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Evaluation of megadose vitamin therapy in an experimental brain tumor
Evaluation of Megadose Vitamin Therapy in an Experimental Brain Tumor Samuel D. Newell, Jr., B.S., John Kapp, M.D., Ph.D., and John H. Romfh, Ph.D.
A mixture of vitamins C and B12 in high dosage, which has mental design of Poydock and associates [8], applying it to been reported to eradicate ascites tumors in rats, was brain tumors instead of ascites tumors. tested for its antineoplastic effect against the L9 glioma in Fisher CDF strain rats. No difference in survival time Materials and Methods between animals receiving the vitamin mixture and con- The Brain Tumor Model trols could be demonstrated. Possible reasons for the dif- The tumor used in this study was the L9 gliosarcoma grown ferent response to therapy in the two experimental t u m o r in a monolayer cell culture in Eagle's basal medium and systems are discussed. 10% fetal calf serum supplemented with 398 /,g/ml Newell SD Jr, KappJ, RomfhIH: Evaluationof glutamine, 80.5 units/ml penicillin, and 80.5 mg/ml strepmegadosevitamin therapyin an experimentalbrain tomycin. The monolayer was harvested with 0.25% trypsin tumor. Surg Neurol 16:161-164, 1981 EDTA and suspended in Gey's balanced salt solution at a concentration of 4 x 106 cells/ml. In 1974 and 1976 Cameron and Pauling stimulated interest These cells were transplanted into a group of 35 adult in the vitamin therapy of cancer with their suggestion that male CDF rats using the methods of Barker and associates ascorbic acid was an important factor in host defense [1]. The rats were anesthetized with sodium pentobarbital against cancer [3, 4]. Numerous mechanisms by which (60 milligrams per kilogram of body weight) and placed in a vitamin C might act to inhibit cancer have been sug- stereotactic head holder. Following the atlas of K6nig and gested, including increasing stromal integrity of normal Klippel [7], a drill hole was made in the skull 4.0 mm antissue, stimulating lymphocytes to a higher level of terior to the frontal zero plane and 2.5 mm to the left of the immunocompetence, inducing "favorable change in the midline with a No. 5 dental burr. A stainless steel, 0.40 x steroid environment," inhibiting hyaluronidase activity in 0.0625 inch screw with a 0.03 inch, center-drilled hole was malignant cells, increasing antiviral activity, and interfer- screwed into the hole in the skull. A blunted 22-gauge ing with the metabolism of malignant cells [3, 4, 9, 11]. It needle was fashioned with a stop so that when the needle has also been suggested that vitamin C is selectively con- was inserted through the hole in the screw, the tip of the centrated in tumors and may form cytotoxic amounts of needle was allowed to extend exactly 3 mm beyond hydrogen peroxide within the tumor as a byproduct of oxi- the end of the screw. By means of this needle and a dation [2, 9, 11]. microliter syringe, 0.1 /,1 of the cell suspension (a total In 1979 Poydock and co-workers [8] reported a 100% kill of approximately 40,000 cells) was injected into the rate of ascites tumors in mice treated with large doses of brain of the rat. The hole in the screw was then plugged vitamins C and B~a. The absolute results that they reported with bone wax, and the skin was closed by suture. made this a remarkable study that necessitated further investigation. A search of the literature dealing specifically Preparation of Vitamin Mixture with vitamins and tumors of the brain did not prove fruit- The injectable vitamin mixture was prepared as described ful. by Poydock and co-workers [8]. Ascorbic acid, calcium asThis experiment was developed to follow the experi- corbate, and cyanocobalamin were obtained in pure crystalline form from Sigma Chemical Company.* The vitamin solution was prepared by dissolving 0.1 gm cyanocobalamin From the Departmentsof Neurosurgeryand Anatomy, Universityof Misin 10 ml distilled water, and adding 0.2 gm ascorbic acid to sissippi MedicalCenter, Jackson, MI. this. Just prior to injection, one part of this mixture was Address reprint requeststo Dr. John Kapp, Departmentof Neurosurgery, Universityof MississippiSchoolof Medicine, 2500N. State St., Jackson, added to three parts of a 5% calcium ascorbate solution. MS 39216. The pH of this final mixture was 4.9, and each milliliter Key words: neoplasm;ascorbicacid; vitamin B,2;orthomoleculartherapy; brain tumor. *St. Louis, MO. 0090-3019/81/080161-04501.25 © 1981 by Little, Brown and Company (Inc.)
