L-Arginine supplementation enhanced expression of glucose transporter (GLUT 1) in sickle cell anaemia subjects in the steady state

Abstract

L-Arginine may have therapeutic value in the management of sickle cell disease and diabetes mellitus. There is very little information on the interaction of GLUT 1 and L-Arginine in sickle cell disease subjects. This study compared the blood levels of Glut 1, fasting blood glucose (FBG) and fasting insulin (FIns) in non-sickle cell anaemia (HbAA) and sickle cell anaemia (HbSS) subjects in the steady state before and following L-Arginine supplementation (1 g/day for 6 weeks). Nitric oxide metabolites, (NO_X), catalase, superoxide dismutase and glutathione peroxidase were also measured in each group of subjects. Correlation coefficients between change (Δ) in Glut 1 and change (Δ) in FBG, Fins, NO_X and antioxidant enzymes respectively were calculated. Before supplementation, Glut 1, NO_X, GP_X and CAT were significantly higher in HbAA subjects while FIns, FBG and MDA were higher in HbSS subjects. In both groups, supplementation significantly increased NO_X, Glut 1 and antioxidant enzymes but decreased MDA. Supplementation increased FIns in HbAA but decreased FBG and FIns in HbSS subjects. In both groups of subjects, ΔGLUT 1 correlated positively with ΔNOX, antioxidant enzymes and Δ[R] but negatively with ΔMDA. ΔGLUT 1 correlated negatively with ΔFBG and ΔFins in HbSS but positively in HbAA. Study thus showed that in the steady state HbSS subjects had lower GLUT 1 but elevated FBG and Fins levels than HbAA subjects. Additionally, L-Arginine increased GLUT I and antioxidant enzymes but decreased Fins, FBG and MDA in HbSS subjects.

Highlights

• •Little information on the interaction between GLUT1 and L-Arginine in HbSS subjects.
• •Blood level of GLUT1 was higher in HbAA than in HbSS subjects.
• •Arginine increased GLUT1 in HbAA and HbSS subjects.
• •Arginine decreased fasting insulin and fasting glucose in HbSS subjects but increased fasting glucose in HbAA subjects.

1. Introduction

L-Arginine is a basic amino acid and a potential therapeutic agent in the management of sickle cell disease (Eleuterio et al., 2019) and diabetes mellitus (Stancic et al., 2012). L-Arginine plays important roles in several metabolic processes such as ammonia detoxification, immune modulation and secretion of hormones (Barbosa et al., 2006). It is also a substrate for the nitric oxide synthase enzyme (NOS) and therefore the precursor of nitric oxide (NO) (Wu, 2009). Evidence from literature suggests that the effects of L-Arginine supplementation on either diabetes mellitus or sickle cell disease patients may be mediated through a common pathway – the NO pathway. In diabetes mellitus, L-Arginine had been reported to possess pancreatic β-cell regenerative capacity (Stancic et al., 2012) resulting in increased insulin secretion. The positive effects of L-Arginine on diabetes mellitus are thought to be mediated by NO exerting its effects on endothelium-dependent relaxation (Böger and Ron, 2005). In sickle cell disease, the effects of L-arginine are also thought to be mediated by NO (Eleuterio et al., 2019; Jaja et al., 2020). In sickle cell anaemia subjects, L-Arginine supplementation increased antioxidant enzymes levels, (Little et al., 2009; Kehinde et al., 2015), nitric oxide availability (Morris et al., 2000), enhanced blood trace metals levels (Ogungbemi et al., 2018), caused a significant reduction in total opioid use and pain in children (Morris et al., 2013) and adults (Eleuterio et al., 2019).

Although diabetes mellitus is a rare finding among SCD sufferers (Akinlade et al., 2018), some authors had reported elevated fasting blood glucose, insulin and homeostasis model assessment of insulin resistance (HOMA-IR) in sickle cell disease (SCD) patients as compared with non-sickle cell disease sufferers (Al-Sultan et al. (2010; Jaja et al., 2020). In addition, Jaja et al. (2020) reported that in sickle cell anaemia (HbSS) subjects supplementation with L-Arginine reduced the elevated fasting glucose and insulin levels but did not affect the impaired glucose and insulin responses to oral glucose tolerance test (OGTT). On the other hand, Akinlade et al. (2014) reported similar insulin level but lower level of fasting glucose in SCA subjects compared to non sickle cell anaemia (HbAA) subjects. They also showed that patients with SCA had similar insulin sensitivity status as HbAA individuals.

