- Email: [email protected]
CIRCADIAN VARIATION IN HUMAN BRAIN ENZYMES
753 known pressor factors. We do not feel, however, that the two cases presented necessarily support this claim. When renal-vein-renin ratios are close, especially with low renin levels, determinations of renal blood-flow may be required to establish whether a kidney is secreting renin inappropriately. The development of new inhibitors of angiotensin 11 formation or true competitive antagonists of this vasoactive peptide may help to select more reliably patients with renin-
dependent hypertension. Department of Medicine,
General Infirmary, Leeds LS1 3EX
S. G. BALL R. D. THOMAS M. R. LEE
THE SEARCH FOR "MENOTOXIN"
SIR,- There are enough resemblances between plant and human biochemistry to make "menotoxin" (an acetone-soluble substance extractable from menstrual fluid and toxic to plants’-3) more than just a curiosity. In physiological amounts any "toxic" effects it may have on the menstruating woman herself are much less than the dramatic effects demonstrable on some plants, but if menotoxin could be isolated subtler effects on the human body might be sought. First, however, a more reliable plant test system is needed. Crude extracts containing mainly lipid material were made4 from menstrual dressings provided by students not taking oral contraceptives and, as controls, from unused tampons and towels. The method of extraction excludes bacterial contamination at this stage. The succulent plant Kalanchae blosfeldtiana may be induced to flower by exposure to a short-day regimen, followed by transfer to a long-day regimen. Individual plants may produce scores of flowers all in a similar stage of development.Flowers which are removed from the plant and incubated in humidity chambers at 250C with their stalks in a solution containing antibiotics (chloramphenicol and streptomycin, each at 25 g/ml) and the plant-growth substance kinetin (5 pLmoI/1) remain healthy for up to 20 days. Eventually, the petals lose their red colour and turn brown, probably because of the oxidation of polyphenols released during cell lysis. This loss of pigmentation followed by lysis and browning is similar to that seen in ageing leaves; and, as in leaves, senescence of Kalanchl1e flowers is much accelerated by the plant-growth substance abscisic acid (at 1 mmol/1). Senescence of excised Kalanchoe flowers was also much accelerated by menstrual extracts (made up in antibiotic-kinetin solution), whereas extracts from unused tampons did not have this effect. The acceleration was not due to acidity (pH 5-4) because menstrual extracts buffered at pH 7-4 had similar effects to unbuffered extracts, and control solutions buffered at pH 5.4 did not accelerate senescence. Nor was it due to more rapid growth of microorganisms in menstrual extracts, for, when we incubated flowers in solutions containing no antibiotics and sucrose (0-2mol/1) as an extra carbon source, the resultant heavy microbial contamination did not accelerate flower senescence. We conclude that a component of menstrual extracts causes an acceleration of senescence in excised Kalanchöe flowers. That much is fact; what follows is largely speculation. Since high concentrations of prostaglandin E1 (P.G. E1) induce the development of plant acid phosphatasecould "menotoxin" be one or more of the prostaglandins6 found in menstrual extracts? We tested prostaglandins E l’ E2’ F 1 cx, and F:zcx in concentrations similar to those found in menstrual extracts, and found them inactive on our system; so were p.G.A.1, A2. and 1. Reid, H. E. Lancet, 1974, 1, 988. 2. David, G. ibid. p. 1172. 3. Pickles, V. R. ibid. p. 1292. 4. Chtheroe, H. J., Pickles, V. R. J. Physiol. Lond. 1961, 156, 255. 5. Curry, S. C., Galsky, A. G. Plant Cell Physiol. Tokyo, 16, 799. 6. Pickles, V. R., Hall, W. J., Best, F. A., Smith, G. N. J. Obstet. Commonw. 1965, 72, 185.
many other prostaglandins and related substances not yet tested. An alternative approach might be to seek in menstrual extracts substances known for their toxic effects on plants. One possibility is fusaric acid, a plant-growth inhibitor produced by fungi and sometimes used for treatment of hypertension because it inhibits dopamine-p-hydroxylase. A concentration of 1 mmol/1 was clearly active in our experimental system, having effects similar to those of abscisic acid and the menstrual extracts. Hartmann and Keller-Tescfikel found that 600 mg of fusaric acid followed by three 500 mg doses of oral levodopa produced temporary effects that might be called psychotomimetic. So far as we know, fusaric (5-butylpicolinic) acid has not been identified as a product of human or mammalian metabolism, although picolinic acid is an isomer of nicotinic acid. We are not suggesting that the mood changes before or during menstruation are exactly like those found by Hartmann and Keller-Teschke, and even less that the menotoxin is fusaric acid-indeed, there is no evidence that the menotoxin is a single substance or that the menstruating uterus is its only human source. However, the example of fusaric acid does show the possibility of there being substances capable of interacting both with plant and human metabolism, in a manner relevant to the mood changes in the menstrual but there
B,,
phase. Departments of Botany and University College,
Br.
