Biochemical Changes in Fatty Acids , Hydrocarbons and Sterols as well as Total Lipids of Albino Rats Ingested some Synthetic Colourants and Flavourants as Food Additives

The present study was carried out to investigate the effect of ingested synthetic food colourants or flavourants on total lipids content feces. The feces of rats ingested synthetic food colourants or flavourants has more lipids than that of control feces. In treated rat feces some fatty acids were disappeared (C19) or appeared (C14) while the other were increased (C15, C20, C18:2) or decreased (C6) when compared with the control feces. Most of unsaturated fatty acids (un SFA) might bind with the synthetic food colourants or flavourants and secreted in feces, but less consumed than saturated fatty acids (SFA) which, observed on hydrocarbon components, but sterols including cholesterol were more secreted by synthetic food additives treatments. Generally, the increase of total lipids and lipid fractions in feces such as unsaturated fatty acids (un SFA), total sterols (TS) especially cholesterol maybe due to their abilities to bind with food additives and form complex which secreted in feces.


Introduction
Indeed literature has been revealed few reports, which dealt with metabolic changes and other side effects of synthetic food additive.However, several synthetic organic materials are natural constituents of edible food.Acceptance of these substances has been hindered by problems involving assessment of the net benefit of their use, some individuals stressing the absence of any attendant preservative or nutrient function and others often overlooking any possible hazards to health (Lerner and Lerner 2011;Becerril et al., 2013).Artificial additives have been pilloried as possible toxic hazards, branded as potential sensitizer (Mister and Hathcock 2012).Progress has been in the testing program of many synthetic food additives, to overcome the "lack of sufficiently comprehensive biological evidence of safety" so common (FAO/WHO 1974;Branen et al., 2001;Msagati 2013).Synthetic food flavourants and colourants are considered the most important class of food additives.They have been used in candy, soft drinks, foods, pharmaceuticals and cosmotics for many years, comparatively little work on their metabolic action was published (Gultekin et al.,2013;Martyn et al.,2013).With increasing awareness of possible health hazards associated with their use, however, more attention has been focused the biological activity (Brown et al., 1980;Borzelleaca et al., 1983;Abdel-Rahim et al., 1992;Lodi et al., 2011;Alger et al., 2013).Further investigations were carried out on both synthetic food flavourants and colourants to evaluate the acceptability of these compounds as food additives for human and animal feeding (Tsuji et al., 1978;Ramadan and El-Damhogy 1994).
It must be born in mind that, with the exception of a few special cases of hypersensitivity to a variety of chemicals including certain additives, no direct evidence has emerged to incriminate any synthetic food additives among the factors responsible for serious illness in man (Lucova et al., 2013), about 20% of synthetic food colourants and more of synthetic food flavourants are absorbed from gut (Gaur et al., 2003;Sarikaya et al., 2012), moreover, they possess a wide range of biological and physiological effects on the metabolism of animals (FAO/WHO 1974;Abdel-Rahim et al., 1992;Tanaka 2006;Tanaka et al., 2008;Gao et al., 2011).
There is not available data in literature about the effect of synthetic food additives on lipid and their fractions in feces (This work is the first of its kind in this area in Egypt and perhaps in all countries of the world).For that, the aim of the present work was to carry out some biochemical studies on rats orally administrated some synthetic food colourants and flavourants in a trial to evaluate their effects on the patterns of fatty acids (saturated and unsaturated), sterols, hydrocarbons composition as well as total lipids content in rat feces.
was equipped with DB-23 (J & W 122-2362) 25 μ capillary column, 60 m × 0.25 mm ID, 0.15 µ m.The operating conditions for gas chromatography were as follows: injector temperature 250 °C, carrier gas: helium at 30 cm/sec, measured at 150 °C, oven temperature 50 °C for 4 min, 150 °C for 4 min and held at 250 °C until the chromatogram was completed.The detector temperature was 280°C.Mass spectroscopy operating parameters were electron ionization at 70 ev, accelerating voltage 10 kV and scan M/Z range from 50 to 500.National Institute of Standards and Technology (NIST) library according to Jiang et al. (2006).

