ORIGINAL ARTICLE

Omega-3 and Omega-6 salivary fatty acids as markers of dietary fat quality: A cross-sectional study in Argentina

Acidos grasos salivales Omega-3 y Omega-6 como marcadores de la calidad lipidica de la dieta: un estudio de corte transversal en Argentina

 

María D. Defagó^1,2, Nilda R. Perovic², Mirta A. Valentich¹, Gastón Repossi¹, Adriana B. Actis^1,3

¹ Universidad Nacional de Córdoba, Instituto de Investigaciones en Ciencias de la Salud (INICSA), CONICET, Córdoba, Argentina.
² Universidad Nacional de Córdoba, Facultad de Ciencias Médicas Escuela de Nutrición, Córdoba, Argentina.
³ Universidad Nacional de Córdoba, Facultad de Odontología, Cátedra B de Anatomía, Córdoba, Argentina.

 

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ABSTRACT

The use of saliva for analyzing biological compounds has recently been expanded. The aim of this study was to analyze the correlation between specific dietary sources of n-3 and n-6 fatty acids (FA) and their salivary levels to evaluate their role as intake markers.
Seventy-nine healthy volunteers were included. A validated food frequency questionnaire was used for data collection and Interfood v.1.3 software was employed to quantify food intake. Salivary samples were collected following international standards and FA profile was determined by gas liquid-chromatography. Multiple linear regression analyses were performed for dependent variables (salivary FA profile) to detect independent associations with n-3 and n-6 FA food source intake, adjusted by age, gender, body-mass index, total energy intake, regular exercise, alcohol intake and smoking. Salivary concentrations of alpha-linolenic acid (ALA) 18:3 n-3 were significantly associated with nuts intake (p=0.05, 95% CI 0.02-0.07, p=0.04). Salivary concentrations of linoleic acid (LA) 18:2 n-6 and arachidonic acid (AA) 20:4 n-6 were associated with the intake of n-6 vegetable oils and red meat, cold meat and viscera (p=-0.80, 95% CI 0.06-0.09p=0.03; P=-0.40, 95% CI 0.30-0.50, p=0.02, respectively).
This study supports the hypothesis that salivary concentrations of n-3 and n-6 FA are related to food intake. Monitoring dietary FA though salivary markers is relevant for nutrition epidemiology and for prevention and management of several diseases related to fat intake.

Key words: Ftty acids; Diet; Saliva; Biomarkers.

RESUMEN

El uso de biomarcadores salivales está en continua expansión. El objetivo de este estudio fue analizar la asociación entre fuentes alimentarias de ácidos grasos (AG) n-3 y n-6 y sus concentraciones salivales como marcador de ingesta. Participaron 79 voluntarios sanos. Se aplicó un cuestionario validado de frecuencia de consumo alimentario y el software Interfood v.1.3 para su procesamiento. Las muestras salivales se recogieron según estándares internacionales y se determinó el perfil de AG salivales por cromatografía gaseosa. Se desarrolló un modelo de regresión lineal múltiple ajustado por sexo, edad, índice de masa corporal, valor energético total, actividad física, consumo de tabaco y alcohol para analizar la asociación entre el perfil de AG salivales y la ingesta de alimentos fuente de AG n-3 y n-6.
Las concentraciones salivales del AG alfa-linolénico (ALA) 18:3 n-3 se asociaron positivamente con la ingesta de nueces (p=0.05, IC 95% 0.02-0.07, p=0.04), mientras que las concentraciones salivales de ácido linoleico (AL) 18:2 n-6 y araquidónico (AA) 20:4 n-6 se asociaron con el consumo de aceites ricos en n-6 (p=0.80, 95% IC 0.06-0.09p=0.03) y de carnes rojas, fiambres y embutidos y visceras, (p=0.40, IC 95% 0.30-0.50, p=0.02). De acuerdo a estos resultados, las concentraciones salivales de AG n-3 y n-6 se relacionan a la ingesta de sus alimentos fuente. El monitoreo de la ingesta lipídica a través de biomarcadores salivales constituye un aporte a la epidemiología nutricional y a la prevención y tratamiento de patologías vinculadas a la ingesta de grasas.

Palabras clave: Ácidos grasos; Saliva; Dieta; Biomarcadores.

