Absorption Kinetics of the Main Conjugated Linoleic Acid Isomers in

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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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Journal of Agricultural and Food Chemistry

Absorption kinetics of the main CLA isomers in commercial rich oil after oral administration in rats

Luís M. Rodríguez-Alcalá1,3, Irma Ares2, Javier Fontecha1, María-Rosa Martínez-Larrañaga2, Arturo Anadón2 and María-Aránzazu Martínez2*

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 María-Aránzazu Martínez, E-mail: [email protected] 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 CLA-rich oil (Tonalin TG-80), rumenic acid (RA) and C18:2 trans 10, cis 12. Isomer plasma disposition after single oral dose of 3000 mg Tonalin TG-80/kg, containing 1200 mg/kg of each isomer, was studied in rats. Isomer plasma concentrations were determined by GC-FID. Plasma kinetics showed rapid oral absorption of RA and C18:2 trans 10, cis 12 (t½a 0.34 ± 0.09 and 0.53 ± 0.01 h) and slow elimination (t½β 25.68 ± 3.29 and 18.12 ± 1.71 h); maximal isomer plasma concentrations (Cmax) 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 pre-clinical kinetic study in rats help to design future studies in humans for evaluating CLA isomer dose-response

Keywords: CLA isomers; RA; C18:2 trans 10, cis 12; oral absorption; plasma disposition; rats

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Journal of Agricultural and Food Chemistry

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INTRODUCTION

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Conjugated linoleic acid (CLA) refers to a group of polyunsatured fatty acids that exist

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as positional and stereo-isomers of conjugated linoleic acid (18:2). CLA is located

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primarily in foods such as beef, lamb and dairy foods. Numerous beneficial biological

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actions have been assigned to CLA.1,2,3 There is evidence that diets containing CLA

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reduce adiposity in experimental animals and humans, although the potential

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mechanisms by which CLA exert their effects through lipid metabolism parameters,

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glucose metabolism and insulin sensitivity are not yet fully understood. In experimental

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animals, it was also found that in pregnant rats after intake of a synthetic mixture of

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CLA, the birthed pups had longer tails (pointing out to skeletal growth), weightier

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gastrocnemius and soleus muscles, while similar adipose mass but smaller adipocyte

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size.4 These data indicate that dietary CLA may also be valuable to certain growth

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parameters.

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In the literature, much attention has been aimed at these two main CLA isomers

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contained in CLA-rich oils and their bioactivity. Rumenic acid (RA; C18:2 cis 9, trans

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11) is the main CLA compound in dairy fat, associated to anticarcinogenic,

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antiatherogenic, antidiabetic properties and stimulation of the immune system5,6 while

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C18:2 trans 10, cis 12 seems to reduce body fat7 and is mostly present in these CLA

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synthetic mixtures. Thus, in the present market there are available commercial CLA-

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rich oils, obtained from the alkali isomerization of linoleic acid (LA) oils,8 with a level

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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

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well as 5.6 g per100 g CLA oil of other minor isomers.9

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Recent studies found that commercial CLA oil added to powder milk (0.96

g/kg

body

weight/day)

and

fed

to

rats

with

an

induced-by-diet 3

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hypercholesterolemic status can effectively decrease the triglycerides concentration in

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blood while oral intake (0.46 g/kg body weight/day) of the oil alone decreased body

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fat.10 Moreover, works carried out on humans using a dose of 90 g cheese/day (0.60 g

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CLA/day) found increased plasma concentrations of RA, trans-vaccenic (TVA), alpha-

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linolenic (ALA), arachidonic acid (AA) and eicosapentaenoic acid (EPA) together with

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7% reduction in the level of LDL-cholesterol and endocannabinoid anandamid;11 similar

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effects were described when assaying 0.80 g commercial CLA oil/day. In further

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investigations, these later authors observed that feeding obese Zucker rats with a 1 %

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CLA diet increased the concentration of docosahexaenoic fatty acid (DHA), N-

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oleoylethanolamide (OEA) and N-palmitoylethanolamide (PEA) in liver.12 Such

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compounds are associated to the induction of PPARα, a regulator of genes implicated in

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peroxisomal β-oxidation, which seems to be activated by CLA.13

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However, despite these above mentioned results, at the current moment, it also

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exists an intense debate about the bioactivity of CLA isomers. Some further human and

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animal research works reported contradictory effects without a clear association

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between CLA intake and positive biological effects.14,15 Thus, overweight subjects

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receiving dosages of 1.8 and 3.6 g/day showed regain of fat-free mass after weight

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loss.16 This could be due to factors such as doses tested in humans must be much higher

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than those tested in animals, metabolic differences associated among species or related

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to the protocol used in the study. It has to be noted that the considered effective dose of

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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.17 Moreover, McGowan et al.,18

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reported that doses of 7.5 g CLA oil/day at least during 10 days in women reduced

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expression of S14 ( regulator of the Acetyl-CoA carboxylase) and Ki-67 (proliferation

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marker) in breast cancer tissue. However, data about dose-response properties with

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respect to plasma concentrations of CLA and cancer risk modulation are very scarce.

