Review Article
Plasma Albumin Alterations in Gastrointestinal Cancer Patients
Inta Kalnina1*, Tija Zvagule1 and Jelena Kirilova2
1Department of Chemistry, Daugavpils University, Latvia
2Department of Chemistry, RJga Stradins University, Latvia
*Corresponding author: Inta Kalnina, Department of Chemistry, Daugavpils University, Latvia
Published: 18 Oct, 2016
Cite this article as: Kalnina I, Zvagule T, Kirilova J. Plasma Albumin Alterations in Gastrointestinal Cancer Patients. Clin Oncol. 2016; 1: 1113.
Abstract
Albumin plays a prime role in most functional characteristics of plasma and immune state of
organism. Patients with gastrointestinal cancer exhibit a poorly functioning immune system. This
study work investigated the possibility of using the fluorescent probe ABM for detecting structural
and functional alterations of blood plasma albumin in pathological states. The aim of this research
work was to evaluate the changes of blood albumin in correlation with some immunological
parameters in patients with gastrointestinal cancer. 46 patients with gastrointestinal cancer and
24 healthy donors were examined in this study. Lymphocyte subpopulations were determined
by flow cytometry using the appropriate specific monoclonal antibodies in the peripheral blood.
Patients were tested one day before the operation and ten days after surgical treatment. The
fluorescent probe - amino derivative of benzanthrone, ABM (developed at Daugavpils University,
Daugavpils, Latvia) was used to characterize the blood plasma albumin. Before surgical treatments,
the average ABM intensity in the patients' blood plasma was decreased compared to that seen
with the samples from the healthy donors. The lymphocyte distribution among subsets differed
not only between observed groups of patients, but also before and after surgical treatment. ABM
fluorescence intensity was found to correlate with the absolute number of CD38+ cells and with
some immunological parameters both before and after surgical treatment. To conclude, measures
of ABM fluorescence values for lymphocytes and blood plasma could potentially be used in clinical
immunological screening (instead of more expensive routine test) to provide a snapshot of immune
status of individuals.
Keywords: Blood plasma albumin; Immune diagnostics; Fluorescent probe ABM;
Gastrointestinal cancer; Lymphocytes
Introduction
The principal function of plasma albumin is to transport a wide variety of fatty acids and metabolites via the main binding regions [1,2]. Membrane damage in different pathologies may be a consequence not only of lipid peroxidation, but also of protein alterations. Albumin plays a prime role in most functional characteristics of plasma and immune state of organism [3]. The fluorescent probe -aminoderivative of benzanthrone, ABM (developed at Riga Technical University, Riga, Latvia) was used to characterize the blood plasma albumin. Patients with gastrointestinal cancer exhibit a poorly functioning immune system, that is characterized by decrease in T-lymphocyte proliferation [4,5] and reduced CD4+/ CD8+ ratio [6,7]. This research investigated the possibility of using the fluorescent probe ABM for detecting structural and functional alterations of blood plasma albumin in pathological states. ABM reflects such properties of lymphocytes as physical-chemical state of their membrane, membrane micro viscosity, and proliferating activity of lymphocytes, lipid metabolic activity and some phenotypical characteristics of lymphocytes. The examined changes of the studied parameters could be used for assessing the alterations of the cellular mechanisms of immunity, and, therefore, can be applied as preliminary screening test in immune diagnostics.
Aim
The aim of our study work was to evaluate the changes of blood albumin in correlation with some immunological parameters (T-lymphocytes counts, CD4+/CD8+ ratio etc.) in patients with gastrointestinal cancer.
Material and Methods
ABM
A 46 patients with gastrointestinal cancer (30 colorectal in stages II-IV; 16 gastric in stage III) and 24 healthy donors were examined in this study. Lymphocyte
subpopulations: CDS* common T-cells, CD4* helper cells, CDS*
cytotoxic cells, CD16+ natural killer cells, CD38* activated T- and
B-cells, also activated natural killer cells were determined by flow
cytometry using the appropriate specific monoclonal antibodies
(Becton Dickenson, USA) in the peripheral blood. These patients
were tested one day before the operation and ten days after surgical
treatment. Plasma albumin, as in earlier studies, was detected using the
ABM fluorescent probe [8-10] developed in Daugavpils University,
Daugavpils, Latvia. In the current study, blood plasma (200-fold
diluted) or cells (5* 106 per assay) incubated without probe were
used as each patient's personal "blank" in each experiment. The ABM
(resulting concentration in sample=19.6 pmol/1) was added into 1 ml
aliquot of the patient's blood plasma (or 1 ml solution containing the
cells) at temperature of 18-20°C and the mixture then was allowed to
sit for ~ 5 min. The time interval between cell/plasma isolation and
fluorescence measurement was held constant for all samples (i.e.,
3 hr). Resulting fluorescence parameters were then registered on a
Spectrofluo JY3 spectrofluorometer (ISA JobinYvon Instruments S.
