Chapter Three……………….……………….……Materials and Methods
MATERIALS AND METHODS
3.1. MATERIALS
3.1.1. SUBJECTS OF THE STUDY
The patients enrolled in this study were taken from the out patient
department attending the internal medicine consultancy clinic and those
undergoing haemodialysis at the dialysis unit of Merjan teaching Hospital
at the Babylon Province. These patients were categorized for their age,
gender, weight, height, smoking, nutritional and health status. The study
was extended from June, 2010 to January, 2011.
The history of these patients include the following :
 Past medical history may reveal a disease which is known to be
associated with anemia (e.g rheumatoid arthritis or previous
surgery of the bowel).
 Family
history
and
ethnic
background
to
exclude
haemoglobinopathies and hereditary spherocytosis.
 A drug history (e.g. aspirin and anti-inflammatory drugs) or drugs
that may cause haemolysis or aplasia.
 A thorough gastrointestinal history is important, looking for
symptoms indicating blood loss.
 Menorrhagia is a common cause of anemia in females still
menstruating, so women should always be asked about their
periods.
The sample of this study consist of 100 female patients suffered
from iron deficiency anemia and is divided into two main groups. The
first group involved 50 patients diagnosed having IDA by clinical
laboratory test and have normal renal functions. Their age was
(mean ± standard deviation, 33 ± 8.9) and their BMI ranges (19.05-34.04
kg/m2).
30
Chapter Three……………….……………….……Materials and Methods
The second group also consists of 50 patients suffered from
chronic renal failure (CRF) and anemia. Their age was (M ± SD, 49 ±
11.4) and BMI ranges from (15.12-33.9 Kg/m2).
The first and second groups were further subdivided into 2 subgroups,
each of which consists of 25 patients. A daily oral dose of Vit. C equal to
500mg and 1000mg were given to each subgroup. The same above doses
of Vit. C were also administered to each 25 patients of the second group
(50 patients with chronic renal failure and suffered from IDA).
Anthropometric data for women included in the study are shown in
table (1). The study also involved a fifty control subjects.
Table (1) : Anthropometric data for women included in the study
Anthropometric
IDA patients with
IDA Patients
Control
data
normal renal
with CRF
Groups n= 50
function n = 50
n=50
Age ( years)
33± 8.9
49± 11.4
38.7 ± 8.2
34.04 ± 12.04
33.9 ± 10.12
30.3± 8.9
( means ± SD)
BMI ( Kg/ m2)
( Means ± SD)
3.1.2. EQUIPMENTS AND KITS
3.1.2.1. VITAMIN C SUPPLEMENTATION
The used vitamin C was synthesized as oral tablet containing (500
mg) manufactured by Julphar Pharmacticual company (Dubai United
Arab Emarates).
31
Chapter Three……………….……………….……Materials and Methods
3.1.2.2 HEIGHT AND WRIGHT MEASURMENT APPARATUS
The height of the patient is measured by using a tap measure. The
weight is measured by using the manual and weight scale apparatus.
Measurement of height and weight for each patient was done to calculate
their body mass index (BMI) according to the following equation :
BMI = Weight (Kg)/Height (m2) (Eknoyan & Garabed, 2008).
3.1.2.3. CHEMICHAL KITS
By using different enzyme techniques for determination the
concentrations of the following :
 Serum Urea (Urea-Kit S 180, Bio Merieux, France).
 Serum Creatinine (SYRBIO Diagnostic Reagents For Laboratories,
Syria).
 Serum Iron (Iron liquicolor Kit, Human, Germany)
 Serum Total Iron binding Capacty (REF, 1137010) Linear
Chemicals, Spain.
 Serum Ferritin (Accu-Bind, ELISA Microwells, Ferritin Product,
USA).
 Serum Erythropoietin (DRG, EPO (erythropoietin) ELISA. DRG
international Inc., USA.
 Serum vitamin C level kit.
