- Email: [email protected]
TRYPTOPHAN METABOLISM IN CARCINOMA OF THE BREAST
239
by partial gastrectomy or by vagotomy and drainage operations is affected to much the same degree by diet and by ABO blood-group and secretor status as it is in healthy
upon
individuals. We thank Mr. J. H. Conyers, Mr. R. A. Hall, and Mr. J. K. Willson-Pepper for permission to study patients under their care; Prof. Paul Fourman for the serum-total-alkaline-phosphatase estimations ; and Mrs. M. A. Pybus for secretarial assistance. Requests for reprints should be addressed to M. J. S. L., M.R.C. Statistical Research Unit, University College Hospital Medical School, 115 Gower Street, London W.C.I. REFERENCES
Arfors, K. E., Beckman, L., Lundin, L. G. (1963) Acta genet. 13, 89, 366. Bamford, K. F., Harris, H., Luffman, J. E., Robson, E. B., Cleghorn, T. E. (19b5) Lancet, i, 530. Beckman, L. (1964) Acta genet. 14, 286. Boyd, W. C., Shapleigh, E. (1954) Blood, 9, 1195. Clark, C. G., Crooks, J., Dawson, A. D., Mitchell, P. E. G. (1964) Lancet, i, 734.
Deller, D. J., Begley, M. D. (1963) Australas. Ann. Med. 12, 282. Jones, C. T., Williams, J. A., Cox, E. V., Meynell, M. J., Cooke, W. T.,
Stammers, F. A. R. (1962) Lancet, ii, 425. Langman, M. J. S., Leuthold, E., Robson, E. B., Harris, J., Luffman, J. E.,
Harris, H. (1966) Nature, Lond. 212, 41. Morgan, D. B., Paterson, C. R., Woods, C. G., Pulvertaft, C. N., Fourman, P. (1965) Lancet, ii, 1085. Shreffler, D. C. (1965) Am. J. hum. genet. 17, 71. Yong, J. M. (1966) Lancet, i, 1132.
TRYPTOPHAN METABOLISM IN CARCINOMA OF THE BREAST D. P. ROSE
carcinoma of the breast. Results have been obtained in patients treated by mastectomy alone, and compared with those of patients treated by oophorectomy and mastectomy, and with a group of controls.
Patients, Controls, and Methods The controls subjects were 12 female members of the hospital staff and 3 female patients who had no evidence of malignant, hepatic, or renal disease, who were not anaemic or in congestive cardiac failure, and who were not taking any form of hormone therapy (table i). The 44 patients had all been treated for carcinoma of the breast. At the time of this investigation they were well, and showed no evidence of tumour recurrence. In 20 cases mastectomy alone had been performed, and in 24 the initial treatment had been by oophorectomy and mastectomy. Most of the patients had had postoperative radiotherapy, but in no case had this been given within three months of the investigation. None were receiving hormone therapy. 3-hydroxykynurenine (H.K.), xanthurenic’ acid (x..a.), and 3-hydroxyanthranilic acid (H.A.) were determined in urine collected after the administration of a tryptophan load. The women fasted overnight and the next morning 5 g. (24,500 µmoles) L-tryptophan was given orally, suspended in a glass of milk. Urine was collected for eight hours after the tryptophan had been given, and the total volume was then measured and a sample was kept at - 20"C until the determinations were carried out. X.A. was determined by thin-layer chromatography on cellulose (Walsh 1965), and H.K. and H.A. were measured by the column-chromatography procedures of Brown and Price (1956) as modified by Heeley (1965). The urines were not hydrolysed before analysis. Results
M.D. Sheff. LECTURER IN CHEMICAL
From the
PATHOLOGY,
UNIVERSITY OF SHEFFIELD
University Department of Chemical Pathology, Royal Infirmary, Sheffield 6 The excretions of three
tryptophan metabolites, 3-hydroxykynurenine, xanthurenic acid, and 3-hydroxyanthranilic acid, have been determined in the urine of 15 controls, 20 patients treated for cancer of the breast by mastectomy alone, and 24 patients treated by oophorectomy and mastectomy. High excretions of metabolites occurred in 13 of the patients treated by, mastectomy alone. The abnormal tryptophan metabolism may be the result of increased oestrogen activity in some Summary
patients with breast
cancer.
