- Email: [email protected]
Plasma oxypurines in gastric and colorectal cancer
Biomed & Pharmacother (1990)44.403-l-07
(DRhevier,Paris
M Lorenzi2, B Porcelli’, D Vannonil, R Leoncinil, M Pizzichini’, R Pagan?, E Marinellol’
A Di Stefano*,
tistituto di CitimicclBiologica;
21s&itutodi Clinica Chirurgica Universird di Siena. Pian dei Mantel&d 44, 53100 Siena. Italy
(Received 2 October 1989; accepted 6 Augast 1999)
Summary - Reverse phase high performance liquid cbr~atography has been used for the determination of plasma hypoxanthine, xanthme, and uric acid in notrnaI subjects and in patients with gastric and cotorectal cancer. Plasma oxypurines are significantly elevated in either type of cancer, while uric acid concentration is only higher in gastric crmcer.The variations are related to the stage of the tumors, and the physiopathology of their occnrrence is discussed. oxyputines I gastrtc cancer I colorectal cancer R&urn&- Taux d’oxypurines plasmatiques dans les canceelS gastriques ou colorectaux. Nous avow mestud ks tutu d’hypomttthine, de xunfhine et d’ncidt? urique plasmatiques chez des sujets atteints de cancer gastrique ou cokmctal. L’hypoxanthine est significativement dlevke &ns le plasma pour les dew types de tumew>la xamthine est &ev&e, mais de fugon twn sigttificative~ tandis qtte la concentration de I’acide urique est augment& wdquenzent chez ks sujets atteints de &mew gastrique. L-es v~i~.o~ sent mises en corr~i&tionavec le stade des twtreurs;ia phys~~~t~lo~ie de leur incidence est dismttfe. oxyptutues / cancer gastrhpte I cancer eotoreetal
Introduction The levels of oxypurines have been extensively investigated in biological fluids in many diseases [2, 7-11, 13, 16, 23-251, However, their determination, until a few years ago, presented several problems, as the analytics methods used were of dubious sensitivity and specificity [1, 4, 5, 14, 221. Using a very sensitive and specific high performance liquid chromatographic method, we measured the plasma levels and urine concentrations of both the 2 purine bases derived by nucleotide catabolism (hypoxanthine and xanthine), and of uric acid, in patients affected by gout, rheumatoid arthritis, leukemia and several forms of neoplasia [6, 17-211. Little data, however, is available concerning the behavior of the oxypurines in neoplasia. * Correspondenceand reprints
We have attempted to obtain more information regarding the behavior of these compounds in cancer patients and to investigate their possible diagnostic role. In fact, it is reasonable to conceive that, especially in pathologies characterized by a rapid cellular p~life~tio~, such as lymphopro~ife~tive diseases and tumors, an accelerated pmine turnover should be reflected in altered levels of purines in biological fluids. We have extended some preliminary observations Vi, 181 regarding plasma concentrations of hypoxantbine, xanthine and uric acid in gastric and colorectal cancer: their levels have been compared with tumor stages and grades.