3.1.2.4.AUTOMATIC BLOOD ANALYZER (HORIBA, GERMANY)
To obtain a full blood count, anticoagulated blood is processed
through automatic blood analyzer which use a variety of technologies
(particle-size, radiofrequency and laser instrumentation) to measure the
haematological parameters. These include the number of circulating
blood cells and platelets, the proportion of whole blood volume occupied
32
Chapter Three……………….……………….……Materials and Methods
by red cells (the haematocrit Hct) and the red cells indices which give
information about the size of red cells (mean corpuscle volume, MCV)
and the amount of haemoglobin present in the red cells (mean corpuscular
hemoglobin, MCH) and (mean corpuscular hemoglobin concentration,
MCHC, Craig et al., 2006).
3.2. BLOOD COLLECTION
Before Vit. C supplementation, blood sample was collected by
using 10 ml. syringe (MEDECO inject) from the antecubital vein at 9.00
AM in the morning. The second sample was collected after 2 weeks of
Vit. C administration and the third sample, after 4 weeks of Vit. C
treatment by patient.
From each blood sample collected 2.5 ml was taken into a test tube
mounted with Potassium-Ethylene Diamine Tetra Acetic acid (K2-EDTA)
that acts as an anticoagulant. About 50 microliter ( L) of this blood was
incorporated through automated blood analyzer to demonstrate the
following blood parameters :
 Red blood corpuscles Count-RBCs count (cells/mm3).
 Level of hemoglobin in blood (g/dl).
 Packed cell volume (PCV%).
 Mean corpuscle volume-(MCV) (femtoliter, fl).
 Mean corpuscular hemoglobin (MCH, Picogram).
 Mean corpuscle hemoglobin concentration (MCHC)(g/dl).
 Total leukocytes Count-TLC (cells/mm3).
The
remaining
blood
volume
was
collected
in
plain
noncoagulated test tube (AFCO-DISPO) put in a centrifuge for 5 minutes
with centrifugal force at a rate of 5000 RPM (round per minute) to get
better precipitation of blood cells and the supernatant serum was aspirated
33
Chapter Three……………….……………….……Materials and Methods
by a micropipette to be used for estimation of the following chemical
parameters :
3.2.1. DETERMINATION OF SERUM UREA :
Urea-kit enables end point enzymatic determination of urea
concentrations (Urease – modified Berthelot reaction) in human urine,
serum or plasma. Urease hydrolyzes urea by producing ammonium
(Fawcett & Scott, 1960).
(urea+H2O
Urease
2NH3+CO2).
In an alkaline medium, the ammonium ions react with salicylate
and
hypochlorite
to
form
a
green
colored
indophenols
(2,2 dicarboxylindophenol). The reaction is catalyzed by the sodium
nitroprusside :
NH4+salicylate+hypochlorite
indophenol .
The color intensity is proportional to urea concentration in the
sample (Patton & Crouch, 1977).
The range of expected value for urea concentration in adult was
(15-45 mg/dl) (2.5-7.5 mmol/l) (Metais et al., 1990).
3.2.2. DETERMINATIOAN OF SERUM CREATININE
Creatinine in alkaline picrate solution forms a color complex. The
kit for this estimation contain sodium hydroxide (1.6 mol/l), picric acid
(35 mmol/l) and a deproteinization agent (trichloroacetic acid TCA 10%).
The deproteinization procedure is done by mixing the serum sample with
(TCA 10%, 0.5 ml), centrifuge well at 2500 rpm for 10 minutes, the
supernatant is mixed with reagent mixture. The mixture let stands for 20
34
Chapter Three……………….……………….……Materials and Methods
minutes at room temperature, then the optical density of the sample and
standard against blank is read (Henry, 1974).
Concentration of creatinine :
× Standard Concentration O.D (optical density)
The reference values for serum creatinine are 0.6-1.36mg/dl for
men and 0.5-0.9 mg/dl for women (Walker, 2010).
3.2.3. SERUM IRON
The kit for determination of serum iron (Garcic, 1979) contain
chromazurol B and cetyltrimethylammonium-bromide (CTMA) to form a
colored ternary complex with maximum absorbance at 623 nanometer.
The intensity of the color produced is directly proportional to the
concentration of iron in the sample. About 50 l of the sample and
standard solution mixed with 1000 l of the reagent, incubate for 15
minutes at 25ْc. Measure the absorbance of the sample and the standard
against the reagent blank within 60 minutes (callahan, 1982).
