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Endocrine function and haemoglobinopathies: Relation between the sickle cell gene and circulating plasma levels of testosterone, luteinising hormone (LH) and follicle stimulating hormone (FSH) in adult males
269
Clinica Chimica Acta, @ ElsevieriNorth-Holland
CCA
105 (1980) 269-273 Biomedical Press
1431
ENDOCRINE FUNCTION AND HAEMOGLOBINOPATHIES: RELATION BETWEEN THE SICKLE CELL GENE AND CIRCULATING PLASRIIA LEVELS OF TESTOSTERONE, LUTEINISING HORh4ONE (LH) AND FOLLICLE STIMULATING HORh~~NE (FSH) IN ADULT MALES
O.A.
DADA
* and
Department (Received
E.U.
of Chemical January
21st,
NDUKA Pathology,
Ilniuersity
of Ibadan,
Ibadan
(Nigeria)
1980)
Summary
Circulating plasma levels of testosterone, luteinising hormone and follicle stimulating hormone were determined in adult Nigerian males who have haemoglobin (Hb) genotypes HbAA, AS, AC, SC and SS. The levels of the three hormones were significantly lower in homozygous sickle cell disease (HbSS) compared to the other groups, whereas the levels of testosterone and luteinising hormone were significantly reduced in HbSC heterozygotes compared to HbAS heterozygotes only. These results are suggestive of an association of secondary hypogonadism with haemoglobin S disease.
Introduction
Earlier studies on the stability of haemoglobin (Hb) gene frequencies in West Africa indicated a greater potential fertility of unions in which one parent carries a single abnormal haemogIobin gene (Hb heterozygotes -AS and -AC) [ 1,2]. More recent studies have also indicated higher fertility in HbAS compared to the normal HbAA males [3]. In contrast, homozygous sickle cell disease (HbSS) is associated with impairment of sexual development and abnormalities of gonadal function [4,5]. Apart from these studies, there have been no direct comparisons of the circulating plasma levels of the reproductive hormones in individuals of different haemoglobin genotypes. In the present study, the circulating levels of testosterone (T), luteinising hormone (LH) and follicle stimulating hormone (FSH) were determined in adult males with haemoglobin genotypes AA, AS, AC, SC, SS and CC.
* To
whom
correspondence
should
be addressed.
270
Materials and methods Subjects
The subjects with haemoglobin genotypes AA, AS and AC were adult (aged 20-45 years) male Nigerian blood donors at the University College Hospital, Ibadan. Subjects with haemoglobin variants SS, SC and CC were adult male also aged 20-45 years) patients attending the haemoglobinopathy clinics of the haematology department of the same hospital. Blood samples were collected from these patients during periods of established remission. In all subjects, blood was collected between 09.00 and 11.00 a.m. and immediately centrifuged and stored frozen at -20°C until analysed. The haemoglobin genotypes of all samples were confirmed by electrophoresis on cellulose acetate strips [6]. Radioimmun~assay
of testosterone
~l,2,6,7-3H]~stosterone (s-a. 105 Ci/mmol) was purchased from the RadioChemical Centre, Amersham, U.K., and purified by chromatography on columns of Sephadex LH-20 [7] before use. Antiserum raised against testosterone-3(o-carboxymethyl) oxime-bovine serum albumin was supplied by the World Health Organisation, Matched Reagents Programme (MRP). Information supplied by the MRP showed that the antiserum exhibited 14% cross-reaction with 5~-d~ydrotestosterone, 0.8% with A4-androstenedione and 6% with 5cuandrostanediol. Testosterone was assayed by radioimmunoassay in duplicate samples of plasma (50 ~1 + 50 yl 0.1 mol/l phosphate buffer, pH 7.2, containing 0.14 mol/l NaCl, 0.1% thiomersal and 1% gelatin) which was extracted with 5 ml peroxide-free diethyl ether. The extracts were dried under nitrogen. 