Hyperhomocysteinemia and folate levels in normal healthy Nigerians living in Zaria: Subanalysis of ABU homocysteine cross-sectional survey.

Background: This study aimed to determine the prevalence of hyperhomocysteinemia and folate status in a sample of normal healthy Nigerians living in Zaria as well as assess the relationship between homocysteine, folate, and blood pressure (BP) levels.
Methods: It was a cross-sectional analytical study carried out among 65 normal healthy volunteers aged 18-65 years. Participants were randomly selected from willing patient escorts, hospital employees, and willing staff presenting at the Ahmadu Bello University Medical Centre, Zaria and Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. The percentage of participants who had high homocysteine levels as well as their plasma folate status was determined.
Results: There were 9.2% with hyperhomocysteinemia >15 μmol/L and 51% with hyperhomocysteinemia >10 μmol/L. The mean plasma homocysteine level was 10.8 ± 2.7 μmol/L with male and female values of 10.7 ± 2.6 and 10.8 ± 2.8, respectively (P = 0.87). The mean plasma folate level was high (116.7 ± 44.0 ng/mL) with male value of (111.5 ± 44.9 ng/mL) which did not differ significantly (P = 0.37) from that of females (121.4 ± 43.3 ng/mL). Homocysteine showed a positive significant (P = 0.01) relationship with folate but not with BP's (P > 0.05).
Conclusion: There is a high prevalence of hyperhomocysteinemia in normal healthy Northern-Nigerians which cannot be accounted for by suboptimal folate levels. Hyperhomocysteinemia may not be a risk factor for cardiovascular disease in normal healthy Nigerians despite its high levels as it showed no significant relationship with BP.

INTRODUCTION

Homocysteine is a sulfur-containing amino acid with chemical formula HSCH2 CH2 CH (NH2) CO2H which is derived from the metabolism of methionine, an essential amino acid obtained from dietary protein such as meat, seafoods, eggs, and dairy products.[1] It is metabolized through two vitamin-dependent pathways. First, a re-methylation pathway which occurs in all body tissues, wherein vitamin B12, folate, and methyl group from N-5-methyl-tetrahydrofolate co-enzyme are utilized, converting 50% of homocysteine back to methionine.[2] A second re-methylation pathway also takes place in the kidney and liver using betaine, vitamin B2, and magnesium rather than folate and vitamin B12.[123] Second, a transsulfuration pathway which requires vitamin B6 and pyridoxal-5-phosphate in the presence of an enzyme cystathionine-β-synthase, to convert the remaining homocysteine to cysteine and taurine.[124] The cysteine is then utilized by the body to form a powerful antioxidant called glutathione which acts to preserve cellular processes from oxidative damage.[14] Therefore, the absence or mutation of the enzymes involved in this pathway or the deficiency of these vitamins can result in impaired metabolism and consequent hyperhomocysteinemia.[23]

Several other factors interact with homocysteine metabolism resulting in increased levels. These include demographic factors such as increasing age, male sex, postmenopausal status; metabolic factors such as diabetes, hypothyroidism, hyperlipidemia, renal failure; and social factors like chronic alcoholism and smoking, all leading to raised blood levels.[23] Certain medications such as fenofibrate, methotrexate, anticonvulsants (phenytoin and carbamazepine), cholesterol-lowering agents (nicotinic acid, cholestyramine, and colestipol), cyclosporine, metformin, levodopa, sulfonamides, antacids interact with homocysteine metabolism resulting in raised blood levels.[123] Inflammatory conditions such as systemic lupus erythematosus, hyperproliferative disorders as well as high dietary intake of coffee lead to hyperhomocysteinemia.[235] Obesity has also been linked with higher homocysteine (Hcy) levels.[13]

Furthermore, there are myriads of diseases that have been linked with high homocysteine levels. Over 49 years ago, McCully discovered the initial link between homocysteine and atherosclerotic vascular disease.[6] This paved the way for several studies on homocysteine showing its link with cardiovascular diseases (CVD) in the United States (US) and European countries and subsequently in the sub-Saharan Africa viz-a-viz: CVD such as coronary artery disease,[278] stroke,[391011] hypertension,[41213] and cardiac failure.[14] Other diseases include neurodegenerative disorders like multiple sclerosis,[15] dementia, and Alzheimer's disease;[16] congenital defects like neural tube defects;[17] polycystic ovarian syndrome and pregnancy-related complications such as preeclampsia, eclampsia, placenta abruption, and multiple pregnancy losses [341819] among others. Hyperhomocysteinemia is a risk factor for various diseases especially CVD and studies from the United States of America (USA) and European countries reported a 5%–10% prevalence of hyperhomocysteinemia in their general population and 25% among people with vascular diseases.[8912181920] The prevalence in the general population documented in sub-Saharan Africa and South-West Nigeria is similar to that outside Nigeria albeit different partition limits.[41013] There is no consensus definition for hyperhomocysteinemia with different partition levels used in various studies; previously 15 μmol/L was used, however, presently experts recommend an upper limit of 10 μmol/L as homocysteine was found to be atherogenic at such lower levels.[212223]

