Lipids (2014) 49:225–233 DOI 10.1007/s11745-013-3873-4
ORIGINAL ARTICLE
Lymphatic Transport of a-Linolenic Acid and Its Conversion to Long Chain n-3 Fatty Acids in Rats Fed Microemulsions of Linseed Oil D. Sugasini • V. C. Devaraj • Mullangi Ramesh B. R. Lokesh
•
Received: 28 August 2013 / Accepted: 27 November 2013 / Published online: 14 December 2013 Ó AOCS 2013
Abstract In the present study we evaluated the uptake of ALA and its conversion to EPA ? DHA in rats given linseed oil (LSO) in native form or as a microemulsion in whey protein or in lipoid. In a single oral dose study in which rats maintained on rodent pellets deficient in x-3 fatty acids were intubated with 0.35 g LSO in lipoid, the amount of ALA present in lymph was increased reaching a maximum concentration of 16.23 mg/ml at 2.5 h. The amount of ALA present in lymph was increased to a maximum level of 10.95 mg/ml at 4 h in rats given LSO as a microemulsion in whey protein. When LSO was given without emulsification, the amount of ALA present in lymph was found to reach a maximum level of 7.08 mg/ml at 6 h. A similar result was observed when weaning rats were intubated with 0.15 g of LSO per day for a period of 60 days. Higher levels of ALA by 41 and 103 % were observed in lymph lipids of rats given microemulsions of LSO in whey protein and in lipoid respectively as compared to rats given LSO without pre-emulsification. Very little conversion of ALA to EPA and DHA was observed in lymph lipids but higher amounts of EPA ? DHA was observed in liver and serum of rats given LSO in microemulsion form. This study indicated that ALA concentration in lymph lipids was increased when LSO was given in microemulsion form in lipoid and further conversion to EPA and DHA was facilitated in liver and serum. D. Sugasini B. R. Lokesh (&) Department of Lipid Science and Traditional Foods, CSIR-Central Food Technological Research Institute, Mysore 570 020, India e-mail: [email protected] V. C. Devaraj M. Ramesh Department of Drug Metabolism and Pharmacokinetics, Jubilant Biosys, Bangalore 560 022, India
Keywords n-3 Fatty acids \ nutrition Edible oils \ nutrition Fat absorption \ physiology Lipid absorption \ physiology Docosahexaenoic acid \ specific lipids Fatty acids \ specific lipids n-3 Fatty acids \ specific lipids Abbreviations LSO Linseed oil LA Linoleic acid (18:2, n-6) ARA Arachidonic acid (20:4, n-6) ALA a-Linolenic acid (18:3, n-3) EPA Eicosapentaenoic acid (20:5, n-3) DHA Docosahexaenoic acid (22:6, n-3)
Introduction Dietary lipids are absorbed in the small intestine and this is facilitated by complex physico-chemical interactions and enzymatic-mediated processes. Lipids are emulsified by bile salts which are then acted upon by lipases. The hydrolysed products are absorbed by the intestinal epithelial cells [1]. Digestion of fats depends on the molecular structure of the lipids, the interfacial composition of the lipid emulsions and the droplet size of the emulsified oils [2, 3].The lipids that are absorbed from the intestine are either directly transported to the liver via the portal system or reassembled to form chylomicrons that are transported to the liver via the lymphatic system [4]. The rate of appearance and extent of lipids present in the lymphatic system following fat intake gives an indication of the bioavailability of ingested lipids [1, 5]. Recently we have demonstrated in rats that the bioavailability of a-linolenic acid (ALA) from linseed oil (LSO) is significantly enhanced when given in microemulsion form in
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whey protein or in lipoid. Further we also observed that there is a significant conversion of a-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma and liver lipids when linseed oil was fed to rats as microemulsions in lipoid [6]. It is not clear whether this conversion of a-linolenic acid to long chain n-3 fatty acids takes place in the intestine or after reaching the liver. It is also interesting to study whether the increased bioavailability of a-linolenic acid given in microemulsion form reflects on its enhanced presence and also the rapidity by which it is transported via the lymphatic system. Therefore the present study is aimed at measuring the kinetics of linseed oil absorption from the intestine and its appearance in the lymphatic system when given without any pre-emulsification (native form) or as a microemulsion in whey protein or in lipoid. Further, the conversion of ALA to EPA and DHA was followed in lymph, liver and serum after giving LSO without pre-emulsification or in microemulsion form.
Materials and Methods
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house facility at the Central Food Technological Research Institute in Mysore, India. Rats were acclimatized for 5 days before starting the experiments. Animals had free access to water and fed pelleted rodent diets (Sai durga feeds and Foods, Bangalore, India). Lymph fluid was collected from these rats as described by Bollman et al. [7] and Combe et al. [8]. Rats were anaesthetized with ketamine/xylazine (90 mg and 10 mg/Kg body wt respectively). After opening the abdominal wall (a fundus region of the stomach) a polyethylene catheter (0.86 mm inner diameter and 1.27 mm outer diameter) was inserted into the main mesenteric lymph duct and fixed with a pursestring suture [9]. The rats were placed in individual cages. After 2 h, 0.35 g of LSO in native or emulsion form were administered through intubation which was followed by 0.5 ml of water. Lymph was collected at different intervals of time up to 24 h in polypropylene tubes kept in an ice bath. The flow rate of lymph was found to range from 0.25 to 0.3 ml/h. All animal experiments conformed to the guidelines for the handling of laboratory animals and approved by the Institutional Animals Ethics Committee recognized by the Government of India.
Materials Edible grade linseed oil (LSO) was provided by Kamani flax omega industries, Mumbai, India. Lipoid S75-3 (69 % phosphatidylcholine and 10 % phosphatidyl ethanolamine) was a gift from Lipoid (Ludwigshafen, Germany). Sodium chloride and potassium chloride were purchased from Sisco Research Laboratories, Mumbai, India. ALA, EPA, DHA, dipalmitoyl phosphatidylcholine were procured from the Sigma Chemical Company, St. Louis, MO, USA. All the solvents used in experiments were of analytical grade and distilled before use.
Methods Preparation of Microemulsions of LSO The protein based emulsions of LSO using whey protein and phospholipid-based emulsions of LSO using lipoids were prepared as described earlier by Sugasini and Lokesh [6]. LSO which is not subjected to any pre-emulsification step was designated as native oil. Animal Experiments Collection of Lymph After Giving LSO in Native or Microemulsion Form to Rats Through Intubation: Single Oral Dose Study Male wistar rats (Out Breed-Wistar. IND-cft (2c), weighing around 225 g were maintained in an approved animal
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Absorption Kinetics of ALA The absorption kinetics (lymph response) parameter of ALA was calculated as a non-compartmental analysis section of Winnonlin version 3.1 (Pharsight Corporation, CA, USA). The area under the concentration–time curve (AUC0–t) was calculated by the trapezoidal method with linear interpolation, whereas the peak concentration (Cmax) and the time to reach Cmax (Tmax) were directly taken from the analyzed data. ! n1 X ðmði þ 1Þ þ miÞ:ti AUC ¼ 2 i¼1 with mi denoting the single measurements, ti denoting the time distance between the measurements, and n denoting the total number of measurements. Dietary Studies Weaning male Wistar rats [Out Breed-Wistar, IND-cft (2c)] weighing 40 ± 4 g were grouped (six rats in each group) by random distribution and housed in individual cages, under a 12-h light/dark cycle, in an approved animal house facility at the Central Food Technological Research Institute in Mysore, India. The rats were fed standard rodent pellets (Sai Durga Feeds and Foods, Bangalore, India) for 60 days. Growth of the rats was monitored by weighing at regular intervals. The rats had free access to food and water throughout the study. The rats were also
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administered orally by gavage with 0.15 g of LSO in native form or as emulsions in whey protein or lipoid once a day at 10 am for 60 days. After 60 days, lymph was collected from each rat as described earlier.
Total phospholipids were quantitated by the ferrous ammonium thiocyanate method of Stewart [12] using dipalmitoyl phosphatidylcholine (10–100 lg) as the reference standard.
Total Lipid Extraction from Lymph and Serum
Statistical Analysis
Total lipid was extracted from lymph or serum using the Bligh and Dyer [10] method. Lymph or serum (0.5 ml) was taken in a stoppered tube and 2 ml of methanol was added and vortexed for 30 s. Chloroform (1 ml) was added and shaken well for 30 s. Again chloroform (1 ml) and water (1 ml) were added and vortexed well for 30 s. The extract was filtered and the filtrate was allowed to settle for phase separation. The aqueous methanolic layer was removed by aspiration and the chloroform layer containing the total lipid was used for further analysis. The total lipids in the liver were extracted as described earlier [6].