12 of the 24
patients
treated
by oophorectomy excreted less tryptophan metabolites than did the controls. Increased excretions by 8 of this group may have been due to increased adrenal tion of oestrogens after oophorectomy.
produc-
Introduction of CARCINOMA the breast is one of the hormonedependent tumours. Oophorectomy, adrenalectomy, and hypophysectomy, and the administration of sex hormones, all have an established place in the treatment of breast cancer, and it is generally believed that hormones have an important role in the aetiology of this disease, although this has yet to be defined. Tryptophan is metabolised to nicotinic acid in the liver by way of a number of intermediate compounds (fig. 1), and I have found that the administration of oestrogens causes an increase in the excretion of several of these metabolites in urine (Rose 1966). In view of this hormonal effect on tryptophan metabolism I decided to investigate the excretion of metabolites in the urine of women with
The results are shown in tables i-m. 13 of the 20 patients treated by mastectomy alone excreted at least one of the metabolites in larger amounts than did the controls, and in 5 cases (nos. 20, 25, 32, 33, and 34) the excretions of all three compounds were above the highest figure for the controls. In only one of these patients was the amount of excreted metabolites less than the lowest figure for the control groups (no. 35, x.A.). 8 of the 24 oophorectomised patients had raised excretions of one or more metabolites, but in only 2 (nos. 55 and 59) were there increases in all three metabolite excretions. However, 12 patients in this group excreted one or more of the derivatives in amounts which were less than
240
excreted increased amounts of H.A. Abnormalities of
tryptophan
meta-
in with patients carcinoma of the bladder. Boyland and Williams (1955) found increased amounts of kynurenine, anthranilic acid, H.K., and H.A. in the urine of such patients. Brown et al. (1955) measured the excretions of eight metabolites, and found that bladder
bolism
occur
some
Subjects 1-12 were healthy, female members of the hospital staff. The diagnoses in the 3 patients were: femoral hernia (no. 13); resolved cholecystitis (no. 14); and fissure-in-ano (no. 15).
those excreted by any of the controls. H.K. was the metabolite most frequently affected by oophorectomy; low excretions were found in 10 patients. In 5 of these 10 there was also a reduced excretion of one or both of the other two metabolites. The total amount of the tryptophan dose excreted as H.K., x.A., and H.A. by each of the subjects studied is shown in fig. 2. With 1 exception (no. 9) the controls formed a fairly homogeneous group. The total excretions range from 400 to 1000 .mole. Both groups of patients, however, show a marked scatter of the results. This is particularly so of the oophorectomised women, due to the low total excretions by 10 of this group. Discussion An abnormal excretion of tryptophan metabolites was found in over half of the patients who had had a mastectomy performed, without oophorectomy, for cancer of the breast. All of these patients seemed to be free from recurrence of breast cancer, and were otherwise well. It seems unlikely, therefore, that the abnormalities were due to the tumour itself. Increased excretions of metabolites after a tryptophan load have been noted previously in carcinoma of the breast (Price et al. 1955, Price 1958), but the numbers and details of these cases were not published. Khalafallah and Abul-Fadl (1964) found all of 3 patients with breast cancer, studied after a tryptophan load, TABLE
II-EXCRETION OF TRYPTOPHAN METABOLITES BY PATIENTS TREATED BY MASTECTOMY ALONE
tumours were as-
sociated with abnormal excretions of kynurenine,
acetylkynurenine, and kynurenic
Fig. 2-The total amount of the tryptophan dose excreted as H.K., X.A., and H.A. by the controls, by patients treated by mastectomy alone, and by patients treated by mastectomy and oophorectomy.
