Download were measured in the sera of depressed and panic

Evaluation of Sialic acid and Cholesterol in Sera of Patients With
Colorectal Carcinoma and Benign Tumor .
Mehdi Mohammed Reda , M.Sc.
Department of Biochemistry , College of Medicine , Kufa University
: ‫الخالصة‬
‫ممر‬
‫سم‬
‫تممق اسممست ياممحامس لممسيي اممسس س و كا ساممحلوا ك ممر لألمما اوخ ي واسحممد و‬
.‫ ين الشوسص اص سء‬13 ‫قا ان و احقسق ألس د ا‬
‫ و‬TSA ‫) ممر‬P<0.01 ‫مممس ي يامممد‬
‫ممر يلألمما قمملنسن قا ممان و اممحقسق نويممس‬
‫قا مان و امحقسق‬
‫يسودم‬
‫سم‬
‫مر اوخ ي‬
‫ب كم‬. (P<0.05) ‫كا ساحلوا ك ر‬
‫ك مس ال‬-‫ و كا سامحلوا ك مر يقسخةمد بساشموسص اصم سء‬BSA ,TSA ‫مس ي يامد ر‬
.‫لألسحسن يقسخةد بساشوسص اص سء‬
‫س حسن‬
‫لسممل ممالف‬
‫ وك‬BSA
‫ب ي ودا‬
‫ ر كال‬LSA ,FSA ‫ماد أي تغسل ي ياي ر اسق‬
Abstract :
Thirty one cases from each type of colorectal carcinoma , benign tumor
,and healthy individuals were subjected to study.The levels of sialid acid and
total cholesterol are evaluated to obtain clear profile of changes in these
biochemical parameters.
The study showed the following results:
* In colorectal carcinoma : There are significant increase (p<0.01) in
serum total sialic acid (TSA), bound sialic acid (BSA),and total cholesterol
p<o.o5 as compared with the control group.
* In benign tumor : There are no significant increase in serum TSA,BSA and
total cholesterol as compared with control group.
[1]
* There are no significant changes
in free sialic acid (FSA) and lipid
associated sialic acid (LSA) in both patients as compared with control
group.
Introduction:
The term sialic acids denotes a member of a family comprising more than
20 natural derivatives of neuraminic acid, an acidic amino sugar in pyranose
form with nine carbon atoms (1). Unsubstituted neuraminic acid does not
occur naturally and the amino group and hydroxyl groups may be substituted
by different groups. The preferred localization of sialic acids is the outer cell
membrane where these sugars often occur in high concentration and are
components of glycoproteins, gangliosides
or polysaccharides. The
electronegative charge of a human erythrocyte, for example, is mainly due to a
dense coat of about 20 million sialic acid molecules (2). Sialic acids are found
usually in the terminal position of oligosaccharide chains, but in gangliosides
and some glycoproteins they also occur in the side position of oligosaccharide
chains. These structural differences, together with the multiple forms of sialic
acids, give the sialic acid part of oligosaccharide chains enormous structural
diversity. The resulting biological implications are mirrored in the observation
that the type of cells, their functional or developmental stages and malignancy
determine the nature of linkage and density of sialic acid molecules on cell
surface (2,3,4). It has been found that many glycoproteins and glycolipids are
increased in sera and malignant tissues of patients with various types of
cancers. Sialic acids- are the predominant carbohydrate of these compounds
(5). The relevance of sialic acids to the tumor cell is apparent from the
increased sialylation and sialytransferase activity observed in many cancer
cells (3). Sialic acid containing glycosphingolipids could be a microglial
activator (6) and it modulates cell-cell and cell matrix interactions (7).Some
[2]
tumors have been repoted to have elevated contents of sialic acid ,including
human tumors of the colon , lung and prostate (8,9). However marked
elevation of sialic acid has been indicated in plasma of patients with head and
neck ,breast, colorectal, lung, prostate and bladder cancer (8,10).
Cholesterol is widely distributed in all tissues ,but it is especially abundant in
the nervous system, where it is important for many aspects of cellular
structure and function (11). During the last decades, conflicting data have
been obtained on the relationship between the serum cholesterol level and the
incidence of colorectal cancer (12).The total amount of fat in the diet ,whether
high or low, isn’t really linked with disease. Bad fats increase the risk for
certain disease and good fats lower the risk (13). Heart disease is not the only
condition that has been linked with fat intake. Researches speculate an
association between dietary fat and certain cancers. As with breast cancer,
international comparisons initially suggested an association between total
dietary fat intake and colon cancer risk (14).
Materials and methods:
Patients:
This study was conducted during the period from November 2003until the
end of 2004 in Hepatology and Gastroenterology Teaching Hospital .
The subjects were thirty one patients with colorectal carcinoma aged (2575year) , and thirty one patient with benign colorectal tumor aged (22-70year).