Results are expressed as means ± SD for each experimental group. The data were analyzed by one-way ANOVA followed by a post hoc Tukey test to compare the control and treatment groups; p values \0.05 were considered as statistically significant. All statistical analysis was performed using SPSS statistical software package version 17.0. Winnonlin version 3.1 (Pharsight Corporation, CA, USA) was also used to generate the pharmacokinetic parameter AUC needed in data analysis.
Fatty Acid (FA) Composition Fatty acid composition of emulsified oils, dietary lipids, serum, lymph and liver lipids were analyzed as methyl esters using gas liquid chromatography [Fisons, with a flame ionization detector (FID) fitted with a fused silica capillary column 25 m 9 0.25 mm (Parma bond FFAP-DF -0.25, Machery-Negal GmbH co., Duren, Germany)]. The methyl esters of fatty acids were prepared using 14 % BF3/ MeOH as described by Morrison and Smith [11]. The operating conditions set for gas liquid chromatography were: column temperature 220 °C, injection temperature 230 °C, and detector temperature 240 °C. Nitrogen was used as the carrier gas. Individual fatty acids were identified by comparison with the retention times of standards (Nu-Check-Prep, Inc, Elysian MN, USA) and were quantified by an online Shimadzu Chromatopack CR6A integrator. Pentadecanoic acid was used as an internal standard for quantitating individual fatty acids. Estimation of Triacylglycerol, Total Cholesterol and Phospholipids Triacylglycerol levels in the lymph were measured by an enzymatic method using Agappe Diagnostic kits, Kerala, India. The instructions given by the manufacturer were followed for estimating triacylglycerol levels. Total cholesterol in the lymph was also measured by an enzymatic method using diagnostic kits supplied by Agappe, Kerala, India. The procedure provided by the manufacturer of the kits was followed for estimating cholesterol.
Results Fatty Acid Composition of Oils Given by Gavage for the Short Term and the Long Term Study Fatty acid composition of LSO given without pre-emulsification or given as microemulsified oil are reported earlier [6]. No significant differences in the fatty acid composition of oil provided without emulsion or that provided in microemulsion form was observed. The fatty acid composition of background diet given in pelleted form was 39 % linoleic acid, 33 % oleic acid, 23 % palmitic acid and 4.3 % stearic acid. No n-3 PUFA was present in background diet. Rats used for the single dose study were given 0.35 g of LSO in native form (without emulsion formation) or as emulsion form through gavage containing 116 ± 3.9 mg a-linolenic acid (ALA) and 29 ± 1.2 mg of linoleic acid (LA). In single dose studies, higher amounts of LSO were provided as compared to that given in dietary studies for clear visualization of lymph flow. Rats were given 0.15 g of LSO by intubation in dietary studies which provided 46 ± 2.8 mg of ALA and 13 ± 0.6 mg of LA per day for 60 days. Lymphatic Absorption of LSO in Rats Given Native and Emulsified Oils The amount of LSO absorbed into lymph was followed at different intervals of time up to 24 h (Fig. 1). The amount of lipid in lymph increased linearly and reached a maximum at 2.5 h (Tmax) when rats were given LSO as a microemulsion with lipoid. The amount of lipid in lymph increased reaching a maximum at 4 h (Tmax) in rats given LSO as a microemulsion with whey protein. The amount of lipid in lymph increased reaching a maximum at 6 h (Tmax)
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Fig. 1 Total lipid in lymph of rats given 0.35 g of LSO in the native or emulsified form. Lymph was collected up to 24 h from six rats in each group and the lipid were extracted and quantified. Results are expressed as means ± SD
in rats given LSO without emulsion formation. Thus the time taken by LSO to reach the maximum amount in lymph is significantly reduced when given as microemulsions in lipoid or whey protein as compared to oil given without pre-emulsion formation. At 2.5 h, the maximum amount of LSO was observed in lymph when given as a microemulsion in lipoid. At this stage, the amount of total lipid observed in lymph of rats given LSO in a whey protein emulsion or in native form was found to be lower by 46 and 68 % respectively as compared to those given in lipoid (Fig. 1). LSO given in a whey protein emulsion reached maximum accumulation in lymph by 4 h, but the amount of LSO at this stage was still less by 22 % as compared to the maximum amount that was achieved when LSO was given as emulsions in lipoid. LSO given without emulsion formation reached a maximum level in lymph at 6 h. Here again the maximum amount of LSO found in the lymph was lower by 38 % as compared to the maximum amount that was achieved when LSO was given in lipoid. Thus emulsions of LSO in lipoid not only increased the rate but also the extent of absorption when compared to that given in whey protein or without pre-emulsion formation. Lymphatic Uptake of n-3 Fatty Acids in Rats Given LSO in Native or Microemulsion Form The amount of ALA present in lymph lipids after the ingestion of LSO in native or emulsified form was studied at different intervals of time (Fig. 2). The ALA of lymph increased rapidly and reached a maximum at 2.5 h in rats given LSO as a microemulsion in lipoid. Similarly the amount of ALA present in lymph increased reaching a maximum at 4 h in rats given LSO as a microemulsion in whey protein. The amount of ALA present in lymph
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Fig. 2 Alpha linolenic acid (ALA) absorption in lymph of rats given 0.35 g of LSO in native or emulsified form. Lymph was collected for up to 24 h from six rats in each group and ALA in the total lipids was estimated. Results are means ± SD
Table 1 Maximum ALA concentration (Cmax), time required to reach Cmax (Tmax) in lymph ALA Tmax (h)
AUC0–24 h (mg 9 h/ml)
7.08 ± 0.4a
6
43.29 ± 5.82a
b
4
61.52 ± 7.96b
c
2.5
90.71 ± 9.5c
ALA Cmax (mg/ml) LSO native LSO in whey protein LSO in lipoid
10.95 ± 0.6
16.23 ± 1.2
Rats were given 0.35 g of linseed oil in native or emulsified form in whey protein or in lipoid Lymph was collected for 24 h from six rats per group. The lipid was extracted and evaluated for n-3 PUFA. Means in a column with different superscript letters differ significantly at P \ 0.05 for a specific parameter measured, Results are expressed as means ± SD
increased reaching a maximum at 6 h in rats given LSO in native form. The amount of ALA present in lymph at maximum absorption time was 7.08, 10.95 and 16.23 mg/ ml for rats given LSO in native form and as microemulsions with whey protein and lipoid respectively (Table 1). Thus a significantly higher amount of ALA by 54.6 and 129 % was present in lymph at the maximum time of absorption in rats given LSO as microemulsions in whey protein or with lipoid respectively. The area under the curve (AUC) which gives an estimate of the bioavailability of ALA was significantly higher for rats given LSO in a microemulsion as compared to rats given LSO in non-emulsified form. The AUC for ALA was found to be 43.29, 61.52 and 90.71 mg/ml lymph when LSO was given in native, as microemulsion in whey protein and lipoid respectively (Table 1). There was also
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Table 2 Body weight, lymph volume and total lipid content in lymph of rats fed linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
Body weight gain (g/rat)
297 ± 9
312 ± 13
309 ± 8
Total lymph fluid (ml for 24 h)
7.1 ± 0.3
7.4 ± 0.2
7.7 ± 0.2
Total lipid (mg lipid/ml of lymph)
8.3 ± 0.2a
9.5 ± 0.2b
11.6 ± 0.3c
Table 3 Lipid composition of lymph of rats given Linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
Triacylglycerol (mg/dl)
142 ± 7.4
145 ± 7.8
151 ± 6.1
Cholesterol (mg/dl)
23.8 ± 2.7
24.5 ± 3.2
27.9 ± 2.9
Phospholipids(mg/dl)
33.5 ± 3.8
34.2 ± 5.0
42.8 ± 4.1
Values are means ± SD, n = 6 Table 4 Fatty acid composition (wt%) of lymph in rats given linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
16:0
23.6 ± 2.5
22.5 ± 1.8
19.2 ± 2.9
16:1(n-7)
3.0 ± 0.2
3.1 ± 0.3
2.5 ± 0.4
18:0 18:1(n-9)
4.2 ± 0.3 19.8 ± 1.2
3.6 ± 0.4 20.2 ± 1.0
3.1 ± 0.3 21.5 ± 1.3
18:2(n-6)
20.1 ± 1.5c
16.8 ± 1.2b
12.4 ± 1.0a
18:3(n-3)
17.5 ± 1.6
a
b
24.7 ± 2.2
35.5 ± 2.8c
11.3 ± 0.5
c
b
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05 for a specific parameter measured
marginal conversion of ALA to EPA and DHA. The amount of EPA present in lymph of rats given LSO in native, LSO in microemulsion form with whey protein or with lipoid was found to be 0.2, 0.29 and 0.82 mg respectively. Similarly the amount of DHA present in LSO in native, LSO in microemulsion form with whey protein or with lipoid was found to be 0.1, 0.22 and 0.47 mg respectively. Thus very little conversion of ALA from LSO to long chain n-3 PUFA was observed during absorption of lipids in the intestine.
