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Tuberous sclerosis: Hydroxyproline content in urine and tissues
Tuberous Sclerosis: Hydroxyproline Content in Urine and Tissues Harumi Tanaka, MD and Masataka Arima, MD To elucidate the nature of an overabundance of collagen seen on microscopic examination in tuberous sclerosis (TS), the hydroxyproline content in tissues and urine was determined. TS tissues of 5 patients were obtained on necropsy or plastic surgery. Urine was collected from 10 patients with TS and 19 controls. Tumors in kidney, pancreas, lung and heart but not brain contained more hydroxyproline than the surrounding tissues. In brain with the lowest hydroxyproline content, the tumor showed reduced hydroxyproline compared to normal. Collagen sheet in TS skin and shagreen patch showed the same hydroxyproline content as control skin. The urinary hydroxyproline: creatinine ratios of the patients with TS were all higher than those in age-matched controls with or without anticonvulsant treatment except for congenital muscular dystrophy. The higher content of hydroxyproline in several affected tissues and urine of patients with TS might indicate that an increase in collagen synthesis occurs in TS. Tanaka H, Arima M. Tuberous sclerosis: hydroxyproline content in urine and tissues. Brain Dev 1981;3: 81-5
Tuberous sclerosis (TS) is characterized by the triad of epilepsy, mental retardation and adenoma sebaceum. This is not a rare disease with autosomal dominant transmission, but the incidence of sporadic cases is high [1]. Although several biochemical studies on urine, serum and tissues have been done, no clear evidence for a fundamental biochemical anomaly in this disease has been found until now [2-7] . The presence of collagen proliferation and alteration in skin lesions of patients with TS by microscopical investigation [8,9] prompted us From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication: November 28, 1980. Accepted for pUblication: February 23, 1981.
Key words: Tuberous sclerosis, hydroxyproline, col· lagen, shagreen patch. Correspondence address: Dr. Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Qgawa-Higashi-Machi, Kodaira, Tokyo 187, Japan.
to investigate the possibility of an error in connective tissue metabolism. In a previous study we investigated the origin of collagen proliferation by measuring collagenase in peripheral leucocytes, but no indication of abnormal activity of collagenase in TS was seen [10]. Based on this observation, we suggested the collagen prolifetation in TS might indicate an increased synthesis of collagen. The following report presents the findings of hydroxyproline content in tissues and urine of patients with TS as a marker of collagen synthesis. Subjects Brain, kidney, pancreas, lung and heart tissues were obtained postmortem from 4 patients with TS. The specimens had been fixed in formalin for various periods between 1 week and 7 years; case 1 for 9 months, case 2 for 6 years and 11 months, case 3 for 6 months and case 4 for 1 week. The affected subjects were diagnosed by. the presence of the characteristic pathological findings including those of brain,
Table I
Summary of cases for urinary analysis
Age (years)
1- 4 5- 7 10-13 15-16 20-
Total
Control
TS
Normal Pathological
Total
M
F
M
F
M
F
0 1
2 0 0 2
0 0 0 1 2
3 2 4 0 0
0
1 1
2 0 4 0 0
1 0 0
7 4 11 2 5
4
6
5
3
9
2
29
M: male, F: female.