We examined 27 healthy control subjects, 30 patients with gastric and 47 with colorectal cancer: all with pri-
M Lorenzi et al tal cancer. The number of cases examined at each stage is reported in tables I and IL Hepatic metastases were present in 12 cases, peritoneal in 4. All blood samples for the determination of purine bases and uric acid were taken at 8 am from fasting patients. Plasma samples were kept at -80°C until HPLC analysis. The blood drawn to measure purine bases was rapidly transferred into cooled and heparinized tubes, then centrifuged at 4°C (3 000 rpm for 15 min). The determinations of purine bases and of uric acid were carried out by HPLC using a Beckman mod 332, at a
mary tumors. None had a history or clinical evidence of kidney diseases or alteration of purine metabolism; their levels of urea and creatinine were normal and urine sediment presented no pathological findings. All patients with primary tumors were operated (explorative or palliative 32%, radical 68%). The “histopathological grading” of adenocarcinoma was determined generally and results are reported in tables I and II (Grade 1: well and moderately differentiated; Grade 2: poorly differentiated, Grade 3: mutinous adenocarcinoma). The patients were staged according to the UICC classification [15] for the gastric and to Dukes [3] for the colorec-
Table I. Plasma oxypurines Qtmol/l) and uric acid (mmol/l) levels in patients with gastric cancer. Results are the mean f SD. Tumor feature
No of patients
Hypoxanthine
Xanthine
Uric acid
Stage I Stage II Stage III StageIV
5 5 8 12
1.78 2.58 2.20 4.16
Grade1 Grade 2 Grade 3
12 8 4
3.18 f 0.80** 3.66 f 1.03** 2.35 + l.lO**
0.53 f 0.14 1.01 St 0.50** 1.63 & l.Ol**
0.19 f 0.04 0.21 * 0.02** 0.22 + 0.12
sizeSCIll
11 15
2.12 f 0.45** 3.84 + 0.90**
0.72 f 0.40 0.44 f 0.10
0.21 f 0.14* 0.17 f 0.02
Metastases absent Metastases uresent
25 5
3.31 z!z0.60** 2.21 f 0.70**
0.85 f 0.20* 0.58 z!z0.10
0.19 * 0.02** 0.18 + 0.06
f + + f
0.30** 0.90** 0.40** l.lO**
1.16 0.77 0.80 0.66
f + + f
0.88* 0.30* 0.50 0.10**
0.27 0.11 0.18 0.18
z!z0.12** f 0.04 + 0.02* f O-02*
* P c 0.05. ** P c 0.01, compared with normal
Table II. Plasma oxypurines (pmol/l) and uric acid (mmoljl) levels in patients with colorectal cancer. Results are the mean f SD. Tumor feature
No of patients
Hypoxanthine
0.80** 0.50** 0.70** 1.50**
0.76 1.47 0.96 0.97
f f & It
0.40** 0.50** 0.40** 0.50**
Uric acid
0.12 0.17 0.13 0.19
+ f f f
0.02 0.03** 0.01** 0.04**
Stage A Stage B Stage C Stage D
11 17 9 10
3.69 2.76 2.78 2.30
Grade 1
Grade 2 Grade 3
28 4 5
3.18 + 0.50** 2.66 + 0.71** 2.16 f 0.64**
1.33 + 0.40** 0.83 + 0.50** 1.04 f 0.60**
0.14 f 0.02 0.22 + 0.20 0.16 f 0.06
Sizec5cm Size>5cnl
25 16
3.11 i 0.50** 2.90 f 0.50
1.06 f 0.30** 1.27 z!z0.10
0.15 f 0.02 0.15 f 0.02
Metastases absent Metastases present
36 11
3.10 f 0.30** 2.24 f 0.40**
1.56 f 0.50** 0.86 f 0.50
0.15 f 0.02 0.17 It 0.03
* P < 0.05. ** P c 0.01, compared with normal
+ f f +
Xanthine
Plasma oxypurines in gastric and colorectal cancer fixed wavelength of 254 nm, with a Supelcosil column LC-18 (5 pm) 4.6 mm x 25 cm. 0.5 ml of plasma was added to 0.1 ml 4 N HClQ. and, after centrifugation at 3 000 rpm for 10 min. the clear supernatant was neutralized by adding 0.02 ml of 5 N KOH. The plasma samples were eluted with a 0.01 M KaHPO4 buffer (pH 5.5) and a linear gradient between O-25% methanol for 30 mim the flow rate was 1 ml/min. Under these chromatographic conditons, the retention times of uric acid, hypoxanthine and xanthine were: 6.6, 11.1 and 12.4 min, respectively (fig 1). As the peak of guanine exhibited the same retention time of hypoxanthine, each sample was submitted to a duplicate run, before and after treatment with 0.015 ml of xanthine oxidase (Boehringer Manheim Gmbh. 20 U/ml) in order to eliminate the hypoxanthine peak, while maintaining the guanine peak.