20:4(n-6) 20:5(n-3)
a
0.3 ± 0.05
4.3 ± 0.3a a
0.9 ± 0.1b
b
0.4 ± 0.05
22:6(n-3)
0.2 ± 0.02
0.3 ± 0.03
0.6 ± 0.1c
Total n-6
31.4c
25.4b
16.7a
Total n-3
a
b
37c
b
0.45a
n-6/n-3
a
8.4 ± 0.8
18.0
c
1.74
25.8 0.98
Dietary Studies
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
Rats were given daily 0.15 g of LSO by intubation in native form or in microemulsion form in whey protein or in lipoid for a period of 60 days. Body weight, the total amount of lymph fluid collected for 24 h and the total lipids in lymph was estimated. No significant differences in body weight and volume of lymph fluid in rats given LSO in microemulsion form or non-emulsified form were observed (Table 2). However significant increases in the total lipid content by 14 and 39.7 % were observed in lymph of rats given LSO in microemulsion form in whey protein or lipoid respectively as compared to those given LSO in non emulsified form (Table 2).
Fatty Acid Composition of Lymph of Rats Given LSO in Native or Microemulsion Form
Lymph Lipids in Rats Given LSO in Native or Microemulsion Form There were no significant differences in triacylglycerol, cholesterol and phospholipid content of lymph in rats given LSO in emulsion form with whey protein as compared to rats given LSO without emulsification. However lymph of rats given LSO in lipoid showed a marginally higher level of triacylglycerols and phospholipids as compared to those given LSO in native form but it was not statistically significant (Table 3).
Lymphatic lipids in rats given LSO contained significant amounts of ALA (Table 4). The rats given LSO in microemulsion forms in whey protein or in lipoid contained increased amounts of ALA by 41 and 102.8 % respectively when compared to rats given LSO without emulsification. Thus there was a significant increase in ALA content in rats given LSO in lipoid (F(2, 15) = 62.59, P \ 0.05**) as compared to those given LSO without emulsification or those given in whey protein. The n-6 to n-3 fatty acid ratio in lymphatic lipids given LSO, LSO in whey protein and in lipoid was 1.74, 0.98 and 0.45 respectively. A marginal increase in long chain n-3 PUFA, EPA and DHA was observed in lymph lipids of rats given LSO as microemulsions in whey protein or lipoid (Table 4). This result is in agreement with that observed in the single oral dose study. Liver Fatty Acid Composition of Rats Given LSO in Native and Microemulsion Form Liver lipids contained n-3 fatty acids in rats given LSO. The rats given LSO, LSO in microemulsion with whey
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Table 5 Fatty acid composition (wt%) of liver lipids in rats given linseed oil (LSO) native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Table 6 Fatty acid composition (wt%) of serum lipids in rats given linseed oil (LSO) native or microemulsion form for 60 days
Emulsion in lipoid
Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
16:0
22.6 ± 1.4
22.0 ± 1.7
20.2 ± 2.1
16:0
27.5 ± 1.8
25.8 ± 2.3
25.1 ± 2.0
16:1(n-7)
2.9 ± 0.4
2.5 ± 0.3
2.3 ± 0.3
16:1(n-7)
2.1 ± 0.2
1.9 ± 0.3
2.0 ± 0.4
18:0 18:1(n-9)
10.3 ± 1.3 25.9 ± 1.2a
11.3 ± 0.5 27.2 ± 0.9a,b
9.2 ± 1.0 28.6 ± 1.4b
18:0 18:1(n-9)
7.8 ± 0.8 29.3 ± 1.0
8.4 ± 0.3 30.7 ± 1.4
7.5 ± 0.6 31.5 ± 2.1
18:2(n-6)
21.7 ± 1.0c
18.7 ± 1.2b
14.3 ± 1.1a
18:2(n-6)
19.1 ± 1.3a
18.4 ± 1.0a
13.9 ± 1.5b
18:3(n-3)
2.3 ± 0.3
a
3.3 ± 0.2 c
b
4.6 ± 0.4 b
11.3 ± 0.4
20:5(n-3)
1.2 ± 0.2
a
22:5(n-6)
0.7 ± 0.02b
0.5 ± 0.07b
0.4 ± 0.06a
22:5(n-3)
0.2 ± 0.02
a
0.3 ± 0.06
a
b
0.9 ± 0.08
a
b
Total n-6 PUFA
33.7
Total n-3 PUFA
a
n-6/n-3
4.6
7.33
c
2.4 ± 0.3
1.6 ± 0.1 29.4 7.6
c
b
b
3.87
b
b
8.1 ± 0.3 6.5 ± 0.6
0.6 ± 0.04 c
5.2 ± 0.5 22.8 16.9
c
1.35
a
9.7 ± 0.6
c
20:5(n-3)
0.9 ± 0.1
a
22:5(n-6)
0.6 ± 0.2a
20:4(n-6)
c
a
2.0 ± 0.3
a
18:3(n-3)
a
20:4(n-6)
22:6(n-3)
10.2 ± 0.5
c
22:5(n-3)
0.2 ± 0.03
22:6(n-3)
a
0.8 ± 0.1
Total (n-6)
29.4
Total (n-3)
a
n-6/n-3
3.9
7.54
c
2.8 ± 0.2
b
4.1 ± 0.5c
8.4 ± 0.4
b
6.2 ± 0.3a
1.6 ± 0.2
b
4.9 ± 0.3c
0.4 ± 0.1a a
0.3 ± 0.06 1.3 ± 0.1 27.2 6.0
c
b
b
4.53
b
0.1 ± 0.02b b
0.5 ± 0.05b 4.2 ± 0.4c 20.2a 13.7c
b
1.47a
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
protein or in lipoid showed ALA to an extent of 2.3, 3.3 and 4.6 % of total fatty acids (Table 5). The EPA content in rats given LSO, LSO microemulsion in whey protein or in lipoid were found to be 1.2, 2.4 and 6.5 % respectively. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained DHA in liver lipids to an extent of 0.9, 1.6 and 5.2 % of total fatty acids (Table 5). This indicated that ALA from LSO, LSO as microemulsions in whey protein or in lipoid was converted to EPA and DHA in liver. A significantly higher amount of conversion being observed when LSO was given as emulsions in lipoid. The n-6 to n-3 fatty acid ratio in liver lipids given LSO, LSO in whey protein or in lipoid were 7.33, 3.87 and 1.35 respectively indicating a higher efficacy of microemulsified oils in increasing n-3 fatty acid levels by replacing n-6 fatty acids in liver lipids (Table 5).
respectively (Table 6). This indicated that ALA from LSO was not only deposited in serum lipids but was also converted to EPA and DHA with higher amounts of conversion being observed when LSO was given as an emulsion in lipoid. The n-6 to n-3 fatty acid ratio in serum lipids given LSO, LSO in whey protein or in lipoid were 7.54, 4.53 and 1.47 respectively indicating higher efficacy of microemulsified oils in increasing n-3 fatty acid levels by replacing n-6 fatty acids in serum lipids (Table 6). A significant increase in ALA content was observed in rats given LSO as a microemulsion in lipoid (F(2, 15) = 100.29, P \ 0.05**) when compared to those given LSO without emulsification. A significant amount of EPA and DHA was also found in the serum of rats given LSO as a microemulsion in lipoid compared to those given LSO in native form (F(2, 15) = 280.45, P \ 0.05**); (F(2, 15) = 341.51, P \ 0.05**)(Table 6). This result indicated that ALA provided in microemulsion with lipoid is converted to long chain n-3 PUFA with greater efficiency which was subsequently observed in serum lipids.
