1. Introduction
Defects in either intraluminal lipid digestion or uptake and transport of its digestive products across the gut barrier may lead to lipid malabsorption [
1,
2]. Clinically, lipid malabsorption is well recognized by steatorrhea and may bear severe consequences if the enzymatic activity in the upper intestine drops below 10% of what is considered normal [
2,
3]. Another example is cystic fibrosis, an autosomal recessive genetic disorder affecting multiple organ systems, most critically the lungs. However, it also exhibits pancreatic insufficiency that leads to malabsorption of dietary lipids. Enzyme replacement therapy though helpful still shows a certain degree of steatorrhea which indicates additional underlying causes of malabsorption. Indeed, it has been suggested that this type of persistent malabsorption is not only due to insufficient enzymes but also due to incomplete intraluminal solubilization or reduced enterocyte uptake, or both [
2]. Another clinical scenario of lipid malabsorption is observed after intestinal surgeries. Gastrojejunostomy, ileostomy and similar surgeries may lead to reduced surface area for absorption, thereby creating a malabsorption condition. Likewise, cholecystectomy may also have an impact due to an impaired secretion of the bile required for emulsification of lipids for efficient lipolytic activity. In these and other malabsorption conditions, management is imperative as lipids are not only a source of energy, but also of bioactive nutrients. Minor lipids found in the human diet, for example, α-linolenic acid (ALA, 18:3
n-3) and linoleic acid (LA, 18:2
n-6) are truly essential because humans lack the enzymes required for their biosynthesis. These FA also are precursors to other biologically important LC-PUFA such as arachidonic acid (ARA, 20:4
n-6), eicosapentaenoic acid (EPA, 20:5
n-3) and docosahexaenoic acid (DHA, 22:6
n-3) that are proposed to have multiple benefits.
The bulk of dietary lipid is contributed by triacylglycerols (TAG). Distribution and composition of fatty acids (FA) in dietary TAG has an impact on their bioavailability and subsequently their exerted function on the body. During digestion, dietary TAG encounters the effects of gastrointestinal lipases resulting in formation of
sn-2 monoacylglycerol (MAG) and two free FA that are absorbed by enterocytes [
4]. Within enterocytes, the
sn-2 MAG are reacylated to TAG and are released into lymphatic circulation via chylomicrons. FA released from the
sn-1 and
sn-3 positions often have different metabolic fates than FA at the
sn-2 position as they are retained on the glycerol backbone in absence of
sn-2 specific lipolytic enzymes. Therefore they cross apical barrier as 2-MAG, exerting different solubility than free FA [
5]. The metabolic fates and hydrolysis rates depend on the FA chain length, nature of the FA in the TAG molecule and stereospecific (
sn) location on the TAG [
4,
6]. Indeed studies have shown that the use of interesterified lipids with modified FA composition (
i.e., palmitic acid, stearic acid) at the
sn-2 position may affect the amount absorbed [
1,
7].
Researchers have employed different strategies of preclinical models to study lipid malabsorption. One employs partial small bowel resection in rats [
8] to reduce absorptive surface area. Alternatively, if the interest is primarily in cystic fibrosis, a knockout mice model of cystic fibrosis transmembrane conductance regulator (CFTR) may be used [
9]. Finally, a lipase inhibiting compound like Orlistat (40% lower lipid absorption) may be used to create lipid malabsorption like condition when dietary lipids are provided as TAG in rats [
6,
10]. It is imperative that the selection of the model should be commensurate with the hypothesis being tested.
Current strategies to combat lipid malabsorption may include dietary supplements, intestinal enzyme therapy and treating underlying causes such as inflammation. Alternatively, dietary solutions can be sought to enhance absorption in deficient conditions. In case of enzyme insufficiency, administration of partially digested TAG, such as MAG, may help intraluminal solubilization and enterocyte uptake. Therefore, the objective of the present study was to identify if MAG provided in different forms are potential vehicles of fatty acids in conditions of low lipases activity.
