Maturitas, 15 (1992) 113-119
113
Elsevier Scientific Publishers Ireland Ltd. MAT 00689
Skin collagen changes related to age and hormone replacement therapy C. Castelo-Branco, Department
M. Duran and J. Gonzhlez-Merlo
ofGynaecology and Obstetrics,
Hospital Clinic i Provincial de Barcelona School of Medicine, University of Barcelona, 08036-Barcelona (Spain)
(Received July 18, 1991; revision received October 17, 1991; accepted October 20, 1991)
A total of 76 nulliparous women who had been hospitalized for minor operations, classified according to age group (by decade from 20s to 60s) and 118 postmenopausal women randomly allocated to one of four groups were studied. In all, 312 skin biopsies were taken from the lower abdomen at 0 and 12 months and the skin collagen changes noted. Collagen content decreased significantly with age beyond the 40s (P c 0.001) and after the menopause (P < 0.01). The decrease was preventable by the use of hormone replacement therapy. All the therapeutic regimens induced increases in skin collagen content, whereas in the control group a significant decrease was observed (P < 0.05).
Key words: oestrogens; skin; collagen; age; menopause
Introduction The skin, like all other tissues, undergoes regressive changes with age (loss of elasticity, reduction in epidermal thickness, elastoic degeneration, etc.). The lack of interest generated by the study of these changes can be attributed firstly to the tendency to associate aging with degenerative disorders, such as cardiovascular diseases, cancer and osteoporosis, which together are responsible for the majority of deaths in people over 50, and secondly, as Kligman and Balin have pointed out [1], to the fact that no one after all dies of old skin! Studies [2,3] have shown that skin collagen is affected by hypo-oestrogenism and that the skin collagen content declines in the years following the menopause [4-61. The thickness of the skin, which was one of the aspects considered in these studies, decreased in a parallel manner after the menopause. The aims of our study were to determine how the amount of skin collagen varies according to age and time since menopause and to evaluate the effects of different hormone replacement therapy (HRT) regimens on skin collagen. Correspondence to: C. Castelo-Branco MD, Dept. of Gynaecology & Obstetrics, Hospital Clinic i Provincial de Barcelona, c/Villarroel 170, 08036_Barcelona, Spain. 0378-5122/92/$05.00
0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
114 TABLE I SKIN COLLAGEN, AGE AND TIME SINCE MENOPAUSE Decade
n
Age (years)
20 30 40 50 60
15 20 15 15 11
25.3 35.7 45.7 53.2 68.2
f f f f +
2.7 3.4 2.3 2.9 3.1
Mean elapsed time since menopause (months)
Skin collagen (fig collagen/ mg protein)
6.8 f 13.8 5.7 f 7.4 212.0 f 53.7
117.2 f 6.3 117.7 f 5.6 119.6 zt 8.9 114.2 zt 9.5 108.0 zt 7.2
Subjects and Methods In order to assess the effect of age and time since menopause on the amount of skin collagen, we studied 76 nulliparous women who had been admitted for minor operations, dividing them into age groups for this purpose (Table I). To evaluate the skin collagen changes brought about by HRT, 118 postmenopausal women who had undergone hysterectomy and bilateral oophorectomy were allocated at random to one of four different study groups. The four groups received 0.625 mg/day conjugated equine oestrogens (CEE) over a 25-day cycle each month (n = 28), 50 &day transdermal 17/3-oestradiol over a 24day cycle each month (n = 28), 0.625 mg/day CEE every day of the month (n = 32), and no treatment (n = 30), respectively. The untreated subjects constituted the control group. All the treated patients also received 2.5 mg/day medroxyprogesterone acetate sequentially for the last 12 days of each cycle. In all patients punch biopsy skin specimens, 4 mm in diameter, were taken from a non-sun-exposed site on the lower abdomen (4 cm above the pubic symphysis). In the four study groups, biopsies were taken at the beginning of the study and one year later. The second biopsy was taken from the same skin area but away from the immediate location of the first. In total, 3 12 samples were taken and processed in accordance with the calorimetric method described by Lopez de Leon and Rojkind [7]. The collagen content results are expressed as pg collagen/mg protein. The significance of differences within groups was assessed by the paired t-test and between groups by analysis of variance. Pearson’s correlation coefficients (r) were calculated and the regression lines delimiting the confidence intervals were plotted. The results were considered significant where P < 0.05. ReSllltS Age and time since menopause
The quantity of skin collagen plotted against age gave a curve whose peak corresponded to patients aged between 20 and 40 (Fig. 1). Thereafter it showed highly
20
30
50
40 Age
60
(years)
Collagen
-
Fig. 1. Skin collagen changes with age.
