Pruebas de la interfaz de usuario (IU)

Thirty-two female mixed breed rabbits (Oryctolagus cunniculus), of seven months of age, weighing between 900 and 1256 g with mean weight of 1065 ± 45.00 g were obtained from Goshen Farm Enterprises, a commercial rabbit breeding farm, in Oshogbo, Osun State, Nigeria. The animals were individually housed under the standard housing condition (22oC, light/ darkness cycle 12/12 hours) in a wire mesh rabbit hutches in the Animal House of the Department of Bioche- mistry, Adekunle Ajasin University, Akungba- Akoko, Ondo State, Nigeria. This study was approved by the local Institutional Animal Ethics Committee and was performed in accordance with the National Research Council’s guide for the care and use of laboratory animals.

The animals were fed ad libitum with pelletized commercially prepared grower feed containing 15% crude protein, 7% fat, 10% crude fiber, 1.0% calcium, 0.35% phosphorus and 2550 Kcal/kg of metabolisable energy. After two weeks of physiological adjustment period, the rabbits were randomly assigned into four groups of eight rabbits each. Group 1 received no MSG, group 2 received 1 mg, group 3 received 2 mg, and group 4 received

European Journal of Biological Research 2016; 6 (3): 218-225 4 mg per kilogram body weight of 40% aqueous

solution of MSG, prior to feeding every 48 hrs for nine weeks, using a rat gavage needle constituting the control, low dose group, medium dose group and high dose group, respectively. The experimental design adopted for this research was Completely Randomized Design (CRD).

Parameters on daily feed consumption and weekly weight gains were monitored. The feed consumption for each animal was measured daily by difference between the daily feed supplied and leftover, and live weight changes of the animals were determined weekly as the weight difference in comparison to the weight in the previous week. At the end of nine weeks of MSG administration, blood samples were obtained from 16 rabbits, consisting of four rabbits from each group for haematological and serum biochemical analyses.

2.2. Haematological and serum biochemical analyses

Haematological parameters (erythrocyte counts, total leukocyte counts, packed cell volume (PCV), haemoglobin (Hb) concentrations and the blood constants - mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) were determined as described by Ewuola and Egbunike [14], while the biochemical parameters (total serum protein, albumin, globulin and albumin - globulin ratio) were determined as earlier descri- bed [15]. Also the serum free cholesterol was deter- mined by enzymatic colorimetric methods using commercial test kits (Quimica Clinica Applicada, S.A.), while the serum enzymes, alanine amino- transferase (ALT), alkaline phosphatase (ALP) and aspartate aminotransferase (AST), were obtained using the Randox Laboratories Ltd, UK test kits. All determinations were carried out in duplicate.

2.3. Statistical analysis

Data obtained were analyzed by one-way analysis of variance (ANOVA) procedure of SAS [16]. The treatment means were compared using the Duncan procedure of the same software and result given p values of < 0.05 were considered significantly different.

3. RESULTS

The mean weekly feed intake of rabbits fed varied doses of MSG are as shown in Figure 1. The mean feed intake of the rabbits in this study significantly increased (p<0.05) proportionately throughout the experimental period with increased doses of MSG administered.

Weight gains of female rabbits administered with varied doses of monosodium glutamate are as shown in Table 1. Significant differences (p<0.05) in body weight gains and feed conversion efficiency were observed. The final weight gained by the rabbits significantly increased (p<0.05) with increased doses of MSG administered, with percent weight changes in rabbits administered with low dose of MSG not significantly (p>0.05) different from the control, which was however significantly (p<0.05) lower than those administered with medium and high doses of MSG. The rabbits administered with low dose of MSG were more efficient in feed conversion compared with those administered with other doses, including the control.

Figure 1. Feed intake of rabbits administered with varied doses of monosodium glutamate.

Table 2 shows the mean haematological parameters of both the control and rabbits administered varied concentrations of aqueous solution of MSG. There was no significant difference (p > 0.05) between the mean PCV, platelets, RBC, haemoglobin, MCV, MCH, and MCHC of the control and MSG-treated rabbits. However, there was significant difference between the mean white blood cell (WBC) counts of the control and those administered aqueous solution of

European Journal of Biological Research 2016; 6 (3): 218-225 MSG. The mean WBC values of those administered

aqueous MSG were generally higher than those of the control.

