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FT-IR ANALYSIS OF DERIDE HUMAN SERUM FOR DETERMINING

ALBUMIN CONCENTRATION IN IRAQI PATIENTS WITH RENAL

FAILURE *Israa G. Zainal & **Ameer S. Radi

*AL-Mustansiriya University ,College Of Science ,Chemistry Department.

israaz@yahoo.com, AL-Kademiyah Hospital **,ameer.sltan@yahoo.com.

Abstract

This study attempt to evaluate the spectral difference in serum albumin level between healthy and patients with renal failure pre and post dialysis using two methods kit assay & IR spectroscopy . The results showed a non significant

difference between the level of albumin measured by kit assay & IR spectroscopy to all studied groups . Correlation

studies indicated that there were a positive significant correlation between albumin assayed by kit assay & IR

spectroscopy [(r = 0.59 , p = 0.004 ), (r = 0.638 , p = 0.001) and ( r = 0.478 , p = 0.024 )] in control and pre ,post

dialysis patients respectively .

Keywords: Chronic renal failure, Serum albumin, FT-IR spectroscopy

Introduction

The kidney plays a central role in the

regulation of plasma concentrations of

low molecular weight proteins and human

serum albumin(1)

.Renal failure occurs

where there is damage in the kidneys that

impairs their ability to filter and remove

waste products from the blood. Diagnosis

of renal failure is made by collection of

blood and urine samples for analysis (2)

.

Proteins or albumin in serum is

commonly tested to monitor liver and

renal conditions .Not surprisingly ,

therefore , serum and blood analysis

become one of the most common tests

performed and all of the major dissolved

metabolites are routinely in clinical

laboratory.Most of these analyses are

carried out with a photometric methods

using an enzymatic reactions .The

photometric measurement method is well

established and easily available. However

, the chemical process such as enzymatic

reaction requires expensive reagents and a

relatively large volume of the sample.

More than 50 of the operating cost of

clinical analyzers in the hospital is

devoted to the expenses of reagents.

Recently , there have been some reports

on the optical spectroscopic method for

the quantification of major metabolites in

human blood , serum , tissue and other

substrates(3- 6)

.Infrared spectroscopy offers

an approach to clinical analysis that is

conceptually very appealing . Whereas

countless assays rely on the use of chemical

agents to " recognize " the analyte of

interest and to react with the analyze to

produce specific color changes .IR-based

analysis is found on the rich IR absorption

patterns that characterize the analytes

themselves. These absorption patterns

provide the basis to distinguish among the

constituents and to separately quantify

them. The most obvious distinguishing

feature is that no reagents are required. In

addition , IR-based analytical methods

require very small sample volumes

(typically micro liters) , show good

precision over the entire physiological range

, and are well suited for automation(7- 9)

. The

aim of the present study is to find the

spectral difference in serum albumin level

between healthy and renal failure (pre and

post dialysis) blood sera using kit assay &

FTIR spectroscopic technique

.

Materials and methods

Blood samples were collected from (22)

patients (12 males and 10 females) , age

range between 20 to 70 years with chronic

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renal failure undergoing hemodialysis(

HD) . Renal failure blood samples before

and after dialysis were collected from AL-

Kademiyah Teaching Hospital . Healthy

group samples were collected from twenty

two subjects , with the same age range.

FT-IR measurements :

Eighty micro liter of serum were diluted

with twenty micro liter of four mg/L

aqueous potassium thiocyanate (KSCN)

solution , then , thirty five micro liter of

each diluted samples were spread evenly

over the surface of AgCl cell . All the

specimens were air dried for 30 minutes

to measure the IR spectra . Infrared

spectra in the region 4000-400 cm-1

were

recorded by FT-IR 8488S (SHIMAD24)

spectrophotometer .

Serum Albumin level Albumin level was measured by

colorimetric method using a kit supplied

by Spinreact .

Statistical analysis Descriptive statistics were used in analyzing

the patients characteristics and laboratory

parameters for each groups. In addition ,

unpaired student t – test was used to assess

group differences , where appropriate .A

statistical significant difference was

accepted as p value less than 0.05 . All the

statistical analyses in this study were made

using SPSS 10.0 for windows program.