J. A. BRYANT
of Physiology,
D. G. HEATHCOTE V. R. PICKLES
P.O. Box 78, Cardiff CF1 1XL
CIRCADIAN VARIATION IN HUMAN BRAIN ENZYMES
SIR,-While investigating the effects of various factors
on
cerebral enzyme activities in
patients with no neurological or psychiatric abnormality we found that enzyme activity varied significantly with time of death. Most of the enzymes so far measured in the frontal cortex (including glutamate decarboxylase, choline acetyltransferase, hexokinase, and acetylcholinesterase) appear to undergo fluctuations throughout the 24 h period. The size and phase of these changes differed in the various enzymes. Data for acetylcholinesterase, which hydrolyses the putative neurotransmitter acetylcholine, are shown in the table. The ACETYLCHOLINESTERASE ACTIVITIES IN FRONTAL CORTEX (BRODMANN AREA 10) OF NORMAL SUBJECTS
* Estimated by the method of Ellman
et
al.’ and
expressed
as
mean-rS.E.M.
f Difference (estimated by (P<0.02).
the Student’s
t
test) significantly different
activity is significantly raised in the middle of the day (3rd period) compared with the middle of the night (lst period, P<0.02). The difference did not seem to be due to differences in factors unrelated to the time of death, such as age, sampling delay, type of terminal illness, and terminal state of consciousness, none of which varied extensively within the four groups. enzyme
On the basis of these 7. 1.
Gynœc.
are
findings and of reports2-4
that animal
Hartmann, E., Keller-Teschke, M. Lancet, 1977, i, 37. Ellman, G. L., Courtney, K. D., Andres, V., Featherstone, R. H. Biochem. Pharmac. 1961, 7, 88. 2. Moudgil, V. K., Kanungo, M. S. Comp. gen. Pharmac. 1973, 4, 127. 3. Von Mayersbach, H. in Chronobiology (edited by L. E. Sheving, M. D. Halberg, and J. E. Pauly); p. 19. Stuttgart, 1974. 4. Mohan, C., Radha, E. Life Sci. 1974, 15, 231.
754 brain acetylcholinesterase undergoes circadian alterations, we suggest that the time of death may merit consideration in studies of necropsy brain tissue designed to investigate mental disorders. In addition, determination of the circadian fluctuation in putative neurotransmitter systems may be of value in the investigation of mental disorders. Thus, in depression, for example, the characteristic diurnal mood variation may be linked to fluctuations in one or more specific neurotransmitter activities. Department of Pathology, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE
ELAINE K. PERRY ROBERT H. PERRY MICHAEL J. TAYLOR BERNARD E. TOMLINSON
SEASONALITY IN DOWN SYNDROME
SIR,-Dr Janerich and Professor Jacobson (March 5, p. 515) discuss seasonality of Down syndrome births, as pre-
study from Jerusalem,l and put forward an endocrinological explanation for seasonality and the aetiology of Down syndrome. Noting a similarity between monthly conception-rates of Down syndrome fetuses and monthly variation in restradiol-receptor levels in mammary tumours, they suggest that the endocrine factors which cause the changes in restradiol-receptor concentration also affect the meiotic process. While this hypothesis is interesting, it needs to be considered critically. First, seasonality in Down syndrome has not been consistently demonstrated .2 For example, McDonaldfound no association with month of birth in a study of 2398 Down syndromes cases in Quebec. Haynes et al. found no seasonal pattern for Down syndrome births in a 37-year period in Rochester, Minnesota. Robinson (cited by Smith and Berg2), sented in
a
on the other hand, found that there was no difference in incidence between the periods May to October and November to April for births to women over 35 but, for women under 35 there was a marked increase in incidence in the May-October period. The total number of cases was only 36. In the Jerusalem studyl seasonal peaks in incidence were demonstrated in the spring and autumn but the total number of cases was only 103. These data and others reviewed by Smith and Berg do not support a strong relationship between season of birth and the incidence of Down syndrome. A second point is the association between Down syndrome and advanced maternal age. This is by far the most consistent epidemiological finding in Down syndrome. Any hypothesis on the aetiology of Down syndrome must take this into account, and the endocrinological explanation proposed would be more convincing if an association between restradiol-receptor levels and age could be demonstrated. In Robinson’s studyz seasonality of Down syndrome births was restricted to younger mothers, supporting the suggestion that women who give birth to a Down syndrome child at a young age should be considered separately when aetiological factors are being sought.’ Since young mothers account for only a small proportion of Down syndrome births, a process possibly unique to them cannot entirely explain the aetiology, hence the importance of looking at age-related changes in the suggested endocrinological mechanism.
