ft-ir analysis of deride human serum for …
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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.
[email protected], AL-Kademiyah Hospital **,[email protected].
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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3
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.
References
1- Donadio, E., Piccolomini, F., Dimuccio,
V., Felicioli, A., Balestreri ,E., Cianti ,R.,
Armini, A., Felicioli ,R., Donadio, C.,
"Serum albumin fragmentation in end stage
renal disease patients – a pilot study
".,Clinic. Chem. Lab. Med. 2009, 47, 1373-
1379.
2- Devi, T.S.R., Gunasekaran, S., Hudson,
J.W., Joybell, S.A., "Analysis on renal
failure patients blood samples :
characterization and efficacy study"., Indian
J. Of Science and Technology. 2009,2,46-
50.
3-Budinova,G ., Salva, J., Volka K.,"
Application of molecular spectroscopy in the
mid-infrared region to the determination of
glucose and cholesterol in whole blood and
in blood serum"., Appl.
Spectrsc.1997,51,631-639.
4- Janatsch,G ., Kruse, J.D.," Multivariate
calibration for assays in clinical chemistry
using attenuated total reflection infrared
spectra of human blood plasma".,
Anal.Chem .1989,61,2016.
5- Cadet, F., Robert, C ., Offmann, N.D.,"
Simultaneous determination of sugars by
multivariate analysis applied to mid-infrared
spectra of biological samples "., Appl.
Spectrsc.1997,51,369-375.
5
6
6- Vonach, R., Buschmann, J., Falkowaki,
R., Schindler, R., Lendle, B., Keller, R.,"
Application of mid-infrared transmission
spectrometry to the direct determination of
glucose in whole blood"., Appl.
Spectrsc.1998,51,820-822.
7-Heise, H.M ., Morbach, R., Koschinsky,
T., Gries, F.A., "Multicomponent assay for
blood substrates in human plasma by mid-
infrared spectroscopy and its evaluation of
clinical analysis".,
Appl.Spectroscop.1994,48,85-89.
8- Budinova, G., Salva, J., Volka K.,
"Application of molecular spectroscopy in
the mid-infrared region to the determination
of glucose and cholesterol in whole blood
and in blood serum".,
Appl.Spectroscop,1997,51,631-35.
9- Khalil, O.S., "spectroscopic and clinical
aspects of non invasive glucose
measurements"., Clin.Chem.1999.45,165-
177.
10- Sankari, G., Krishna, M.E.,
Jayakumaran, S., Gunasekaran, S., Vishnu,
P.V., Shyama, S., Subramaniam, S.,
Surapaneni, K.M.," Analysis of serum
immunoglobulins using fourier transform
infrared spectral measurements"., Biology
and Medicine.2010,2,42-48.
11- Devi1,T.S.R., Gunasekaran, S., Wesley,
H. J ., Sarone, A .J.," Analysis on renal
failure patients blood samples:
characterization and efficacy study"., Indian
Journal of Science and
Technology.2009,2,0974-6846.
12- Cyril, P., Georges, C., André, C.,
Gérard, D.," Plasma protein contents
determined by fourier transform infrared
spectrometry"., Clinical
Chemistry.2001,47,730-7.
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