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INTRODUCTION

The relationship between diet and the risk of developing chronic diseases has been documented in recent decades. Nutritional epidemiology, focusing on dietary intake, involves the development of several tools to estimate calories, proteins, carbohydrates, fats, vitamins, minerals, etc., such as diet records, dietary recalls and food frequency questionnaires (FFQ). The FFQ is one of the most commonly used methods because it provides insight into the regular dietary intake of a population over time and is relatively cheap, quick and easy to use¹. Although this progress in dietary recall tools is promising and cost-effective, the methods for assessing dietary intake are still not without intake error, a commonly cited research limitation. The combination of different methods (e.g. administration of different questionnaires and assessment of biomarker levels) could reduce respondent burden and reporting bias²³.

Blood is the most commonly used biological fluid for biomarker determination and is sensitive to dietary intake. Urine is a good indicator of hydrosoluble compound intake, since their concentration in urine depends on the nutrient saturation degree in tissues^4,5. The use of saliva for analyzing biological compounds to evaluate nutritional status has recently been expanded^6-8. Collecting saliva is non-invasive, more comfortable than venipuncture and requires no special equipment, and it is easily stored prior to analysis^9-11.

Fatty acid (FA) intake is reflected in a wide variety of biological tissue samples, such as serum and subcutaneous adipose tissue^12,13. A previous study showed a significant increase of α-linolenic acid 18:3 n-3 (ALA) in saliva from vegetarians or semivegetarians compared to people who consumed a mixed diet rich in animal products, who had significantly higher levels of salivary arachidonic acid 20:4 n-6 (AA)¹⁴. This finding could be related to the significant intake of food rich in n-3 essential fatty acids (EFA), such as soy, dried fruits, sunflower and corn oils.

Competition between metabolic pathways may lead to changes in FA composition independent of dietary content. It is reasonable to expect that the best markers of dietary intake would be FA that cannot be endogenously synthetized such as trans, linoleic acid 18:2 n-6 (LA) and ALA. Other FAs whose synthesis depends on EFA intake include eicosapentaenoic acid 20:5 n-3 (EPA) and docosahexaenoic acid 22:6 n-3 (DHA), also present in fish and seafood¹⁵. EFA are important compounds which play a role in the complex biological process of inflammation. EFA and their metabolites are known to have pro- and anti-inflammatory actions and to regulate gene expression, enzyme activity, immune response and gluconeogenesis¹⁶.

We hypothesized that FA salivary levels may be affected by dietary sources of FA. The main aim of this study was to analyze the correlation between specific dietary sources of n-3 and n-6 FA and salivary levels of EFA and derivatives, to evaluate their role as intake markers.

MATERIALS AND METHODS

Study participants

A cross-sectional study was carried out involving non-probability sampling of 79 male and female adults aged eighteen to eighty years who visited two hospitals in Córdoba (Privado and Córdoba), Argentina for a routine check-up. Subjects on special diets and those with digestive and/or metabolic dysfunction (such as celiac disease, lactose intolerance or diabetes) were excluded. All participants provided informed consent to participate in this study. The study complies with the Helsinki Declaration and was carried out according to the guidelines for the protection of the volunteers' rights. It was approved by the Institutional Ethics Committees (Córdoba, Argentina).

Dietary assessment

Participants were interviewed by two well-trained professional nutritionists. A validated qualitative and quantitative FFQ was used to collect data. It included questions related to 257 types of food and drinks consumed, with emphasis on food sources of fat¹⁷. This instrument includes questions on intake of whole and low-fat dairy products, cheese, eggs, beef, poultry, pork, fish, canned fish, seafood, viscera, sausages, vegetables, tomato derivatives, herbs, fruits, nuts, legumes, cereals, bakery products, animal fat, oils, seasonings, sugar, confectionery, pastry, beverages (alcoholic and non-alcoholic), snacks, ice cream and soy products. The frequency references used were the number of times each item was consumed per month, per week or per day and never. Portion size was described as small, medium or large using a photographic guide to help subjects understand these definitions¹⁸.

Food data were processed using a software package called Interfood v.1.3, to produce information in grams/day of each food item consumed¹⁹. This software is an open-source program that has three basic components: dietary intake FFQ; a database of common foods and their composition considering 131 substances (macro- and micronutrients and phytochemicals); and a relational database that links the FFQ data with the food database.

