Cholecalciferol (vitamin D3) and retinyl palmitate (vitamin A) display different solubilization capacities in mixed micelle solutions: effect of interactions with mixed micelle components and of cholecalciferol self-associating properties.

Damien P. Prévéraud, Charles Desmarchelier, David Chapron, Ali Makky, Véronique Rosilio, Patrick Borel

► To cite this version:

Damien P. Prévéraud, Charles Desmarchelier, David Chapron, Ali Makky, Véronique Rosilio, et al. Cholecalciferol (vitamin D3) and retinyl palmitate (vitamin A) display different solubilization capacities in mixed micelle solutions: effect of interactions with mixed micelle components and of cholecalciferol self-associating properties. 16. Fat Soluble Vitamins Congress - March 21-23, 2017 - Paris, Société Française des Vitamines et Biofacteurs (SFVB). FRA., Mar 2017, Paris, France. 130 p. hal-01595512

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16th **Fat**
**Soluble**
**Vitamins**
CONGRESS

Damien P. Prévéraud

CHOLECALCIFEROL (VITAMIN D3) AND RETINYL PALMITATE (VITAMIN A) DISPLAY DIFFERENT SOLUBILIZATION CAPACITIES IN MIXED MICELLE SOLUTIONS:

* EFFECT OF INTERACTIONS WITH MIXED MICELLE COMPONENTS AND OF CHOLECALCIFEROL SELF-ASSOCIATING PROPERTIES
# Vitamins A and D in food of animal origin

<figure><img src="image_3.png" /></figure>

Vitamin A → retinyl palmitate (RP)

<figure><img src="image_4.png" /></figure>

Molecular Formula
Average mass
Log P

$$C_{36}H_{60}O_2$$
524.86 Da
$$14.83 \pm 0.45$$

<figure><img src="image_5.png" /></figure>

Vitamin D → cholecalciferol (D3)

<figure><img src="image_6.png" /></figure>

Molecular Formula
Average mass
Log P

$$C_{27}H_{44}O$$
384.638 Da
$$9.72 \pm 0.27$$

<figure><img src="image_7.png" /></figure>

Digestion process in the upper gastrointestinal tract (¹):

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

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

Micellization is a key step for bioavailability

<footnote n="1">¹Borel, P., Caillaud, D. and Cano, N. J. (2013) *Crit. Rev. Food Sci. Nutr.* 55.; ²Reboul, E. and Borel, P. (2011) *Prog. Lipid Res.* 50.</footnote>
# Interactions with mixed micelles components

<figure><img src="image_10.png" /><figcaption>mixed micelles</figcaption></figure>

<table><thead><tr><th rowspan="2">components</th><th colspan="3">ORIGIN</th></tr><tr><th>diet</th><th>bile</th><th>digestion products</th></tr></thead><tbody><tr><td>Bile salts</td><td></td><td>✓</td><td></td></tr><tr><td>Cholesterol</td><td>✓</td><td>✓</td><td></td></tr><tr><td>Phospholipids</td><td>✓</td><td>✓</td><td></td></tr><tr><td>Lysophospholipids</td><td></td><td>✓</td><td>✓</td></tr><tr><td>Fatty acids</td><td></td><td></td><td>✓</td></tr><tr><td>Monoglycerides</td><td></td><td></td><td>✓</td></tr></tbody></table>

☐ Mixed micelle lipid composition affects the solubility of fat-soluble vitamins:

- Substitution of lysophospholipids by phospholipids = lymphatic absorption of vitamin E ↗ in rat (Koo and Noh, 2001 J Nutr)

- Change in FA profile (chain length and saturation degree) = absorption of D3 ↗ in perfused rat (Hollander, 1978 Gut)

- Addition of oleic and linoleic acids = absorption of D3 ↗ (Hollander, 1978 Gut)

**Objectives:**

(i) compare the relative solubility of D3 and RP in the aqueous phase rich in mixed micelles

(ii) study the interactions between these vitamins and the mixed micelle components

<figure><img src="image_11.png" /></figure>
Methods

<figure><img src="image_12.png" /></figure>
# Incorporation efficiency of RP and D3 in mixed micelles

