Research Paper - Clinical Investigation (2017) Volume 7, Issue 1

Liver enzymes in patients diagnosed with non-alcoholic fatty liver disease (NAFLD) in Veracruz: a comparative analysis with the literature

Corresponding Author:
Octavio Carvajal- Zarrabal
Biochemical and Nutrition Chemistry Area
University of Veracruz, SS Juan Pablo II s/n, 94294 Boca del Río, Ver., Mexico
E-mail: [email protected]

Submitted: 28 April 2017; Accepted: 09 May 2017; Published online: 16 May 2017


Background: Up to the present, NAFLD diagnosis has been established through the invasive method of biopsy. The search for a non-invasive alternative, for example biomarkers, is a motive for research. Previous studies have shown that for diabetic or obese patients with NAFLD, the profile of liver enzymes as NAFLD biomarkers has so far not defined pathological liver condition as such. This study aim was comparatively analyze the results of previous research on liver function enzymes of obese patients with NAFLD and to corroborate them with this type of patient in Veracruz. Methods and Findings: Forty hospital patients were recruited and classified according to their body mass index (BMI) into 4 groups: Group A (Overweight), Group B (Grade I obesity), Group C (Grade II obesity) and Group D (Grade III obesity), leaving aside those who had a history of hepatitis or consumption of alcoholic beberages. A survey was conducted on these patients to determine sex, signs of liver disease, medicine consumption; laboratory studies which included glucose, total cholesterol, triglycerides, alanine aminotranferase (ALT), aspartate aminotransferase (AST), Alkaline phosphatase (ALP), total protein, and total bilirrubin were also determined. Glucose levels were directly related to BMI, and the most frequent disorder was elevation of ALT levels (72.5%), higher than AST (25%) and ALP (45%). Some variables of the lipid profile showed a significant (P<0.05) elevation of triglycerides (85%) and highly significant (P<0.01) raised cholesterol levels (82.5%). Conclusions: The study shows that there are alterations in liver enzymes levels and highlight the importance of gender, BMI and dyslipidemia to assess the risk of individuals with NAFLD, which reaffirm the association with the disease.


Hepatic enzymes; Non-alcoholic fatty liver disease; Obesity; Type 2 diabetes


Fatty liver disease is defined as the accumulation of macrovesicular fat that increases liver weight by 5-10%. It encompasses a spectrum of disorders ranging from fat accumulation or steatosis and hepatic inflammation (steatohepatitis), to fibrosis and even cirrhosis, with all the complications that entail, such as portal hypertension and hepatocellular carcinoma [1,2]

There are two major groups of fatty liver, either alcoholic or non-alcoholic [3-5]. The latter, known as non-alcoholic fatty liver disease (NAFLD), describes the accumulation of lipids in hepatocytes of subjects who drink little or no alcohol. It is mainly associated with metabolic diseases such as type 2 diabetes mellitus, obesity, dyslipidemia and other components of the metabolic syndrome [6]. Alcohol consumption deemed as the definition of non-association with alcohol in men is less than 40 mL per day and less than 20 mL per day for women [4,7]. NAFLD prevalence has been increasing in recent decades. Although the NAFLD condition in Mexico has not been clearly defined, there are reports where it is detected in 10.3% of the general population and 18.5% of the diabetic population [1]. NAFLD is characterized by the persistent rise in hepatic enzymes, without association to excessive consumption of alcohol; it is asymptomatic, with specific ultrasound and histological characteristics, when there is an excess of fat within the hepatocyte [4,7]. The diagnosis is usually made by a Para clinical study, where there is a persistence of elevated liver enzymes. Particular alanine amino transferase (ALT) and aspartate amino transferase (AST) are present and through hepatic ultrasound it is detected an increase of the refringence with respect to the kidney. However, it is the liver biopsy that allows diagnosis confirmation and the study of the degree of severity [7-9].

Classically significant risk groups where a condition of NAFLD could be diagnosed have been described as diabetic, obese or dyslipidemic [10-12]. Today it is known that NAFLD can also occur in people who have no apparent risk factors, but whose fat distribution is altered, as in the case of subjects who have a tendency to predominantly abdominal fat distribution, still being of slim build; so the clinical spectrum is wider than anyone originally thought [7].