Patients were evaluated by full medical history to exclude any existing
systemic disease that may affect the parameters of the study, particularly
diabetes melitus, liver disease, renal disease, hypertension and chronic drug
[3]
intake. Thirty one apparently healthy subjects aged (20-50 years) were
selected as control group.
Blood Samples:
Ten milliliters of venous blood samples were taken from each patient and
control. The samples were transferred into clean plain tube. Haemolyzed
samples were discarded. The blood was left at room temperature for 10
minutes for clotting, centrifuged 3000 rpm for 10 minutes, then serum was
separated and stored at -20 c until the time for analysis.
Determination of Total Sialic Acid (TSA) :
TSA was measured using Svennerholm (15) method as modified by
George etal (16):
Resorcinol Reagent Preparation:
Six hundred milligrams of resorcinol was dissolved in 60mlof 28% HC1,
40ml of water and 25 micromole of copper sulfate.
Assay:
In brief, 20 uL of serum was diluted into 500u.L in a test tube with
distilled water. Then 0.1 ml of 0.04M periodic acid solution was added. The
mixture was thoroughly mixed and allowed to stand in an ice bath for 20
minutes. After the addition of 1.25 ml of resorcinol reagent, the solution was
mixed and placed in an ice for 5 minutes. The solution then heated at 100°C
for 15 minutes, cooled in tap water and 1.25 ml of tertiary butyl alcohol
solution(95%) was added. After vigorous mixing, the tubes were pooled in a
37°C water bath for 3 minutes, cooled at room temperature and the absorbance
was measured by UV-visible Spectrophotometer(PYE- UNICAM 8600) at
630 nm. The concentration was obtained from the standard curve developed
[4]
from different concentrations of n-acetyl neuraminic acid (NANA) as shown
in Figure (1).
Determination of Lipid Associated Sialic Acid (LSA):
LSA is measured according to the modified procedure of Katopodis and
Stock (17):
Fifty micro liters of serum were diluted with 150 uL of distilled water and
transferred to crushed ice . The diluted serum were extracted by 3ml of
chloroform :methanol (2:1 v/v) at (4-5°C) and vortexed for thirty seconds.
The lipid extract was partitioned with 0.5 ml of cold distilled water and
mixed by repeatedly inverting the tubes for thirty seconds. After centirfuging
the tubes for 5 minutes at room temperature at 2500 rpm, one ml of tube
solution was transfered upper aqueous layer containing LSA into a new test
tube. The aqueous layer containing LSA was precipitated with 50u.L of
phosphotungstic acid (Ig/1ml) and after mixing, then it was left stand at room
temperature for 5 minutes and was centrifuge for 5 minutes at 2500 rpm and
the supernatant was removed by suction. One milliliter of distilled water was
added to the precipitate and the mixture was vortexed until the precipitate is in
suspension without gross particles (about 1 minute).One milliliter of
resorcinol reagent was added to the tube, mixed and placed in boiling water
for exactly 15 minutes. After 15 minutes, tubes was transfered to an ice and
water bath and was left for 10 minutes. To the ice cold tube add 2ml butyl
acetate-(n-butanol)85:15 v/v mixture was added at room temperature,
vortexed and centrifuged for 5 minutes at 2500 rpm the extracted blue color
was read at 580 nm and the amount of lipid bound sialic acid was determined
by use of a standard curve developed from a standard sample of n-acetyl
neuraminic acid and use of this formula:
LSA mg/dL =
X*100000
y*50*1000
[5]
where X= Concentration of NANA obtained from standard curve .
y= 1 ml. of supernatant: volume of entire supernatant (In the original paper it
had been 1/1.3)
Determination of Bound Sialic Acid (BSA):
Chromogen formed by periodate oxidation is destroyed at 37°C,whereas
the chromogen obtained from the oxidation of the glycosidically bound sialic
acid is stable at this temperature (16). BSA was estimated using the protocol
mentioned for TSA except that the oxidation step is carried out at 37°C not at
0°C.
Determination of Free Sialic Acid (FSA):
Free sialic acid was determined from the difference of the level indicated
with the oxidation at 0°C and that obtained by oxidation at 37°C as
demonstrated for TSA and BSA.
Determination of Cholesterol:
Total serum cholesterol was measured by cholesterol kit (PAP
100bioMerieux), using an enzymatic method (18).
The principle of this
method was to analysis the cholesterol ester to the cholesterol and fatty acids,
and then oxidized it to get the quinoemine:
cholesterol esterase
Cholesterol ester
cholesterol + fatty acids
cholesterol oxidase
2H2O2 +phenol+4-aminoantipyrin
Peroxidase
quinoemine+4H2O
'The absorbance was measured by UV-Visible spectrophotometer(PYEUNICAM 8600) at 500 nm after five minutes at 37°C.The intensity of the
[6]
color produced was directly proportional to the total cholesterol concentration
in the sample.