Glucose transporters (GLUTs) are membrane-associated carrier proteins that mediate the transport of glucose. GLUT I - a facilitative transporter protein – present on all tissues is highly expressed on the surface of red blood cells. It is a part of the insulin signaling pathway involved in different biological functions like cellular metabolism, energy regulation and stress response. Thus, it catalyses and is essential in the transport and provision of glucose for energy production in red blood cells, brain, muscle and adipose tissue (Pragallapati and Manyam, 2019). It is also associated with the transport of vitamins and other sugars like galactose, mannose and glucosamine (Shah et al., 2012). GLUT I expression is regulated by blood glucose concentration. In brain, it is upregulated in hypoglycaemic states. During oxidative stress, GLUT I is involved in the maintenance of insulin action and the regulation of reactive oxygen species (ROS) (Andrisse et al., 2014). GLUT I has also been associated with pathologies including diabetes mellitus and sickle cell disease. In diabetic rats, chronic hyperglycemia down regulated GLUT1 expression suggesting an adaptive reaction of the body to prevent excessive glucose entering the cell that may lead to cell damage (Leãoa et al., 2020). Glut 1 expression had been found to be higher on the surface of red blood cells of hydroxyurea-treated sickle cell disease (HbSS) patients than on non-sickle cell disease (HbAA) subjects. In addition, there was a strong positive correlation between GLUT 1 expression and haemoglobin F (HbF) (Paikari et al., 2019).

It is not known how L-Arginine supplementation may affect GLUT 1 expression in sickle cell anemia subjects in the steady state. This study compared the blood levels of GLUT1 in HbAA and HbSS subjects in the steady state and investigated the effect of a 6-week supplementation with L-Arginine on GLUT 1 expression, fasting insulin and fasting blood glucose levels in these subjects.

2. Materials and methods

Forty (40) non sickle cell anaemia (HbAA) subjects (20 males and 20 females) and forty (40) sickle cell anaemia (HbSS) subjects (20 males and 20 female) were studied. HbSS subjects were volunteers that regularly attended the adult Out-Patients’ Sickle Cell Clinic of Lagos University Teaching Hospital (LUTH), Lagos, Nigeria. The sample size was determined based on the equation of Eng et al. (Eng, 2003).

N = 4σ² (Z_crit + Z_pwr)² ÷ D²

Where N = 60 = total sample size required for the two groups

σ = 1.51 = assumed standard deviation for each group

Z_crit = 1.96 = standard deviation at α = 0.05

Z_pwr = 0.842 = desired statistical power for the study

D = 1 = minimum expected difference between two means (Eng et al., 2003)

They were in the steady state and without a history of diabetes mellitus, hypertension, human immunodeficiency virus (HIV), hepatitis, or blood transfusion six months prior to the study. HbAA subjects (electrophoretically determined) served as control and were students of tertiary institutions in Lagos, Nigeria. The Lagos University Teaching Hospital Health Research Ethics Committee gave approval for the study (ADM/DCST/HREC/APP/1359). Each subject gave an informed consent.

On presenting to the laboratory, anthropometric measurements - age (years) height (centimeter) and weight (kilogram) of each subject was recorded. Each subject presented to the laboratory after a 12-hr over-night fast. After withdrawing 3 mL of blood from an ante-cubital vein, one (1) milliliter was put into EDTA bottle for the measurement of haematologic parameters - red blood cell (RBC) count, packed cell volume (PCV), haemoglobin concentration ([Hb]), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular volume (MCV)). The remaining 2 mL was put into plain sample bottle for the estimation of the following: fasting blood glucose, plasma insulin, L-arginine [R] concentration, antioxidant enzymes (CAT, SOD, GP_X) levels, nitric oxide metabolites (NO_X) level, malondialdehyde (MDA) and GLUT 1 concentration. GLUT 1 expression was assayed in erythrocytes. Serum was obtained by centrifuging blood at 3000 rpm for 15 min. L-Arginine (NOW FOODS, Dallas, USA) (1 g/day for six weeks) was then administered to each subject (Kehinde et al., 2015). Measurements were made again at the end of 6 weeks.