heart and kidney. They were of both sexes and ranged in age from 0 to 18 years; case 1 a 10month-old male, case 2 a 2-year-old male, case 3 a 18-year-old female and case 4 a premature baby. In these cases both tumors and the surrounding, apparently normal tissues were analyzed. Control collagen sheet was obtained postmortem from abdominal skin of a 22-yearold male (case 5), and dorsal skin collagen sheet in shagreen patch and in apparently normal skin was obtained at plastic surgery from a 24-yearold male (case 6). The skin specimens had been stored at -30°C for several months. Urine samples were collected in the morning from 10 patients with TS and 19 controls. Ages and the sex of these cases are summarized in Table 1. Control subjects included both normal and pathological subjects who were diagnosed as epilepsy, mental deficiency, cerebral palsy, Wilson's disease, Lesch-Nyhan syndrome and congenital muscular dystrophy. Methods The tissue was dissected, weighed and homogenized repeatedly in water with a Teflon pestle (20-100 mg tissue per 1-2 ml water). The hydroxyproline assay was carried out with whole homogenate of tissues. Urine samples were immediately transferred to screw-capped tubes or bottles without preservatives and stored at 4°C until analysis. Excluding alkaline urine, analysis of urine was performed within 2 days after collection. The intake of gelatine-containing foods was checked for all individuals before collection of urine. All assays were performed in duplicate and repeated as many times as 82 Brain & Development, VoIJ, No 1, 1981
possible. Tissue homogenate and urine were hydrolyzed in 6 N HCI in sealed tubes at 124°C for 3 hours. After hydrolysis, 50 mg of Norit "A" (carbon decolorizing, American Norit Co.) was added to the hydrolysate. After mixing the hydrolysate, the solution was collected by centrifugation at 3,000 rpm for 5 min. After 0.5-1.0 ml of colorless supernatant was dried in a water bath, and 0.5-5.0 ml of 0.001 N HCI was added and well mixed, an aliquot of solution was analyzed for hydroxyproline using the spectrophotometric method of Bergman and Loxley [11] with minor modification. To 0.3 ml of 0.001 N HCI solution, 0.3 ml of oxidant solution was added and the solution was well mixed. 3.9 ml of Ehrlich's reagent solution (in the proportion of 1 g of aldehyde to 3 ml of acid) was added, mixed, and the tube was heated for 20 min at 60°C in a water bath. After the tube was cooled for 2 to 3 min in running tap water, the absorbance against water was measured at 558 mJ1. Creatinine analysis was performed on all urine specimens by alkaline picrate technique [12]. Hydroxyproline: creatinine ratios were calculated by dividing mg of hydroxyproline per dl by mg of creatinine per dl. Results The hydroxyproline content of tissues is summarized in Table 2. Tumors in kidney, pancreas, lung and heart but not brain contained more hydroxyproline than the surrounding normal tissues, ranging from 105% for heart to 383% for polycystic kidney. In brain tissues which showed the lowest content of hydroxyproline, the tumors revealed reduced hydroxyproline compared to control areas. On the other hand, collagen sheet from TS skin and shagreen patch showed the same hydroxyproline content as age-matched control skin collagen sheet. The urinary hydroxyproline: creatinine ratios for normal and pathological controls and patients with TS from 1 year to 50 years of age are presented in Fig 1. There was a decrease in the values of the ratio from 1 to 4 years of age. The ratios then remained nearly constant to 13 years when a decrease with increasing age again occurred. No Significant difference in the ratio was observed between males and females in any age group. In further evaluation, the ratios for
Table 2 Hydroxyproline content in tissues of TS
Table 3 Comparison of hydroxyproline: creatinine ratio in urine of two age groups of TS and controls
A. Formalin fixed tissues of TS ().lg)
Tissue
Case Normal no.
Tumor
1.44 ( 57) 2.51 (grey matter) ( 98) 1.47 (white-grey matter) 1.17 ( 53) 2.19 2 (grey matter) ( 57) 2.05 (white-grey matter) (180) 148 Kidney 3 82 (383) 314 1 (polycystic kidney) (174) 153 Pancreas 3 88 (119) 458 Lung 3 385 (191) 164 86 Heart (138) 145 3 105 (105) 117 4 111 1
Brain
Control
5 6
3.14
Skin
3.13 (100) 2.92 ( 93)
Hydroxyproline content of whole homogenate is expressed as J.l.g or mg per 100 mg wet weight (% of control). See text for each case.
•
0:
00
0.150 0.091
± ±
0.016 (4) 0.025 (9) #
.
a
. 5
.