Therefore, if hyperhomocysteinemia is detected early in the apparently normal healthy population, it may be modified by lifestyle changes such as exercise, avoidance of alcohol and smoking, intake of high fiber foods that are rich in fruits and vegetables and low in salt and cholesterol.[5] It may also provide an avenue for incorporation of public health intervention policies.[5] Notwithstanding, there is a paucity of data on homocysteine assessment in normal healthy adult Nigerian population with few studies emanating from Akande et al.,[4] Glew et al.,[20] Alkali et al.,[11] and Ajuluchukwu et al.,[13] with the latter three studies comparing homocysteine in patients with various CVD against normal healthy controls.

Furthermore, inadequate dietary intake of sources of B-vitamins might be a strong determinant of hyperhomocysteinemia,[5] but its relevance on an apparently healthy population as determined by the prevalence of hyperhomocysteinemia associated with inadequate folate intake has barely been studied in Nigeria. This study was therefore aimed to determine the prevalence of hyperhomocysteinemia and plasma folate statuses in a sample of normal healthy Nigerians living in Zaria based on the new recommended partition limit as well as assess the relationship between homocysteine, folate, and blood pressure (BP) levels in normotensive individuals.

METHODS

Design

The study was a cross-sectional analytical study carried out between January 2016 and March 2016 among 65 normal healthy volunteers aged 18–65 years who were randomly selected from willing patient escorts to the large hall of the Ahmadu Bello University (ABU) Medical Centre, Zaria as well as the out-patient department of ABU Teaching Hospital, Zaria, Nigeria during the primary survey, respectively. Other volunteers were from hospital employees and willing staff of both hospitals. The study was a subsect of the ABU Homocysteine Survey which was a randomized controlled study done on hypertensive subjects and controls in Zaria, Nigeria.[24] In that study, an additional cross-sectional survey of normal healthy volunteers was surveyed to determine baseline homocysteine and folate levels in the general population.

Ethical clearance was derived from the Health Research Ethical Committee, Ministry of Health, Kaduna, with Reference number: MOH/ADM/744/VOL. 1/369 and all participants gave written informed consent.

Inclusion criteria

Inclusion criteria were healthy adult controls >18 years old with BP <140/90 mmHg and no history of previously diagnosed diabetes mellitus.

Exclusion criteria

Exclusion criteria included patients with renal failure (serum creatinine >3 g/dL or glomerular filtration rate [GFR] <60 ml/min) as determined by Cockcroft-Gault equation;[25] historical evidence of heart failure, stroke/transient ischemic attack or heart attack; excessive caffeine use; chronic folic acid, vitamin B12 and vitamin B6 supplementation; sickle cell disease or pregnancy; current tobacco and excessive alcohol intake as well as use of drugs known to affect homocysteine metabolism viz-a-viz: methotrexate, anticonvulsants, nitrous oxide, sulfadoxine-pyrimethamine, penicillamine, and contraceptives.[11324] Current smokers, chronic alcoholics, and patients with diabetes mellitus (determined historically and by fasting blood glucose [FBG] >7 mmol/L) were also excluded.[113]

Study size

Where:

n = Desired sample size

z = Standard normal deviation at 95% confidence interval (CI) (1.96)

p = Prevalence of hyperhomocysteinemia in normal healthy Nigerians which is 5%[413] using data from a hospital-based study done by Akande et al.[4] and Ajuluchukwu et al.[13] hence P = 5% =0.05

q = 1–p = 1–0.05 = 0.95, d = degree of accuracy at 0.05

With an estimated attrition rate of approximately 5%–10% (non-response and indeterminate results), i.e., 5/100 × 60–10/100 × 60 = 3–6, the estimated sample size required was approximately 63–66 which is the minimum number required hence a sample size of 65 individuals was obtained.

Hypothesis

There is low prevalence of hyperhomocysteinemia and low folate levels in normal healthy Nigerians living in Zaria

There is no relationship between plasma homocysteine and plasma folate levels in normal healthy Nigerians living in Zaria

There is no relationship between plasma homocysteine and BP levels in Nigerians living in Zaria.

