NMR-Based Lipidomic Approach To Evaluate Controlled Dietary

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NMR-based lipidomic approach to evaluate controlled dietary intake of lipids in adipose tissue of a rat mammary tumor model Lobna Ouldamer, Lydie Nadal-Desbarats, Stephan Chevalier, Gilles Body, Caroline Goupille, and Philippe Bougnoux J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00788 • Publication Date (Web): 12 Jan 2016 Downloaded from http://pubs.acs.org on January 12, 2016

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Journal of Proteome Research

NMR-based lipidomic approach to evaluate controlled dietary intake of lipids in adipose tissue of a rat mammary tumor model Lobna Ouldamer1,2,3,*, Lydie Nadal-Desbarats3,4, Stephan Chevalier

2,3

, Gilles Body

1,3

,

Caroline Goupille2,5, Philippe Bougnoux2,3,5

Authors’ affiliations: 1

Department of Gynecology, Centre Hospitalier Universitaire de Tours, Hôpital Bretonneau. 2

boulevard Tonnellé, 37044 Tours, France; 2INSERM UMR1069, 10 boulevard Tonnellé, 37044 Tours, France; 3François-Rabelais University, 10 boulevard Tonnellé, 37044 Tours, France; 4 INSERM UMR930, 10 boulevard Tonnellé, 37044 Tours, France; 5 Department of Oncology, Centre Hospitalier Régional Universitaire de Tours. Hôpital Bretonneau, 2 boulevard Tonnellé, Tours, France

Corresponding author*: Lobna Ouldamer Inserm UMR 1069 10 Boulevard Tonnellé 37044 Tours (France) Phone: +33 2 47 36 6179 Fax: +33 2 47 36 62 26 Email: [email protected]

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Abstract Fatty acids composition of adipose tissue may provide information on the nutritional part of the risk or evolution of breast cancer. To determine whether 1H-NMR of adipose tissue provides information on the nature of the diet consumed, a dietary intervention with increasing percentage of polyunsaturated n-3 docosahexaenoic acid (DHA 22:6n-3, provided as DHASCO oil) was applied to a rat model of N-Nitroso N-Methyl Urea-induced mammary tumors. Spectra of the lipid extracts were obtained from adipose tissues in five groups of Sprague-Dawley rats fed with a diet containing 7% peanut/rapeseed enriched with 8%(w/w) of an oil without (palm oil) or with low (1%), moderate (3%) or high (8%) DHASCO content. A control group received a basal diet with 15% peanut/rapeseed representative of the “Western” diet. After 5 months of those 5 controlled diets, adipose tissue was collected for analysis of the lipid extract using both 1HNMR analysis on an 11.7T spectrometer and gas chromatography considered as gold standard. 1

H-NMR analysis showed a dose-dependent increase in DHA in the lipid extract of adipose

tissues, a commensurate decrease in n-6 polyunsaturated fatty acids in the three DHA groups which allowed to follow n-6/n-3 ratio changes. The highest n-6/n-3 ratio was observed in the control “western diet” group compared to the other diet groups. The integrated spectral regions showed separation between groups thereby documenting a specific NMR lipid profile corresponding to each dietary intervention. Those diet-dependent NMR lipid profiles were consistent with that obtained with gas chromatography analyzes of the same samples. This study is a proof of concept highlighting the potential use of 1H-NMR approach to evaluate dietary intervention in biopsies of adipose tissues.

Keywords: Docosahexaenoic acid; 1H-NMR spectroscopy; Dose-effect; lipidomics.


