency for the Safety of Health Products) and registered at Clinical Trials
534 (NCT02099032, NCT02146339). The clinical trials were conducted at the Human Nutrition Research
535 Centre Rhône-Alpes (CRNH-RA; Lyon, France) and at the Human Nutrition Research Centre
536 Auvergne (CRNH-A; Clermont-Ferrand, France) according to the Second Declaration of Helsinki and
537 the French Huriet-Serusclat law. All data reported in the current article were obtained from samples
538 stored in the biobank during the clinical studies, for which participants gave a written consent in order
539 prior to inclusion in the study to use the samples for further metabolic analyses. All authors had access
540 to the study data and reviewed and approved the final manuscript.

541

542 Author Contributions

543 MLB: conceptualization, validation, formal analysis, investigation, data curation, writing - original
544 draft, visualization; CV: conceptualization, methodology, validation, formal analysis, investigation,
545 data curation, writing - original draft, visualization; ECom and LJ-C: methodology, formal analysis,
546 investigation, data curation; MLe: conceptualization, validation, formal analysis, investigation, data
curation; FJ: methodology, validation, formal analysis, investigation; MT, SP, EL, A-EB, KB, JD, AD
547 and CC: investigation; EM: methodology, validation, investigation; CR: formal analysis, data curation;
548 PG, NL and ECot: provided essential resources; AB-D: validation, formal analysis, investigation;
549 MLa: conceptualization; SL-P: conceptualization, methodology, validation, investigation; LO:
550 methodology, formal analysis, data curation; HV: contributed to results interpretation and revised
551 manuscript; CM-B: conceptualization, methodology; DC: conceptualization, methodology, validation,
552 formal analysis, investigation, writing - original draft, data visualization; M-CM: conceptualization,
553 methodology, writing - original draft, data visualization, project administration, supervision and
554 primary responsibility for final article content. All authors read, revised and approved the final
555 manuscript.
556

557
558

559 Acknowledgements

560 The authors would like to thank the volunteers of VALOBAB-C and VALOBAB-D trials, Dr N
561 Feugier (CRNH-RA) and A Prulière (CRNH-A) for volunteer recruitment/follow-up; C Maitrepierre,
562 J Peyrat, E Bain (CRNH-RA), Carla Domingues-Faria, Adeline Blot (CRNH-A), D Provenchère, H
563 Parrot, and N Meunier (CRNH-A) for clinical and technical help; M Sothier (CRNH-RA), A Caille, N
564 Lyon-Belgy (CRNH-A) for help in dietary analysis; A Faure, M Cervantes and S Gonin (HCL CBS),
565 P Calmard and J Rivière (UNH) for help in lipid analyses, E Blond (HCL CBS) for stool collection
566 and storage management; C Buisson (CarMeN) for technical assistance in stool lipid extraction; C
567 Jouve (UNH) for technical assistance in blood lipid analysis; A Wauquier for technical assistance on
568 microbiota analysis; K Raynal (ACTALIA Produits Laitiers) and JP Bodin (ENILIA-ENSMIC) for
569 their respective work on test cheese elaboration, analysis and related management; D Kalnin (Philolao)
570 for help in the management of cream cheese randomization; Professor Y François (HCL Lyon Sud),
571 Professor D Pezet and B Gillet and F Morel-Laporte (Clermont Hospital) for screening ileostomy
572 patients list; G Gesan-Guiziou, JY Gassi and F Gaucheron (STLO, Plateforme Lait) for butterserum
573 process setup and assistance in transferring this PL enrichment process to ENILIA; C Bourlieu (STLO)
574 for contribution in buttermilk PL analysis; V Plattner (HCL) and the clinical research department for
575 trials' monitoring; C Oudin for her skillful assistance in ANR VALOBAB project coordination.
576 Members of the steering committee of ANR VALOBAB project (partners CarMeN/INRA,
577 UNH/UCA, STLO/INRA, ENILIA-ENSMIC, ITERG, MEDIS, CNIEL) are acknowledged for useful
578 discussions. F Laugerette and A Penhoat are acknowledged for their contribution to the high-fat diet
579 study in mice. The authors would like to thank CNIEL (French Dairy Interbranch Organization) for
580 financial support. M Le Barz acknowledges a postdoctoral grant from Société Francophone du Diabète.
581 C Vors and L Joumard-Cubizolles acknowledge ANR for postdoctoral fellowship. M Lecomte thanks
582 Région Rhône-Alpes ARC1 for PhD grant. MC Michalski acknowledges a grant from Groupe Lipides et Nutrition. Part of the artwork in the graphical abstract and Figure 1 has been designed using resources from Flaticon.com, Servier Medical Art and FreePik. We thank Antoine Serafin for elaborating human gut physiology artwork for the Figure 1 and the graphical abstract.

