Absorption Kinetics of the Main Conjugated Linoleic Acid Isomers in Commercial-Rich Oil after Oral Administration in Rats

Article abstract of DOI:10.1021/acs.jafc.7b02143

This study aimed to assess the oral absorption and plasma kinetics of two main isomers contained in commercial conjugated linoleic acid (CLA)-rich oil (Tonalin TG-80), rumenic acid (RA), and C18:2 trans-10, cis-12. The isomer plasma disposition after the single oral dose of 3000 mg of Tonalin TG-80/kg, containing 1200 mg/kg of each isomer, was studied in rats. The isomer plasma concentrations were determined by gas chromatography with flame ionization detection. The plasma kinetics showed rapid oral absorption of RA and C18:2 trans-10, cis-12 (t_1/2a 0.34 ± 0.09 and 0.53 ± 0.01 h) and slow elimination (t_1/2β 25.68 ± 3.29 and 18.12 ± 1.71 h); the maximal isomer plasma concentrations (C_max) of 8.48 ± 0.98 and 7.67 ± 0.80 μg mL^-1, respectively, were estimated at 2.08 ± 0.14 and 2.26 ± 0.11 h. Our results from a preclinical kinetic study in rats help to design future studies in humans for evaluating the CLA isomer dose-response.

Full text of DOI:10.1021/acs.jafc.7b02143

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Article
Absorption kinetics of the main CLA isomers in
commercial rich oil after oral administration in rats
Luis M. Rodríguez-Alcalá, Irma Ares, Javier Fontecha, María-Rosa
Martínez-Larrañaga, Arturo Anadon, and María-Aránzazu Martínez
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02143 • Publication Date (Web): 09 Aug 2017
Downloaded from http://pubs.acs.org on August 11, 2017
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Page 1 of 27
Journal of Agricultural and Food Chemistry
Absorption kinetics of the main CLA isomers in commercial rich oil
after oral administration in rats
1,3
2
1
Luís M. Rodríguez-Alcalá , Irma Ares , Javier Fontecha , María-Rosa
2
2
2*
Martínez-Larrañaga , Arturo Anadón and María-Aránzazu Martínez
1
Instituto de Investigación en Ciencias de la Alimentación (CIAL, CSIC-UAM), C/
Nicolás Cabrera 9, Campus de Cantoblanco de la Universidad Autónoma de Madrid,
28049 Madrid, Spain
2
Department of Toxicology and Pharmacology, Faculty of Veterinary Medicine,
Universidad Complutense de Madrid, 28040 Madrid, Spain
3
Centro de Investigación en Recursos Naturales y Sustentabilidad (CIRENYS),
Universidad Bernardo O' Higgins, Fábrica Nº 1990, segundo piso, Santiago de Chile,
Chile.
_
____________________________________________________________________
Correspondence to: Professor MaríaꢀAránzazu Martínez,
*
Eꢀmail: arantxam@vet.ucm.es
Telephone: +34 91 3943834
_____________________________________________________________________
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Abstract
This study aimed to assess oral absorption and plasma kinetics of two main isomers
contained in commercial CLAꢀrich oil (Tonalin TGꢀ80), rumenic acid (RA) and C18:2
trans 10, cis 12. Isomer plasma disposition after single oral dose of 3000 mg Tonalin
TGꢀ80/kg, containing 1200 mg/kg of each isomer, was studied in rats. Isomer plasma
concentrations were determined by GCꢀFID. Plasma kinetics showed rapid oral
absorption of RA and C18:2 trans 10, cis 12 (t½ 0.34 ± 0.09 and 0.53 ± 0.01 h) and
a
β
slow elimination (t½ 25.68 ± 3.29 and 18.12 ± 1.71 h); maximal isomer plasma
ꢀ1
concentrations (Cmax) of 8.48 ± 0.98 and 7.67 ± 0.80 µg mL , respectively, were
estimated at 2.08 ± 0.14 and 2.26 ± 0.11 h. Our results from a preꢀclinical kinetic study
in rats help to design future studies in humans for evaluating CLA isomer doseꢀresponse
Keywords: CLA isomers; RA; C18:2 trans 10, cis 12; oral absorption; plasma
disposition; rats
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INTRODUCTION
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Conjugated linoleic acid (CLA) refers to a group of polyunsatured fatty acids that exist
as positional and stereoꢀisomers of conjugated linoleic acid (18:2). CLA is located
primarily in foods such as beef, lamb and dairy foods. Numerous beneficial biological
1
,2,3
actions have been assigned to CLA.