A., Longjumeau, France) at an excitation wavelength of 470 nm and
an emission wavelength of 520-700 nm. To register luminescence, the
sample was placed in a cuvette (1 * 10 * 40 mm3) fixed at an angle of 30°
to the excitation light beam. Fluorescence intensity was then recorded
and reported in terms of arbitrary units (AU). The final intensity value
for each patient's sample was then generated after accounting for (i.e.,
subtracting out) the value associated with their personal "blank";
this approach thereby eliminated any potential contributions from
any autofluorescing constituents in the plasma sample. Data were
analyzed using either Student T-test or Wilcoxon-Mann-Whitney
U-test. Significance was considered at < 0.05 [11].
Table 1
Table 1
Spectral characteristics of ABM and subpopulations of T-lymphocytes in colorectal cancer patients.
Table 2
Table 2
Spectral characteristics of ABM and sub-populations of T-lymphocytes in gastric cancer patients.
Results
ABM binding with plasma albumin
In the gastrointestinal cancer patients, the ABM emission spectra
maximum (i.e. 650 nm) in patients' blood plasma was not altered
in comparison to that seen in healthy donors. In contrast, with the
respect to fluorescence intensity, before surgical treatments, the
average ABM intensity in the patients' blood plasma was decreased
compared to that seen with the samples from the healthy donors.
Specifically, the fluorescence in colorectal and gastric groups (Table 1 and 2) were decreased by 23% and 22%, respectively. At 10 days
after their operations, the average ABM fluorescence intensity in
colorectal and gastric groups was decreased further by 13.9% and
10.3%, respectively (Table 1 and 2).
The lymphocyte distribution among subsets differed not only
between observed groups of patients, but also before and after
surgical treatment. Results among the patients of colorectal cancer
group (Table 1) indicate that, before surgery, the absolute count
CD38+ cells, and the CD4+/ CD8+ ratio were significantly decreased
(by 84%, and 33%, respectively) compared to corresponding control
subject value. Somewhat unexpected, relative percentages of Ci)16+
cells in this group were greater (by 25%). After surgical treatment, the
absolute count of lymphocytes and CD16+ cells were reduced by 18%
and 53%, respectively. The absolute count of CD38+ cells and CD4+/
CD8+ ratio increased (by 205% and 22%, respectively) compared to
pre-surgical values, but did not reach control levels.
The results from the gastric cancer group patients (Table 2 show,
that before surgery, the absolute count of CD38+ cells and lymphocytes
were each increased (by 15.7% and 24.1%, respectively) compared
to control values. After surgery, the absolute count of lymphocytes
was reduced (by 18.2%) relatively to pre surgery levels. This shift was
sufficient enough to yield a value that was now lower than control
levels (by 5.2%). As with the results in the other patient subgroup, the
absolute count of CD38+ cells was decreased and CD4+/ CD8+ ratio
was increased (by 45.9% and 21.9%, respectively) compared to the
pre-surgical values, but did not reach control levels.
These results (different ABM spectral characteristics) can be
explained by different structural characteristics of albumin in observed
patient groups. ABM fluorescence in pre-operation in colorectal and
gastric patient groups was found to correlate with the absolute count
of CD38+ cells (r=+0.926, r=+0.798, respectively). Before surgical
treatment in gastric cancer group it was held with respect to the
relative count of CD8+ cells (r=-0.804). After the operations both
CD4+/CD8+ ratio increases in both groups of gastrointestinal cancer
as compared with pre-operation indices. Absolute count of CD38+
cells in gastric cancer patients blood was observed to be decreased,
but in colorectal patients-increased relative to pre-operation values.