100 ~1 buffer was added to the residue plus 100 ~1.(5000 cpm) of a solution in buffer of [ 1,2,6,7-3H]testosterone and 100 ~1 antiserum solution (final dilution 1 : 200 000). Standards were in the range O-500 pg/tube. After incubation at 30°C for 1 h, free and bound steroids were separated using 0.5 ml dextrancoated charcoal (0.5% activated charcoal, 0.05% dextran in assay buffer) and centrifuged for 5 min at 500 X g. The supematant (unbound fraction) was decanted into vials, 1 ml ethanol and 10 ml scintillation cocktail (4.5 g PPO per litre toluene) were added and the amount of radioactivity determined in a Packard tricarb liquid scintillation counter (Model 3375). Radioimmunoassay
of hLH and hFSH
Standard preparations of human pituitary luteinising hormone (hLH), 1st International Reference Preparation (code number 68/40), and human pituitary follicle stimulating hormone (hFSH) (code number 69/104) were obtained from the National Institute of Biological Standards and Control, London, U.K. Antisera to hLH and hFSH were obtained in freeze-dried form in sealed ampoules from the MRP. Information supplied by the MRP showed that the hLH antiserum exhibited 50% cross-reaction against hLH p subunit; hLH o! subunit, 5%, hFSH, 0.5%; and hTSH, 0.6%; the hFSH antiserum showed cross-reaction with hLH, 0.1%; hCG, 0.1%; and hTSH, 0.5%. Preparations of hLH, and hFSH labelled with “‘1 by the lactoperoxidase method (tracers) were obtained from the Swiss Federal Institute for Reactor Research, Switzerland. The assay procedure employed 100 ~1 plasma which was added to 100 ~1
271
antiserum solution (final dilution l/l 750 000), 100 ~1 of the tracer (10 000 cpm) and 400 ~1 phosphate buffer, pH 7.4, containing 0.14 mol/l NaCI, 0.1% thiomersal and 5% bovine serum albumin. The mixture was incubated at 4°C for 48 h after which 100 ,~l (1 : 26 dilution) antirabbit gammaglobulin (2nd antibody) was added and the reaction terminated by centrifugation (1500 X g, 30 min) after 24 h incubation at 4°C. The amount of radioactivity was determined in the precipitate (bound fraction) in a Packard gamma counter model. The amount of each hormone in plasma samples was calculated from a linearised log&-log plot of standard curves 181. The inter- and intra-assay variations were, for testosterone 11.2% and 9.2% respectively, for hLH, 16.4% and 12.7% and for hFSH 14.9 and 10.5%. Results The mean levels of testosterone, LH and FSH together with their 95% confidence limits are shown in Table I. Analysis of variance of the data established significant differences between the groups shown in Table II. The mean levels of T, LH and FSH shown for HbAA subjects are not significantly different from those of Hb-AS, -AC and -SC subjects. However, the mean levels of these hormones are significantly lower in Hb-SS subjects compared to the other groups (p < 0.01 for T and LH and p < 0.05 for FSH). A comparison between two heterozygote groups (AS vs. SC) showed that T and LH, but not FSH, levels are significantly higher in -AS individuals (p < 0.05). No other comparisons showed significant differences. Only one subject with homozygous haemoglobin C (HbCC disease) was available for the study and in this subject the values of the three hormones are outside the 95% confidence limits of the mean values for Hb-AA subjects. However, more data are required before any conclusions can be drawn from this.
TABLE
I
MEAN
PLASMA
ADULT
MALES
Haemoglobin
AA
AS
SS
SC
AC
cc
LEVELS WITH
genotype
AND
95%
DIFFERENT
CONFIDENCE
LIMITS
HAEMOGLOBIN No.
TESTOSTERONE,
LH
AND
FSH
Testosterone
LH
subjects
(nmof/l)
(I.U.
