DETERMINING THE POSSIBILITY OF ENZYMATIC TEST USING IN EARLY DIAGNOSIS OF GASTRIC CANCER

 

Biochemistry

 Borzenko B.G., 

PhD, professor of chemistry department of the Donetsk National Medical University, named after M. Gorky (DonNMU),

Vasilenko I.V.,

PhD, professor, head of the pathologic anatomy department of the DonNMU,

Bakurova E.M.,

 associate professor of chemistry department of the DonNMU,

Mironova K.A.,

head of the laboratory assistants of the chemistry department of theDonNMU,

Sh. A. Warsalee, student of the DonNMU 

 

DETERMINING THE POSSIBILITY OF ENZYMATIC TEST USING 

IN EARLY DIAGNOSIS OF GASTRIC CANCER

The enzymes activity of nucleotide metabolism study in tissues of gastric mucosa and serum in peptic ulcer and gastric cancer patients. It is established that, simultaneously increased activity of thymidine kinase, thymidine phosphorylase in peri-ulcerous zone, there serum activity are increased too (p<0.001, p <0.05, respectively). Due to those changes in the activity these enzymes in the blood reflect the features of the enzymatic changes in the tissues. The activity of enzymes in the presence of chronic atrophic gastritis, precancerous changes in the mucosa (intestinal metaplasia, dysplasia), is similar to the activity in cancer. The results showed that, the more pronounced cooperative effect changes in the activity of enzymes studied, the higher the risk of neoplastic transformation in patients with peptic ulcer disease. Investigation of changes in the activity of these enzymes in the serum in the dynamics can be used for monitoring patients at high risk of cancer developing.

Key words: chronic atrophic gastritis, peptic ulcer, metaplasia, dysplasia, gastric cancer, thymidine kinase, thymidine phosphorylase

Gastric mucosa (GM) – intensively regenerating tissue with highly proliferative activity. But the possibility of malignant gastric ulcers, according to various researchers, is small. However, the occurrence of ulcers in disregenerative processes such as epithelial dysplasia  are precancerous process [1]. Not accidentally, the disorders of the ratio of processes of tissue proliferation and apoptosis takes place, as in the pathogenesis of peptic ulcer (PU), and gastric cancer (GC). The level of tissue proliferation is largely determined by the metabolism of  DNA  and its predecessors. It is established that the endogenous pool of pyrimidine DNA precursors - thymidylate incorporation directly correlates with the proliferative capacity of tissue [4, 6]. The level of thymidine is regulated by key enzymes of «salvage way» synthesis - thymidine phosphorylase (TP), which catalyses the reversible reaction conversion of thymidine, and thymidine kinase (TK), consisting of thymidine into thymidylate (TMP). Thymidine phosphorylase works as a phosphorylase (catabolic, TPc) and transferase (anabolic, TPan) activity [4]. The latter, which is also growth factor in the angiogenesis of human gastric cancer, was first identified in platelets (PD-ECGF) [7]. Along with these effects, TP has anti-apoptosis action [2, 3]. It is known that the activity of TK directly correlates with the intensity of cell proliferation [5, 6]. The purpose of this investigation is to study the relative age-dependant activity of the TK, TP in patients with PU and in patients with GC .

Materials and methods. The material for investigation was the blood serum of 80 patients with PU in the age of 30 to 60 years old, 45 patients were examined with GC in age from 40 to 60 years.  The activity of tissue enzymes studied in the postoperative material from 30 patients with GC and 30 patients with ulcerous disease. As control, were used serum of 40 healthy individuals of similar age (30 – 60 ) without gastroduodenal pathology, and  distant from the tumour tissue or ulcer (Locus morbi) gastric mucous. TK activity was determined by a standard method [4] in a certain modification, using as substrate C14-Thymidine (Russia). Radioactivity drive diethyl-amino-ethylcellulose paper (Limbro, UK)  were counted in scintillation on the biological scintillation counter (SBS-2). TK activity was expressed as nanomole  thymidine phosphate formed for 1 hour per 1 mg of serum protein. The activity of TPc and TPan determined spectrophotometrically [4] the optical density of thymine (thymidine)  changing. Activity was expressed as nanomole of thymidine  (thymine), formed in 1 minute per 1 mg of protein.  Protein concentration was determined by the method of Lowry [8 ]. Statistical treatment of results was performed using parametric and nonparametric methods.

Results and discussion. It is known that the risk of cancer increases with age. It is possible that age-dependent increase in the incidence of stomach cancer contributes to more frequent development of dysplasia of the gastric mucosa in the elderly. We have previously investigated age-sensitive enzyme activity. Therefore, as in the control group and in the pathological group, activity of the enzymes were studied relative to age.  

Study of serum activity of TK, TPan, TPc was performed in age from 30 to 60 years. A significant increase in the activity of TK was established (see Table 1), which directly correlates with the age of healthy peoples. TK activity in patients with PU in the age of 30 to 50 years significantly increased as compared to control. In gastric cancer the maximum increase in TK (4-fold compared to normal) was observed in patients 40-49 years of age. It grows up to 8,15 ± 0,02 nmol / mg · hr.

