Manuscript accepted on : 28-Sep-19
Published online on: 30-09-2019
Plagiarism Check: Yes
Reviewed by: Kulvinder Kaur
Second Review by: Francisco Solano
Sawsan Hassan Mahassni 
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Corresponding Author E-mail: sawsanmahassni@hotmail.com
DOI : http://dx.doi.org/10.13005/bbra/2769
ABSTRACT: Saudi Arabia has one of the highest adult overweight and obesity rates, especially in females, leading to increased mortality, morbidity, infections, and risk for many diseases. This study determined the counts and percents of lymphocyte subtypes (CD3, CD4, CD8, and CD16 +CD56 cells) and serum IgG, IgA, and IgM concentrations in blood samples collected from sixty-four Saudi female university employees with an age range of 24-52 years. There is only one other study on the counts/numbers of lymphocyte subtypes in overweight and obese Saudi females. Anthropometric measurements were used to categorize the subjects into groups according to the body mass index (BMI), waist-to-hip ratio (WHR), and waist circumference (WC). Results were all compared to the controls. Antibody concentrations were not significantly different. The CD3and CD4 counts were significantly higher for the obese BMI group and the overweight and obese BMI, respectively. The high-risk WHR group had a significantly lower CD 3% and a significantly higher CD16 + CD56 count. The high-risk WC group had significantly higher CD3 and CD4 counts and a significantly lower CD16 + CD56%. Thus, obesity leads to changes in the cellular adaptive and innate immune systems, while not affecting the humoral adaptive immune system.
KEYWORDS: Adaptive Immunity; Antibodies; Innate Immunity; Obesity; Overweight; Saudi Females
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Introduction
Obesity and overweight are considered major health problems in most countries of the world with developed and rich countries being affected more than poorer or underdeveloped countries. In addition, most countries are experiencing increasing rates of both overweight and obesity, with obesity being more widespread than underweight worldwide, with the exception of some African sub-Saharan and Asian countries.1 Overweight and obesity have been increasing in the Middle East for the last few decades, possibly due to factors linked to changing lifestyles and modernization. Rates for overweight and obesity among adults in Saudi Arabia are among the highest in the Middle East.2 In Saudi Arabia, according to the Saudi Arabia profile published in 2016 by the World Health Organization (WHO),3 69.2% of females and 67.5% of males are overweight whereas39.5% of females and 29.5 of males are obese. Obesity and overweight lead to increased morbidity and mortality, higher infection rates, worse symptoms for some diseases, and an increased risk for many diseases, such as cardiovascular diseases, dyslipidemia, diabetes, hypertension, metabolic syndrome, liver diseases, kidney diseases, osteoarthritis, many types of cancers, and some psychiatric illnesses. In addition, obesity and overweight are linked to many disruptions of and effects on different systems in the body including the immune system.4-9
The most commonly used anthropometric adiposity measures are the body mass index (BMI), the waist-to-hip ratio (WHR), and the waist circumference (WC). They are simple ways that allow measurement of the level of overweight or obesity with simple readily available tools. In addition, they help to specify the distribution of fat in the body and thus enabling to determine the presence of upper body obesity, which is highly associated with visceral fat, or lower body obesity. Upper or central obesity, which is more common in men than women, is highly linked to many of the ill health and obesity-related diseases that are more prevalent in people with this body shape. On the other hand, lower body obesity, more prevalent in women and leads to the pear shape that is common in adult women, leads to lower morbidity and obesity-related diseases.
The determination of counts and percents of lymphocyte subsets in overweight and obese subjects helps to ascertain the state of the immune system in these individuals and the extent to which the immune system is affected by increased weight. Additionally, specific lymphocyte types indicate the type(s) of immunity that may be affected by increased weight. Therefore, CD3 cells, or T lymphocytes, are concerned with cellular adaptive immunity with the CD4 subtype, mainly helper T cells, being involved in enhancing or helping the adaptive (acquired) immune system, while the CD8 subtype, which is cytotoxic/suppressor cells, having roles in killing unwanted or infected cells, or suppressing an adaptive reaction. CD19, or B cells, are the major cells of the humoral adaptive immune system and the cells from which plasma cells mature and finally produce antibodies. Finally, CD16 +CD56 cells, or natural killer (NK) cells, are important in the innate immune system. Thus, these cells give a good picture of the state of the immune system and may explain the altered immune response in subjects with unhealthy weight and their high incidence of diseases and mortality.