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contained 2.5 mg cyanocobalamin, 5.0 mg ascorbic acid, and 37.5 mg calcium ascorbate for a total vitamin concentration of 45.0 mg vitamin/ml. The Vitamin Experiment Fifteen days after the transplantation of the tumor cells, 1 rat was autopsied, and histological sections of the brain were studied to determine that a growing tumor was present. Also on day 15, the vitamin injections were begun. The vitamin solution was administered intraperitoneally at a dosage of 360 mg vitamin/kg body weight daily for seven days. Fifteen of the 34 rats received the vitamin injections, while the remaining 19 were controls, receiving an equivalent volume of physiological saline solution intraperitoneally. The survival times of the rats were recorded, after which the animals were autopsied and the brains were removed and studied grossly and microscopically.
Results The number of days that each rat lived after the transplantation of the tumor cells was recorded as the survival time. This survival time was used as an index of comparison of the test animals to the controls. Figure 1 shows the distribution of the survival times of the animals. The mean survival time of the control group was 31.16 days, and the standard deviation from the mean was 5.47. The range was 25 to 47 days, and the median was 30. In the group of animals treated with the vitamin mixture, the mean survival time was 30.80 days, and the standard deviation was 5.32. The range for this group was 24 to 44 days, and the median was 30. Gross examination and histological preparations (Fig. 2) of each brain showed that there was a viable tumor present in each rat.
Discussion The dose of vitamins used in this experiment is equivalent to 1.2 gm of vitamin B,e and 22.8 gln vitamin C per day in a 70-kg human. If the vitamins were effective in killing or retarding the growth of the tumor cells, there should have been a statistically significant increase in the survival time of the test rats over that of the control rats. The data clearly indicate that there was no such increased survival. Therefore, we conclude that the vitamin mixture used by Poydock and associates [8] is not effective against this brain tumor in rats. There are several possible explanations for the difference in results, other than variable sensitivity of individual tumors to the drug. The rats in this study were given the same vitamin mixture intraperitoneally as outlined by Poydock and associates [8]. The pH of this solution was 4.9. The tumor cells of Poydock and co-workers were implanted intraperitoneally and followed 24 hours later by the first of seven successive daily injections of this acidic solution into the peritoneum.
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Fig. 1. Survival time from day of tumor transplantation in animals receiving megadose vitamin therapy and control animals.
It is possible that the tumor cells could not withstand such an exposure to a low pH. This problem should not be encountered in this brain tumor model because of the rat's physiological buffering system. The second possibility involves the establishment of the tumor, or lack thereof, in the peritoneum. Poydock and associates [8] began the vitamin treatments 24 hours after injection of the tumor cells into the peritoneum. It may be suggested that these cells were not in their animals long enough to establish themselves as viable, growing tumor cells and were therefore quite vulnerable. This method of treatment so soon after the cells are implanted does not correlate well with a clinical situation. However, cells implanted into the brain tumor model were allowed to grow for 15 days before treatment was begun. Barker and associates [ 1] found in their studies that any treatment within the first week after implantation interferes with the establishment of tumor; the second week of growth is logarithmic, while beyond day 14 the tumor exhibits growth characteristics found in a clinical situation. If treatment is started after day 14, the tumor has had a chance to establish itself; hence, the correlation with a clinical situation is better. A third possibility is that due to the blood-brain barrier, the vitamin mixture did not gain access to the brain tumor used in our study. Kidd and associates [6] found the level of vitamin B,2 in cerebrospinal fluid to have a range of 0 to 50 pg/ml in a group consisting of 43 patients. Only 1 patient had no detectable B,.,, and the mean was 21 pg/ml. In norreal serum, B,e binds to alpha-globulin, and as Clausen [5] reported, larger-molecular.weight alpha~-macroglobulins have been detected in cerebrospinal fluid. Therefore, serum-bound B,., should cross the blood-brain barrier. The findings of Kidd and co-workers [6] suggest a selective control of passage of vitamin B,2 across the blood-brain barrier. As for vitamin C, Spector and associates [10] attribute the transport of ascorbic acid into the brain to a mechanism located largely in the choroid plexus.