There is evidence that diets containing CLA
reduce adiposity in experimental animals and humans, although the potential
mechanisms by which CLA exert their effects through lipid metabolism parameters,
glucose metabolism and insulin sensitivity are not yet fully understood. In experimental
animals, it was also found that in pregnant rats after intake of a synthetic mixture of
CLA, the birthed pups had longer tails (pointing out to skeletal growth), weightier
gastrocnemius and soleus muscles, while similar adipose mass but smaller adipocyte
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size. These data indicate that dietary CLA may also be valuable to certain growth
parameters.
In the literature, much attention has been aimed at these two main CLA isomers
contained in CLAꢀrich oils and their bioactivity. Rumenic acid (RA; C18:2 cis 9, trans
11) is the main CLA compound in dairy fat, associated to anticarcinogenic,
5
,6
antiatherogenic, antidiabetic properties and stimulation of the immune system while
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C18:2 trans 10, cis 12 seems to reduce body fat and is mostly present in these CLA
synthetic mixtures. Thus, in the present market there are available commercial CLAꢀ
8
rich oils, obtained from the alkali isomerization of linoleic acid (LA) oils, with a level
of 77.7 g CLA per 100 g oil consisting in a 1:1 ratio of RA and C18:2 trans 10, cis 12 as
9
well as 5.6 g per100 g CLA oil of other minor isomers.
Recent studies found that commercial CLA oil added to powder milk
(0.96 g/kg body weight/day) and fed to rats with an inducedꢀbyꢀdiet
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hypercholesterolemic status can effectively decrease the triglycerides concentration in
blood while oral intake (0.46 g/kg body weight/day) of the oil alone decreased body
10
fat. Moreover, works carried out on humans using a dose of 90 g cheese/day (0.60 g
CLA/day) found increased plasma concentrations of RA, transꢀvaccenic (TVA), alphaꢀ
linolenic (ALA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) together with
11
7% reduction in the level of LDLꢀcholesterol and endocannabinoid anandamid; similar
effects were described when assaying 0.80 g commercial CLA oil/day. In further
investigations, these later authors observed that feeding obese Zucker rats with a 1 %
CLA diet increased the concentration of docosahexaenoic fatty acid (DHA), Nꢀ
12
oleoylethanolamide (OEA) and Nꢀpalmitoylethanolamide (PEA) in liver. Such
compounds are associated to the induction of PPARα, a regulator of genes implicated in
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peroxisomal βꢀoxidation, which seems to be activated by CLA.
However, despite these above mentioned results, at the current moment, it also
exists an intense debate about the bioactivity of CLA isomers. Some further human and
animal research works reported contradictory effects without a clear association
1
4,15
between CLA intake and positive biological effects.
Thus, overweight subjects
receiving dosages of 1.8 and 3.6 g/day showed regain of fatꢀfree mass after weight
16
loss. This could be due to factors such as doses tested in humans must be much higher
than those tested in animals, metabolic differences associated among species or related
to the protocol used in the study. It has to be noted that the considered effective dose of
CLA in humans (3 g CLA/day) was proposed after extrapolation of data obtained from
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animal studies focused in prevention of breast cancer. Moreover, McGowan et al.,
reported that doses of 7.5 g CLA oil/day at least during 10 days in women reduced
expression of S14 ( regulator of the AcetylꢀCoA carboxylase) and Kiꢀ67 (proliferation
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marker) in breast cancer tissue. However, data about doseꢀresponse properties with
respect to plasma concentrations of CLA and cancer risk modulation are very scarce.
Therefore, CLA concentrations have to be used with caution. The European
Food Safety Authority (EFSA) suggested that CLA intake (up to 6 months) does not
seem to impair insulin sensitivity, blood glucose control or liver function; however,
although the reported effects on blood lipids are unlikely to negatively impact on
cardiovascular disease risk, longꢀterm effects of CLA intake have not been sufficiently
19
assessed in humans. Some studies assaying the proposed safe doses reported
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increment of inflammatory markers or LDL/HDL and total cholesterol/HDL ratios,
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hepatotoxicity and insulin resistance pointing out to C18:2 trans 10, cis 12.
However, these negative actions may be isolated cases, as they have not been reported
in other intervention studies.