Most research studies on obesity and overweight in Saudi Arabia are epidemiological in nature. Studies4-8 have been done on the effects of overweight and obesity on different health-related parameters and cells of the immune system in Saudi females. Worldwide, studies9-15on the effects of overweight and obesity on the immune system and on specific types of lymphocytes are few and contradictory. After an extensive search on the internet, only one study4 was found on counts/percentages of lymphocyte subtypes in the circulation in overweight and obese Saudi adult females. Thus, the current study aimed to fill this knowledge gap by determining the counts and percents of immune system cells and the concentrations of antibodies in overweight and obese Saudi female university workers compared to healthy weight subjects.
Materials and Methods
Subjects, Anthropometric Measurements, Sample Collection, and Categorizations Of Subjects
Sixty-four randomly has chosen Saudi female university employees, ages 24-52 years, were recruited from the King Abdulaziz University, Jeddah, Saudi Arabia. All subjects signed a consent form for participating in this study. None of the subjects was pregnant or menstruating at the time of blood sample collection; had any chronic diseases such as diabetes, high blood pressure, and heart diseases; nor on any medications.
Blood samples were collected from the subjects into sodium heparin vacutainer tubes for the determination of the types of lymphocyte subsets. In addition, blood samples were collected in plain vacutainer tubes for the determination of antibody concentrations. These tubes were centrifuged after clot formation and serum was subsequently collected. In addition, anthropometric measurements (weight, height, and waist and hips circumferences) were obtained at the same time of blood collection.
The BMI cut off values for the BMI groups were: below 18.5 kg/m2 for the underweight BMI group, 18.5-24.9kg/m2 for the healthy BMI group, 25-29.9kg/m2 for the overweight BMI group, and 30-39.9kg/m2 for the obese BMI group. None of the subjects was morbidly obese. For the WHR groups, the low-risk group subjects had WHRs below or equal to 0.8, the moderate risk group was WHRs between 0.81 and 0.85, and finally, the high-risk group was subjects with a WHR above 0.85. WCs below 82.5 cm were in the WC low-risk group, a WC between 82.5 and 88.9 cm was in the moderate-risk group, and a WC above 88.9 cm was in the high-risk group.
Determination of counts and percents of lymphocyte subsets
The counts and percents of the lymphocyte subsets were determined in whole blood using a BD FACSC anto II flow cytometer (Becton Dickinson Company, CA, USA) using the BD Multitest 6-color TBNK reagent (BD Biosciences, CA, USA) for determining the CD markers on the surface of cells.
Determination of serum IgG, IgA, and IgM concentrations
Serum concentrations of IgG, IgA, and IgM antibodies were determined on a BN II system nephelometric analyzer (Siemens, Germany) using N antisera to human immunoglobulins (IgG, IgA, and IgM) (Siemens, Germany).
Statistical analysis
Statistical and analytical analyses of the results were done using the SPSS Statistics statistical program (version 20). To test for the presence of statistically significant differences between the groups, the one-way ANOVA test was used for the normally distributed parameters while the Kruskal-Wallis H test was used for the non-normally distributed parameters. For the post hoc tests, the LSD test was used for the normally distributed parameters and the Dunnett T3test was used for the non-normally distributed parameters. The resulting P values indicate whether the differences between the groups is considered significant (P < 0.05), highly significant (P < 0.01), or non-significant(P ≥ 0.05).
Results
Subjects and Categorizations
The subjects’ ages ranged from a minimum of 24 years to a maximum of 52 years. The 64 subjects had a mean age of 32.9 years and a standard deviation of 8.5 years. The weights of the subjects had a range of 39-103 kg and a mean of 64.9 kg with a standard deviation of 15.2 kg. The heights of the subjects ranged from 145.5 cm to 171 cm with a mean and standard deviation of 159.1 ± 5.6 cm. Categorizing the subjects into the BMI, WHR, and WC groups (Table 1) led to most subjects being in the healthy BMI group and the low-risk WHR and WC groups.