acid. Later these workers found that about half of patients with bladder cancer have raised excretions of these three metabolites, and of H.K. (Brown et al. 1960). However, unlike some of the cases of breast cancer, the excretions of X.A. were normal. CEstrogen-treated patients excrete large quantities of H.K., x.A., and H.A. in urine collected after a tryptophan load, and it has been suggested that this is due to hormoneinduced increases in the levels of hepatic enzymes concerned in the metabolism of tryptophan to nicotinic acid (Rose 1966). A second factor may be a lack of pyridoxal5-phosphate coenzyme relative to the increased capacity of tryptophan oxygenase and kynurenine 3-hydroxylase to convert the aminoacid to H.K. (fig. 1), and this may be why TABLE
OF TRYPTOPHAN METABOLITES BY PATIENTS TREATED BY MASTECTOMY AND OOPHORECTOMY
III-EXCRETION
241
the rises in the excretions ofH.K. and x.A. are usually much greater than that of H.A. The abnormal metabolism of tryptophan by 13 of the 20 patients who were studied after treatment by mastectomy alone may be a reflection of an increased secretion of oestrogen or, as has been suggested by Hayward (1964), The increased a defective production of androgens. excretions of tryptophan metabolites could not have been a consequence of mastectomy since I have also observed them in untreated patients with carcinoma of the breast
restricted to the skeletal system, seems to be an exception, and this suggests that the abnormal gene is suppressed in skin fibroblasts in this condition. Although these studies have been concerned primarily with the inherited disorders of mucopolysaccharide metabolism, the methodology appears applicable to other genetic disorders, associated with an intracellular accumulation of a normal or abnormal metabolic product. Introduction
THE inherited disorders of mucopolysaccharide (unpublished). metabolism are, by convention, classified into six separate 12 of the 24 patients who had been treated by oophorecsyndromes based on clinical, biochemical, and genetic tomy and mastectomy excreted low amounts of one or more of the three metabolites. These results suggest that studies; in proposing his classification, McKusick (1965) the removal of oestrogen activity brings about a reduction suggested that the heterogeneity existing in this group of in the capacity for conversion of tryptophan to nicotinic mucopolysaccharide disorders could be further elucidated acid. Lojkin (1956) has shown that oophorectomy causes by investigations at the cellular level. We have shown a marked drop in the urinary excretions of Nl-methyl(Danes and Beam 1966a) that under certain conditions the nicotinamide and nicotinic acid in rats, and that this genetic defect can be recognised in cell culture of skin change is reversed by the administration of oestrone and fibroblasts from patients with the Hurler (1919) and progesterone. 8 of the oophorectomised patients had Hunter (1917) syndromes. When the fibroblasts of raised metabolite excretions. This could have resulted patients with these diseases were stained with the metafrom an increase in oestrogen production by the adrenal chromatic dye toluidine-blue 0, the cytoplasm stained red glands, which has been shown to occur in a proportion of indicating the presence of increased cellular mucowomen after oophorectomy (Bulbrook et al. 1958). polysaccharides. Moreover, the degree of cellular metachromasia correlated with the intracellular mucoI thank Dr. G. M. King for allowing me to study his patients. polysaccharide content determined quantitatively (Danes REFERENCES and Bearn 1966b). We have extended these observations Boyland, E., Williams, D. C. (1955) Biochem. J. 60, p. 5. to the other known mucopolysaccharidoses and suggest M. biol. 985. R. Chem. (1956) J. 219, Brown, R., Price, J. Satter, E. J., Wear, J. B. (1960) Acta Un. int. Cancr. 16, 299. that cellular metachromasia can be used as a genetic Wear, J. B. (1955) Proc. Am. Ass. Cancer Res. 2, 7. marker for investigating the mode of inheritance of these Bulbrook, R. D., Greenwood, F. C., Hadfield, G. J., Scowen, E. F. (1958) — —
— —
Br. med. J. ii, 7. L. (1964) Br. J. Surg. 51, 224. Heeley, A. F. (1965) Clin. Sci. 29, 465. Khalafallah, A. S., Abul-Fadl, M. A. M. (1964) Br. J. Cancer, 18, 592. Lojkin, M. E. (1956) J. Nutr. 59, 443. Price, J. M. (1958) Univ. Mich. med. Bull. 24, 461. Brown, R. R., Curreri, A. R., McIver, F. A. (1955) Clin. Res. Proc. 3, 201. Rose, D. P. (1966) Clin. Sci. 31, 265. Walsh, M. P. (1965) Clin. chim. Acta, 11, 263.
Hayward, J.
—
CELLULAR METACHROMASIA, A GENETIC MARKER FOR STUDYING THE MUCOPOLYSACCHARIDOSES B. SHANNON DANES M.D.