Serum Fatty Acid Composition of Rats Given LSO in Native and Microemulsion Form Serum lipids contained n-3 fatty acids in rats given LSO, LSO as microemulsion with whey protein or with lipoid. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained ALA to an extent of 2.0, 2.8 and 4.1 % of total fatty acids respectively (Table 6). The EPA content in rats given LSO, LSO as microemulsions in whey protein or in lipoid were found to be 0.9, 1.6 and 4.9 % of total fatty acid respectively. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained DHA to an extent of 0.8, 1.3 and 4.2 % of total fatty acid
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Discussion The rate and extent of lipid accumulation in the lymph is an indication for the bioavailability of ingested lipids. This also reflects on the postprandial rise in triacylglycerol levels in the plasma. Earlier we demonstrated that LSO in the microemulsion form with whey protein or lipoid is absorbed at a higher rate from intestinal everted sacs. A
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significant increase in n-3 fatty acid accumulation was observed in plasma and liver lipids when emulsified LSO was fed to rats for a period of 30 days [6]. We made similar observations for increased uptake of n-3 PUFA when pre emulsified fish oil was fed to rats for a period of 30 days [13]. Garaiova et al. [14] made similar observations when they fed healthy adults with a mixture of fish oil, borage oil and flaxseed oil. An increase in ALA, EPA and DHA to an extent of 1.52, 2.97 and 2.26 fold was observed when the mixture of oils was given after pre-emulsification as compared to those given oil without emulsification. Interestingly, in this study, other than n-3 fatty acids levels, none of the other fatty acids changed. Cansell et al. [15] observed that liposomes made from marine lipids can be utilized as oral route vectors for enhanced absorption of n-3 PUFA. Fatty acid absorption in liposome form was found to be 98 % as compared to that observed with native fish oil which showed 73 % absorption. In this study it was also observed that the proportion of DHA found in lymph was 78 % when given in liposome form as compared to 47 % observed for DHA when native oil was given. The phospholipid based liposomes favoured higher absorption of n-3 PUFA [15, 16]. Recently Haug et al. [17] observed that absorption of EPA and DHA was enhanced by 100–105 % when fish oil was given in the form of droplets trapped in a gelatin matrix (gelled emulsions) as compared to those given as soft gels. The Tmax for their appearance in plasma was 2 h when given as emulsions which was delayed to 6 h when given as soft gels [17]. These studies indicated that pre-emulsification of lipids apart from that which takes place with bile salts in the intestine enhances the absorption of total lipids in the gastrointestinal tract. Of particular interest is a significant increase in the uptake of n-3 PUFA over other fatty acids which needs to be probed further. It is not only the emulsified form but also the size of the emulsions that significantly altered the rate of absorption of lipids. Micro- and nano-emulsions of lipids were better absorbed as compared to native oils [6, 18, 19]. In addition, the presence of co-substrates also altered the lymphatic absorption of n-3 PUFA. The lymphatic transport of ALA was enhanced by 24 % when co-administered with palmitic acid. This increment in ALA absorption was attributed to enhanced intracellular re-esterification of ALA into triacylglycerol and subsequent incorporation into chylomicrons. This was not observed when palmitate was co-administered with medium chain fatty acids. The partitioning of long chain fatty acids to the lymphatic system was dependent on the polarity of fatty acids and conditions that prevailed in the luminal milieu during fatty acid absorption [20, 21]. In addition to these factors, the positions of fatty acids in the triacylglycerols of the dietary lipids also affect the absorption of fatty acids. n-3 PUFA (EPA ? DHA) are
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more readily and efficiently absorbed when they are located at the sn-2 position of triacylglycerol compared to when they were present in triacylglycerols having random fatty acid distribution in all the three positions [22, 23]. The absorption of DHA associated with the sn-2 position of phospholipids was significantly enhanced in preterm infants given formulated milk [24, 25]. Our results from the present investigation are in general agreement with observations reported in the literature. LSO in microemulsion form in whey protein or in lipoid was absorbed at a faster rate and in increased amounts as compared to LSO given without pre-emulsification. The rate and extent of LSO/ALA accumulation in lymph following ingestion in various forms was followed over a period of 24 h. Tmax an indicator for rate of absorption was found to be 6, 4 and 2.5 h when LSO was given without pre-emulsification or as microemulsions in whey protein and in lipoid respectively. The Cmax measured from the area under the curve by using the trapezoidal method indicated that the total amount of ALA of lymph was increased by 55 and 129 % when LSO was given after emulsifying in whey protein and lipoid respectively. Similar results were observed by Couedelo et al. [26] who followed lymphatic absorption of ALA in rats fed flaxseed in non-emulsified form or as an emulsion in deoxycholate and lecithin. ALA in emulsified form was taken at a higher rate with Tmax of 3 h and Cmax of 14 mg/ml as compared to bulk oil absorption with a Tmax of 5 h and a Cmax of 9 mg/ ml. These values are closer to the values reported in the present investigation for ALA absorption when LSO was given in whey protein or in lipoid. LSO given in lipoid emulsions had a faster and higher rate of transport in lymph. Lipoid is a phospholipid based emulsifying material with 69 % lecithin and 10 % phosphatidylethanolamine. It has been shown in many experiments that phospholipid based emulsions deliver n-3 PUFA more efficiently than TAG-associated n-3 PUFA [15, 24, 27]. Further, lipoid emulsions were smaller in size (246–249 nm) than emulsions formed in whey protein (700–704 nm) or other binding materials like gum acacia (1,092–1,093 nm) [6, 13]. This again indicated that nano-emulsions and emulsions with a smaller size are absorbed at a faster rate and in higher amounts. Another objective of the present work was to trace the conversion of ALA to long chain n-3 PUFA (EPA ? DHA) when LSO was given as microemulsions. There was a marginal increase in the conversion of ALA to EPA and DHA when lymph lipids were evaluated following the ingestion of LSO in native or microemulsion forms by rats. Chen and Nileson [28] have shown that rat intestinal villi and crypt cells contain D6 and D5 desaturase which can convert ALA to longer chain n-3 PUFA. However, these enzymes metabolized very small amounts of
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dietary linolenic acid to long chain n-3 PUFA. It was further shown that a highly active acyl CoA: 1acyl sn-glycerol 3 phosphocholine acyl transferase in crypt cells favored the retention of PUFA in phospholipids limiting the ALA conversion to long chain PUFA [28–30]. The lipids in the form of chylomicrons are transported to the liver which contains active desaturases and elongase systems. Following LSO intake for a period of 60 days, we observed that rat liver contained ALA, EPA and DHA to an extent of 2.3, 1.2 and 0.9 wt%. When LSO was given as a microemulsion with whey protein the corresponding values for ALA, EPA and DHA were 3.3, 2.4 and 1.6 wt%. However when LSO was given as a microemulsion with lipoid the values for ALA, EPA and DHA in liver lipids were 4.6, 6.5 and 5.2 wt%. This indicated that n-3 PUFA levels were increased by 1.66-fold and 3.7-fold in liver when LSO was given as a microemulsion in whey protein and lipoid respectively as compared to that observed in rats given LSO without emulsion formation. Similar results were also found for n-3 PUFA levels in serum lipids. This indicated that ALA from LSO is absorbed at a faster rate and in higher amounts when given as microemulsions but very little conversion of ALA to EPA and DHA took place in the intestine. The concentration of ALA was increased significantly when LSO was given as microemulsions to rats as compared to those given LSO in native form. We had shown earlier that when given at higher concentrations, ALA is converted to EPA and DHA in the liver and other tissues [31]. However, our studies relied on the measurement of unlabelled fatty acids as a proxy for conversion of ALA to long chain n-3 PUFA metabolites. It has also been reported that a major portion of ALA undergoes b-oxidation and will not be available for further metabolism [32, 33]. It is also known that when a lower amount of essential fatty acid (EFA) is available, the oxidation pathway is lowered but conversion of EFA to long chain PUFA is significantly enhanced [34]. The normal diet given to rats in our studies was deficient with respect to n-3 PUFA. It is also possible that ALA in the sn-2 position of TAG is protected by microemulsion formation and made available for pathways other than b-oxidation such as chylomicron formation and for desaturation and elongation in the liver. LSO contains 28.4 mol % 18:3 in the sn-2 position [26]. Therefore, higher levels of EPA and DHA observed in the serum of rats given LSO in microemulsion form may be a result of higher concentration of ALA delivered to the liver via a microemulsion form. In conclusion, these studies indicated that microemulsions of LSO in phospholipid based lipoid are absorbed at a faster rate with higher efficiency. It was also observed that the conversion of ALA to long chain n-3 PUFA is taking place more efficiently in the liver than in the intestine and subsequently higher levels of EPA and DHA are observed
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in the liver as well as in the serum of rats given ALA-rich LSO in microemulsion form. Acknowledgments The authors acknowledge the keen interest and encouragement of Prof. Ram Rajasekharan, Director, Central Food Technological Research Institute for this work. D. Sugasini acknowledges the grant of a Senior Research Fellowship by the Indian Council of Medical Research, New Delhi, India. The authors acknowledge the help of Nugehally Srinivas, Jubilant biosciences, Bangalore in giving training in collecting lymph fluids from rats. Conflict of interest
There is no conflict of interest.