Screening evaluation and data collection

The individuals underwent a detailed medical history, and data collection was by well-structured interviewer-administered questionnaire carried out by the author along with four trained assisting medical doctors. The most important data obtained during the screening was: the biodata (address, age, sex, tribe, and religion); detailed 24 h dietary recall; family and social history including smoking and alcohol history. Physical examination, anthropometric measurements (subjects weight, height, and body mass index (BMI) were calculated in weight [kg]/height2 [m2]), and BPs, were determined in the healthy controls. BPs were measured using Accoson Mercury Sphygmomanometer, twice in the left arm of seated subjects previously rested for 5 min and by standard protocol. The mean of the two readings was used.[26] The normal BP was defined by the absence of a history of hypertension and systolic BP (SBP) <140 mmHg or diastolic BP (DBP) <90 mmHg.[26]

Blood sample collection

Blood samples for plasma homocysteine and folate levels were collected from the antecubital vein of either arm, after an overnight fast and without tourniquet application between 7 a. m. and 9 a. m. The blood was divided into two 5 ml aliquots and put into labeled potassium ethylenediaminetetraacetic acid-containing plastic vacutainer tubes and plain specimen bottles respectively. A drop (0.6 TIU/ml or 500 Kallikrein inactivator U/ml) of aprotinin (trasylol®) had been previously added to the test tubes. These were taken to the Immunology laboratory of the major tertiary hospital within 4 h of collection in ice cubes, where they were centrifuged at 1800 revolutions per minute for 20 min and plasma separated within 1–2 h. These were divided into aliquots in cryovials and stored at −70°C in the Anti-retroviral laboratory till analyzed.[27] Other investigations such as serum electrolyte, urea and creatinine and FBG were also assayed in the chemical pathology laboratory of the same hospital using the Chenray 120 automated clinical chemistry auto-analyzer.

The measurement of plasma homocysteine and folate

The Human direct homocysteine enzyme-linked immunosorbent assay kit (ELISA-Elabscience Biotechnology Co., Ltd., WuHan, P. R. C. with Lot No: AK0016JULI5066 and Catalog No: E-EL-HO156), was used for in vitro quantitative determination of human homocysteine in plasma according to the manufacturer's manual and based on the Elisa principle.[2427] The concentration of homocysteine in the samples was determined by comparing the optical density of samples to the standard curve.[2427] The folic acid ELISA kit-Elabscience Biotechnology Co. Ltd., WuHan, P. R. C. with Lot No: AK0016JULI5067 and Catalog No: E-EL-0009 was used for the in vitro quantitative determination of plasma folate levels in accordance with the manufacturer's manual.[28] Laboratory analysis was done by the laboratory scientist and the lead author and in one batch under the same atmospheric condition. Partition limit for plasma homocysteine was 10 μmol/L [212223] and the previous partition limit of 15 μmol/L was also additionally applied.[413] The partition limit for plasma folate was 20 ng/mL.[24]

Data analysis

Data were validated and analyzed by Statistical Package for Social Sciences (SPSS) version 16-software (SPSS Inc., Chicago, IL, USA). There was no missing data as the questionnaire was interviewer-administered by the lead author and four trained medical doctors and laboratory analysis were done for all the 65 individuals. Categorical variables were presented as numbers and percentages with difference determined using Chi-square. Numerical variables were presented as mean ± standard deviation. Independent Student's t-test was used to compare Hcy, folate, BMI, GFR, packed cell volume (PCV), urea and creatinine in males and females. Pearson's correlation was used to determine the relationship between Hcy, folate and BP. To further study the factors contributing to hyperhomocysteinemia and its relationship with folate, dependent variable Hcy was categorized into 0 if level was <10 μmol/L and 1 if values were >10 μmol/L. The independent variables were categorized as age (1 if <30 years, 2 if 30–45 years, 3 if 45–65, 4 if >65 years); sex (1 if male and 2 if female); BMI (1 if <18, 2 if 18–24.9 kg/m2, 3 if 25–29.9 kg/m2, 4 if >30 kg/m2); vegetables and fruits in daily diet (1 if Yes and 2 if No); family history (1 if positive history of hypertension and 2 if no such history); SBP (1 if <120 mmHg, 2 if 120 mmHg, 3 if 120–139 mmHg); DBP (1 if <80 mmHg, 2 if 80 mmHg, and 3 if 80–89 mmHg) and folate (1 if <100 ng/dL [low normal], 2 if >100 ng/dL [high normal]). Binary Multiple Regression analysis was then applied to examine the interaction of age, sex, BMI, FPG, vegetables/fruits in daily diet and family history with homocysteine and folate as well as BP. In addition, Hcy was also recoded as 1 if level was <15 μmol/L and 2 if >15 μmol/L to determine its distribution among both sexes at a higher partition level. The level of statistical significance was assumed to be P ≤ 0.05 at 95% CI.