Collection of saliva samples

Saliva was collected from 8:00 to 10:00 a.m. after a minimum of six to eight hours fasting. With the subject leaning forward, unstimulated but spontaneously flowing whole saliva was collected in sterile plastic test tubes (5 mL or more). Prior to saliva collection, the study subjects did not consume any drinks and were at rest with no previous physical activity, tooth brushing and/or oral rinse⁹. Samples were frozen at -20°C until they were processed by centrifuging at 80xg at 5°C for 30 minutes. The supernatant was analyzed.

Fatty acids analysis

Total salivary lipids were extracted with chloroformmethanol 2:1 according to Folch's method and FA were methylated by sodium methoxide^20,21. Salivary FA was separated, identified and quantified using a capillary column (BPX 70.30 m long, 0.25 mm ID, 0.25 um film, Phenomenex, Torrance, USA) in a gas chromatograph (Clarus 500 Perkin Elmer, Waltham, USA). Oven temperature began at 170°C, rising 1.2°C/min up to 240°C, with a total chromatographic run time of 30 minutes²². The free FA were identified using commercial standards. Values, expressed as a percentage of detected FA, corresponded to the mean of two chromatographic runs on each sample.

Assessment of other variables

Certified personnel following standardized procedures took all additional measurements. Body weight and height were measured using a professional mechanical scale equipped with calibrated stadiometer. Participants were in underwear and without shoes.

Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters (kg/m²). Other variables such as physical activity and alcohol and tobacco use were also evaluated.

Statistical analysis

Baseline demographic characteristics were calculated as means and standard deviations for continuous variables or as numbers and percentages for categorical variables. Gender differences in variables of lifestyle status, food intake and salivary FA concentrations were compared by Wilcoxon's test for continuous variables and Fisher's test for categorical variables. Multiple linear regression analyses were performed for dependent variables (salivary FA profile) to detect independent associations with n-3 and n-6 FA food source intake in grams/day (fish and seafood, nuts, vegetables oils rich in n-6, and red meat, cold meat and viscera), adjusted by age, gender, BMI, total energy intake, regular exercise, alcohol intake and smoking. All tests were twosided and significance was considered at p<0.05. All analyses were performed with Stata® statistical software package (v.11.0).

RESULTS

Table 1 shows the basic characteristics of the study subjects. Mean age was 57.7 years for men and 49.2 years for women. 84.7% of men and 72.2% of women were current drinkers and 57.9% of men and 41.1% of women were current smokers. Average body mass index (BMI) was similar in men and women (27.7 and 25.5 kg/m², respectively). Regular physical activity was low in both men and women (47.3% and 45.1%, respectively). Calorie intake was 2678 ± 105 kcal/day for men and 2468 ± 387 kcal/day for women.

Table 1: Characteristics of study subjects.

Dietary fat source profile is shown in Figure 1. Overall, the foods most often consumed were dairy products, red meat, confectionary and stuffed pasta. Regarding specific dietary n-6 FA food source intake, sunflower, soybean and com oils were the oils most frequently consumed (oil average 21 ± 3 g/day) followed by red meat (84 ± 12 g/day). Total intake of red meat, viscera and cold meats was 120 ± 21 g/day). Regarding n-3 FA food source, fish, seafood and derivatives intake was 11 ± 5 g/day.

Fig. 1: Fat dietary source intake, grams per day.

Values are mean ± standard deviation for continuous variables and number (percentage) for categorical variables.

Hake, mackerel, dorado and tuna were the most common fish species in the diet. Nuts intake (5± 2 g/day) included mainly walnuts and peanuts. Nonsignificant statistical differences were found for food intake, alcohol and tobacco use and physical activity by gender (p>0.05).

Eighteen salivary FAs were analyzed by gas chromatography. Table 2 shows salivary FA composition. Non-significant statistical differences were found between men and women (p>0.05).

Table 2: Salivary fatty acid profile*.

The results of multivariate analyses are shown in Table 3. Salivary concentrations of ALA 18:3 n-3 were significantly associated with the intake of nuts (P=0.05, 95% CI 0.02-0.07, p=0.04). On the other hand, salivary concentrations of LA 18:2 n-6 and AA 20:4 n-6 were associated with vegetable oils and with red meat, cold meat and viscera intakes, respectively (P=0.80, 95% CI 0.06-0.09 p=0.03; P=0.40, 95% CI 0.30-0.50, p=0.02, respectively).

Table 3: Linear regression analysis (B coefficient) by food intake.