<figure><img src="image_13.png" /></figure>

Desmarchelier *et al.* (2013) *MNFR*

<figure><img src="image_14.png" /></figure>

☐ Vitamin analysis by HPLC

<figure><img src="image_15.png" /></figure>

Société Française
Vitamines & Biofacteurs
interactions between RP and D3, and the mixed micelle
components

☐ Surface tension measurement:

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

→ Critical Micellar Concentration (CMC)

☐ Surface pressure measurement:

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

-> Compression isotherms

→ Surface compressional modulus

$$
K = -A \frac{d\pi}{dA}
$$

☐ Cryo-TEM (Transmission Electron Microscopy) analysis:

<figure><img src="image_22.png" /></figure>

→ Electron micrographs

<figure><img src="image_23.png" /></figure>

Société Française
Vitamines & Biofacteurs
# Results
Solubilization of RP and D3 in mixed micelle solutions

<figure><img src="image_24.png" /></figure>

☐ D3 solubilization was linear (R²=0.98, regression slope=0.71) and higher than that of RP, which reached a plateau with a maximum concentration around 125 µM
Interfacial behaviour of micelle components

<figure><img src="image_25.png" /></figure>

LDP showed better surface parameters (CMC and surface tension) than NaTC

LDP aimed to improved NaTC surface tension and CMC by inserting well in NaTC domain

D3 and RP solubilization depend on their capacity to penetrate the LDP and NaTC domains

<figure><img src="image_26.png" /></figure>
Interfacial behaviour of the 2 vitamins and lipids

<figure><img src="image_27.png" /></figure>

<table><thead><tr><th>Monolayer</th><th>A<sub>c</sub> (Å<sup>2</sup>)</th><th>π<sub>c</sub> (mN/m)</th><th>K<sub>max</sub> (mN/m)</th></tr></thead><tbody><tr><td>PC</td><td>54.0</td><td>40.0</td><td>112.7</td></tr><tr><td>LysoPC</td><td>32.0</td><td>36.0</td><td>44.8</td></tr><tr><td>Monoolein</td><td>27.0</td><td>41.0</td><td>94.7</td></tr><tr><td>Oleic acid</td><td>26.0</td><td>37.3</td><td>96.5</td></tr><tr><td>Cholesterol</td><td>38.0</td><td>44.4</td><td>939.8</td></tr><tr><td>LDP mixture</td><td>29.0</td><td>41.0</td><td>115.4</td></tr><tr><td>D<sub>3</sub></td><td>35.2</td><td>38.0</td><td>187.4</td></tr><tr><td>RP</td><td>56.0</td><td>16.2</td><td>42.8</td></tr></tbody></table>

A<sub>c</sub>: collapse molecular area, π<sub>c</sub>: collapse surface pressure.

K<sub>max</sub>: maximum compressibility modulus.

Based on K<sub>max</sub>, the lipid monolayers can be classified into **poorly organized** (K < 100 mN/m, for lyso-PC, monoolein, and oleic acid), **liquid condensed** (100 < K < 250 mN/m, for POPC and the LDP mixture) and **highly rigid monolayers** (K > 250 mN/m, for cholesterol)

D3 and LDP exhibit similar interfacial behavior, whereas RP show very different behavior compared to the micelle lipids

<figure><img src="image_28.png" /></figure>
Interaction of the 2 vitamins with NaTC and LDP

<table><thead><tr><th>Sample</th><th>C<sub>micelle</sub><br>(mM)</th><th>C<sub>vitamin</sub><br>(mM)</th><th>Surface tension<br>(mN/m)</th></tr></thead><tbody><tr><td>NaTC micelles</td><td>5</td><td>0</td><td>44.9 ± 0.1</td></tr><tr><td>NaTC micelles + D3</td><td>5</td><td>0.1</td><td>36.8 ± 0.5</td></tr><tr><td>NaTC micelles + RP</td><td>5</td><td>0.1</td><td>37.9 ± 0.5</td></tr></tbody></table>

☐ NaTC micelle barely affect by D3 or RP.