Then, it is clear that to obtain reliable data about the biological effects of RA and
C18:2 trans 10, cis 12, these compounds have to be tested on the basis of their oral
absorption and plasma disposition, but at the current moment such information is
scarce. In a previous investigation, the authors of this work performed a study to
characterize for the first time the absorption of RA, TVA (a precursor of RA in the
mammary gland) and ALA (an essential polyunsaturated fatty acid [PUFA]) in rats after
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a single oral dose of a goat milk fat naturally enriched in these fatty acids. The results
showed that TVA, RA and ALA were quickly absorbed and slowly eliminated; the
maximum concentrations were identified in liver > plasma > erythrocyte. The
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importance of these research works is highlighted by data reported by Jedidi et al.,
assaying the in vitro intestinal absorption of fatty acids from a 1 % or 3.5 % fat milk
using an in vitro simulator and where the fatty acid bioaccesibility depended on chain
length, presence of double bonds and the percentage of fat. Moreover, elsewhere it was
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reported that ad libitum administration to SpragueꢀDawley rats of diets (84 g fat/kg diet)
containing 0.5% of CLA (RA and C18:2 trans 10, cis 12; 1:1) during 16 weeks resulted
in a higher accumulation of RA than C18:2 trans 10, cis 12 in liver despite their
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chemical similarities. Beyond absorption, few studies in experimental animals and
humans have also been conducted to determine differential biological effects of CLA in
relation to individual isomer dose, interval and duration of dietary CLA.
Therefore, the safety and efficacy in the use of these compounds require of in
vivo kinetic studies, as they will lead to a better adjustment of the experimental dosage
in future functional experiments. To date, plasma kinetic studies of CLA isomers are
scarce. Thus, the aim of the present research work was to evaluate oral absorption and
kinetic plasma behaviour of two main CLA isomers (RA and C18:2 trans 10, cis 12)
contained in a commercial CLAꢀrich oil (Tonalin TGꢀ80) after its administration to rats
measuring isomer plasma concentrations using a sensitive analytical method.
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MATERIALS AND METHODS
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Chemicals. N,NꢀDimethylformamide (HPLC grade), hexane (HPLC grade),
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methanol (HPLC grade) and sulfuric acid (98 %) were purchased from LABꢀSCAN
(Dublin, Ireland). Sodium bicarbonate (99 %), sodium methoxide (95 %) and
tritridecanoin (99 %) were obtained from Sigma (St. Louis, Missouri, USA). Glyceryl
tritridecanoate (99 %; TGꢀC13) and CLA isomers (purity 99 %; RA and C18:2 trans 10,
cis 12) were purchased from NuꢀChek Prep,Inc.(Elysian,Minnesota,USA). Commercial
CLAꢀrich oil (Tonalin TGꢀ80; 80 % g CLA per 100 oil, 40 % RA and 40 % C18:2 trans
10, cis 12 according to own data) was purchased from BASF (Cognis, Illertissen,
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Germany). All other chemicals were of the highest quality grade and provided from
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Animals and experimental design. The study was analyzed and approved
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02 by the Ethics Committee on Animal Research of the Complutense University of Madrid.
03 Adult male Wistar rats (Charles River Inc., Margate, Kent, UK; 220ꢀ250 g) were used
04 in this study. The rats were individually housed in polycarbonate cages in a silent room
05 under a controlled temperature (22 ± 2 ºC), relative humidity (50 ± 10%) and 12 h
06 light/dark cycle (light from 08.00 to 20.00 h), with solid food (A04 rodent diet,
07 Scientific Animal Food & Engineering, SAFE, Augy, France) and water ad libitum. The
08 rats were randomly divided into seven experimental groups: six groups of 10 animals
09 each treated with Tonalin TGꢀ80 orally, and one blank or control group of 5 animals
10 without any treatment. Animals were dispossessed of food for 12 h before single oral
11 dose of 3000 mg Tonalin TGꢀ80/kg body weight, but were supplied water ad libitum.
12 Before dose administration, Tonalin TGꢀ80 was kept in a water bath set to 30ºC to reach
3
13 a liquid state (897 kg per m ) and was dispensed by gavage in a volume of 0.84 mL
14 CLA oil/rat of 250 g body weight. The oral Tonalin TGꢀ80 dose used of 3000 mg/kg
15 body weight was equivalent to 1200 mg/kg body weight of each CLA isomer. Oral dose
16 of 3000 mg Tonalin TGꢀ80/kg body weight was chosed based on preceding experiments
17 where the dose and route of administration did not show any undesirable effects,
18 abnormal clinical signs as well as changes in behavioral, body weight, or in food and
19 water consumption in the animals (data not showed). Moreover, this dose was also
20 estimated to provide plasma concentrations of the CLA isomers to be above the level of
21 limit of quantification (LOQ) of the analytical method for both CLA isomers (RA and
22 C18:2 trans 10, cis 12).
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Animals were sacrificed by cervical dislocation (one animal at each time) and
24 then blood samples at 15 min, 30 min, 1, 2, 4, 6, 8, 12, 24 and 48 h after oral dose of
25 Tonalin TGꢀ80 were collected in heparinized tubes. Plasma was separated by
26 centrifugation and stored frozen at ꢀ80ºC until analysed.