19
19.5
4.5
17.6-21.4
3.7-5.3
3.8-5.0
22.5
4.3
4.3
18.8-26.2
3.3-5.3
3.7-5.0
9.4
2.0
3.1
8.1-10.9
1.4-2.6
2.0-4.1
17.4
3.2
3.6
13.0-21.8
2.1-4.3
1.2-5.0
20.7
3.8
3.3
19.9-21.5
2.3-4.8
1.6-5.1
14.5
0.7
1.4
40
19
12
7
1
of
OF
GENOTYPES FSH II)
(I.U./l) 4.4
IN
272
TABLE
II
DIFFERENCES THE
ANALYSIS
BETWEEN OF
THE
VARIANCE
Hormone -~
contrasts
Testosterone
AA
vs AS.
AA
YS SS
LH
FSH
HORMONE AND
THE
-AC,
vs AS,
SC,
AC
AA
vs As,
AC,
SC
AA
vs SS vs AS,
ss
“S SC
AA
vs AS,
AA
vs SS
AS
vsSC
SS
vs
SS
vsSC.AC
TABLE
I AS
ESTABLISHED
BY
CONTRASTS
*
P 0.01 N.S.
P 0.01 P 0.05 P 0.01
AC
P 0.05 AC,
SC
N.S. P 0.05 N.S.
AS
P 0.05
-____ * Not
IN
APPROPRIATE
P 0.05
vs SC
vssc
OF
I’ 0.01
AS
AS
SHOWN
Significance N.S.
SC
SS
SS
LEVELS CALCULATION
N.S. .-._-
..--
. ..-
significant.
Discussion The results of the present study have shown that circulating plasma levels of T, LH, and FSH are si~ificantly reduced in individu~s with homozygous sickle cell (SS) disease. The low levels of these hormones may reflect hypogonadal function secondary to hypopituitary function. Hypopotuitarism has previously been reported in HbSS disease as resulting from intravascular thrombosis and pituitary infarction [9,10]. These results are in contrast to those of Abbasi et al. [5] who found in 14 adult males with HbSS disease lower than normal levels of androgens, together with elevated basal LH levels and an exaggerated LH response to gonadotrophin releasing hormone (GnRH), findings which are indicative of primary hypogonadal function. The reasons for the discrepancies between these two results are not clear yet, but they both provide some evidence that hypogonadal function is a feature of HbSS disease. In heterozygous HbSC disease, which is associated in affected individu~s with morbidity though often of a milder nature than HbSS disease [ 111, a clear-cut picture did not emerge. Levels of the three hormones were not significantly different from those of HbAA subjects but were found to be lower than those of Hb-AS heterozygotes. In the Hb-AS and -AC heterozygotes, levels of testosterone and gonadotrophins were in the same range as for normal HbAA individuals. The present results suggest that differences in circulating plasma gonadotrophins and gonadal hormones may not be responsible for the different fertility patterns in male AS and AC heterozygotes compared to normal AA individuals [ 1-3 1. A possible effect of a tropical environment on the circulating levels of testosterone in Zambian African males compared to men from temperate regions has been suggested [ 12]. It is of interest in this respect to note that the IeveIs of
273
testosterone in normal HbAA males in this study are in the same range as those reported from other temperate countries [ 13,141. Acknowledgements This study was supported by a grant from the World Health Organisation Expanded Programme of Research Training in Human Reproduction. We thank Professor W.A. Isaacs-Sodeye for permission to study his patients; the staff of the blood bank for their cooperation and Professors B. Kwaku Adadevoh and D.A. Olatunbosun for their interest and encouragement of this work. References 1
Roberts.
D.F.
and
Boyo,
A.E.
(1960)
Ann.
Hum.
2
Roberts,
D.F.
and
Boyo,
A.E.
(1962)
Hum.
Biol.
3
Van
Ros.
G.
Proceedings
in
Haemoglobinopathy.
of a Workshop,
4
Olanbiwonnu,
N.O..
