- Email: [email protected]
SERUM EQUILIN AND CONJUGATED EQUINE ŒSTROGENS
547 have been excluded from the calculations. Analysis by both Spearman rank and Kendall rank correlation methods (parametric statistics are invalid because of the non-gaussian distribution of the data) shows no significant relationship between serum ferritin levels and age in the remaining 12 patients. Hæmatology Division, Institute of Medical and Veterinary Science Adelaide, South Australia 5000
R. W. BLUNDEN B. M. DUNCAN
SERUM EQUILIN AND CONJUGATED EQUINE ŒSTROGENS
SIR,-Many technical, metabolic, and
clinical issues
are
by the paper from Dr Whittaker and his colleagues (Jan. 5, p. 14). They suggest that the grossly increased and very variable serum concentration of equilin and its improbable raised
relation
and oestradiol in are due to some unusual property of the metabolism of this hormone in women. To support this statement they refer to our report on the metabolism of 3H-equilin in man.’ They misinterpret our data as indicating that after the intravenous injection of 3H-equilin there is a slow urinary excretion and persistently high serum levels of the hormone 24 h after injection. However, the data to which they refer deal with total radioactivity rather than with "unconjugated equilin" which they claim to measure in their radioimmunoassay. We found in the plasma only 3% of’ the total dose injected after 24 h, and by 72 h almost no radioactivity (10-12 cpm/ml) is present in the serum. During this period (72 h) over 72% of the injected dose was recovered in the urine exclusive of any enterohepatic circulation or excretion in the faeces. Furthermore, Whittaker et al. have not taken into account the rapid metabolism of equilin reported by us.’ Thus the radioactive materials found in the blood and urine are mainly reduced and conjugated metabolites of equilin. For example, 20 min after injection of equilin the concentration of equilin sulphate is twice that of equilin and this increased to 50 times the level of equilin after 90 min. Some months ago we shared with one of this group (Dr P. D. G. Dean) other dataZ relating to the metabolism of 3H-equilin, 3-equilin sulphate and premarin given both intravenously and by mouth. In the latter experiments we found that the serum concentration of unconjugated cestrone and of unconjugated equilin continued to reflect the ratio of the sulphates found in the premarin used in these experiments. The half-life of unconjugated equilin after the injection of 10 mg premarin intravenously was approximately 43 min while that of oestrone was 63 min. After oral premarin 10 mg (i.e., 2-5 mg equilin sulphate) we found the maximum concentration of free equilin in plasma to be 560 pg/ml (2090 pmol/1) at 4 h. At 8 h the serum concentration was half of the peak value and after 24 h only trace amounts could be found. While this pattern is similar (a 4 h peak) to that reported by Whittaker et al. the absolute values which they found after administering only 1-25 mg premarin (0-3mg equilin sulphate) are very much higher (6753 pg/ml and 1635 pg/ml at 4 and 24 h). It is not likely that different rates of absorption could explain these differences and it is not even remotely possible that the absorption would be sufficiently slow to explain the presence of equilin in the serum 13 weeks after the last oral dose. women
Normal females
Steroid sulphatase
activity
Affected males
Hetero-
zygotes
in cultural fibroblasts.
the normal or the defective X chromosome. An exception seems to be the steroid sulphatase locus which escapes inactivation.’ Theoretically, then, detection should be easier for this enzyme defect than for other X-linked enzyme defects. We have re-examined this question using dehydroepiandrosterone sulphate (DHEAS) as substrate. With DHEAS we have found enzyme activities to be about 100 times higher than they are with cholesteryl sulphate. And this property has enabled us to demonstrate that steroid sulphatase activities are significantly higher in females than in males.8 As the figure shows, enzyme activities in fibroblasts of five unrelated obligate heterozygotes (ascertained by two affected sons, one affected son plus positive family history, or affected father) and ten normal females do not overlap. In heterozygotes, there is remarkably little interindividual variation, which fits into the concept of non-inactivation of the gene. With DHEAS carrier detection is possible for steroid sulphatase deficiency. Previous failures to detect heterozygotes are probably due to unsuitable substrates rather than biological variation. Institute of Human Genetics and Anthropology,
University of Freiburg, D-7800 Freiburg, West Germany Dermatological Hospital, University of Münster
C. R. MÜLLER B. MIGL H. H. ROPERS R. HAPPLE
IRON OVERLOAD AND AGE IN &bgr;-THALASSÆMIA SIR,-Dr Lagos and colleagues (Jan. 26, p. 204) claim that iron overload, as measured by serum ferritin, is positively related to age in patients with p-thalasseemia intermedia. While there is indeed a significant statistical relationship between serum ferritin and age in these 15 patients, 3 of them had received blood transfusions. Since the number of transfusions is correlated with serum ferritin levels,’ these 3 patients should 7. Shapiro LJ, Mohandas T, Weiss R, Romeo G. Non-inactivation of an X chromosome locus in man. Science 1979; 204: 1224-26. 8. Müller CR, Migl B, Traupe, Ropers HH. X-linked steroid sulfatase: Evidence for different gene dosage in males and females. Hum Genet (in 1.
