Establishment of Reference Values for Serum Dehyroepiandrosterone-sulphate (DHEA-S) in healthy Adults in Yenagoa, Bayelsa State, Nigeria.

Ezeiruaku, F. C. & Baro, J. O.: Department of Medical Laboratory Science, Faculty of Basic Medical Sciences,
College of Health Sciences, Niger Delta University, Bayelsa State, Nigeria.
Okeke, C.U.:Department of Prosthetics and Orthotics, School of Health Technology, Federal University of Technology, Owerri, Imo State, Nigeria.

All correspondence to: Ezeiruaku, F. C., Department of Medical Laboratory Science, Faculty of Basic Medical Sciences, College of Health Sciences, Niger Delta University, Bayelsa State, Nigeria.

ABSTRACT:

Dehyroepiandrosterone (DHEA) and its sulphate (DHEA-S) are the most abundant sex steroid hormones in humans. It is present in the blood of both men and women and can be metabolized by the body into testosterone and androstenedione, or can be changed into the female hormone, oestrogen. Measurement of serum DHEA (S) is a useful marker of adrenal androgen synthesis and its level in the serum of both men and women has been helpful in the diagnosis and treatment of adrenal androgen dysfunction and infertility. Establishment of laboratory reference range is a useful tool for the interpretation of individuals result. This study was designed to establish the normal level of this androgen hormone (DHEA-S) in the serum of apparently health adult persons in Bayelsa state, Niger Delta region of Nigeria with respect to age and gender. This is with the aim to establish the DHEAS hormone ranges and if there is any significant difference in the level of the hormone with respect to age and gender. Enzymes linked immunosorbent Assay (ELISA) method was used to estimate the levels of the hormone. From the result, the level of the serum DHEA-S showed no significant difference (P<0.05) in the gender, but there was a significant decrease with increase in age. The graph slope from the study showed that DHEAS in men decline faster than that of the female with inclining age. The results indicate adrenal insufficiency with increase in age for both male and female.

Key Words: Dehydroepiandrosterone, Hormones, Steroids, Adrenal gland, Androgens, Aging, Reference.