Newell et al: Vitamin Therapy in Experimental Brain Tumor
A
B
Fig. 2. The L.g brain tumor in coronal section of the brain (A) and at higher magnification ( B ) , showing invasion along perivascular spaces into adjacent brain tissue. (A, H&E; ×lO before 40% reduction. B, H&E; ×lO0.)
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Exclusion by the blood-brain barrier would not insure exclusion from the tumor, however, since the blood-tumor barrier may be more permeable than the normal bloodbrain barrier. The question of accessibility of drug to tumor is at best partially answered in most forms of brain tumor chemotherapy. The important point is whether or not the systemically administered drug is effective in eradicating the tumor. Variability in the sensitivity of individual tumors to drugs is well known. Also, nonspecific stimulation of host defense mechanisms may enhance the effectiveness of other modalities of cancer therapy. Nevertheless, based on the results of this study, it does not appear that administration of megadoses of vitamins B~ and C will improve results in the treatment of malignant brain tumors.
This work was partially supported by an institutional grant from The American Cancer Society. The tumor line was originally supplied by W. H. Sweet, P. T. Kornblith, J. R. Messer, and B. O. Whitman of the Massachusetts General Hospital, Boston; it was given to us by M. Barker, Brain Tumor Research Center, University of California, San Francisco.
References 1. Barker M, Hoshino T, Gurcay O, Wilson CB, Nielson SL, Downie R, Eliason J: Development of an animal brain tumor model and its response to therapy with 1,3-bis(2-chloroethyl)-l-nitrosourea. Cancer Res 33:976-983, 1973 2. Benade L, Howard T, Brick D: Synergistic killing of Ehrlich ascites carcinoma cells by ascorbate and 3-amino-l,2,4 trizole. Oncology 23:33-34, 1969 3. Cameron E, Pauling L: The orthomolecular treatment of cancer: I. The role of ascorbic acid in host resistance. Chem Biol Interact
9:273-283, 1974 4. Cameron E, Pauling L: Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci USA 73:3685-3689, 1976 5. Clausen J: The serum proteins in normal and pathological spinal fluids. Acta Psychiatr Scand [Suppl] 35:12-22, 1960 6. Kidd HM, Gould CEG, Thomas JW: Free and total vitamin B~2 in cerebrospinal fluid. Can Med Assoc J 88:876-881, 1963 7. K/SnigJFR, Klippel RA: The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams & Wilkins, 1963 8. Poydock ME, Fardon JC, Gallina D, Ferro V, Heher C: Inhibiting effect of vitamins C and B~2on the mitotic activity of ascites tumors. Exp Cell Biol 47:210-217, 1979 9. Prasad KN: Modulation of the effects of tumor therapeutic agents by vitamin C. Life Sci 27:275-280, 1980 10. Spector R, Lorenzo AV: Ascorbic acid homeostasis in the central nervous system. Am J Physiol 225:757-763, 1973 11. Wilson CWM: Clinical pharmacologicalaspectsof ascorbicacid. Ann NY Acad Sci 258:355-376, 1975
Editorial Note: We Need Your Help Recently one of the readers of Surgical Neurology suggested that it would be well to discontinue the biographical notes and historical material which have been published in the journal since its beginning. This has come as something of a surprise as all other comments regarding this material have been complimentary. Nevertheless, this single adverse comment raises in our minds the question as to whether this type of material is of continuing interest to our readers. Perhaps the time has come to discontinue the publication of these biographical notes about those individuals who have made significant contributions to neurological surgery. Obviously the editors cannot judge this matter satisfactorily by themselves. We need the guidance of our readers. Therefore, we are soliciting comments on this question. We shall appreciate receiving letters from our readers as to whether we should continue publishing biographical and historical material. Paul C. Bucy, M.D., Editor
