Intermediary Changes

The sample size was calculated using the Fishers formula.

SAMPLE SIZE CALCULATION

The minimum sample size was calculated as follows:

n = Z²pq d² Where:

n = estimated sample size

Z = 1.96 (at 95% confidence limit) p = estimated disease prevalence d = desired precision limit of 10% (0.1) q = 1 – p

The prevalence of malnutrition in chronic kidney disease in Nigeria is put at 43.2% ¹¹ Therefore, p will be 0.43.

n = 1.96² x 0.43 x 0.57 = 94.15 (0.1)²

The minimum sample size of subjects was calculated as 94 patients. For the purpose of this study the sample size was 110 patients. 56 age and gender matched subjects were used as controls.

SAMPLE TECHNIQUE AND PATIENTS CLASSIFICATION

PATIENT SELECTION

Patients with CKD in stages 3 to 4 attending the nephrology clinic were consecutively enrolled and patients who satisfied the inclusion criteria were recruited.

INCLUSION CRITERIA

CASES:

 Patients attending the nephrology clinic with CKD stage 3 and 4 (eGFR 15-59ml/min).

 Patients aged between 18 and 75 years.

 Patients who consented to the study CONTROLS

Fifty six (56) apparently healthy age and sex matched subjects who volunteered including members of staff and clients visiting the hospital for routine medical tests with no evidence of CKD were recruited as controls.

Inclusion criteria for controls 1. Age between 18 and 75 years 2. No history of chronic kidney disease 3. No history of hypertension

4. No history of diabetes

5. No obvious infection or inflammation

PATIENTS EXCLUSION CRITERIA

 Stage 5 CKD patients

 Patients with nephrotic syndrome

 Patients with evidence of liver disease

 Patients with evidence of congestive cardiac failure

 Patients on steroid therapy and NSAIDs

 Patients with recognizable infection or inflammatory diseases like arthritis

 Patients with a neurological deficit

PRE-STUDY COUNSELLING / ETHICAL ISSUE

Approval for this study was obtained from the ethical research committee of the Lagos State University Teaching Hospital, Ikeja (LASUTH) (Appendix). Subjects were briefed about the purpose of the research, procedure and the potential benefits of the study. Informed written consent was obtained from the subjects.

PROCEDURE AND MEASUREMENTS

Each patient had 3 visits within the study period 1^st Visit

Patients attending the nephrology out-patient clinic in stages 3 and 4 CKD who met the inclusion criteria and agreed to participate in the study were recruited during the first visit.

CKD was confirmed by the presence of eGFR < 60ml/min on 2 separate occasions 3 months apart. The patients had the process and benefits of the study explained to them and written informed consent was obtained afterwards. The subjects were scheduled for the next visit within 1-2 weeks.

2^nd visit

Anthropometric measurements were taken at this visit. The weight was measured using a Seca 761 Class IIII floor scale (England) with subjects wearing light clothes, no headgear or footwear. Measurements were taken to the nearest 0.5kg. The height was measured with the subject standing erect with his/her back against a standing meter rule such that the occiput, back and the heels were in contact with the meter rule. Measurements were taken to the nearest 0.05 meters. Body mass index was calculated by the formula:

BMI = weight (kg) Height² (m²)

Blood pressure was taken after 10 minutes of the patient being seated using an Accusson mercury sphygmomanometer. Patients were seated comfortably, feet on the ground and the arm to be measured at the level of the heart and free from any constrictive clothing.

Sphygmomanometers with the appropriate cuff size were used, 2 blood pressure

measurements were taken 5 minutes apart and the mean taken as the blood pressure of the subjects. The onset of the 1^st Korotkoff sound was used to determine the systolic blood pressure while its disappearance determined the diastolic blood pressure. A patient was hypertensive if the blood pressure was greater than 140/90mmHg, had a history of hypertension or the patient was on anti-hypertensive medications.