2.1. Estimation of some haematological parameters

Haematological indices - red blood cell count (RBC count, million/mm³), packed cell volume (PCV, %) haemoglobin concentration [Hb, g/dL], mean corpuscular volume (MCV, fL), mean corpuscular haemoglobin (MCH, pg), mean corpuscular haemoglobin concentration (MCHC, g/dL) and glycated haemoglobin (%) were determined using an automated counter (Mindray BC 2800 Haematology Automated System, China).

2.2. Determination of plasma L-Arginine concentration ([R, μmol/L])

An assay kit (Oxford Biomedical Research, U. S. A.) was used in estimation of L-Arginine concentration.

2.3. Estimation of malondialdehyde

Malondialdehyde, (MDA), level in blood was determined using the thiobarbituric acid method that is based on the principle that lipid peroxides condense with 1-methyl-2-phenyl indole under acidic conditions (Titus et al., 2004).

2.4. Determination of serum levels of antioxidant enzymes

Serum catalase (CAT) and superoxide dismutase (SOD) levels were assayed as described by Rukkumani et al. (2004) while serum glutathione peroxidase (GPx) level was measured as described by Ellman (1959).

2.5. Determination of nitric oxide metabolite (NO_X)

Nitric oxide metabolite was determined using commercial assay kit (Oxford Research, USA) (Sun et al., 2003).

2.5.1. Measurement of [GLUT 1] in plasma

GLUT 1 concentration was determined using assay kit (Kono biotech Co., Ltd, China) as described by Domittile et al. (2017).

2.5.2. Measurement of GLUT 1 expression in erythrocytes

GLUT 1 expression was assayed in red blood cells using Western Blot (Thomas et al., 2009) at the Institute for Advanced Medical Research and Training (IAMRAT), University College Hospital, Ibadan, Nigeria.

2.6. Determination of blood glucose

Fasting blood glucose was determined by using the ONETOUCH Ultra 2 Blood glucose monitoring system (LIFESCAN, CHINA). Fasting blood glucose was determined after a 12-hr fast.

2.7. Statistical analyses

Results are presented as mean ± SEM. Statistical analyses were made using Graph pad prism 5. Statistical comparisons were made using the Student's t-test or the paired Student's t-test as appropriate and significance level was adopted at p < 0.05 for each analysis. Spearman's rank correlation coefficients (r) were calculated between change in nitric oxide metabolites (ΔNOx) or Glut1 (ΔGlut1) or L-Arginine (ΔR) and change (Δ) in each of the other measured variables.

3. Results

Physical and haematological characteristics of the subjects are presented in Table 1. The table shows that while mean age and mean height were similar in both groups, mean weight and mean body mass index (BMI) were significantly less in HbSS subjects (p < 0.05 in each case). Hematocrit (Hct%), red blood cell count (RBC) and hemoglobin (Hb) were significantly lower in HbSS than in HbAA subjects (p < 0.001 in each case).

Table 1.

Comparison of Physical and some haematological parameters of subjects.

Parameters	HbAA (n = 40)	HbSS (n = 40)	p level
	(±SEM)	(±SEM)
Age (years)	30.7 ± 1.0	27.5 ± 1.2	NS
Height (cm)	172.5 ± 2.0	167.5 ± 2.0	NS
Weight (kg)	69.2 ± 1.0	59.8 ± 1.1	<0.01
BMI	23.1 ± 0.6	21.8 ± 0.6	<0.05
Hct (%)	40.3 ± 1.3	26.1 ± 1.1	<0.001
RBC (106/μL)	4.8 ± 0.2	3.2 ± 0.2	<0.001
Hb (g/dL)	13.7 ± 0.4	8.9 ± 0.3	<0.001

Table 2 shows that before arginine supplementation, plasma arginine (R) in both groups of subjects was similar while plasma GP_X, NO_X, and Glut 1 were higher in HbAA than in HbSS subjects (p < 0.05, p < 0.001, p < 0.001 respectively). The table also shows that MDA was significantly higher in HbSS subjects than in HbAA subjects. (Table 2; a Vs b).

Table 2.

Comparison of measured parameters between HbAA and HbSS subjects.