RESULTS

The top-line results, medical pro forma/questionnaire and ethical clearance can be found in supplementary files.

Subject participation

Consecutively, a total of 80 normal healthy controls were randomly screened at the ABU Medical Centre, Zaria as well as ABUTH, Zaria, Nigeria from January 2016 to March 2016. Of these, 65 individuals met eligibility criteria, were enrolled, had complete data collected which was analyzed. The remaining 15 individuals were excluded: Eight individuals on account of incidental finding of raised (BP >140/90 mmHg); four on account of incidental finding of raised FBG >7 mmol/L and three on account of history of the current smoking [Figure 1].

Figure 1

Flow Chart of Events in Homocysteine and Folate Study on Normal Healthy controls: A subanalysis of the ABU Homocysteine Survey 2016. n = Number of subjects; FBG = Fasting Blood Glucose

The sociodemographic characteristic of the normal healthy controls

Table 1 presents the sociodemographic characteristic of the study participants. There were more females, 34 (52.3%) than males, 31 (47.7%) though not statistically significantly (P = 0.71) different. The mean age of the subjects was 41.9 ± 12.1 years falling within the 4th decade and there was no significant difference (P = 0.83), between that of males and females. There were more subjects within the middle age [Table 1]. More subjects, 37 (56.9%) ate fruits and vegetables in their daily diet in both males and females with no statistically significant (P = 0.41) difference. Majority, 51 (78.5%) had no family history of hypertension in both groups.

Table 1

Sociodemographic characteristics of the normal healthy controls

Variables	Individuals (%)			P
	Males (n=31)	Females (n=34)	Total
Sex	31 (47.7)	34 (52.3)	65 (100)	0.71
Age	41.6±13.9	42.2±10.4	41.9±12.1	0.83
Very young (<30 years)	6 (19.4)	5 (14.7)	11 (16.9)	0.80
Young (3-45 years)	10 (42.3)	14 (41.2)	24 (36.9)
Middle age (45-65 years)	13 (41.9)	14 (41.2)	27 (41.5)
Elderly (>65 years)	2 (6.5)	1 (2.9)	3 (4.6)
Tribe
Hausa	23 (74.2)	19 (55.9)	42 (64.6)	0.49
Yoruba	3 (9.7)	6 (17.6)	9 (13.8)
Igbo	2 (6.5)	4 (11.8)	6 (9.2)
Others	3 (9.7)	5 (14.7)	8 (12.3)
Religion
Christianity	20 (64.5)	24 (70.6)	44 (67.7)	0.60
Islam	11 (25.5)	10 (29.4)	21 (32.3)
Fruits/vegetables daily diet
Yes	16 (51.6)	21 (61.8)	37 (56.9)	0.41
No	15 (48.4)	13 (38.2)	28 (43.1)
Family history of hypertension
Yes	4 (12.9)	10 (29.4)	14 (21.5)	0.11
No	27 (87.1)	24 (70.6)	51 (78.5)

Nonsignificant at P>0.05. Difference between the two groups by Chi-square

Clinical and laboratory parameters of the normal healthy controls

Table 2 presents data on the clinical and laboratory parameters of the study population. The female subjects trended towards higher BMI than males though with no statistically (P = 0.88) significant difference. There was a significant (P = 0.001) difference in height between the two groups with males being taller than females. There was no statistically significant, (P = 0.29 and P = 0.88) difference in the SBP and DBP of males and females, respectively. The mean plasma homocysteine was 10.8 ± 2.7 μmol/L, and there was no statistically significant (P = 0.87) difference in homocysteine levels between males and females. The mean plasma folate level was 116.7 ± 44.0 ng/mL with no statistically significant (P = 0.37) difference between that of males and females. There was a significant (P < 0.001) difference in the PCV between males and females with higher values in males. The males had higher creatinine levels than females with significant difference (P < 0.001). The mean GFR was 107.9 ± 12.9 ml/min/1.73 m2 which was within normal range and did not differ statistically (P = 0.82) significantly between males and females [Table 2].