press). Letsky EA,
Miller F, Worwood M, Flynn DM. Serum ferritin in children with thalassæmia regularly transfused. J Clin Pathol 1974; 27: 652-55.
to
the concentration of
cestrone
taking conjugated equine cestrogens
1. Bhavnani BR, Woolever CA. Alternate pathways of steroid biosynthesis in the origin, metabolism and biological effects of ring B unsaturated oestrogens. In: Hobkirk R, ed. Steroid biochemistry: vol II. Boca Raton, Florida: C.R.C. Press, 1979: 1-50. 2. Bhavnani BR, Woolever CA. Absorption and metabolism of equine estrogens in postmenopausal women and normal men. Society for Gynecologic Investigation 26th annual meeting (San Diego, California, March, 1979):
abstrp. 9 (no. 12). 3. Ruder HJ, Loriaux L, Lipsett MB. Estrone sulfate: Production metabolism in man. J Clin Invest 1972, 51: 1020.
rate
and
548
.
Our pharmacokinetic studies of equilin metabolism in humans and pregnant mares leads us to believe that the metabolism of equilin in the human is much the same as that of other cestrogens, including oestrone. Our experience in the development of the radioimmunoassay indicates that not only cross-reactions with other steroids but also a non-specific and erratic effect of human plasma and serum on the assay systems lead to very high and variable results unless the hormone is isolated and purified before assay. All of the values to which we refer in our publications were obtained by this technique and have been corrected for procedural losses. Another related problem is the purity of the 3H-equilin used in the assay. The freshly synthesised material which we received from New England Nuclear was only 80% pure and required rigorous purification before use. The report by Whittaker et al. contains readily recognised examples of the need for rigid control of the assay system. Using the stated 5.5% cross-reaction of the cestrone antibody with equilin and the values reported in tables i and n, we calculate that the reported concentrations of equilin can account for up to 88% of the average values given for oestrone in table i and for up to 102% (average 88%) of the oestrone found in the serum of women taking premarin plus norethisterone. Does norethisterone alter both the endogenous production and the absorption and metabolism of exogenous oestrone in such a way that no unconjugated oestrone is present in these samples ? If this is true, what is the source of the large amounts of unconjugated cestradiol Whittaker et al. found? There is potential for error in the cross-reaction of equilin antibody with oestrone sulphate (4.4%) and equilin sulphate (4.9%). The serum concentration of oestrone sulphate is approximately 10 times that of unconjugated -oestrone3 while that of equilin sulphate is up to 50 times that of equilin.’ In the absence of any data on the serum concentrations of these sulphates it is not possible to calculate their contributions to the free equilin values reported. Cross-reactions of the equilin antibody with other more polar metabolites and their conjugates is not discussed. Our experience indicates that not only is this another real source of error in the assay but also that these compounds account for greater than 90% of the ring B unsaturated oestrogens found in the serum and in the urine after tritiated equilin is given.’1 A further comment on this extraordinary report relates to the biological activity of equilin. Whittaker et al. note that it is a potent oestrogen. Bioassay data’ indicate that its activity is similar to that of oestrone. Our metabolic studies 1,2 show that women metabolise equilin in a manner similar to the metabolism of cestrone. It is amazing what Whittaker et al. found significant amounts of unconjugated equilin 13 weeks after the last dose of premarin; what is even more bizarre is that this hormone appeared to have no biological activity as indicated by the gonadotropin levels. In view of our own experience and that reported by others’-3 we do not accept the inference in the closing statement of this paper that equilin should be regarded as different from other cestrogens. The data presented in this report must be regarded with caution until the validity of the assay is proved. We hope that this team’s forthcoming papers will deal with this issue in some detail. Department of Obstetrics and Gynecology,
Faculty of Medicine, McMaster University, Hamilton, Ontario, Canada
C. A. WOOLEVER B. R. BHAVNANI
their 3H-equilin. We assume that this is because immunoassay methods are still "in preparation".’1 The details of our immunoassay were accepted for publica-
infusions of own
tion before the Lancet paper was even started, but will not appear until the spring.2 We will substantiate the major assay points briefly. Pooled pre-treatment serum from the women was added to the standard curves in each assay; we also did recoveries using a spiked sample from this pre-treatment serum. The intra-assay coefficient of variation, at 9.6%, was as good as that for other routine steroid assays; the purity of the 3H-equilin was established by two separate thin layer chromatographic techniques and by high-pressure liquid chromatography. The cross-reaction of our antibody was measured against nine other related oestrogens and their conjugates and found to be less than 1% in all cases. With the low degree of cross-reactivity of equilin sulphate and oestrone sulphate (4-5%), they would have to be present in large amounts to contribute significantly ; even if they did this it would not invalidate our clinical warning because equilin sulphate can be converted to equilin, probably by the oestrogen sulphatase present in human