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Therefore, CLA concentrations have to be used with caution. The European

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Food Safety Authority (EFSA) suggested that CLA intake (up to 6 months) does not

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seem to impair insulin sensitivity, blood glucose control or liver function; however,

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although the reported effects on blood lipids are unlikely to negatively impact on

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cardiovascular disease risk, long-term effects of CLA intake have not been sufficiently

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assessed in humans.19 Some studies assaying the proposed safe doses reported

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increment of inflammatory markers20 or LDL/HDL and total cholesterol/HDL ratios,21

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hepatotoxicity22 and insulin resistance23 pointing out to C18:2 trans 10, cis 12.

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However, these negative actions may be isolated cases, as they have not been reported

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in other intervention studies.

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Then, it is clear that to obtain reliable data about the biological effects of RA and

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C18:2 trans 10, cis 12, these compounds have to be tested on the basis of their oral

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absorption and plasma disposition, but at the current moment such information is

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scarce. In a previous investigation, the authors of this work performed a study to

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characterize for the first time the absorption of RA, TVA (a precursor of RA in the

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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.24 The results

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showed that TVA, RA and ALA were quickly absorbed and slowly eliminated; the

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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.,25

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assaying the in vitro intestinal absorption of fatty acids from a 1 % or 3.5 % fat milk

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using an in vitro simulator and where the fatty acid bioaccesibility depended on chain

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length, presence of double bonds and the percentage of fat. Moreover, elsewhere it was

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reported that ad libitum administration to Sprague-Dawley rats of diets (84 g fat/kg diet)

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containing 0.5% of CLA (RA and C18:2 trans 10, cis 12; 1:1) during 16 weeks resulted

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in a higher accumulation of RA than C18:2 trans 10, cis 12 in liver despite their

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chemical similarities.26 Beyond absorption, few studies in experimental animals and

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humans have also been conducted to determine differential biological effects of CLA in

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relation to individual isomer dose, interval and duration of dietary CLA.

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Therefore, the safety and efficacy in the use of these compounds require of in

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vivo kinetic studies, as they will lead to a better adjustment of the experimental dosage

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in future functional experiments. To date, plasma kinetic studies of CLA isomers are

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scarce. Thus, the aim of the present research work was to evaluate oral absorption and

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kinetic plasma behaviour of two main CLA isomers (RA and C18:2 trans 10, cis 12)

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contained in a commercial CLA-rich oil (Tonalin TG-80) after its administration to rats

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measuring isomer plasma concentrations using a sensitive analytical method.

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MATERIALS AND METHODS

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Chemicals. N,N-Dimethylformamide (HPLC grade), hexane (HPLC grade),

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methanol (HPLC grade) and sulfuric acid (98 %) were purchased from LAB-SCAN

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(Dublin, Ireland). Sodium bicarbonate (99 %), sodium methoxide (95 %) and

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tritridecanoin (99 %) were obtained from Sigma (St. Louis, Missouri, USA). Glyceryl

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tritridecanoate (99 %; TG-C13) and CLA isomers (purity 99 %; RA and C18:2 trans 10,

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cis 12) were purchased from Nu-Chek Prep,Inc.(Elysian,Minnesota,USA). Commercial

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CLA-rich oil (Tonalin TG-80; 80 % g CLA per 100 oil, 40 % RA and 40 % C18:2 trans

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10, cis 12 according to own data) was purchased from BASF (Cognis, Illertissen,

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99 100

Germany). All other chemicals were of the highest quality grade and provided from commercial sources.

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Animals and experimental design. The study was analyzed and approved

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by the Ethics Committee on Animal Research of the Complutense University of Madrid.

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Adult male Wistar rats (Charles River Inc., Margate, Kent, UK; 220-250 g) were used

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in this study. The rats were individually housed in polycarbonate cages in a silent room

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under a controlled temperature (22 ± 2 ºC), relative humidity (50 ± 10%) and 12 h

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light/dark cycle (light from 08.00 to 20.00 h), with solid food (A04 rodent diet,

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Scientific Animal Food & Engineering, SAFE, Augy, France) and water ad libitum. The

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rats were randomly divided into seven experimental groups: six groups of 10 animals

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each treated with Tonalin TG-80 orally, and one blank or control group of 5 animals

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without any treatment. Animals were dispossessed of food for 12 h before single oral

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dose of 3000 mg Tonalin TG-80/kg body weight, but were supplied water ad libitum.