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ORIGINAL ARTICLE
Lymphatic Transport of a-Linolenic Acid and Its Conversion to Long Chain n-3 Fatty Acids in Rats Fed Microemulsions of Linseed Oil D. Sugasini • V. C. Devaraj • Mullangi Ramesh B. R. Lokesh
•
Received: 28 August 2013 / Accepted: 27 November 2013 / Published online: 14 December 2013 Ó AOCS 2013
Abstract In the present study we evaluated the uptake of ALA and its conversion to EPA ? DHA in rats given linseed oil (LSO) in native form or as a microemulsion in whey protein or in lipoid. In a single oral dose study in which rats maintained on rodent pellets deficient in x-3 fatty acids were intubated with 0.35 g LSO in lipoid, the amount of ALA present in lymph was increased reaching a maximum concentration of 16.23 mg/ml at 2.5 h. The amount of ALA present in lymph was increased to a maximum level of 10.95 mg/ml at 4 h in rats given LSO as a microemulsion in whey protein. When LSO was given without emulsification, the amount of ALA present in lymph was found to reach a maximum level of 7.08 mg/ml at 6 h. A similar result was observed when weaning rats were intubated with 0.15 g of LSO per day for a period of 60 days. Higher levels of ALA by 41 and 103 % were observed in lymph lipids of rats given microemulsions of LSO in whey protein and in lipoid respectively as compared to rats given LSO without pre-emulsification. Very little conversion of ALA to EPA and DHA was observed in lymph lipids but higher amounts of EPA ? DHA was observed in liver and serum of rats given LSO in microemulsion form. This study indicated that ALA concentration in lymph lipids was increased when LSO was given in microemulsion form in lipoid and further conversion to EPA and DHA was facilitated in liver and serum. D. Sugasini B. R. Lokesh (&) Department of Lipid Science and Traditional Foods, CSIR-Central Food Technological Research Institute, Mysore 570 020, India e-mail: [email protected] V. C. Devaraj M. Ramesh Department of Drug Metabolism and Pharmacokinetics, Jubilant Biosys, Bangalore 560 022, India
Keywords n-3 Fatty acids \ nutrition Edible oils \ nutrition Fat absorption \ physiology Lipid absorption \ physiology Docosahexaenoic acid \ specific lipids Fatty acids \ specific lipids n-3 Fatty acids \ specific lipids Abbreviations LSO Linseed oil LA Linoleic acid (18:2, n-6) ARA Arachidonic acid (20:4, n-6) ALA a-Linolenic acid (18:3, n-3) EPA Eicosapentaenoic acid (20:5, n-3) DHA Docosahexaenoic acid (22:6, n-3)
Introduction Dietary lipids are absorbed in the small intestine and this is facilitated by complex physico-chemical interactions and enzymatic-mediated processes. Lipids are emulsified by bile salts which are then acted upon by lipases. The hydrolysed products are absorbed by the intestinal epithelial cells [1]. Digestion of fats depends on the molecular structure of the lipids, the interfacial composition of the lipid emulsions and the droplet size of the emulsified oils [2, 3].The lipids that are absorbed from the intestine are either directly transported to the liver via the portal system or reassembled to form chylomicrons that are transported to the liver via the lymphatic system [4]. The rate of appearance and extent of lipids present in the lymphatic system following fat intake gives an indication of the bioavailability of ingested lipids [1, 5]. Recently we have demonstrated in rats that the bioavailability of a-linolenic acid (ALA) from linseed oil (LSO) is significantly enhanced when given in microemulsion form in
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whey protein or in lipoid. Further we also observed that there is a significant conversion of a-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plasma and liver lipids when linseed oil was fed to rats as microemulsions in lipoid [6]. It is not clear whether this conversion of a-linolenic acid to long chain n-3 fatty acids takes place in the intestine or after reaching the liver. It is also interesting to study whether the increased bioavailability of a-linolenic acid given in microemulsion form reflects on its enhanced presence and also the rapidity by which it is transported via the lymphatic system. Therefore the present study is aimed at measuring the kinetics of linseed oil absorption from the intestine and its appearance in the lymphatic system when given without any pre-emulsification (native form) or as a microemulsion in whey protein or in lipoid. Further, the conversion of ALA to EPA and DHA was followed in lymph, liver and serum after giving LSO without pre-emulsification or in microemulsion form.
Materials and Methods
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house facility at the Central Food Technological Research Institute in Mysore, India. Rats were acclimatized for 5 days before starting the experiments. Animals had free access to water and fed pelleted rodent diets (Sai durga feeds and Foods, Bangalore, India). Lymph fluid was collected from these rats as described by Bollman et al. [7] and Combe et al. [8]. Rats were anaesthetized with ketamine/xylazine (90 mg and 10 mg/Kg body wt respectively). After opening the abdominal wall (a fundus region of the stomach) a polyethylene catheter (0.86 mm inner diameter and 1.27 mm outer diameter) was inserted into the main mesenteric lymph duct and fixed with a pursestring suture [9]. The rats were placed in individual cages. After 2 h, 0.35 g of LSO in native or emulsion form were administered through intubation which was followed by 0.5 ml of water. Lymph was collected at different intervals of time up to 24 h in polypropylene tubes kept in an ice bath. The flow rate of lymph was found to range from 0.25 to 0.3 ml/h. All animal experiments conformed to the guidelines for the handling of laboratory animals and approved by the Institutional Animals Ethics Committee recognized by the Government of India.
Materials Edible grade linseed oil (LSO) was provided by Kamani flax omega industries, Mumbai, India. Lipoid S75-3 (69 % phosphatidylcholine and 10 % phosphatidyl ethanolamine) was a gift from Lipoid (Ludwigshafen, Germany). Sodium chloride and potassium chloride were purchased from Sisco Research Laboratories, Mumbai, India. ALA, EPA, DHA, dipalmitoyl phosphatidylcholine were procured from the Sigma Chemical Company, St. Louis, MO, USA. All the solvents used in experiments were of analytical grade and distilled before use.
Methods Preparation of Microemulsions of LSO The protein based emulsions of LSO using whey protein and phospholipid-based emulsions of LSO using lipoids were prepared as described earlier by Sugasini and Lokesh [6]. LSO which is not subjected to any pre-emulsification step was designated as native oil. Animal Experiments Collection of Lymph After Giving LSO in Native or Microemulsion Form to Rats Through Intubation: Single Oral Dose Study Male wistar rats (Out Breed-Wistar. IND-cft (2c), weighing around 225 g were maintained in an approved animal
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Absorption Kinetics of ALA The absorption kinetics (lymph response) parameter of ALA was calculated as a non-compartmental analysis section of Winnonlin version 3.1 (Pharsight Corporation, CA, USA). The area under the concentration–time curve (AUC0–t) was calculated by the trapezoidal method with linear interpolation, whereas the peak concentration (Cmax) and the time to reach Cmax (Tmax) were directly taken from the analyzed data. ! n1 X ðmði þ 1Þ þ miÞ:ti AUC ¼ 2 i¼1 with mi denoting the single measurements, ti denoting the time distance between the measurements, and n denoting the total number of measurements. Dietary Studies Weaning male Wistar rats [Out Breed-Wistar, IND-cft (2c)] weighing 40 ± 4 g were grouped (six rats in each group) by random distribution and housed in individual cages, under a 12-h light/dark cycle, in an approved animal house facility at the Central Food Technological Research Institute in Mysore, India. The rats were fed standard rodent pellets (Sai Durga Feeds and Foods, Bangalore, India) for 60 days. Growth of the rats was monitored by weighing at regular intervals. The rats had free access to food and water throughout the study. The rats were also
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administered orally by gavage with 0.15 g of LSO in native form or as emulsions in whey protein or lipoid once a day at 10 am for 60 days. After 60 days, lymph was collected from each rat as described earlier.
Total phospholipids were quantitated by the ferrous ammonium thiocyanate method of Stewart [12] using dipalmitoyl phosphatidylcholine (10–100 lg) as the reference standard.
Total Lipid Extraction from Lymph and Serum
Statistical Analysis
Total lipid was extracted from lymph or serum using the Bligh and Dyer [10] method. Lymph or serum (0.5 ml) was taken in a stoppered tube and 2 ml of methanol was added and vortexed for 30 s. Chloroform (1 ml) was added and shaken well for 30 s. Again chloroform (1 ml) and water (1 ml) were added and vortexed well for 30 s. The extract was filtered and the filtrate was allowed to settle for phase separation. The aqueous methanolic layer was removed by aspiration and the chloroform layer containing the total lipid was used for further analysis. The total lipids in the liver were extracted as described earlier [6].