Up to the present, NAFLD diagnosis has been established through the invasive method of biopsy. The search for a non-invasive alternative, for example biomarkers, is a motive for research. Previous studies have shown that for diabetic or obese patients with NAFLD, the profile of liver enzymes as NAFLD biomarkers has so far not defined pathological liver condition as such. Therefore, the objective of this study was to comparatively analyze the results of previous research on liver function enzymes in obese patients with NAFLD and to corroborate them with this type of patients in Veracruz ISSSTE General Hospital.


Selection and processing of biological samples

The frequency of liver enzymes was evaluated using clinical files and the Munich Alcoholism Test (MALT) for patients of the Veracruz ISSSTE General Hospital with a confirmed non-alcoholic fatty liver diagnosis. Essential information concerning to risk factors such as sex, anthropometric measurements and the presence of diabetes were also collected. The group was made up of 21 men and 19 women. They were classified according to body mass index (BMI) into 4 groups: Group A (n=7), overweight patients with fatty liver; Group B (n=8), patients with fatty liver disease and grade I obesity; Group C (n=17), patients with fatty liver disease and grade II obesity, and Group D (n=8), patients with fatty liver disease and grade III obesity.

The eligibility criterion used to define and classify obesity according to the World Health Organization (WHO, 2000) was body mass index (BMI, kg/m2): Normal weight (18.5-24.9); Overweight (25-29.9); Grade 1 Obesity (30–34.9), Grade II Obesity (35-39.9), Grade III Obesity (≥40). Additionally, the MALT test was used to classify the patient as non-alcoholic, a noninclusion criterion considered to be patients with ≥11 points. Other considerations were not having suffered from hepatitis for at least six months previously, not to be in treatments using contraceptives, paracetamol, aspirin, gold salts, phenylbutazone and non-steroid antihistamines, and, in general, non-use of hepatotoxic drugs.

The participants were informed regarding the study and after clarifying the explanation of the protocol, fasting by all subjects was required before sampling. A sample of 10 ml venous blood was taken from the antecubital vein and placed in tubes. Blood was centrifuged at 35,000 rpm for 15 min to retrieve the serum and immediately the analyte profile was determined. The study was conducted according to the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Veracruz ISSSTE General Hospital.


Glucose was determined by the glucose oxidase method; total cholesterol (TC), triglyceride (TG), total protein, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were determined with COBAS c 501 (Roche Diagnostic, USA) through enzymatic colorimetric methods using BioMérieux (Paris, France) kits.

Statistical analysis

Data are expressed as the mean ± standard deviation (¯ ± SD). Statistical significance was determined by ANOVA procedures, with post hoc Tukey multiple range tests for comparison of ranges (P < 0.05). Data were analyzed with a 2011 IBM© SPSS© 20 version statistical package.


General characteristics of the study population

Table 1 shows the general characteristics and biochemical markers in patients diagnosed with NAFLD. A total of 40 patients were included, 21 men (52.5%) and 19 women (47.5%). Average body mass index (BMI) was 35.4±4.6. Most of the patients were overweight (7, 17.5%) and some degree of obesity (33, 82.5%) according to the criteria lay down by the World Health Organization (WHO). The presence of diabetes and two elements of dyslipidemia was found in 19 cases (47.5%), 34 (85%) with hypertriglyceridemia and 33 (82.5%) with hypercholesterolemia respectively. Average levels of ALT, AST, and ALP were (in U/L) 48.8±12.9, 36.4±11.8, and 211.4±82.9, respectively. BT and PT levels were within normal ranges (1.0±0.3 and 7.3±0.6 mg/dL respectively).

