1. Introduction
Supplementing methionine (Met) to broiler diets in order to balance the amino acids in accordance to the broilers demand is a common practice. However, Met has several import functions apart from its role as a proteinogenic amino acid [
1]. For instance, S-adenosyl-Met—a metabolite of Met—is acting as a precursor of homocysteine, from which cysteine (Cys) is formed via the trans-sulfuration pathway [
2]. Cys is moreover required for the formation of taurine and glutathione (GSH), both of which have important functions as antioxidants [
3]. Dietary supplementation of Met could therefore improve the antioxidant defense system of the body via increased concentrations of these Met metabolites. According to the role of Met in the antioxidant system, it has been shown that an insufficient supply of dietary-sulfur-containing amino acids (SAA) attenuates the antioxidant system in broilers [
4,
5]. The aim of present study is to investigate the hypothesis that supplementation of Met in excess of the requirement enhances the antioxidant system in broilers. It is well-known that some commonly used parameters of lipid peroxidation, such as concentrations of thiobarbituric acid-reactive substances (TBARS), are relatively unspecific parameters of lipid peroxidation [
6]. In contrast to TBARS, oxidation products of cholesterol (cholesterol oxidation products, COPs) and phytosterols (phytosterol oxidation products, POPs) are much more specific indicators of lipid oxidation [
7,
8]. Moreover, COPs and POPs are relevant with respect to human health as they promote the development of several diseases including coronary heart disease or cancer [
9,
10]. COPs and POPs are formed predominately during heat-processing of foods containing cholesterol or phytosterols, respectively [
11,
12,
13,
14]. In order to investigate the hypothesis that supplementation of Met in broilers reduces the formation of COPs and POPs, we determined the concentrations of individual COPs and POPs in heat-processed thigh muscle of broilers.
4. Discussions
This study investigated the effect of dietary DLM supplementation on the antioxidant system in broilers. In contrast to our recent study, which investigated the effect of Met supplementation to a diet in which the Met concentration was 15% below NRC requirements [
5], we used basal diets which were still below those recommended by the breeder but met requirements according to NRC [
17]. The finding that animal performance in the present study did not differ between the three groups of broilers implies that the Met + Cys concentration in the basal diets was already sufficient for maximum growth under the specific conditions in this experiment. However, it should be noted that the small number of replications for assessment of growth performance (n = 3) does not yield results with a sufficient statistical power. Therefore, the performance data should be interpreted carefully. Moreover, it should be noted that the effect of the treatment on growth performance was not the primary aim of this study.
The data of this study show that dietary Met concentrations which are 15–20% above the breeder´s recommendations, compared to diets 15–20% below those recommendations, have only minor effects on the concentrations of tocopherols and vitamin C in the plasma, liver, and thigh muscle of broilers. The only exception of this was an increase of the concentration of vitamin C in thigh muscle of the broilers of group DLM 1. As concentrations of antioxidants such as tocopherols or vitamin C are expected to be lowered under condition of oxidative stress due to an increased consumption by reaction with ROS [
29], it is expected that a reduction of oxidative stress would lead to increased concentrations of these antioxidants. Thus, these results would indicate that supplementation of DLM in slight excess of the recommendations has limited effects on the antioxidant system. However, we observed a significant increase of GSH in liver and thigh muscle of broilers supplemented with DLM. This observation might be due to the fact that GSH forms a storage pool of readily available Cys, which is increased when Met availability increases [
2]. Previous studies have already shown that supplementation of Met increases the formation of GSH in liver and thigh muscle of broilers [
4,
5,
15,
30,
31,
32]. GSH is one of the major water-soluble antioxidants in the body, and thus an increased GSH concentration could imply that the antioxidant status was improved by DLM supplementation. In contrast to the suggestion of an increased antioxidant status of broilers supplemented with DLM, concentrations of TBARS in plasma, liver, and thigh muscle remained unchanged by DLM supplementation. The concentrations of TBARS are often determined in studies dealing with oxidative stress as an indicator of lipid peroxidation. However, it should be noted that TBARS represent a relatively unspecific parameter of lipid peroxidation due to the fact that thiobarbituric acid reacts with a variety of aldehydes and breakdown products of proteins and carbohydrates [
6]. Thus, the observation that TBARS concentrations were largely unaffected by the treatment must not necessarily be contradictory to the suggestion that supplementation of DLM caused an improvement of the antioxidant status.