Parameters	Pre-Supplement		Post-Supplement		p value
	HbAA (n = 40)	HbSS (n = 40)	HbAA (n = 40)	HbAA (n = 40)
	(a)	(b)	(c)	(d)	(a)Vs(b)	(a)Vs(c)	(b)Vs(d)	(c)Vs(d)
[R] (μmol/L)	65.1 ± 2.0	63.7 ± 1.5	72.6 ± 1.9	86.3 ± 6.2	NS	<0.01	<0.01	0.05
FBG (mmol/L)	4.7 ± 0.2	6.2 ± 0.3	5.0 ± 0.1	5.7 ± 0.2	<0.05	NS	<0.05	<0.05
Fins (μU/mL)	1.2 ± 0.3	16.2 ± 3.6	5.8 ± 1.2	1.9 ± 0.3	<0.001	<0.01	<0.001	<0.01
HbA1c (%)	5.3 ± 0.2	3.5 ± 0.3	4.3 ± 0.3	2.6 ± 0.2	<0.001	<0.05	<0.05	0.001
MDA (μmol/mg prot)	28.7 ± 2.8	42.0 ± 3.9	14.7 ± 1.4	10.4 ± 0.9	<0.01	<0.05	<0.01	0.001
GPx (U/mg prot)	2.5 ± 0.3	1.0 ± 0.1	3.5 ± 0.4	1.7 ± 0.3	<0.01	<0.01	<0.05	<0.05
CAT (U/mg prot)	3.6 ± 0.6	0.9 ± 0.3	7.0 ± 0.9	2.0 ± 0.6	<0.001	<0.01	<0.05	<0.01
SOD (U/mg prot)	2.4 ± 0.1	2.7 ± 0.1	4.0 ± 0.4	4.3 ± 0.4	<0.05	NS	<0.01	<0.05
NOX (μM/L)	54.2 ± 2.4	45.4 ± 3.3	78.2 ± 4.8	77.1 ± 7.4	<0.05	<0.001	<0.001	NS
Glut 1 (pg/mL)	121.1 ± 9.5	84.0 ± 3.1	156.4 ± 11.9	110.5 ± 7.2	<0.05	<0.01	<0.01	<0.05

Key: [R] = Arginine concentration; FBG = Fasting blood glucose; Fins – Fasting blood insulin; HbA1c = Glycated hemoglobin; MDA Malondialdehyde; GPx = Glutathione peroxidase; CAT = Catalase; SOD – Superoxide dismutase; NOx = Nitric oxide metabolites.

In HbAA subjects, arginine supplementation significantly elevated all parameters except MDA that significantly fell (p < 0.01) (Table 2; a Vs c). In HbSS subjects, supplementation reduced MDA (p < 0.001) but significantly elevated [R], GP_X, NO_X and Glut 1. (See Table 2; b Vs d).

Fig. 1 shows the expression of Glu1 in the erythrocytes of each group of subjects as shown by the band colour intensity as assayed by Western Blot. Bands 1–3 were obtained after supplementation with L-Arginine HbSS subjects, while bands 4–7 were obtained from HbAA subjects after supplementation with arginine. Bands 8–10 were obtained from HbAA subjects before supplementation while bands 11–12 were obtained from HbSS subjects before supplementation with L-Arginine. L-Arginine supplementation increased Glut 1 expression in both groups of subjects as indicated by enhanced band colour intensity in bands 1–7 as against bands 8–12.

Fig. 1.

Expression of Glu1 in the erythrocytes of HbSS and HbAA subjects.

Bands 1–3 and Bands 4–7 were obtained from HbSS and HbAA subjects respectively after L-arginine supplementation. Pre-supplementation bands are bands 8–10 and band 11 and 12 for HbSS and HbAA respectively.

Table 3 shows correlation coefficients (r) calculated between change in plasma nitric oxide metabolites (ΔNO_X) or Glut 1 (ΔGlut 1) with the other measured parameters in the subjects. In each of the groups, Δ[R] positively and significantly correlated with ΔNO_X or ΔGlut 1. In each group of subjects, ΔNO_X was positively and significantly related to ΔGlut 1 and ΔGPx but negatively with ΔMDA. In each group of subjects ΔGlut 1 was positively correlated with GPx but negatively with ΔMDA.

Table 3.

Correlation coefficients calculated between the measured variables.