Table 1: Adiposity measures categorizations for the subjects.
| Groups | Range | N | N% of total | Minimum | Maximum | Mean | ± SD |
| BMI** (kg/m2) | |||||||
| Underweight | < 18.5 | 7 | 10.9 | 15.4 | 18.4 | 16.7 | 1.3 |
| Healthy | 18.5-24.9 | 21 | 32.8 | 18.7 | 24.8 | 21.4 | 1.9 |
| Overweight | 25-29.9 | 18 | 28.1 | 25.1 | 29.6 | 27.4 | 1.4 |
| Obese | 30-39.9 | 18 | 28.1 | 30.0 | 37.6 | 32.2 | 2.1 |
| Total | 64 | 100.0 | 15.4 | 37.6 | 25.6 | 5.5 | |
| WHR** | |||||||
| Low | ≤ 0.80 | 34 | 54.8 | 0.47 | 0.80 | 0.7 | 0.1 |
| Moderate | 0.81-0.85 | 12 | 19.4 | 0.81 | 0.85 | 0.8 | 0.0 |
| High | > 0.85 | 16 | 25.8 | 0.86 | 1.21 | 0.9 | 0.1 |
| Total | 62 | 100.0 | 0.47 | 1.21 | 0.8 | 0.1 | |
| WC* (cm) | |||||||
| Low | < 82.5 | 28 | 45.2 | 49.5 | 82.0 | 71.5 | 7.3 |
| Moderate | 82.5-88.9 | 13 | 21.0 | 82.5 | 88.0 | 85.3 | 2.0 |
| High | > 88.9 | 21 | 33.9 | 89.0 | 116.0 | 98.3 | 7.5 |
| Total | 62 | 100.0 | 49.5 | 116.0 | 83.5 | 13.6 | |
| *ANOVA one-way test, **Kruskal-Wallis test
N: Number of subjects |
|||||||
Counts and percents of lymphocyte subsets and antibody concentrations
For the BMI categorization, the mean CD3 and CD4 counts (Table 2) and mean CD8 percents (Table 3) were significantly different between the groups. As for the WHR groups, the mean CD16 +CD56 counts (Table 2) and CD3 percents (Table 3) were significantly different between the groups. Finally, for the WC groups, the mean CD3 and CD4 counts (Table 2) and mean CD16 +CD56 percents(Table 3) were significantly different between the groups. The remaining cell counts and percents for each obesity measure were not significantly different between the respective groups. Finally, the mean IgG, IgA, and IgM concentrations (Table 4) were not significantly different between each of the BMI, WHR, and WC groups.
Table 2: Statistical analysis for mean lymphocyte cell counts for the subjects using the BMI, WHR, and WC.
| BMI | WHR | WC | ||||||||||
| Cell counts | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value |
| CD3* | Underweight | 1.6908 | 0.7853 | 0.023S | Low | 1.9135 | 0.5554 | 0.873 | Low | 1.7596 | 0.5608 | 0.046S |
| Healthy | 1.7195 | 0.4636 | Moderate | 1.9656 | 0.7257 | Moderate | 1.9911 | 0.3980 | ||||
| Overweight | 2.0434 | 0.4965 | High | 2.0042 | 0.5537 | High | 2.1696 | 0.6413 | ||||
| Obese | 2.2172 | 0.5665 | ||||||||||
| CD4* | Underweight | 0.9391 | 0.5071 | 0.015S | Low | 1.1573 | 0.3363 | 0.954 | Low | 1.0642 | 0.3529 | 0.042S |
| Healthy | 1.0475 | 0.2854 | Moderate | 1.1673 | 0.4193 | Moderate | 1.1563 | 0.2854 | ||||
| Overweight | 1.2666 | 0.2968 | High | 1.1900 | 0.3294 | High | 1.3127 | 0.3333 | ||||
| Obese | 1.2999 | 0.2949 | ||||||||||
