Limits of restrictions and one-sided limits Sometimes we work with the function defined on a subset.

23 CONTROLS

Controls were age and sex matched healthy individuals without kidney disease in the hospital environment that met the inclusion criteria. They were recruited from among doctors, nurses, pharmacists, clerks and medical students in the hospital in a case: control ratio of 1:1, which is 160 controls. The controls were apparently healthy individuals who had no symptoms suggestive of renal disease such as nocturia, pedal or facial swelling or frothiness of urine.

INCLUSION CRITERIA FOR CONTROL SUBJECTS

 Age and sex matched individuals alongside the CKD patients.

 Individuals with normal renal function

EXCLUSION CRITERIA FOR CONTROL SUBJECTS

 Proteinuric individuals

 Individuals on medications such as; beta blockers, alpha blockers anti-coagulants, steroids contraceptive pills, and thiazides.

 Individuals with features of or being managed for chronic obstructive liver disease, hypothyroidism or nephrotic syndrome

 Hypertensive or diabetic individuals

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sought. A researcher administered questionnaire (shown in appendix) was completed. Data on physical characteristics such as weight, height, hip and waist circumference were recorded. Weight before breakfast was measured in kilograms using hospital health scale ZT-120 with patients putting on light clothing without foot wears. The height was measured in meters using the same scale. The body mass index BMI defined in metres-squared as weight in kilogram divided by the square of patient’s height in meters was calculated. The waist circumference in centimetres before breakfast was measured in the horizontal plane at the level of the natural waist line taken to be at the umbilicus using a non-stretchable tape, the hip circumference in centimetres was also measured in the horizontal plane at the level of maximum diameter and waist hip ratio calculated. Blood pressure was measured using the Accosson® mercury sphygmomanometer.

All subjects were instructed to observe an overnight fast for 10-12 hours before the day of sample collection and a sample bottle was given for collection of early morning mid-stream urine. Method of collection was explained to avoid contamination, after the washing of hands, female patients were to spread the labia of the vagina and male patients withdraw the foreskin of the glans. The external genitalia was to be washed and wiped dry with a paper towel , and the midstream urine collected after discarding the first portion

Each subject had the following investigations done;

i. Serum creatinine from which GFR was estimated using the Cockcroft-Gault formula

ii. Haematocrit level

25 iii. Fasting blood glucose (FBG) iv. Fasting serum lipids

v. Renal USS vi. Urinalysis

Fasting serum lipid profile: estimation

In all cases, Blood was collected in plain bottles, each sample was subsequently centrifuged and the clear supernatant pipetted into plain containers stored at −20^oC until analysed.

Measurement of Total Cholesterol was done using Cholesterol Oxidase/Peroxidase Reaction

Principle - Free and esterified cholesterol in the sample originates by means of the coupled reactions below, a coloured complex measured by spectrophotometry.

Cholesterol ester +H2O cholesterol esterase Cholesterol + Fatty acid Cholesrerol +2H2O cholesterol oxidase Cholestenone + 2H2O + O2 2H2O2 + 4-aminoantipyrine phenol peroxidase Quinoneimine +4H2O

Cholesterol concentration in the sample was calculated by the following formula:

sample C standard C

standard A

sample

A  

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Measurement of HDL-cholesterol was done using the Phosphotungstate/

Mq – Chol. Oxidase/Peroxidase Reaction

Principle - Very low density lipoprotein (VLDL) and LDL-Cholesterol in the sample Precipitate with phosphotungstate and magnesium ions. The supernatant contains HDL which is spectrophotometrically measured by means of the coupled reaction of oxidase/ peroxidase stated in the case of total cholesterol.

HDL- Cholesterol concentration in the sample was calculated thus

sample C factor dilution

sample standard

C standard A

sample

A   

Measurement of Triglyceride was done using the Glycerol phosphate oxidase/ peroxidase reaction.

Principle - TG in the sample originates by means of the coupled reactions described below, a colored complex that can be measured spectrophotometrically.

TG + H2O lipase glycerol Glycerol +Fatty acids Glycerol + ATP glycerol kinase Glycerol-3-P +ADP

Glycerol -3-P + O2 G-3 oxidase Dihydroxyacetone –P +H2O2

2H2O2 + 4- aminoantipyrine chlorophenol peroxidase Quinoneimmine + 4H2O The concentration of TG is calculated thus:

sample C standard C

standard A

sample

A  

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LDL- cholesterol was calculated using the frieldwald formula. This is valid as long as triglyceride level is less than 400mg/dl, however no case or control had Tg level greater than 400mg/dl and so this formula was used.