127
Analysis of plasma samples. Plasma sample preparation. The
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28 concentrations of RA and C18:2 trans 10, cis 12 in plasma were determined as fatty acid
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29 methyl esters (FAME) using a oneꢀstep procedure according to CastroꢀGomez et al.,
30 with some modifications: 500 µL of plasma sample were spiked with 100 µL of
31 glyceryl tritridecanoate (1.44 µg/mL). Transterification reaction was carried out by
32 adding 2.5 mL methanolic sodium methoxide 0.5 M at 80 ºC during 10 min while
33 afterwards an esterification reaction was accomplished using 1.66 mL of N,Nꢀ
34 dimethylformamide and 3 mL 1M H
35 cooling samples in ice, FAME were extracted using 1 mL of hexane and 1 min vortex
36 (waiting 5 min for the separation of layers), then adding 7,5 mL 6 % w/v Na CO and
2 4
SO in methanol at 100 ºC for 30 min. After
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37 finally 3500 rpm, 10 min. at room temperature. Upper phase was collected and placed
38 into a vial to be analysed by gas chromatography with flame ionization detector (GCꢀ
39 FID).
40
To prepare the standard curves, RA and C18:2 trans 10, cis 12 were diluted in
41 hexane to final concentrations into a range of 0.006 to 300 µg/mL in rat control plasma.
42 Promptly after adding the CLA isomer solution to the plasma, they were prepared for
43 analysis as above mentioned. The calibration curves were performed the same day as
44 the rat treated samples.
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FAME analysis. The obtained FAME fraction, was injected (1 µL; 10:1 split
1
46 ratio) into a gas chromatograph (GC) 6890 Agilent (Palo Alto, CA, USA) coupled to a
47 flame ionization detector (FID) and a mass spectrometer (MS; Agilent 5973N) coupled
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48 to a CPꢀSil88 capillary column (100 m x 0.25 mm i.d. x 0.2 mm, Chrompack,
49 Middelburg, The Netherlands) with working conditions according to RodríguezꢀAlcalá
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50 et al. The injection volume was 1 ꢁL. MS was used to identify and FID to quantify the
51 CLA isomers by comparing the CLA oil supplement (Tonalin TGꢀ80; FAME prepared
52 from 5 mg as described in analysis of plasma samples) and CLA standards. In these
53 conditions, LOD was 0.009 µg/mL and LOQ was 0.03 µg/mL.
154
Data analysis. Plasma CLA isomers (RA and C18:2 trans 10, cis 12)
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55 concentrations versus time curves were fitted to twoꢀcompartment model using the
56 computer program Phoenix (Version 7.0; Pharsight Corporation, Mountain View, CA,
57 USA). This model was established for best fit based on a smaller value for the Akaike’s
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58 Information Criterion. The twoꢀcompartment model was the best fit for the two CLA
59 isomers and it was used to determine the kinetic parameters.
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Plasma concentration versus time curves of both CLA isomers after a single oral
61 dose of Tonalin TGꢀ80 were fitted to the following exponential equation:
ꢀ
αt
ꢀβt
–Kat
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C = A
1
× e + A
2
× e – A
3
× e
63 where C is the plasma concentration of the compound; A
1
2 3
, A and A are mathematical
64 coefficients; α is the hybrid rate constant for the distribution phase; β is the hybrid rate
65 constant for the elimination terminal phase; and Ka the firstꢀorder absorption rate
66 constant, and t is the time.
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Absorption halfꢀlife (t1/2a), halfꢀlife of α phase (t1/2α), halfꢀlife of β phase (t1/2β),
68 distribution rate constants for transfer of the CLA isomer from the central to the
69 peripheral compartment (K12) and from the peripheral to the central compartment (K21),
70 and the elimination rate constant (K ) were calculated by use of standard equations as
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71 described.
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The area under the concentrationꢀtime curves (AUC) and the mean residence
73 time (MRT) were calculated as follows:
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AUC = (A
1
/α) + (A
2
3 a
/β) – (A /K )
2
2
2
MRT = [(A
1
/α ) + (A
2
/β ) – (A
3
/K
a
)] (1/AUC)
Maximum plasma concentration (Cmax) and the interval (time) from oral
77 administration until Cmax was detected (Tmax) was also estimated directly from the
78 concentration versus time curve.