STAIDAIDS
405
Quantitative determination of the bases was obtained by comparing the surface of the peaks with suitable calibration curves. Peaks were identified through the evaluation of retention time. co-elution of known standards, and disappearance after treatment with xanthine oxidase. Statistical analysis of data was carried out using an analysis of variance (F-test).
Results The pIasma hypoxanthine, xanthine and uric acid levels in controls, in gastric and colorectaf cancer are shown in table III. Tables I and II show the same parameters, in relation to stage, grade and tumors in all 77 cancer patients. Hypoxanthine
The average plasma levels of hypoxanthine are significantly higher in patients with gastric and colorectal ca.ncer, than in the controls (P c 0.01). When stage, grade, size and metastases of the neoplasia are considered, significant increases of hypoxanthine are evident (P c 0.01) in gastric cancer. All mean values also increase significantly in colorectal cancer except for size > 5 cm. Xanthine
Fig 1. Chromatograms of a standard mixture (uric acid, hypoxanthine and xanthine) and of a plasma sample. In brackets: the retention times. Chromatographic conditions are reported in the text.
This base also shows significative variations in cancer compared to controls (P c 0.05). For gastric tumors the increase is more evident at stage I and grades 2 and 3, while in colorectal tumors the levels are enhanced in all stages and grades.
Plasma levels in hypoxantlrhre,xanthine (pmol/l), uric acid [mmol/l)in gastricaudin coioreCtal Cancf~.Resultsa~ the mean + SD. -
Table ILL
No
of putients
Age
Hypoxanthine
Xanthine
Uric acid
Normalsubjects
27
19-49
0.65 f 0.09
0.28 f 0.07
0.14 f 0.01
Gastric cancer
30
25-85
2.98 f 0.40**
0.80 f 0.19*
0.19 f 0.02 ** (25)
Colorectal cancer
47
42-86
2.90 + 0.30**
1.11 * 0.24*
0.16 + 0.013
* P c 0.05. ** P c 0.01 in comparisonto the controls
(38)
(DRhevier,Paris
M Lorenzi2, B Porcelli’, D Vannonil, R Leoncinil, M Pizzichini’, R Pagan?, E Marinellol’
A Di Stefano*,
tistituto di CitimicclBiologica;
21s&itutodi Clinica Chirurgica Universird di Siena. Pian dei Mantel&d 44, 53100 Siena. Italy
(Received 2 October 1989; accepted 6 Augast 1999)
Summary - Reverse phase high performance liquid cbr~atography has been used for the determination of plasma hypoxanthine, xanthme, and uric acid in notrnaI subjects and in patients with gastric and cotorectal cancer. Plasma oxypurines are significantly elevated in either type of cancer, while uric acid concentration is only higher in gastric crmcer.The variations are related to the stage of the tumors, and the physiopathology of their occnrrence is discussed. oxyputines I gastrtc cancer I colorectal cancer R&urn&- Taux d’oxypurines plasmatiques dans les canceelS gastriques ou colorectaux. Nous avow mestud ks tutu d’hypomttthine, de xunfhine et d’ncidt? urique plasmatiques chez des sujets atteints de cancer gastrique ou cokmctal. L’hypoxanthine est significativement dlevke &ns le plasma pour les dew types de tumew>la xamthine est &ev&e, mais de fugon twn sigttificative~ tandis qtte la concentration de I’acide urique est augment& wdquenzent chez ks sujets atteints de &mew gastrique. L-es v~i~.o~ sent mises en corr~i&tionavec le stade des twtreurs;ia phys~~~t~lo~ie de leur incidence est dismttfe. oxyptutues / cancer gastrhpte I cancer eotoreetal
Introduction The levels of oxypurines have been extensively investigated in biological fluids in many diseases [2, 7-11, 13, 16, 23-251, However, their determination, until a few years ago, presented several problems, as the analytics methods used were of dubious sensitivity and specificity [1, 4, 5, 14, 221. Using a very sensitive and specific high performance liquid chromatographic method, we measured the plasma levels and urine concentrations of both the 2 purine bases derived by nucleotide catabolism (hypoxanthine and xanthine), and of uric acid, in patients affected by gout, rheumatoid arthritis, leukemia and several forms of neoplasia [6, 17-211. Little data, however, is available concerning the behavior of the oxypurines in neoplasia. * Correspondenceand reprints
We have attempted to obtain more information regarding the behavior of these compounds in cancer patients and to investigate their possible diagnostic role. In fact, it is reasonable to conceive that, especially in pathologies characterized by a rapid cellular p~life~tio~, such as lymphopro~ife~tive diseases and tumors, an accelerated pmine turnover should be reflected in altered levels of purines in biological fluids. We have extended some preliminary observations Vi, 181 regarding plasma concentrations of hypoxantbine, xanthine and uric acid in gastric and colorectal cancer: their levels have been compared with tumor stages and grades.