• • i ... . 0"
10
It is well-known that collagen is a unique pro-
• o· 15
50
Fig 1 Urinary hydroxyproline: creatinine ratios of controls and TS from 1 to 50 years of age. /j. male normal control, It male pathological control, 0 female normal control, ¢ female pathological control, II.male TS, • female TS, it indicates the case with congenital
DMP.
0.214 ± 0.117 (2) 0.142 ± 0.040 (6)
Discussion
•
" "f
1- 4 years 5-13 years
Control
both sexes were calculated together. The ratios of TS were all above the control range except for one child. The only patient exhibiting a very high ratio, 0.454, was a one-year-old girl with congenital muscular dystrophy and low urinary creatinine excretion. This high ratio was omitted from further calculation. Comparison of the ratios of TS and controls from two age groups is presented in Table 3. In both age groups TS showed higher ratios than controls. In the 5- to 13-year age group the difference in the mean ratio was significant by the t test. Nine of our 10 patients with TS received anticonvulsant therapy and only one aged 2 years had not been treated until urine analysis. The relationship between anticonvulsant therapy and the ratio was evaluated. In the 1- to 4-year age group the ratio of 0.297 in the patient without anticonvulsants was not lower than the other TS. In the 5- to 13-year age group no difference was observed in the ratio of control groups between 3 epilepsy with anticonvulsants (mean value ± SD of 0.095 ± 0.042) and epilepsy without anticonvulsants (0.10) .
TS
Shagreen 6 patch
TS
Values are expressed as mean ± SD (cases). # means significantly different from the control group (p < 0.01). See Table 1 and text for each case.
B. Frozen skin collagen sheet (mg)
Case no.
Age group
tein, in that there is the presence of the unusual amino acids, hydroxyproline and hydroxylysine (13) . The early experiments also showed that hydroxyproline in collagen could arise from proline but not from hydroxyproline which was administered to animals [14, 15] indicating that free hydroxyproline was not the source of collagen hydroxyproline. But nearly all the hydroxyproline in vertebrates is found in collagen and the only source of this amino acid appears to be the hydroxylation of "bound"
Tanaka et al: Hydroxyproline in tuberous sclerosis 83
proline in situ. In the present study we determined the hydroxyproline content in tissues as a marker of collagen protein and that in urine as an excretory product derived from the soluble and insoluble collagen of both soft and calcified tissues [16, 17] . Although for analysis of hydroxyproline in tissues defatted material is usually used, we attempted to use the crude whole homogenate for the following reasons: 1) There were formalin-fixed tissues ofTS for assaying the hydroxyproline. 2) These tissues were very small sections. 3) Our purpose for analyzing the hydroxyproline was for a relative comparison between a tumor and the normal surrounding tissue. With our procedures the measured values by repeated analyses almost agreed. Our results indicate that there is an excess of hydroxyproline in tumors of hydroxyproline-rich tissues, when compared with the surrounding normal tissues, but not in brain where hydroxyproline is hardly detectable. Furthermore the skin collagen sheet and shagreen patch from TS showed the same hydroxyproline content as that of controls. Based on the findings it is assumed that collagen deposition as observed histologically in TS tissues [8,9,18, 19,20] depends on the nature of the underlying tissues and that the overgrowth of collagen in the affected site is one of the most clear signs of TS. In the present study we could not analyze angiofibroma. The report by Fisher et al [7] showing that angiofibroma contained not more but less collagen than normal skin does not contradict our conclusion. To avoid difficulties in collecting 24-hour urine specimens from young children, Allison et al [21] presented evidence that the variability of the ratio of hydroxyproline: creatinine between individuals was much less than that of corresponding 24-hour urinary hydroxyproline values. Furthermore the early morning specimen was suggested to be the best sample [22] . Kulkarni et al found that a child consuming gelatin showed a slight increase in hydroxyproline excretion but that the variation in the hydroxyproline: creatinine ratio was not greater than the average variation found in individual samples [22]. The results obtained in this study were in agreement with those of several authors [21, 23] in that at all ages the ratio is essentially equivalent in both sexes and that the ratio depends on the growth rate. 84 Brain & Development, Vol 3, No 1, 1981
