Download Prolactin and Growth Hormone: Molecular

Review Article
Ann Clin Biochem 1990; 27: 542-550
Prolactin and growth hormone: molecular heterogeneity and
measurement in serum
Caroline R Smith * and M R Norman
From the Department of Clinical Biochemistry, King's College School of Medicine, Denmark Hil/,
London SE5, UK
SUMMARY. The pituitary hormones prolactin and growth hormone are related
single-chain polypeptides. Both hormones exist in the circulation in several molecular
forms, and this heterogeneity may account for some of the complex and sometimes
contradictory actions, in vivo and in vitro, of both hormones. It may also lead to
problems with quantitation by immunoassays and discrepancies between the results
given by assays using different antibodies. Modified forms of the hormones may have
markedly different activity in bioassays from that of the parent hormone, but the
clinical significance of this is unclear. In this review we summarize what is known
about the molecular heterogeneity of the hormones and briefly discuss the implications for clinical biochemists.
Additional key phrases: somatotropin; somatomammotropin
Over the last 10 years there have been major
developments in understanding the relationship
between the structure and function of the pituitary peptide hormones. The glycoprotein hormones follitropin (FSH) and lutropin (LH), for
example, are now known to exist in the circulation in a number of different forms depending on
the composition of their carbohydrate units."?
Activity in immunoassays may be determined
primarily by the peptide chain and thus be
unrepresentative of the biological activity of the
hormone, which is modified by the carbohydrate
group.Y
TABLE
1.
The importance of molecular heterogeneity in
the physiology of human prolactin (hPRL) and
human growth hormone (hGH) has only recently
been appreciated. Major sources of molecular
heterogeneity of both hormones are summarized
in Tables 1 and 2.
mE JipRL AND hGH GENE FAMILY
The hGH and hPRL are believed to have evolved
as a result of duplications of a common ancestral
gene. Studies of other animal growth hormones
have shown, however, that hGH differs consider-
Major forms of hGH in serum
Approx. MW
Variant
(kD)
Gene/Chromosome
Comments
Monomeric pituitary hGH
hGH 20kD
'Big' hGH
'Big-big' hGH
22
20
40-50
>60
hGH-N/17
hGH-N/l7
hGH-N/l7
hGH-N/17
Fragments
<20
hGH-N/17
Placental hGH
22
24,26
hGH-V/17
hGH-V/17
Approx. 75% of total immunoactive hGH
Approx. 16% total immunoactive hGH
Dimers
(a) Aggregates
(b) hGH binding-protein complex
Present in serum in basal state
?Formed in tissues
Secreted by placenta
Glycosylated forms of placental hGH
·Present address: Department of Rheumatology, The Whittington Hospital, Highgate Hill, London N19, UK.
Correspondence: Dr M R Norman.
542
Prolactin and growth hormone
TABLE
2.
543
Major forms of hPRL in serum
Approx. MW
Variant
(kD)
Gene/Chromosome
Comments
Main component of hPRL reference
preparations but only 30% serum hPRL
in basal state
Major component of serum hPRL in
basal state
Reduced in hyper-prolactinaemic states
Predominently dimers
< 20% serum hPRL
Aggregates
< 5% serum hPRL
Found in serum, ?formed in tissues
Monomeric pituitary hPRL
23
hPRL/6
Glycosylated hPRL (G-hPRL)
25
hPRL/6
'Big' hPRL
50-60
hPRL/6
'Big big' hPRL
Fragments
> !OO
16,8, etc.
hPRL/6
hPRL!6
ably from any of the non-primate GHs in both
amino-acid sequence and biological activity, with
hGH the only GH with substantial lactogenic
activity. Although hPRL is less different from
most non-primate PRLs, rat PRL differs considerably in amino-acid sequence from other
mammalian PRLs. 5
The hPRL gene appears to be present 'as a
single copy and is located on chromosome 6.6
The hGH gene cluster is found on chromosome
17,7 and probably consists of the normal human
pituitary hGH gene (hGH-N), three human
placental lactogen (hPL) genes, one of which
may be non-functional, and the hGH-variant
(hGH-V) gene. 8•9 All these genes have in common
a five-exon structure and each encodes a protein
product of 22-26 kD molecular weight.
The expression of the hGH-N gene is restricted
to the somatotrophs of the anterior pituitary;
expression of the hPL genes is restricted to the
syncytiotrophoblast cells of the placenta. The
hGH-V gene has recently been shown to be expressed in the placenta." The hPRL gene is expressed in the pituitary lactotrophs, the decidua
of the placenta and in luteal phase endometrium.
2). There is considerable (42%) homology
between the coding sequences of the hPRL and
hGH genes. The structures of both hormones are
stabilized by intra-chain disulphide bonds.
The three-dimensional structure of a
genetically-engineered variant of porcine GH
was determined recently:" a large proportion of
the molecule was found to consist of four IXhelices folded to form a compact globular structure. In view of the similarities in amino-acid
sequence, it is likely that hGH and hPRL assume
a similar shape. Elegant studies by Cunningham
et al. 15•16 of the effects on binding characteristics
of mutations introduced at various points have
allowed the mapping of receptor and monoclonal
antibody binding sites on the hGH molecule. The
receptor binding-site consists of three sequences,
MOLECULAR STRUCfURE OF hPRL AND
hGH
hPRL is a single-chain polypeptide with an
approximate molecular weight (MW) of 23 kD
(Fig. 1). Amino-acid sequencing of hPRL
derived from a pool of normal pituitaries suggested a 198 amino-acid sequence. I I More recently, sequencing of hPRL cDNA from pituitary
adenoma tissue has revealed an almost identical
199 amino-acid sequence. 12 hGH is also a singlechain protein, with the major circulating form
having 191 residues and a MW of 22 kD 1J (Fig.