The serum proteins of female rabbits exposed to different doses of monosodium glutamate are as

shown in Table 3. The Table showed significant (p < 0.05) reduction in the total protein, albumin and globulin values of rabbits administered with MSG compared to the control.

Table 1. Weight gain of female rabbits administered with varied doses of MSG (Mean ± SEM) Parameters Control Low dose

(1 mg/kg) Medium dose (2 mg/kg) High dose (4 mg/kg) Initial weight (g) 1109.90±28.87 1106.00±20.41 1104.25±44.65 1102.86±86.05 Final weight (g) 1370.83±28.85d 1374.09±20.27c 1547.15±45.91b 1687.82±86.07a Change in weight (%) 23.51±1.09c 24.24±0.32c 40.11±2.83b 53.04±1.99a Feed Conversion Efficiency 3.15±0.26a 2.94±0.43a 4.35±0.85b 5.11±0.52b abc_{: Means with similar alphabetical superscripts on the same row do not differ significantly (P>0.05) from each other.}

Table 2. Haematological parameters of female rabbits administered with varied doses of MSG (Mean ± SEM) Parameters Control Low dose

(1 mg/kg)

Medium dose (2 mg/kg)

High dose (4 mg/kg) Packed cell volume (%) 32.10 ± 0.91b 37.50 ± 0.50a 34.90 ± 0.53b 29.33 ± 0.52c Red blood cell (x106/mm3) 4.76 ± 1.27 5.58 ± 0.42 5.37 ± 0.85 4.55 ± 0.78 Haemoglobin g/L 99.50 ± 3.05b 117.50 ± 2.05a 98.7 ± 1.54b 93.30 ± 1.64c Platelets (x103 /mm3) 115.50 ± 4.65 170.50 ± 4.25 164.5 ± 0.92 140.00 ± 6.60 White blood cell (x103/mm3) 12.75 ± 0.15b 13.70 ± 1.40a 13.58 ± 1.53a 13.53 ± 1.43a Mean corpuscular volume (µ3) 67.44 ± 7.16 67.20 ± 12.5 64.99 ± 6.24 64.46 ± 6.78 MCH (µµg) 20.90 ± 2.42 21.06 ± 1.25 18.38 ± 1.81 19.85 ± 2.10 MCHC (%) 4.00 ± 0.34 3.133 ± 0.10 3.54 ± 0.34 3.08 ± 0.31 abc

: Means with similar alphabetical superscripts on the same row do not differ significantly (P>0.05) from each other.

Table 3. Serum proteins of female rabbits administered with varied doses of MSG (Mean ± SEM) Parameters Control Low dose

(1 mg/kg) Medium dose (2 mg/kg) High dose (4 mg/kg) Total Protein (g/L) 6.90±31a 6.53±0.15b 6.18±0.10b 6.10±0.21b Albumin (g/L) 3.93±0.27a 3.77±0.27b 3.53±0.27c 3.40±0.32c Globulin (g/L) 2.97±0.38a 2.76±0.25b 2.65±0.31b 2.70±0.30b Albumin-Globulin ratio 1.32±0.25a 1.37±0.26a 1.33±0.28a 1.26±0.28a ab_{: Means with similar alphabetical superscripts on the same row do not differ significantly (P>0.05) from each other.}

Table 4. Serum enzyme activities and cholesterol of female rabbits administered with varied doses of monosodium glutamate (Mean ± SEM)

Parameters Control Low dose (1 mg/kg)

Medium dose (2 mg/kg)

High dose (4 mg/kg) Aspartate Aminotransferase (IU/L) 36.70±6.70b 40.00±5.80b 42.50±7.50a 46.70±3.80a Alanine Aminotransferase (IU/L) 36.70±0.33b 36.70±0.88b 45.00±0.87a 46.70±0.33a Alkaline Phosphate (IU/L) 88.50±15.82c 101.76±2.03b 154.00±5.88a 183.67±20.2a Cholesterol (mg/dl) 105.33±17.17b 106±12.06b 117.25±9.14ab 126.67±9.61a ab_{: Means with similar alphabetical superscripts on the same row do not differ significantly (P>0.05) from each other.}