Results

The present study was conducted to

evaluate the sensitivity and accuracy of

mid-IR spectroscopy in the determination of

serum albumin. The IR-based quantification

methods were calibrated by comparison

with the results provided by kit assay.

Table (1) shown the levels of serum

albumin as (mean± SD) g/L in pre& post-

dialysis patients with CRF and control

group , using two methods kit assay & IR

spectroscopy .

Table (1): Serum albumin levels in pre& post-dialysis patients with CRF and control group

using two methods ( kit & IR spectroscopy ).

methods No. Mean±SD(g/L) P value

Kit-control 22 38.500±2.483 p>0.05

IR-control 22 36.522±2.457

Kit-pre-dialysis 22 31.363±6.059 p>0.05

IR-pre-dialysis 22 33.281±4.739

Kit-post- dialysis 22 30.345±6.515 p>0.05

IR-post-dialysis 22 29.163±5.716

Results showed a non significant

difference between the level of albumin

measured by kit assay & IR spectroscopy

to all studied groups .A representative FT-

IR absorption

spectrum of serum samples were shown in

Figures ( 1 , 2 & 3 ) for all studied groups.

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Fig(1) FTIR spectrum of a healthy human serum sample

Fig(2)FTIR spectrum of a renal failure patient pre-dialysis

Fig(3)FTIR spectrum of a renal failure patient post-dialysis

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Figure (4):Correlation between Kit-albumin& IR-albumin in control

Figure (5):Correlation between Kit-albumin& IR-albumin in pre-dialysis

Figure (6): Correlation between Kit-albumin& IR-albumin in post-dialysis

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Correlation studies indicated that there were

a positive significant correlation between

albumin assayed by kit & IR spectroscopy [

(r = 0.59 , p = 0.004 ), (r = 0.638 , p = 0.001)

and ( r = 0.478 , p = 0.024 )] in control and

pre ,post dialysis patients respectively ,

figures ( 4,5 and 6) .

Discussion

Results in this study showed a non

significant difference between the level of

albumin measured by kit assay & IR

spectroscopy to all studied groups .

A vibration band assignment is

done with the idea of the group frequencies

of the various analytes present in the sample.

The spectral region (3600 – 3000) cm-¹

comprises of C-H, O-H and N-H stretching

vibrations of the protein. The prominent

absorption peak 3300 cm-¹ is due to the N-H

stretching mode (amide A) of proteins. The

asymmetric and symmetric stretching C-H

vibrations of methyl and methylene group

are found to be present around 2930 – 2875

cm-¹.The strong absorption band at 1650 cm-

1 correspond to C=O stretching vibrations

(amide I) whereas the vibration band at 1542

cm-¹ is attributed as amide II arising of N-H

bending vibrations strongly coupled with C-

N stretching of proteins.

The absorption peaks in the region

(1400-1200) cm-¹ arise due to the C-H

deformation of methyl and methylene group

of the proteins. The asymmetric and

symmetric P-O stretching vibrations are

found to be around 1245 cm-¹ and 956 cm-¹

respectively. The spectral region 1250-925

cm-¹ is predominantly occupied by C-O-C

asymmetric and symmetric vibrations of

phospholipids of proteins(10)

.

Devi1 T et al(11)

, Concluded A

systematic approach has been made using

FTIR spectroscopic technique to study the

spectral difference between healthy and

renal failure patients blood samples and also

to find the efficacy of peritoneal dialysis on

renal failures. The spectral results are well

supported by the clinical values(11)

.

Cyril P et al(12)

,observed that determine

the concentrations of various proteins in

plasma on the basis of their most

characteristic IR absorption peaks. For

albumin, the best correlation with results

obtained by a comparison method was found

using the N-H absorption region (1600-

1480) cm-¹ common to all plasma proteins

and concluded that the FT-IR spectrometry

is a useful tool for determining

concentrations of multiplebiomolecules in

micro samples of plasma.

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