120
115
110 1
I
I
I
5
10
15
I
I
I
20
25
Time since menopause
-
Collagen
+
I
30
35
(TSM) in years
Collagen
Fig. 2. Skin collagen changes realted to time since menopause.
I
40
(TSM P 12)
I
45
50
116 TABLE II INITIAL CLINICAL DATA Group
n
Age (years)
Mean elapsed time since menopause (months)
Weight (kg)
CEE cy n-S CEE co Control
28 28 32 30
46.3 * 5.2 41.6 f 5.3 46.3 f 6.1 48.1 ?? 4.3
21.2 ?? 13.1 18.3 zk 13.2 22.5 zt 12.7 20.0 ?? 11.4
68.5 68.8 67.3 61.3
f zt zt f
2.2 5.6 7.1 5.2
CEE cy, conjugated equine oestrogens cyclically; TTS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously. Values are means f SD.
significant (I = 0.6143, P < 0.001) inverse correlation with chronological age. When the relationship between time since menopause and skin collagen content was evaluated, it was again found to be statistically significant (r = 0.5098, P < 0.01; Fig. 2). Skin collagen and HRT No significant differences between the four groups were found as regards the in-
itial clinical data and baseline values (Tables II and III). When the skin collagen content after 12 months of treatment was assessed an increase was observed in all the HRT groups, whereas the control group, as expected, showed a significant decrease of almost 3.2% (P < 0.05; Fig. 3). In the group which received the cyclical CEE regimen an increase of less than 2% was seen (2.0 pg collagen/mg protein), the smallest registered in any of the treated groups. The group on the continuous CEE regimen showed a slightly higher increase (3.0%, P < 0.05), but it was in the transdermal group that the greatest increase was observed, namely 5.1% (5.8 &mg, P c 0.01). When the results after 12 months of treatment were compared significant differences were found between the treated (transdermal and continuous CEE) and the untreated patients. The difference proved to be greatest in the case of the transdermal group (7.0 pg collagen/mg protein, P < 0.01). TABLE III SKIN COLLAGEN CONTENT AT BASELINE AND AFTER 12 MONTHS Group
Collagen at 0 months
Collagen after 12 months
Change (%)
CEE cy (1) l-l-S (2) CEE co (3) Control (4)
115.1 zt 0.8 114.0 f 1.2 114.4 ?? 0.9 116.5 f 1.1
117.1 f 119.8 zt 117.8 f 112.8 f
+1.8 +5.1* +3.0** -3.2**
0.7 0.6 1.0 0.9
Within groups: *P < 0.01, **P < 0.05. Between groups: 2-4 P < 0.01, 3-4 P < 0.05. Values are means?? S.D. expressed as cg collagemmg protein CEE cy, conjugated equine oestrogens cyclically; TTS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously.
117
TTS
CEE cy m
Initial
CEE
m
co
Con troi
After 12 months
Fig. 3. Changes in skin collagen content related to hormone replacement therapy. CEE cy, conjugated equine oestrogens cyclically; ITS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously.