Determination of unsaponifiable profile by GC-MS
The unsaponifiable fractions were finally collected in ether and taken to dryness under vacuum.The residue was analyzed using the gas chromatograph HP 5890 (Hewlett Packard) equipped with the MS detector (MSD 5970), EI, 70 ev and fitted with a capillarycolumn DB-1701 (12 m x 0.18 mm × 0.4 mm; J&W Scientific).The column temperature was programmed from 260 to 300 °C while injection temperature was set at 280 °C.Helium was the carrier gas at a flow rate of 0.7 cm 3 /min.Identification of peaks was based on the retention time of standard substances and MS spectra.Analyses were run in triplicate.Calculations of percent composition of demethylhydrocarbons and demethylsterol fractions were based on the peak area.

Statistical analysis
All experimental results were expressed as means ± S.D. Analysis of variance was performed by ANOVA procedures.The results with P < 0.05 were regarded to be statistically significant.Data were statistically analyzed using Costate Statistical Package (Anonymous, 1989).

Results and Discussion
Effect of synthetic food colourants and flavourants on the patterns of fatty acids (saturated and unsaturated), sterols, hydrocarbons composition as well as total lipids, total saturated acids (SFA), total unsaturated fatty acids (USFA), total sterols (TS) and total hydrocarbons (TH) contents were investigated and the results are tabulated in Table (1-6).

Synthetic food colourants
As shown in Table (1), the ratios USFA/SFA and TS/TH were 0.11 and 0.14 for control rat feces, respectively, while total lipids were 8.12%.On the other hand, the ratio un SFA/SFA of rat feces ingested synthetic food colourants was higher than that control, whereas Ponceau 4R gave the highest effect, followed by brown chocolate, while Yellow 2G gave the lowest one, but TS/TH ratio was lower than that control.In case of total lipids, their levels in rat feces ingested the synthetic food colourants were almost two times more than control rat feces.From the above results in Table (1), it was suggested that, unSFA and TS of control feces were consumed and absorbed into intestinal tract, while SFA and TH were markedly increased and secreted in rat feces.From results of treated rat feces with synthetic colourants, it was concluded that, no great difference was observed in SFA between treated rat feces with colourants and control ones, while the increment of unSFA of treated rat feces, relative to control, may be due to their abilities

Animals, diets and treatments
A total of 70 adult male albino rats 80 days old, average weight 90-100g were kept under normal healthy laboratory conditions and fed normal diet which consisted of casein 15%, cotton seed oil 10%, cellulose 5%, salt mixture 4% (Hegested et al., 1941), vitamin mixture 1% (Compbell, 1961) and corn starch 65%.Diet and water were supplied ad libitum for a period of month.
The rats were divided into 7 groups; the first group (10 rats) served as control and fed on synthetic additives samples free diets.The other six groups (each of 10 rats) were ingested by stomach tube a dose of 0.4 g/kg diet every 48 hours of each of synthetic food colourants (Yellow: 2G, Ponceau 4R and chocolate) for a period of one month (FAO/Who, 1974).Faces of each group of rats were collected and subjected for analysis.Also, cotton seed oil of diet was analyzed as comparative study with investigated samples.

Lipid extraction
The lipid materials of rat feces were extracted using choloform-methanol mixture (2:1, v/v) according to method of Bligh and Dyer (1959).Lipid content was expressed as g/100 g feces.

Separation of fatty acids and unsaponifiables from lipid samples
Lipid material was saponified with methanolic KOH (40 %, w/v) for 24 h at room temperature according to Ahmed et al. (1986).The unsaponifiables were extracted three times with ether.The aqueous layer was acidified with HCl (1:1, v/v) and the liberated fatty acids were extracted three times with ether.The combined extracts of unsaponifiables and fatty acids were washed several times with distilled water and then dried over anhydrous sodium sulfate.

Methylation of lipid materials
The standard and the sample fatty acids were converted to methyl esters using ethereal solution of diazomethane according to Vogel (1975).