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Introduction The prospect of primary prevention of breast cancer by nutrition requires identification of the population at risk. Previous identification of a significant inverse association between individual levels of docosahexaenoic acid (DHA 22:6n-3) in breast adipose tissue and the risk of breast cancer suggested a role for dietary long chain n-3 polyunsaturated fatty acids (PUFA) 1. However, the influence of other fatty acids with respect to the risk of breast cancer led to define a composite biomarker taking into account the contribution of fatty acid on the risk of breast cancer 2. Diet has also been involved in tertiary prevention of breast cancer. Dietary lipids have been long studied in association with breast cancer survival and recurrence. Using adipose tissue as a biomarker, the risk of metastases was found to be associated with a low level of alphalinolenic acid, the essential n-3 polyunsaturated fatty acid 3. Rock et al, recapitulated evidence that dietary lipids may influence local or distant recurrences and in turn influence survival 4. Chlebowski et al, in a controlled randomized trial of nearly 2.500 women already treated for an early breast cancer, have shown that a dietary intervention with reduction of the dietary fat intake led to a reduction of the relapse rate 5. Thus, quantitative and/or qualitative change in total dietary intake can modify breast cancer outcome. Identifying which nutrients or dietary interventions are protective is therefore a major clinical interest. Fatty acid composition of the white adipose tissue remains the most reliable qualitative biomarker of previous dietary intake of fatty acids because of its slow turnover time (2–3 years) in weight-stable individuals 6. Analysis of adipose tissue fatty acid profile has been based on conventional analytical procedure such as gas chromatography (GC), which remains a time-consuming method. Proton magnetic resonance spectroscopy (1H-NMR) of adipose tissue lipids represents an appealing alternative approach, which could circumvent the limitations of GC analysis 7. 1H-NMR provides precise determination of biochemical and metabolic profiles in a single spectrum but no study to date has validated this approach in terms of biomarker of past dietary intake of lipids to replace standard lipid biochemical techniques. There is therefore a need to determine first whether 1H-NMR of adipose tissue might provide information on the nature of the lipid diet and long-term modification in the diet. A dietary intervention with five oils conditioned with different percentage of DHA was applied to a well-established rat model developing primary breast tumor 8. The impact of each


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dietary intervention on adipose tissues was ascertained both by 1H-NMR spectra, to evaluate its performance in such situation, and by GC to determine the precise composition in fatty acids in the same white adipose biopsy. The comparison of the information provided by the fatty acid composition analyzed by GC and that obtained by the 1H-NMR spectroscopy lipid profile shows that 1H-NMR spectroscopy of adipose tissue is effective in discriminating different DHA-enriched or unsaturated-enriched diets and in identifying the nature of past diets.


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Materials and Methods: Study design: Experiments were approved by the local Institutional Animal Care and Use Committee (IACUC of the University Hospital of Tours) and all procedures followed the guidelines recommended by the EU for the care and use of laboratory animals. Thirty-five female Sprague-Dawley rats (Harlan, Gannat, France), purchased when they were 43 days old, were acclimatized for five days using standardized laboratory conditions with a 12h-light/12h-dark cycle in a room with a temperature- (20-22°C) and humidity-controlled environment (7678%). Mammary tumors were chemically induced in the rats when they were 48 days old (mammary gland maturation time) by a subcutaneous injection (25 mg/kg body weight) of a single dose of N-Nitroso N-Methyl Urea (NMU, Sigma-Aldrich Co, France), as previously described 8. These mammary tumors were mostly adenomatous or papillary carcinomas tumor detection and growth was followed every week by palpation and caliper measurement. A median number of three mammary tumors developed in rats for each dietary group, mammary tumors gradually appeared between 2 and 5 months after NMU injection at a median interval of 51 days (range 28-119). This interval was similar between groups. When tumor size reached 2-3 cm2, rat were ethically sacrificed. This staggering of tumor appearance led to echeloned sacrifices and to a mean time of dietary intervention of 125 ± 30 days. Diet: At the time of arrival, all rats received standard kibbles for rodent animals (Harlan, France). Three days after NMU injection, rats were randomly assigned to different groups and two experiments were set up: 1- A dose effect experiment, consisting of four groups of six to eight rats fed with a basal diet containing 7% of a mixture of peanut and rapeseed oils. This basal diet was supplemented daily with 8% palm oil (Société Industrielle des Oléagineux. St Laurent Blangy, France) containing high saturated and mono-unsaturated fatty acid levels, under isocaloric conditions. Three other groups were supplemented with DHA at 1% DHASCO – 7 % Palm oil (low DHA: L-DHA), 3% DHASCO -5% Palm oil (moderate: M-DHA) and 8% DHASCO (high: H-DHA), in order to maintain isocaloric conditions at 15% of total lipids. DHA was provided as microalgae-purified triglycerides, containing 40% DHA (DHASCO oil, DSM Nutritional Products, Columbia, MD. USA). Fatty acid composition of DHASCO oil (expressed as % area)


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was: 14:0, 12.45%; 16:0, 7.36%; 18:1 n-9, 21.18%; 18:2n-6, 1.09%; 18:3n-3,

NMR-Based Lipidomic Approach To Evaluate Controlled Dietary

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