In terms of quantity, collagen is the major component of the skin. To study it, we need a simple, accurate method that allows us to determine how much is present in small quantities of tissue. Brincat [2-6,8] recently calculated the collagen content in accordance with the methods described by Neuman [9] and Woessner [lo], but the total error involved was 11.4% [2]. In a previous study we described the method we had used to determine the amount of collagen in small samples of skin with an error of close to 5% [ll]. Although, traditionally, more importance is attached to time since menopause rather than age as a cause of the phenomena associated with oestrogen deficiency [12-161, we found in our study that the closest correlation was with chronological rather than postmenopausal age. We believe this was due to the fact that the majority of the postmenopausal women aged between 40 and 55 in this study (over 60%) had recently undergone surgical menopause and had therefore spent very little time in the postmenopause (Table I). In such patients ovarian function remains virtually intact until the moment that surgery takes place, which is why they were practically completely unaffected by the relative hormone deficiency observable during the perimenopause. The effects of oestrogen deficiency were consequently restricted to the short period of time between surgery and participation in the study. In our results the statistical significance of the correlation between time since menopause
118
and collagen content had to be carefully assessed, since it could have been included in the chronological age variable, which is much stronger. Moreover, when patients who have been postmenopausal for longer than 1 year are considered, the reduction in skin collagen content is more pronounced and the gradient of the line is steeper than in the case of chronological age (Fig. 1). The results in the control group and those obtained by studying the development of skin collagen with age show that skin collagen loss increases markedly in the initial postmenopausal years. These findings are consistent with those reported by Punnonen [ 171, to the effect that the greatest reduction in skin thickness was observed in the years immediately following bilateral oophorectomy. Previous studies have set out to evaluate several oral HRT regimens, but have not demonstrated any important differences between them [17-191. On the basis of our results we are warranted in suggesting that daily administration is more effective than cyclical regimens, since it produces a greater increase in the amount of collagen present. Even though a small increase (1.8%) could be detected in the case of the cyclical CEE regimen, it was much smaller than the margin of error of the method, so it cannot be known for certain whether it was a real increase. Our results also confirm the value of the transdermal route for preventing collagen loss. A previous study by Brincat [4] also pointed to the greater effectiveness of the percutaneous route compared with other parenteral routes (implants). Our study compared treated and untreated patients and so paralleled that carried out in Dulwich [3]. The quantity of skin collagen proved to be significantly higher in HRT patients than in women of the same age who received no treatment. Moreover, the differences observed at 12 months between the HRT groups and the control group are greater in every case than those detected within groups of treated patients. The results support the clinical judgement that the skin of postmenopausal patients who have taken HRT appears to deteriorate less as a result of aging. References Kligman AM, Balin AK. Aging of human skin. In: Balin AK, Kligman AM, eds. Aging and the skin. Raven Press, New York, 1989, l-8. Brincat M, Kabalan S, Studd JWW, Moniz CF, de Trafford J, Montgomery J. A study of the decrease of skin collagen content, skin thickness and bone mass in the postmenopausal woman. Obstet Gynecol 1987; 70: 840-845. Brincat M, Moniz CF, Studd JWW, Darby AJ, Magos A, Cooper D. Sex hormones and skin collagen content in postmenopausal women. Br Med J 1983; 287: 1337-1338. Brincat M, Versi E, Moniz CJ, Magos A, de Trafford J, Studd JWW. Skin collagen changes in postmenopausal women receiving different regimens of estrogen therapy. Obstet Gynecol 1987; 70: 123-127. Brincat M, Moniz CF, Kabalan S, Versi E, O’Dowd T, Magos AL, Montgomery J, Studd JWW. Decline in skin collagen content and metacarpal index after the menopause and its prevention with sex hormone replacement. Br J Obstet Gynaecol 1987; 94: 126-129. Brincat M, Moniz CF, Studd JWW, Darby AJ, Magos A, Emburey G, Versi E. The long-term effects of the menopause and of administration of sex hormones on skin collagen and skin thickness. Br J Obstet Gynaecol 1985; 92: 256-259. Lopez de Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-tixed parafftnembedded sections. J Histochem Cytochem 1985; 33: 737-743.