S.iron = 100 ×
( mol/L).
The reference values for serum iron is 10.6-32 mol/l for male and
6.6-28 mol/L for female (Walker, 2010).
3.2.4. DETERMINATION OF SERUM TOTAL IRON BINDING
CAPACITY (TIBC)
Serum iron is bound to transferrin, but only about one third of the
iron binding sites are saturated with iron. The unsaturated iron-binding
capacity of transferrin (UIBC) denotes the available iron-binding sites of
serum. The amount of iron that serum transferrin can bind when
35
Chapter Three……………….……………….……Materials and Methods
completely saturated with an excess of Fe+3 is the total iron-binding
capacity (TIBC). The kit of TIBC contains 2 reagents, an iron solution
about (500
g/dl) Fe+3 (89.5
mol/l) and the other reagent consists of
magnesium carbonate powder.
The method measures the TIBC (Zak & Epstein, 1978) by first
saturating the transferrin with an excess of Fe+3. The remaining iron is
adsorbed with magnesium carbonate and once the binding process is
complete, the quelator is removed by centrifugation, and an assay for iron
content performed on the supernatant. From this measurement the TIBC
value is obtained. The unsaturated iron-binding capacity (UIBC) is
calculated by subtracting the TIBC from the serum iron value.
TIBC = g/dl supernat. ×3 (Dilution Factor)
UIBC = TIBC-Serum Iron.
Transferrin saturation(%) =
The reference value for TIBC in adults serum ranged 250-425
g/dl (45-76 mol/l) (Tietz, 1995).
3.2.5. DETERMINATION OF SERUM FERRITIN
Circulating ferritin levels have been used as a valuable tool in the
differential diagnosis of anemia due to iron deficiency and anemias due to
other chronic disorders and its level has been used to monitor, noninvasively the erosions of iron storage during pregnancy and in patients
undergoing haemodialysis (Anonymous, 1995).
The kit of ferritin has the following reagents :
 Ferritin
calibrators-1ml/vial,
human
serum
based
were
calibrated using a reference preparation.
 Ferritin Biotin Reagent containing biotinylated monoclonal
mouse IgG in buffer, dye, and preservative.
36
Chapter Three……………….……………….……Materials and Methods
 Ferritin Enzyme Reagent containing Horseradish peroxides
(HRP) labeled anti-ferritin IgG in buffer.
 Streptavidin coated plate coated with streptavidin and packaged
in an aluminum bag with drying agent.
 Wash solution containing a surfactant in buffered saline.
 Substrate A containing tetramethylbenzidine in buffer.
 Substrate B containing hydrogen peroxide (H2O2) in buffer.
 Stop solution, a vial containing a strong acid (1N HCL).
In this method (Jandal, 1996), ferritin calibrator, patient specimen
or control is first added to a streptavidin coated well. Biotinylated
monoclonal antibody (specific for ferritin) is added and the reactants
mixed. Reaction results between the biotinylated ferritin antibody and
native ferritin to form an immune complex that is deposited on the
streptavidin coated wells. The excess serum proteins are washed away via
a wash step. Another ferritin specific antibody, labeled with an enzyme, is
added to the wells. The enzyme labeled antibody binds to the ferritin
already immobilized on the well. Excess enzyme is washed off via a wash
step. A color is generated by the addition of a substrate. The intensity of
the color generation is directly proportional to the concentration of the
ferritin in the sample.
Approximate reference ranges for serum ferritin were (20-300
ng/ml) for males and (14-150 ng/ml) for females (Walker, 2010).
3.2.6. DETERMINATION OF SERUM ERYTHROPOIETIN (EPO)
The DRG EPO ELISA kit is intended for the quantitative
determination of EPO in human serum. This assay is for in vitro
diagnostic use, as an aid in the diagnosis of anemias and polycythemias.