The patients with TS showed a higher hydroxyproline: creatinine ratio than the age-matched controls at all ages with or without anticonvulsant treatment. Several studies on the levels of urinary excretion of hydroxyproline in relation to pathological conditions have been published [13, 24, 25]. An elevated level of urinary hydroxyproline was reported in growing children and patients with acromegaly, thyrotoxicosis and hyperparathyroidism [13, 24]. Growth hormone probably influences stimulation of collagen synthesis, thyroid hormone stimulates both collagen synthesis and degradation, and parathyroid hormone also influences both the synthesis and degradation of bone matrix collagen [13]. To our knowledge, constant hormonal disturbance in TS is not observed. Furthermore in patients with Paget disease, Marfan syndrome, osteoporosis and scleromyxoedema as well as in patients suffering from "hydroxylation deficiency," increased excretion of hydroxyproline was reported [24, 26]. Patients in the former group showed well substantiated alterations in the collagen metabolism of bone or skin. On the other hand the elevated excretion of urinary hydroxyproline was detected in only one of seven patients with scleroderma where collagen deposition can be demonstrated only histologically [13]. Although the basic mechanism promoting the accumulation of collagen in TS tissues is not known, the elevated urinary hydroxyproline: creatinine ratio suggests that the quantitative alterations in collagen metabolism in TS may be large enough to be detected in the urine. Acknowledgments The authors are grateful to the following Drs. for supplying us with specimens from tuberous sclerosis subjects: Dr. T. Aoki, Dr. N. Fukunaga (Toho University), Dr. S. Ikeda, Dr. K. Shiota (Saitama Medical College), Dr. T. Takao (Tottori University), and Dr. K. Misugi (Yokohama City University). This study was supported in part by Grant No. 80-05-02 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan. References 1. Fleury P, de Groot WP, Delleman JW, Verbeeten B, Frankenmolen-Witkiezwicz 1M. Tuberous sclerosis: the iJ;lcidence of sporadic cases versus familial cases. Brain Dev 1980;2: 107-117. 2. Carpenter DG, Carter CH, Brinson EM, McCarty DA. Biochemical studies of tuberous sclerosis. I.
3.
4.
5. 6. 7.
8.
9. 10.
11.
12. 13.
14.
A survey of some protein-bound carbohydrates and amino acids. J Pediatr 1964;65:124-126. Fischer MH, Fortune JS, Gerritsen T. Serum proteins and alkaline phosphatase levels in patients with tuberous sclerosis. Am J Ment Defic 1974; 78:674-677 . Rundle AT, Atkin J, Sudell B. Serum and tissue proteins in tuberous sclerosis. I. Serum and redcell polymorphic systems. Hum Genet 1975;27: 15-22. Rundle AT, Atkin J. Serum O!,-macroglobulin levels in tuberose sclerosis. J Ment Detic Res 1976;20:231-236. Rundle AT. Plasma acute-phase reactant proteins in tuberose sclerosis. J Ment Defic Res 1976;20: 237-242. Fisher MH, Fortune JS, Foster SH, Gilbert EF. Chemical analysis of an angiofibroma from a patient with tuberous sclerosis. J Ment Defic Res 1977;21 :251-261. Nickel WR, Reed WB. Tuberous sclerosis. Special reference to the microscopic alterations in the cutaneous hamartomas. Arch DermatoI1962;85: 209-226. Ranneberg KM, Richter I-E. Zur Ultrastruktur des Pflastersteinnaevus bei M. Pringle. Arch Dermatol Forsch 1972;243:318-325. Tanaka H, Arima M, Hasegawa H, Ohno K. Biochemical studies on collagen and inorganic substances in tuberous sclerosis. Brain Dev 1979; 1:218. Bergman I, Loxley R. Two improved and simplified methods for the spectrophotometric determination of hydroxyproline. Anal Chem 1963 ;35: 1961-1965. Bonsnes RW, Taussky HH. On the colorimetric determination of creatinine by the Jaffe reaction. J Bioi Chem 1945;158:581-591. Sjoerdsma A, Udenfriend S, Keiser H, LeRoy EC. Hydroxyproline and collagen metabolism. Clinical implications. Ann Intern Med 1965;63:672694. Stetten MR. Some aspects of the metabolism of hydroxyproline, studied with the aid of isotopic nitrogen. J Bioi Chem 1949;181:31-37.