We examined 27 healthy control subjects, 30 patients with gastric and 47 with colorectal cancer: all with pri-
M Lorenzi et al tal cancer. The number of cases examined at each stage is reported in tables I and IL Hepatic metastases were present in 12 cases, peritoneal in 4. All blood samples for the determination of purine bases and uric acid were taken at 8 am from fasting patients. Plasma samples were kept at -80°C until HPLC analysis. The blood drawn to measure purine bases was rapidly transferred into cooled and heparinized tubes, then centrifuged at 4°C (3 000 rpm for 15 min). The determinations of purine bases and of uric acid were carried out by HPLC using a Beckman mod 332, at a
mary tumors. None had a history or clinical evidence of kidney diseases or alteration of purine metabolism; their levels of urea and creatinine were normal and urine sediment presented no pathological findings. All patients with primary tumors were operated (explorative or palliative 32%, radical 68%). The “histopathological grading” of adenocarcinoma was determined generally and results are reported in tables I and II (Grade 1: well and moderately differentiated; Grade 2: poorly differentiated, Grade 3: mutinous adenocarcinoma). The patients were staged according to the UICC classification [15] for the gastric and to Dukes [3] for the colorec-
Table I. Plasma oxypurines Qtmol/l) and uric acid (mmol/l) levels in patients with gastric cancer. Results are the mean f SD. Tumor feature
No of patients
Hypoxanthine
Xanthine
Uric acid
Stage I Stage II Stage III StageIV
5 5 8 12
1.78 2.58 2.20 4.16
Grade1 Grade 2 Grade 3
12 8 4
3.18 f 0.80** 3.66 f 1.03** 2.35 + l.lO**
0.53 f 0.14 1.01 St 0.50** 1.63 & l.Ol**
0.19 f 0.04 0.21 * 0.02** 0.22 + 0.12
size
11 15
2.12 f 0.45** 3.84 + 0.90**
0.72 f 0.40 0.44 f 0.10
0.21 f 0.14* 0.17 f 0.02
Metastases absent Metastases uresent
25 5
3.31 z!z0.60** 2.21 f 0.70**
0.85 f 0.20* 0.58 z!z0.10
0.19 * 0.02** 0.18 + 0.06
f + + f
0.30** 0.90** 0.40** l.lO**
1.16 0.77 0.80 0.66
f + + f
0.88* 0.30* 0.50 0.10**
0.27 0.11 0.18 0.18
z!z0.12** f 0.04 + 0.02* f O-02*
* P c 0.05. ** P c 0.01, compared with normal
Table II. Plasma oxypurines (pmol/l) and uric acid (mmoljl) levels in patients with colorectal cancer. Results are the mean f SD. Tumor feature
No of patients
Hypoxanthine
0.80** 0.50** 0.70** 1.50**
0.76 1.47 0.96 0.97
f f & It
0.40** 0.50** 0.40** 0.50**
Uric acid
0.12 0.17 0.13 0.19
+ f f f
0.02 0.03** 0.01** 0.04**
Stage A Stage B Stage C Stage D
11 17 9 10
3.69 2.76 2.78 2.30
Grade 1
Grade 2 Grade 3
28 4 5
3.18 + 0.50** 2.66 + 0.71** 2.16 f 0.64**
1.33 + 0.40** 0.83 + 0.50** 1.04 f 0.60**
0.14 f 0.02 0.22 + 0.20 0.16 f 0.06
Sizec5cm Size>5cnl
25 16
3.11 i 0.50** 2.90 f 0.50
1.06 f 0.30** 1.27 z!z0.10
0.15 f 0.02 0.15 f 0.02
Metastases absent Metastases present
36 11
3.10 f 0.30** 2.24 f 0.40**
1.56 f 0.50** 0.86 f 0.50