Determination of fatty acid composition by (GC-MS)
The fatty acid methyl esters were determined by GC-MS using Trace GC Model 2000 series produced by Thermo equipped with Selective Detector Mass Spectroscopy Model SSQ 7000 produced by Finnigan.This equipment was interfaced via HP chemstation version A 02.12 software (Hewlett-Packard.Avondale, PA).The gas chromatography to bind with synthetic colourants in a complex from secreted in feces.Also, the results are indicated that, TS of treated rat samples are consumed and absorbed of Ponceau 4R and brown chocolate, relative to control rat feces, while the increase of TH in the treated feces may be due to form a new complex with synthetic colourants which secreted in feces.Generally, total lipids are higher in treated rat feces than in control, it means that, synthetic colourants may be linked with different lipid fractions which secreted in feces.

Synthetic food flavourants
As shown in Table (4), it was observed that, SFA are the major fatty acids either in control or in treated rat feces with synthetic food flavourants, since their levels were ranged from 76.85 to 90.35%, while USFA levels of treated rat samples (except banana flavourant treatment) are two times more than that of control rat feces.Also, the ratio of unSFA/SFA under the effect of synthetic flavourants gave the same trend as of colourants treatment.Total sterols (TS) in treated rat feces with banana flavourant is higher than that of control rat feces, while strawberry flavourant represents the highest value, but chocolate flavourant gave the lowest one, relative to control.No significant difference was observed in TH between control and treated rat feces except of strawberry flavourant treatment.In treated rat feces with banana and strawberry flavourans, ratio of TS/TH was higher than that of control feces, while the chocolate flavourant gave the lowest one.Levels of total lipids in treated rat feces with synthetic food flavourants are about two times or more higher than that of control.From the above results in Table ( 4), it was concluded that, large amounts of synthetic food flavourants may be bound with unSFA and TS to give a new complex secreted in feces when compared with control feces.Generally, this trend was observed in total lipids which secreted in and lipid fractions which secreted in feces via bound with synthetic flavourants (Chen and Ni 2009;Ohtsuki et al., 2012;Pundir and Rawal 2013).
From obtained results in Tables (1-6) it was observed that, synthetic food additives (colourants and flavourants) have similar effects on lipid fractions, these may be due to that, synthetic food colourants and flavourants have several function groups such as -OH, -COOH, -N=N-, -NH2, HSO4, …..etc (FAO/ WHO, 1974;Ferreira et al., 2000;Stolz et al., 2001;Chudgar and Oakes 2003;Isaac et al., 2006).These synthetic food additives were bound with the different lipid fractions to prevent the absorption of them including cholesterol through intestinal tract and their secretion in feces.The appearance or disappearance and increase or decrease of some lipid fractions which observed in the present work may be due to intestinal microflora (Rencuzogullari et al., 2001;Vally et al., 2009;Garcia-Gavin et al., 2012).These microorganisms have different biological systems for degradation or synthesis of lipids (Dawes and Sutherland, 1976;Jawetz et al., 1978;Brul and Coote, 1999;Tajkarimi et al., 2010) which may be altered synthetic food additives (Grasso et al., 1974;Hveland-Smith andCombos, 1980 and1982;Maha, 1990;Zhang and Ma 2013).These data are confirmed by those of FAO/WHO (1974); Abdel-Rahim et al. (1992) and Beezhold et al. (2014).On the other hand, the elevation of total lipids and lipid fractions in treated rat feces were paralleled with decrease of them in blood and liver (Abdel-Rahim et al., 1989;Caroch et al., 2014).

Conclusions
Finally, other efforts must be supported to investigate the capability and safety of these tested synthetic colourants and flavourants as hypocholestrolimic and hypolipidimic agents for lipid fractions.Also, more investigations are required to assess the significance of the present findings with regard to general undesirable effects of synthetic additives in different organisms.

Table 1 .
Effect of synthetic food colourants on total lipids, saturated, unsaturated fatty acids, sterols and hydrocarbons of rat feces

Table 2 .
Relative percentage of fatty acids in rat feces affected by synthetic food colourants

Table 3 .
Relative percentage of unsaponifiable matters in rat feces affected by synthetic food colourants

Table 5 .
Relative percentage of fatty acids in rat feces affected by synthetic food flavourants

Table 6 .
Relative percentage of unsaponifiable matters in rat feces affected by synthetic food flavourants

Table 4 .
Effect of synthetic food flavourants on total lipids, saturated, unsaturated fatty acids, sterols and hydrocarbons of rat feces