endometrium.4 The cross-reaction of equilin in the cestrone assay is not strictly relevant; the figure of 3-5% is based upon the estimate of the ratio of each steroid required to produce 50% inhibition of the zero binding.5 This is not a rigid relationship, particularly at high concentrationsand cannot be used to assess the possible contribution of a minor cross-reacting steroid in the determination of another with any accuracy. As Woolever and Bhavnani are aware’ (p. 26), it is difficult to compare pharmacodynamic data from short-term i.v. injections in men with those from long-term oral administration in postmenopausal women. They are also selective in the use of their own data; their reference to a half-life of 43 min for equilin after injection derives from their immunoassay of free cestrone and equilin (methodology unpublished), in samples taken over only 80 min. Using 3H-equilin they reported a halflife of 5.2h for equilin and comment "It is evident that the activity remains in the circulation of man for a much longer period than it does in the pregnant mare" (p. 36). Their suggestion from preliminary data (p. 40) that "the free steroid is readily sulphated after intravenous injections" (p. 41) should also be contrasted with their conclusion that "equilin sulphate can be rapidly metabolised to the free steroid found in the serum" (p. 41). They further point out that in humans "equilin and equilin sulphate are more potent than oestrone and cestrone sulphate" (p. 31) and later refer to them as "unique steroids" in their influence on the reproductive and other systems (p. 32). Their current position on the differences between equilin and other oestrogens, as indicated in their letter, is not supported by Ruder et al.,7 since that paper does not refer to equilin at all. Woolever and Bhavnani’ have previously noted that "equilin and its metabolites are retained in the blood and tissues of the human much longer than in the pregmare" (p.42). We reported that during active therapy with all forms of hormone replacement therapy FSH and LH levels were supnant
1. Bhavnani BR, Woolever CA. Alternate pathways of steroid biosynthesis and the origin, metabolism and biological effects of ring B unsaturated estrogens. In: Hobkirk R, ed. Steroid biochemistry II. Florida: C.R.C Press, 1979: 1-50. 2. Morgan MRA, Whittaker PG, Fuller BP, Dean PDG. A radioimmunoassay for equilin in post-menopausal plasma: plasma levels of equilin determined after oral administration of conjugated equine estrogens (Premarin). J
Steroid Biochem (in press).
PG, Morgan MRA, Dean PDG, Cameron HD, Lind T. Serum equilin, oestrone and oestradiol levels in post-menopausal women receiving conjugated equine estrogens (’Premarin’). Lancet 1980; i: 14-16. 4. Gurpide E, Stolee A, Tseng L. Quantitative studies of tissue uptake and disposition of hormones. Acta Endocrinol 1971; 157: 247-78. 5. Abraham GE. Solid phase radioimmunoassay of estradiol-17&bgr; J Clin Endoc3. Whittaker
*** This letter has been shown to Dr Lind and his colleagues, whose reply follows.-ED.L. SIR,-We agree with Professor Woolever and Dr Bhavnani that data based upon immunoassay techniques should be rigidly controlled, so we are surprised that their criticisms are largely based upon single studies with intravenous injections or
rinol Metab 1969; 29: 866-70. 6. Hunter WM. Discussion. In: Cameron EHD, Hillier SG, Griffiths K, eds Steroid immunoassay. Cardiff: Alpha Omega, 1975: 75. 7. Ruder HJ, Loriaux L, Lipsett MG. Estrone sulphate: Production rate and metabolism in man. J Clin Invest 1972: 51: 1020-33.
549
pressed to a small extent but never to values found in premenopausal women;8 the fact that after therapy was discontinued the gonadotrophins returned to pre-treatment levels is not therefore surprising as no-one has shown that the concentrations of circulating equilin we found will inhibit gonadotrophin release. But in any case their effect upon gonadotrophin secretion probably does not reflect biopotency of equilin in
M.R.C. Human Reproduction Group, Princess Mary Maternity Hospital, Newcastle upon Tyne NE2 3BD
P. G. WHITTAKER T. LIND
an annual incidence of 1 or 2 cases in the whole of the U.K. H. A. Edmondson and others identified 42 patients through the University of California department of pathology over a twenty year period. Out of 34 patients traced, 17 had been on the pill for more than five years and 17 for less than five years. From these figures an inspired guess would lead me to suggest that the incidence of pill-associated liver tumours in Britain would be of the order of 5-10 cases per year or 1-2 cases per year for every million women on the pill. If the K.C.H. workers’ recommendation were to be accepted, then each of the 5.94 million women on the pill would have her abdomen palpated and ESR and liver function tests done every 6 months. Even if this check-up costs only jl (a ludicrously low estimate) for all of nearly 12 million examinations, the early diagnosis of 1 pill-related hepatic tumour would cost about /1 million. I can suggest many more worthwhile destinations for N.H.S. funds. A more reasonable conclusion to the paper would have been to advise liver function tests and ESRs in any woman on the pill who complains of upper abdominal or lower chest pain.