→ D3 and RP insert into NaTC domain (same effect on its organization)

<figure><img src="image_29.png" /></figure>

☐ For LDP/D3, surface pressure was controlled by LDP

☐ For LDP/RP, surface pressure was controlled by RP

<figure><img src="image_30.png" /></figure>

☐ D3 conferred a better rigidity of LDP monolayers than RP:

→ Micelles are more stable

→ Better capacity to solubilize at low concentration

<figure><img src="image_31.png" /></figure>
# Interaction of the 2 vitamins with mixed micelle

→ Is that also true for higher concentration ?

<table><thead><tr><th>Sample</th><th>C<sub>micelle</sub> (mM)</th><th>C<sub>vitamin</sub> (mM)</th><th>Surface tension (mN/m)</th></tr></thead><tbody><tr><td>Mixed micelles</td><td>6.1</td><td>0</td><td>29.0 ± 0.2</td></tr><tr><td>Mixed micelles + D3</td><td>6.1</td><td>0.1</td><td>31.6 ± 0.1</td></tr><tr><td>Mixed micelles + D3</td><td>6.1</td><td>1</td><td>30.5 ± 0.5</td></tr><tr><td>Mixed micelles + RP</td><td>6.1</td><td>0.1</td><td>29.5 ± 0.2</td></tr><tr><td>Mixed micelles + RP</td><td>6.1</td><td>1</td><td>31.8 ± 0.5</td></tr></tbody></table>

☐ Low impact of vitamin concentration in mixed micelles
cholecalciferol self-associating properties

☐ Since (i) D3 showed an interfacial behavior similar to that of the lipid mixture, and since (ii) it could be solubilized at very high concentrations in an aqueous phase rich in mixed micelles, then its self-assembling properties were more specifically investigated:

<figure><img src="image_32.png" /></figure>

☐ D3 can self-associate to form micelles at concentrations ≥ 0.1 mM

☐ D3 forms various organized systems at higher concentrations (1 mM):
Fibers (nanotubes), aggregates
Conclusion

<figure><img src="image_33.png" /></figure>
# Summary of Findings - Conclusion

* D3 displays a higher solubility in mixed micelles than RP... WHY ?

* Solubility of vitamins in mixed micelles is function of their interfacial behavior and affinity with micelle components:

* Even if $[LDP] < [NaTC]$ in micelle, solubility of RP and D3 is dependent of their capacity to insert into both NaTC and LDP domains:

* D3 has similar behavior than LDP, whereas RP is very different

* Both RP and D3 can insert into NaTC

* the different abilities of the two vitamins to solubilize into mixed micelles is based on their interactions with micelle components,

* but it is also due to the fact that, conversely to RP, D3 can self-associate in structures that are readily soluble in an aqueous phase.

* Whether these D3 self-assemblies are available for absorption by the intestinal cell needs further studies.

<figure><img src="image_34.png" /></figure>
# Cholecalciferol (vitamin D3) and Retinyl Palmitate (vitamin A) display different solubilization capacities in mixed micelle solutions: effect of interactions with mixed micelle components and of cholecalciferol self-associating properties

Damien P. Prévéraud<fnref n="1" />*, Charles Desmarchelier², David Chapron³, Ali Makky³, Véronique Rosilio³, Patrick Borel²

¹Adisseo France SAS, Centre of Expertise and Research in Nutrition, Commentry, France; ²UMR 1260 INRA/1062 INSERM/Aix-Marseille University, "Nutrition, Obesity and Risk of Thrombosis", Marseille, France; ³University Paris-Sud, CNRS UMR 8612, Institut Galien Paris Sud, Châtenay-Malabry, France; *damien.preveraud@adisseo.com

## Scope

- Vitamin A (as retinyl palmitate, RP) and vitamin D (as cholecalciferol, D3): the 2 main fat-soluble vitamins found in food and animal origin. These vitamins in the human upper gastrointestinal tract during digestion is assumed to follow that of dietary lipids<sup>1,3</sup>; this includes emulsification, solubilization in mixed micelles, diffusion across the unstirred layer and transfer to the enterocyte<sup>2</sup>.

- Solubilization in mixed micelles (micellarization): a key step for their bioavailability.