Division of Epidemiology, School of Public Health, University of California, Los Angeles, Los Angeles, California 90024, U.S.A.
SERUM-25-HYDROXYCHOLECALCIFEROL AND RENAL OSTEODYSTROPHY
reported low concentrations of serum-25-hydroxycholecalciferol (25-H.c.c.) in chronic renal SIR,—Eastwood
al.have
a relation between low concentration values and histomorphometric evidence of osteomalacia. The low serum-24-H.c.c. was thought to be caused by a disturbance in the hepatic transformation of vitamin D due to induction of hepatic microsomal enzymes. In a study of renal osteodystrophy in 26 patients with chronic renal failure (not on haemodialysis and not receiving vitamin-D supplements) we measured the serum concentrations of 25-H.c.c., calcium, immunoreactive parathyroid hormone (P.T.H.),2 alkaline phosphatase, and creatinine. Histomorphometric analysis of transiliac bone-biopsy specimens, obtained after double labelling with tetracycline, was done on undecalcified bone sections using the point-count principle.3 4 Serum-25-H.c.c. was measured by a competitive protein-binding assay’ with a minor modification in the chromatographic step. The coefficient of variation of repeated measurements at 35nmol/l was 13-5%. The sensitivity in the routine assay was 3.5 nmol/l.
failure and found
HISTOMORPHOMETRIC AND BIOCHEMICAL VALUES IN
26
(MEAN +- S.D.)
PATIENTS WITH CHRONIC RENAL FAILURE
* Data from 134 normal Danish
Data from 11 normal Danish
subjects. subjects.
Like Eastwood et al.’ we divided our patients into three groups according to the severity of the mineralisation defect as expressed by calcification fronts which were normal (>62%) (6 patients) or 30-62% (10 patients) or <30% (10 patients). The results are shown in the table. With the decrease in calcification front the osteoid volume increased (R= 062, P<0.01) as did the osteoid covered surfaces. The osteoid volume exceeded 3 S.D. of normal mean in 9 of 10 patients in group 3 and in 5 of 10 patients in group 2. The mean serum-25-H.C.C. of all 26 patients (78 nmol/l) was slightly higher than the mean value found in 596 normal Danish subjects. The mean serum-25-H.C.C. was lower (P<0.01) in group 1 than in group 2 and 3. No patient had a serum-25-H.c.c. below 15 nmoi/1. No correlation was found between serum-25-H.c.c. and the de-
LOWELL E. SEVER 1.
Harlap, S. Am. J. Epidem. 1974, 99, 210. Smith, G. F.n Berg, J. M. Down’s Anomaly. Edinburgh, 1976. McDonald, A. D. Teratology, 1972, 6, 1. Haynes, S. G., Gibson, J. B., Kurland, L. T. Neurology, Minneapolis, 1974, 24, 691. 5. Emanuel, I., Sever. L. E., Milham, S., Thuline, H. C. Lancet, 1972, ii, 361.
1. 2. 3. 4.
et
2. 3.
4. 5.