586

587 **Data sharing statement.** According to French law on the publication of biomedical research/clinical trials, we are not allowed to make the clinical database publicly available on the web, nor send it to third parties, nor to make visible the location of the study associated with the database.

589
590 REFERENCES

591

592 1. Iqbal J, Walsh MT, Hammad SM, Hussain MM. Sphingolipids and lipoproteins in health and
metabolic disorders. *Trends Endocrinol Metab* 2017;28(7):506–518.

594 2. Norris GH, Blesso CN. Dietary and Endogenous Sphingolipid Metabolism in Chronic
Inflammation. *Nutrients* 2017;9(11). doi:10.3390/nu9111180

596 3. Duan R-D. Physiological functions and clinical implications of sphingolipids in the gut. *Journal of Digestive Diseases* 2011;12(2):60–70.

598 4. Gault CR, Obeid LM, Hannun YA. An overview of sphingolipid metabolism: from synthesis to
breakdown. *Adv. Exp. Med. Biol.* 2010;688:1–23.

600 5. Hannun YA, Obeid LM. Sphingolipids and their metabolism in physiology and disease. *Nat Rev Mol Cell Biol* 2018;19(3):175–191.

602 6. Meikle PJ, Summers SA. Sphingolipids and phospholipids in insulin resistance and related
metabolic disorders. *Nat Rev Endocrinol* 2017;13(2):79–91.

604 7. Gorden DL et al. Biomarkers of NAFLD progression: a lipidomics approach to an epidemic. *J Lipid Res* 2015;56(3):722–736.

606 8. Borodzicz S, Czarzasta K, Kuch M, Cudnoch-Jedrzejewska A. Sphingolipids in cardiovascular
diseases and metabolic disorders. *Lipids Health Dis* 2015;14:55.

608 9. Mantovani A, Dugo C. Ceramides and risk of major adverse cardiovascular events: A meta-analysis of longitudinal studies. *Journal of Clinical Lipidology* 2020;14(2):176–185.

610 10. de Carvalho LP et al. Plasma Ceramides as Prognostic Biomarkers and Their Arterial and
Myocardial Tissue Correlates in Acute Myocardial Infarction. *JACC Basic Transl Sci*
2018;3(2):163–175.

613 11. Wang DD et al. Plasma Ceramides, Mediterranean Diet, and Incident Cardiovascular Disease in
614 the PREDIMED Trial (Prevención con Dieta Mediterránea).. *Circulation* 2017;135(21):2028–2040.

615 12. Laaksonen R. Identifying new Risk Markers and Potential Targets for Coronary Artery Disease:

616 The Value of the Lipidome and Metabolome.. *Cardiovasc Drugs Ther* 2016;30(1):19–32.

617 13. Jiang XC et al. Plasma sphingomyelin level as a risk factor for coronary artery disease..

618 *Arterioscler Thromb Vasc Biol* 2000;20(12):2614–2618.

619 14. Choi S, Snider AJ. Sphingolipids in High Fat Diet and Obesity-Related Diseases.. *Mediators*

620 *Inflamm* 2015;2015:520618.

621 15. Hilvo M, Vasile VC, Donato LJ, Hurme R, Laaksonen R. Ceramides and Ceramide Scores:

622 Clinical Applications for Cardiometabolic Risk Stratification. *Front. Endocrinol.* 2020;11:570628.

623 16. Le Barz M, Boulet MM, Calzada C, Cheillan D, Michalski M-C. Alterations of endogenous

624 sphingolipid metabolism in cardiometabolic diseases: Towards novel therapeutic approaches.

625 *Biochimie* 2020;169:133–143.

626 17. Vesper H et al. Sphingolipids in food and the emerging importance of sphingolipids to nutrition..

627 *J Nutr* 1999;129(7):1239–1250.