ABM fluorescence intensity correlated with some immunological
parameters both before and after surgical treatment. Before operation in all patient groups fluorescence intensity correlated with the number of CD38+ cells (p <0.05), supporting the view that fluorescence
intensity depends on the level of activated lymphocytes. In both
groups (colorectal and gastric cancer) the ABM fluorescence in cell
suspension was found to correlate with ratio CD4/CD8 (r =+0.843,
r=+0.756, respectively).
Discussion
It is known that different disease-associated biomarkers that
bind to albumin can cause changes of the protein structure and
function. Tumour cells release a variety of bioactive proteins
and peptide fragments into the blood that may reflect important
disease-related information. It may reveal effects of the tumour on
the organism itself. Tumour-derived proteins enter the circulation
and bind to transport proteins. Sequestration of these peptides by
carrier proteins (albumin) protects these markers from clearance and
helps amplify their concentration in the circulation [12]. In cancer,
the blood plasma content of important unsaturated fatty acid (i.e.,
oleic, arachidonic acids) is increased -these natural constituents and
their counterparts also increasingly occupy binding sites on albumin
[5,13]; and, albumin can bind an array of drugs, including ibuprofen,
indomethacin, etc. (and their metabolites) commonly ingested by
cancer patients [13]. Some conformational alterations in albumin are
observed at early stages of most cancers. In late stages of cancer, the
levels of pathological metabolites increase and the albumin ultimately
cannot bind them all. These induced structural/functional alterations
in the albumin could then manifest as "shifts" away from the normal
"main" binding sites with high affinity for the ABM to other sites with
lower affinities and specificities. Such shifts would be in agreement
with the observations of others [2,14,15] that albumin molecules
are known to contain different binding sites (i.e., classes) that differ
in affinity [5,10,16] for ABM and various other probes. Albumin
is a single source of ABM fluorescence in blood plasma. Probe
binds to albumin with high selectivity. The results of the current
investigation are also in agreement with Electron Spin Resonance
(ESR) spectroscopy studies to measure structural/functional changes
in serum albumin of cancer patients [12]. Analysis of the ESR spectra,
using spin probe, revealed substantial differences in spectrum
variables between patients and healthy individuals. For example, the
fraction corresponding to patients and the increasing width of the
spectral line indicated an alteration in albumin conformation that
limited the movement of spin probe bound to this site. In general,
the ESR spectral line shape allowed several different values to be
deduced, e.g., albumin capacity to bind spin probe, polarity of spin
probe binding site, and probe mobility [12].
We observed that before the operation the accounts of lymphocytes
and natural killer cells (CD16+) were increased in patients with
gastrointestinal cancer in comparison with healthy donors. Absolute
count of CD38* cells (the CD38+ cells mainly consist of activated Tand
B-lymphocytes) increased with an increase of F in cell suspension
in colorectal cancer patient group, but in gastric patient group
decreases relative to pre-operation values. The numbers of natural
killer (CD16+) cells was not related with ABM fluorescence intensity
F and the functional activity of the whole lymphocyte subpopulation.
Increase of ABM fluorescence intensity in cell suspension after
the operation is due to compensatory rise of functional activity in
response to a decrease in the count of the lymphocytes subsets.
Changes in the fluorescence parameters of the cancer patients'
lymphocytes could be reflective of changes in one/more inherent characteristics of their cells. In the studies here, the phenotypical
character of the cells was evaluated by examining changes in the
lymphocyte populations (i.e., their numbers) themselves. These flow
cytometry studies indicated that there were significant changes in
lymphocyte (and subpopulations) levels among the gastrointestinal
cancer patients, unfortunately, the variations in total lymphocytes
levels never clearly and consistently tallied the corresponding changes
in ABM fluorescences (Table 1 and 2).
Conclusion
Our results suggest the measures of ABM fluorescence intensity values for lymphocytes and blood plasma could potentially be used in clinical immunological screening (instead of more expensive routine test) to provide a snapshot of immune status in these cancer patients. Fluorescence behavior of ABM could be of use as potential indicators of alterations of cellular immunity in individuals. Compared to many commonly used diagnostic protocols this fluorescence-based method is less expensive and not very time consuming, technically simple and 100 times more sensitive than standard absorbance based method.
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