The kit components are the followings :
37
Chapter Three……………….……………….……Materials and Methods
 Reagent (1) : Biotinylated EPO Antibody (mouse monoclonal
anti-human EPO)
 Reagent (2) : Peroxidase (Enzyme) labeled EPO antibody
(mouse monoclonal anti-human EPO)
 Reagent A : ELISA wash concentrate (saline with surfactant
with preservative ciprofloxacin)
 Reagent B : TMB substrate (tetramethylbenzidine).
 Stopping solution : ELISA Stop solution (1N sulfuric acid)
 Microplate : One holder with streptavidin coated strips.
 Calibrators : lypholized synthetic human-EPO is a buffered
protein solution.
The DRG EPO immunoassay is a two-site ELISA (Enzyme-linked
Immunosorbant Assay) for the measurement of the biologically active
165 amino acid chain of EPO. It utilizes two different mouse monoclonal
antibody to human EPO specific for well-defined regions on the EPO
molecule. One mouse monoclonal antibody to human EPO is biotinylated
and the other antibody to human EPO is labeled with horseradish
peroxidase (HRP) for detection.
Streptavidin
Biotinylated Anti-EPO
(mouse monoclonal)
EPO
HRP
conjugated Anti-EPO
(mouse monoclonal)
In this method (Garcia et al, 1982), calibrators, controls or patient
samples are simultaneously incubated with the enzyme labeled antibody
and a biotin coupled antibody in a streptavidin-coated microplate well.
By the end of the assay incubation, the microwell is washed to remove
unbound components and the enzyme bound to the solid phase is
incubated with the substrate (tetramethylbenzidine). An acidic stopping
solution is then added to stop the reaction and converts the color to
38
Chapter Three……………….……………….……Materials and Methods
yellow. The intensity of the yellow color is directly proportional to the
concentration of EPO in the sample. A dose response curve of absorbance
of the solution in the wells within 10 minutes, using a microplate reader
set at (450 nm) against 250
l of distilled or deionized water.
Concentrations of EPO present in the controls and patient samples are
determind directly from this curve. The DRG-standards have been
calibrated against the World Health Organization (WHO) erythropoietin
international standard that consists of recombianant DNA derived EPO.
The reference ranges for EPO in serum were 4.3-32.9 m /ml
(Miller et al., 1985).
3.2.7. DETERMINATION OF THE LEVEL OF VITAMIN C IN
PLASMA (mg/dl)
Plasma ascorbate was measured by a method described by
Gowenlock etal, (1988). To estimate the plasma ascorbate, the following
steps are followed :
a) An aliquot of 0.5ml plasma in a test tube was mixed with 2ml of
(Metaphosphoric Acid), the test tube with its content was placed in
a centrifuge at a speed of 2500/RPM, for 10 minutes.
b) About 1.2ml of the supernatant was slowly added into a test tube
containing 0.4ml of the reagent (DTCS) (Dinitrophenyl hydrazinethiourea-Copper Sulfate reagent). After thoroughly and slowly
mixing, the mixture was placed in water bath at 37cْ for 3 hours.
After that the test tube was transferred to ice bath and allow to
stand for 10 minutes.
c) The colored supernatant was transferred to another test tube
containing 2ml of (cold sulfuric acid).
d) The absorbance of the colored supernatant was measured by
spectrophotometer, through the followings :
39
Chapter Three……………….……………….……Materials and Methods
 Calibration of the spectrophotometer to read absorbance
at (520 nm) against a blank constituted with distilled
water.
 Recording the concentration of the sample.
 The concentration was obtained by a standard curve and
this value was byproduct by (5) to get the concentration of
the vitamin C in 100ml of plasma.
The reference value for plasma vitamin C was 0.4-1.5 mg/dl
(Gowenlock et al., 1988).
3.3. STATISTICAL ANALYSIS :
Statistical analysis was performed using the computer Software
Statistical Package of Social Science (SPSS 7.5 , SPSS Inc. , Chicago ,
USA). All Data were expressed as mean ± SD. Analysis of variance
followed by pairwise multiple comparison was used for comparison of
more than two mean. A within group comparison among post - treatment
values and baselines was analyzed by analysis of variance for repeated
measures , followed by paired Student’s T-test.P-value of less than 0.05
was considered as statistically significant (Woolson, 1987).
40