628 18. Drouin-Chartier J-P et al. Comprehensive Review of the Impact of Dairy Foods and Dairy Fat on

629 Cardiometabolic Risk123. *Adv Nutr* 2016;7(6):1041–1051.

630 19. Thorning TK et al. Whole dairy matrix or single nutrients in assessment of health effects: current

631 evidence and knowledge gaps [Internet]. *The American Journal of Clinical Nutrition* [published

632 online ahead of print: April 12, 2017]; doi:10.3945/ajcn.116.151548

633 20. Norris GH, Jiang C, Ryan J, Porter CM, Blesso CN. Milk sphingomyelin improves lipid

634 metabolism and alters gut microbiota in high fat diet-fed mice.. *J Nutr Biochem* 2016;30:93–101.

635 21. Lecomte M et al. Milk polar lipids affect in vitro digestive lipolysis and postprandial lipid

636 metabolism in mice.. *J Nutr* 2015;145(8):1770–1777.

637 22. Lecomte M et al. Dietary emulsifiers from milk and soybean differently impact adiposity and
638 inflammation in association with modulation of colonic goblet cells in high-fat fed mice.. *Mol Nutr Food Res* 2016;60(3):609-620.

640 23. Eckhardt ERM, Wang DQ -H., Donovan JM, Carey MC. Dietary sphingomyelin suppresses intestinal cholesterol absorption by decreasing thermodynamic activity of cholesterol monomers.
641
642 *Gastroenterology* 2002;122(4):948-956.

643 24. Wat E et al. Dietary phospholipid-rich dairy milk extract reduces hepatomegaly, hepatic steatosis
644 and hyperlipidemia in mice fed a high-fat diet.. *Atherosclerosis* 2009;205(1):144-150.

645 25. Milard M et al. Acute effects of milk polar lipids on intestinal tight junction expression: towards
646 an impact of sphingomyelin through the regulation of IL-8 secretion?. *J. Nutr. Biochem.*
647 2019;65:128-138.

648 26. Milard M et al. Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated
649 Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids. *Mol Nutr Food Res*
650 2019;63(4):e1801078.

651 27. Bourlieu-Lacanal C et al. Polar lipid composition of bioactive dairy co-products buttermilk and
652 butterserums: emphasis on sphingolipid and ceramide isoforms. *Food Chemistry* 2018;240:67-74.

653 28. Conway V, Gauthier SF, Pouliot Y. Buttermilk: Much more than a source of milk phospholipids.
654 *Animal Frontiers* 2014;4(2):44-51.

655 29. Vors C et al. Milk polar lipids reduce lipid cardiovascular risk factors in overweight
656 postmenopausal women: towards a gut sphingomyelin-cholesterol interplay. *Gut* 2020;69(3):487.

657 30. Nyberg L, Nilsson Å, Lundgren P, Duan R-D. Localization and capacity of sphingomyelin
658 digestion in the rat intestinal tract. *The Journal of Nutritional Biochemistry* 1997;8(3):112-118.

659 31. Chaurasia B, Summers SA. Ceramides - lipotoxic inducers of metabolic disorders.. *Trends Endocrinol Metab* 2015;26(10):538-550.

661 32. Galadari S, Rahman A, Pallichankandy S, Galadari A, Thayyullathil F. Role of ceramide in
662 diabetes mellitus: evidence and mechanisms. *Lipids Health Dis* 2013;12:98.

663 33. Bergman BC et al. Serum sphingolipids: relationships to insulin sensitivity and changes with

664 exercise in humans. *Am J Physiol Endocrinol Metab* 2015;309(4):E398-408.

665 34. Nilsson A, Duan R-D. Absorption and lipoprotein transport of sphingomyelin. *J Lipid Res* 2006;47(1):154-171.

667 35. Nilsson A, Duan RD. Alkaline sphingomyelinases and ceramidases of the gastrointestinal tract.

668 *Chem Phys Lipids* 1999;102(1-2):97-105.

669 36. Ohlsson L et al. Sphingolipids in human ileostomy content after meals containing milk

670 sphingomyelin. *The American Journal of Clinical Nutrition* 2010;91(3):672-678.

671 37. Trošť K et al. Describing the fecal metabolome in cryogenically collected samples from healthy

672 participants. *Scientific Reports* 2020;10(1):885.

673 38. Bowden J et al. Harmonizing Lipidomics: NIST Interlaboratory Comparison Exercise for

674 Lipidomics Using SRM 1950-metabolites in Frozen Human Plasma. *Journal of Lipid Research*

675 2017;58(12):2275-2288.

676 39. Olsen I, Jantzen E. Sphingolipids in Bacteria and Fungi. *Anaerobe* 2001;7(2):103-112.