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Differences in pharmacokinetic data between RA and C18:2 trans 10 cis 12,
80 were analyzed for statistical significance by the paired Student’s tꢀtest (GraphPad Prism
81 6). Differences of P<0.05 were considered significant.
82
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83 RESULTS
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Quantitative determination of CLA isomers (RA and C18:2 trans
186 10, cis 12) in plasma samples. One of the main challenges in the analytical
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87 determination of fatty acids from biological samples is the utilization of timeꢀ
88 consuming and cumbersome isolation and preparation steps, especially in studies with a
89 high number of samples. Thus, procedures where lipids are isolated and transformed
90 into FAME in one singleꢀstep (direct derivatization, DT) are therefore highly
91 interesting. However, some of the most commonly assayed DT procedures only use acid
32
92 catalysts as reagent (unsuitable for transesterification reactions) or those combining
33
93 transesterification/esterification reactions utilize boron trifluoride, known to be
34
94 unstable and producing of artefacts. Furthermore, boron trifluoride is only available
95 as commercial preparations usually at 10ꢀ14 % in methanol.
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The method assayed in the presented study has been previously reported and
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97 optimized for the analysis of CLA isomers in biological samples. According to this
98 procedure, acylglycerols are transesterified in a reaction catalysed using 0.5 M sodium
99 methoxide, while esterification of free fatty acids is carried out using methanolic
00 sulfuric acid (1 M). This latter reagent has the advantage of being prepared in the
01 laboratory. Under these conditions, it was found no alteration of the FAME profile
02 essentially for CLA isomers, the best response factor values from short chain fatty acids
03 to PUFA as well as high accuracy in the quantification of total cholesterol and
04 derivatization performance thus showing a great realiability in the analysis of the total
27
05 fatty acid composition in both biological and food samples.
06 In the current work, analysis (mean values of three analyses) of the assayed
07 Tonalin TGꢀ80 revealed that RA and C18:2 trans 10 cis 12ꢀCLA represented 40.4 ±
08 0.18 g/100 g oil and 39.9 ± 0.06 g/100 g oil, respectively. CLA cis, cis isomers were
09 0.06 ± 0.01 g/100 g oil and CLA trans, trans isomers were 1.00 ± 0.07 g/100 g oil.
10 Linoleic acid was also detected (0.20 ± 0.03 g/100 g oil) together with C18:2 cis 9 trans
11 12 (0.20 ± 0.01 g/100 g oil), C18:2 trans 9 cis 12 (0.19 ± 0.01 g/100 g oil), C18:2 trans
12 9 trans 12 (0.10 ± 0.01 g/100 g oil) while linolenic acid (C18:3 cis 9 cis 12 cis 15) was
13 below 0.05 ± 0.01 g/100 g oil. As monounsaturated fatty acid, it was detected oleic acid
14 (C18:1 cis 9; 13.6 ± 0.75 g/100 g oil) and C18:1 cis 11 (0.81 ± 0.06 g/100 g oil) and as
15 saturated fatty acid moieties, the fraction was composed of stearic (2.66 ± 0.13 g/100 g
16 oil) and palmitic (0.88 ± 0.03 g/100 g oil).
2
2
2
2
2
2
2
2
2
2
2
2
17
Calibration curves supplied linear results over the RA and C18:2 trans 10 cis 12ꢀ
2
18 CLA isomer concentration from 0.006 to 300 µg/mL in plasma (R = 0.996). Figure 1
19 shows the chromatographs obtained from the analysis of rat plasma samples in the
20 current study collected at time 0 and 2 h after oral dose of Tonalin TGꢀ80 (3000 mg
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21 Tonalin TGꢀ80/kg body weight). It can be observed how the CLA isomers, RA and
22 C18:2 trans 10, cis 12 were detected according the above mentioned derivatization and
2
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23 chromatographic conditions.
The analytical method was selective for those
24 compounds; no endogenous interference was detected on the chromatograms. The
25 recovery was determined as the percentage between the concentrations measured in a
26 blank plasma sample spiked with each isomer in triplicate using two isomer levels. The
27 recovery values ranged from 94.7 % to 99.9 %. The reproducibility and repeatability
28 were estimated by the analyses of five spiked control plasma in three different days
29 separated by 10 days in the case of reproducibility and consecutive analyses to calculate
30 repeatability. These values were expressed by standard relative deviation of the peak
31 areas of the five analyses, and gave a result of 3.5 % and 5 % for reproducibility and
32 repeatability, respectively. For both CLA isomers, the detection limit (LOD) was
33 estimated to be 0.009 µg/mL [concentration obtained from the equation 3σ /S (σ:
34 standard deviation of the yꢀintercept of the regression line; S: slope of the calibration
35 curve)] while the limit of quantification (LOQ) was estimated at 0.03 µg/mL (obtained
35
36 from the equation 10σ /S) as elsewhere recommended.