Department of Physiology, University of Manchester
M. R. A. MORGAN
Buchan Lodge,
Department of Biochemistry, University of Liverpool
P. D. G. DEAN
terms of human endometrial response. Indeed it was the potential of equilin for long-term action as an endometrial stimulator that prompted our comment that it would seem prudent to discontinue ’Premarin’ after 12 months. Furthermore, this therapy time-span has the total support of Ayerst Laboratories, the manufacturers of premarin, as indicated in their recent advertisements.9 If our findings are confirmed by other workers using immunoassay methods it might be’necessary to suggest that, in view of the other preparations available, premarin should not be prescribed at all.
of oral-contraceptiveassociated liver tumour Dr Neuberger and colleagues (Feb. 9, p. 273) state that for two patients they could not ascertain the name of the contraceptive that had been used for at least 5 years. Did these patients have no pharmacists or general practitioners ? Neuberger et al. state that "Eight of our patients had been using the pill for 5 years or longer". The table shows that one woman had been on the pill for 2 years and that two others had used it for 4 years. Continuous use of the pill for 5 years is said to increase the risk of liver tumour "5-fold", while "The risk of a woman on oral contraceptives developing liver tumour is very low". Neuberger et al. shun absolute figures: how low? They give no indication at all of how important this risk is when set against, for instance, the risk of lung cancer in smokers or of fatal accidents in motorists. This paper could have been more informative. Rijksinstituut voor de Volksgezondheid, 3720 BA Bilthoven, Netherlends
cases
J.
BORST
SIR,-Dr Neuberger and his colleagues describe 10 cases of hepatic tumour in women on oral contraceptives collected over a period of 10 years at King’s College Hospital from a probable population of at least 10 million people. I take exception to their recommendation that "routine six month screening should include examination of the abdomen for hepatomegaly as well as estimation of ESR and liver function tests". This may be clinically sound in the context of a specialist liver unit but seems financially naive for the N.H.S. as a whole. Neuberger et al. cite two papers in their discussion of the incidence of pill-associated liver tumours. M. P. Vessey and his colleagues described hepatic tumours in association with the pill as "extremely rare" and found only 1 case in a study which involved seven years of morbidity in the 5 million population of Scotland and 299 000 women-years of pill use in the R.C.G.P. study and the Oxford record linkage study, suggest8. Lind T, Cameron E, Hunter WM, et al. A prospective, controlled trial ofsix forms of hormone replacement therapy given to post-menopausal women. Br J ObstetGynœcol1979; 86:suppl 3. 9. Ayerst Laboratories. Summary of prescribing information. Am J Obstet
Gynecol1979: 135:xxiv.
7 Pedwell Way, Norham, Berwick
LIVER TUMOURS AND ORAL CONTRACEPTIVES
SiR,-In their description of ten
ing
upon Tweed
G. A. C. BINNIE
ELECTROPHYSIOLOGY AND AVOIDANCE OF INVASIVE NEURORADIOLOGY IN MULTIPLE SCLEROSIS
SIR,-I agree with Professor Mastaglia and his colleagues (Jan. 19, p. 144) that visual evoked responses (VEP) and other electrophysiological techniques are valuable in the investigation of patients with multiple sclerosis (MS) but a note of caution is needed, as a recent patient illustrates. A 58-year-old man was admitted with a 12-month history of clumsiness of both hands, particularly the right, causing difficulty in fastening buttons and tying shoelaces. He had also noted he was dragging his right leg. There was no history of previous neurological episodes and in particular no visual symptoms. The cranial nerves were normal and visual acuity was not impaired. Proprioception was impaired in both hands and the arm reflexes were symmetrically increased. There was mild weakness of the right leg and the right plantar response was extensor. All the reflexes were increased but more so on the right with knee and ankle clonus. Sensation in the legs was normal. His symptoms and signs could, on clinical grounds, be explained by a cervical cord lesion and, despite the late age of onset, demyelination had to be considered. The VEP was 120 ms in the right eye (normal 106-$±7-5 5 ms) and normal in the left eye. Auditory evoked responses were also normal. Plain X-rays of the cervical spine showed marked narrowing at C3/4 and C4/5 disc spaces. A metrizamide myelogram demonstrated severe cervical cord compression from cervical spondylosis both anteriorly and posteriorly at C3/4, C4/5 and C5/6. The cerebrospinal fluid protein content, including the IgG component, was normal and the cell count was not increased. A cervical laminectomy was recommended and at operation the myelogram findings were confirmed. MS and cervical spondylosis with myelopathy may coexist, particularly in the older patient. MS is untreatable but cervical spondylosis may be improved or its progression checked by surgery. Reliance on the VEP might have deprived this patient of potential benefit. As with any other investigation, the VEP must be interpreted in the clinical context. Invasive neuroradiology must not be too readily abandoned in the older patient who, on clinical grounds, has a solitary spinal cord lesion.
