Study site
This study was carried out at the National Diabetes Management and Research Centre (NDMRC), Accra, Ghana. It serves as the Diabetes Clinic of the Korle-Bu Teaching Hospital (KBTH). The KBTH is a 2000-bed tertiary teaching hospital with about 200 admissions per day. The hospital covers all medical specialties and provides referral healthcare services to an estimated population of 24 million Ghanaians. The NDMRC is designed to manage diabetic conditions of patients in Ghana. The Centre also supports and enhances the national effort in diabetes, its complications, and related endocrine and metabolic diseases through interdisciplinary research.
Study population
The study population comprised Diabetes Mellitus patients aged 30 to 80 years who attended the NDMRC and consented to participate in the study. A volunteer group without Diabetes Mellitus and of the same age-brackets were enrolled as controls.
Ethical approval
Ethical approval was obtained from the Ethics and Protocol Review Committee of the School of Allied Health Sciences, University of Ghana prior to the enrolment of participants.
Study design
This was an analytical cross-sectional study with age-matched comparison controls. The study was conducted from February through May 2014. Two sampling approaches were used. First, an active case-finding network was organized with visits to the NDMRC at Korle-Bu. Physician-identified patients with known type-1 or type-2 diabetes attending the center for review and management of various diabetic conditions were referred to the research team for screening and inclusion in the study after informed consent was obtained. Diabetic patients enrolled into the study were categorized as cases. Prior to enrollment, the nature, purpose and potential risks of the study were explained to all subjects. Patients’ case history was ascertained through physician-assisted review of medical folders. Cases were recruited based on the following screening criteria: 30–80 years of age, pre-prandial capillary plasma glucose levels of 3.9 – 7.2 mmol/l and mean plasma glucose of 8.6 mmol/l correlating with achievement of target glycated haemoglobin (HbA1c) of <7 % as per the [9] criteria. Overall 66 patients were enrolled to participate in the study. Forty-four other subjects without diabetes were prospectively recruited as controls within the same study period. The control group were aged-matched volunteers accompanying cases to the diabetic center. The same biochemical criteria were examined in the controls subjects. Controls had fasting plasma glucose <5.6 mmol/L or two-hour glucose during OGTT < 7.8 mmol/L. Second, venous blood samples were collected from diabetics and non-diabetic controls and analyzed for various haematological changes.
Exclusion criteria
Pregnant and lactating women were excluded from the study. Patients with medical conditions such as infections, cerebrovascular diseases, ischaemic heart disease and malignancies excluded from the study. Other exclusion criteria were smoking and alcoholism.
Study conduct
Haematological indices and fasting glucose analysis
After an overnight fast, 5 ml of venous blood was collected from each participant. Three milliliters of which was put into an EDTA tube for haematological indices and SOD activity analysis and 2 ml was put into a fluoride tube for fasting plasma glucose (FPG) analysis using an automated analyzer (A25 Biosytems, Canada). The haematological indices were determined by performing a full blood count (FBC) using an automated haematology analyzer (Sysmex KX-21 N, Germany). The following haematological indices were assessed: Haemoglobin, Haematocrit, Mean cell volume, white cell count (including lymphocyte and neutrophil count); platelet count. The rest of the blood sample was then centrifuged at 4000 rpm for 10 min to separate plasma from the red blood cells. The stored red blood cells were used for further work involving SOD activity determination.
Measurement of sod activity as an index of oxidative stress status
Briefly, the SOD activity levels were determined by using SOD Assay kit (Cayman Chemicals, Michigan, USA). Whole blood obtained by venipuncture from the participants was centrifuged at 4000 rpm for 10mins, and plasma carefully separated. Two hundred and fifty (250) microliters of erythrocytes obtained from the packed cells was lysed with thousand (1,000) microliters of ice-cold deionized distilled water and centrifuged at 6,000 rpm for 20 min. Thousand microliters (1000 μl) of the supernatant (erythrocyte lysate) was collected for assay and stored on ice. The supernatant fluid was then diluted by a factor of 100 with sample buffer, and 10 μl of the diluted solution used to assay Cu/ZnSOD (Copper/Zinc Superoxide dismutase) activity as described by the kit manufacturer.
Data analysis
Data obtained was put into a database and analyzed statistically using Statistical Package for Social Sciences (SPSS) 20.0 software for windows. Summary statistics was presented using the descriptive statistics of mean and standard deviation for numerical variables and proportions for categorical variables. Independent t-test or analysis of variance (ANOVA) and Pearson’s correlation analysis were used to determine the mean differences and the correlation between oxidative stress and haematological parameters respectively. A p-value less than 0.05 was interpreted as significant.