A mixture of vitamins C and B12 in high dosage, which has mental design of Poydock and associates [8], applying it to been reported to eradicate ascites tumors in rats, was brain tumors instead of ascites tumors. tested for its antineoplastic effect against the L9 glioma in Fisher CDF strain rats. No difference in survival time Materials and Methods between animals receiving the vitamin mixture and con- The Brain Tumor Model trols could be demonstrated. Possible reasons for the dif- The tumor used in this study was the L9 gliosarcoma grown ferent response to therapy in the two experimental t u m o r in a monolayer cell culture in Eagle's basal medium and systems are discussed. 10% fetal calf serum supplemented with 398 /,g/ml Newell SD Jr, KappJ, RomfhIH: Evaluationof glutamine, 80.5 units/ml penicillin, and 80.5 mg/ml strepmegadosevitamin therapyin an experimentalbrain tomycin. The monolayer was harvested with 0.25% trypsin tumor. Surg Neurol 16:161-164, 1981 EDTA and suspended in Gey's balanced salt solution at a concentration of 4 x 106 cells/ml. In 1974 and 1976 Cameron and Pauling stimulated interest These cells were transplanted into a group of 35 adult in the vitamin therapy of cancer with their suggestion that male CDF rats using the methods of Barker and associates ascorbic acid was an important factor in host defense [1]. The rats were anesthetized with sodium pentobarbital against cancer [3, 4]. Numerous mechanisms by which (60 milligrams per kilogram of body weight) and placed in a vitamin C might act to inhibit cancer have been sug- stereotactic head holder. Following the atlas of K6nig and gested, including increasing stromal integrity of normal Klippel [7], a drill hole was made in the skull 4.0 mm antissue, stimulating lymphocytes to a higher level of terior to the frontal zero plane and 2.5 mm to the left of the immunocompetence, inducing "favorable change in the midline with a No. 5 dental burr. A stainless steel, 0.40 x steroid environment," inhibiting hyaluronidase activity in 0.0625 inch screw with a 0.03 inch, center-drilled hole was malignant cells, increasing antiviral activity, and interfer- screwed into the hole in the skull. A blunted 22-gauge ing with the metabolism of malignant cells [3, 4, 9, 11]. It needle was fashioned with a stop so that when the needle has also been suggested that vitamin C is selectively con- was inserted through the hole in the screw, the tip of the centrated in tumors and may form cytotoxic amounts of needle was allowed to extend exactly 3 mm beyond hydrogen peroxide within the tumor as a byproduct of oxi- the end of the screw. By means of this needle and a dation [2, 9, 11]. microliter syringe, 0.1 /,1 of the cell suspension (a total In 1979 Poydock and co-workers [8] reported a 100% kill of approximately 40,000 cells) was injected into the rate of ascites tumors in mice treated with large doses of brain of the rat. The hole in the screw was then plugged vitamins C and B~a. The absolute results that they reported with bone wax, and the skin was closed by suture. made this a remarkable study that necessitated further investigation. A search of the literature dealing specifically Preparation of Vitamin Mixture with vitamins and tumors of the brain did not prove fruit- The injectable vitamin mixture was prepared as described ful. by Poydock and co-workers [8]. Ascorbic acid, calcium asThis experiment was developed to follow the experi- corbate, and cyanocobalamin were obtained in pure crystalline form from Sigma Chemical Company.* The vitamin solution was prepared by dissolving 0.1 gm cyanocobalamin From the Departmentsof Neurosurgeryand Anatomy, Universityof Misin 10 ml distilled water, and adding 0.2 gm ascorbic acid to sissippi MedicalCenter, Jackson, MI. this. Just prior to injection, one part of this mixture was Address reprint requeststo Dr. John Kapp, Departmentof Neurosurgery, Universityof MississippiSchoolof Medicine, 2500N. State St., Jackson, added to three parts of a 5% calcium ascorbate solution. MS 39216. The pH of this final mixture was 4.9, and each milliliter Key words: neoplasm;ascorbicacid; vitamin B,2;orthomoleculartherapy; brain tumor. *St. Louis, MO. 0090-3019/81/080161-04501.25 © 1981 by Little, Brown and Company (Inc.)