Columbia,
Ph.D. Iowa
ASSISTANT PROFESSOR AND ASSOCIATE PHYSICIAN, THE ROCKEFELLER UNIVERSITY, NEW YORK CITY
diseases. Genetic Material and Methods Several families with different genetic mucopolysaccharidoses (see table) have been investigated. Although the patients were diagnosed on clinical and radiological grounds, the urinary excretion of mucopolysaccharides was determined in all patients except those with the Morquio syndrome (Brailsford 1929, Morquio 1929). Specimens of skin were obtained from patients, from certain relatives considered to be carriers by pedigree studies, and from healthy individuals. The method used to establish the cell lines, the preparation of cytological slides, and the subsequent staining of the cells with the metachromatic dye, toluidine-blue 0, have been described in detail elsewhere (Danes and Beam 1966a). Cytological evaluation of all preparations was based on the examination of a hundred fields each containing approximately 100 cells. Since the fibroblast cultures derived from skin of healthy individuals showed essentially no cellular metachromasia (Danes and Beam 1966a), the results have been recorded as either positive or negative.
ALEXANDER G. BEARN M.D. Lond., M.R.C.P., M.R.C.P.E PROFESSOR AND CHAIRMAN, DEPARTMENT OF MEDICINE, UNIVERSITY MEDICAL COLLEGE, AND PHYSICIAN-IN-CHIEF, YORK
From the
CORNELL
THE NEW
HOSPITAL, NEW YORK CITY
Rockefeller University and the Rockefeller University Hospital, New York City
Cell culture of skin fibroblasts seems to offer a means by which inherited disorders of mucopolysaccharide metabolism may be investigated. Since skin fibroblasts are readily available by biopsy and grow well in culture, a cell involved in the metabolic defect can be examined under controlled conditions. Cellular metachromasia, as evidenced by red cytoplasm on staining with toluidine-blue O, seems to be a reliable genetic marker for investigating mucopolysaccharidoses. The Morquio syndrome, in which the abnormalities are Summary
METACHROMASIA OF SKIN FIBROBLASTS GROWN IN CELL CULTURE FROM FAMILIES WITH THE GENETIC MUCOPOLYSACCHARIDOSES
by partial gastrectomy or by vagotomy and drainage operations is affected to much the same degree by diet and by ABO blood-group and secretor status as it is in healthy
upon
individuals. We thank Mr. J. H. Conyers, Mr. R. A. Hall, and Mr. J. K. Willson-Pepper for permission to study patients under their care; Prof. Paul Fourman for the serum-total-alkaline-phosphatase estimations ; and Mrs. M. A. Pybus for secretarial assistance. Requests for reprints should be addressed to M. J. S. L., M.R.C. Statistical Research Unit, University College Hospital Medical School, 115 Gower Street, London W.C.I. REFERENCES
Arfors, K. E., Beckman, L., Lundin, L. G. (1963) Acta genet. 13, 89, 366. Bamford, K. F., Harris, H., Luffman, J. E., Robson, E. B., Cleghorn, T. E. (19b5) Lancet, i, 530. Beckman, L. (1964) Acta genet. 14, 286. Boyd, W. C., Shapleigh, E. (1954) Blood, 9, 1195. Clark, C. G., Crooks, J., Dawson, A. D., Mitchell, P. E. G. (1964) Lancet, i, 734.
Deller, D. J., Begley, M. D. (1963) Australas. Ann. Med. 12, 282. Jones, C. T., Williams, J. A., Cox, E. V., Meynell, M. J., Cooke, W. T.,
Stammers, F. A. R. (1962) Lancet, ii, 425. Langman, M. J. S., Leuthold, E., Robson, E. B., Harris, J., Luffman, J. E.,
Harris, H. (1966) Nature, Lond. 212, 41. Morgan, D. B., Paterson, C. R., Woods, C. G., Pulvertaft, C. N., Fourman, P. (1965) Lancet, ii, 1085. Shreffler, D. C. (1965) Am. J. hum. genet. 17, 71. Yong, J. M. (1966) Lancet, i, 1132.
TRYPTOPHAN METABOLISM IN CARCINOMA OF THE BREAST D. P. ROSE
carcinoma of the breast. Results have been obtained in patients treated by mastectomy alone, and compared with those of patients treated by oophorectomy and mastectomy, and with a group of controls.