Eastwood, J. B., Harris, E., Stamp, T. C. B., de Wardener, H. E. Lancet, 1976, ii, 1210. Christensen, M. S. Scand. J. clin. Lab. Invest. 1976, 36, 313. Meunier, P., Edouard, C., Courpron, P., Toussaint, F. in Vitamin D and Problems Related to Uremic Bone Disease (edited by de Gruyter); p. 149. Berlin, 1975. Frost, H. M. Calc. Tiss. Res. 1969, 3, 211. Haddad, J. G., Chyu, K. J. J. clin. Endocr. Metab. 1971, 33, 992.
dependent hypertension. Department of Medicine,
General Infirmary, Leeds LS1 3EX
S. G. BALL R. D. THOMAS M. R. LEE
THE SEARCH FOR "MENOTOXIN"
SIR,- There are enough resemblances between plant and human biochemistry to make "menotoxin" (an acetone-soluble substance extractable from menstrual fluid and toxic to plants’-3) more than just a curiosity. In physiological amounts any "toxic" effects it may have on the menstruating woman herself are much less than the dramatic effects demonstrable on some plants, but if menotoxin could be isolated subtler effects on the human body might be sought. First, however, a more reliable plant test system is needed. Crude extracts containing mainly lipid material were made4 from menstrual dressings provided by students not taking oral contraceptives and, as controls, from unused tampons and towels. The method of extraction excludes bacterial contamination at this stage. The succulent plant Kalanchae blosfeldtiana may be induced to flower by exposure to a short-day regimen, followed by transfer to a long-day regimen. Individual plants may produce scores of flowers all in a similar stage of development.Flowers which are removed from the plant and incubated in humidity chambers at 250C with their stalks in a solution containing antibiotics (chloramphenicol and streptomycin, each at 25 g/ml) and the plant-growth substance kinetin (5 pLmoI/1) remain healthy for up to 20 days. Eventually, the petals lose their red colour and turn brown, probably because of the oxidation of polyphenols released during cell lysis. This loss of pigmentation followed by lysis and browning is similar to that seen in ageing leaves; and, as in leaves, senescence of Kalanchl1e flowers is much accelerated by the plant-growth substance abscisic acid (at 1 mmol/1). Senescence of excised Kalanchoe flowers was also much accelerated by menstrual extracts (made up in antibiotic-kinetin solution), whereas extracts from unused tampons did not have this effect. The acceleration was not due to acidity (pH 5-4) because menstrual extracts buffered at pH 7-4 had similar effects to unbuffered extracts, and control solutions buffered at pH 5.4 did not accelerate senescence. Nor was it due to more rapid growth of microorganisms in menstrual extracts, for, when we incubated flowers in solutions containing no antibiotics and sucrose (0-2mol/1) as an extra carbon source, the resultant heavy microbial contamination did not accelerate flower senescence. We conclude that a component of menstrual extracts causes an acceleration of senescence in excised Kalanchöe flowers. That much is fact; what follows is largely speculation. Since high concentrations of prostaglandin E1 (P.G. E1) induce the development of plant acid phosphatasecould "menotoxin" be one or more of the prostaglandins6 found in menstrual extracts? We tested prostaglandins E l’ E2’ F 1 cx, and F:zcx in concentrations similar to those found in menstrual extracts, and found them inactive on our system; so were p.G.A.1, A2. and 1. Reid, H. E. Lancet, 1974, 1, 988. 2. David, G. ibid. p. 1172. 3. Pickles, V. R. ibid. p. 1292. 4. Chtheroe, H. J., Pickles, V. R. J. Physiol. Lond. 1961, 156, 255. 5. Curry, S. C., Galsky, A. G. Plant Cell Physiol. Tokyo, 16, 799. 6. Pickles, V. R., Hall, W. J., Best, F. A., Smith, G. N. J. Obstet. Commonw. 1965, 72, 185.
many other prostaglandins and related substances not yet tested. An alternative approach might be to seek in menstrual extracts substances known for their toxic effects on plants. One possibility is fusaric acid, a plant-growth inhibitor produced by fungi and sometimes used for treatment of hypertension because it inhibits dopamine-p-hydroxylase. A concentration of 1 mmol/1 was clearly active in our experimental system, having effects similar to those of abscisic acid and the menstrual extracts. Hartmann and Keller-Tescfikel found that 600 mg of fusaric acid followed by three 500 mg doses of oral levodopa produced temporary effects that might be called psychotomimetic. So far as we know, fusaric (5-butylpicolinic) acid has not been identified as a product of human or mammalian metabolism, although picolinic acid is an isomer of nicotinic acid. We are not suggesting that the mood changes before or during menstruation are exactly like those found by Hartmann and Keller-Teschke, and even less that the menotoxin is fusaric acid-indeed, there is no evidence that the menotoxin is a single substance or that the menstruating uterus is its only human source. However, the example of fusaric acid does show the possibility of there being substances capable of interacting both with plant and human metabolism, in a manner relevant to the mood changes in the menstrual but there
B,,
phase. Departments of Botany and University College,
Br.