5
Abbasi,
Prasad.
A.A.,
L. and
A.O.,
of Ibadan.
Frasier,
Ortega.
J.,
S.D.
24,
375-387
20-37
Reproduction
University
Penny.
Genet. 34,
and
Contraception
(Kwaku
Adadevoh.
B.,
ed.).
in press (1975)
Conego.
E.
and
J. Pediatr.
87,
Oberleas,
D.
459-464 (1976)
Ann.
Int.
Med.
85.
601-
605 6
Dacie,
7
Brenner,
P.F.,
8
Rodbard.
D.,
9
Wintrobe.
M.M.
(1967)
in Clinical
(1968)
Br. J. Clin.
Allen.
D.M.
J.W.
and
Adadevoh,
B.K.
11
Nechelles,
T.F..
Synthesis,
12
Briggs,
M.H.
13
Knorr,
D.,
S.M.
(1968)
R.,
Cekan,
Bridson.
10
and
Lewis,
Guerrero,
pp. and
W. and
56-61,
Brig@,
Bidlingmaier,
Practical Z. and
Rayford.
and
Haematology,
Diczfalusy,
P.L.
(1969)
Haematology, Pratt.
22,
Fink&
J. Lab.
pp.
(1972)
F.,
Acta
(1969)
Endocrinol. 0..
pp.
512-513.
Steroids Med.
22,
74,
Lea and
Churchill,
London
775-794
770-781
Febiger,
Philadelphia
442443
H.E.
Butenandt.
ed..
Clin.
687-755,
In Clinical
Appleton-Century-Crofts, M.
4th
E. (1973)
New 70.
Fend&
H.
Disorders
of Haemoglobin
Structure
York 619424 and
Ehrt-Wehle,
R.
(1974)
Acta
Endocrinol.
75,181-194 14
Sjoberg, 2.131-137
B.,
de la Tome.
B.,
Hedman,
M.,
Falkay,
G.
and
Diczfalusy.
E. (1979)
J. Endocrinol.
Invest.
Clinica Chimica Acta, @ ElsevieriNorth-Holland
CCA
105 (1980) 269-273 Biomedical Press
1431
ENDOCRINE FUNCTION AND HAEMOGLOBINOPATHIES: RELATION BETWEEN THE SICKLE CELL GENE AND CIRCULATING PLASRIIA LEVELS OF TESTOSTERONE, LUTEINISING HORh4ONE (LH) AND FOLLICLE STIMULATING HORh~~NE (FSH) IN ADULT MALES
O.A.
DADA
* and
Department (Received
E.U.
of Chemical January
21st,
NDUKA Pathology,
Ilniuersity
of Ibadan,
Ibadan
(Nigeria)
1980)
Summary
Circulating plasma levels of testosterone, luteinising hormone and follicle stimulating hormone were determined in adult Nigerian males who have haemoglobin (Hb) genotypes HbAA, AS, AC, SC and SS. The levels of the three hormones were significantly lower in homozygous sickle cell disease (HbSS) compared to the other groups, whereas the levels of testosterone and luteinising hormone were significantly reduced in HbSC heterozygotes compared to HbAS heterozygotes only. These results are suggestive of an association of secondary hypogonadism with haemoglobin S disease.
Introduction
Earlier studies on the stability of haemoglobin (Hb) gene frequencies in West Africa indicated a greater potential fertility of unions in which one parent carries a single abnormal haemogIobin gene (Hb heterozygotes -AS and -AC) [ 1,2]. More recent studies have also indicated higher fertility in HbAS compared to the normal HbAA males [3]. In contrast, homozygous sickle cell disease (HbSS) is associated with impairment of sexual development and abnormalities of gonadal function [4,5]. Apart from these studies, there have been no direct comparisons of the circulating plasma levels of the reproductive hormones in individuals of different haemoglobin genotypes. In the present study, the circulating levels of testosterone (T), luteinising hormone (LH) and follicle stimulating hormone (FSH) were determined in adult males with haemoglobin genotypes AA, AS, AC, SC, SS and CC.