Patients, Controls, and Methods The controls subjects were 12 female members of the hospital staff and 3 female patients who had no evidence of malignant, hepatic, or renal disease, who were not anaemic or in congestive cardiac failure, and who were not taking any form of hormone therapy (table i). The 44 patients had all been treated for carcinoma of the breast. At the time of this investigation they were well, and showed no evidence of tumour recurrence. In 20 cases mastectomy alone had been performed, and in 24 the initial treatment had been by oophorectomy and mastectomy. Most of the patients had had postoperative radiotherapy, but in no case had this been given within three months of the investigation. None were receiving hormone therapy. 3-hydroxykynurenine (H.K.), xanthurenic’ acid (x..a.), and 3-hydroxyanthranilic acid (H.A.) were determined in urine collected after the administration of a tryptophan load. The women fasted overnight and the next morning 5 g. (24,500 µmoles) L-tryptophan was given orally, suspended in a glass of milk. Urine was collected for eight hours after the tryptophan had been given, and the total volume was then measured and a sample was kept at - 20"C until the determinations were carried out. X.A. was determined by thin-layer chromatography on cellulose (Walsh 1965), and H.K. and H.A. were measured by the column-chromatography procedures of Brown and Price (1956) as modified by Heeley (1965). The urines were not hydrolysed before analysis. Results
M.D. Sheff. LECTURER IN CHEMICAL
From the
PATHOLOGY,
UNIVERSITY OF SHEFFIELD
University Department of Chemical Pathology, Royal Infirmary, Sheffield 6 The excretions of three
tryptophan metabolites, 3-hydroxykynurenine, xanthurenic acid, and 3-hydroxyanthranilic acid, have been determined in the urine of 15 controls, 20 patients treated for cancer of the breast by mastectomy alone, and 24 patients treated by oophorectomy and mastectomy. High excretions of metabolites occurred in 13 of the patients treated by, mastectomy alone. The abnormal tryptophan metabolism may be the result of increased oestrogen activity in some Summary
patients with breast
cancer.
12 of the 24
patients
treated
by oophorectomy excreted less tryptophan metabolites than did the controls. Increased excretions by 8 of this group may have been due to increased adrenal tion of oestrogens after oophorectomy.
produc-
Introduction of CARCINOMA the breast is one of the hormonedependent tumours. Oophorectomy, adrenalectomy, and hypophysectomy, and the administration of sex hormones, all have an established place in the treatment of breast cancer, and it is generally believed that hormones have an important role in the aetiology of this disease, although this has yet to be defined. Tryptophan is metabolised to nicotinic acid in the liver by way of a number of intermediate compounds (fig. 1), and I have found that the administration of oestrogens causes an increase in the excretion of several of these metabolites in urine (Rose 1966). In view of this hormonal effect on tryptophan metabolism I decided to investigate the excretion of metabolites in the urine of women with
The results are shown in tables i-m. 13 of the 20 patients treated by mastectomy alone excreted at least one of the metabolites in larger amounts than did the controls, and in 5 cases (nos. 20, 25, 32, 33, and 34) the excretions of all three compounds were above the highest figure for the controls. In only one of these patients was the amount of excreted metabolites less than the lowest figure for the control groups (no. 35, x.A.). 8 of the 24 oophorectomised patients had raised excretions of one or more metabolites, but in only 2 (nos. 55 and 59) were there increases in all three metabolite excretions. However, 12 patients in this group excreted one or more of the derivatives in amounts which were less than
240
excreted increased amounts of H.A. Abnormalities of
tryptophan
meta-
in with patients carcinoma of the bladder. Boyland and Williams (1955) found increased amounts of kynurenine, anthranilic acid, H.K., and H.A. in the urine of such patients. Brown et al. (1955) measured the excretions of eight metabolites, and found that bladder
bolism
occur
some
Subjects 1-12 were healthy, female members of the hospital staff. The diagnoses in the 3 patients were: femoral hernia (no. 13); resolved cholecystitis (no. 14); and fissure-in-ano (no. 15).