0.15 f 0.02 0.17 It 0.03
* P < 0.05. ** P c 0.01, compared with normal
+ f f +
Xanthine
Plasma oxypurines in gastric and colorectal cancer fixed wavelength of 254 nm, with a Supelcosil column LC-18 (5 pm) 4.6 mm x 25 cm. 0.5 ml of plasma was added to 0.1 ml 4 N HClQ. and, after centrifugation at 3 000 rpm for 10 min. the clear supernatant was neutralized by adding 0.02 ml of 5 N KOH. The plasma samples were eluted with a 0.01 M KaHPO4 buffer (pH 5.5) and a linear gradient between O-25% methanol for 30 mim the flow rate was 1 ml/min. Under these chromatographic conditons, the retention times of uric acid, hypoxanthine and xanthine were: 6.6, 11.1 and 12.4 min, respectively (fig 1). As the peak of guanine exhibited the same retention time of hypoxanthine, each sample was submitted to a duplicate run, before and after treatment with 0.015 ml of xanthine oxidase (Boehringer Manheim Gmbh. 20 U/ml) in order to eliminate the hypoxanthine peak, while maintaining the guanine peak.
STAIDAIDS
405
Quantitative determination of the bases was obtained by comparing the surface of the peaks with suitable calibration curves. Peaks were identified through the evaluation of retention time. co-elution of known standards, and disappearance after treatment with xanthine oxidase. Statistical analysis of data was carried out using an analysis of variance (F-test).
Results The pIasma hypoxanthine, xanthine and uric acid levels in controls, in gastric and colorectaf cancer are shown in table III. Tables I and II show the same parameters, in relation to stage, grade and tumors in all 77 cancer patients. Hypoxanthine
The average plasma levels of hypoxanthine are significantly higher in patients with gastric and colorectal ca.ncer, than in the controls (P c 0.01). When stage, grade, size and metastases of the neoplasia are considered, significant increases of hypoxanthine are evident (P c 0.01) in gastric cancer. All mean values also increase significantly in colorectal cancer except for size > 5 cm. Xanthine
Fig 1. Chromatograms of a standard mixture (uric acid, hypoxanthine and xanthine) and of a plasma sample. In brackets: the retention times. Chromatographic conditions are reported in the text.
This base also shows significative variations in cancer compared to controls (P c 0.05). For gastric tumors the increase is more evident at stage I and grades 2 and 3, while in colorectal tumors the levels are enhanced in all stages and grades.
Plasma levels in hypoxantlrhre,xanthine (pmol/l), uric acid [mmol/l)in gastricaudin coioreCtal Cancf~.Resultsa~ the mean + SD. -
Table ILL
No
of putients
Age
Hypoxanthine
Xanthine
Uric acid
Normalsubjects
27
19-49
0.65 f 0.09
0.28 f 0.07
0.14 f 0.01
Gastric cancer
30
25-85
2.98 f 0.40**
0.80 f 0.19*
0.19 f 0.02 ** (25)
Colorectal cancer
47
42-86
2.90 + 0.30**
1.11 * 0.24*
0.16 + 0.013
* P c 0.05. ** P c 0.01 in comparisonto the controls
(38)