- Mixed micelles are mainly made of a mixture of bile lipids (bile salts, cholesterol, phytolipids, lysophospholipids, monoglycerides).

- Mixed micelle lipid composition has been shown to significantly affect vitamin solubility<sup>3</sup> and the possibility to be used to question the need of mixed micelles for its solubilization in the aqueous environment of the intestinal tract lumen<sup>6-7</sup>.

☐ This study was designed to compare in vitro the relative solubility of D3 and RP in the intestinal lumen during digestion and to study by surface tension and surface pressure measurements the interactions between these vitamins and the mixed micelle composition.

## Methods

* Synthesis of mixed micelles and measurement of D₃ and RE incorporation efficiency by HPLC

<figure><img src="image_35.png" /></figure>

* Surface tension measurement and determination of the critical micelle concentration (cmc)

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

Tensiometer K10 (Kross)

* Analysis of the molecular interactions between micelle components and vitamins by surface pressure measurements (Langmuir trough)

<figure><img src="image_38.png" /></figure>

* Cryo-TEM (Transmission Electron Microscopy) analysis

<figure><img src="image_39.png" /></figure>

Come and see
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Oslo room (Level -1)

<table><caption>Figure 3. Interaction of D3 and RP with NaTC</caption><thead><tr><th>Component of solute</th><th>Micelle solution concentration (mM)</th><th>Vitamin concentration (nm)</th><th>Surface tension (mN/m)</th></tr></thead><tbody><tr><td>NaTC micelles</td><td>0</td><td>0.1</td><td>44.9 ± 0.1</td></tr><tr><td>NaTC micelles + RP</td><td>0.5</td><td>0.1</td><td>34.8 ± 0.1</td></tr><tr><td>NaTC micelles + D3</td><td>0.5</td><td>0.1</td><td>37.1 ± 0.3</td></tr></tbody></table>

<figure><img src="image_40.png" /><figcaption>Figure 5. Self-assembling of D3 at concentrations higher than 1 µM</figcaption></figure>

<figure><img src="image_41.png" /><figcaption>→ Co-existing molecular assemblies in 1 mM pure D3 hydrated system.</figcaption></figure>

## Conclusion

The results obtained show that D3 displays a higher solubility in mixed micelles than RP. This difference is mainly due to the different abilities of the two vitamins to solubilize into mixed micelles based on their interactions with micelle components, but it is also due to the fact that, conversely to RP, D3 can self-associate in structures that are readily soluble in an aqueous phase. Whether these D3 self-assemblies are available for absorption by the intestinal cell needs further studies.

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<footnote n="1">¹Borel, P., Caillaud, D., and Cano, N. J. (2013) Crit. Rev. Food Sci. Nutr. 55: ⁵Reboul, E., and Borel, P. (2011) Prog. Lipid Res. 50: ⁵Koo, S. I., and Noh, S. K. (2001) J. Nutr. 131: ⁴Hollander, D., Muralidhara, S., and Zimmerman, A. (1978) Gut 19: ⁵Meredith, S. C., Bolt, M. J., and Rosenberg, I. H. (1984) J. Colloid Interface Sci. 99: ⁶Rautureau, M., and Rambaud, J. C. (1981) Gut 22: ⁷Maislos, M., and Shany, S. (1987) Isr. J. Med. Sci. 23</footnote>
Damien P. Prévéraud

ADISSEO
A Bluestar Company

Centre of Expertise and Research
in Nutrition, Commentry (France)

Charles Desmarchelier,
Patrick Borel

<figure><img src="image_42.png" /></figure>

INRA
SCIENCE & IMPACT

<figure><img src="image_43.png" /></figure>

Inserm

<figure><img src="image_44.png" /></figure>

Aix*Marseille
université

UMR 1260 INRA/1062
INSERM/Aix-Marseille University,
"Nutrition, Obesity and Risk of
Thrombosis", Marseille (France)

David Chapron, Ali Makky,
Véronique Rosilio

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>

CNRS UMR 8612, Institut Galien
Paris Sud, University Paris-Sud,
Châtenay-Malabry (France)