Department ofNeurology, Salford Royal Hospital, Salford M60 9EP
A. C. YOUNG
R. W. BLUNDEN B. M. DUNCAN
SERUM EQUILIN AND CONJUGATED EQUINE ŒSTROGENS
SIR,-Many technical, metabolic, and
clinical issues
are
by the paper from Dr Whittaker and his colleagues (Jan. 5, p. 14). They suggest that the grossly increased and very variable serum concentration of equilin and its improbable raised
relation
and oestradiol in are due to some unusual property of the metabolism of this hormone in women. To support this statement they refer to our report on the metabolism of 3H-equilin in man.’ They misinterpret our data as indicating that after the intravenous injection of 3H-equilin there is a slow urinary excretion and persistently high serum levels of the hormone 24 h after injection. However, the data to which they refer deal with total radioactivity rather than with "unconjugated equilin" which they claim to measure in their radioimmunoassay. We found in the plasma only 3% of’ the total dose injected after 24 h, and by 72 h almost no radioactivity (10-12 cpm/ml) is present in the serum. During this period (72 h) over 72% of the injected dose was recovered in the urine exclusive of any enterohepatic circulation or excretion in the faeces. Furthermore, Whittaker et al. have not taken into account the rapid metabolism of equilin reported by us.’ Thus the radioactive materials found in the blood and urine are mainly reduced and conjugated metabolites of equilin. For example, 20 min after injection of equilin the concentration of equilin sulphate is twice that of equilin and this increased to 50 times the level of equilin after 90 min. Some months ago we shared with one of this group (Dr P. D. G. Dean) other dataZ relating to the metabolism of 3H-equilin, 3-equilin sulphate and premarin given both intravenously and by mouth. In the latter experiments we found that the serum concentration of unconjugated cestrone and of unconjugated equilin continued to reflect the ratio of the sulphates found in the premarin used in these experiments. The half-life of unconjugated equilin after the injection of 10 mg premarin intravenously was approximately 43 min while that of oestrone was 63 min. After oral premarin 10 mg (i.e., 2-5 mg equilin sulphate) we found the maximum concentration of free equilin in plasma to be 560 pg/ml (2090 pmol/1) at 4 h. At 8 h the serum concentration was half of the peak value and after 24 h only trace amounts could be found. While this pattern is similar (a 4 h peak) to that reported by Whittaker et al. the absolute values which they found after administering only 1-25 mg premarin (0-3mg equilin sulphate) are very much higher (6753 pg/ml and 1635 pg/ml at 4 and 24 h). It is not likely that different rates of absorption could explain these differences and it is not even remotely possible that the absorption would be sufficiently slow to explain the presence of equilin in the serum 13 weeks after the last oral dose. women
Normal females
Steroid sulphatase
activity
Affected males
Hetero-
zygotes
in cultural fibroblasts.
the normal or the defective X chromosome. An exception seems to be the steroid sulphatase locus which escapes inactivation.’ Theoretically, then, detection should be easier for this enzyme defect than for other X-linked enzyme defects. We have re-examined this question using dehydroepiandrosterone sulphate (DHEAS) as substrate. With DHEAS we have found enzyme activities to be about 100 times higher than they are with cholesteryl sulphate. And this property has enabled us to demonstrate that steroid sulphatase activities are significantly higher in females than in males.8 As the figure shows, enzyme activities in fibroblasts of five unrelated obligate heterozygotes (ascertained by two affected sons, one affected son plus positive family history, or affected father) and ten normal females do not overlap. In heterozygotes, there is remarkably little interindividual variation, which fits into the concept of non-inactivation of the gene. With DHEAS carrier detection is possible for steroid sulphatase deficiency. Previous failures to detect heterozygotes are probably due to unsuitable substrates rather than biological variation. Institute of Human Genetics and Anthropology,
University of Freiburg, D-7800 Freiburg, West Germany Dermatological Hospital, University of Münster
C. R. MÜLLER B. MIGL H. H. ROPERS R. HAPPLE
IRON OVERLOAD AND AGE IN &bgr;-THALASSÆMIA SIR,-Dr Lagos and colleagues (Jan. 26, p. 204) claim that iron overload, as measured by serum ferritin, is positively related to age in patients with p-thalasseemia intermedia. While there is indeed a significant statistical relationship between serum ferritin and age in these 15 patients, 3 of them had received blood transfusions. Since the number of transfusions is correlated with serum ferritin levels,’ these 3 patients should 7. Shapiro LJ, Mohandas T, Weiss R, Romeo G. Non-inactivation of an X chromosome locus in man. Science 1979; 204: 1224-26. 8. Müller CR, Migl B, Traupe, Ropers HH. X-linked steroid sulfatase: Evidence for different gene dosage in males and females. Hum Genet (in 1.