15. Peterkofsky B, Udenfriend S. Conversion of proline to collagen hydroxyproline in a cell-free system from chick embryo. J Bioi Chem 1963; 238:3966-3977. 16. Lindstedt S, Prockop DJ. Isotopic studies on urinary hydroxyproline as evidence for rapidly catabolized forms of collagen in the young rat. J Bioi Chem 1961;236:1399-1403. 17. Prockop DJ. Isotopic studies on collagen degradation and the urine excretion of hydroxyproline. J Clin Invest 1964;43:453460. 18. Dammert K, Niemi KM. Naevus elasticus (elastoma juvenile Weidman) and naevus collagenicus lumbosacralis in Pringle's disease. Dermatologica 1968;137:3645. 19. O'Callaghan TJ, Edwards JA, Tobin M, Mookerjee BK. Tuberous sclerosis with striking renal involvement in a family. Arch Intern Med 1975; 135:1082-1087. 20. Inglis K. The nature and origin of smooth musclelike neoplastic tissue in renal tumors of the tuberous sclerosis complex. Cancer 1960;13:602611. 21. Allison DJ, Walker A, Smith QT. Urinary hydroxyproline: creatinine ratio of normal humans at various ages. Gin Chim Acta 1966; 14:729-734. 22. Kulkarni M, Kilgore L. Diurnal variations of hydroxyproline and creatinine excretion in children. Am J Clin Nutr 1973;26:1069-1072. 23. Yoneyama K. Urinary hydroxyproline as an index of growth potential. I. A study on 1-6 year old infants. Public Health Jpn (Tokyo) 1974;21: 623-626. 24. Nusgens B, Lapiere CM. The relationship between proline and hydroxyproline urinary excretion in human as an index of collagen catabolism. Gin Chim Acta 1973;48:203-211. 25. Wharton BA, Gough G, Williams A, Kitts S, Pennock CA. Urinary total hydroxyproline: creatinine ratio. Range of normal, and clinical application in British children. Arch Dis Child 1972;47:74-79. 26. Sjoerdsma A, Davidson JD, Udenfriend S, Mitoma C. Increased excretion of hydroxyproline in Marfan's syndrome. Lancet 1958;2:994.
Tanaka et al: Hydroxyproline in tuberous sclerosis 85
Tuberous sclerosis (TS) is characterized by the triad of epilepsy, mental retardation and adenoma sebaceum. This is not a rare disease with autosomal dominant transmission, but the incidence of sporadic cases is high [1]. Although several biochemical studies on urine, serum and tissues have been done, no clear evidence for a fundamental biochemical anomaly in this disease has been found until now [2-7] . The presence of collagen proliferation and alteration in skin lesions of patients with TS by microscopical investigation [8,9] prompted us From the Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Kodaira, Tokyo. Received for publication: November 28, 1980. Accepted for pUblication: February 23, 1981.
Key words: Tuberous sclerosis, hydroxyproline, col· lagen, shagreen patch. Correspondence address: Dr. Harumi Tanaka, Division of Child Neurology, National Center for Nervous, Mental and Muscular Disorders, Qgawa-Higashi-Machi, Kodaira, Tokyo 187, Japan.