Table 2

Clinical and laboratory parameters of the normal healthy controls

Variables	Males (n=31), n (%)	Females (n=34), n (%)	Total (n=65)	P
Mean weight (kg)	70.8±11.3	68.7±11.3	69.7±12.9	0.50
BMI (kg/m2)	25.5±3.1	25.7±4.7	25.6±4.0	0.88
Underweight (<18 kg/m2)	1 (3.2)	2 (5.9)
Normal (18-24.9 kg/m2)	11 (35.5)	12 (35.3)		0.54
Overweight (25-29.9 kg/m2)	16 (51.6)	13 (38.2)
Obese (>30 kg/m2)	3 (9.7)	7 (20.6)
Mean height (m)	1.7±0.09	1.6±0.07	1.6±0.09	0.001*
SBP (mmHg)	121.9±9.6	119.2±10.7	120.5±10.1	0.29
DBP (mmHg)	77.1±7.4	76.9±8.4	77.0±7.8	0.88
Plasma homocysteine (µmol/L)	10.7±2.6	10.8±2.8	10.8±2.7	0.87
Plasma folate (ng/mL)	111.5±44.9	121.4±43.3	116.7±44.0	0.37
PCV (%)	44.9±3.8	38.2±2.9	41.4±4.7	<0.001†
FBG (mmol/L)	5.6±1.0	5.5±0.9	5.6±1.0	0.72
GFR (ml/min/1.73 m2)	107.5±14.0	108.2±12.1	107.9±12.9	0.82
Urea (mmol/L)	5.1±1.6	5.09±1.8	5.1±1.7	0.95
Creatinine (µmol/L)	100.1±11.9	65.0±14.3	81.7±22.0	<0.001†

Difference between two groups by Independent Student’s t-test. *Level of significance is at P<0.01, †Level of significance at P<0.001. BMI=Body mass index, SBP=Systolic blood pressure, DBP=Diastolic blood pressure, FBG=Fasting blood glucose, PCV=Packed cell volume, GFR=Glomerular filtration rate

Classification of homocysteine and sex distribution between normal healthy controls using different partition limits

Table 3 presents data on the classification of plasma homocysteinemia into normal (<10 μmol/L) and high (>10 μmol/L) as well as the sex distribution of homocysteine between normal healthy controls. There were more, 33 (51%) of the normal healthy controls who had hyperhomocysteinemia by the current definition consisting of 16 (48.5%) males and 17 (51.5%) females with no statistically significant (P = 0.90) difference. The Odds ratio of females having hyperhomocysteinemia than normal homocysteine levels was 1.03 (95% CI, 0.65–1.64) while that of males was 0.97 (95% CI, 0.58–1.61) [Table 3].

Table 3

Classification of homocysteine and sex distribution among healthy controls using different partition limits

Plasmahomocysteine	Males (n=31), n (%)	OR	95% CI, lower-upper	Females (n=34), n (%)	OR	95% CI, lower-upper	Total (n=65), n (%)	Combined OR	95% CI, lower-upper	P
>10.0 µmol/L (high levels)	16 (48.5)	0.97	0.58-1.61	17 (51.5)	1.03	0.65-1.64	33 (51.0)	0.94	0.35-2.48	0.90
>15.0 µmol/L (high levels)	2 (33.3)	1.48	0.46-4.71	4 (66.7)	0.76	0.41-1.42	6 (9.2)	1.90	0.33-11.38	0.46

OR and 95% CI. Non-significance at P>0.05. n=Number of individuals, OR=Odds ratio, CI=Confidence interval

Using the previously recommended partition limit for homocysteine at 15 μmol/L [Table 3], there were 6 (9.2%) of the patients with hyperhomocysteinemia with more females 4 (66.7%) than males 2 (33.3%). The odds ratio of males having hyperhomocysteinemia was 1.48 (95% CI, 0.46–4.71) which appeared greater than the Odds ratio of females, 0.76 (95% CI, 0.41–1.42) over normohomocysteinemia though this was not statistically significant (P = 0.46). The odds of both having hyperhomocysteinemia was 1.9 (95% C, 0.33–11.38) [Table 3].

Pearson correlation between plasma homocysteine, plasma folate, and blood pressure levels in normal healthy controls

Table 4 presents the correlation between plasma homocysteine, plasma folate and BPs both systolic and diastolic. There was no statistically significant (P = 0.08, r = 0.2) correlation of homocysteine with plasma folate using Pearson's correlation. Homocysteine also showed no significant (P = 0.55 and P = 0.74) correlation with SBP and DBP respectively in the normal healthy controls.