press). Letsky EA,
Miller F, Worwood M, Flynn DM. Serum ferritin in children with thalassæmia regularly transfused. J Clin Pathol 1974; 27: 652-55.
to
the concentration of
cestrone
taking conjugated equine cestrogens
1. Bhavnani BR, Woolever CA. Alternate pathways of steroid biosynthesis in the origin, metabolism and biological effects of ring B unsaturated oestrogens. In: Hobkirk R, ed. Steroid biochemistry: vol II. Boca Raton, Florida: C.R.C. Press, 1979: 1-50. 2. Bhavnani BR, Woolever CA. Absorption and metabolism of equine estrogens in postmenopausal women and normal men. Society for Gynecologic Investigation 26th annual meeting (San Diego, California, March, 1979):
abstrp. 9 (no. 12). 3. Ruder HJ, Loriaux L, Lipsett MB. Estrone sulfate: Production metabolism in man. J Clin Invest 1972, 51: 1020.
rate
and
548
.
Our pharmacokinetic studies of equilin metabolism in humans and pregnant mares leads us to believe that the metabolism of equilin in the human is much the same as that of other cestrogens, including oestrone. Our experience in the development of the radioimmunoassay indicates that not only cross-reactions with other steroids but also a non-specific and erratic effect of human plasma and serum on the assay systems lead to very high and variable results unless the hormone is isolated and purified before assay. All of the values to which we refer in our publications were obtained by this technique and have been corrected for procedural losses. Another related problem is the purity of the 3H-equilin used in the assay. The freshly synthesised material which we received from New England Nuclear was only 80% pure and required rigorous purification before use. The report by Whittaker et al. contains readily recognised examples of the need for rigid control of the assay system. Using the stated 5.5% cross-reaction of the cestrone antibody with equilin and the values reported in tables i and n, we calculate that the reported concentrations of equilin can account for up to 88% of the average values given for oestrone in table i and for up to 102% (average 88%) of the oestrone found in the serum of women taking premarin plus norethisterone. Does norethisterone alter both the endogenous production and the absorption and metabolism of exogenous oestrone in such a way that no unconjugated oestrone is present in these samples ? If this is true, what is the source of the large amounts of unconjugated cestradiol Whittaker et al. found? There is potential for error in the cross-reaction of equilin antibody with oestrone sulphate (4.4%) and equilin sulphate (4.9%). The serum concentration of oestrone sulphate is approximately 10 times that of unconjugated -oestrone3 while that of equilin sulphate is up to 50 times that of equilin.’ In the absence of any data on the serum concentrations of these sulphates it is not possible to calculate their contributions to the free equilin values reported. Cross-reactions of the equilin antibody with other more polar metabolites and their conjugates is not discussed. Our experience indicates that not only is this another real source of error in the assay but also that these compounds account for greater than 90% of the ring B unsaturated oestrogens found in the serum and in the urine after tritiated equilin is given.’1 A further comment on this extraordinary report relates to the biological activity of equilin. Whittaker et al. note that it is a potent oestrogen. Bioassay data’ indicate that its activity is similar to that of oestrone. Our metabolic studies 1,2 show that women metabolise equilin in a manner similar to the metabolism of cestrone. It is amazing what Whittaker et al. found significant amounts of unconjugated equilin 13 weeks after the last dose of premarin; what is even more bizarre is that this hormone appeared to have no biological activity as indicated by the gonadotropin levels. In view of our own experience and that reported by others’-3 we do not accept the inference in the closing statement of this paper that equilin should be regarded as different from other cestrogens. The data presented in this report must be regarded with caution until the validity of the assay is proved. We hope that this team’s forthcoming papers will deal with this issue in some detail. Department of Obstetrics and Gynecology,
Faculty of Medicine, McMaster University, Hamilton, Ontario, Canada
C. A. WOOLEVER B. R. BHAVNANI
their 3H-equilin. We assume that this is because immunoassay methods are still "in preparation".’1 The details of our immunoassay were accepted for publica-
infusions of own
tion before the Lancet paper was even started, but will not appear until the spring.2 We will substantiate the major assay points briefly. Pooled pre-treatment serum from the women was added to the standard curves in each assay; we also did recoveries using a spiked sample from this pre-treatment serum. The intra-assay coefficient of variation, at 9.6%, was as good as that for other routine steroid assays; the purity of the 3H-equilin was established by two separate thin layer chromatographic techniques and by high-pressure liquid chromatography. The cross-reaction of our antibody was measured against nine other related oestrogens and their conjugates and found to be less than 1% in all cases. With the low degree of cross-reactivity of equilin sulphate and oestrone sulphate (4-5%), they would have to be present in large amounts to contribute significantly ; even if they did this it would not invalidate our clinical warning because equilin sulphate can be converted to equilin, probably by the oestrogen sulphatase present in human
