INTRODUCTION:

A sensitive and specific blood test for early detection and subsequently for the management of cancer patients would be of great clinical value; however the quest for such a test is still ongoing. Increased levels of enzymes, glycoproteins, hormones, oncofoetal proteins and peptides have been considered as potential tumor markers for helping in screening, diagnosis, staging, and prognosis and monitoring of cancer treatment. Alternations in the cell surface during transformation of normal cell to a malignant are well known. Alterations in serum plasma levels of the Total Sialic Acid (TSA) and Lipid Associated Sialic Acid (LASA) levels have been reported to be useful in early diagnosis of cancer as well as in the management of the patients with various malignancies¹.

Studies of cancer cells have revealed alteration in cell surface and change in plasma membranes in form of sialic acid content of glycoproteins and glycolipids as possible markers for these cancer cells producing them.

Other studies of serum or plasma total or lipid associated sialic acid content has been studied in patients with malignant melanomas, carcinoma of the ovary, colorectal cancer, breast cancer and other cancers and leukemia. Several glycosyltransferase levels have been reported to be abnormal in human cancer².

Other studies concluded the significant elevation in serum (TSA) total sialic acid LASA and TSA/TP. TSA and TSA/TP were superior to LASA in differentiating solid tumors and leukemia from normal and controls. Also a descending trend in TSA, TSA/TP was noted after successful treatment of the malignancy, on the other hand, children will ALL and infection had a significantly higher serum TSA and TSA/TP levels compared with children infections disease only³.

This study was achieved to compare the levels of ALL, LASA, TSA, and TPA between normal people (control) and with patients who suffers from leukemia and Hodgkin’s.

MATERIALS AND METHODS:

Sera specimens:

Blood samples were collected from the patients and controls donors by venipuncture. The blood samples were left for 20 minutes at room temperature, after coagulation, the serum was separated using centrifuge at 3000 rpm for 10 minutes, and kept at -20°C.

Determination of the total proteins (TP) in sera:

Protein content in sera of the patients was determined using Lowry method, by Bovine Serum Albumin (BSA) as a standard protein⁴.

Determination of total sialic acid (TSA) levels in the sera:

TSA was assayed using Katapoids et al. method⁵.

Determination of Lipid-Associated Sialic Acid (LASA) levels in sera:

LASA was assayed in serum samples using the method that described by Katapodis et al.⁵ The assay consists of the following steps:

1- 150𝛍l of deionized water was added to 50𝛍l of serum sample; the tube was vortexes for 5 seconds and placed on ice.

2- 3ml of cold chloroform/methanol solution at 4°C was added to step solution and after mixing them, a 0.5 ml of cold deionized water was added, the tubes were centrifuged for 5 minutes at 3000rpm at room temperature.

3- One ml of the resulting upper layer of step 2 solution was transferred to another tube, and 50𝛍l of phosphor tungestic acid solution were added, mixed and allowed to stand at room temperature for 5 minutes.

4- The assay tubes were centrifuged for 5 minutes at 3000rpm, the supernant fluid was removed and the remaining precipitate was dissolved in one ml of deionized water.

5- To each assay tube, 1ml of resorcinol reagent was added, then the tubes were placed in a boiling water for exactly 15 minutes, then for 10minutes on ice.

6- Two ml of butyl acetate/methanol reagent was added, the assay mixtures were vortexes and centrifuged for 10 minutes at 3000rpm. The extracted chromphore was read at 580 nm, against water.

Statistical methods:

The results for TSA, LASA, TSA/TP ratio were analyzed statistically by values expressed as mean±SD. The level of significance was determined by student’s test⁶.

RESULTS AND DISCUSSION:

Serum samples were collected from the patients and the control as shown in table 1, and the standard curve (figure 1) was obtained by plotting the absorbance against the corresponding concentration of the standard protein, and was used to determine the unknown protein concentration of the serum sample.

Table 1: The host information of the patients and control subject studied

Group	Case	No. of cases	Female	Male	Age (years)
I	Leukemia	152	61	91	13-65
A	ALL	79	34	45	13-26
B	CLL	21	15	6	50-65
C	AML	7	5	2	14-30
D	CML	45	20	25	28-50
II	Hodgkin’s	26	12	14	26-55
III	Normal (control)	12	5	7	35-60
IV	Nonmalignant	37	15	22	20-60

ALL = Acute Lymphocytic Leukemia

AML = Acute Myelocytic Leukemia

CLL = Chronic Lymphocytic Leukemia

CML = Chronic Myelocytic Leukemia

Figure 1: The standard curve for determination of the total protein concentration

Table 2 shows the mean values ± SD of the four biomarkers TP, TSA, LASA, TSA/TP that obtained from sera of the control and the groups of the patients (leukemia and Hodgkin’s). From table 2 it is clear that there are no significant differences, when compared to the mean value of normal and pathological controls and usually, the mean values of total protein in sera of all groups still in the normal range, but the levels of TP were generally increased in leukemia and Hodgkin’s in comparison with healthy group. The elevation of total protein levels in leukemia and Hodgkin’s diseases are due to the destruction of blast cells⁷.