Table 4

Correlation between plasma homocysteine, plasma folate and blood pressure levels in normal healthy controls (n=65)

Variables	Plasma homocysteine
	r	P
Plasma folate	0.20	0.08
SBP	−0.08	0.55
DBP	−0.04	0.74

Nonsignificance at P>0.05. r=Pearsons correlation coefficient, SBP=Systolic blood pressure, DBP=Diastolic blood pressure

Relationship between plasma homocysteine, plasma folate, and blood pressure levels in normal healthy controls

Table 5 presents data on the relationship between plasma homocysteine, plasma folate, and BPs using the multiple Binary Logistic Regression analysis. Plasma homocysteine showed a significant (P = 0.01) positive relationship with plasma folate with tautology, Odds ratio (OR): 1.02 (95% CI, 1.00–1.03) in the unadjusted model. It, however, showed no significant relationship with plasma folate when adjusted for age, sex, BMI, SBP/DBP, family history of hypertension, vegetables and fruits in daily diet, FBG, urea, creatinine, GFR, weight, height, and PCV.

Table 5

Relationship between homocysteine, plasma folate, and blood pressure levels in normal healthy controls

Variable	Homocysteine levels
	OR	95% CI	P
Unadjusted model
Plasma folate	1.02	1.00-1.03	0.01*
SBP	1.72	0.44-6.70	0.38
DBP	1.08	1.03-1.13	0.43
Adjusted model
Plasma folate†	1.02	0.99-1.06	0.12
SBPǂ	2.60	0.6-11.30	0.20
DBPǂ	2.60	0.36-18.62	0.34

Multiple Binary Logistic Regression analysis. *Level of significance at P<0.05, †Adjusted for age, sex, BMI, GFR, urea, creatinine, weight, height, SBP, DBP, family history of hypertension, vegetables/fruits in daily diet, plasma folate, PCV, FBG, ǂAdjusted for above parameters except SBP and DBP. OR=Odds ratio, CI=Confidence interval, GFR=Glomerular filtration rate, PCV=Packed cell volume, FBG=Fasting blood glucose, BMI=Body mass index, SBP=Systolic blood pressure, DBP=Diastolic blood pressure

Homocysteine showed no significant (P = 0.38 and P = 0.43) relationship with SBP and DBP respectively in the unadjusted model of the normal healthy controls [Table 5]. Following adjustment for age, sex, BMI, folate, family history of hypertension, vegetables and fruits in daily diet, PCV, FBG, weight, height, GFR, urea and creatinine, homocysteine also showed no significant (P = 0.2 and P = 0.34) relationship with blood pressures both systolic and diastolic, respectively [Table 5].

DISCUSSION

The mean plasma homocysteine level (10.8 ± 2.7 μmol/L) found in the present study was similar to previous reports:[41113] Ajuluchukwu et al. in South-Western Nigeria reported mean Hcy of 8.29 ± 2.4 μmol/L though lower than that of this study;[13] Akande et al. in North Central, Nigeria reported mean Hcy of 11.29 ± 3.6 μmol/L [4] while Alkali et al. documented mean Hcy of 13.1 ± 4.5 μmol/L in North East, Nigeria.[11] Glew et al. documented higher values from Abuja, Jos and Fulani tribe of Northern Nigeria.[29] Reports from sub-Saharan Africa was 8.4 ± 2.4 μmol/L in South African blacks [18] and that from US blacks was 8.3 ± 2.7 μmol/L [30] which was lower than that of this study. There are conflicting findings with regards to racial differences in mean homocysteine levels. Previous reports outside Nigeria showed higher mean homocysteine levels in whites compared to blacks.[183132] Yet another study in Asian Indians living in the US in comparison to Caucasians showed significantly (P < 0.0001) higher mean homocysteine levels (14 ± 6.5 μmol/L versus 8.7 ± 3.6 μmol/L) in the Asian Indians than the Caucasians.[33] The reason for these disparities could be due to variations in case definition, blood sampling, sample size, and methods used.[34]