endometrium.4 The cross-reaction of equilin in the cestrone assay is not strictly relevant; the figure of 3-5% is based upon the estimate of the ratio of each steroid required to produce 50% inhibition of the zero binding.5 This is not a rigid relationship, particularly at high concentrationsand cannot be used to assess the possible contribution of a minor cross-reacting steroid in the determination of another with any accuracy. As Woolever and Bhavnani are aware’ (p. 26), it is difficult to compare pharmacodynamic data from short-term i.v. injections in men with those from long-term oral administration in postmenopausal women. They are also selective in the use of their own data; their reference to a half-life of 43 min for equilin after injection derives from their immunoassay of free cestrone and equilin (methodology unpublished), in samples taken over only 80 min. Using 3H-equilin they reported a halflife of 5.2h for equilin and comment "It is evident that the activity remains in the circulation of man for a much longer period than it does in the pregnant mare" (p. 36). Their suggestion from preliminary data (p. 40) that "the free steroid is readily sulphated after intravenous injections" (p. 41) should also be contrasted with their conclusion that "equilin sulphate can be rapidly metabolised to the free steroid found in the serum" (p. 41). They further point out that in humans "equilin and equilin sulphate are more potent than oestrone and cestrone sulphate" (p. 31) and later refer to them as "unique steroids" in their influence on the reproductive and other systems (p. 32). Their current position on the differences between equilin and other oestrogens, as indicated in their letter, is not supported by Ruder et al.,7 since that paper does not refer to equilin at all. Woolever and Bhavnani’ have previously noted that "equilin and its metabolites are retained in the blood and tissues of the human much longer than in the pregmare" (p.42). We reported that during active therapy with all forms of hormone replacement therapy FSH and LH levels were supnant
1. Bhavnani BR, Woolever CA. Alternate pathways of steroid biosynthesis and the origin, metabolism and biological effects of ring B unsaturated estrogens. In: Hobkirk R, ed. Steroid biochemistry II. Florida: C.R.C Press, 1979: 1-50. 2. Morgan MRA, Whittaker PG, Fuller BP, Dean PDG. A radioimmunoassay for equilin in post-menopausal plasma: plasma levels of equilin determined after oral administration of conjugated equine estrogens (Premarin). J
Steroid Biochem (in press).
PG, Morgan MRA, Dean PDG, Cameron HD, Lind T. Serum equilin, oestrone and oestradiol levels in post-menopausal women receiving conjugated equine estrogens (’Premarin’). Lancet 1980; i: 14-16. 4. Gurpide E, Stolee A, Tseng L. Quantitative studies of tissue uptake and disposition of hormones. Acta Endocrinol 1971; 157: 247-78. 5. Abraham GE. Solid phase radioimmunoassay of estradiol-17&bgr; J Clin Endoc3. Whittaker
*** This letter has been shown to Dr Lind and his colleagues, whose reply follows.-ED.L. SIR,-We agree with Professor Woolever and Dr Bhavnani that data based upon immunoassay techniques should be rigidly controlled, so we are surprised that their criticisms are largely based upon single studies with intravenous injections or
rinol Metab 1969; 29: 866-70. 6. Hunter WM. Discussion. In: Cameron EHD, Hillier SG, Griffiths K, eds Steroid immunoassay. Cardiff: Alpha Omega, 1975: 75. 7. Ruder HJ, Loriaux L, Lipsett MG. Estrone sulphate: Production rate and metabolism in man. J Clin Invest 1972: 51: 1020-33.
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pressed to a small extent but never to values found in premenopausal women;8 the fact that after therapy was discontinued the gonadotrophins returned to pre-treatment levels is not therefore surprising as no-one has shown that the concentrations of circulating equilin we found will inhibit gonadotrophin release. But in any case their effect upon gonadotrophin secretion probably does not reflect biopotency of equilin in
M.R.C. Human Reproduction Group, Princess Mary Maternity Hospital, Newcastle upon Tyne NE2 3BD
P. G. WHITTAKER T. LIND
an annual incidence of 1 or 2 cases in the whole of the U.K. H. A. Edmondson and others identified 42 patients through the University of California department of pathology over a twenty year period. Out of 34 patients traced, 17 had been on the pill for more than five years and 17 for less than five years. From these figures an inspired guess would lead me to suggest that the incidence of pill-associated liver tumours in Britain would be of the order of 5-10 cases per year or 1-2 cases per year for every million women on the pill. If the K.C.H. workers’ recommendation were to be accepted, then each of the 5.94 million women on the pill would have her abdomen palpated and ESR and liver function tests done every 6 months. Even if this check-up costs only jl (a ludicrously low estimate) for all of nearly 12 million examinations, the early diagnosis of 1 pill-related hepatic tumour would cost about /1 million. I can suggest many more worthwhile destinations for N.H.S. funds. A more reasonable conclusion to the paper would have been to advise liver function tests and ESRs in any woman on the pill who complains of upper abdominal or lower chest pain.