INTRODUCTION
Dehyroepiandrosterone and its sulphate (DHEA-S) is the most important human C-19 steroid (Buttler et al., 2013) and it’s a precursor for androgens and estrogens (Longcope, 1996). DHEA is secreted by the
adrenal gland with small fraction from brain and production is at least partly controlled by
adrenocorticotropic hormone (ACTH). DHEAS is the most abundant steroid in humans with serum concentrations 250 – 500 times higher than DHEA, 100 – 500 times higher than testosterone and 1000 – 10,000 times higher than estradiol (Labine et al., 2004). DHEA-S is desulphated enzymatically to produce DHEA, which is in turn converted into various estrogenic and androgenic compounds. According to Hammer et al., (2005), because the two forms are easily inter converted, it is hard to discuss one without the other and it’s common to refer to them as DHEA (S). Both hormones are albumin bound, but binding of DHEA-S is much higher (Gao et al., 2005). In gonads and several other tissues, most notably skin, steroid sulfatases can convert DHEA-S back to DHEA, which can then be metabolised to stronger androgens and to estrogens (Maeda et al., 2002). However, only the non-sulphated (free) DHEA shows biological activity. Dehyroepiandrosterone (DHEA) and its sulphates are
synthesized and secreted primarily by the Zona veticularis of the adrenal cortex (Chadwick et al., 2005). The adrenal gland is the sole source of these steroids in women whereas in men, the testes secrete 5% of DHEAS and 10-25% of DHEA (Bird et al., 1984). DHEA and DHEAS serve as the precursors to approximately 50% of androgens in adult men, 75% of active estrogens in premenopausal women and 100% of active estrogens in post menopausal women (Longcope, 1996). Circulating levels of DHEA(S) decline after until about the age of five, and then start to rise a few years before sexual maturation begins. Levels peaks around the age of 20 to 30 years and then decline to only 20-30% of peak levels by the age of 70 to 80 years (Kahonen et al., 2000). Because of the coincidence between the natural decline of DHEA(S) levels with age and the onset of diseases associated with aging process, a great deal of research has been directed into the examination of the roles that both hormones play in the body, and to the possibility of supplementary levels to slow or reverse the aging process. These studies have severally reported widespread effect for the hormones, but a molecular mechanism of action has not yet been isolated for either DHEA or its sulphate form. It remains unclear therefore, whether either compound has a physiological role other than serving as a precursor molecule (Perrini et al., 2005). Recent views point out that this lack of understanding about the mode of action of the two hormones along with the problem in the design and analysis of certain studies; have led to discrepancies among the findings.
The test for DHEA(S) is performed widely these days to check the function of the two adrenal glands. This is done in women who show signs of having excess male hormones. Some of these signs are male body changes, excess hair growth, irregular periods and problems associated with becoming pregnant, low Libido and not having sex satisfaction (Perrini et al., 2005). Serum DHEA levels may be increased in the following conditions, polycystic ovary syndrome (PCOS), Androgen Secreting Adrenal Adenomas, Adrenal insufficiency and post Menopausal Vulvovaginal atrophy (Genges, 2004). DHEAS has appeared to be useful in the retardation of the aging process in humans. DHEA levels can become a
standard diagnostic tool in the prediction of disease, mortality and life span. Therefore DHEA levels are directly related to the probability of dying in humans. This has led to its widespread use in so many countries (Leowattana, 2004). Many methods apply in the laboratory measurement of DHEAS in this part of the world. These include competitive binding assays (Lewis et al., 1996), chemiluminescence immunoassays (Elmlinger et al., 2002), Radioimmunoassay (Remer et al., 1994), measurement of serum or plasma DHEAS using the enzyme immunoassay (Dhar et al., 1985) and the Chromatography systems (Nakajima et al., 1998). All the methods however have its drawbacks and in this study of DHEA-S, the Enzyme linked immunosorbent assay (ELISA) method was used because of its cost effectiveness and availability. It is a good idea to be a partner in the management of our hormonal health. The more we know about it, the better off we will be; one thing that can be of enormous assistance in understanding our hormonal treatment and hormonal drug administration is to simply know what the normal reference ranges are for the various hormones. It becomes necessary for every laboratory and regions with regard to race to establish its own normal reference ranges, utilizing a particular method of assay. This makes effective interpretation of patient’s results, cum management of various hormonal disorders.

Materials/methods
Study area:
The study was carried out in Bayelsa State, Niger Delta region of Nigeria. The state covered an area of about 8,150 sq meters and with a population of about 1.7 million people (Nigeria census, 2006) of which 43% are adults. Blood samples were collected from subjects and volunteers from the Niger Delta University environment, Southern Ijaw Local Government area, Yenagoa municipal area and its environs.
Study population:
A total of one thousand, five hundred (1500) subjects were used for this study. This was distributed into four age groups of 18-30 , 31-50 , 51-70 with 200 subjects each and >70 years with 150 subjects for both male and female. The sex and age of the subjects were obtained through the administered questionnaire. The individuals excluded from the study were subjects identified with different endocrine dysfunction (Stephen et al., 2008) and those taking DHEA drugs and vitamins or supplements that contain DHEA/DHEAS for certain reasons. The apparently healthy subjects were selected for the study Sample collection and preparation: About 5.0ml of blood were collected from each subject using the standard venepuncture technique. The samples were discharged into a clot activator tube and centrifuged for 10minutes at 3,000 rpm. The serum collected was stored frozen at -200c and analysis was done within 10 days of collection. The subject’s age and gender were noted after collection with history of having endocrine disorder and or on vitamins/supplements containing DHEA(S). The essence and details of the study were explained to the subjects and informed consent was obtained from them before sample collection. Ethical approval was gotten from the ethical committee of the University for this Study.
Sampling method:
To achieve this reference range establishment, a nonrandom sampling of the different subjects from the
population was used with respect to age and gender (Lumsden and Mullen, 1978; Alex et al., 2010; Mikael, 2014). The questionnaire administered took into consideration the subjects with various endocrine dysfunctions as established. The study was carried out between April, 2012 and May, 2015.
Method of assay:
The study was carried out with a method that has been established to give a precise range of results in hormonal assay (Amballi et al., 2007). The enzyme linked immunosorbent assay (ELISA) method was used for the Dehydroepiandrosterone sulphate hormone estimation (Dhar et al., 1985). The Elisa test is used on the principle of solid phase enzyme linked immunosorbent assay, where the antibody to be measured is incubated with specific antigen coupled to a solid phase (Uotila et al., 1951., Peter et al., 2001). The product kit was acquired from Micro Well Laboratories, USA.
The components of the ELISA were specifically designed to analyse the DHEAS hormone. It applies to in-vitro quantitative determination of the hormone concentration in serum (Georges, 2014). The DHEA-S measurement was preferred because of its stability in the blood and a better marker in clinical practise because it has a longer half life with no circadian variety.
Statistical analysis:
In order to establish the range of reference values for this study, the Gaussian techniques was employed. The data are expressed as mean + standard deviation and normal ranges. Correlation between the groups studied was tested using the tool of ANOVA. The standard t-test; pair two samples for mean was used. 95% confidence level (P<0.05) were used and considered significant.