There was a very high prevalence of hyperhomocysteinemia in this study, with 51% of normal healthy controls having high plasma homocysteine >10 μmol/L. Further application of the previous partition limit of 15 μmol/L still showed a higher prevalence of hyperhomocysteinemia (9.2%) in the general healthy Nigerians resident in the Northern part of the country compared to that of 5% reported previously locally.[413] Reference values for homocysteine are difficult to establish; Previously, over the past two decades, hyperhomocysteinemia was said to occur with Hcy level >15 μmol/L.[891218] The NHANES III study done in the US established that in a healthy population, higher homocysteine values in the presence of adequate plasma vitamin concentrations and absence of renal insufficiency can be regarded as hyperhomocysteinemia.[35] Based on these criteria, hyperhomocysteinemia was defined for values greater than 11.4 μmol/L in males and 10.4 μmol/L in females.[3235] Malinow et al. found that the risk for carotid artery wall thickening increased significantly with plasma homocysteine concentrations >10.5 μmol/L,[36] while Selhub et al. also found that the risk of arterial stenosis was elevated in subjects with homocysteine concentrations >11.4 μmol/L.[37] Therefore, a higher cutoff point to define hyperhomocysteinemia was regarded as being too high, as homocysteine appeared to be atherogenic at lower plasma concentrations.[38] Hence, some experts suggested that normal tHcy levels should be <10 μmol/L,[2122] while some other expert opinion suggested that with such a cutoff, 30%–50% of the general population would be defined as “hyperhomocysteinaemic;” with higher proportions among CVD patients and the elderly.[23]

The very high prevalence of hyperhomocysteinemia (>10 μmol/L) in this study is similar to some previous report.[32] However, it is contrary to reports from the US and European countries which documented 5%–10% of the general population,[2038] as well as some Nigerian study which documented 5% prevalence.[413] The reason for the disparity may be attributed to the different cut-points used in the different studies with higher partition limits in the Nigerian study.[413] However, with the previous higher cut-point of hyperhomocysteinemia (>15 μmol/L), the prevalence rate fell within the global prevalence of 5%–10% of the general population.[8912181920]

Other reasons for the higher levels when compared to studies done in Europe and USA based on the current recommended definition may be due to variations in plasma levels of folate, vitamin B12 and B6 in each country's population.[32] Plasma folate deficiency was not common in this study population similar to some other studies.[3234394041] The mean plasma folate concentration was 116.7 ± 44 ng/mL with range from 36 to 278 ng/mL. Using the standard reference range for the US of 2–20 ng/mL or 4.5–45.3 nmol/L,[42] there were no subjects with low folate levels in this study. This contradicts report of folate deficiency as a common cause of hyperhomocysteinemia in the US and European countries (two-third of population),[3843] as well as 37% folate deficiency in North Chinese population and 31%–40% folate deficiency in urban and rural Costa Rica women.[32]

The high plasma folate may be attributed to high intake of folic acid from food supplements and type of diet in this study population. A larger proportion of the subjects (56.7%) had fruits and vegetables in their daily diet, which could have resulted to the appearance of unmetabolized folic acid in plasma as reported from previous studies.[394041] Furthermore, the inhabitants of the Northern part of the Nigeria consume diets which consist of grains, beans (”pete wake”), peanuts in form of “kuli kuli” and milk in form of “fura da nono” as complementary protein which are rich sources of vitamins and folate.[44] The Hausa's which constituted majority (64.6%) of the sample population of this study also eat fresh vegetables such as spinach, carrots, lettuce, garden egg (”yalo”) and pumpkin.[44] They also add to their soups several vegetables like okra called “miyan kubewa;” green leafy vegetables of various types referred to as “miyan tauhe,” “miyan kuka,” and “miyan zogale” as well as bitter leaf called “miyan shuwaka” in Hausa language. These vegetables are cultivated and produced en-mass as farm products from their local villages, neighboring towns and States such as Kudan, Hunkuyi, Zaria; Dan Mahawayi village, Zaria; Sauyoyi Bijimi, Biye, Zaria; Ikara Local Government Area, Kaduna; Gadan Gaya, Kaduna; Danja, Katsina and Kwanan Gafa, Kano. Therefore these vegetables are readily available and affordable for consumption of the people and are largely transported to the Southern parts of the country for commercial purposes.[44]

Other B-vitamins such as vitamin B12 and B6 were not assayed in this study hence further investigation regarding their role in homocysteine metabolism in this population need to be determined in subsequent studies. Furthermore, polymorphism(s) of the enzymes involved in homocysteine metabolism may explain why certain individuals have hyperhomocysteinemia despite a normal vitamin status;[38] however, this was not determined in this study hence cannot be accounted for. The renal function of the subjects were within normal and had no significant (P = 0.86) difference between males and females as renal dysfunction has been shown to be associated with hyperhomocysteinemia.[2]