677 40. Geiger O, Padilla-Gómez J, López-Lara IM. Bacterial Sphingolipids and Sulfonolipids [Internet].

678 In: Geiger O ed. *Biogenesis of Fatty Acids, Lipids and Membranes*. Cham: Springer International

679 Publishing; 2019:123-137

680 41. Lee M-T, Le HH, Johnson EL. Dietary sphinganine is selectively assimilated by members of the

681 mammalian gut microbiome. *J Lipid Res* [published online ahead of print: July 9, 2020];

682 doi:10.1194/jlr.RA120000950

683 42. Milard M et al. Milk Polar Lipids in a High-Fat Diet Can Prevent Body Weight Gain: Modulated

684 Abundance of Gut Bacteria in Relation with Fecal Loss of Specific Fatty Acids. *Mol Nutr Food Res*

685 2019;63(4):e1801078.
686 43. Norris GH, Jiang C, Ryan J, Porter CM, Blesso CN. Milk sphingomyelin improves lipid
687 metabolism and alters gut microbiota in high fat diet-fed mice. *J Nutr Biochem* 2016;30:93–101.

688 44. Heaver SL, Johnson EL, Ley RE. Sphingolipids in host-microbial interactions. *Curr Opin
689 Microbiol* 2018;43:92–99.

690 45. Pan W et al. Elevation of ceramide and activation of secretory acid sphingomyelinase in patients
691 with acute coronary syndromes. *Coron Artery Dis* 2014;25(3):230–235.

692 46. Abdel Hadi L, Di Vito C, Riboni L. Fostering Inflammatory Bowel Disease: Sphingolipid
693 Strategies to Join Forces. *Mediators of Inflammation* 2016;2016:13.

694 47. Feng D, Ohlsson L, Ling W, Nilsson A, Duan R-D. Generating ceramide from sphingomyelin by
695 alkaline sphingomyelinase in the gut enhances sphingomyelin-induced inhibition of cholesterol
696 uptake in Caco-2 cells. *Dig Dis Sci* 2010;55(12):3377–3383.

697 48. Zhang P et al. Alkaline sphingomyelinase (NPP7) promotes cholesterol absorption by affecting
698 sphingomyelin levels in the gut: A study with NPP7 knockout mice. *American Journal of
699 Physiology-Gastrointestinal and Liver Physiology* 2014;306(10):G903–G908.

700 49. Norris GH, Blesso CN. Dietary sphingolipids: potential for management of dyslipidemia and
701 nonalcoholic fatty liver disease. *Nutr Rev* 2017;75(4):274–285.

702 50. Norris GH, Porter CM, Jiang C, Millar CL, Blesso CN. Dietary sphingomyelin attenuates hepatic
703 steatosis and adipose tissue inflammation in high-fat-diet-induced obese mice. *J Nutr Biochem*
704 2017;40:36–43.

705 51. Chung RWS et al. Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal
706 cholesterol absorption in high-fat-fed mice. *PLoS One* 2013;8(2):e55949–e55949.

707 52. Hammad SM et al. Blood sphingolipidomics in healthy humans: impact of sample collection
708 methodology. *J Lipid Res* 2010;51(10):3074–3087.

709 53. Eich C et al. Changes in membrane sphingolipid composition modulate dynamics and adhesion
710 of integrin nanoclusters. *Scientific Reports* 2016;6:20693.

711 54. Le Barz M, Boulet MM, Calzada C, Cheillan D, Michalski M-C. Alterations of endogenous

712 sphingolipid metabolism in cardiometabolic diseases: Towards novel therapeutic approaches.

713 *Biochimie* 2020;169:133–143.

714 55. Gassi JY et al. Preparation and characterisation of a milk polar lipids enriched ingredient from

715 fresh industrial liquid butter serum: Combination of physico-chemical modifications and

716 technological treatments. *International Dairy Journal* 2016;52:26–34.

717 56. Vors C et al. Modulating absorption and postprandial handling of dietary fatty acids by

718 structuring fat in the meal: a randomized crossover clinical trial. *Am J Clin Nutr* 2013;97(1):23–36.

719 57. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total

720 lipides from animal tissues. *J Biol Chem* 1957;226(1):497–509.

721 58. Becart I, Chevalier C, Biesse JP. Quantitative analysis of phospholipids by HPLC with a light

722 scattering evaporating detector – application to raw materials for cosmetic use. *Journal of High*

723 *Resolution Chromatography* 1990;13(2):126–129.