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238
Pharmacokinetics of RA and C18:2 trans 10, cis 12-CLA isomer.
239 In the plasma samples collected at time 0, i.e. before oral dose of Tonalin TGꢀ80 no RA
240 or C18:2 trans 10, cis 12 were detected However, after oral dose of Tonalin TGꢀ80,
241 these CLA isomers were detected between 15 min and 48 h (Figure 2). Mean plasma
242 concentrations of CLA isomers after single oral dose of Tonalin TGꢀ80 are presented in
243 Figure 2. Good fit of the observed data for a twoꢀcompartment open model was found.
244 Values of the kinetic parameters that described absorption, distribution and elimination
245 of CLA isomers after single oral dose of Tonalin TGꢀ80 are summarized in Table 1.
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When administered orally Tonalin TGꢀ80, the CLA isomers C18:2 trans 10, cis
2
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2
2
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2
2
2
2
2
2
2
2
47 12 and RA were rapidly but poorly absorbed. The absorption halfꢀlife (t1/2a) was
48 calculated to be more rapid for RA than for C18:2 trans 10, cis 12 (0.34 ± 0.09 and 0.53
49 ± 0.01 h, respectively) P<0.01, but the maximal plasma concentration estimated for RA
50 and C18:2 trans 10, cis 12ꢀCLA isomer was not significantly different (Cmax, 8.48 ±
51 0.98 and 7.67 ± 0.80 µg/mL, respectively), as well as the area under the concentrationꢀ
52 time curve (AUC 98.81 ± 9.74 and 83.83 ± 10.30 mg h/L, respectively).
53
RA was distributed more slowly after oral dosing than C18:2 trans 10, cis 12
54 (distribution halfꢀlife [t1/2α] was 2.56 ± 0.34 and 1.92 ± 0.43 h, respectively), P<0.05.
55 The elimination halfꢀlife (t1/2β) of RA was somewhat greater than that calculated for
56 C18:2 trans 10, cis 12ꢀCLA isomer (P< 0.01). The elimination halfꢀlife (t1/2β) was 25.68
57 ± 3.29 and 18.12 ± 1.71 h, respectively, evidencing a slow final disappearance of both
58 CLA isomers from blood. This was also sustained by the values observed for the MRT,
59 which were 25.53 ± 2.72 h for RA and 19.77 ± 1.54 h for C18:2 trans 10, cis 12ꢀCLA
60 isomer (Table 1).
61
262 DISCUSSION
2
2
2
2
2
2
2
2
63
64 The limited available data on the in vivo oral absorption of CLA isomers creates a
65 difficulty to understand the physiological effects of these substances as food
66 ingredients. More works are needed to elucidate oral doses of CLA isomers that could
67 be recommended for the intakes of CLA to improve health. The focus of the efficacy
68 and safety assessments of many functional ingredients relies on animal (particularly
69 rodents) and human studies. Kinetic characteristics combined with dynamic patterns
70 must be contemplated for establish the efficiency and safety of CLA consumption. Preꢀ
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71 clinical pharmacokinetic information in an animal model is essential to project further
72 pharmacokinetic studies in humans.
73
To our knowledge, the present work is the first to describe the plasma kinetics of
74 two CLA isomers (RA, and C18:2 trans 10, cis 12) in rats after single oral dose of a
75 commercial CLA oil supplement (Tonalin TGꢀ80) using an unbiased analytical method
76 in order to quantify plasma concentrations for evaluating the pharmacokinetic profile of
77 these CLA isomers. The analytical method used is reliable and sensitive and allows an
78 accurate information of the plasma kinetic disposition of the RA and C18:2 trans 10, cis
79 12 in rats.
80
This study reports the kinetics of the RA and C18:2 trans 10, cis 12ꢀCLA isomer
81 after a single oral dose of a commercial CLA oil supplement (3000 mg Tonalin TGꢀ
82 80/kg body weight, equivalent to 1200 mg/kg body weight of each CLA isomer).