N Sex BMI BMI Classification Diabetes Glucose (mg/mL) TC (mg/dL) TG
ALT (U/L) AST (U/L) ALP (U/L) BT (mg/dL) PT (mg/dL)
1 M 28 Overweight NO 89 278 189 46 35 367 0.65 7.9
2 M 29 Overweight NO 96 204 156 35 25 156 0.99 6.9
3 M 28 Overweight NO 100 187 145 36 24 158 0.93 7.9
4 F 29 Overweight Yes 110 190 146 39 38 145 0.92 6.4
5 M 28 Overweight NO 85 199 123 36 32 90 0.95 7.7
6 M 29 Overweight NO 92 185 134 25 21 95 0.97 7.5
7 F 24 Overweight NO 98 178 145 36 32 279 0.78 7.0
8 F 33 Obesity grade 1 Yes 135 189 139 26 20 129 0.83 7.9
9 M 34 Obesity grade 1 Yes 143 178 289 76 68 79 0.89 6.4
10 F 33 Obesity grade 1 Yes 176 299 204 47 40 115 0.78 8.3
11 M 31 Obesity grade 1 NO 94 246 278 76 63 289 1.9 6.8
12 F 32 Obesity grade 1 NO 89 267 278 54 35 325 0.85 8.1
13 F 33 Obesity grade 1 NO 90 256 289 49 31 267 1.4 7.2
14 F 34 Obesity grade 1 NO 91 354 167 48 36 210 0.82 8.1
15 F 34 Obesity grade 1 NO 103 310 199 34 24 156 0.92 7.6
16 F 36 Obesity grade II NO 98 256 190 43 34 256 1.2 6.9
17 M 39 Obesity grade II Yes 167 300 256 49 41 245 0.89 6.9
18 F 35 Obesity grade II NO 85 367 278 59 45 289 0.73 6.0
19 M 37 Obesity grade II Yes 149 296 256 46 35 167 0.94 7.2
20 M 35 Obesity grade II NO 100 345 210 80 69 278 1.6 7.4
21 F 37 Obesity grade II NO 91 345 299 65 53 305 0.79 6.8
22 M 37 Obesity grade II Yes 118 270 200 37 25 182 1.5 6.9
23 M 36 Obesity grade II Yes 134 289 256 39 21 164 0.78 6.9
24 M 35 Obesity grade II NO 98 367 245 36 26 174 0.85 8.6
25 F 38 Obesity grade II NO 89 327 198 58 47 278 0.93 7.6
26 M 39 Obesity grade II Yes 110 338 167 47 39 229 0.65 8.2
27 M 39 Obesity grade II NO 102 311 178 45 32 189 0.72 7.4
28 M 37 Obesity grade II Yes 189 342 156 47 29 200 1.1 6.5
29 F 36 Obesity grade II NO 87 365 164 43 27 196 0.69 7.3
30 F 35 Obesity grade II NO 102 278 162 47 41 289 0.78 7.3
31 F 35 Obesity grade II Yes 145 270 189 57 43 295 0.89 6.9
32 F 38 Obesity grade II NO 102 324 256 32 19 132 1.1 7.3
33 M 41 Obesity grade III Yes 135 200 178 56 39 56 1.1 7.1
34 M 42 Obesity grade III Yes 135 295 165 59 45 304 1.3 7.5
35 F 41 Obesity grade III Yes 165 365 345 58 32 289 0.81 7.4
36 M 42 Obesity grade III Yes 206 210 200 58 38 56 1.1 7.4
37 M 41 Obesity grade III Yes 135 340 300 56 39 289 0.95 7.1
38 F 42 Obesity grade III Yes 148 365 310 59 39 304 1.07 7.5
39 M 42 Obesity grade III Yes 135 300 170 56 38 141 1.1 7.1
40 F 41 Obesity grade III Yes 145 365 165 59 39 289 1.1 7.5

Table 1. General characteristics and blood markers in patients diagnosed with NAFLD

Distribution of the patients in the study of the Veracruz ISSSTE General Hospital by BMI

Table 2 shows the distribution of patients with NAFLD classified by BMI. 17.5 percent are overweight (Group A), followed by 20% presenting grade I obesity (Group B), 42.5% with grade II obesity (Group C) and 20% with grade III obesity (Group D).

  Body Mass Index
  Gender Group A Overweight Group B Grade I Obesity Group C Grade II Obesity Group D Grade III Obesity    
  n % n % n % n % n %
Male 5 12.5 2 5.0 9 22.5 5 12.5 21 52.5
Female 2 5.0 6 15.0 8 20.0 3 7.5 19 47.5
Total 7 17.5 8 20.0 17 42.5 8 20.0 40 100

Table 2. Distribution of patients with non-alcoholic fatty liver disease grouped according to body mass index.

Biochemical parameters in patients with non-alcoholic fatty liver

Table 3 shows the results found for glycemic levels, lipid profile and liver function enzyme profile in patients with non-alcoholic fatty liver.