Furthermore, the high prevalence of hyperhomocysteinemia in the normal healthy Nigerians may appear to be a risk factor for cardiovascular disease;[4101113] however, this cannot be inferred from this study as Hcy showed no significant (P > 0.05) relationship with blood pressure in both unadjusted and adjusted Binary Logistic models. With westernization and epidemiologic transition in Africa especially in Nigeria being the giant of Africa, it was expected that there will be a rise in incidence of myocardial infarction in sub-Saharan Africa, but there is rather a lower incidence of coronary artery disease observed in black people living in Africa.[38] The aftermath of the unique efficiency of methionine and homocysteine metabolism in blacks may partly explain why blacks are resistant to coronary heart disease despite high homocysteine levels.[38]

On a further note, homocysteine showed no significant correlation with plasma folate using Pearson's correlation analysis. However, in the unadjusted model of the multiple Binary Logistic Regression analysis, homocysteine showed a significant (P = 0.01) positive relationship with plasma folate. The odd of folate being related to hyperhomocysteinemia was OR, 1.02 (92% CI, 1.00–1.03) in the combined male and female model. However, there was no such relationship in the adjusted model of confounding variables. Studies have shown an inverse relationship between homocysteine and plasma folate which was attributed to low dietary intake of folic acid.[12] Some other study done in elderly showed high homocysteine and high folate in presence of vitamin B12 deficiency.[333745] The lack of inverse relationship in this study further buttresses the lack of folate deficiency as a cause of hyperhomocysteinemia in this study. Hence, other factors should be evaluated as the cause of hyperhomocysteinemia.

There was also no significant (P > 0.05) relationship of homocysteine with SBP and DBP by Pearson's correlation and multiple binary logistic regression. Other investigators have detected a positive relationship between Hcy and BP with a significant correlation with high blood pressure in previous studies.[433] The Hordaland homocysteine study carried out on 12,000 Scandinavians showed a weakly positive correlation of Hcy with both SBP and DBP, which was stronger in the elderly.[8] The present study was however done on normal healthy controls with mean age in the 4th decade (41.9 ± 12.1 mmHg) who had normal mean systolic (120.5 ± 10.1 mmHg) and diastolic blood pressures (77.0 ± 7.8 mmHg) which did not differ significantly (P = 0.29 and P = 0.88) between the male and female individuals, respectively. There were no hypertensive individuals in this study hence may explain the lack of correlation of homocysteine with blood pressure.

CONCLUSION

This study has shown high mean plasma homocysteine levels and high prevalence of hyperhomocysteinemia in normal healthy Nigerians resident in Zaria. The high homocysteine levels could not be accounted for by folate deficiency as there were high mean plasma folate concentrations. The study showed a positive relationship of homocysteine with folate in the unadjusted model and no relationship when adjusted for confounders. Hyperhomocysteinemia may not be a risk factor for cardiovascular disease in the general Nigerian population despite high levels as it showed no significant relationship with blood pressure.

Recommendation/Limitation

The high prevalence of hyperhomocysteinemia (Hcy >10 μmol/L) detected early in the apparently normal healthy sample population of Nigerians resident in Zaria may appear to signify a high risk for development of CVD however further studies relating homocysteine with echocardiographic structure and function should be carried out in normal healthy Nigerians risk (currently concluded) as well as carotid intimal thickening, to determine if this high level translates to cardiovascular risk.

Proactively, it is recommended that the government should institute public intervention policies at all tiers of government with aim to educate the Nigerian Africans on the importance of lifestyle changes such as exercise, avoidance of alcohol/smoking, intake of high fibre, low salt and low cholesterol-containing foods, rich in fruits and vegetables, as recommended by the World Health Organization. Folate supplementation is not recommended for the general population of Nigerians living in Zaria except for pregnant women and sickle cell disease patients as recommended by the National Committee for Non-communicable Disease Survey, Nigeria, as plasma folate levels were high in this study.[24]

Larger population and longitudinal-based studies should be carried out in Nigeria across all geopolitical zones to determine whether there is a similar high prevalence of hyperhomocysteinemia associated with high folate levels, as this study is limited due to its cross-sectional nature. The further limitation might be the sample size however several similar studies with valid conclusions locally and internationally have used similar or smaller sample sizes.[141113203034] Other B-vitamins should be assayed in subsequent studies in Nigeria (presently on-going) and genetic studies should be carried out (presently on-going) to determine whether there is any genetic mutation of the enzymes involved in the metabolism of homocysteine in Nigerian Africans.

Financial support and sponsorship

“This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.” MicroNova Pharmaceuticals Nigerian Limited and Emzor Pharmaceuticals Industries Limited provided drugs used in the primary ABU Homocysteine randomized placebo-controlled research study.

Conflicts of interest

There are no conflicts of interest.