The results in table 2 reveal that the mean values of sera TSA in leukemia and Hodgkin’s patients were elevated (P<0.001 an P<0.01, respectively) in comparison to normal healthy and pathological controls (P<0.001 and P<0.001 respectively). However the comparison of sera TSA for different types of leukemia (ALL, AML, CLL, and CML), the leukemia and Hodgkin’s patients show high level of TSA as compared with pathological controls and normal healthy. There was highly significant (P<0.001) difference between the serum TSA in (leukemia, Hodgkin’s) patients and normal healthy and even control groups, and no significant difference in sera TSA level in the four types of leukemia and Hodgkin’s are observed. It is clear from table 2 that the mean values of total sialic acid to total protein ratio (TSA/TP) in sera of leukemia and Hodgkin’s patients were significantly elevated (P<0.01) as compared to the normal healthy and control as shown in table 2.

Accordingly, the TSA/TP ratio could be useful markers for detecting malignancies. Some authors demonstrated a significant rise in TSA/TP and other biomarkers levels in cancer patients⁸. The cell death protein may induce local cellular and vascular reactions which may cause changes in the composition of extra cellular fluid⁹.

Table 3 shows the specificity and sensitivity of TSA test in normal individuals and patients with leukemia, Hodgkin’s and control. The specificity was calculated as the number of cases having TSA levels less or equal the upper limit of normal (73 mg/dL) divided by the total number of cases. The sensitivity of TSA test was calculated by dividing the total number of cases which have TSA levels upper than normal limit (73 mg/dL) divided by the total number of cases. The results from table 3 shows that more than 85% all kind of leukemia patients had elevated values of TSA, also Hodgkin’s had nearly the same elevated values, on the other, hand the normal and the control group the specificity test of TSA was 59.4% and the normal group 100%.

Table 3: Specificity and sensitivity of total sialic acid measurement

Group	No. of cases	Specificity %*	Sensitivity %**
ALL	79	12.65	87.34
AML	7	14.4	88.8
CLL	21	9.5	90.4
CML	45	11.1	85.5
Hodgkin’s	26	15.3	84.6
Normal	12	100	-
Control	37	59.45	40.54

* The number of cases has TSA values 73 mg/dL divided by the total number of cases by 100

** The number of cases has TSA values > 73 mg/dL divided by the total number of cases by 100

ALL = Acute Lymphocytic Leukemia

AML = Acute Myelocytic Leukemia

CLL = Chronic Lymphocytic Leukemia

CML = Chronic Myelocytic Leukemia

The standard curve was obtained (figure 2) by plotting the absorbance at 580 nm against the corresponding concentrations of standard sialic acid solutions which was used to determine the TSA levels in the serum samples. LASA levels in sera samples were determined using the standard sialic acid curve in figure 2.

Figure 2: the standard curve for determination of total sialic acid concentration

Many glycoproteins and glycolipids from malignant cells differ in carbohydrate composition from those found in normal cells since many of these glycoconjgates contain terminal sialic acid which can be shed into the circulation¹⁰.

Cell surface glycoproteins and glycolipids have altered cell to cell recognition; adhesion, antigenicity, and invasiveness in malignant conditions¹⁰. Increased sialic acid levels on the surface of transformed or malignant cell have been shown to be released into the circulation¹¹. The potential role of sialic acid in the mechanism of tumor cells by interfering with the immune response of the host and that sialic acid content appears to be correlated with metastatic ability in variety tumor cells¹².

Total sialic acid TSA is of great interest as a marker of malignancy although it has been demonstrated not to be specific for any type of cancer. Serum TSA levels have been found to be elevated in a number of different cancers. Studies by Shamberger and Dnistrain on adults affected with neoplasms of the hemopoietic system, point the usefulness of determining sialic acid in monitoring the neoplastic process^{3, 13}.

Increasing in the concentration of sialic acid was noted in neaplasmas of the lungs, gall bladder, melanoma, and neoplasm of the female sex organs, the increases in TSA level will increase the neoplasm during metastasis¹⁴.

Normalization of sialic acid concentration after treatment of the disease constitutes a favorable prognosis while its increase creates suspicion of relapse of the disease¹¹.

In patients affected by ovarian cancer it was noted that concentrations exceeding (120 mg/dL) were related to poor prognosis. Comparing the results in figure 3 with staging of the leukemia ALL, CMLL diseases showed a good agreement between total sialic acid concentration and course disease⁸.

Figure 3: TSA concentration in different stages of leukemia patients

Increases TSA levels after the end of treatment in comparison with the control group may be a result of minimal residual disease. In cases of relapse we observed again an increase of TSA concentration, its value remain high if the treatment failed. Figure 3 shows that TSA elevated to high level in last stage before death, for ALL and CML patients, 3 patients with ALL and 1 patient with CML, a serial determination for TSA was made one of ALL patient who died and the CML patient also who died with disease, had a significantly who elevated in TSA. This gives us a good tool for prognosis and monitoring stages of disease and the response for treatment. Because TSA is not a specific marker for a given type of neoplasm, changes in concentration can only be interpreted together with the clinical picture of the disease^{3, 8}.