Parameters	HbAA	HbSS
ΔNOX Vs ΔGlut 1	0.86***	0.84***
ΔNOX Vs ΔCAT	0.76**	0.86***
ΔNOX Vs ΔSOD	0.78**	0.80***
ΔNOX Vs ΔGPX	0.76**	0.78**
ΔNOX Vs ΔFBG	0.78**	0.82***
ΔNOX Vs ΔFins	0.86***	−0.81***
ΔNOX Vs ΔMDA	−0.60*	−0.70**
ΔGlut 1 Vs ΔCAT	0.75**	0.70**
ΔGlut 1 Vs ΔSOD	0.76**	0.66*
ΔGlut 1 Vs ΔGPX	0.78**	0.70**
ΔGlut 1 Vs ΔFBG	0.94***	−0.90***
ΔGlut 1 Vs ΔFins	0.74**	−0.82***
ΔGlut 1 Vs ΔMDA	−0.82***	−0.78**
Δ[R] Vs ΔGlut 1	0.68*	0.74**
Δ[R] Vs ΔNOx	0.70*	0.75**
Δ[R] Vs ΔHbA1c	−0.60*	−0.27

Key: * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

4. Discussion

Glucose transporters accomplish the movement of glucose from the extracellular space (deriving from the blood stream) into cells. In this study plasma glucose transporter (Glut 1) was found to be higher in HbAA than in HbSS subjects. Qualitative (Fig. 1) and quantitative (Table 2) assessments showed that arginine supplementation elevated plasma Glut 1 levels in HbSS and HbAA subjects. This result is similar to an earlier one which showed that Glut 1 level was higher in hydroxyurea-treated HbSS patients than in their HbAA counterparts that did not take hydroxyurea (Paikari et al., 2019). This is important following reports of the combination of hydroxyurea and arginine in the management of sickle cell disease patients (Little et al., 2009; Eleuterio et al., 2019; Temiye et al., 2020).

In the steady state, our results showed that fasting blood glucose and fasting insulin levels were higher in HbSS than in HbAA subjects suggesting some degree of insulin resistance. This is in agreement with earlier reports (Al-Sultan et al., 2010; Jaja et al., 2020). Supplementation with L-Arginine reduced these parameters in HbSS subjects but increased fasting insulin in HbAA subjects. The effect of L-Arginine on fasting insulin in HbAA subjects was expected since L-Arginine is an insulin secretagogue (Gannon and Nuttall, 2010; Stancic et al., 2012). Potentiation of insulin secretion by L-Arginine had been linked to a rise in intracellular calcium (Nobuyoshi et al., 2006). It is not clear by what mechanism L-Arginine caused the lowering of glucose and insulin in HbSS subjects. However, following L-Arginine supplementation, change (Δ) in GLUT 1 level correlated negatively with change (Δ) in fasting blood glucose and change (Δ) in fasting insulin respectively in HbSS subjects suggesting that elevated GLUT 1 may have accounted for the low level of blood glucose following an enhanced transport of glucose into cells. Immunocytochemical studies had shown that although insulin does not affect GLUT 1 expression, it induces a translocation of the transporter to the plasma membrane accounting for the increased transport of glucose into the cell (Cifuentes et al., 2011).

This study also shows that glycated hemoglobin was lower in HbSS than in HbAA subjects. Sickle red cells have a much shorter lifespan than normal red blood cells. Shorter RBC survival in sickle cell disease results in less time for hemoglobin to be exposed to glycation, reducing HbA1c concentration (Klonoff, 2020). This may explain the lower HbA1c values obtained in this study. In both groups of subjects, L-Arginine supplementation reduced the level of glycation suggesting that L-Arginine may play some role in glycemic control in sickle cell disease management.

Results of this study confirm earlier reports that in the steady state blood levels of NO_X and antioxidant enzymes, (CAT, SOD and GP_X) were higher in HbAA than in HbSS subjects, while MDA was higher in HbSS than in HbAA subjects (Little et al., 2009; Jaja et al., 2016). Thus in steady-state HbSS subjects, lower GLUT I level, elevated fasting glucose and fasting insulin were associated with elevated reactive oxygen species resulting in increased oxidative stress. L-Arginine supplementation elevated NOx, antioxidant enzymes and GLUT 1 (as earlier noted) but decreased oxidative stress in these subjects. It had been suggested that GLUT1 may play a role in regulation of reactive oxygen species and could contribute to maintenance of insulin action in the presence of oxidative stress (Andrisse et al., 2014).