DISCUSSION

This study showed a significant correlation between salivary ALA 18:3 n-3, LA 18:2 n-6 and AA 20:4 n-6, and dietary sources in a healthy adult population. Although the presence of FA in adipose tissue, serum or plasma is accepted as a reliable biomarker of their intake in food, there is still scanty literature referring to the correlation between dietary and salivary lipids^{23 24}.

Salivary LA 18:2 n-6 and ALA 18:3 n-3 would be expected to have the strongest association with dietary intake because they cannot be synthetized endogenously. In our study, nut intake, a source of n-3 FA, was associated with 18:3 n-3 salivary levels. Nuts are natural foods rich in unsaturated FA; most nuts contain substantial amounts of monounsaturated FA, while walnuts are especially rich in both n-6 and n-3 FA²⁵. Although there is no evidence about nuts intake and salivary lipids, frequent consumption of nuts is related with lower concentrationsof inflammation markers and is also recommended by the WHO Guideline on Sugar Intake for Adults and Children for dental health professionals^26-28. The American Heart Association promotes the consumption of four servings (30-40 gram per portion) per week of nuts, seeds or legumes²⁹. The study population shows inadequate intake of nuts according recommendations, but even though it was low, it was reflected in the salivary n-3 profile. On the other hand, we found no association between fish intake and salivary levels of long chain n-3 FAs due to the very low fish intake recorded, which was considerably lower than current guidelines (100 grams, twice a week). In this respect, other authors have shown a correlation between fish intake and plasma n-3 FA^30,31.

Our results showed significant association between oil intake -mainly sunflower oil, followed by soybean and corn oils- and salivary LA 18:2 n-6. Previous studies have reported high intake of sunflower oil in Argentina³². The aforementioned oils are the main sources of 18:2 n-6 FA^33,34. The effects of 18:2 n-6 on health and disease are still controversial. An imbalance in the n-3:n-6 ratio, produced by an increased n-6 consumption or a reduced n-3 intake, may promote the endogenous synthesis of inflammatory molecules³⁵.

Significant association was observed between red meat intake, cold meat and viscera and AA 20:4 n-6 salivary concentrations. The presence of this FA in human saliva as related to diet and inflammatory conditions has been previously demonstrated ^10,36. Westernized diets characterized by high intake of red meat and processed foods have been related to inflammation biomarkers such as leukotrienes-2 and thromboxanes-4, which are associated with cardiovascular diseases and certain types of cancer³⁷. EFA metabolites, derived by lipoxygenase and cyclooxygenase enzymatic pathways, have been quantified in human mixed saliva as well as in saliva fractions. The level of free AA and the quantitative hydroxyeicosatetraenoic acid (HETE) act as markers for the inflammatory processes occurring in the oral mucosa and in saliva in response to the development of squamous cell carcinoma³⁸. In addition, pro-inflammatory lipid mediator precursors have been detected as potential markers for aggressive periodontitis³⁹. In this sense, monitoring dietary FAs such as LA and ALA and their metabolites is a relevant strategy in the prevention and management of several fat intake-related diseases, not for only oral diseases, but also cardiovascular disorders, diabetes and tumors such as prostate and mammary, among others.

Secretion of saliva plays an important role in numerous significant biological processes. Although some salivary components are well characterized, data for characterizing salivary lipids and their functions is scarce and controversial. However, the qualitative and quantitative content of salivary lipids may change in pathological states (e.g. cystic fibrosis and diabetes), and according our results, due to nutritional exposure⁴⁰. Thus, salivary lipids could be used for validating dietary measurement or as markers of food consumption.

Some limitations of the present study must be considered. It is cross-sectional design and therefore we cannot infer causality from these results. In addition, these findings cannot yet be generalized due to the sample size and the fact that a particular population was analyzed.

In conclusion, the present study supports the hypothesis that salivary concentrations of n-3 and n-6 FA are related to the intake of certain foods. Further studies are needed to confirm these results and to determine the causal direction of this relationship.

FUNDING

This work was supported by a grant from the Secretaría de Ciencia y Tecnologia, Universidad Nacional de Córdoba, Argentina, Number RHCS 2472/10.

CORRESPONDENCE

Dra. María Daniela Defagó

Instituto de Investigaciones en Ciencias de la Salud (INICSA), CONICET. Enrique Barros esq. Enfermera Gordillo, Ciudad Universitaria, Córdoba (5000), Argentina. danieladefago@hotmail.com

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