Non-alcoholic fatty liver groups
Parameter Group A Group B Group C Group D
Glucose (mg/dL)
Triglycerides (mg/dL)
Total cholesterol (mg/dL)
Total protein (mg/dL)
Total bilirubin (mg/dL)
Aspartate aminotransferase (U/L)
Alanine aminotransferase (U/L)
Alkaline phosphatase (U/L)
96 ± 80
148 ± 21
203 ± 34
7.3 ± 0.6
0.9 ± 0.1
30 ± 6.3
36.1 ± 6.2
184.3 ± 102
115 ± 32
230 ± 60*
262 ± 60*
7.6 ± 0.7
1 ± 0.4
39.6 ± 17.3
51.3 ± 17.7
196.3 ± 90.1
116 ± 30
215 ± 45
317 ± 36**
7.2 ± 0.6
0.9 ± 0.3
36.8 ± 12.7
48.8 ± 11.9
227.5 ± 55.5
150 ± 25
229 ± 76*
305 ± 68**
7.3 ± 0.2
1.1 ± 0.1
38.6 ± 3.5
57.6 ± 1.4
216 ± 112

Table 3. Glucose, lipid profile, and liver enzymes in patients with non-alcoholic fatty liver disease.

Overweight patients with non-alcoholic fatty liver (Group A)

This group was made up of a total of 7 patients, 5 male and 2 female, all overweight and a BMI average of 28±1.8 Kg/m2. Within this group, two patients presented alterations outside normal ranges in the concentrations of glucose, triglycerides and cholesterol (110, 173, 241 mg/dL, respectively) and in the levels of ALT (46 U/L) and alkaline phosphatase (367 and 279 U/L) for one and two patients respectively. However, the average levels of triglycerides were significantly lower (P≤0.05) in comparison with groups B and D. Additionally, cholesterol levels were significantly lower (P≤0.05) in relation to group B, and very significantly lower (P≤0.01) in comparison to groups C and D.

Patients with non-alcoholic fatty liver disease and grade I obesity (Group B)

This group was made up of 8 patients, 2 male, and 6 female, all with grade I obesity and a BMI average of 33±1.1 Kg/m2. In this group, 3 patients were diagnosed with diabetes, with glucose levels between the ranges of 135-176 mg/dL, an alteration in ALT enzymes (47-76 U/L) in 6 patients, in AST levels (63 and 68 U/L) in 2 patients, for alkaline phosphatase (267-325 U/L) in 3 patients; and for total bilirubin (1.4, 1.9 U/L) in 2 patients. However, when comparing the average levels among study groups, no significant difference was found for any of these parameters or total proteins. On the other hand, almost all patients except one presented hypertriglyceridemia (167-289 mg/dL), whose average of triglycerides (230 ± 60 mg/dL) increased significantly (P<0.05) in relation to groups A and C but not for group D. In addition 6 patients presented hypercholesterolemia (246-354 mg/dL), whose average levels of cholesterol (262±60 mg/dL) increased significantly (P<0.05) in relation to group A. However, when compared to groups C and D (P<0.01 difference compared to group A), these group B cholesterol levels were lower in significance, P<0.05.

Patients with non-alcoholic fatty liver disease and grade II obesity (Group C)

This group was made up of 17 patients, 9 male, and 8 female, all with grade II obesity and an average BMI of 37±1.5 Kg/m2. Seven patients were diagnosed with diabetes, the levels of which were in the range of 110-189 mg/dL. In this group, all patients presented dyslipidemia, whose average triglyceride and cholesterol levels were raised in comparison to group A. However, the triglycerides in this group were significantly lower (P<0.05) than those of groups B and D, but not of Group A. Contrary to this, cholesterol levels increased significantly (P<0.01) compared to group A, but not to groups B and D. Liver enzyme levels were altered: ALT, 43-80 U/L for 13 patients, AST, 41-69 U/L for 7 patients, alkaline phosphatase, 245-305 U/L for 8 patients and total bilirubin 1.2-1.6 mg/dL for 4 patients. However, no significant differences between the average values of these parameters and levels of total protein were found when study groups were compared.