Generally MAG oils can be produced by esterification of FA with glycerol followed by short-path distillation purification [
11]. MAG can occur as two different type of positional isomers:
sn-1(3)-MAG and
sn-2-MAG. Unsaturated
sn-2-MAG are not stable and readily isomerized to give rise to
sn-1(3)-MAG [
12]. Therefore,
sn-2-MAG cannot be used in feeding experiment as free form and must be chemically protected in order to remain stable during the trial. In the present study, two protective agents have been used to stabilize
sn-2 MAG: esterification with acetic acid which leads to formation of diacethyl-MAG derivatives and protection with vanillin giving rise to the formation of an acetal (
Figure 1). In addition to these protected
sn-2-MAG variants we also compared a free MAG by feeding rats receiving Orlistat. We assessed the accretion of the FA of interest, EPA in plasma and RBC for 21 days.
Figure 1.
Chemical structures and the name of the compounds tested in the present study: (A) Vanillin acetal of sn-2-MAG; (B) 1,3-diacetyl-2-MAG; (C) sn-1(3)-MAG; R indicates a fatty acid as eicosapentaenoic (EPA) or docosahexaenoic acid (DHA).
Figure 1.
Chemical structures and the name of the compounds tested in the present study: (A) Vanillin acetal of sn-2-MAG; (B) 1,3-diacetyl-2-MAG; (C) sn-1(3)-MAG; R indicates a fatty acid as eicosapentaenoic (EPA) or docosahexaenoic acid (DHA).
2. Methods and Materials
2.1. animals and Experimental Diet
All experimental procedures involving animals were approved by the cantonal veterinary office in Switzerland and revised by the internal ethical committee of the Nestlé Research Center. Male Wistar rats (n = 30, 270 ± 5 g) were housed in independent cages (Euro standard Type III H: 425 × 266 × 185 mm, floor area 800 cm2, Techniplast, Switzerland), received tap water and diets ad libitum for 21 days. The animals were randomly divided into five groups.
Fish Oil (FO) diet consisted of AIN 93M base diet supplemented with fish oil (Sofinol S.A., Manno, Switzerland), high oleic sunflower oil (HOSO, Sofinol S.A., Manno, Switzerland) and cocoa butter (Gerkens Cacao
®, Deventer, The Netherlands) as a lipid source. Total lipid content was 20 g/100 g diet (approximately 40% of the energy), see
Table 1. Diets Fish oil + Orlistat (FO + O), free MAG + O,
sn-2 MAG vanillin acetal + Orlistat (Vanil + O) and Acetylated
sn-2 MAG + Orlistat (Acetyl + O) were AIN 93M base diet supplemented with FO, HOSO and either MAG Vanillin Acetal (Stepan Co., Northfield, IL, USA), Diacetylated MAG (Stepan Co., Northfield, IL, USA) or free MAG (Cognis GmbH, Illertissen, Germany) + Orlistat (MAG + O) containing 95.8% of α-MAG, as previously characterized [
9] (
Figure 1), plus 400 mg of Orlistat/kg of diet as listed in
Table 1. The diets were prepared by mixing all the ingredients except Orlistat. The homogenized powders were dried at a low temperature and stored in a small sachet under vacuum at −20 °C. These special precautions were taken to avoid oxidative degradation of
n-3 LC-PUFA. All animals were fed with a paste (water/powder diet, 1:1 w/w) prepared freshly every day. For the groups receiving Orlistat, the drug was added to the diet at 400 mg/kg every time the paste was prepared. FA composition of diets is described in
Table 2.
Table 1.
Composition of the experimental diets (g/kg of dry matter).
Table 1.