119
8 Brincat M, Versi E, O’Dowd T, Moniz CF, Magos A, Kabalan S, Studd JWW. Skin collagen 9 10 11 12 13 14 15 16 17 18
19
changes in post-menopausal women receiving oestradiol gel. Maturitas 1987; 9: 1-5. Neuman RE, Logan MA. The determination of collagen and elastin in tissues. J Biol Chem 1950; 186: 549-556. Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1960; 93: 440-447. Castelo-Branco C, Valderrama R, Osuna C, Gonzalez-Merlo J. Measurement of collagen changes with aging in punch skin biopsies: Evaluation of a calorimetric method. Rev Diag Biol (in press). Riis BJ, Thomsen K, Str#n V, Christiansen C. The effect of percutaneous estradiol and natural progesterone on post-menopausal bone loss. Am J Obstet Gynecol 1987; 156: 61-65. Christiansen C, Riis BJ, R@ibro P. Prediction of rapid bone loss in postmenopausal women. Lancet 1987; i: 1105-I 108. Smith R. Osteoporosis: cause and management. Br Med J 1987; 294: 329-332. Evans RA. The diagnosis of postmenopausal osteoporosis. Med J Aust 1987; 146: 285-286. Lindsay R. Estrogens in the prevention and treatment of osteoporosis. Schweiz Med Wschr 1989; 119: 1806-1810. Punnonen R. Effect of castration and peroral therapy on skin. Acta Obstet Gynecol Stand 1973; Suppl 21: l-45. Punnonen R. The effect of castration and peroral oestrogen therapy on the epidermal mitotic activity in women. In: Basic actions of sex steroids on target organs. Basel: Karger, 1971: 254. Punnonen R. On the effect of castration and peroral estrogen therapy on the skin. Acta Obstet Gynecol Stand 1971; Suppl 50: 32-40.
113
Elsevier Scientific Publishers Ireland Ltd. MAT 00689
Skin collagen changes related to age and hormone replacement therapy C. Castelo-Branco, Department
M. Duran and J. Gonzhlez-Merlo
ofGynaecology and Obstetrics,
Hospital Clinic i Provincial de Barcelona School of Medicine, University of Barcelona, 08036-Barcelona (Spain)
(Received July 18, 1991; revision received October 17, 1991; accepted October 20, 1991)
A total of 76 nulliparous women who had been hospitalized for minor operations, classified according to age group (by decade from 20s to 60s) and 118 postmenopausal women randomly allocated to one of four groups were studied. In all, 312 skin biopsies were taken from the lower abdomen at 0 and 12 months and the skin collagen changes noted. Collagen content decreased significantly with age beyond the 40s (P c 0.001) and after the menopause (P < 0.01). The decrease was preventable by the use of hormone replacement therapy. All the therapeutic regimens induced increases in skin collagen content, whereas in the control group a significant decrease was observed (P < 0.05).