J. A. BRYANT
of Physiology,
D. G. HEATHCOTE V. R. PICKLES
P.O. Box 78, Cardiff CF1 1XL
CIRCADIAN VARIATION IN HUMAN BRAIN ENZYMES
SIR,-While investigating the effects of various factors
on
cerebral enzyme activities in
patients with no neurological or psychiatric abnormality we found that enzyme activity varied significantly with time of death. Most of the enzymes so far measured in the frontal cortex (including glutamate decarboxylase, choline acetyltransferase, hexokinase, and acetylcholinesterase) appear to undergo fluctuations throughout the 24 h period. The size and phase of these changes differed in the various enzymes. Data for acetylcholinesterase, which hydrolyses the putative neurotransmitter acetylcholine, are shown in the table. The ACETYLCHOLINESTERASE ACTIVITIES IN FRONTAL CORTEX (BRODMANN AREA 10) OF NORMAL SUBJECTS
* Estimated by the method of Ellman
et
al.’ and
expressed
as
mean-rS.E.M.
f Difference (estimated by (P<0.02).
the Student’s
t
test) significantly different
activity is significantly raised in the middle of the day (3rd period) compared with the middle of the night (lst period, P<0.02). The difference did not seem to be due to differences in factors unrelated to the time of death, such as age, sampling delay, type of terminal illness, and terminal state of consciousness, none of which varied extensively within the four groups. enzyme
On the basis of these 7. 1.
Gynœc.
are
findings and of reports2-4
that animal
Hartmann, E., Keller-Teschke, M. Lancet, 1977, i, 37. Ellman, G. L., Courtney, K. D., Andres, V., Featherstone, R. H. Biochem. Pharmac. 1961, 7, 88. 2. Moudgil, V. K., Kanungo, M. S. Comp. gen. Pharmac. 1973, 4, 127. 3. Von Mayersbach, H. in Chronobiology (edited by L. E. Sheving, M. D. Halberg, and J. E. Pauly); p. 19. Stuttgart, 1974. 4. Mohan, C., Radha, E. Life Sci. 1974, 15, 231.
754 brain acetylcholinesterase undergoes circadian alterations, we suggest that the time of death may merit consideration in studies of necropsy brain tissue designed to investigate mental disorders. In addition, determination of the circadian fluctuation in putative neurotransmitter systems may be of value in the investigation of mental disorders. Thus, in depression, for example, the characteristic diurnal mood variation may be linked to fluctuations in one or more specific neurotransmitter activities. Department of Pathology, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE
ELAINE K. PERRY ROBERT H. PERRY MICHAEL J. TAYLOR BERNARD E. TOMLINSON
SEASONALITY IN DOWN SYNDROME
SIR,-Dr Janerich and Professor Jacobson (March 5, p. 515) discuss seasonality of Down syndrome births, as pre-
study from Jerusalem,l and put forward an endocrinological explanation for seasonality and the aetiology of Down syndrome. Noting a similarity between monthly conception-rates of Down syndrome fetuses and monthly variation in restradiol-receptor levels in mammary tumours, they suggest that the endocrine factors which cause the changes in restradiol-receptor concentration also affect the meiotic process. While this hypothesis is interesting, it needs to be considered critically. First, seasonality in Down syndrome has not been consistently demonstrated .2 For example, McDonaldfound no association with month of birth in a study of 2398 Down syndromes cases in Quebec. Haynes et al. found no seasonal pattern for Down syndrome births in a 37-year period in Rochester, Minnesota. Robinson (cited by Smith and Berg2), sented in
a
on the other hand, found that there was no difference in incidence between the periods May to October and November to April for births to women over 35 but, for women under 35 there was a marked increase in incidence in the May-October period. The total number of cases was only 36. In the Jerusalem studyl seasonal peaks in incidence were demonstrated in the spring and autumn but the total number of cases was only 103. These data and others reviewed by Smith and Berg do not support a strong relationship between season of birth and the incidence of Down syndrome. A second point is the association between Down syndrome and advanced maternal age. This is by far the most consistent epidemiological finding in Down syndrome. Any hypothesis on the aetiology of Down syndrome must take this into account, and the endocrinological explanation proposed would be more convincing if an association between restradiol-receptor levels and age could be demonstrated. In Robinson’s studyz seasonality of Down syndrome births was restricted to younger mothers, supporting the suggestion that women who give birth to a Down syndrome child at a young age should be considered separately when aetiological factors are being sought.’ Since young mothers account for only a small proportion of Down syndrome births, a process possibly unique to them cannot entirely explain the aetiology, hence the importance of looking at age-related changes in the suggested endocrinological mechanism.