Composition of the experimental diets (g/kg of dry matter).
| Nutrients (g/kg) | FO | FO + O | Vanil + O | Acetyl + O | MAG + O |
|---|
| Lipids (Total) | 200 | 200 | 200 | 200 | 200 |
| Cocoa butter | 100 | 100 | 100 | 100 | 100 |
| High Oleic Sunflower oil | 72 | 72 | 60 | 70 | 79 |
| Fish oil | 28 | 28 | - | - | - |
| MAG Vanillin Acetal | - | - | 40 | - | - |
| Diacetylated MAG | - | - | - | 30 | - |
| MAG | - | - | - | - | 21 |
| EPA (20:5 n-3) | 4.4 | 4.4 | 4.4 | 4.2 | 4.7 |
| DHA (22:6 n-3) | 2.9 | 2.9 | 7.4 | 3.6 | 3.1 |
| Orlistat | - | 0. 4 | 0. 4 | 0. 4 | 0. 4 |
| Corn starch | 461 | 461 | 461 | 461 | 461 |
| α-Casein | 140 | 140 | 140 | 140 | 140 |
| Sucrose | 100 | 100 | 100 | 100 | 100 |
| Cellulose | 50 | 50 | 50 | 50 | 50 |
| Mineral mix AIN-93M | 35 | 35 | 35 | 35 | 35 |
| Vitamin mix AIN-93M | 10 | 10 | 10 | 10 | 10 |
| l-cysteine | 1.8 | 1.8 | 1.8 | 1.8 | 1.8 |
| Choline bitartrate | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| Butylhydroxytoluene | 0.008 | 0.008 | 0.008 | 0.008 | 0.008 |
Table 2.
Weight Parameters, Dietary Intake and Apparent Lipid Absorption in rats fed different oils for 21 days.
Table 2.
Weight Parameters, Dietary Intake and Apparent Lipid Absorption in rats fed different oils for 21 days.
| Experimental Groups |
|---|
| FO | FO + O | Vanil + O | Acetyl + O | MAG + O |
|---|
| Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD | Mean ± SD |
|---|
| Final body weight (g) | 317.7 ± 13.1 | 310.6 ± 7.6 | 314.6 ± 4.2 | 316.5 ± 8.8 | 310.4 ± 13.6 |
| Weight gain (g/day) | 4.4 ± 0.6 | 4.0 ± 0.3 | 4.2 ± 0.7 | 4.6 ± 0.3 | 4.1 ± 0.5 |
| Fat Mass (g) | Day 0 | 30.7 ± 1.6 | 30.1 ± 0.4 | 29.0 ± 1.8 | 29.1 ± 0.7 | 29.4 ± 0.8 |
| Day 21 | 54.4 ± 4.0 | 46.1 ± 1.3 | 49.6 ± 2.7 | 52.3 ± 3.0 | 50.2 ± 5.6 |
| Lean Mass (g) | Day 0 | 179.0 ± 5.6 | 181.2 ± 3.7 | 181.6 ± 3.5 | 178.6 ± 4.6 | 178.6 ± 4.7 |
| Day 21 | 223.8 ± 8.8 | 225.2 ± 5.8 | 226.3 ± 3.1 | 224.8 ± 4.9 | 220.1 ± 7.7 |
| Lipid Intake (g/day) | 4.1 ± 0.2 | 4.6 ± 0.2 | 5.2 ± 0.1 * | 4.9 ± 0.3 | 4.9 ± 0.3 |
| Daily Food Intake (g/day) | 20.7 ± 0.8 | 22.8 ± 0.9 | 25.0 ± 0.6 * | 23.9 ± 1.4 | 23.9 ± 1.7 |
| Fecal Lipid excretion (g/day) | 0.43 ± 0.05 *** | 2.49 ± 0.30 | 3.10 ± 0.17 *** | 1.97 ± 0.21 *** | 3.16 ± 0.31 *** |
| Apparent Lipid absorption (%) | 89.5 ± 0.8 *** | 44.8 ± 4.7 | 37.1 ± 2.1 *** | 58.5 ± 2.5 *** | 32.9 ± 3.6 *** |
| EPA Intake (mg/day) | 91.08 ± 3.5 | 100.32 ± 4.2 | 110.0 ± 3.2 | 100.38 ± 6.2 | 112.33 ± 7.5 |
| DHA Intake (mg/day) | 60.03 ± 3.5 | 66.12 ± 4.2 | 185 ± 3.2 *** | 86.04 ± 6.2 ** | 74.09 ± 5.3 |
2.2. Experimental Design
Body weight was recorded twice a week for 21 days. Food intake was recorded five times a week. Body composition (fat and lean mass) was determined in animals in triplicate by quantitative nuclear magnetic resonance spectroscopy (Echo MRI-4in 1/500™; Echo Medical Systems, Houston, TX, USA), before the diet intervention (Day 0) and the day of the necropsy (Day 21). For determination of the fecal lipid excretion, animals were placed in metabolic cages for 48 h starting at day 17. Apparent lipid absorption was calculated using lipid intake and excretion data after feces were collected for 48 h and the food intake was monitored over this time period.