those excreted by any of the controls. H.K. was the metabolite most frequently affected by oophorectomy; low excretions were found in 10 patients. In 5 of these 10 there was also a reduced excretion of one or both of the other two metabolites. The total amount of the tryptophan dose excreted as H.K., x.A., and H.A. by each of the subjects studied is shown in fig. 2. With 1 exception (no. 9) the controls formed a fairly homogeneous group. The total excretions range from 400 to 1000 .mole. Both groups of patients, however, show a marked scatter of the results. This is particularly so of the oophorectomised women, due to the low total excretions by 10 of this group. Discussion An abnormal excretion of tryptophan metabolites was found in over half of the patients who had had a mastectomy performed, without oophorectomy, for cancer of the breast. All of these patients seemed to be free from recurrence of breast cancer, and were otherwise well. It seems unlikely, therefore, that the abnormalities were due to the tumour itself. Increased excretions of metabolites after a tryptophan load have been noted previously in carcinoma of the breast (Price et al. 1955, Price 1958), but the numbers and details of these cases were not published. Khalafallah and Abul-Fadl (1964) found all of 3 patients with breast cancer, studied after a tryptophan load, TABLE
II-EXCRETION OF TRYPTOPHAN METABOLITES BY PATIENTS TREATED BY MASTECTOMY ALONE
tumours were as-
sociated with abnormal excretions of kynurenine,
acetylkynurenine, and kynurenic
Fig. 2-The total amount of the tryptophan dose excreted as H.K., X.A., and H.A. by the controls, by patients treated by mastectomy alone, and by patients treated by mastectomy and oophorectomy.
acid. Later these workers found that about half of patients with bladder cancer have raised excretions of these three metabolites, and of H.K. (Brown et al. 1960). However, unlike some of the cases of breast cancer, the excretions of X.A. were normal. CEstrogen-treated patients excrete large quantities of H.K., x.A., and H.A. in urine collected after a tryptophan load, and it has been suggested that this is due to hormoneinduced increases in the levels of hepatic enzymes concerned in the metabolism of tryptophan to nicotinic acid (Rose 1966). A second factor may be a lack of pyridoxal5-phosphate coenzyme relative to the increased capacity of tryptophan oxygenase and kynurenine 3-hydroxylase to convert the aminoacid to H.K. (fig. 1), and this may be why TABLE
OF TRYPTOPHAN METABOLITES BY PATIENTS TREATED BY MASTECTOMY AND OOPHORECTOMY
III-EXCRETION
241
the rises in the excretions ofH.K. and x.A. are usually much greater than that of H.A. The abnormal metabolism of tryptophan by 13 of the 20 patients who were studied after treatment by mastectomy alone may be a reflection of an increased secretion of oestrogen or, as has been suggested by Hayward (1964), The increased a defective production of androgens. excretions of tryptophan metabolites could not have been a consequence of mastectomy since I have also observed them in untreated patients with carcinoma of the breast
restricted to the skeletal system, seems to be an exception, and this suggests that the abnormal gene is suppressed in skin fibroblasts in this condition. Although these studies have been concerned primarily with the inherited disorders of mucopolysaccharide metabolism, the methodology appears applicable to other genetic disorders, associated with an intracellular accumulation of a normal or abnormal metabolic product. Introduction
THE inherited disorders of mucopolysaccharide (unpublished). metabolism are, by convention, classified into six separate 12 of the 24 patients who had been treated by oophorecsyndromes based on clinical, biochemical, and genetic tomy and mastectomy excreted low amounts of one or more of the three metabolites. These results suggest that studies; in proposing his classification, McKusick (1965) the removal of oestrogen activity brings about a reduction suggested that the heterogeneity existing in this group of in the capacity for conversion of tryptophan to nicotinic mucopolysaccharide disorders could be further elucidated acid. Lojkin (1956) has shown that oophorectomy causes by investigations at the cellular level. We have shown a marked drop in the urinary excretions of Nl-methyl(Danes and Beam 1966a) that under certain conditions the nicotinamide and nicotinic acid in rats, and that this genetic defect can be recognised in cell culture of skin change is reversed by the administration of oestrone and fibroblasts from patients with the Hurler (1919) and progesterone. 