Results are expressed as means ± SD for each experimental group. The data were analyzed by one-way ANOVA followed by a post hoc Tukey test to compare the control and treatment groups; p values \0.05 were considered as statistically significant. All statistical analysis was performed using SPSS statistical software package version 17.0. Winnonlin version 3.1 (Pharsight Corporation, CA, USA) was also used to generate the pharmacokinetic parameter AUC needed in data analysis.
Fatty Acid (FA) Composition Fatty acid composition of emulsified oils, dietary lipids, serum, lymph and liver lipids were analyzed as methyl esters using gas liquid chromatography [Fisons, with a flame ionization detector (FID) fitted with a fused silica capillary column 25 m 9 0.25 mm (Parma bond FFAP-DF -0.25, Machery-Negal GmbH co., Duren, Germany)]. The methyl esters of fatty acids were prepared using 14 % BF3/ MeOH as described by Morrison and Smith [11]. The operating conditions set for gas liquid chromatography were: column temperature 220 °C, injection temperature 230 °C, and detector temperature 240 °C. Nitrogen was used as the carrier gas. Individual fatty acids were identified by comparison with the retention times of standards (Nu-Check-Prep, Inc, Elysian MN, USA) and were quantified by an online Shimadzu Chromatopack CR6A integrator. Pentadecanoic acid was used as an internal standard for quantitating individual fatty acids. Estimation of Triacylglycerol, Total Cholesterol and Phospholipids Triacylglycerol levels in the lymph were measured by an enzymatic method using Agappe Diagnostic kits, Kerala, India. The instructions given by the manufacturer were followed for estimating triacylglycerol levels. Total cholesterol in the lymph was also measured by an enzymatic method using diagnostic kits supplied by Agappe, Kerala, India. The procedure provided by the manufacturer of the kits was followed for estimating cholesterol.
Results Fatty Acid Composition of Oils Given by Gavage for the Short Term and the Long Term Study Fatty acid composition of LSO given without pre-emulsification or given as microemulsified oil are reported earlier [6]. No significant differences in the fatty acid composition of oil provided without emulsion or that provided in microemulsion form was observed. The fatty acid composition of background diet given in pelleted form was 39 % linoleic acid, 33 % oleic acid, 23 % palmitic acid and 4.3 % stearic acid. No n-3 PUFA was present in background diet. Rats used for the single dose study were given 0.35 g of LSO in native form (without emulsion formation) or as emulsion form through gavage containing 116 ± 3.9 mg a-linolenic acid (ALA) and 29 ± 1.2 mg of linoleic acid (LA). In single dose studies, higher amounts of LSO were provided as compared to that given in dietary studies for clear visualization of lymph flow. Rats were given 0.15 g of LSO by intubation in dietary studies which provided 46 ± 2.8 mg of ALA and 13 ± 0.6 mg of LA per day for 60 days. Lymphatic Absorption of LSO in Rats Given Native and Emulsified Oils The amount of LSO absorbed into lymph was followed at different intervals of time up to 24 h (Fig. 1). The amount of lipid in lymph increased linearly and reached a maximum at 2.5 h (Tmax) when rats were given LSO as a microemulsion with lipoid. The amount of lipid in lymph increased reaching a maximum at 4 h (Tmax) in rats given LSO as a microemulsion with whey protein. The amount of lipid in lymph increased reaching a maximum at 6 h (Tmax)
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Fig. 1 Total lipid in lymph of rats given 0.35 g of LSO in the native or emulsified form. Lymph was collected up to 24 h from six rats in each group and the lipid were extracted and quantified. Results are expressed as means ± SD
in rats given LSO without emulsion formation. Thus the time taken by LSO to reach the maximum amount in lymph is significantly reduced when given as microemulsions in lipoid or whey protein as compared to oil given without pre-emulsion formation. At 2.5 h, the maximum amount of LSO was observed in lymph when given as a microemulsion in lipoid. At this stage, the amount of total lipid observed in lymph of rats given LSO in a whey protein emulsion or in native form was found to be lower by 46 and 68 % respectively as compared to those given in lipoid (Fig. 1). LSO given in a whey protein emulsion reached maximum accumulation in lymph by 4 h, but the amount of LSO at this stage was still less by 22 % as compared to the maximum amount that was achieved when LSO was given as emulsions in lipoid. LSO given without emulsion formation reached a maximum level in lymph at 6 h. Here again the maximum amount of LSO found in the lymph was lower by 38 % as compared to the maximum amount that was achieved when LSO was given in lipoid. Thus emulsions of LSO in lipoid not only increased the rate but also the extent of absorption when compared to that given in whey protein or without pre-emulsion formation. Lymphatic Uptake of n-3 Fatty Acids in Rats Given LSO in Native or Microemulsion Form The amount of ALA present in lymph lipids after the ingestion of LSO in native or emulsified form was studied at different intervals of time (Fig. 2). The ALA of lymph increased rapidly and reached a maximum at 2.5 h in rats given LSO as a microemulsion in lipoid. Similarly the amount of ALA present in lymph increased reaching a maximum at 4 h in rats given LSO as a microemulsion in whey protein. The amount of ALA present in lymph
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Fig. 2 Alpha linolenic acid (ALA) absorption in lymph of rats given 0.35 g of LSO in native or emulsified form. Lymph was collected for up to 24 h from six rats in each group and ALA in the total lipids was estimated. Results are means ± SD
Table 1 Maximum ALA concentration (Cmax), time required to reach Cmax (Tmax) in lymph ALA Tmax (h)
AUC0–24 h (mg 9 h/ml)
7.08 ± 0.4a
6
43.29 ± 5.82a
b
4
61.52 ± 7.96b
c
2.5
90.71 ± 9.5c
ALA Cmax (mg/ml) LSO native LSO in whey protein LSO in lipoid
10.95 ± 0.6
16.23 ± 1.2
Rats were given 0.35 g of linseed oil in native or emulsified form in whey protein or in lipoid Lymph was collected for 24 h from six rats per group. The lipid was extracted and evaluated for n-3 PUFA. Means in a column with different superscript letters differ significantly at P \ 0.05 for a specific parameter measured, Results are expressed as means ± SD
increased reaching a maximum at 6 h in rats given LSO in native form. The amount of ALA present in lymph at maximum absorption time was 7.08, 10.95 and 16.23 mg/ ml for rats given LSO in native form and as microemulsions with whey protein and lipoid respectively (Table 1). Thus a significantly higher amount of ALA by 54.6 and 129 % was present in lymph at the maximum time of absorption in rats given LSO as microemulsions in whey protein or with lipoid respectively. The area under the curve (AUC) which gives an estimate of the bioavailability of ALA was significantly higher for rats given LSO in a microemulsion as compared to rats given LSO in non-emulsified form. The AUC for ALA was found to be 43.29, 61.52 and 90.71 mg/ml lymph when LSO was given in native, as microemulsion in whey protein and lipoid respectively (Table 1). There was also
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Table 2 Body weight, lymph volume and total lipid content in lymph of rats fed linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
Body weight gain (g/rat)
297 ± 9
312 ± 13
309 ± 8
Total lymph fluid (ml for 24 h)
7.1 ± 0.3
7.4 ± 0.2
7.7 ± 0.2
Total lipid (mg lipid/ml of lymph)
8.3 ± 0.2a
9.5 ± 0.2b
11.6 ± 0.3c
Table 3 Lipid composition of lymph of rats given Linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
Triacylglycerol (mg/dl)
142 ± 7.4
145 ± 7.8
151 ± 6.1
Cholesterol (mg/dl)
23.8 ± 2.7
24.5 ± 3.2
27.9 ± 2.9
Phospholipids(mg/dl)
33.5 ± 3.8
34.2 ± 5.0
42.8 ± 4.1
Values are means ± SD, n = 6 Table 4 Fatty acid composition (wt%) of lymph in rats given linseed oil (LSO) in native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
16:0
23.6 ± 2.5
22.5 ± 1.8
19.2 ± 2.9
16:1(n-7)
3.0 ± 0.2
3.1 ± 0.3
2.5 ± 0.4
18:0 18:1(n-9)
4.2 ± 0.3 19.8 ± 1.2
3.6 ± 0.4 20.2 ± 1.0
3.1 ± 0.3 21.5 ± 1.3
18:2(n-6)
20.1 ± 1.5c
16.8 ± 1.2b
12.4 ± 1.0a
18:3(n-3)
17.5 ± 1.6
a
b
24.7 ± 2.2
35.5 ± 2.8c
11.3 ± 0.5
c
b
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05 for a specific parameter measured
marginal conversion of ALA to EPA and DHA. The amount of EPA present in lymph of rats given LSO in native, LSO in microemulsion form with whey protein or with lipoid was found to be 0.2, 0.29 and 0.82 mg respectively. Similarly the amount of DHA present in LSO in native, LSO in microemulsion form with whey protein or with lipoid was found to be 0.1, 0.22 and 0.47 mg respectively. Thus very little conversion of ALA from LSO to long chain n-3 PUFA was observed during absorption of lipids in the intestine.