In both groups of subjects, significantly positive correlation coefficients were calculated between change in nitric oxide metabolites (ΔNO_X) and change in GLUT 1 level (ΔGlut 1), change in GLUT 1 level (ΔGlut 1) and change in each of the antioxidant enzymes and between change in nitric oxide metabolites (ΔNO_X) and change in each of the antioxidant enzymes following L-Arginine supplementation. Thus, elevated antioxidant enzyme or nitric oxide levels were positively related to elevated Glut 1 levels in HbAA and HbSS subjects following L-Arginine supplementation. On the other hand, change in GLUT 1 level (ΔGLUT I) or change in nitric oxide metabolites (ΔNOx) correlated negatively with change in malondialdehyde level (ΔMDA). GLUT I levels in blood of HbAA and HbSS subjects may therefore be enhanced with a concomitant enhancement of NOx and antioxidant levels. In summary, L-Arginine supplementation in HbAA and HbSS subjects led to enhancement of blood GLUT I, CAT, SOD, GPx and NOx levels and a lowering of MDA level in blood. L-Arginine therefore could be acting through a combination of pathways. Firstly, results of this study support that the nitric oxide pathway as earlier suggested (Stancic et al., 2012; Bakshi and Morris, 2016) may mediate L-Arginine effect. L-Arginine is a substrate for the nitric oxide synthase enzyme (NOS) and consequently the biological precursor of nitric oxide (NO) (Wu, 2009). Secondly, since L-Arginine is an antioxidant, it may also be acting through its antioxidant effect by reducing oxidative stress burden (Little et al., 2009; Jaja et al., 2016).

Potentially, L-Arginine may have use in various types of diseases – sickle cell disease (Jaja, 2017; Jaja et al., 2020), hypertension, ischemic heart disease, heart failure, atherosclerosis and diabetes mellitus (Szlas et al., 2022). Although L-Arginine supplementation may be beneficial in the management of diabetes mellitus (Stancic et al., 2012), studies investigating the effect of L-Arginine supplementation on glucose metabolism in sickle cell disease are scanty (Jaja et al., 2020). Thus the mechanisms of regulatory effects of L-Arginine on carbohydrate metabolism in sickle cell disease are not fully understood. For instance, in this study, while L-Arginine had little or no effect on FBG in HbAA subjects it significantly reduced FBG in HbSS subjects. Also, L-Arginine significantly elevated fasting insulin in HbAA subjects (because it is an insulin secretagogue) but had the opposite effect in HbSS subjects.

Different investigators had used different doses and duration for the supplementation of L-Arginine in the management of carbohydrate metabolism disorders (Szlas et al., 2022) or in management of sickle cell disease (Kehinde et al., 2015). Although L-Arginine is found in food, doses of 3–8 g/d are considered safe and not to cause acute pharmacologic effects in humans (Böger, 2007). In this study, we used a dose of 1 g/day for a duration of 6 weeks. It is not clear what effect higher doses will produce in HbSS subjects. L-Arginine may exert some side effects when supplemented in supra physiological doses and for long periods of time. Oral doses of L-Arginine-HCl (>9 g/day) had been associated with nausea and diarrhoea (Grimble, 2007; Stancic et al., 2012). Higher doses could also cause anaphylaxis apart from inducing changes in numerous chemicals and electrolytes in the blood, including potassium (Stancic et al., 2012). It had also been reported that in T2DM-free adults, higher dietary L-Arginine intake or levels may increase risk of T2DM and it may have an independent role in T2DM development (Mirmiran et al., 2021).

In conclusion, the novel finding of this study was that in the steady state glucose transporter (Glut 1) level was higher in HbAA than in HbSS subjects and L-Arginine supplementation increased the glucose transporter levels in both groups of subjects. Following L-Arginine supplementation, elevated GLUT 1 level may have caused increased entry of glucose into cells accounting for the reduction in the elevated fasting glucose seen in HbSS subjects.

Funding

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

CRediT authorship contribution statement

W.A. Saka: Conceptualization, Methodology, Data acquisition, Data curation, Funding acquisition. C.N. Anigbogu: Conceptualization, Supervision. M.O. Kehinde: Conceptualization, Provision of participants. S.I. Jaja: Conceptualization, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.