724 59. Rombaut R, Camp JV, Dewettinck K. Analysis of phospho- and sphingolipids in dairy products

725 by a new HPLC method. *J Dairy Sci* 2005;88(2):482–488.

726 60. Kyrklund T. Two procedures to remove polar contaminants from a crude brain lipid extract by

727 using prepacked reversed-phase columns. *Lipids* 1987;22(4):274–277.

728 61. Sullards MC, Liu Y, Chen Y, Merrill AH. Analysis of mammalian sphingolipids by liquid

729 chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry

730 (TIMS). *Biochim Biophys Acta* 2011;1811(11):838–853.
<figure><img src="image_5.png" /><figcaption>**Figure 1:** Design of VALOBAB-C and VALOBAB-D clinical trials and graphical summary of analyses performed on predefined secondary outcomes. In the VALOBAB-C clinical trial, 58 postmenopausal women were supplemented with test cream cheese containing either 0, 3 or 5 g of milk PL during 4 weeks. In the VALOBAB-D trial, 4 ileostomy patients were subjected to the acute consumption of the 3 test cheeses following a cross-over study design. In both trials, during the exploratory visit, overnight fasted volunteers received a standardized breakfast rich in fat and sugars at time 0 and a meal containing the test cream cheese at time 240 min of the postprandial period. Tables and Figures reporting specific results are listed. Cer: ceramides; CMRF: chylomicron-rich fraction; MFGM: milk fat globule membrane; PL: polar lipids; SP: sphingolipids; SM: sphingomyelins; TAG: triacylglycerols. Molecular structures were drawn using the LIPIDMAPS® tool.</figcaption></figure>

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<figure><img src="image_6.png" /><figcaption>**Figure 2:** Major correlations between the impacts of milk PL supplementation on blood lipids and on serum SM and Cer (VALOBAB-C). (A-C) and (G): Spearman's correlations between blood lipids and serum SM and Cer species. All data are expressed as ΔV2-V1 at fasting (yellow: all groups were considered for the analysis (A and G) (n = 30); blue: the 2 groups supplemented with either 3 or 5g-milk PL only (n = 20); orange: the control group only (n = 10). For panels (A-C) and (G), asterisks in bold represent correlations that remain significant after adjustment for clinical center, quartiles of volunteer age and waist circumference. Graphs illustrating specific Spearman's correlations between the intervention impact on C16+18 SM species and on LDL-C (D), Total C (E) and ApoB48 (F); between C20+22 Cer species proportions (%) and LDL-C (H), and Total C (I); between C24-26 Cer species proportions (%) and LDL-C (J), and Total C (K). Apo: apolipoprotein; C: cholesterol; Cer: ceramides; CTL: control; HDL: high density lipoprotein; LDL: low density lipoprotein; NEFA: non-esterified fatty acids; PL: polar lipids; SM: sphingomyelin; TAG: triacylglycerols.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 3:** Milk PL supplementation during 4 weeks modulate SM and Cer molecular composition of plasma CMRF (see complements in Supplemental Table 3 and Supplemental Figure 2, VALOBAB-C trial). Kinetics of ΔV2-V1 CMRF SM and Cer normalized by CMRF TAG content (A and B, respectively). Molecular composition analysis of specific SM and Cer species in CMRF after normalization by CMRF TAG content: C16:1 SM (C), C20:1 SM (D), C24:1 SM (E), C16:1 Cer (F), C22:0 Cer (G), C24:1 Cer (H). Data are presented as mean ± SEM (n = 6 / group). The vertical dotted line represents the intake of the meal including the control or milk PL-rich dairy (according to group). The *P*<sub>group</sub> and *P*<sub>posthoc</sub> are shown for the postprandial period from 120 to 480 min. Statistical analysis was done using a linear mixed model followed by Tukey-Kramer's *post hoc* test. *P*<sub>posthoc</sub> corresponds altogether to *P*<sub>CTL versus 3g-PL</sub>, *P*<sub>CTL versus 5g-PL</sub> and *P*<sub>3g versus 5g-PL</sub>. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer: ceramides; CMRF: chylomicron-rich fraction; CTL: control; PL: polar lipids; SM: sphingomyelins; TAG: triacylglycerols.</figcaption></figure>

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<figure><img src="image_8.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_10.png" /></figure>

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770 **Figure 4: Milk PL ingestion modulate SM and Cer species in ileal efflux in ileostomy patients**