* To
whom
correspondence
should
be addressed.
270
Materials and methods Subjects
The subjects with haemoglobin genotypes AA, AS and AC were adult (aged 20-45 years) male Nigerian blood donors at the University College Hospital, Ibadan. Subjects with haemoglobin variants SS, SC and CC were adult male also aged 20-45 years) patients attending the haemoglobinopathy clinics of the haematology department of the same hospital. Blood samples were collected from these patients during periods of established remission. In all subjects, blood was collected between 09.00 and 11.00 a.m. and immediately centrifuged and stored frozen at -20°C until analysed. The haemoglobin genotypes of all samples were confirmed by electrophoresis on cellulose acetate strips [6]. Radioimmun~assay
of testosterone
~l,2,6,7-3H]~stosterone (s-a. 105 Ci/mmol) was purchased from the RadioChemical Centre, Amersham, U.K., and purified by chromatography on columns of Sephadex LH-20 [7] before use. Antiserum raised against testosterone-3(o-carboxymethyl) oxime-bovine serum albumin was supplied by the World Health Organisation, Matched Reagents Programme (MRP). Information supplied by the MRP showed that the antiserum exhibited 14% cross-reaction with 5~-d~ydrotestosterone, 0.8% with A4-androstenedione and 6% with 5cuandrostanediol. Testosterone was assayed by radioimmunoassay in duplicate samples of plasma (50 ~1 + 50 yl 0.1 mol/l phosphate buffer, pH 7.2, containing 0.14 mol/l NaCl, 0.1% thiomersal and 1% gelatin) which was extracted with 5 ml peroxide-free diethyl ether. The extracts were dried under nitrogen. 100 ~1 buffer was added to the residue plus 100 ~1.(5000 cpm) of a solution in buffer of [ 1,2,6,7-3H]testosterone and 100 ~1 antiserum solution (final dilution 1 : 200 000). Standards were in the range O-500 pg/tube. After incubation at 30°C for 1 h, free and bound steroids were separated using 0.5 ml dextrancoated charcoal (0.5% activated charcoal, 0.05% dextran in assay buffer) and centrifuged for 5 min at 500 X g. The supematant (unbound fraction) was decanted into vials, 1 ml ethanol and 10 ml scintillation cocktail (4.5 g PPO per litre toluene) were added and the amount of radioactivity determined in a Packard tricarb liquid scintillation counter (Model 3375). Radioimmunoassay
of hLH and hFSH
Standard preparations of human pituitary luteinising hormone (hLH), 1st International Reference Preparation (code number 68/40), and human pituitary follicle stimulating hormone (hFSH) (code number 69/104) were obtained from the National Institute of Biological Standards and Control, London, U.K. Antisera to hLH and hFSH were obtained in freeze-dried form in sealed ampoules from the MRP. Information supplied by the MRP showed that the hLH antiserum exhibited 50% cross-reaction against hLH p subunit; hLH o! subunit, 5%, hFSH, 0.5%; and hTSH, 0.6%; the hFSH antiserum showed cross-reaction with hLH, 0.1%; hCG, 0.1%; and hTSH, 0.5%. Preparations of hLH, and hFSH labelled with “‘1 by the lactoperoxidase method (tracers) were obtained from the Swiss Federal Institute for Reactor Research, Switzerland. The assay procedure employed 100 ~1 plasma which was added to 100 ~1