European Journal of Biological Research 2016; 6 (3): 218-225 Significant effect of MSG levels in female

rabbits administered with varied doses on serum enzymes and cholesterol are indicated in Table 4. The activities of the serum enzymes and levels of cholesterol significantly increased (p < 0.05) with increase in the MSG levels administered. The serum AST and ALP activities of rabbits administered 2 and 4 mg MSG/kg body weight were significantly higher (p < 0.05) than the serum activities of these enzymes in rabbits in control and low dose MSG. The activities of the ALT in the sera of the rabbits significantly increased (p < 0.05) with increase in the MSG levels administered. The mean cholesterol level in rabbits administered the high MSG dose, which was not significantly different (p > 0.05) from those administered the medium MSG dose, was however significantly higher (p < 0.05) than the cholesterol levels of those on the low MSG dose and the control.

4. DISCUSSION

Appetite is a complex mixture of physio- logical and psychological phenomena which include feelings of hunger, total energy intake, ingestion of particular nutrients, distribution and sizes of meals, specific cravings and food preferences [17]. Results of this study which showed that MSG, at 40% concentration, had significant effects on the relative feed intake of the animals with proportional increase in feed intake with increasing doses of MSG per kg of body weight throughout the period of the MSG administration could be attributed to the effect of the administered MSG. The pattern of feed consump- tion was directly related to the concentrations of MSG administered. However, since the rabbits were fed ad libitum, and the diets were isocaloric and isonitrogenous, the differences in the feed con- sumption which apparently reflected in the weight gain by the animals could not be to the increased palatability of the food but rather the MSG effect on the appetite control center of the brain. Halpern [18] reported increased food palatability with appropriate concentrations of MSG while Moore [19] reported that through the stimulation of the orosensory receptors, monosodium glutamate influences the appetite positively and induces weight gain in addition to improving the palatability of meals. Since the MSG was not added to the diets, the

increased feed intake and the resultant proportionate increase in the weight gained by the animals could be due to improved appetite enhancement as a result of the positive influence of MSG on the appetite control center of the brain as observed in rats admi- nistered MSG subcutaneously by Reddy et al. [20].

Increased body weight was reported in rats administered 15 to 30 mg MSG /kg (equivalent to 1 and 2 g/person) by Falalieieva et al. [21]. Similarly, increased body weight in rats administered MSG have been reported by several authors [1, 2, 22, 23]. These results showed that MSG is detrimental to the normal feeding physiology by abnormally inducing appetite by, probably modulating the appetite control centre of the brain. Appropriate concen- trations of MSG have been reported to improve palatability of meals [18] and thus influence the appetite centre positively with resultant increase in body weight [24]. The results of the present investigation in which significant increase in final body weight of the rabbits administered low dose of MSG (1 mg/kg) is an indication that MSG, even at low doses, is capable of producing alterations in the body weight. Therefore, the maximum dose of MSG should be reconsidered taking into account its influence on feed consumption and resultant excessive weight gain.

Although, MSG improved the PCV and Hb concentration of the rabbits at the low-dose level (2 mg MSG/kg b.wt), it should, however, be noted that the mean PCV and Hb values of rabbits administered with 4 mg MSG/kg b.wt were lower than the range values of 31.0-48.6 % (for PCV) and 98.0-158 g/L (for Hb) for normal female rabbit reported by Mitruka and Rawnsley [25]. Similarly, the WBC values of the rabbits administered varied levels of MSG obtained in this study were above the 6.00-13.0 x 103/mm3 reported by Mitruka and Rawnsley [25] for inbred New Zealand White rabbits. The significantly higher WBC values observed in rabbits administered varied levels of MSG compared with the control suggests that MSG could impair the normal function of the body immune system to increase body susceptibility to infections.