Key words: oestrogens; skin; collagen; age; menopause
Introduction The skin, like all other tissues, undergoes regressive changes with age (loss of elasticity, reduction in epidermal thickness, elastoic degeneration, etc.). The lack of interest generated by the study of these changes can be attributed firstly to the tendency to associate aging with degenerative disorders, such as cardiovascular diseases, cancer and osteoporosis, which together are responsible for the majority of deaths in people over 50, and secondly, as Kligman and Balin have pointed out [1], to the fact that no one after all dies of old skin! Studies [2,3] have shown that skin collagen is affected by hypo-oestrogenism and that the skin collagen content declines in the years following the menopause [4-61. The thickness of the skin, which was one of the aspects considered in these studies, decreased in a parallel manner after the menopause. The aims of our study were to determine how the amount of skin collagen varies according to age and time since menopause and to evaluate the effects of different hormone replacement therapy (HRT) regimens on skin collagen. Correspondence to: C. Castelo-Branco MD, Dept. of Gynaecology & Obstetrics, Hospital Clinic i Provincial de Barcelona, c/Villarroel 170, 08036_Barcelona, Spain. 0378-5122/92/$05.00
0 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
114 TABLE I SKIN COLLAGEN, AGE AND TIME SINCE MENOPAUSE Decade
n
Age (years)
20 30 40 50 60
15 20 15 15 11
25.3 35.7 45.7 53.2 68.2
f f f f +
2.7 3.4 2.3 2.9 3.1
Mean elapsed time since menopause (months)
Skin collagen (fig collagen/ mg protein)
6.8 f 13.8 5.7 f 7.4 212.0 f 53.7
117.2 f 6.3 117.7 f 5.6 119.6 zt 8.9 114.2 zt 9.5 108.0 zt 7.2
Subjects and Methods In order to assess the effect of age and time since menopause on the amount of skin collagen, we studied 76 nulliparous women who had been admitted for minor operations, dividing them into age groups for this purpose (Table I). To evaluate the skin collagen changes brought about by HRT, 118 postmenopausal women who had undergone hysterectomy and bilateral oophorectomy were allocated at random to one of four different study groups. The four groups received 0.625 mg/day conjugated equine oestrogens (CEE) over a 25-day cycle each month (n = 28), 50 &day transdermal 17/3-oestradiol over a 24day cycle each month (n = 28), 0.625 mg/day CEE every day of the month (n = 32), and no treatment (n = 30), respectively. The untreated subjects constituted the control group. All the treated patients also received 2.5 mg/day medroxyprogesterone acetate sequentially for the last 12 days of each cycle. In all patients punch biopsy skin specimens, 4 mm in diameter, were taken from a non-sun-exposed site on the lower abdomen (4 cm above the pubic symphysis). In the four study groups, biopsies were taken at the beginning of the study and one year later. The second biopsy was taken from the same skin area but away from the immediate location of the first. In total, 3 12 samples were taken and processed in accordance with the calorimetric method described by Lopez de Leon and Rojkind [7]. The collagen content results are expressed as pg collagen/mg protein. The significance of differences within groups was assessed by the paired t-test and between groups by analysis of variance. Pearson’s correlation coefficients (r) were calculated and the regression lines delimiting the confidence intervals were plotted. The results were considered significant where P < 0.05. ReSllltS Age and time since menopause
The quantity of skin collagen plotted against age gave a curve whose peak corresponded to patients aged between 20 and 40 (Fig. 1). Thereafter it showed highly
20
30
50
40 Age
60
(years)
Collagen
-
Fig. 1. Skin collagen changes with age.
120
115
110 1
I
I
I
5
10
15
I
I
I
20
25
Time since menopause
-
Collagen
+
I
30
35
(TSM) in years
Collagen
Fig. 2. Skin collagen changes realted to time since menopause.
I
40
(TSM P 12)
I
45
50
116 TABLE II INITIAL CLINICAL DATA Group
n
Age (years)
Mean elapsed time since menopause (months)
Weight (kg)
CEE cy n-S CEE co Control
28 28 32 30
46.3 * 5.2 41.6 f 5.3 46.3 f 6.1 48.1 ?? 4.3
21.2 ?? 13.1 18.3 zk 13.2 22.5 zt 12.7 20.0 ?? 11.4
68.5 68.8 67.3 61.3
f zt zt f
2.2 5.6 7.1 5.2
CEE cy, conjugated equine oestrogens cyclically; TTS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously. Values are means f SD.