771 (see complements in Supplemental Figure 3, VALOBAB-D trial). Cumulated enrichment over 0-

772 480 min of total SM and Cer (A). Molecular composition of ileal content efflux after 8 h of

773 accumulation in SM (B) and Cer (C) species. Data are expressed in µmol and presented as mean ±

774 SEM (n=4/group) and empty circles represent individual values. Statistical analysis was done using

775 one-way ANOVA followed by Tukey's *post hoc* test (normal data) or Friedman's test followed by

776 Dunn's *post hoc* test (non-normal data). Letters "a" and "b" indicate statistically different intervention

777 effects between groups as calculated by *post hoc* analysis. Results are presented based on the

778 assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer:

779 ceramides; CTL: control; PL: polar lipids; SP: sphingolipids; SM: sphingomyelins.
<figure><img src="image_11.png" /><figcaption>**Figure 5:** Effect of milk PL supplementation during 4 weeks on SM and Cer species excreted in feces (see complements in Supplemental Table 4, VALOBAB-C trial). Molecular composition of SM (A) and Cer (B) in fecal samples (ΔV2-V1). Data are presented as mean ± SEM (control, n = 9; 3g-PL, n = 7; 5g-PL, n = 8) and expressed in µmol/ g of lyophilized feces. Empty circles represent individual values. Variations of specific SP species present in fecal samples were also determined and expressed as percentage of total SM and Cer, respectively: C16:0 SM (C), C22:0 SM (D), C24:0 SM (E), C16:1 SM (F), C22:1 SM (G), C24:1 SM (H), C16:0 Cer (I), C22:0 Cer (J), C24:0 Cer (K), C16:1 Cer (L), C22:1 Cer (M) and C24:1 Cer species (N) (ΔV2-V1). Statistical analysis was done using non-parametric analysis (non-normal data). Letters “a” and “b” indicate statistically different intervention effects between groups as calculated by *post hoc* analysis. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. Cer: ceramides; CTL: control; PL: polar lipids; SM: sphingomyelins.</figcaption></figure>