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Before dose administration, Tonalin TG-80 was kept in a water bath set to 30ºC to reach

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a liquid state (897 kg per m3) and was dispensed by gavage in a volume of 0.84 mL

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CLA oil/rat of 250 g body weight. The oral Tonalin TG-80 dose used of 3000 mg/kg

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body weight was equivalent to 1200 mg/kg body weight of each CLA isomer. Oral dose

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of 3000 mg Tonalin TG-80/kg body weight was chosed based on preceding experiments

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where the dose and route of administration did not show any undesirable effects,

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abnormal clinical signs as well as changes in behavioral, body weight, or in food and

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water consumption in the animals (data not showed). Moreover, this dose was also

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estimated to provide plasma concentrations of the CLA isomers to be above the level of

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limit of quantification (LOQ) of the analytical method for both CLA isomers (RA and

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C18:2 trans 10, cis 12).

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Animals were sacrificed by cervical dislocation (one animal at each time) and

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then blood samples at 15 min, 30 min, 1, 2, 4, 6, 8, 12, 24 and 48 h after oral dose of

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Tonalin TG-80 were collected in heparinized tubes. Plasma was separated by

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centrifugation and stored frozen at -80ºC until analysed.

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Analysis of plasma samples. Plasma sample preparation. The

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concentrations of RA and C18:2 trans 10, cis 12 in plasma were determined as fatty acid

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methyl esters (FAME) using a one-step procedure according to Castro-Gomez et al.,27

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with some modifications: 500 µL of plasma sample were spiked with 100 µL of

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glyceryl tritridecanoate (1.44 µg/mL). Transterification reaction was carried out by

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adding 2.5 mL methanolic sodium methoxide 0.5 M at 80 ºC during 10 min while

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afterwards an esterification reaction was accomplished using 1.66 mL of N,N-

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dimethylformamide and 3 mL 1M H2SO4 in methanol at 100 ºC for 30 min. After

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cooling samples in ice, FAME were extracted using 1 mL of hexane and 1 min vortex

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(waiting 5 min for the separation of layers), then adding 7,5 mL 6 % w/v Na2CO3 and

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finally 3500 rpm, 10 min. at room temperature. Upper phase was collected and placed

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into a vial to be analysed by gas chromatography with flame ionization detector (GC-

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FID).

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To prepare the standard curves, RA and C18:2 trans 10, cis 12 were diluted in

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hexane to final concentrations into a range of 0.006 to 300 µg/mL in rat control plasma.

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Promptly after adding the CLA isomer solution to the plasma, they were prepared for

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analysis as above mentioned. The calibration curves were performed the same day as

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the rat treated samples.

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FAME analysis. The obtained FAME fraction, was injected (1 µL; 10:1 split

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ratio) into a gas chromatograph (GC) 6890 Agilent (Palo Alto, CA, USA) coupled to a

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flame ionization detector (FID) and a mass spectrometer (MS; Agilent 5973N) coupled 8 ACS Paragon Plus Environment

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to a CP-Sil88 capillary column (100 m x 0.25 mm i.d. x 0.2 mm, Chrompack,

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Middelburg, The Netherlands) with working conditions according to Rodríguez-Alcalá

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et al.28 The injection volume was 1 µL. MS was used to identify and FID to quantify the

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CLA isomers by comparing the CLA oil supplement (Tonalin TG-80; FAME prepared

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from 5 mg as described in analysis of plasma samples) and CLA standards. In these

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conditions, LOD was 0.009 µg/mL and LOQ was 0.03 µg/mL.

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Data analysis. Plasma CLA isomers (RA and C18:2 trans 10, cis 12)

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concentrations versus time curves were fitted to two-compartment model using the

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computer program Phoenix (Version 7.0; Pharsight Corporation, Mountain View, CA,

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USA). This model was established for best fit based on a smaller value for the Akaike’s

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Information Criterion.29 The two-compartment model was the best fit for the two CLA

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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 dose of Tonalin TG-80 were fitted to the following exponential equation:

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C = A1 × e -αt + A2 × e -βt – A3 × e –Kat

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where C is the plasma concentration of the compound; A1, A2 and A3 are mathematical

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coefficients; α is the hybrid rate constant for the distribution phase; β is the hybrid rate

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constant for the elimination terminal phase; and Ka the first-order absorption rate

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constant, and t is the time.

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Absorption half-life (t1/2a), half-life of α phase (t1/2α), half-life of β phase (t1/2β),

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distribution rate constants for transfer of the CLA isomer from the central to the

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peripheral compartment (K12) and from the peripheral to the central compartment (K21),

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and the elimination rate constant (K10) were calculated by use of standard equations as

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described.30,31

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The area under the concentration-time curves (AUC) and the mean residence

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time (MRT) were calculated as follows:

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AUC = (A1/α) + (A2/β) – (A3/Ka)

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MRT = [(A1/α2) + (A2/β2) – (A3/Ka2)] (1/AUC)

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Maximum plasma concentration (Cmax) and the interval (time) from oral

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administration until Cmax was detected (Tmax) was also estimated directly from the

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concentration versus time curve.

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Differences in pharmacokinetic data between RA and C18:2 trans 10 cis 12,

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were analyzed for statistical significance by the paired Student’s t-test (GraphPad Prism

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6). Differences of P0.05; **P