significant (I = 0.6143, P < 0.001) inverse correlation with chronological age. When the relationship between time since menopause and skin collagen content was evaluated, it was again found to be statistically significant (r = 0.5098, P < 0.01; Fig. 2). Skin collagen and HRT No significant differences between the four groups were found as regards the in-
itial clinical data and baseline values (Tables II and III). When the skin collagen content after 12 months of treatment was assessed an increase was observed in all the HRT groups, whereas the control group, as expected, showed a significant decrease of almost 3.2% (P < 0.05; Fig. 3). In the group which received the cyclical CEE regimen an increase of less than 2% was seen (2.0 pg collagen/mg protein), the smallest registered in any of the treated groups. The group on the continuous CEE regimen showed a slightly higher increase (3.0%, P < 0.05), but it was in the transdermal group that the greatest increase was observed, namely 5.1% (5.8 &mg, P c 0.01). When the results after 12 months of treatment were compared significant differences were found between the treated (transdermal and continuous CEE) and the untreated patients. The difference proved to be greatest in the case of the transdermal group (7.0 pg collagen/mg protein, P < 0.01). TABLE III SKIN COLLAGEN CONTENT AT BASELINE AND AFTER 12 MONTHS Group
Collagen at 0 months
Collagen after 12 months
Change (%)
CEE cy (1) l-l-S (2) CEE co (3) Control (4)
115.1 zt 0.8 114.0 f 1.2 114.4 ?? 0.9 116.5 f 1.1
117.1 f 119.8 zt 117.8 f 112.8 f
+1.8 +5.1* +3.0** -3.2**
0.7 0.6 1.0 0.9
Within groups: *P < 0.01, **P < 0.05. Between groups: 2-4 P < 0.01, 3-4 P < 0.05. Values are means?? S.D. expressed as cg collagemmg protein CEE cy, conjugated equine oestrogens cyclically; TTS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously.
117
TTS
CEE cy m
Initial
CEE
m
co
Con troi
After 12 months
Fig. 3. Changes in skin collagen content related to hormone replacement therapy. CEE cy, conjugated equine oestrogens cyclically; ITS, transdermal therapeutic system; CEE co, conjugated equine oestrogens continuously.
In terms of quantity, collagen is the major component of the skin. To study it, we need a simple, accurate method that allows us to determine how much is present in small quantities of tissue. Brincat [2-6,8] recently calculated the collagen content in accordance with the methods described by Neuman [9] and Woessner [lo], but the total error involved was 11.4% [2]. In a previous study we described the method we had used to determine the amount of collagen in small samples of skin with an error of close to 5% [ll]. Although, traditionally, more importance is attached to time since menopause rather than age as a cause of the phenomena associated with oestrogen deficiency [12-161, we found in our study that the closest correlation was with chronological rather than postmenopausal age. We believe this was due to the fact that the majority of the postmenopausal women aged between 40 and 55 in this study (over 60%) had recently undergone surgical menopause and had therefore spent very little time in the postmenopause (Table I). In such patients ovarian function remains virtually intact until the moment that surgery takes place, which is why they were practically completely unaffected by the relative hormone deficiency observable during the perimenopause. The effects of oestrogen deficiency were consequently restricted to the short period of time between surgery and participation in the study. In our results the statistical significance of the correlation between time since menopause
118
and collagen content had to be carefully assessed, since it could have been included in the chronological age variable, which is much stronger. Moreover, when patients who have been postmenopausal for longer than 1 year are considered, the reduction in skin collagen content is more pronounced and the gradient of the line is steeper than in the case of chronological age (Fig. 1). The results in the control group and those obtained by studying the development of skin collagen with age show that skin collagen loss increases markedly in the initial postmenopausal years. These findings are consistent with those reported by Punnonen [ 171, to the effect that the greatest reduction in skin thickness was observed in