Determination of lipid-associated sialic acid (LASA) levels in sera of patients with leukemia, Hodgkin's and non-cancer individuals:

Serum lipid-associated sialic acid (LASA) levels were determined in normal healthy persons and in non-cancer (control) patients, using the method of Katapodis et al., (189). Table 4 shows the mean value of LASA in four types of leukemia, Hodgkin's, control and normal individuals, and the comparison of the mean values of sera LASA between these groups. The result in this tale reveal over all elevation in LASA levels of each group of cancer compared with normal healthy and control individuals.

The increased LASA levels in sera with leukemia, Hodgkin's were statistically significant when compared with those obtained from normal healthy and control patients (0.001 and 0.001 respectively). Table 4 was calculated by the value of 19 mg/dL as the upper limit of normal. In general, high sensitivity was observed 100% for all types of leukemia and 96.1% for Hodgkin's, 72.9% for control (non-malignant) and 33.3% for normal healthy.

Table 4: specificity and sensitivity of LASA in leukemia, Hodgkin's normal, and control (malignant) patients

Group	No. of cases	Specificity %	Sensitivity %
ALL	79	Zero	100
AML	7	Zero	100
CLL	21	Zero	100
CML	45	Zero	100
Hodgkin's	26	2.85	96.15
Non-malignant	37	17.10	72,90
Normal healthy	12	66.60	33.30

* Specificity was calculated by division the value of LASA cases by 19.84 mg/dL multiplied by 100

** Sensitivity was calculated by the division the value of LASA cases by 19.84 mg/dL multiplied by 100

ALL = Acute Lymphocytic Leukemia

AML = Acute Myelocytic Leukemia

CLL = Chronic Lymphocytic Leukemia

CML = Chronic Myelocytic Leukemia

hus serum LASA has been suggested as useful tumor marker, more satisfactory than TSA. LASA is expected to behave as a non-specific tumor marker after shedding, and that is an important aspect of the turnover of normal cell surface constituents which occurs principally in both normal and malignant growing cell¹⁵.

Schutter et al¹⁴, measured the LASA levels in eight different cancers (lung, colon, ovarian, prostate, leukemia, thyroid, pancreas, adrenal and gastrointestinal cancer), the results were compared were normal with normal healthy. It shows that LASA is useful marker as a prognostic determinant in a variety of neoplastic condition. A serial determination of LASA was made for 3 colon, one prostate and one ovarian cancer patients, the two colon cancer patients who died with the metastatic disease had significantly elevated LASA values, while the prostate and ovarian cancer who died their LASA was decreased after surgery (60). Some researchers used TSA and TSA/TP and LASA in diagnosis of children with various malignancies, a significantly elevated them, and the researchers also observed the decreasing after successful treatment^14,15.

In this research LASA appear to be useful marker in distinguishing between healthy individuals and cancer patients, since LASA exhibits a high sensitivity. The combined use of it with other markers may provide a high degree of marker positively. Figure 4 shows a serial determination of LASA that was made in four leukemia patients, 3 ALL and 1 CML, the data show that one of the ALL and CML patient who died with disease had significantly elevated LASA values and on another hand the LASA had slightly decreased in patients who were responding to chemotherapy, the LASA levels of leukemia is > Hodgkin’s^{8, 15}.

Figure 4: LASA concentration in different stages of leukemia patients

CONFLICT OF INTEREST:

The author declares no conflict of interest.

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Received on 24.07.2020 Modified on 14.10.2021

Research J. Pharm. and Tech. 2022; 15(6):2650-2654.

DOI: 10.52711/0974-360X.2022.00443

Group	Samples No.	TP mg/Dl ± SD	TSA mg/dL ±SD	LASA mg/dL ±SD	TSA/TP ± SD
Leukemia	-	-	-	-	-
ALL	79	7.33 ± 0.93	91.1 ± 12.15	37.17 ± 14.22	13.14 ± 4.30
AML	45	7.54 ± 0.46	91.67 ± 16.30	35.02 ± 14.20	11.64 ± 3.20
CLL	21	7.36 ± 1.14	85.28 ± 11.48	34.16 ± 12.60	11.21 ± 3.53
CML	7	7.61 ± 0.28	91.42 ± 14.35	41.42 ± 10.69	13.8 ± 4.50
Hodgkin’s	26	7.22 ± 0.53	80.65 ± 11.45	32.23 ± 10.27	11.11 ± 3.25
Pthol. controls	37	7.30 ± 0.79	59.86 ± 10.80	24.60 ± 6.72	8.81 ± 0.47
Normal controls	12	7.09 ± 0.34	48.83 ± 8.02	20.66 ± 3.82	7.14 ± 0.48