RESULTS

The results of the Serum DHEA-S levels in the adults male and female in Yenagoa, Niger Delta region of Nigeria is as Presented in the tables and graphs below.

Table 1: The Mean+ S.D serum DHEA-S in the adult female and their ranges with respect to age groups.

Table 2: The Mean+ S.D serum DHEA-S in the adult male and their ranges with respect to age groups.

DISCUSSION

Dehydroepiandrosterone sulphate (DHEAS) has been described as a fountain of youth in that it appears to be the hormone that retards the aging process in humans (Leowattana, 2004; Chadwick et al., 2005). This has lead to uncontrolled use of the DHEA-S all over the world, particularly by the aged. This study was carried out to establish the normal ranges in adult men and women in Yenagoa, Bayelsa State, Niger Delta Region of Nigeria because of its clinical implication in fertility treatment. Most often the test is performed to check the function of the two adrenal glands which is the major sources of androgens in humans. The interpretation of medical laboratory data is an example of decision making by comparison. Therefore, reference values are needed for all tests performed in the clinical laboratory, not only from healthy individuals but from patients with relevant diseases. This is because a patient’s laboratory result simply is not medically useful if appropriate data for comparison are lacking. Data collected during the medical interview, clinical examination and supplementary investigations according to Mikael, 2014,
must be interpreted by comparison with reference data, which might be based on reasoning using advanced knowledge of normal and pathologic anatomy, physiology, biochemistry and other areas of medical sciences. The results obtained from the study (tables 1, 2,) showed that the serum DHEA-S levels declines with inclining age. The statistical test of significance at (P<0.05), 95% confidence level showed no significance difference between the age bracket 18-30 and 31-50. There is a significant decrease with this two age groups and 51-70 and greater than 70 years. The serum DHEA-S values for men were statistically not significantly different from that obtained for women at this confidence level when the age groups were matched. The graph slope from the study showed that DHEAS in men decreases faster than that of the female. This study is in comparison with the work done by Nafziger et al., 1998; Kahonen et al., 2000 and Perrini et al., 2005 that showed the declining levels if DHEA-S with age. The decrease in DHEA-S with age might be as a result of decrease muscle mass, bone mass and adrenal insufficiency (Elmlinger et al., 2003). A gradual decline throughout life result from “adrenopause” contributing few percentage (Nakamoto, 2006) (Etieme-Emile et al., 2000). The skin which is also known to produce DHEA-S becomes elastic (skin elasticity) as one age leading to diminished DHEAS levels (Nafziger et al., 1998).

CONCLUSION

Thanks to the goodwill of the subjects and volunteers used for this reference study. The subjects in this study eager to collaborate in this effort of public health importance, were in such good condition, particularly the subjects over 70 years by giving out their blood sample. The study established the ranges of DHEA-S in the serum of male and female adults in Yenagoa, Bayelsa State, Nigeria with the levels declining as the subject’s ages and differences not statistically significant in the gender, but differs
significantly in the age groups. The results indicate adrenal insufficiency with increase in age for both male and female.

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