the years immediately following bilateral oophorectomy. Previous studies have set out to evaluate several oral HRT regimens, but have not demonstrated any important differences between them [17-191. On the basis of our results we are warranted in suggesting that daily administration is more effective than cyclical regimens, since it produces a greater increase in the amount of collagen present. Even though a small increase (1.8%) could be detected in the case of the cyclical CEE regimen, it was much smaller than the margin of error of the method, so it cannot be known for certain whether it was a real increase. Our results also confirm the value of the transdermal route for preventing collagen loss. A previous study by Brincat [4] also pointed to the greater effectiveness of the percutaneous route compared with other parenteral routes (implants). Our study compared treated and untreated patients and so paralleled that carried out in Dulwich [3]. The quantity of skin collagen proved to be significantly higher in HRT patients than in women of the same age who received no treatment. Moreover, the differences observed at 12 months between the HRT groups and the control group are greater in every case than those detected within groups of treated patients. The results support the clinical judgement that the skin of postmenopausal patients who have taken HRT appears to deteriorate less as a result of aging. References Kligman AM, Balin AK. Aging of human skin. In: Balin AK, Kligman AM, eds. Aging and the skin. Raven Press, New York, 1989, l-8. Brincat M, Kabalan S, Studd JWW, Moniz CF, de Trafford J, Montgomery J. A study of the decrease of skin collagen content, skin thickness and bone mass in the postmenopausal woman. Obstet Gynecol 1987; 70: 840-845. Brincat M, Moniz CF, Studd JWW, Darby AJ, Magos A, Cooper D. Sex hormones and skin collagen content in postmenopausal women. Br Med J 1983; 287: 1337-1338. Brincat M, Versi E, Moniz CJ, Magos A, de Trafford J, Studd JWW. Skin collagen changes in postmenopausal women receiving different regimens of estrogen therapy. Obstet Gynecol 1987; 70: 123-127. Brincat M, Moniz CF, Kabalan S, Versi E, O’Dowd T, Magos AL, Montgomery J, Studd JWW. Decline in skin collagen content and metacarpal index after the menopause and its prevention with sex hormone replacement. Br J Obstet Gynaecol 1987; 94: 126-129. Brincat M, Moniz CF, Studd JWW, Darby AJ, Magos A, Emburey G, Versi E. The long-term effects of the menopause and of administration of sex hormones on skin collagen and skin thickness. Br J Obstet Gynaecol 1985; 92: 256-259. Lopez de Leon A, Rojkind M. A simple micromethod for collagen and total protein determination in formalin-tixed parafftnembedded sections. J Histochem Cytochem 1985; 33: 737-743.
119
8 Brincat M, Versi E, O’Dowd T, Moniz CF, Magos A, Kabalan S, Studd JWW. Skin collagen 9 10 11 12 13 14 15 16 17 18
19
changes in post-menopausal women receiving oestradiol gel. Maturitas 1987; 9: 1-5. Neuman RE, Logan MA. The determination of collagen and elastin in tissues. J Biol Chem 1950; 186: 549-556. Woessner JF Jr. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1960; 93: 440-447. Castelo-Branco C, Valderrama R, Osuna C, Gonzalez-Merlo J. Measurement of collagen changes with aging in punch skin biopsies: Evaluation of a calorimetric method. Rev Diag Biol (in press). Riis BJ, Thomsen K, Str#n V, Christiansen C. The effect of percutaneous estradiol and natural progesterone on post-menopausal bone loss. Am J Obstet Gynecol 1987; 156: 61-65. Christiansen C, Riis BJ, R@ibro P. Prediction of rapid bone loss in postmenopausal women. Lancet 1987; i: 1105-I 108. Smith R. Osteoporosis: cause and management. Br Med J 1987; 294: 329-332. Evans RA. The diagnosis of postmenopausal osteoporosis. Med J Aust 1987; 146: 285-286. Lindsay R. Estrogens in the prevention and treatment of osteoporosis. Schweiz Med Wschr 1989; 119: 1806-1810. Punnonen R. Effect of castration and peroral therapy on skin. Acta Obstet Gynecol Stand 1973; Suppl 21: l-45. Punnonen R. The effect of castration and peroral oestrogen therapy on the epidermal mitotic activity in women. In: Basic actions of sex steroids on target organs. Basel: Karger, 1971: 254. Punnonen R. On the effect of castration and peroral estrogen therapy on the skin. Acta Obstet Gynecol Stand 1971; Suppl 50: 32-40.
