Journal of Surgical Oncology 16:37-41 (1981)

Evaluation of Whole Blood Catalase Estimation for Diagnosis of Malignancy .......................................................................................... ..........................................................................................

JOHN FITZPATRICK, MD, ARVIND BHARGAVA, PhD, RAMEZ BEDWANI, MD, and MARCEL GAGNON, PhD

Whole blood catalase levels were estimated using a disc flotation method in 209 random patients with a wide variety of malignancies. Fifty patients had received no treatment, and the remainder, although having undergone prior therapy, had recurrent or metastatic disease at the time of the study. No rela- tionship was found between the presence of cancer and catalase levels. A direct relationship was found for catalase with hemoglobin levels in both normal and patients’ samples. Whole blood catalase is of no value in diagnosis and monitor- ing of cancer. The decreased catalase values found here and reported previously by others are the result of low hemoglobin levels found in many patients with cancer.

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Key words: whole blood, catalase, malignancy

INTRODUCTION

Catalase, an iron-containing hemoprotein, is the enzyme that decomposes hydrogen peroxide to water and oxygen and is present in largest amounts in liver, kidney, and red blood cells [ l , 2 ,3 ,4] . Previous reports [ l , 2 ,3 ,5 ,6 ] have demonstrated reduction in normal liver tissue catalase activity in rats and mice bearing a wide variety of tumors, though no difference from normal was observed in whole blood catalase activity [2 ,6] . Similar reduction in human liver catalase activity has been shown in association with ad- vanced cancer [4,7]. Decreased blood catalase activity also has been reported in patients with a variety of untreated cancers [5]. It was further suggested [5] that there was a posi- tive association between the recurrence of tumor following surgery and blood catalase

From the Departments of Laboratory Medicine and Epidemiology, Roswell Park Memorial Institute, Buffalo, and Centre de Recherches en Sciences AppliquCs a l’Alimentation, Universitk du Quebec a Montreal.

Address reprint requests to John Fitzpatrick, MD, Department of Laboratory Medicine, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, NY 14263.

0022-4790/81/1601-0037$01.40 @ 1981 Alan R. Liss, Inc.

38 Fitzpatrick et a1

levels. Most tests used for measurement of catalase activity have been time consuming and cumbersome, requiring rigid temperature and pressure controls. The following study was therefore begun to see if measurement of whole blood catalase activity using a simple test system [9] was indeed a practical and useful diagnostic and monitoring test for cancer.

MATERIALS AND METHODS

A total of 209 random patients with a variety of malignancies, including carcinomas of lung, breast, stomach, colon, uterus, cervix, and kidney; malignant melanoma; lymphoma; and acute and chronic leukemia, were studied. Fifty patients had received no previous ther- apy, and 159 were receiving or had previously received treatment, including surgery, radia- tion therapy, or chemotherapy, but had either local recurrent or metastatic disease at the time of sampling.

anticoagulant or venous blood anticoagulated with ethylenediaminetetraacetic acid using the disc flotation method of Gagnon et a1 [9].

For this method, whole blood (10 pl) was lysed in 1 .O ml of distilled water contain- ing 0.02% ethanol to arrest decrease in catalase activity during storage [ lo ] . After 10 min, 14 ml of 0.01 M phosphate buffer, pH 8.4, was added. Absorbant paper discs 12.7 mm in diameter were then saturated with the solution of blood. Excess drops adhering to the disc were removed, and the disc was dropped into a cuvette (16-mm diameter and 125 mm in height) containing 5 ml of freshly prepared 3.0% hydrogen peroxide solution. The cuvette was inserted into a patent automatic timing device (Catalasimetre, Allca Instrument Co., Ltd., Montreal, Canada), and the time of fall and rise to the surface of the solution was recorded in seconds. The oxygen liberated by catalase action from the hydrogen peroxide adheres to the disc, causing it to float to the surface. Each patient sample was measured in triplicate, and the mean of the results recorded as the disc flotation time (seconds). Discs moistened in distilled water (0.02% ethanol) and phosphate buffer served as controls. In solutions containing catalase, only the log 10 of the disc flotation time bears a straight- line relationship to log 10 catalase concentration. Results were expressed in seconds (disc flotation time) with a shorter time expressing a greater catalase concentration and vice versa.

(disc flotation time) of cases and controls according to their hemoglobin level; a P-value maximum of 0.05 was used for significance. A two-way analysis of variance [ l 11 was ap- plied to exclude intergroup and intragroup differences and to show the effect of cancer and/or hemoglobin level on catalase value. Pearson’s correlation coefficient was applied to test for the relationshp between hemoglobin levels and catalase values of normal con- trols and cancer cases. Confidence limits for each catalase value corresponding to each hemoglobin level were also calculated.

Whole blood catalase estimation was performed on either capillary blood with no

Student’s t-test was applied to test for the difference between mean values of catalase

RESULTS

No difference was found in catalase activity between finger-stick and venous anti- coagulated blood in simultaneous analysis of samples from 10 normal donors and 30 can- cer patients. As venous samples were easier to obtain, anticoagulated blood was used in all studies.