| CD8** | Underweight | 0.7207 | 0.2714 | 0.066 | Low | 0.7121 | 0.2786 | 0.628 | Low | 0.6455 | 0.2685 | 0.120 |
| Healthy | 0.6114 | 0.2328 | Moderate | 0.7081 | 0.3731 | Moderate | 0.7673 | 0.2173 | ||||
| Overweight | 0.7122 | 0.2353 | High | 0.7514 | 0.2395 | High | 0.7944 | 0.3286 | ||||
| Obese | 0.8524 | 0.3388 | ||||||||||
| CD19** | Underweight | 0.0478 | 0.0430 | 0.394 | Low | 0.0468 | 0.0227 | 0.644 | Low | 0.0497 | 0.0253 | 0.883 |
| Healthy | 0.0526 | 0.0190 | Moderate | 0.0519 | 0.0213 | Moderate | 0.0465 | 0.0176 | ||||
| Overweight | 0.0447 | 0.0191 | High | 0.0477 | 0.0275 | High | 0.0467 | 0.0248 | ||||
| Obese | 0.0457 | 0.0222 | ||||||||||
| CD16 + CD56** | Underweight | 0.3196 | 0.1924 | 0.686 | Low | 0.2523 | 0.1324 | 0.018S | Low | 0.2920 | 0.1320 | 0.261 |
| Healthy | 0.2723 | 0.1275 | Moderate | 0.2113 | 0.0983 | Moderate | 0.2507 | 0.1508 | ||||
| Overweight | 0.2918 | 0.1860 | High | 0.3538 | 0.1628 | High | 0.2544 | 0.1549 | ||||
| Obese | 0.2511 | 0.1462 | ||||||||||
| *ANOVA one-way test, **Kruskal-Wallis test, S: significant difference | ||||||||||||
Table 3: Statistical analysis for mean lymphocyte cell percents for the subjects using the BMI, WHR, and WC.
| BMI | WHR | WC | ||||||||||
| Cell% | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value |
| CD3%* | Underweight | 78 | 5 | 0.843 | Low | 78 | 5 | 0.027S | Low | 76 | 5 | 0.401 |
| Healthy | 76 | 4 | Moderate | 79 | 5 | Moderate | 79 | 5 | ||||
| Overweight | 78 | 8 | High | 74 | 9 | High | 78 | 9 | ||||
| Obese | 78 | 9 | ||||||||||
| CD4%* | Underweight | 42 | 5 | 0.146 | Low | 47 | 6 | 0.177 | Low | 46 | 6 | 0.609 |
| Healthy | 47 | 6 | Moderate | 47 | 5 | Moderate | 46 | 6 | ||||
| Overweight | 48 | 7 | High | 44 | 7 | High | 48 | 7 | ||||
| Obese | 46 | 7 | ||||||||||
| CD8%* | Underweight | 35 | 5 | 0.041S | Low | 29 | 7 | 0.816 | Low | 28 | 7 | 0.401 |
| Healthy | 27 | 5 | Moderate | 28 | 8 | Moderate | 31 | 8 | ||||
| Overweight | 27 | 7 | High | 28 | 6 | High | 28 | 7 | ||||
| Obese | 30 | 8 | ||||||||||
| CD19%** | Underweight | 7 | 3 | 0.103 | Low | 10 | 4 | 0.522 | Low | 10 | 4 | 0.509 |
| Healthy | 11 | 4 | Moderate | 12 | 4 | Moderate | 11 | 4 | ||||
| Overweight | 11 | 4 | High | 12 | 7 | High | 12 | 6 | ||||
| Obese | 13 | 6 | ||||||||||
| CD16 + CD56%* | Underweight | 14 | 5 | 0.103 | Low | 11 | 5 | 0.060 | Low | 13 | 5 | 0.020S |
| Healthy | 12 | 5 | Moderate | 9 | 3 | Moderate | 10 | 4 | ||||
| Overweight | 11 | 6 | High | 13 | 5 | High | 9 | 5 | ||||
| Obese | 9 | 5 | ||||||||||
| *ANOVA one-way test, **Kruskal-Wallis test, S: significant difference | ||||||||||||
Table 4: Statistical analysis for mean immunoglobulin concentrations for the subjects using the BMI, WHR, and WC.