271
antiserum solution (final dilution l/l 750 000), 100 ~1 of the tracer (10 000 cpm) and 400 ~1 phosphate buffer, pH 7.4, containing 0.14 mol/l NaCI, 0.1% thiomersal and 5% bovine serum albumin. The mixture was incubated at 4°C for 48 h after which 100 ,~l (1 : 26 dilution) antirabbit gammaglobulin (2nd antibody) was added and the reaction terminated by centrifugation (1500 X g, 30 min) after 24 h incubation at 4°C. The amount of radioactivity was determined in the precipitate (bound fraction) in a Packard gamma counter model. The amount of each hormone in plasma samples was calculated from a linearised log&-log plot of standard curves 181. The inter- and intra-assay variations were, for testosterone 11.2% and 9.2% respectively, for hLH, 16.4% and 12.7% and for hFSH 14.9 and 10.5%. Results The mean levels of testosterone, LH and FSH together with their 95% confidence limits are shown in Table I. Analysis of variance of the data established significant differences between the groups shown in Table II. The mean levels of T, LH and FSH shown for HbAA subjects are not significantly different from those of Hb-AS, -AC and -SC subjects. However, the mean levels of these hormones are significantly lower in Hb-SS subjects compared to the other groups (p < 0.01 for T and LH and p < 0.05 for FSH). A comparison between two heterozygote groups (AS vs. SC) showed that T and LH, but not FSH, levels are significantly higher in -AS individuals (p < 0.05). No other comparisons showed significant differences. Only one subject with homozygous haemoglobin C (HbCC disease) was available for the study and in this subject the values of the three hormones are outside the 95% confidence limits of the mean values for Hb-AA subjects. However, more data are required before any conclusions can be drawn from this.
TABLE
I
MEAN
PLASMA
ADULT
MALES
Haemoglobin
AA
AS
SS
SC
AC
cc
LEVELS WITH
genotype
AND
95%
DIFFERENT
CONFIDENCE
LIMITS
HAEMOGLOBIN No.
TESTOSTERONE,
LH
AND
FSH
Testosterone
LH
subjects
(nmof/l)
(I.U.
19
19.5
4.5
17.6-21.4
3.7-5.3
3.8-5.0
22.5
4.3
4.3
18.8-26.2
3.3-5.3
3.7-5.0
9.4
2.0
3.1
8.1-10.9
1.4-2.6
2.0-4.1
17.4
3.2
3.6
13.0-21.8
2.1-4.3
1.2-5.0
20.7
3.8
3.3
19.9-21.5
2.3-4.8
1.6-5.1
14.5
0.7
1.4
40
19
12
7
1
of
OF
GENOTYPES FSH II)
(I.U./l) 4.4
IN
272
TABLE
II
DIFFERENCES THE
ANALYSIS
BETWEEN OF
THE
VARIANCE
Hormone -~
contrasts
Testosterone
AA
vs AS.
AA
YS SS
LH
FSH
HORMONE AND
THE
-AC,
vs AS,
SC,
AC
AA
vs As,
AC,
SC
AA
vs SS vs AS,
ss
“S SC
AA
vs AS,
AA
vs SS
AS
vsSC
SS
vs
SS
vsSC.AC
TABLE
I AS
ESTABLISHED
BY
CONTRASTS
*
P 0.01 N.S.
P 0.01 P 0.05 P 0.01
AC
P 0.05 AC,
SC
N.S. P 0.05 N.S.
AS
P 0.05
-____ * Not
IN
APPROPRIATE
P 0.05
vs SC
vssc
OF
I’ 0.01
AS
AS
SHOWN
Significance N.S.
SC
SS
SS
LEVELS CALCULATION
N.S. .-._-
..--
. ..-
significant.