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<table><caption>Table 1: Impact of 4-week supplementation with milk PL on serum phospholipids and molecular composition of serum SM and Cer species (see complements in Supplemental Table 2).</caption><thead><tr><th rowspan="2"></th><th colspan="2">CTL</th><th colspan="2">3g-PL</th><th colspan="2">5g-PL</th><th rowspan="2">P<sub>group</sub></th><th rowspan="2">P<sub>PL</sub></th></tr><tr><th>V1</th><th>ΔV2-V1</th><th>V1</th><th>ΔV2-V1</th><th>V1</th><th>ΔV2-V1</th></tr></thead><tbody><tr><td colspan="9"><strong>Serum phospholipids (µg / 100 µL)</strong></td></tr><tr><td><strong>Total phospholipids</strong></td><td>227.56±8.68</td><td>-7.02±5.31</td><td>220.47±5.31</td><td>-6.69±5.04</td><td>230.34±5.89</td><td>-18.37±3.74</td><td>0.97</td><td>0.34</td></tr><tr><td><strong>PC</strong></td><td>150.28±5.33</td><td>-2.63±3.58</td><td>143.76±3.87</td><td>-3.51±3.61</td><td>154.49±3.89</td><td>-12.51±2.91</td><td>0.078</td><td>0.2</td></tr><tr><td><strong>PE</strong></td><td>4.94±0.36</td><td>-0.14±0.16</td><td>5.15±0.31</td><td>-0.36±0.28</td><td>5.43±0.28</td><td>-0.28±0.19</td><td>0.76</td><td>0.49</td></tr><tr><td><strong>PI</strong></td><td>19.17±1.92</td><td>-1.03±1.20</td><td>19.54±1.60</td><td>-0.84±1.32</td><td>18.83±1.61</td><td>-2.07±1.56</td><td>0.12</td><td>0.79</td></tr><tr><td><strong>LysoPC</strong></td><td>9.57±0.63</td><td>-0.32±0.38</td><td>8.99±0.47</td><td>-0.08±0.33</td><td>9.38±0.48</td><td>-0.75±0.34</td><td>0.38</td><td>0.81</td></tr><tr><td><strong>SM</strong></td><td>43.61±1.74</td><td>-2.9±1.16</td><td>43.03±1.55</td><td>-1.90±1.20</td><td>42.2±1.87</td><td>-2.76±0.90</td><td>0.79</td><td>0.68</td></tr><tr><td><strong>SM/PC</strong></td><td>0.29±0.01</td><td>-0.02±0.01</td><td>0.30±0.01</td><td>-0.01±0.01</td><td>0.27±0.01</td><td>0.00±0.01</td><td>0.098</td><td>0.069</td></tr><tr><td colspan="9"><strong>Molecular composition of serum SM (µmol / L)</strong></td></tr><tr><td><strong>Total SM</strong></td><td>368.11±29.41</td><td>4.35±24.82</td><td>347.52±20.81</td><td>-25.19±22.15</td><td>405.18±24.22</td><td>-36.37±13.31</td><td>0.37</td><td>0.17</td></tr><tr><td><strong>C16:0 SM</strong></td><td>121.11±14.14</td><td>1.95±7.74</td><td>114.84±10.01</td><td>-7.05±6.31</td><td>131.49±14.10</td><td>-12.44±3.10</td><td>0.25</td><td>0.12</td></tr><tr><td><strong>C16:1 SM</strong></td><td>17.82±2.04</td><td>0.44±0.97<sup>a</sup></td><td>17.29±1.49</td><td>-0.64±0.84<sup>a,b</sup></td><td>20.03±2.12</td><td>-3.35±0.50<sup>b</sup></td><td><strong>0.007</strong><sup>†</sup></td><td><strong>0.029</strong></td></tr><tr><td><strong>C18:0 SM</strong></td><td>28.48±2.78</td><td>0.81±1.40</td><td>27.62±2.20</td><td>-2.44±1.40</td><td>31.67±2.88</td><td>-3.40±1.66</td><td>0.13</td><td><strong>0.048</strong></td></tr><tr><td><strong>C18:1 SM</strong></td><td>13.94±1.69</td><td>1.02±0.87<sup>a</sup></td><td>13.78±1.01</td><td>-2.21±0.69<sup>a,b</sup></td><td>14.78±1.44</td><td>-2.99±0.79<sup>b</sup></td><td><strong>0.003</strong></td><td><strong>0.0007</strong><sup>†</sup></td></tr><tr><td><strong>C20:0 SM</strong></td><td>17.83±1.37</td><td>0.79±1.64</td><td>17.72±1.39</td><td>0.73±1.40</td><td>21.12±1.06</td><td>1.29±0.78</td><td>0.95</td><td>0.89</td></tr><tr><td><strong>C20:1 SM</strong></td><td>7.79±0.59</td><td>0.62±0.61<sup>a</sup></td><td>7.62±0.42</td><td>-0.71±0.5<sup>a,b</sup></td><td>8.87±0.59</td><td>-1.57±0.48<sup>b</sup></td><td><strong>0.025</strong></td><td><strong>0.013</strong></td></tr><tr><td><strong>C22:0 SM</strong></td><td>30.94±2.77</td><td>1.00±2.85</td><td>28.10±1.86</td><td>0.43±2.52</td><td>35.59±1.31</td><td>0.36±1.81</td><td>0.98</td><td>0.8</td></tr><tr><td><strong>C22:1 SM</strong></td><td>28.30±1.80</td><td>0.69±2.03</td><td>27.31±1.75</td><td>-0.27±2.42</td><td>33.16±1.37</td><td>-1.58±1.31</td><td>0.72</td><td>0.87</td></tr><tr><td><strong>C24:0 