The significantly lower serum protein values observed in rabbits administered MSG compared with the control is an indication of the negative impact of the flavour enhancer on protein intake

European Journal of Biological Research 2016; 6 (3): 218-225 and/or utilization in the animals. The values of the

serum proteins observed in this study for all the animals, irrespective of the MSG levels were however, within the range values for normal rabbit reported in the literature [25]. Tawfik and Al-Badr [2] reported significantly decreased serum proteins administered MSG at 0.6 and 1.6 mg/g body weight for 14 days. Similarly, Madiha et al. [1] observed significantly reduced serum proteins in rats admi- nistered 1 g MSG/kg body weight for 8 weeks. The inhibitory effect of MSG on protein profile is in agreement with the finding of Newairy et al. [26]. The intestine regulates the uptake of amino acids, and the liver plays a significant role in regulating protein metabolism. So, the significant decrease in the concentrations of serum proteins observed in the animals treated with MSG in this study, particularly the albumin, could be attributed on one hand to reduced uptake of dietary protein by the intestinal mucosa, as there is growing apprehension that the excellent bleaching properties of MSG could be harmful or injurious to the stomach mucosa [4]. Also, it could be due to alterations in the protein synthesis in the liver, as reported by Cherroret et al. [27], due to the damaging effect of MSG on liver cells as confirmed by the increase in the activities of serum transferases in this study or increase rate of protein degradation.

Apart from the ALP value of the animals administered the high dose of 3 mg MSG/kg b. wt that was higher than the ALP range of 4.10-16.1 IU/L, all the activities of the serum enzymes examined were within the ranges reported for normal rabbit by Mitruka and Rawnsley [25]. In accordance with the results obtained in this present study are the findings of elevated AST and γ glutamyletransferase (GGT) [2] and ALT, AST and ALP [1] in rats administered 1 g MSG/kg body weight for 8 weeks, suggesting disturbances in metabolism affecting the liver function resulting in elevation of transferases’ levels. The ALT enzyme is a sensitive marker of liver damage [28]. Therefore, the increase in the serum ALT activity might suggest an indication of liver damage. According to Tawfik and Al-Badr [2], MSG could dissociate easily to release free glutamate that its diminution produces ammonium ion (that could be toxic unless detoxified in the liver via the reactions of the urea cycle. Thus, the possible NH4

+

overload

that may occur as a result of an increased level of glutamate following MSG intake could damage the liver, resulting in enzyme leakage that may lead to observed elevation in their activities. The enzymes are released into the circulating blood only after damage to liver structural integrity [29].

The seemingly dose-dependent increase in serum cholesterol in this study is in an agreement with significant increase in total serum cholesterol reported in rats administered MSG at 0.5 and 1.0 g per day for 28 days [30]. According to these authors, it is possible that MSG was able to increase the activities of 3-hydroxyl-3-methylglutaryl coen- zyme A (HMG CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis with consequent increased synthesis of cholesterol in the MSG treated rats. Thomas et al. [22] reported hyper- lipidaemia with significantly elevated levels of serum triacylglycerol and cholesterol in MSG treated rats and proposed that a shift in glucose metabolism towards lipogenesis might account for the hyperlipidaemia. Collison et al. [31] reported increased expression of several genes implicated in adipocytes differentiation, elevated serum free fatty acids and triglycerides in mice treated with low dose (0.64 g/l) MSG for 32 weeks, which the authors hypothesized that it could be the consequence of deregulation between liver and adipose tissue.

5. CONCLUSION

This study has shown that MSG, even at low doses, is capable of producing alterations in the body weight which may result in obesity. The results of this study suggest that MSG has both hepatotoxic and dyslipidaemia effects due to alterations in both aminotransferase activities and lipid profile, even at low doses. Hence MSG, though a flavour enhancer food additive, must be carefully used in food preparation due to its influence on food consumption resulting in alterations in weight gain, and its negative impacts on serum proteins, activities of serum enzymes as well as lipid profile. Thus, it could be concluded that MSG may be hepatotoxic at a low dose, hence should be avoided during the treatment of liver disorders.

European Journal of Biological Research 2016; 6 (3): 218-225 AUTHORS’ CONTRIBUTION

FAG conceived and designed the experiment, analysed and interpreted the data and prepared the manuscript; ORO and IMA were involved in the acquisition of the data and writing of the manuscript, while RAOG-A provided technical support. The final manuscript has been read and approved by all authors.