to investigate the possibility of an error in connective tissue metabolism. In a previous study we investigated the origin of collagen proliferation by measuring collagenase in peripheral leucocytes, but no indication of abnormal activity of collagenase in TS was seen [10]. Based on this observation, we suggested the collagen prolifetation in TS might indicate an increased synthesis of collagen. The following report presents the findings of hydroxyproline content in tissues and urine of patients with TS as a marker of collagen synthesis. Subjects Brain, kidney, pancreas, lung and heart tissues were obtained postmortem from 4 patients with TS. The specimens had been fixed in formalin for various periods between 1 week and 7 years; case 1 for 9 months, case 2 for 6 years and 11 months, case 3 for 6 months and case 4 for 1 week. The affected subjects were diagnosed by. the presence of the characteristic pathological findings including those of brain,
Table I
Summary of cases for urinary analysis
Age (years)
1- 4 5- 7 10-13 15-16 20-
Total
Control
TS
Normal Pathological
Total
M
F
M
F
M
F
0 1
2 0 0 2
0 0 0 1 2
3 2 4 0 0
0
1 1
2 0 4 0 0
1 0 0
7 4 11 2 5
4
6
5
3
9
2
29
M: male, F: female.
heart and kidney. They were of both sexes and ranged in age from 0 to 18 years; case 1 a 10month-old male, case 2 a 2-year-old male, case 3 a 18-year-old female and case 4 a premature baby. In these cases both tumors and the surrounding, apparently normal tissues were analyzed. Control collagen sheet was obtained postmortem from abdominal skin of a 22-yearold male (case 5), and dorsal skin collagen sheet in shagreen patch and in apparently normal skin was obtained at plastic surgery from a 24-yearold male (case 6). The skin specimens had been stored at -30°C for several months. Urine samples were collected in the morning from 10 patients with TS and 19 controls. Ages and the sex of these cases are summarized in Table 1. Control subjects included both normal and pathological subjects who were diagnosed as epilepsy, mental deficiency, cerebral palsy, Wilson's disease, Lesch-Nyhan syndrome and congenital muscular dystrophy. Methods The tissue was dissected, weighed and homogenized repeatedly in water with a Teflon pestle (20-100 mg tissue per 1-2 ml water). The hydroxyproline assay was carried out with whole homogenate of tissues. Urine samples were immediately transferred to screw-capped tubes or bottles without preservatives and stored at 4°C until analysis. Excluding alkaline urine, analysis of urine was performed within 2 days after collection. The intake of gelatine-containing foods was checked for all individuals before collection of urine. All assays were performed in duplicate and repeated as many times as 82 Brain & Development, VoIJ, No 1, 1981
possible. Tissue homogenate and urine were hydrolyzed in 6 N HCI in sealed tubes at 124°C for 3 hours. After hydrolysis, 50 mg of Norit "A" (carbon decolorizing, American Norit Co.) was added to the hydrolysate. After mixing the hydrolysate, the solution was collected by centrifugation at 3,000 rpm for 5 min. After 0.5-1.0 ml of colorless supernatant was dried in a water bath, and 0.5-5.0 ml of 0.001 N HCI was added and well mixed, an aliquot of solution was analyzed for hydroxyproline using the spectrophotometric method of Bergman and Loxley [11] with minor modification. To 0.3 ml of 0.001 N HCI solution, 0.3 ml of oxidant solution was added and the solution was well mixed. 3.9 ml of Ehrlich's reagent solution (in the proportion of 1 g of aldehyde to 3 ml of acid) was added, mixed, and the tube was heated for 20 min at 60°C in a water bath. After the tube was cooled for 2 to 3 min in running tap water, the absorbance against water was measured at 558 mJ1. Creatinine analysis was performed on all urine specimens by alkaline picrate technique [12]. Hydroxyproline: creatinine ratios were calculated by dividing mg of hydroxyproline per dl by mg of creatinine per dl. Results The hydroxyproline content of tissues is summarized in Table 2. Tumors in kidney, pancreas, lung and heart but not brain contained more hydroxyproline than the surrounding normal tissues, ranging from 105% for heart to 383% for polycystic kidney. In brain tissues which showed the lowest content of hydroxyproline, the tumors revealed reduced hydroxyproline compared to control areas. On the other hand, collagen sheet from TS skin and shagreen patch showed the same hydroxyproline content as age-matched control skin collagen sheet. The urinary hydroxyproline: creatinine ratios for normal and pathological controls and patients with TS from 1 year to 50 years of age are presented in Fig 1. There was a decrease in the values of the ratio from 1 to 4 years of age. The ratios then remained nearly constant to 13 years when a decrease with increasing age again occurred. No Significant difference in the ratio was observed between males and females in any age group. In further evaluation, the ratios for
Table 2 Hydroxyproline content in tissues of TS
Table 3 Comparison of hydroxyproline: creatinine ratio in urine of two age groups of TS and controls
A. Formalin fixed tissues of TS ().lg)
Tissue
Case Normal no.