 

 

 

Table 1. Age-dependent activity thymidine metabolism enzymes in the serum of blood in healthy and diseases 

Age,

years

ТК,   nmol/h·mg 

control

PU

GC

30-39

1,36±0,05

3,72±0,52*

-

40-49

2,17±0,14

3,15±0,28*

8,15±0,02**

50-59

5,44±1,10

6,01±0,17

6,82±1,02

 

TPan ,   nmol/min·mg 

control

PU

GC

30-39

9,88±0,68

11,21±2,38

-

40-49

9,90±0,98

14,01±2,24*

18,40±1,88**

50-59

9,25±0,78

12,18±1,50*

16,15±2,54*

 

TPc ,   nmol/min·mg 

control

PU

GC

30-39

41,61±4,03

27,06±3,98

-

40-49

43,84±3,20

25,68±4,21*

17,64±2,30***

50-59

46,21±2,44

22,73±3,98**

13,47±1,32***

 

 

*- р < 0,05, ** - р < 0,01, *** - р < 0,001 in comparison with control

In the control group studied, the activity of TPan did not change significantly. In patients with PU, and GC were found an increase in enzyme activity.


In studying the activity of TPc, no significant age-related changes in healthy subjects were noted. In patients with PU and GC after 40 years, the enzyme activity in serum is reduced by 1.8 and 3 times respectively.

The observed change in activity of metabolic enzymes thymidine in patients with peptic ulcer disease and gastric cancer at the age of 40-60 years can lead to an increase in the endogenous pool of DNA precursors and, therefore, to an increase in the intensity of its synthesis and the rate of tissue proliferation.

Cluster analysis show a different intensity changes in enzyme activity in uncomplicated (PU group 1) compared with ulcerative disease against with atrophic and precancerous lesions change in the gastric mucous membrane, usually from complications (PU group 2). Since rates of enzyme activity were distributed among 4 clusters (C1, C2, C3, C4).Between control groups and GC (C1 and C4) are maximal differences, a group of patients with ulcerative disease is heterogeneous. The values ​​of enzyme activity among the patients of the 1st and 2nd groups, PU (or clusters C2 and C3) was significantly different, with the latter indicators of activity are close to those in GC. Shifts of enzymatic activity are most pronounced in clusters C3 and C4, i.e., in cancer and in complicated peptic ulcer disease that occurs on the background of atrophy of the mucosa, precancerous changes. These biochemical studies confirmed pathologic study.
              In subsequent studies, investigations of the comparative enzymes activity  in gastric mucosal biopsies from ulcerous zone,  tumours and remote areas of gastric mucosa (GM) from Locus morbi at the age of 40-60 years (Table 2).

 

 

 

Table 2. The tissues enzymes activity

 

Диагноз

ТPаn

ТPc

GM

control

Locus

morbi

GM

control

Locus

morbi

 PU  1st  group

 (n = 10)

37,68 ±

4,18

62,00 ±

7,13**

63,89 ±

5,96

33,84 ±

2,95**

PU 

2nd group

(n = 10)

40,05 ±

5,96

83,75 ±

6,22***

62,04 ±

6,40

25,50 ±

2,50***

 GC

(n = 20)

40,46 ±

4,77

88,01 ±

5,32***

57,82 ±

7,99

21,92 ±

4,60**







 

*- р < 0,05, ** - р < 0,01, *** - р < 0,001 in comparison with control

In biopsies from periulcerous zone as compared to remote areas of mucous membrane (GM), reduced the activity of  ТPc in patients BU group 1 was 1.9 times,  the TPan activity increased by 1.6 times. The most characteristic feature in the subsequent pathomorphologic  study of this group was the presence of inflammation of the shaft with deposit of fibrin lymphohistiocytal infiltration in the peri ulcerous zone, the presence of gastritis without signs of atrophy (see Fig.1).

FIG. 1. The pathomorphologic  changes in gastric mucous in PU 1st group

In group 2, patients with active PU had highest TPan activity (an increased 2-fold) and had minimal TPc activity (decreased on average by 2.4 times). In the pathomorphologic study was found the development of ulcerative process in the background of chronic atrophic gastritis, intestinal metaplasia, dysplasia of varying degree (see Fig. 2). Such a shift in thymidine phosphorylase activity was observed in stomach cancer. TPc reduced it to 2.6, and for TPan increased by 2.2 times.

FIG. 2. The pathomorphologic changes in gastric mucous in PU 2d group

The results showed that by screening ulcer patients and carrying out research activity of serum TK, TP because their shifts are parallel to changes in activity in the tissues.

At present, reliable data on the development of cancer from chronic  ulcers and the frequency of malignant gastric ulcers can be obtained only by dynamic monitoring of patients using gastric biopsy.  The study of the proposed enzymes in serum may reasonably select a group of cancer risk among ulcer patients.

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