Cholesterol, a natural compound in animal tissues located mainly in cell membranes, is susceptible to oxidation, which results in the formation of COPs. The formation of COPs in animal tissues is strongly increased by heat-processing. It has been observed that concentrations of COPs in breast or thigh muscle of broilers are increased 10- to 40-fold by heating at a temperature of 180 °C for 20 min [
11]. The formation of COPs is not only a valid indicator of the susceptibility of a tissue towards lipid peroxidation, but COPs are also relevant from a nutritional point of view, as their intake in humans is associated with the risk of various diseases, such as cardiovascular disease or cancer [
10]. We observed that supplementation of DLM led to a reduction of the concentrations of individual and total COPs in heat-processed thigh muscle. This finding agrees with a previous study which has shown that supplementation of DLM reduces the formation of COPs in heat-processed thigh muscle in broilers subjected to heat stress [
15]. The inverse correlations between the concentration of GSH and vitamin C in thigh muscle and the concentrations of COPs in heat-processed thigh muscle indicates that the reduced formation of COPs was due to increased concentrations of those antioxidants in the muscle. The concentrations of total COPs in heat-processed thigh, being in the range between 10 and 30 nmol/g sample in the three treatment groups, were in the same order of magnitude as those detected in cooked broiler meat considered in other studies [
33,
34]. Although the concentrations of COPs found in heated thigh muscle are much lower than in, e.g., dried egg products, heat-processed meat products contribute considerably to the intake of COPs in consumers [
35]. Thus, the reduction of the concentration of COPs by supplementation of DLM might be regarded as a favorable effect, not only with respect to animal health, but also with respect to product quality of broiler meat.
In this study, we also determined the concentrations of phytosterols and POPs in heat-processed thigh muscle. Phytosterols are very similar to cholesterol with respect to their structures and functions [
36]. We were able to detect several phytosterols in thigh muscle of broilers, with campesterol, sitosterol, and campestanol as the major components. The concentration of total phytosterols, being around 150 nmol/g (equivalent to 5.8 mg/100 g) in heat-processed thigh muscle of the control broilers, is much lower than in most plant foods, such as in cereals (50–100 mg/100 g [
37]) or vegetable oils (150–600 mg/100 g [
38]). Thus, broiler meat will contribute less to the total daily intake of phytosterols in Western diets which is commonly around 200 to 400 mg at a daily energy intake of 2000 kcal [
39,
40,
41]. Phytosterols are regarded as beneficial compounds in human nutrition, as they are able to reduce the absorption of cholesterol in the intestine, although significant effects on plasma cholesterol concentration are only reached by supplementation of 1.5 to 3 g [
42,
43]. Like cholesterol, phytosterols are susceptible to autoxidation, with 7α-hydroxy-, 7β-hydroxy-, and 7-keto derivatives being the main oxidation products [
36]. In the present study, we were able to detect oxidation products of campesterol and sitosterol in heat-processed thigh muscle. Among the POPs identified, 7-keto derivatives of campesterol and sitosterol showed the greatest concentrations in heated broiler thigh muscle. This agrees with observations that 7-keto derivatives are the most abundant oxidation products of phytosterols in various products [
14]. In our study, around 7–8 mol% of sitosterol and 1.2–1.5 mol% of campesterol in thigh muscle were oxidized during heat-processing. These data show that the phytosterols in the broiler thigh muscle were highly susceptible to oxidation during heat-processing. We observed that the oxidation of campesterol to 7α-OH campesterol, 7ß-campesterol, and 7-keto campesterol in heat-processed broiler thigh muscle was reduced by DLM supplementation. Correlation analysis revealed an inverse relationship between the concentration of vitamin C in thigh muscle and the concentrations of POPs in heat-processed thigh muscle, indicating that the slightly increased concentrations of vitamin C in muscle of broilers supplemented with DLM in comparison the control broilers could be responsible for a reduced formation of POPs. Although POPs are poorly absorbed in the small intestine, they are suggested, like COPs, to be atherogenic, and therefore their intake via foods requires attention [
9,
44]. However, it has to be noted the concentrations of total POPs in heat-processed thigh muscle being in the range between 3.5 and 4 pmol per g (equivalent to 0.875 to 1.00 µg/100 g) of thigh muscle are much lower than in other foods, such as plant oils (0.5 to 7 mg/100 g in different samples of corn oil or sunflower oil), or products baked with margarines, such as cookies or muffins (0.2–0.7 mg/100 g) [
13,
45]. Thus, the consumption of heat-processed broiler meat is uncritical with respect to the ingestion of POPs.