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Surgical Neurology Vol 16 No 2 August 1981
contained 2.5 mg cyanocobalamin, 5.0 mg ascorbic acid, and 37.5 mg calcium ascorbate for a total vitamin concentration of 45.0 mg vitamin/ml. The Vitamin Experiment Fifteen days after the transplantation of the tumor cells, 1 rat was autopsied, and histological sections of the brain were studied to determine that a growing tumor was present. Also on day 15, the vitamin injections were begun. The vitamin solution was administered intraperitoneally at a dosage of 360 mg vitamin/kg body weight daily for seven days. Fifteen of the 34 rats received the vitamin injections, while the remaining 19 were controls, receiving an equivalent volume of physiological saline solution intraperitoneally. The survival times of the rats were recorded, after which the animals were autopsied and the brains were removed and studied grossly and microscopically.
Results The number of days that each rat lived after the transplantation of the tumor cells was recorded as the survival time. This survival time was used as an index of comparison of the test animals to the controls. Figure 1 shows the distribution of the survival times of the animals. The mean survival time of the control group was 31.16 days, and the standard deviation from the mean was 5.47. The range was 25 to 47 days, and the median was 30. In the group of animals treated with the vitamin mixture, the mean survival time was 30.80 days, and the standard deviation was 5.32. The range for this group was 24 to 44 days, and the median was 30. Gross examination and histological preparations (Fig. 2) of each brain showed that there was a viable tumor present in each rat.
Discussion The dose of vitamins used in this experiment is equivalent to 1.2 gm of vitamin B,e and 22.8 gln vitamin C per day in a 70-kg human. If the vitamins were effective in killing or retarding the growth of the tumor cells, there should have been a statistically significant increase in the survival time of the test rats over that of the control rats. The data clearly indicate that there was no such increased survival. Therefore, we conclude that the vitamin mixture used by Poydock and associates [8] is not effective against this brain tumor in rats. There are several possible explanations for the difference in results, other than variable sensitivity of individual tumors to the drug. The rats in this study were given the same vitamin mixture intraperitoneally as outlined by Poydock and associates [8]. The pH of this solution was 4.9. The tumor cells of Poydock and co-workers were implanted intraperitoneally and followed 24 hours later by the first of seven successive daily injections of this acidic solution into the peritoneum.
IO
,Is)
~8
animols)
z6. 0
4-
P9
Z O"
DAYS
Fig. 1. Survival time from day of tumor transplantation in animals receiving megadose vitamin therapy and control animals.
It is possible that the tumor cells could not withstand such an exposure to a low pH. This problem should not be encountered in this brain tumor model because of the rat's physiological buffering system. The second possibility involves the establishment of the tumor, or lack thereof, in the peritoneum. Poydock and associates [8] began the vitamin treatments 24 hours after injection of the tumor cells into the peritoneum. It may be suggested that these cells were not in their animals long enough to establish themselves as viable, growing tumor cells and were therefore quite vulnerable. This method of treatment so soon after the cells are implanted does not correlate well with a clinical situation. However, cells implanted into the brain tumor model were allowed to grow for 15 days before treatment was begun. Barker and associates [ 1] found in their studies that any treatment within the first week after implantation interferes with the establishment of tumor; the second week of growth is logarithmic, while beyond day 14 the tumor exhibits growth characteristics found in a clinical situation. If treatment is started after day 14, the tumor has had a chance to establish itself; hence, the correlation with a clinical situation is better. A third possibility is that due to the blood-brain barrier, the vitamin mixture did not gain access to the brain tumor used in our study. Kidd and associates [6] found the level of vitamin B,2 in cerebrospinal fluid to have a range of 0 to 50 pg/ml in a group consisting of 43 patients. Only 1 patient had no detectable B,.,, and the mean was 21 pg/ml. In norreal serum, B,e binds to alpha-globulin, and as Clausen [5] reported, larger-molecular.weight alpha~-macroglobulins have been detected in cerebrospinal fluid. Therefore, serum-bound B,., should cross the blood-brain barrier. The findings of Kidd and co-workers [6] suggest a selective control of passage of vitamin B,2 across the blood-brain barrier. As for vitamin C, Spector and associates [10] attribute the transport of ascorbic acid into the brain to a mechanism located largely in the choroid plexus.