Patients with non-alcoholic fatty liver disease and grade III obesity (Group D)

This group was made up of 8 patients, 5 males and 3 female, all with grade III obesity and an average BMI of 41±0.5 Kg/m2. In this group, patients presented alterations in almost all levels of the evaluated parameters: glucose (135-206 mg/dL), triglycerides and cholesterol (165-345 and 200-365 mg/dL), ALT (56-59 U/L) for all patients respectively, and alkaline phosphatase (289-304 U/L for five patients). However, the average triglyceride (229±76 mg/dL) and cholesterol levels (305±68 mg/dL) were higher in comparison, the former significantly (P<0.05) compared to groups A and C, but not for Group B, and the latter very significantly (P<0.01) in comparison to group A, of higher significance when compared to group B (P<0.01 rather than P<0.05) and of equally very high significance in the case of group C.


NAFLD is a problem of global health that affects 10 to 24% of the general adult population in several countries [12,13]. However, prevalence increases from 57.5% in obese adults [14] to 75% [15]. Prevalence is 2.6% in children, increasing from 22.5 to 52.8% in obese children [16,17]. Increases between 55 and 70% are also found in people with BMI above 30 Kg/m2.

When anthropometric variables were considered, the results of this study reveal that 82.5% of the subjects evaluated showed one of the grades of obesity and 17.5 % were overweight. Studies such as Martinez et al. [18] reported obesity in a range of 40 to 100%; however, Rousch et al. [1] in a similar study in Veracruz, found values of 14 t.6%, extremely low compared to the results found in this study. Contrary to this, Uscategui et al. [19] in his study of patients with NAFLD, reported that 73 percent were obese, noting a higher prevalence of obesity in males. Other studies have shown that the prevalence of NAFLD in obese patients is 4.6 times higher, up to 74% [ 20]. In this sense, the group of patients with NAFLD in this study showed a fourfold prevalence, up to 82.5%, similar to the findings of Bellentani et al. [20] Also, it has been shown that NAFLD increases proportionally with BMI; in this study, eight patients with NAFLD were found to have a BMI >40 Kg/m2.

The number of males was similar to females, 52.5%, and 47.5% respectively, although Gaviria et al. [21] reported a frequency of 73% for females and 27% for males. The results suggest sex is not a risk factor for predisposition to NAFLD. However, population studies in the USA have shown that the prevalence is higher in men than in women [3], similar to what was found in this study, which is in turn consistent with that reported by other authors [22,23].

Glucose levels in patients with NAFLD were related directly to BMI; as BMI increased, so did average glucose levels, but not significantly so. Some authors like Cabrerizo et al. [24] associated diabetes with NAFLD, a fact demonstrated by the results of this study where 47.5% of the subjects had diabetes, patients in Groups C and D showing the greatest numbers. These findings are compatible with the results of other authors where higher NAFLD prevalence has been reported in diabetics compared to the general population [6,25]. However, a study carried out in South India showed that NAFLD prevalence in diabetics was 56.5%, much higher than the results found here [26]. It is obvious that when subjects have both diabetes mellitus and obesity, the risk of NAFLD increases significantly [27].

Liver function enzyme elevation, particularly alanine aminotransferase (ALT), is often the first sign of NAFLD, an increase of one to three times its normal value being observed [28]. In this research, even though no statistical significance was found, the most frequent alteration was ALT level elevation (72.5% patients) with a higher level relative to AST level (25% patients), where similarly elevated values for both transaminases were not found. Contrary to this, Gaviria et al. [21] found hypertransaminasemia AST levels (23.3%) more elevated in comparison with ALT (13.3%) in obese patients with NAFLD, which suggests that bothS enzymes are closely linked to the growing NAFLDassociated obesity epidemic. Studies that have evaluated the etiology of elevated levels of transaminases in the general population of the USA have proposed that elevated levels of ALT, as well as AST, are predictors of the presence of NAFLD [29].

Another abnormality associated with the onset of NAFLD is the elevated levels of alkaline phosphatase. In this regard, a slight increase in levels in 45% total population studied was found, but it was not enough as to be statistically significant when levels between the study groups were compared. These findings are compatible with the results of other authors [1].