Blood was collected into heparinized tubes from the caudal vein after 6 h of food restriction at days 3, 7 and 14. The day of the necropsy (Day 21), animals were anesthetized with isoflurane. Blood was collected from the abdominal aorta, and then RBC and plasma were separated by centrifugation at 626× g for 2 min. Plasma and RBC were stored at −80 °C until lipid analyses were carried out. Liver, brain, retina and spleen were removed and transferred to different vials, flash frozen with liquid nitrogen and stored at −80 °C until further analyses.
2.4. Fatty Acid Methyl Esters Preparation and Analysis by Gas Chromatography
FAMEs in plasma (60 μL) were prepared by mixing sample with a methanolic solution of hydrochloric acid (2 mL, 1.5 N, Supelco, Bellefonte, Palo Alto, CA, USA), methanol (2 mL) and
n-hexane (1 mL) in a test tube. Tubes were then heated at 100 °C for 60 min [
10]. After cooling down to room temperature, water (2 mL) was added and tubes were centrifuged at 2000 rpm for 4 min. The organic phase was collected for GC analyses. Direct methylation of RBC (100 μL) was performed as described for plasma having RBC previously washed with PBS buffer. Tissue fat was methylated from the fat extracted, as described in the previous section.
Analysis of total FAMEs was performed on a 7890 Agilent gas chromatograph (Agilent Technologies, Palo Alto, CA, USA), equipped with a fused-silica BPX-70 capillary column (10 m × 0.1 mm I.D., 0.2 μm film thickness; SGE, Melbourne, Australia). Split injector (25:1) and flame ionization detection (FID) systems were operating at 250 °C. Oven temperature programming was 50 °C isothermal for 0.2 min, increased to 180 °C at 120 °C/min, isothermal for 1 min at this temperature then increased to 220 °C at 20 °C/min and then to 250 °C at 50 °C/min. The carrier gas (H
2) was maintained at a constant 1 mL/min and the acquisition of the FID signal at 100 Hz [
13].
2.5. Statistical Analyses
The main comparisons assessed are the comparisons between the positive control FO + O and the treatment groups Vanil + O, Acetyl + O, and MAG + O. In order to ensure that the addition of Orlistat lowered the absorption of fatty acids, the positive control was also compared to the negative control in this case FO.
Data is presented as means ± standard error of the mean (S.E.M.), except for FA relative percentage in RBC, plasma, liver, spleen, retina and brain where median ± robust SEM are provided. For food intake, fat intake, fecal lipid excretion and apparent lipid absorption (%), an ANOVA and two-sided appropriate contrasts were calculated. For body weight parameters, for days 7, 14 and 21 on body weight, mixed models were performed with body weight at day 3 as covariate, day as continuous variable and subject as random variable. One-sided tests were calculated. For fat mass and lean mass parameters, an ANOVA with parameters at D-3 as covariate was performed. Appropriate one sided contrasts were calculated. For FA in different organs, Kruskal Wallis and exact Wilcoxon tests were performed. One-sided tests were calculated. Statistical calculations were performed using SAS software, version 9.1. Differences were considered as significant at P < 0.05.