Results :
Table (1) showed the different level of sialic acids and total cholesterol in
patient with benign tumor and patient with colorectal carcinoma
in
comparison with healthy controls. TSA and BSA are significantly increased
P<0.01in colorectal carcinoma patients as compared to healthy control. While
the concentration of LSA and FSA in colorectal carcinoma patients showed no
significant difference as compared to healthy controls. The results of serum
sialic acids in patients with benign tumor no significant increase in TSA and
BSA,LSA ,and FSA respectively, as compared to control group. Total
cholesterol is asignificant increase
p< 0.05 in patients with colorectal
carcinoma while ,no significant increase in benign tumor as compared with
control group.
[7]
Table (1):The Biochemical values of the ;colorectal carcinoma, benign
tumor and control groups.
Subjects
Mean  SD
P value
TSA(mg/dl)
Normal controls
61.30
 10.8
Benign Tumor
65.23
 12.72
N.S.
83.0
 11.5
> 0.01
60.5
 9.78
Colorectal carcinoma
BSA(mg/dl)
Normal controls
Benign Tumor
64.04
 11.65
N.S.
Colorectal carcinoma
81.91
 11.58
> 0.01
Normal controls
0.87
 0.31
Benign Tumor
0.98
 0.33
FSA(mg/dl)
Colorectal carcinoma
1.19+ 0.55
N.S.
N.S.
LSA(mg/dl)
Normal control
18.71
 4.22
Benign Tumor
19.85
 4.66
N.S.
Colorectal carcinoma
20.75
 4.79
N.S.
Normal control
4.93
 0.94
Benign Tumor
4.98
 0.96
N.S.
Colorectal carcinoma
5.99
 1.10
< 0.05
Serum cholesterol(mmol/L)
[8]
Absorbance
NANA (ug/ml).
Figure (1) : Calibration Curve of Sialic acid
(N- acetyl neuraminic acid)
Correlation of serum Cholesterol With TSA or LSA:
Figure (2) showed the correlation representation of serum total cholesterol
with LSA and TSA in patient with colorectal carcinoma
as a graphic
representation example and the other correlation coefficient values are shown
in Table (2). There is no significant correlation between both TSA,LSA versus
serum cholesterol in patients with benign tumor, while in patient with
colorectal carcinoma the values is positively correlated between serum TSA
versus serum total cholesterol and no significant correlation between serum
LSA and serum total cholesterol was found.
[9]
Table(2):Correlation coefficients (r-values) of serum TSA and LSA
with serum total cholesterol.
Groups
Cholesterol with TSA
Cholesterol with LSA
Control
0.37
0.29
Benign Tumor
0.13
0.09
Colorectal carcinoma
0.76
0.1
TSA (r=0.76)
S. Conc. (mg/dL)
LSA (r=0.10)
S.Choleesterol (mmol/L)
S.Cholesterol (mmol/ L)
Figure(2): Correlation lines of serum TSA and LSA with serum total
cholesterol in patient with colorectal carcinoma .
[10]
Discussion:
Sialic acids is a terminal component of the reducing end of carbohydrate
chain of glycoproteins and glycolipids including hormones and enzymes
present in serum and tissue and 99% of sialic acid is bound to glycoproteins
and glycolipids (19,20). Several studies have shown that neoplastic
transformation
leads
to
elevation
of
serum NANA
concentration.
Approximately 99% of the TSA found in sera of colorectal cancer patients is
bound to glycoconjugates (21, 22,23,24).In this study total, bound, free, lipid
associated NANA were measured and significant elevation P< 0.01 of TSA
and BSA in patient with colorectal carcinoma was found. The increase of
sialic acid concentration is due to enhancement of sialyltransferase activity in
malignant tumor cells. Thus, the synthesized sialoglycoconjugates will relased
to the extracellular fluid reaching the systemic circulation. This hypothesis is
supported by some experimental evidences.High levels of sialyltranferase
activity have been found in human colon neoplastic tissue it is possible that in
our patients , an increase in sialoglycocojugates content could occur, but these
new synthesized sialoglycoconjugates could be rapidly released from the
tumor into the blood (21) .
Dietary fat is thought to be one of the main risk factors on the basis of
reports of positive correlations between dietary fat intake and increased risks
of cancers of the breast, colon, and prostate (25). Several studies have showed
an association between lipoprotein levels and the presence of adenomas (12).
Cholesterol – caring lipoproteins play central roles in the development of the
disease (14).In this study the estimated total cholesterol have found significant
elevation
p<
o.o5
in
patient
with
colorectal
carcinoma
Several
investigators(25,26,27,28,29) have reported a positive correlation between the
serum total cholesterol level and the risk of colorectal cancer . The correlation
[11]
between TSA and total cholesterol in this study have found a positive
correlation between increased TSA and total cholesterol with increased the
risk of colorectal cancer . Hence the finding of this studies are preliminary in
the correlation between TSA and total cholesterol with colorectal cancer and
may have pathogenic significance for the development of colorectal cancer.
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[12]
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[14]