TITLE: Serum bilirubin, Uric acid biomarkers and their correlation with

glomerular filtration rate among chronic kidney disease patients

RESEARCH QUESTIONS

Does serum bilirubin increase or decrease in CKD patients?

Is there the correlation between serum bilirubin and glomerular filtration rate among CKD
patients?

Is there the correlation between serum uric acid level and estimated glomerular filtration among
CKD patients?

Can serum bilirubin indicate disease progression in CKD diseased patients?

STUDY DESIGN

An Institutional based cross-sectional study design will be carried out among CKD patients on
follow-up at the renal clinic.

DATA COLLECTION AND MEASUREMENT PROCEDURE

Before the actual commencement of data collection, half-day training will be given to all data
collectors by the principal investigator and supervisors. Data will be collected using
questionnaires, anthropometric measurements, record reviews, and blood samples.

Data from the questionnaire, anthropometric measurements, and records are collected by two
BSc nurses working at the chronic follow-up clinic and principal investigator. Laboratory tests
analysis will be performed by laboratory technologists together with the principal investigator.
Furthermore, prevention approaches to COVID-19 transmission such as physical distancing
during an interview, wearing gloves and face masks, hand rubbing with sanitizer before and after
will be strictly followed in every procedure of data collection.
QUESTIONNAIRE

An interviewer-administered structured questionnaire adapted from the WHO Stepwise

instruments and related literature will be used to collect socio-demographic profiles, behavioral
related factors, and clinical data from eligible participants. To keep its consistency, the
questionnaire will be first prepared in English and translated back to both Amharic and Afaan
Oromo local languages then backtranslated to English by a professional expert.

ANTHROPOMETRIC MEASUREMENT

Physical measurements such as weight, height, and BMI will be taken using standardized
methods and adjusted equipment. Weight will be measured in kilograms with light cloths and no
wearing of shoes. Height is measured in centimeters using a vertical scale with no shoes wearing
and in the upright position. After body mass (BMI) calculation is done as weight divided by
height squared (kg/m2), the value of BMI is classified as following: BMI ≤ 18.5
Kg/m2underweight, BMI = 18.5-24.9 Kg/m2 normal weight, BMI = 25-29.9 Kg/m 2 overweight
and BMI ≥ 30 Kg/m2 obese.

BLOOD PRESSURE MEASURE

Blood pressure should be obtained in a standardized fashion and measured using an automated
sphygmomanometer after participants rest for at least five minutes or 30 minutes for those who
take hot drinks like coffee. The cuff should be placed on the flexed arm at the level of the heart
with the correct cuff size (the bladder of the cuff should encircle 80% of the arm). The cuff is
inflated until the radial pulse disappears and as the valve is gradually opened, cuff pressure
slowly decreases. When the cuff's pressure equals the arterial systolic pressure, blood begins to
flow past the cuff and an audible sound is heard using a stethoscope. Thus, the first knocking
sound (Korot koff) is the subject's systolic pressure and when the knocking sound disappears, it
is the diastolic pressure. At least two readings should be obtained five minutes apart. As per the
WHO recommendation, the mean of systolic and diastolic blood pressures is considered for
analysis. Accordingly, participants will be considered as prehypertensive as if systolic BP was
120–139 and diastolic BP 80–89mmHg, and hypertensive as if systolic BP ≥140mmHg and/or
diastolic BP ≥90 mmHg.
BLOOD SAMPLE COLLECTION

After tying up the patient’s upper arm using a tourniquet, 5ml of blood will be drawn from the
antecubital vein using a 5cc syringe by qualified professionals. The blood from each participant
will be collected to serum separator tube and following sample draw immediately investigator
will take a blood sample to the medical biochemistry course unit laboratory for separation of
plasma from the sample. Then after the separated plasma is taken back to the JMC laboratory for
biomarkers analysis by a laboratory technologist with the investigator.