Division of Epidemiology, School of Public Health, University of California, Los Angeles, Los Angeles, California 90024, U.S.A.
SERUM-25-HYDROXYCHOLECALCIFEROL AND RENAL OSTEODYSTROPHY
reported low concentrations of serum-25-hydroxycholecalciferol (25-H.c.c.) in chronic renal SIR,—Eastwood
al.have
a relation between low concentration values and histomorphometric evidence of osteomalacia. The low serum-24-H.c.c. was thought to be caused by a disturbance in the hepatic transformation of vitamin D due to induction of hepatic microsomal enzymes. In a study of renal osteodystrophy in 26 patients with chronic renal failure (not on haemodialysis and not receiving vitamin-D supplements) we measured the serum concentrations of 25-H.c.c., calcium, immunoreactive parathyroid hormone (P.T.H.),2 alkaline phosphatase, and creatinine. Histomorphometric analysis of transiliac bone-biopsy specimens, obtained after double labelling with tetracycline, was done on undecalcified bone sections using the point-count principle.3 4 Serum-25-H.c.c. was measured by a competitive protein-binding assay’ with a minor modification in the chromatographic step. The coefficient of variation of repeated measurements at 35nmol/l was 13-5%. The sensitivity in the routine assay was 3.5 nmol/l.
failure and found
HISTOMORPHOMETRIC AND BIOCHEMICAL VALUES IN
26
(MEAN +- S.D.)
PATIENTS WITH CHRONIC RENAL FAILURE
* Data from 134 normal Danish
Data from 11 normal Danish
subjects. subjects.
Like Eastwood et al.’ we divided our patients into three groups according to the severity of the mineralisation defect as expressed by calcification fronts which were normal (>62%) (6 patients) or 30-62% (10 patients) or <30% (10 patients). The results are shown in the table. With the decrease in calcification front the osteoid volume increased (R= 062, P<0.01) as did the osteoid covered surfaces. The osteoid volume exceeded 3 S.D. of normal mean in 9 of 10 patients in group 3 and in 5 of 10 patients in group 2. The mean serum-25-H.C.C. of all 26 patients (78 nmol/l) was slightly higher than the mean value found in 596 normal Danish subjects. The mean serum-25-H.C.C. was lower (P<0.01) in group 1 than in group 2 and 3. No patient had a serum-25-H.c.c. below 15 nmoi/1. No correlation was found between serum-25-H.c.c. and the de-
LOWELL E. SEVER 1.
Harlap, S. Am. J. Epidem. 1974, 99, 210. Smith, G. F.n Berg, J. M. Down’s Anomaly. Edinburgh, 1976. McDonald, A. D. Teratology, 1972, 6, 1. Haynes, S. G., Gibson, J. B., Kurland, L. T. Neurology, Minneapolis, 1974, 24, 691. 5. Emanuel, I., Sever. L. E., Milham, S., Thuline, H. C. Lancet, 1972, ii, 361.
1. 2. 3. 4.
et
2. 3.
4. 5.
Eastwood, J. B., Harris, E., Stamp, T. C. B., de Wardener, H. E. Lancet, 1976, ii, 1210. Christensen, M. S. Scand. J. clin. Lab. Invest. 1976, 36, 313. Meunier, P., Edouard, C., Courpron, P., Toussaint, F. in Vitamin D and Problems Related to Uremic Bone Disease (edited by de Gruyter); p. 149. Berlin, 1975. Frost, H. M. Calc. Tiss. Res. 1969, 3, 211. Haddad, J. G., Chyu, K. J. J. clin. Endocr. Metab. 1971, 33, 992.