Structured interviewer administered questionnaire, including the subjective global assessment (SGA) was administered by the investigator to determine the history of CKD, history of medication use, the presence of co-morbidities, history of cigarette smoking etc (Appendix 1). SGA is comprised of five components of the history and four signs on physical

examination. The components of the history include: weight loss, change in dietary intake, gastrointestinal symptoms persisting for more than 2 weeks, functional capacity and the disease and its relation to nutritional requirements. Physical examination to look for signs of loss of subcutaneous fat in the triceps and chest wall (mid-axillary line), muscle wasting in

the deltoids and quadriceps muscles, ankle oedema, sacral edema and ascites was done.¹⁴⁵ (Refer Appendix 2).

Ten millilitres of blood was collected from the antecubital fossa of the study subjects after an overnight fast of 8 to 10 hours. Two milliliters of blood was taken into potassium EDTA bottles for Full blood count and processed within 4 hours of collection. Eight millilitres was distributed for clinical chemistry; blood for fasting blood glucose was collected in fluoride oxalate bottles; serum lipids in plain bottles and liver function test in lithium heparin bottles.

Samples were immediately transported to the lab and centrifuged at 3500 rpm for 10 minutes.

The serum was separated and stored in the freezer at -20⁰C. Biochemical analysis was done in one batch at the end of the collection period.

3rd Visit

Results of the investigations were discussed with the patients. Malnourished patients were referred to the dietician and those with traditional cardiovascular risk factors were treated accordingly.

LABORATORY ANALYSIS

Serum creatinine was assayed by the modified Jaffes’s reaction using alkaline picrate. GFR was estimated using the abbreviated modification of diet in renal disease (MDRD) equation.

Conventional serum creatinine was converted to isotope dilution mass spectrometry (IDMS) -based creatinine to give corrected serum creatinine derived from the equation:

Conventional Serum creatinine (mg/dl) = IDMS Serum creatinine (mg/dl) x 1.065 + 0.067¹⁴⁶

Corrected serum creatinine, age at last birthday, gender and race were used to calculate the GFR.

GFR (ml/min/1.73m²) = 186 x Pcr (mg/dl)^-1.154 x Age^-0.203 x 0.742(if female) x 1.210(if black). ¹⁴⁴

Serum albumin was assayed by the bromo-cresol green method for quantitative determination of albumin. This results in the binding of albumin with bromo cresol green and the colour formed is proportional to the concentration of albumin which is measured photometrically between 580-630nm.

C-reactive protein was assayed by the turbidimetric immunoassay method. It measures the antigen-antibody reaction by the end-point method.

Fasting blood glucose was assayed by the Trinders glucose oxidase method; in which glucose in the sample is oxidized to gluconic acid and hydrogen peroxide in the presence of glucose oxidase.

Total cholesterol was assayed by a reagent using the Allain and Roeschlau methods. The resulting chromophore was quantitated at 505nm.

LDL cholesterol was assayed by a method based on the modified polyvinyl sulfonic acid (PVS) and polyethylene-glycol methyl ether (PEGME) coupled classic precipitation method.

LDL-c is released from the PVS/PEGME complex by a specific detergent and is quantified by the Trinder reaction.

HDL cholesterol was assayed by a method based on the modified polyvinyl sulfonic acid (PVS) and polyethylene-glycol methyl ether (PEGME) coupled classic precipitation method.

Reaction of HDL-c with the enzymes produces H2O2 which is detected by the Trinder reaction.

All clinical chemistry analysis was done using Erba XL 600 clinical chemistry analyser (2009, Germany).

Red cell Distribution Width: The samples collected were mixed for 10-15 minutes, after which the RDW was reported as a coefficient of variation of red blood cell volume using a haematology analyser, Mindray Auto-haematology analyzer BC-3000plus (2006-02 Germany).

MAIN OUTCOME MEASURES

1. MALNUTRITION: For the purpose of this study, malnutrition was defined as 2 out of 3 of:

a. Body mass index: < 18.5 kg/m² b. Serum albumin: < 30g/dl

c. Subjective global assessment: SGA stages B and C

2. INFLAMMATION: For the purpose of this study, inflammation was defined as both of:

a. CRP: levels above 10 mg/L.¹³⁵

b. Red cell distribution width: RDW- CV > 14.5%¹⁴⁷