Department of Physiology, University of Manchester
M. R. A. MORGAN
Buchan Lodge,
Department of Biochemistry, University of Liverpool
P. D. G. DEAN
terms of human endometrial response. Indeed it was the potential of equilin for long-term action as an endometrial stimulator that prompted our comment that it would seem prudent to discontinue ’Premarin’ after 12 months. Furthermore, this therapy time-span has the total support of Ayerst Laboratories, the manufacturers of premarin, as indicated in their recent advertisements.9 If our findings are confirmed by other workers using immunoassay methods it might be’necessary to suggest that, in view of the other preparations available, premarin should not be prescribed at all.
of oral-contraceptiveassociated liver tumour Dr Neuberger and colleagues (Feb. 9, p. 273) state that for two patients they could not ascertain the name of the contraceptive that had been used for at least 5 years. Did these patients have no pharmacists or general practitioners ? Neuberger et al. state that "Eight of our patients had been using the pill for 5 years or longer". The table shows that one woman had been on the pill for 2 years and that two others had used it for 4 years. Continuous use of the pill for 5 years is said to increase the risk of liver tumour "5-fold", while "The risk of a woman on oral contraceptives developing liver tumour is very low". Neuberger et al. shun absolute figures: how low? They give no indication at all of how important this risk is when set against, for instance, the risk of lung cancer in smokers or of fatal accidents in motorists. This paper could have been more informative. Rijksinstituut voor de Volksgezondheid, 3720 BA Bilthoven, Netherlends
cases
J.
BORST
SIR,-Dr Neuberger and his colleagues describe 10 cases of hepatic tumour in women on oral contraceptives collected over a period of 10 years at King’s College Hospital from a probable population of at least 10 million people. I take exception to their recommendation that "routine six month screening should include examination of the abdomen for hepatomegaly as well as estimation of ESR and liver function tests". This may be clinically sound in the context of a specialist liver unit but seems financially naive for the N.H.S. as a whole. Neuberger et al. cite two papers in their discussion of the incidence of pill-associated liver tumours. M. P. Vessey and his colleagues described hepatic tumours in association with the pill as "extremely rare" and found only 1 case in a study which involved seven years of morbidity in the 5 million population of Scotland and 299 000 women-years of pill use in the R.C.G.P. study and the Oxford record linkage study, suggest8. Lind T, Cameron E, Hunter WM, et al. A prospective, controlled trial ofsix forms of hormone replacement therapy given to post-menopausal women. Br J ObstetGynœcol1979; 86:suppl 3. 9. Ayerst Laboratories. Summary of prescribing information. Am J Obstet
Gynecol1979: 135:xxiv.
7 Pedwell Way, Norham, Berwick
LIVER TUMOURS AND ORAL CONTRACEPTIVES
SiR,-In their description of ten
ing
upon Tweed
G. A. C. BINNIE
ELECTROPHYSIOLOGY AND AVOIDANCE OF INVASIVE NEURORADIOLOGY IN MULTIPLE SCLEROSIS
SIR,-I agree with Professor Mastaglia and his colleagues (Jan. 19, p. 144) that visual evoked responses (VEP) and other electrophysiological techniques are valuable in the investigation of patients with multiple sclerosis (MS) but a note of caution is needed, as a recent patient illustrates. A 58-year-old man was admitted with a 12-month history of clumsiness of both hands, particularly the right, causing difficulty in fastening buttons and tying shoelaces. He had also noted he was dragging his right leg. There was no history of previous neurological episodes and in particular no visual symptoms. The cranial nerves were normal and visual acuity was not impaired. Proprioception was impaired in both hands and the arm reflexes were symmetrically increased. There was mild weakness of the right leg and the right plantar response was extensor. All the reflexes were increased but more so on the right with knee and ankle clonus. Sensation in the legs was normal. His symptoms and signs could, on clinical grounds, be explained by a cervical cord lesion and, despite the late age of onset, demyelination had to be considered. The VEP was 120 ms in the right eye (normal 106-$±7-5 5 ms) and normal in the left eye. Auditory evoked responses were also normal. Plain X-rays of the cervical spine showed marked narrowing at C3/4 and C4/5 disc spaces. A metrizamide myelogram demonstrated severe cervical cord compression from cervical spondylosis both anteriorly and posteriorly at C3/4, C4/5 and C5/6. The cerebrospinal fluid protein content, including the IgG component, was normal and the cell count was not increased. A cervical laminectomy was recommended and at operation the myelogram findings were confirmed. MS and cervical spondylosis with myelopathy may coexist, particularly in the older patient. MS is untreatable but cervical spondylosis may be improved or its progression checked by surgery. Reliance on the VEP might have deprived this patient of potential benefit. As with any other investigation, the VEP must be interpreted in the clinical context. Invasive neuroradiology must not be too readily abandoned in the older patient who, on clinical grounds, has a solitary spinal cord lesion.
Department ofNeurology, Salford Royal Hospital, Salford M60 9EP
A. C. YOUNG