83 Plasma disposition of RA and C18:2 trans 10, cis 12 after oral administration in rats
84 was adequately described by a twoꢀcompartment open model with a relatively rapid
85 distribution and a slow elimination. When orally administered, CLA isomers (RA and
86 C18:2 trans 10, cis 12) were rapidly but only partially absorbed and slowly eliminated
87 from plasma, as reflects the absorption halfꢀlife of 0.54 and 0.34 h, respectively, and the
88 elimination halfꢀlife of 25.68 and 18.12 h, respectively. The absorption degree of CLA
89 isomers (RA and C18:2 trans 10, cis 12ꢀCLA isomer) was low with a Cmax of 8.48 and
90 7.67 µg/mL at 2.08 and 2.26 h (Tmax), respectively after oral dose of 1200 mg/kg body
91 weight. However, Cmax, AUC and MRT values (Table 1) are important data for CLA
92 isomers, given that they support an extensive metabolism at the site of absorption. The
93 in vitro intestinal absorption of RA and C18:2 trans 10, cis 12ꢀCLA isomer was
94 previously evaluated using a dynamic simulator of the gastrointestinal tract suggesting
2
5
95 that bioavailability was affected by the percentage of fat present , similar results to
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37
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96 that observed in the studies of Fernie et al. , and Gervais et al. . In contrast, Terpstra et
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97 al. demonstrated that administering CLA in the form of triacylglycerols or as free fatty
98 acids to mice had equal effects on body fat and energy expenditure. Similarly, Martin et
3
9
99 al.
in a study conducted in rats to determine intestinal digestibility and lymphatic
00 recovery of glycerol esters of CLA demonstrated that it cannot attribute a distinct
01 cellular accumulation or a difference in the biological activity between the two main
02 CLA isomers (RA and C18:2 trans 10, cis 12) due to selective absorbability. However,
03 Bissonauth et al. (2006) revealed that the digestibility coefficient in hamster was higher
04 for RA than for C18:2 trans 10, cis 12ꢀCLA isomer. In the present study there is no
05 different behaviour in oral absorption between these CLA isomers (Cmax
06 µg/mL or 7.67 ± 0.8 µg/mL respectively). The value of Cmax obtained in the present
07 study for both CLA isomers is a functional CLA concentration based on cell culture,
= 8.48 ± 0.98
,
4
0ꢀ42
08 since tumor cell proliferation is inhibited by exposure to 10ꢀ40 µM.
Furthermore, in
09 previous studies carried out in hamsters, a dose of 600 mg CLA/day during 35 days
10
10 decreased the induced hypertriglyceridemia. Nevertheless, because the CLA intestinal
11 absorption may depend on the fat content of the diet ingested; it would be interesting to
12 investigate in vivo intestinal absorption of CLA isomers as a result of the lipid fraction
13 present in supplement forms of CLA. These observations are encouraging, since CLA
14 must be absorbed efficiently to confer the alleged benefits such as antiꢀdiabetic, antiꢀ
43
15 obesity and anticancer effects. More work is needed on pharmacokinetics (mainly oral
16 absorption and distribution studies) as well as on the mechanisms of RA and C18:2
17 trans 10, cis 12ꢀCLA isomer to better predict the beneficial actions of CLA isomers.
3
3
3
18
In conclusion, the present study in rats reveals that when orally given CLA oil
19 supplement (Tonalin TGꢀ80), RA and C18:2 trans 10, cis 12ꢀCLA isomer are rapidly
20 absorbed into systemic circulation and then distributed throughout the body. Although
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321 the oral absorption degree was low, the maximal plasma concentration obtained seems
322 to be a functional CLA concentration. Moreover RA and C18:2 trans 10, cis 12 were
323 both eliminated from plasma slowly and therefore would be diffused to target tissues to
324 exert systemic effects. The kinetic profile of CLA oil supplement (Tonalin TGꢀ80)
325 observed in this study justify further research on new ways of supplementation in food
326 to increase the oral absorption of CLA isomers. The present pharmacokinetic data of
327 CLA isomers, RA and C18:2 trans 10, cis 12, in an animal model after single oral dose
328 can play a valuable role designing future kinetic studies in humans for evaluating doseꢀ
329 response relationships of these isomers.
330
331 Funding
332
333
This work was supported by Project (ALI- BIRD-CM Program) Ref. S2013/ABI-
334 2728 from Comunidad de Madrid, and Projects Ref. AGL-2011-26713 and Ref.
335 RTA2015-00010-C03-03 from Ministerio de Economía, Industria y Competitividad,
336 Spain.
337 Conflict of interest
338
339
The authors declare that there are no conflicts of interest.
3
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17 Grimble, R. F.; Williams, C. M.; Yaqoob, P.; Calder, P. C. Opposing effects of cisꢀ9,
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19 humans. Am. J. Clin. Nutr. 2004, 80, 614ꢀ620.
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22 1143.