SM</strong></td><td>26.71±2.49</td><td>-0.84±2.63</td><td>23.04±1.6</td><td>-2.12±2.1</td><td>28.14±1.69</td><td>-1.89±1.92</td><td>0.91</td><td>0.67</td></tr><tr><td><strong>C24:1 SM</strong></td><td>74.11±6.12</td><td>-2.01±5.50</td><td>69.28±4.03</td><td>-10.75±5.26</td><td>79.27±4.95</td><td>-10.69±4.28</td><td>0.38</td><td>0.16</td></tr><tr><td><strong>C26:0 SM</strong></td><td>0.39±0.04</td><td>-0.03±0.05</td><td>0.33±0.03</td><td>-0.07±0.05</td><td>0.34±0.02</td><td>0.00±0.04</td><td>0.63</td><td>0.89</td></tr><tr><td><strong>C26:1 SM</strong></td><td>0.69±0.09</td><td>-0.08±0.07</td><td>0.59±0.05</td><td>-0.10±0.05</td><td>0.73±0.08</td><td>-0.10±0.10</td><td>0.98</td><td>0.85</td></tr><tr><td colspan="9"><strong>Molecular composition of serum Cer (µmol / L)</strong></td></tr><tr><td><strong>Total Cer</strong></td><td>8.26±0.54</td><td>0.34±0.59</td><td>8.34±0.66</td><td>0.49±0.79</td><td>10.3±0.77</td><td>-0.64±0.73</td><td>0.49</td><td>0.64</td></tr></tbody></table>
<table><tr><td>16:0 Cer</td><td>0.76±0.07</td><td>-0.01±0.06</td><td>0.71±0.07</td><td>0.09±0.13</td><td>0.86±0.08</td><td>-0.03±0.12</td><td>0.68</td><td>0.77</td></tr><tr><td>16:1 Cer</td><td>0.48±0.05</td><td>-0.05±0.06</td><td>0.41±0.04</td><td>0.02±0.03</td><td>0.45±0.04</td><td>-0.03±0.02</td><td>0.44</td><td>0.33</td></tr><tr><td>18:0 Cer</td><td>0.55±0.02</td><td>-0.02±0.02</td><td>0.58±0.03</td><td>0.07±0.08</td><td>0.65±0.07</td><td>-0.10±0.07</td><td>0.16</td><td>0.92</td></tr><tr><td>18:1 Cer</td><td>0.1±0.01</td><td>-0.01±0.01</td><td>0.09±0.01</td><td>0.01±0.02</td><td>0.11±0.01</td><td>-0.01±0.01</td><td>0.55</td><td>0.61</td></tr><tr><td>20:0 Cer</td><td>0.89±0.1</td><td>-0.07±0.04</td><td>0.90±0.10</td><td>0.06±0.08</td><td>1.00±0.10</td><td>-0.03±0.07</td><td>0.41</td><td>0.30</td></tr><tr><td>20:1 Cer</td><td>0.19±0.08</td><td>0.01±0.03</td><td>0.19±0.08</td><td>0.00±0.01</td><td>0.25±0.09</td><td>0.00±0.03</td><td>0.94</td><td>0.75</td></tr><tr><td>22:0 Cer</td><td>0.89±0.08</td><td>0.09±0.08</td><td>0.93±0.09</td><td>0.11±0.10</td><td>1.25±0.14</td><td>0.00±0.13</td><td>0.74</td><td>0.77</td></tr><tr><td>22:1 Cer</td><td>0.05±0.01</td><td>-0.01±0.01</td><td>0.04±0.00</td><td>0.00±0.00</td><td>0.05±0.00</td><td>0.00±0.01</td><td>0.76</td><td>0.57</td></tr><tr><td>24:0 Cer</td><td>2.71±0.32</td><td>0.30±0.35</td><td>2.72±0.26</td><td>0.21±0.26</td><td>3.54±0.4</td><td>-0.06±0.28</td><td>0.69</td><td>0.55</td></tr><tr><td>24:1 Cer</td><td>1.48±0.13</td><td>0.11±0.08<sup>a</sup></td><td>1.58±0.19</td><td>-0.19±0.08<sup>a,b</sup></td><td>1.96±0.17</td><td>-0.37±0.18<sup>b</sup></td><td><strong>0.033</strong></td><td><strong>0.016</strong></td></tr><tr><td>26:0 Cer</td><td>0.11±0.02</td><td>0.01±0.02</td><td>0.11±0.01</td><td>0.12±0.12</td><td>0.14±0.02</td><td>0.00±0.02</td><td>0.43</td><td>0.57</td></tr><tr><td>26:1 Cer</td><td>0.05±0.01</td><td>-0.01±0.01</td><td>0.05±0.01</td><td>-0.01±0.01</td><td>0.05±0.00</td><td>-0.01±0.01</td><td>0.92</td><td>0.81</td></tr></table>

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Data are presented as mean ± SEM, n = 10 / group. Results are presented based on the assumption of sphingosine d18:1 as the major sphingoid base for determined SM and Cer species. *P* values presented in bold highlight significant intervention effect. *P*<sub>group</sub> represents *P* value associated with group effect as calculated by generalized linear model, while *P*<sub>PL</sub> represents *P* value associated with binary effect of milk PL compared to control. † *P* value remains significant (< 0.05) after adjustment for clinical center, quartiles of volunteer age and waist circumference. <sup>a,b</sup> Different superscript letters indicate statistically different intervention effects between groups as calculated by *post hoc* analysis.