20:4(n-6) 20:5(n-3)
a
0.3 ± 0.05
4.3 ± 0.3a a
0.9 ± 0.1b
b
0.4 ± 0.05
22:6(n-3)
0.2 ± 0.02
0.3 ± 0.03
0.6 ± 0.1c
Total n-6
31.4c
25.4b
16.7a
Total n-3
a
b
37c
b
0.45a
n-6/n-3
a
8.4 ± 0.8
18.0
c
1.74
25.8 0.98
Dietary Studies
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
Rats were given daily 0.15 g of LSO by intubation in native form or in microemulsion form in whey protein or in lipoid for a period of 60 days. Body weight, the total amount of lymph fluid collected for 24 h and the total lipids in lymph was estimated. No significant differences in body weight and volume of lymph fluid in rats given LSO in microemulsion form or non-emulsified form were observed (Table 2). However significant increases in the total lipid content by 14 and 39.7 % were observed in lymph of rats given LSO in microemulsion form in whey protein or lipoid respectively as compared to those given LSO in non emulsified form (Table 2).
Fatty Acid Composition of Lymph of Rats Given LSO in Native or Microemulsion Form
Lymph Lipids in Rats Given LSO in Native or Microemulsion Form There were no significant differences in triacylglycerol, cholesterol and phospholipid content of lymph in rats given LSO in emulsion form with whey protein as compared to rats given LSO without emulsification. However lymph of rats given LSO in lipoid showed a marginally higher level of triacylglycerols and phospholipids as compared to those given LSO in native form but it was not statistically significant (Table 3).
Lymphatic lipids in rats given LSO contained significant amounts of ALA (Table 4). The rats given LSO in microemulsion forms in whey protein or in lipoid contained increased amounts of ALA by 41 and 102.8 % respectively when compared to rats given LSO without emulsification. Thus there was a significant increase in ALA content in rats given LSO in lipoid (F(2, 15) = 62.59, P \ 0.05**) as compared to those given LSO without emulsification or those given in whey protein. The n-6 to n-3 fatty acid ratio in lymphatic lipids given LSO, LSO in whey protein and in lipoid was 1.74, 0.98 and 0.45 respectively. A marginal increase in long chain n-3 PUFA, EPA and DHA was observed in lymph lipids of rats given LSO as microemulsions in whey protein or lipoid (Table 4). This result is in agreement with that observed in the single oral dose study. Liver Fatty Acid Composition of Rats Given LSO in Native and Microemulsion Form Liver lipids contained n-3 fatty acids in rats given LSO. The rats given LSO, LSO in microemulsion with whey
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Table 5 Fatty acid composition (wt%) of liver lipids in rats given linseed oil (LSO) native or microemulsion form for 60 days Oil without emulsion form
Emulsion in whey protein
Table 6 Fatty acid composition (wt%) of serum lipids in rats given linseed oil (LSO) native or microemulsion form for 60 days
Emulsion in lipoid
Oil without emulsion form
Emulsion in whey protein
Emulsion in lipoid
16:0
22.6 ± 1.4
22.0 ± 1.7
20.2 ± 2.1
16:0
27.5 ± 1.8
25.8 ± 2.3
25.1 ± 2.0
16:1(n-7)
2.9 ± 0.4
2.5 ± 0.3
2.3 ± 0.3
16:1(n-7)
2.1 ± 0.2
1.9 ± 0.3
2.0 ± 0.4
18:0 18:1(n-9)
10.3 ± 1.3 25.9 ± 1.2a
11.3 ± 0.5 27.2 ± 0.9a,b
9.2 ± 1.0 28.6 ± 1.4b
18:0 18:1(n-9)
7.8 ± 0.8 29.3 ± 1.0
8.4 ± 0.3 30.7 ± 1.4
7.5 ± 0.6 31.5 ± 2.1
18:2(n-6)
21.7 ± 1.0c
18.7 ± 1.2b
14.3 ± 1.1a
18:2(n-6)
19.1 ± 1.3a
18.4 ± 1.0a
13.9 ± 1.5b
18:3(n-3)
2.3 ± 0.3
a
3.3 ± 0.2 c
b
4.6 ± 0.4 b
11.3 ± 0.4
20:5(n-3)
1.2 ± 0.2
a
22:5(n-6)
0.7 ± 0.02b
0.5 ± 0.07b
0.4 ± 0.06a
22:5(n-3)
0.2 ± 0.02
a
0.3 ± 0.06
a
b
0.9 ± 0.08
a
b
Total n-6 PUFA
33.7
Total n-3 PUFA
a
n-6/n-3
4.6
7.33
c
2.4 ± 0.3
1.6 ± 0.1 29.4 7.6
c
b
b
3.87
b
b
8.1 ± 0.3 6.5 ± 0.6
0.6 ± 0.04 c
5.2 ± 0.5 22.8 16.9
c
1.35
a
9.7 ± 0.6
c
20:5(n-3)
0.9 ± 0.1
a
22:5(n-6)
0.6 ± 0.2a
20:4(n-6)
c
a
2.0 ± 0.3
a
18:3(n-3)
a
20:4(n-6)
22:6(n-3)
10.2 ± 0.5
c
22:5(n-3)
0.2 ± 0.03
22:6(n-3)
a
0.8 ± 0.1
Total (n-6)
29.4
Total (n-3)
a
n-6/n-3
3.9
7.54
c
2.8 ± 0.2
b
4.1 ± 0.5c
8.4 ± 0.4
b
6.2 ± 0.3a
1.6 ± 0.2
b
4.9 ± 0.3c
0.4 ± 0.1a a
0.3 ± 0.06 1.3 ± 0.1 27.2 6.0
c
b
b
4.53
b
0.1 ± 0.02b b
0.5 ± 0.05b 4.2 ± 0.4c 20.2a 13.7c
b
1.47a
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
Values are means ± SD, n = 6; Means in a row with different superscript letters differ significantly at P \ 0.05
protein or in lipoid showed ALA to an extent of 2.3, 3.3 and 4.6 % of total fatty acids (Table 5). The EPA content in rats given LSO, LSO microemulsion in whey protein or in lipoid were found to be 1.2, 2.4 and 6.5 % respectively. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained DHA in liver lipids to an extent of 0.9, 1.6 and 5.2 % of total fatty acids (Table 5). This indicated that ALA from LSO, LSO as microemulsions in whey protein or in lipoid was converted to EPA and DHA in liver. A significantly higher amount of conversion being observed when LSO was given as emulsions in lipoid. The n-6 to n-3 fatty acid ratio in liver lipids given LSO, LSO in whey protein or in lipoid were 7.33, 3.87 and 1.35 respectively indicating a higher efficacy of microemulsified oils in increasing n-3 fatty acid levels by replacing n-6 fatty acids in liver lipids (Table 5).
respectively (Table 6). This indicated that ALA from LSO was not only deposited in serum lipids but was also converted to EPA and DHA with higher amounts of conversion being observed when LSO was given as an emulsion in lipoid. The n-6 to n-3 fatty acid ratio in serum lipids given LSO, LSO in whey protein or in lipoid were 7.54, 4.53 and 1.47 respectively indicating higher efficacy of microemulsified oils in increasing n-3 fatty acid levels by replacing n-6 fatty acids in serum lipids (Table 6). A significant increase in ALA content was observed in rats given LSO as a microemulsion in lipoid (F(2, 15) = 100.29, P \ 0.05**) when compared to those given LSO without emulsification. A significant amount of EPA and DHA was also found in the serum of rats given LSO as a microemulsion in lipoid compared to those given LSO in native form (F(2, 15) = 280.45, P \ 0.05**); (F(2, 15) = 341.51, P \ 0.05**)(Table 6). This result indicated that ALA provided in microemulsion with lipoid is converted to long chain n-3 PUFA with greater efficiency which was subsequently observed in serum lipids.
