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 _____________________________________________________________________

1 ACS Paragon Plus Environment


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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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1

INTRODUCTION

2 3

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

4

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

5

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

6

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

7

reduce adiposity in experimental animals and humans, although the potential

8

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

9

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

10

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

11

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

12

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

13

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

14

parameters.

15

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

16

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

17

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

18

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

19

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-

21

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

22

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

23

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

24 25

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-

30

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

31

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

37

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

51

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

52

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

67

mammary gland) and ALA (an essential polyunsaturated fatty acid [PUFA]) in rats after

68

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

70

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

74

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

82

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)

86

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),

92

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

93

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

94

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

95

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

96

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

97

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

98

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.

101

Animals and experimental design. The study was analyzed and approved

102

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

103

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

104

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,

107

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

108

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

109

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

111

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

113

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

115

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

117

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

118

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

120

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

121

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

122

C18:2 trans 10, cis 12).



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123

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

126

centrifugation and stored frozen at -80ºC until analysed.

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Analysis of plasma samples. Plasma sample preparation. The

128

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

129

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

130

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

131

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

133

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

134

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

135

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

136

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

137

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

138

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

139

FID).

140

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

141

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

142

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

143

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

144

the rat treated samples.

145

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

146

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

147

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

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148

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

149

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

150

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

151

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

152

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

153

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)

155

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

156

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

157

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

158

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

159

isomers and it was used to determine the kinetic parameters.

160 161

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:

162

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

163

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

164

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

165

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

166

constant, and t is the time.

167

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

168

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

169

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

170

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

171

described.30,31



172

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

173

time (MRT) were calculated as follows:

174

AUC = (A1/α) + (A2/β) – (A3/Ka)

175

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

176

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

177

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

178

concentration versus time curve.

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

180

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

181

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

Absorption Kinetics of the Main Conjugated Linoleic Acid Isomers in

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