Tumor
1.44 ( 57) 2.51 (grey matter) ( 98) 1.47 (white-grey matter) 1.17 ( 53) 2.19 2 (grey matter) ( 57) 2.05 (white-grey matter) (180) 148 Kidney 3 82 (383) 314 1 (polycystic kidney) (174) 153 Pancreas 3 88 (119) 458 Lung 3 385 (191) 164 86 Heart (138) 145 3 105 (105) 117 4 111 1
Brain
Control
5 6
3.14
Skin
3.13 (100) 2.92 ( 93)
Hydroxyproline content of whole homogenate is expressed as J.l.g or mg per 100 mg wet weight (% of control). See text for each case.
•
0:
00
0.150 0.091
± ±
0.016 (4) 0.025 (9) #
.
a
. 5
.
• • i ... . 0"
10
It is well-known that collagen is a unique pro-
• o· 15
50
Fig 1 Urinary hydroxyproline: creatinine ratios of controls and TS from 1 to 50 years of age. /j. male normal control, It male pathological control, 0 female normal control, ¢ female pathological control, II.male TS, • female TS, it indicates the case with congenital
DMP.
0.214 ± 0.117 (2) 0.142 ± 0.040 (6)
Discussion
•
" "f
1- 4 years 5-13 years
Control
both sexes were calculated together. The ratios of TS were all above the control range except for one child. The only patient exhibiting a very high ratio, 0.454, was a one-year-old girl with congenital muscular dystrophy and low urinary creatinine excretion. This high ratio was omitted from further calculation. Comparison of the ratios of TS and controls from two age groups is presented in Table 3. In both age groups TS showed higher ratios than controls. In the 5- to 13-year age group the difference in the mean ratio was significant by the t test. Nine of our 10 patients with TS received anticonvulsant therapy and only one aged 2 years had not been treated until urine analysis. The relationship between anticonvulsant therapy and the ratio was evaluated. In the 1- to 4-year age group the ratio of 0.297 in the patient without anticonvulsants was not lower than the other TS. In the 5- to 13-year age group no difference was observed in the ratio of control groups between 3 epilepsy with anticonvulsants (mean value ± SD of 0.095 ± 0.042) and epilepsy without anticonvulsants (0.10) .
TS
Shagreen 6 patch
TS
Values are expressed as mean ± SD (cases). # means significantly different from the control group (p < 0.01). See Table 1 and text for each case.
B. Frozen skin collagen sheet (mg)
Case no.