Serum Fatty Acid Composition of Rats Given LSO in Native and Microemulsion Form Serum lipids contained n-3 fatty acids in rats given LSO, LSO as microemulsion with whey protein or with lipoid. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained ALA to an extent of 2.0, 2.8 and 4.1 % of total fatty acids respectively (Table 6). The EPA content in rats given LSO, LSO as microemulsions in whey protein or in lipoid were found to be 0.9, 1.6 and 4.9 % of total fatty acid respectively. The rats given LSO, LSO as microemulsion in whey protein or in lipoid contained DHA to an extent of 0.8, 1.3 and 4.2 % of total fatty acid
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Discussion The rate and extent of lipid accumulation in the lymph is an indication for the bioavailability of ingested lipids. This also reflects on the postprandial rise in triacylglycerol levels in the plasma. Earlier we demonstrated that LSO in the microemulsion form with whey protein or lipoid is absorbed at a higher rate from intestinal everted sacs. A
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significant increase in n-3 fatty acid accumulation was observed in plasma and liver lipids when emulsified LSO was fed to rats for a period of 30 days [6]. We made similar observations for increased uptake of n-3 PUFA when pre emulsified fish oil was fed to rats for a period of 30 days [13]. Garaiova et al. [14] made similar observations when they fed healthy adults with a mixture of fish oil, borage oil and flaxseed oil. An increase in ALA, EPA and DHA to an extent of 1.52, 2.97 and 2.26 fold was observed when the mixture of oils was given after pre-emulsification as compared to those given oil without emulsification. Interestingly, in this study, other than n-3 fatty acids levels, none of the other fatty acids changed. Cansell et al. [15] observed that liposomes made from marine lipids can be utilized as oral route vectors for enhanced absorption of n-3 PUFA. Fatty acid absorption in liposome form was found to be 98 % as compared to that observed with native fish oil which showed 73 % absorption. In this study it was also observed that the proportion of DHA found in lymph was 78 % when given in liposome form as compared to 47 % observed for DHA when native oil was given. The phospholipid based liposomes favoured higher absorption of n-3 PUFA [15, 16]. Recently Haug et al. [17] observed that absorption of EPA and DHA was enhanced by 100–105 % when fish oil was given in the form of droplets trapped in a gelatin matrix (gelled emulsions) as compared to those given as soft gels. The Tmax for their appearance in plasma was 2 h when given as emulsions which was delayed to 6 h when given as soft gels [17]. These studies indicated that pre-emulsification of lipids apart from that which takes place with bile salts in the intestine enhances the absorption of total lipids in the gastrointestinal tract. Of particular interest is a significant increase in the uptake of n-3 PUFA over other fatty acids which needs to be probed further. It is not only the emulsified form but also the size of the emulsions that significantly altered the rate of absorption of lipids. Micro- and nano-emulsions of lipids were better absorbed as compared to native oils [6, 18, 19]. In addition, the presence of co-substrates also altered the lymphatic absorption of n-3 PUFA. The lymphatic transport of ALA was enhanced by 24 % when co-administered with palmitic acid. This increment in ALA absorption was attributed to enhanced intracellular re-esterification of ALA into triacylglycerol and subsequent incorporation into chylomicrons. This was not observed when palmitate was co-administered with medium chain fatty acids. The partitioning of long chain fatty acids to the lymphatic system was dependent on the polarity of fatty acids and conditions that prevailed in the luminal milieu during fatty acid absorption [20, 21]. In addition to these factors, the positions of fatty acids in the triacylglycerols of the dietary lipids also affect the absorption of fatty acids. n-3 PUFA (EPA ? DHA) are
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more readily and efficiently absorbed when they are located at the sn-2 position of triacylglycerol compared to when they were present in triacylglycerols having random fatty acid distribution in all the three positions [22, 23]. The absorption of DHA associated with the sn-2 position of phospholipids was significantly enhanced in preterm infants given formulated milk [24, 25]. Our results from the present investigation are in general agreement with observations reported in the literature. LSO in microemulsion form in whey protein or in lipoid was absorbed at a faster rate and in increased amounts as compared to LSO given without pre-emulsification. The rate and extent of LSO/ALA accumulation in lymph following ingestion in various forms was followed over a period of 24 h. Tmax an indicator for rate of absorption was found to be 6, 4 and 2.5 h when LSO was given without pre-emulsification or as microemulsions in whey protein and in lipoid respectively. The Cmax measured from the area under the curve by using the trapezoidal method indicated that the total amount of ALA of lymph was increased by 55 and 129 % when LSO was given after emulsifying in whey protein and lipoid respectively. Similar results were observed by Couedelo et al. [26] who followed lymphatic absorption of ALA in rats fed flaxseed in non-emulsified form or as an emulsion in deoxycholate and lecithin. ALA in emulsified form was taken at a higher rate with Tmax of 3 h and Cmax of 14 mg/ml as compared to bulk oil absorption with a Tmax of 5 h and a Cmax of 9 mg/ ml. These values are closer to the values reported in the present investigation for ALA absorption when LSO was given in whey protein or in lipoid. LSO given in lipoid emulsions had a faster and higher rate of transport in lymph. Lipoid is a phospholipid based emulsifying material with 69 % lecithin and 10 % phosphatidylethanolamine. It has been shown in many experiments that phospholipid based emulsions deliver n-3 PUFA more efficiently than TAG-associated n-3 PUFA [15, 24, 27]. Further, lipoid emulsions were smaller in size (246–249 nm) than emulsions formed in whey protein (700–704 nm) or other binding materials like gum acacia (1,092–1,093 nm) [6, 13]. This again indicated that nano-emulsions and emulsions with a smaller size are absorbed at a faster rate and in higher amounts. Another objective of the present work was to trace the conversion of ALA to long chain n-3 PUFA (EPA ? DHA) when LSO was given as microemulsions. There was a marginal increase in the conversion of ALA to EPA and DHA when lymph lipids were evaluated following the ingestion of LSO in native or microemulsion forms by rats. Chen and Nileson [28] have shown that rat intestinal villi and crypt cells contain D6 and D5 desaturase which can convert ALA to longer chain n-3 PUFA. However, these enzymes metabolized very small amounts of
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dietary linolenic acid to long chain n-3 PUFA. It was further shown that a highly active acyl CoA: 1acyl sn-glycerol 3 phosphocholine acyl transferase in crypt cells favored the retention of PUFA in phospholipids limiting the ALA conversion to long chain PUFA [28–30]. The lipids in the form of chylomicrons are transported to the liver which contains active desaturases and elongase systems. Following LSO intake for a period of 60 days, we observed that rat liver contained ALA, EPA and DHA to an extent of 2.3, 1.2 and 0.9 wt%. When LSO was given as a microemulsion with whey protein the corresponding values for ALA, EPA and DHA were 3.3, 2.4 and 1.6 wt%. However when LSO was given as a microemulsion with lipoid the values for ALA, EPA and DHA in liver lipids were 4.6, 6.5 and 5.2 wt%. This indicated that n-3 PUFA levels were increased by 1.66-fold and 3.7-fold in liver when LSO was given as a microemulsion in whey protein and lipoid respectively as compared to that observed in rats given LSO without emulsion formation. Similar results were also found for n-3 PUFA levels in serum lipids. This indicated that ALA from LSO is absorbed at a faster rate and in higher amounts when given as microemulsions but very little conversion of ALA to EPA and DHA took place in the intestine. The concentration of ALA was increased significantly when LSO was given as microemulsions to rats as compared to those given LSO in native form. We had shown earlier that when given at higher concentrations, ALA is converted to EPA and DHA in the liver and other tissues [31]. However, our studies relied on the measurement of unlabelled fatty acids as a proxy for conversion of ALA to long chain n-3 PUFA metabolites. It has also been reported that a major portion of ALA undergoes b-oxidation and will not be available for further metabolism [32, 33]. It is also known that when a lower amount of essential fatty acid (EFA) is available, the oxidation pathway is lowered but conversion of EFA to long chain PUFA is significantly enhanced [34]. The normal diet given to rats in our studies was deficient with respect to n-3 PUFA. It is also possible that ALA in the sn-2 position of TAG is protected by microemulsion formation and made available for pathways other than b-oxidation such as chylomicron formation and for desaturation and elongation in the liver. LSO contains 28.4 mol % 18:3 in the sn-2 position [26]. Therefore, higher levels of EPA and DHA observed in the serum of rats given LSO in microemulsion form may be a result of higher concentration of ALA delivered to the liver via a microemulsion form. In conclusion, these studies indicated that microemulsions of LSO in phospholipid based lipoid are absorbed at a faster rate with higher efficiency. It was also observed that the conversion of ALA to long chain n-3 PUFA is taking place more efficiently in the liver than in the intestine and subsequently higher levels of EPA and DHA are observed
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in the liver as well as in the serum of rats given ALA-rich LSO in microemulsion form. Acknowledgments The authors acknowledge the keen interest and encouragement of Prof. Ram Rajasekharan, Director, Central Food Technological Research Institute for this work. D. Sugasini acknowledges the grant of a Senior Research Fellowship by the Indian Council of Medical Research, New Delhi, India. The authors acknowledge the help of Nugehally Srinivas, Jubilant biosciences, Bangalore in giving training in collecting lymph fluids from rats. Conflict of interest
There is no conflict of interest.
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