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(23)De Almeida, M. M.; De Souza, Y. O.; Luquetti, S. C. P. D.; Sabarense, C. M.;
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26 Garcia, R. M. G. Cisꢀ9, transꢀ11 and transꢀ10,ciꢀ12 CLA mixture does not change body
27 composition, induces insulin resistance and increases serum HDL cholesterol level in
28 rats. J. Oleo Sci. 2015, 64(5), 539ꢀ551.
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(25)Jedidi, H.; Kheadr, E.; Gagnon, F.; Van Calsteren, M. R.; Farnworth, E.; Fliss, I.
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(26)DeGuire, J. R.; Weiler, H. A. Free fatty acid and triacylglycerol forms of CLA
38 isomers are not incorporated equally in the liver but do not lead to differences in bone
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(40) Ou, L.; IP, C.; Lisafeld, B.; Ip, M. M. Conjugated linoleic acid induces
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83 apoptosis of murine mammary tumor cells via Bclꢀ2 loss. Biochem. Biophys. Res.
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(42) Igarashi, M.; Miyazawa, T. The growth inhibitory effect of conjugated linoleic
89 acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid
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93 conjugated linoleic acid. Prog. Lipid Res. 2001, 40, 283ꢀ298.
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FIGURE CAPTIONS
Figure 1. Chromatogram of CLA isomers at time 0 and 2 h after oral administration of
Tonalin TGꢀ80 (3000 mg Tonalin TGꢀ80/kg body weight) in rats.
R
RA (C18:2 cis 9, trans 11) t : 71.98 min.
C18:2 trans 10, cis 12 t : 72.65 min.
R
Figure. 2. Mean plasma concentrations of CLA isomers (RA and C18:2 trans 10, cis
12) after single oral dose of 3000 mg Tonalin TGꢀ80/kg body weight, equivalent to
oral dose of 1200 mg RA/kg body weight (ꢀ) and
oral dose of 1200 mg C18:2 transꢀ10, cisꢀ12/ kg body weight (ꢀ).
Data represent mean ± S.D. values for six rats.
Symbols without bars indicate that S.D. is within the symbols
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Table 1. RA and C18:2 trans 10, cis 12 Pharmacokinetic Parameters in Rats after Single Oral Administration of Tonalin TGꢀ80 (3000 mg/kg
body weigth)
Single Oral Dose of 3000 mg Tonalin TGꢀ80/kg body weight
Parameter
RA
RA
C18:2 trans 10, cis 12
C18:2 trans 10, cis 12
Oral Dose 1200 mg/kg
Oral Dose 1200 mg/kg
Oral Dose 1200 mg/kg
Oral dose 1200 mg/kg
a
b
a
b
Mean ± SD
Median
Mean ± SD
Median
A
A
A
1
2
3
(µg/mL)
10.99 ± 1.29
1.81 ± 0.53
12.80 ± 1.59
0.28 ± 0.04
0.03 ± 0.004
2.14 ± 0.45
0.34 ± 0.09**
2.56 ± 0.34*
25.68 ± 3.29**
0.12 ± 0.03
0.07 ± 0.02
0.11 ± 0.01
98.81 ± 9.74
25.53 ± 2.72**
8.48 ± 0.98
2.08 ± 0.14
10.55
13.93 ± 2.90
2.30 ± 0.65
16.23 ± 3.41
0.37 ± 0.08
0.038 ± 0.004
1.35 ± 0.28
0.53 ± 0.01
1.92 ± 0.43
18.12 ± 1.71
0.16 ± 0.06
0.11 ± 0.02
0.14 ± 0.008
83.83 ± 10.30
19.77 ± 1.54
7.67 ± 0.80
2.26 ± 0.11
13.74
(µg/mL)
(µg/mL)
1.81
2.42
12.36
0.27
16.16
0.38
ꢀ
1
α
β
(h )
ꢀ
1
(h )
0.028
2.14
0.040
1.29
ꢀ1
K
a
(h )
(h)
t
1/2a
1/2α
1/2β
0.32
0.54
t
t
(h)
2.58
1.81
(h)
24.93
0.12
17.34
0.17
ꢀ
1
K
12
K
21
K
10
(h )
ꢀ
1
(h )
0.067
0.11
0.11
ꢀ1
(h )
(mg h/L)
(h)
0.14
AUC
MRT
98.62
24.75
8.45
84.08
19.21
7.69
C
max
max
(µg/mL)
(h)
T
2.07
2.29
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a
Kinetic parameters were calculated from the concentrationꢀtime curves. Data are expressed as mean ± SD (n=6).
Kinetic parameters were calculated from the mean concentrationꢀtime curve.
b
Significantly different (*P>0.05; **P<0.01).
Figure 1
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Figure 2
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