8 of the oophorectomised patients had Hunter (1917) syndromes. When the fibroblasts of raised metabolite excretions. This could have resulted patients with these diseases were stained with the metafrom an increase in oestrogen production by the adrenal chromatic dye toluidine-blue 0, the cytoplasm stained red glands, which has been shown to occur in a proportion of indicating the presence of increased cellular mucowomen after oophorectomy (Bulbrook et al. 1958). polysaccharides. Moreover, the degree of cellular metachromasia correlated with the intracellular mucoI thank Dr. G. M. King for allowing me to study his patients. polysaccharide content determined quantitatively (Danes REFERENCES and Bearn 1966b). We have extended these observations Boyland, E., Williams, D. C. (1955) Biochem. J. 60, p. 5. to the other known mucopolysaccharidoses and suggest M. biol. 985. R. Chem. (1956) J. 219, Brown, R., Price, J. Satter, E. J., Wear, J. B. (1960) Acta Un. int. Cancr. 16, 299. that cellular metachromasia can be used as a genetic Wear, J. B. (1955) Proc. Am. Ass. Cancer Res. 2, 7. marker for investigating the mode of inheritance of these Bulbrook, R. D., Greenwood, F. C., Hadfield, G. J., Scowen, E. F. (1958) — —
— —
Br. med. J. ii, 7. L. (1964) Br. J. Surg. 51, 224. Heeley, A. F. (1965) Clin. Sci. 29, 465. Khalafallah, A. S., Abul-Fadl, M. A. M. (1964) Br. J. Cancer, 18, 592. Lojkin, M. E. (1956) J. Nutr. 59, 443. Price, J. M. (1958) Univ. Mich. med. Bull. 24, 461. Brown, R. R., Curreri, A. R., McIver, F. A. (1955) Clin. Res. Proc. 3, 201. Rose, D. P. (1966) Clin. Sci. 31, 265. Walsh, M. P. (1965) Clin. chim. Acta, 11, 263.
Hayward, J.
—
CELLULAR METACHROMASIA, A GENETIC MARKER FOR STUDYING THE MUCOPOLYSACCHARIDOSES B. SHANNON DANES M.D.
Columbia,
Ph.D. Iowa
ASSISTANT PROFESSOR AND ASSOCIATE PHYSICIAN, THE ROCKEFELLER UNIVERSITY, NEW YORK CITY
diseases. Genetic Material and Methods Several families with different genetic mucopolysaccharidoses (see table) have been investigated. Although the patients were diagnosed on clinical and radiological grounds, the urinary excretion of mucopolysaccharides was determined in all patients except those with the Morquio syndrome (Brailsford 1929, Morquio 1929). Specimens of skin were obtained from patients, from certain relatives considered to be carriers by pedigree studies, and from healthy individuals. The method used to establish the cell lines, the preparation of cytological slides, and the subsequent staining of the cells with the metachromatic dye, toluidine-blue 0, have been described in detail elsewhere (Danes and Beam 1966a). Cytological evaluation of all preparations was based on the examination of a hundred fields each containing approximately 100 cells. Since the fibroblast cultures derived from skin of healthy individuals showed essentially no cellular metachromasia (Danes and Beam 1966a), the results have been recorded as either positive or negative.
ALEXANDER G. BEARN M.D. Lond., M.R.C.P., M.R.C.P.E PROFESSOR AND CHAIRMAN, DEPARTMENT OF MEDICINE, UNIVERSITY MEDICAL COLLEGE, AND PHYSICIAN-IN-CHIEF, YORK
From the
CORNELL
THE NEW
HOSPITAL, NEW YORK CITY
Rockefeller University and the Rockefeller University Hospital, New York City
Cell culture of skin fibroblasts seems to offer a means by which inherited disorders of mucopolysaccharide metabolism may be investigated. Since skin fibroblasts are readily available by biopsy and grow well in culture, a cell involved in the metabolic defect can be examined under controlled conditions. Cellular metachromasia, as evidenced by red cytoplasm on staining with toluidine-blue O, seems to be a reliable genetic marker for investigating mucopolysaccharidoses. The Morquio syndrome, in which the abnormalities are Summary
METACHROMASIA OF SKIN FIBROBLASTS GROWN IN CELL CULTURE FROM FAMILIES WITH THE GENETIC MUCOPOLYSACCHARIDOSES