FRIELDWALD FORMULA

LDL (mg/dl) = TC – (HDL + TG/5)

For every batch of samples analysed, control sera were run alongside and passed.

FBG was measured using an Accucheck glucometer and strips.

A group of age and sex matched control subjects with otherwise normal renal function were also assessed for the above listed parameters.

COLLECTION, PROCESSING AND STORAGE OF BIOLOGIC SAMPLES Each subject, control as well as patient had analysis carried out on a spot urine sample in a sterile container. The samples were tested using the 9 parameter multistix for proteinuria, hematuria

Ten millitres of venous blood was collected by venepuncture, 5ml into a lithium heparin sample bottle for serum creatinine estimation using the Jaffe’s reaction. Creatinine in serum or plasma reacts with alkaline picric acid solution to give a yellow color which was read calorimetrically at 560mm.⁶⁹

GFR was calculated using the Cockcroft Gault formula to enable the staging of CKD.

Cockcroft – Gault equation

 

SCr(mg/dl)

* 72

female if 0.85

* wt(kg)

* Age CrCl 140 



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Results were stratified according to the NKF – DOQ1 classification² into the different stages of CKD using GFR (mL/min/1.73 m²).

Stage 1 - Kidney damage with normal or ↑ GFR≥90 mL/min Stage 2 - Kidney damage with mild ↓ GFR60–89 mL/min Stage 3 - Moderate ↓ GFR30–59 mL/min mL/min

Stage 4 - Severe ↓ GFR15–29 mL/min

Stage 5 - Kidney failure GFR <15 mL/min or dialysis

All blood samples collected were stored at a temperature of less than 10^oC.

THE FOLLOWING DEFINITIONS AND CRITERIA WERE USED TO EVALUATE THE PHYSICAL AND BIOCHEMICAL PARAMETERS

Dyslipidemia was taken as triglyceride levels ˃ 150-400 mg/dL (1.7-4.5mmol/L), total cholesterol > 200mg/dL (>5.2mmol/L), LDL-cholesterol >

135mg/dL (>3.5mmol/L), HDL-cholesterol < 30mg/dL (<0.9 mmol/L) in men or <

40mg/dl (<1.0mmol/L) in women and a ratio of total cholesterol to HDL-cholesterol >5 as defined by WHO¹⁹.

Aetiology of CKD was based on the following:

 Diabetic nephropathy: A diagnosis of diabetes for more than 10 years, presence of large or normal sized kidneys on ultra sound scan in the absence of other possible aetiological factors, diabetic retinal changes

 Hypertensive nephropathy: a history of long standing hypertension, presence of features of long standing hypertension such as thick arterial wall, locomotor brachialis, loud aortic component of the second heart

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sound, stage III hypertensive retinal changes; shrunken kidneys on renal scan and absence of other possible aetiological factors.

 Obstructive nephropathy: presence of oliguria or anuria, dilated calyceal system, with normal to enlarged kidneys on ultrasound in the absence of other possible aetiological factors.

 HIVAN: HIV positive patient, nephrotic range proteinuria, little or no edema, normal to mildly elevated blood pressure.

 Sickle cell nephropathy: sickle cell disease patient, ± nephrotic range proteinuria, normal sized kidneys on ultra sound scan, absence of other possible aetiological factors such as long standing hypertension and diabetes.

 Autosomal dominant polycystic kidney disease (ADPKD): presence of ≥ 3 cysts unilateral or bilateral for patients aged 15 -29 years; ≥3 cysts unilateral or bilateral for patients aged 30 to 39 years, ≥2 cysts in each kidney for patients aged 40 to 59 years and ≥ 4 cysts in each kidney for patients aged 60 and above.

 Chronic glomerulonephritis: shrunken kidneys, active urinary sediments, absence of features of long standing hypertension, in the absence of other possible aetiological factors.

Cardiovascular disease was defined as previously documented medical history/diagnosis by a doctor of CCF, angina, or stroke.

Diabetes mellitus was defined as a previous documented medical history / diagnosis by a doctor or FBS of > 7.0mmol/L or RBS of > 11.1mmol/L in

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symptomatic patients or FBS of > 7.0mmol/L on two occasions in the absence of symptoms.

Obesity was defined as BMI ≥ 30kg/m².