Age group
tein, in that there is the presence of the unusual amino acids, hydroxyproline and hydroxylysine (13) . The early experiments also showed that hydroxyproline in collagen could arise from proline but not from hydroxyproline which was administered to animals [14, 15] indicating that free hydroxyproline was not the source of collagen hydroxyproline. But nearly all the hydroxyproline in vertebrates is found in collagen and the only source of this amino acid appears to be the hydroxylation of "bound"
Tanaka et al: Hydroxyproline in tuberous sclerosis 83
proline in situ. In the present study we determined the hydroxyproline content in tissues as a marker of collagen protein and that in urine as an excretory product derived from the soluble and insoluble collagen of both soft and calcified tissues [16, 17] . Although for analysis of hydroxyproline in tissues defatted material is usually used, we attempted to use the crude whole homogenate for the following reasons: 1) There were formalin-fixed tissues ofTS for assaying the hydroxyproline. 2) These tissues were very small sections. 3) Our purpose for analyzing the hydroxyproline was for a relative comparison between a tumor and the normal surrounding tissue. With our procedures the measured values by repeated analyses almost agreed. Our results indicate that there is an excess of hydroxyproline in tumors of hydroxyproline-rich tissues, when compared with the surrounding normal tissues, but not in brain where hydroxyproline is hardly detectable. Furthermore the skin collagen sheet and shagreen patch from TS showed the same hydroxyproline content as that of controls. Based on the findings it is assumed that collagen deposition as observed histologically in TS tissues [8,9,18, 19,20] depends on the nature of the underlying tissues and that the overgrowth of collagen in the affected site is one of the most clear signs of TS. In the present study we could not analyze angiofibroma. The report by Fisher et al [7] showing that angiofibroma contained not more but less collagen than normal skin does not contradict our conclusion. To avoid difficulties in collecting 24-hour urine specimens from young children, Allison et al [21] presented evidence that the variability of the ratio of hydroxyproline: creatinine between individuals was much less than that of corresponding 24-hour urinary hydroxyproline values. Furthermore the early morning specimen was suggested to be the best sample [22] . Kulkarni et al found that a child consuming gelatin showed a slight increase in hydroxyproline excretion but that the variation in the hydroxyproline: creatinine ratio was not greater than the average variation found in individual samples [22]. The results obtained in this study were in agreement with those of several authors [21, 23] in that at all ages the ratio is essentially equivalent in both sexes and that the ratio depends on the growth rate. 84 Brain & Development, Vol 3, No 1, 1981
The patients with TS showed a higher hydroxyproline: creatinine ratio than the age-matched controls at all ages with or without anticonvulsant treatment. Several studies on the levels of urinary excretion of hydroxyproline in relation to pathological conditions have been published [13, 24, 25]. An elevated level of urinary hydroxyproline was reported in growing children and patients with acromegaly, thyrotoxicosis and hyperparathyroidism [13, 24]. Growth hormone probably influences stimulation of collagen synthesis, thyroid hormone stimulates both collagen synthesis and degradation, and parathyroid hormone also influences both the synthesis and degradation of bone matrix collagen [13]. To our knowledge, constant hormonal disturbance in TS is not observed. Furthermore in patients with Paget disease, Marfan syndrome, osteoporosis and scleromyxoedema as well as in patients suffering from "hydroxylation deficiency," increased excretion of hydroxyproline was reported [24, 26]. Patients in the former group showed well substantiated alterations in the collagen metabolism of bone or skin. On the other hand the elevated excretion of urinary hydroxyproline was detected in only one of seven patients with scleroderma where collagen deposition can be demonstrated only histologically [13]. Although the basic mechanism promoting the accumulation of collagen in TS tissues is not known, the elevated urinary hydroxyproline: creatinine ratio suggests that the quantitative alterations in collagen metabolism in TS may be large enough to be detected in the urine. Acknowledgments The authors are grateful to the following Drs. for supplying us with specimens from tuberous sclerosis subjects: Dr. T. Aoki, Dr. N. Fukunaga (Toho University), Dr. S. Ikeda, Dr. K. Shiota (Saitama Medical College), Dr. T. Takao (Tottori University), and Dr. K. Misugi (Yokohama City University). This study was supported in part by Grant No. 80-05-02 from the National Center for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan. References 1. Fleury P, de Groot WP, Delleman JW, Verbeeten B, Frankenmolen-Witkiezwicz 1M. Tuberous sclerosis: the iJ;lcidence of sporadic cases versus familial cases. Brain Dev 1980;2: 107-117. 2. Carpenter DG, Carter CH, Brinson EM, McCarty DA. Biochemical studies of tuberous sclerosis. I.
3.
4.
5. 6. 7.
8.
9. 10.
11.
12. 13.
14.
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