URINE ANALYSIS (DIPSTICK)

Freshly voided urine will be collected in a clean and dry container. Then, the dry-reagent test
strip technique (dipstick) is used for the semi-quantitative estimation of protein in the urine. It is
based on the principle of protein error in which a specific chromogen immobilized into a pad
reacts with the protein present in the urine and changes the color of the strips from light yellow
to blue green. The change in color is visible to the naked eyes and can be compared with the
color chart for the estimation of the total protein concentration present in the urine sample. The
result was divided into five groups (trace, 1+, 2+, 3+ 4+). The result of 1+ or more is regarded as
proteinuria.

SERUM CREATININE

The alkaline picrate reacts with creatinine to form the orange-colored complex, which is read at
520 nm spectrophotometrically. The concentration is calculated using the following formula:

absorbance of thetest ×2
SCr(mg/dl) = , Normal range of SCr is between 0.3-1.2 mg/dl
absorbanceofstandard

SERUM BILIRUBIN MEASUREMENT

In vitro test for quantitative determination of bilirubin measurement in human serum or plasma
of adult and neonates on Cobas c 111 system.
Principles: Bilirubin reacts with diazotized sulphonic acid to form colored azobilirubin
compound. Unconjugated bilirubin coupled with sulphonic acid in the presence of caffeine
benzoate accelerator. The intensity of color formed is directly proportional to the amount of
bilirubin in the sample. Bilirubin + Diazotized sulphonic acid Colored azobilirubin
compound. Adult and children normal range is <1.3 mg/dL.

SERUM URIC ACID MEASUREMENT

Principle of the method: Serum uric acid will be measured by the uricase method. Uric acid is
oxidized to allantoin and hydrogen peroxide (H 2O2) by the uricase enzyme. Phenolic compound
and 4-amino-antipyrines react with hydrogen peroxide to form red-colored quinonimine dye
complex by the catalytic action of the peroxidase enzyme. The absorbance of light from red color
will be measured at 520nm wavelength and directly proportional to the uric acid concentration
found in the serum sample. The reaction steps will be illustrated as follows
(Uric acid + H2O) Uricase (Allantoin + H2O2), then after Allantoin and H2O2 are formed,

(H2O2 + phenolic compound + 4-amino-antipyrines) Peroxidase Red color quinonimine dye +

H2O
Procedure: About 20 microliter of serum sample will be mixed in a cuvette with about 120mµ
of R1 and 40mµ of R2 then incubated at 370C for five minutes, or at room temperature for 15
minutes. The absorbance of light from the red color will be measured at 520nm wavelength and
is proportional to the uric acid concentration found in the serum sample. The normal value of
serum uric acid is SUA<6mg/dl.

ESTIMATED GLOMERULAR FILTRATION RATE

Estimated GFR will be calculated by the 2009 CKD-EPI equation(101).

For female with SCr≤ 0.7 mg/dl: GFR = 166 x (SCr/0.7) −0.329 (0.993) age

For female with SCr> 0.7 mg/dl: GFR = 166 × (SCr /0.7) −1.209 x (0.993) age

For male with SCr≤ 0.9 mg/dl: GFR = 163 x (SCr / 0.9) −0.411x (0.993) age
For male with SCr> 0.9 mg/dl: GFR = 163 × (SCr / 0.9) −1.209 x (0.993) age.
DATA ENTRY AND ANALYSIS
After the collection of data, it will be properly coded and entered into Epi data version3.1
software and then exported to SPSS version 25 software for analysis. The normality of data will
be tested by Kolmogorov-Shapiro Willi’s test. All continuous and normally distributed data will
be expressed as Mean±SD, where continuous and non-normal data expressed by interquartile
range or median. Categorical data will be presented as proportion or percentage. The difference
in the mean among groups will be analyzed by 1-way ANOVA for normal continuous, Kruskal
Wallis H for non-normal continuous, and by Chi-square(X2) for categorical data respectively.
Pearson correlations will be calculated to see the correlation between total serum bilirubin and
serum uric acid with various factors. Univariable and multivariable linear regression models will
be carried out to test the significance of association. Variables associated at p-value<0.05 will be
considered as statistically significant

RESEARCCH COST

This research is planned to cost 2500USD