Discussion The results of the present study have shown that circulating plasma levels of T, LH, and FSH are si~ificantly reduced in individu~s with homozygous sickle cell (SS) disease. The low levels of these hormones may reflect hypogonadal function secondary to hypopituitary function. Hypopotuitarism has previously been reported in HbSS disease as resulting from intravascular thrombosis and pituitary infarction [9,10]. These results are in contrast to those of Abbasi et al. [5] who found in 14 adult males with HbSS disease lower than normal levels of androgens, together with elevated basal LH levels and an exaggerated LH response to gonadotrophin releasing hormone (GnRH), findings which are indicative of primary hypogonadal function. The reasons for the discrepancies between these two results are not clear yet, but they both provide some evidence that hypogonadal function is a feature of HbSS disease. In heterozygous HbSC disease, which is associated in affected individu~s with morbidity though often of a milder nature than HbSS disease [ 111, a clear-cut picture did not emerge. Levels of the three hormones were not significantly different from those of HbAA subjects but were found to be lower than those of Hb-AS heterozygotes. In the Hb-AS and -AC heterozygotes, levels of testosterone and gonadotrophins were in the same range as for normal HbAA individuals. The present results suggest that differences in circulating plasma gonadotrophins and gonadal hormones may not be responsible for the different fertility patterns in male AS and AC heterozygotes compared to normal AA individuals [ 1-3 1. A possible effect of a tropical environment on the circulating levels of testosterone in Zambian African males compared to men from temperate regions has been suggested [ 12]. It is of interest in this respect to note that the IeveIs of
273
testosterone in normal HbAA males in this study are in the same range as those reported from other temperate countries [ 13,141. Acknowledgements This study was supported by a grant from the World Health Organisation Expanded Programme of Research Training in Human Reproduction. We thank Professor W.A. Isaacs-Sodeye for permission to study his patients; the staff of the blood bank for their cooperation and Professors B. Kwaku Adadevoh and D.A. Olatunbosun for their interest and encouragement of this work. References 1
Roberts.
D.F.
and
Boyo,
A.E.
(1960)
Ann.
Hum.
2
Roberts,
D.F.
and
Boyo,
A.E.
(1962)
Hum.
Biol.
3
Van
Ros.
G.
Proceedings
in
Haemoglobinopathy.
of a Workshop,
4
Olanbiwonnu,
N.O..
5
Abbasi,
Prasad.
A.A.,
L. and
A.O.,
of Ibadan.
Frasier,
Ortega.
J.,
S.D.
24,
375-387
20-37
Reproduction
University
Penny.
Genet. 34,
and
Contraception
(Kwaku
Adadevoh.
B.,
ed.).
in press (1975)
Conego.
E.
and
J. Pediatr.
87,
Oberleas,
D.
459-464 (1976)
Ann.
Int.
Med.
85.
601-
605 6
Dacie,
7
Brenner,
P.F.,
8
Rodbard.
D.,
9
Wintrobe.
M.M.
(1967)
in Clinical
(1968)
Br. J. Clin.
Allen.
D.M.
J.W.
and
Adadevoh,
B.K.
11
Nechelles,
T.F..
Synthesis,
12
Briggs,
M.H.
13
Knorr,
D.,
S.M.
(1968)
R.,
Cekan,
Bridson.
10
and
Lewis,
Guerrero,
pp. and
W. and
56-61,
Brig@,
Bidlingmaier,
Practical Z. and
Rayford.
and
Haematology,
Diczfalusy,
P.L.
(1969)
Haematology, Pratt.
22,
Fink&
J. Lab.
pp.
(1972)
F.,
Acta
(1969)
Endocrinol. 0..
pp.
512-513.
Steroids Med.
22,
74,
Lea and
Churchill,
London
775-794
770-781
Febiger,
Philadelphia
442443
H.E.
Butenandt.
ed..
Clin.
687-755,
In Clinical
Appleton-Century-Crofts, M.
4th
E. (1973)
New 70.
Fend&
H.
Disorders
of Haemoglobin
Structure
York 619424 and
Ehrt-Wehle,
R.
(1974)
Acta
Endocrinol.
75,181-194 14
Sjoberg, 2.131-137
B.,
de la Tome.
B.,
Hedman,
M.,
Falkay,
G.
and
Diczfalusy.
E. (1979)
J. Endocrinol.
Invest.