| BMI | WHR | WC | ||||||||||
| Concentration g/L | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value | Group | Mean | ± SD | P value |
| IgG* | Underweight | 7.79 | 2.97 | 0.477 | Low | 7.43 | 3.10 | 0.510 | Low | 7.81 | 3.06 | 0.204 |
| Healthy | 7.37 | 2.83 | Moderate | 7.59 | 2.77 | Moderate | 7.32 | 2.65 | ||||
| Overweight | 6.71 | 3.15 | High | 6.50 | 2.47 | High | 6.31 | 2.63 | ||||
| Obese | 7.26 | 2.69 | ||||||||||
| IgA* | Underweight | 1.72 | 0.75 | 0.385 | Low | 1.55 | 0.75 | 0.853 | Low | 1.51 | 0.75 | 0.644 |
| Healthy | 1.43 | 0.78 | Moderate | 1.58 | 0.47 | Moderate | 1.74 | 0.54 | ||||
| Overweight | 1.49 | 0.75 | High | 1.67 | 0.83 | High | 1.60 | 0.81 | ||||
| Obese | 1.81 | 0.58 | ||||||||||
| IgM** | Underweight | 1.12 | 0.48 | 0.193 | Low | 0.87 | 0.54 | 0.864 | Low | 0.85 | 0.55 | 0.957 |
| Healthy | 0.72 | 0.34 | Moderate | 0.85 | 0.63 | Moderate | 0.83 | 0.59 | ||||
| Overweight | 0.83 | 0.64 | High | 0.75 | 0.43 | High | 0.82 | 0.48 | ||||
| Obese | 0.86 | 0.57 | ||||||||||
| *ANOVA one-way test, **Kruskal-Wallis test | ||||||||||||
The post hoc analyses were done for significantly different cell counts and percents (Table 5). The mean CD3 count for the obese BMI group and the mean CD4 counts for the overweight and obese BMI groups were significantly higher than the mean counts for the respective healthy BMI groups (controls). The mean CD8 per cent was significantly higher for the underweight BMI group compared to the mean per cent for the control group. As for the post hoc analyses for the WHR groups (Table 5), the mean CD16 +CD56 counts were not significantly different for both the moderate and high risk groups compared to the low risk group (control), while the count for the high-risk group was significantly higher compared to the count for the moderate-risk group. The mean CD3 per cent was significantly lower for the high-risk WHR group compared to the mean per cent for the control. Finally, for the post hoc comparisons for the WC groups, the mean CD3 and CD4 counts for the high-risk group were both significantly higher than the respective mean counts for the control group. The mean CD16 +CD56 per cent for the WC high-risk group was significantly lower than the mean per cent for the control group. The remaining group comparisons for the three adiposity measures were not significantly different compared to the respective control groups.
Table 5: Post hoc statistical analysis for the significantly different parameters for the BMI, WHR, and WC.
| Parameter | Test | G1 | G2 | Mean Difference (G1-G2) | ± SE | P value |
| BMI | ||||||
| CD3 count | LSD | Healthy | Underweight | 0.0288 | 0.2367 | 0.904 |
| Overweight | -0.3238 | 0.1742 | 0.068 | |||
| Obese | -0.4977 | 0.1742 | 0.006S | |||
| CD4 count | LSD | Healthy | Healthy | 0.1085 | 0.1397 | 0.441 |
| Overweight | -0.2191 | 0.1028 | 0.037S | |||
| Obese | -0.2524 | 0.1028 | 0.017S | |||
| CD8% | LSD | Healthy | Underweight | -8 | 3 | 0.007S |
| Overweight | -1 | 2 | 0.776 | |||
| Obese | -3 | 2 | 0.181 | |||
| WHR | ||||||
| CD16 +CD56 count | Dunnett T3 | Low | Moderate | 0.0410 | 0.0363 | 0.600 |
| High | -0.1015 | 0.0466 | 0.110 | |||
| Moderate | High | -0.1426 | 0.0496 | 0.024S | ||
| CD3% | LSD | Low | Moderate | -1 | 2 | 0.655 |
| High | 5 | 2 | 0.016S | |||
| WC | ||||||
| CD3 count | LSD | Low | Moderate | -0.2314 | 0.1885 | 0.225 |
| High | -0.4099 | 0.1622 | 0.014S | |||
| CD4 count | LSD | Low | Moderate | -0.0921 | 0.1119 | 0.414 |
| High | -0.2485 | 0.0963 | 0.012S | |||
| CD16 +CD56% | LSD | Low | Moderate | 3 | 2 | 0.052 |
| High | 4 | 1 | 0.009S | |||
| G1: group 1, G2: group 2 | ||||||
| S: significant difference | ||||||
Discussion
This research study aimed to determine the status of immune system cells and antibodies in Saudi female university workers in Saudi Arabia and to determine the best measure of adiposity to use for this cohort. Studies on immunological parameters in obese and overweight individuals from the local community are important to help determine the effects of overweight and obesity on the immune system to better educate the local population about these effects in the hope of giving more incentives for weight loss and to be able to give better guidelines on healthy weights.