4. Discussion
In the current study, we wanted to explore if protected or free MAG lipids containing EPA and DHA has an influence on absorption and accretion in tissues under lipid malabsorption conditions. We selected three different glyceride derivatives that would, hypothetically require minimum digestion before crossing the gut barrier, and thus have an improved bioavailability of EPA and DHA. The tested molecules were: structured (
A) vanillin acetal of
sn-
2 MAG and (
B) diacetyl derivative of
sn-2 MAG and (C) free MAG mixture of isomers. A fourth group of rats were fed FO as a source of dietary TAG. The diets were made so that the EPA content was comparable (approximately 4.4 g/kg of diet) while DHA levels varied (2.9–7.4 g/kg diet). As expected from our previous experience [
10], blood and tissue levels of EPA and DHA were lower in the Orlistat-fed group that induced malabsorption. In contrast, all experimental molecules were able to restore circulating and tissue levels of EPA in malabsorbing rats. These results suggest that absorption of structured or free MAG does not depend on lipolytic activities impaired by Orlistat. In the case of MAG vanillin acetal, the cleavage of the acetal moiety may happen at acidic pH in the stomach, and the resulting MAG likely being absorbed as such by the enterocytes. However, it is probable that the released
sn-2 MAG isomerize readily in the lumen to
sn-1(3)-isomers before absorption. The acyl migration is due to the fact that primary esters are more stable than the secondary esters. Results with the tested diacetylated MAG suggest that the occurrence of the two acetyl groups does not modify the polarity of the
sn-2 MAG, allowing an efficient uptake by the intestinal cells. These results observed with the two structured MAG derivatives show that these molecules are efficient vehicles of EPA in malabsorption conditions. However, we recognize the limitations observed during production of structured MAG. Ethyl esters are used for the preparation of protected MAG and reaction yield is never complete, leaving them behind as ethyl esters of FA. Indeed, substantial amounts of ethyl ester of FA were detected in our product upon analyses. A part of the ethyl ester fraction can be removed by distillation, but the resulting material is never 100% pure. Therefore, the difference of purity between protected and free MAG may explain part of the difference observed. The free MAG utilized in our study was characterized as having exclusively
sn-1(3) isomers of EPA [
12]. Free MAG showed better absorption efficiencies and accretion to tissues when compared to the protected MAG molecules. It was our assumption that since EPA is esterified to
sn-1(3) positions; it will require action of lipolytic enzymes. Given the fact that Orlistat is not a complete inhibitor of pTGL (pancreatic triglyceride lipases), the available activity might have produced glycerol, thus allowing better micellarization of cleaved EPA for optimal uptake.
Indeed, this is the first study of its kind, where protected molecules of MAG have been tried and, therefore, comparison to existing data is not possible. However, many other strategies have been tried in past to overcome lipid malabsorption. For example, medium-chain triacylglycerols have been used for treatment of impaired intestinal lipolysis, or as a source of energy in parenteral nutrition. However, it increases total cholesterol concentration in primary hypertriglyceridemic subjects [
14]. Alternatively, enzymatic interesterified fish oil with medium-chain FA with decanoic acid (10:0) at
sn-1/3 positions showed an improved lymphatic transport of
n-3 FA at 24 h in malabsorbing rats [
15]. Another approach involves changing the molecular structure of the lipids, when possible. Cyclic FA monomers (CFAM) from linseed oil, acylated in specific positions, were tested in lymphatic FA transport and lipoprotein profile in rats’ lymph [
16]. When structured TAGs were fed in a rat study, the chain length of medium-chain FA located in the primary position did not affect the lymphatic transport of LCFA in the
sn-2 position [
17]. Evidence indicated that 16:0 at the
sn-2 position is absorbed as 2-MAG, and conserved through the process of TAG reassembly in the enterocytes. In piglets, a formula containing synthesized TAG with about 32% of 16:0 in the
sn-2 position showed an increase of the 16:0 in
sn-2 position in plasma and chylomicrons TAG but a reduction of ARA and DHA in phospholipids [
18].