There is evidence that patients with hypertriglyceridemia or dyslipidemia have a 5 to 6 fold increased risk of developing NAFLD with respect to the normal population [3,30]. Rousch et al. [1], Martínez et al. [18] and Cabrerizo et al. [24] agree that the foremost factor in NAFLD prevalence is dyslipidemia. In this study, the most characteristic lipid alterations were the significant elevation of triglyceride levels (85%) and cholesterol (82.5%), biochemical parameters that exhibited a significant difference when compared between study groups. This finding leads to the assumption that lipid regulation, synthesis, and metabolism are altered in this group of patients. This abnormality may be due to an adjustment to the rise of protein binding to the sterol regulatory element SREP- 1 c, a transcription factor of some genes involved in the de novo synthesis of fatty acids; this element inhibits the oxidation of free fatty acids and the stimulation of the fat content in the liver [31-33]. Similarly, the sterol regulatory element SREBP-2 and low-density lipoprotein (LDL) receptors are regulated downwards in subjects with NAFLD, thus inhibiting cholesterol absorption and the synthesis of very high-density lipoprotein (VLDL) in hepatocytes and resulting in a high triglyceride content in the liver [32]. On the other hand, elevated TG levels can alter the lipid profile even more by reducing the cholesterol in lipoproteins of high density (HDL-C) and by increasing dense LDL particles [34]. In addition, a comparison of BMI between study groups revealed a significant difference among them (Group D>C>B>A). Therefore it can be said that BMI is a factor that predisposes people to NAFLD as it is directly associated with obesity. Data from the NHANES-III study on dyslipidemia also reflect a higher prevalence as BMI increases, especially in men.

In clinical practice, it has been reported that serum bilirubin level is related to various diseases. In this study group, total bilirubin levels were within the normal range, without statistical significance. So far the literature had excluded bilirubin as a guiding test for NAFLD. However, a study by Chang et al. [35] revealed that serum conjugated bilirubin levels were significantly associated with a lower incidence of NAFLD. Nevertheless, some authors found that total bilirubin levels are associated in a negative way [36], and still others observed that unconjugated bilirubin levels are rigorously related to NAFLD [37,38], therefore, it is not clear which bilirubin influences NAFLD presence. Unfortunately, in this study, both bilirubins were not measured. It is clear that more research is needed to elucidate the mechanisms underlying this association and to establish the role of bilirubin as a risk marker of non-alcoholic fatty liver disease.

Some limitations of our study deserve to be commented on. Ultrasound diagnosed Non-alcoholic fatty liver disease without histological confirmation of a fatty liver. A more robust sample could allow other factors associated with NAFLD in obese adults to be identified. In addition, the study population had moderate to severe prevalence of obesity and resistance to insulin was not determined; this has a great influence on the physiopathological process, where tumor necrosis factor (TNF) has been shown to increase in the serum of insulin resistant people, type 2 diabetics or android obesity carriers. Recent studies provide new evidence showing that exposure to tobacco smoke and indirect smoke aspiration can accelerate the development of experimental non-alcoholic fatty liver disease [39,40]. Therefore, future research is needed to establish with precision the pathological condition of the liver. However, our sample was representative where there is evidence of similarities with results published in the international literature.


Non-alcoholic fatty liver disease (NAFLD) includes patients who do not have a history of substantial alcohol use. It is characterized by the accumulation in the liver of macro vesicular fat. Among the most relevant findings was that 82.5% of the patients evaluated in this study presented some obesity and the frequency of NAFLD in relation to gender was similar to male and female. Glucose levels were related directly to BMI; as BMI increased so did average glucose values. The alterations in the sick population were elevation in ALT levels, higher than AST levels. A slight increase in ALP levels was observed, as well as significant elevation of triglyceride and cholesterol levels. Therefore, the influence of factors such as gender, BMI, and dyslipidemia that reaffirm the incidence of NAFLD in the population studied cannot be ruled out at the onset of the disease. Since the early detection is needed for timely management and to avoid development into cirrhosis and its complications.


We thank the medical team and clinic of the Veracruz ISSSTE General Hospital in conducting this study.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Competing and Conflicting Interests

The authors stated that they have no conflict of interest regarding the publication of this article.

Author Contributions

We are the team of this research. All authors were involved in literature search, experimental work, and the preparation of manuscript. Noé López-Amador was involved in the study design and the selection of patients; Cirilo Nolasco-Hipólito carried out the statistical analysis of the study; Macario de J Rojas- Jimeno carried out the experiments; Octavio Carvajal- Zarrabal wrote the manuscript, supervised, revised and was involved in the comments made on the draft manuscript. All the authors read and approved the final manuscript.