Whole Blood Catalase and Malignancy 39

No catalase activity was found in normal or patient plasma with all flotation values being above 600 seconds. Because whole blood catalase activity derives mainly from the red blood cell [8] with only minimal contribution from the white cells, whole blood cata- lase would seem related to hemoglobin level. To test for the effect of hemoglobin, dilu- tions of red blood cells of normal controls in their own plasma were prepared to produce a range of hemoglobin values from 6.0 to 16.0 gm/dl. Figure 1 shows individual flotation time readings according to hemoglobin for these normal controls. A linear negative correla- tion was demonstrated between hemoglobin value and flotation time (seconds) in normal controls (r < 0.8), indicating a direct positive correlation between actual catalase value and hemoglobin level with decreasing catalase activity (increased high flotation time) in whole blood associated with decreasing hemoglobin. This significant effect of hemoglobin was therefore taken into account in the subsequent analysis of patient data.

cancer, the 209 patients were divided for analysis into two groups according to whether or not they had previously received any form of therapy. Group I contained 50 patients who had no previous definitive therapy and Group I1 had 159 patients with persistent extensive or recurrent cancer despite therapy.

values (disc flotation time in seconds) of cancer cases in Groups I and II and normal con- trols, taking into account the effect of variation in hemoglobin level. In Group I , there were 38 (76%) cases with a hemoglobin level of 12.0 gm/dl and over, whereas in Group I1 only 70 (44%) had hemoglobin values in this range.

Two-way analysis of variance was undertaken on data in Table I to show the effect of both hemoglobin and the presence of cancer independently on catalase values. When the hemoglobin effect was included, catalase values for all cancer patients in combined

To exclude the possible effects of therapy on blood catalase levels in patients with

Table I shows the distribution of cases together with comparison of mean catalase

;; 2 2 .

r

0

1

Y 2 0 '

I-

I-

2 3 I6 IL

0 14

12 I

0

Fig. 1. Relationship between catalase level (disc flotation time in seconds) and hemoglobin for normal controls between hemoglobin values 6.0 and 16.0 gm/dl.

40 Fitzpatrick et a1

TABLE 1. Comparison of Catalase Levels in Normal Controls and Between Patients With Cancer According to Hemoglobin Value

Hemoglobin Normal controlsa Group Ib Group IIc

- n X n X SE n X SE SE

- - (gm/dl)

6-7.9 11 22.9 0.8 - ~ - 4 17.1 1.5 8-9.9 6 16.0 0.6 5 13.8 0.6 26 14.2 0.7

10-11.9 4 15.9 1.5 7 12.5 0.6 59 12.8 0.2 12-13.9 10 12.5 0.4 20 11.1 0.3 47 11.4 0.3 14-15.9 9 10.5 0.1 18 10.5 0.3 23 10.8 0.3 Total 40 15.6 2.1 50 12.0 0.7 159 13.3 1.1

"n = number of Faniples. X = mean catalase value (sec). SE = standard error of mean. bGroup I = cases having no previous therapy. %roup I1 = cases with prior therapy but with persistent disease.

Groups I and I1 was not significantly different from normal (P < 0.07). Similarly, no inter- group differences between untreated (Group I) and treated (Group 11) patients were found. However, there were significant intragroup differences for catalase values according to hemoglobin level in Group I and Group I1 (P < 0.02) similar to that found in normal con- trols and shown in Figure 1. Thus, catalase activity in both normal and cancer patients is directly dependent upon the actual hemoglobin level and has no relationship to tumor presence, stage, or status of treatment.

DISCUSSION

Previous animal studies [ l , 2 , 3 , 6 ] , while showing a reduction in liver catalase con- tent in rats with widespread tumors not necessarily involving the liver directly, have failed to show an alteration in whole blood catalase level [ 2 , 61. However, Ishikawa et a1 [5] re- ported a decrease in both liver and whole blood catalase in tumor-bearing rats. Removal of the tumor results in a return of both values to normal. The same authors extended their studies to 82 cancer patients and showed that the average value of whole blood catalase was significantly lower. However, 18 patients had no change in catalase, and no account was taken of the level of hemoglobin, hematocrit, or red cell count.

red cells, found no difference from normal for the mean RBC catalase value in 48 patients with various carcinomas. In this study, the effect of hemoglobin on results was negated by the use of packed red cells adjusted to constant hemoglobin level for catalase estima- tion. As in our studies, a linear relationship between hemoglobin and blood catalase was found.

Tudhope [8], in a study of red cell (RBC) catalase using patients' washed and packed

CONCLUSIONS

It is apparent from the present study and that of Tudhope [8] that hemoglobin level is a major determining factor in whole blood catalase level. Ishikawa's study [5] failed to take hemoglobin level into account and thus found a reduction in catalase level in patients with cancer. The present study and the more limited one of Tudhope would suggest that

Whole Blood Catalase and Malignancy 41

there is no relationshp between blood catalase activity and presence of cancer. The previ- ous effect noticed is most likely due to reduced hemoglobin levels frequently found among patients with advanced cancer.

ACKNOWLEDGMENTS

Technical assistance was provided in part through a grant from the Association for

The authors acknowledge the technical help of Daniel Mink, Shiela Lazaration, and Research of Childhood Cancer, Inc.

Lise DubC.

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