For the BMI groups, the mean CD3 cell (T lymphocyte) counts were significantly higher (P = 0.006) for the obese BMI group compared to the healthy (control) group. The mean CD4 cell (T helper lymphocyte) counts were significantly higher for both the overweight (P = 0.037) and obese BMI subjects (P = 0.017) compared to the mean count for the healthy BMI group. Finally, the mean CD8 cell (T suppressor or T regulatory lymphocyte) percent for the underweight BMI was significantly higher (P = 0.007) compared to the mean percent for the control. The remaining cells were not significantly different (P > 0.050) for the BMI groups compared to the control.
These findings agree with the previous finding4 of a significantly higher mean CD4 count for highly obese BMI Saudi female university students compared to healthy weight subjects. In addition, the findings of this previousstudy4 agree with the current findings of no significant differences between the mean CD8 counts, CD4 percents, and CD19 and CD16 +CD56 cell counts and percents for the BMI groups and the control. On the other hand, the current findings disagree with the findings of the above-mentioned study4 of no significant differences for the mean CD3 counts and CD8 percents for the BMI groups compared to the control.
As for the WHR groups, only the mean CD16 +CD56 cell counts and CD3 percents showed significant differences compared to the controls. The mean CD16 +CD56 cell (natural killer lymphocyte) count for the high risk group was significantly higher (P = 0.024) compared to the mean count for the moderate-risk group. On the other hand, both mean counts for the moderate and high risk groups were not significantly different (P > 0.050) from the mean count for the low risk (control) group. The mean CD3 percent for the high risk group was significantly lower (P = 0.016) compared to the mean count for the control.
The current findings agree with those of the above mentioned study on Saudi female university students4 which found no significant differences for the WHR groups for the mean CD8 and CD19 counts and percents, and CD16 +CD56 percents compared to the control. On the other hand, the current findings disagree with the previous findings4 of significantly higher CD3 count and CD4 count and percent for the moderate risk group, and none significantly different CD16 +CD56 counts for both WHR groups compared to the control.
For the WC groups, both the mean CD3 and CD4 cell counts were significantly higher (P = 0.014 and 0.012, respectively) for the high risk group compared to the mean counts for the respective controls. The mean CD16 +CD56 cell percent for the high risk group was significantly lower (P = 0.009) compared to the mean percent for the control group. The remaining cell counts and percents were not significantly different from the controls.
The mean IgG, IgA, and IgM concentrations were not significantly different between each of the BMI, WHR, and WC groups. This agrees with previous studies5 that also showed no significant differences in the concentrations of these antibodies in adolescent Saudi females.
The cohort of the study is considered highly educated since most of the subjects had a college degree or a higher degree. Therefore,the subjects were probably more aware than other cohorts about health and a healthy lifestyle and probably attempted to follow health and diet recommendations. This may explain the minimal differences between overweight and obese subjects compared to the healthy weight ones.
Conclusions
All adiposity measures showed significant changes in CD3 cell (T cells) counts/percents with the highest BMI and WC being associated with significantly higher counts while the highest WHR was associated with a significantly lower per cent. CD4 counts were significantly higher for the overweight and obese BMIs and high-risk WHR. On the other hand, CD16 +CD56 cell percents were significantly lower for the high WC compared to the control while not being significantly different from the controls using the BMI and WHR. Thus, higher obesity, measured by BMI and WC, is associated with higher CD3 and CD4 counts, and lower CD16 +CD56 cell percents compared to the controls. Thus, cellular adaptive immunity and innate immunity are both affected in the obese while humoral adaptive immunity is not.
Funding
No funding was provided for this research study.
Ethical Considerations
Ethical approval for the study and for blood collection was obtained from the Biochemistry Department, King Abdulaziz University, Jeddah, Saudi Arabia.
Conflicts of Interest
The author declares no conflicting interests in this study.
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