Newell et al: Vitamin Therapy in Experimental Brain Tumor
A
B
Fig. 2. The L.g brain tumor in coronal section of the brain (A) and at higher magnification ( B ) , showing invasion along perivascular spaces into adjacent brain tissue. (A, H&E; ×lO before 40% reduction. B, H&E; ×lO0.)
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Exclusion by the blood-brain barrier would not insure exclusion from the tumor, however, since the blood-tumor barrier may be more permeable than the normal bloodbrain barrier. The question of accessibility of drug to tumor is at best partially answered in most forms of brain tumor chemotherapy. The important point is whether or not the systemically administered drug is effective in eradicating the tumor. Variability in the sensitivity of individual tumors to drugs is well known. Also, nonspecific stimulation of host defense mechanisms may enhance the effectiveness of other modalities of cancer therapy. Nevertheless, based on the results of this study, it does not appear that administration of megadoses of vitamins B~ and C will improve results in the treatment of malignant brain tumors.
This work was partially supported by an institutional grant from The American Cancer Society. The tumor line was originally supplied by W. H. Sweet, P. T. Kornblith, J. R. Messer, and B. O. Whitman of the Massachusetts General Hospital, Boston; it was given to us by M. Barker, Brain Tumor Research Center, University of California, San Francisco.
References 1. Barker M, Hoshino T, Gurcay O, Wilson CB, Nielson SL, Downie R, Eliason J: Development of an animal brain tumor model and its response to therapy with 1,3-bis(2-chloroethyl)-l-nitrosourea. Cancer Res 33:976-983, 1973 2. Benade L, Howard T, Brick D: Synergistic killing of Ehrlich ascites carcinoma cells by ascorbate and 3-amino-l,2,4 trizole. Oncology 23:33-34, 1969 3. Cameron E, Pauling L: The orthomolecular treatment of cancer: I. The role of ascorbic acid in host resistance. Chem Biol Interact
9:273-283, 1974 4. Cameron E, Pauling L: Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer. Proc Natl Acad Sci USA 73:3685-3689, 1976 5. Clausen J: The serum proteins in normal and pathological spinal fluids. Acta Psychiatr Scand [Suppl] 35:12-22, 1960 6. Kidd HM, Gould CEG, Thomas JW: Free and total vitamin B~2 in cerebrospinal fluid. Can Med Assoc J 88:876-881, 1963 7. K/SnigJFR, Klippel RA: The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem. Baltimore: Williams & Wilkins, 1963 8. Poydock ME, Fardon JC, Gallina D, Ferro V, Heher C: Inhibiting effect of vitamins C and B~2on the mitotic activity of ascites tumors. Exp Cell Biol 47:210-217, 1979 9. Prasad KN: Modulation of the effects of tumor therapeutic agents by vitamin C. Life Sci 27:275-280, 1980 10. Spector R, Lorenzo AV: Ascorbic acid homeostasis in the central nervous system. Am J Physiol 225:757-763, 1973 11. Wilson CWM: Clinical pharmacologicalaspectsof ascorbicacid. Ann NY Acad Sci 258:355-376, 1975
Editorial Note: We Need Your Help Recently one of the readers of Surgical Neurology suggested that it would be well to discontinue the biographical notes and historical material which have been published in the journal since its beginning. This has come as something of a surprise as all other comments regarding this material have been complimentary. Nevertheless, this single adverse comment raises in our minds the question as to whether this type of material is of continuing interest to our readers. Perhaps the time has come to discontinue the publication of these biographical notes about those individuals who have made significant contributions to neurological surgery. Obviously the editors cannot judge this matter satisfactorily by themselves. We need the guidance of our readers. Therefore, we are soliciting comments on this question. We shall appreciate receiving letters from our readers as to whether we should continue publishing biographical and historical material. Paul C. Bucy, M.D., Editor