COOH
FIGURE 1. Monomeric hPRL. A polypeptide of 199
amino acids with three disulphide bridges.
544
Smith and Norman
arnino-acids.v" It is likely that the recentlydescribed hGH-like peptide found in maternal
serum in the third trimester" represents the
product of the hGH-V gene expressed in the
placenta. It has recently been reported that, as
for the product of the hGH-N gene, alternative
splicing of the hGH-V gene primary transcript
can result in the production of two distinct
proteins (designated hGH-V and hGH_V2).18.19
There is no evidence for more than one hPRL
gene and variant forms of hPRL appear to be
post-translational modifications of the product
of a single gene.
FIGURE 2. Monomeric hGH. A polypeptide of 191
amino acids with two disulphide bridges.
remote in the linear sequence, which are contiguous when the molecule is folded (Fig. 3).
VARIANT FORMS DERIVED FROM
DIFFERENT GENES
The hGH-N gene directs the synthesis of the
22 kD polypeptide, pituitary hGH, and a posttranscriptionally modified form, hGH 2okD , which
is described below and constitutes approximately
10% of pituitary hGH.
The hGH-V gene codes for an hGH-like
hormone which differs from pituitary hGH in 13
3. Three-dimensional structure of hGH
showing the receptor binding site. The structure is based
on that determined for porcine growth hormone," with ex
helices represented by cylinders. Sites ofmutations which
increase ( "") or decrease (.) binding to receptor were
determined by Cunningham and weus." Reproduced by
kind permission ofthe Authors and the Editor of Science.
hGH20kD
Translation ofmRNAs derived from the hGH-N
gene results in synthesis of two distinct polypeptides, hGH m D and hGH 2okD • It is now clear that
the smaller peptide is formed as a result of the use
of an alternative splice-acceptor site within exon
3 of the hGH-N gene,18.20 resulting in an internal
deletion of bases coding for 15 amino acids"
(Fig. 4). The regulation of gene processing by the
two routes is not understood. A third mRNA
variant lacking the entire third exon has also
been detected in pituitary tumour tissue," but the
predicted 17·5 kD protein product has not yet
been definitively identified. There is no evidence
for the existence of analogous hPRL variant
forms.
FIGURE
FIGURE 4.
The 20 kD variant ofhGH. The sequence of
15 amino acids deleted in this variant is represented by a
dashed line.
Pro/actin and growth hormone
POST-TRANSLATIONAL
MODIFICATIONS
Glycosylation
A glycosylated form of hPRL (G-hPRL) is consistently found in human pituitaries and sera."
An oligosaccharide side-chain is attached at
Asn., , resulting in a molecule of apparent MW
25 kD determined by sodium dodecyl sulphate
(SDS) electrophoresis. Recent work shows that
G-hPRL is a major circulating form in normal
sera." We have found that G-hPRL constituted
approximately 70% of total monomeric hPRL in
a study of 42 normal subjects. The proportion of
G-hPRL falls with increasing levels of serum
hPRL, and some patients with extremely high
hPRL levels may have no detectable G-hPRL in
their serum." Very recent work suggests that
there may be more than one form of G-hPRL. 26
G-hPRL is also found in amniotic fluid and is a
secretory product of luteal phase endometrium"
and also of placenta."
The Asn-Gln/Leu-Ser sequence at which Nlinked glycosylation occurs does not occur in
pituitary hGH but is present in the placental
variant. Work by Ray et al. 29 suggests that hGHV is modified by N-linked glycosylation in a
fibroblast cell-line, and there is some evidence
that the placental product is glycosylated in
man."
Charge variants
Charge variants of both hormones, such as
acetylated and deamidated forms, have been
described in serum; it is not clear whether these
represent artefacts or secreted forms."
Aggregates and oligomers
Fractionation of serum by gel filtration consistently reveals three major species of hGH: MW
approximately 20-22 kD (monomeric or little
hGH), 40-50 kD (big hGH) and> 60 kD (big big
hGH).32 Similarly, three species of hPRL are
found in serum with MW 23 kD (monomeric or
little hPRL), 50-60 kD (big hPRL), and
> lOOkD (big big hPRL).33-3s
'Big big' forms of both hormones are mainly
non-covalently bound aggregates.l":" and in the
case of hPRL may be glycosylated." The
complex formed by hGH and its serum binding
protein (see below) probably accounts for most
'big big' hGH. 37 'Big' forms of both hormones
are predominantly dimers,32.38 some disulphidelinked, which appear to be secretory products.
High molecular weight forms of hGH normally
represent approximately 15-50% of total im-
545
munoactive hGH in serum.V The proportion of
'little' hGH has been shown in one study to be
higher in acromegalic patients."
Less than 20% of serum hPRL is present in
high MW forms in sera from both normal
subjects and some patients with hyperprolactinaernia,":" although higher proportions of
high MW forms of hPRL are found in some
patients with pituitary tumours.":" Some women
with hyperprolactinaemia and normal ovarian
function have been shown to have very high
levels of high molecular weight hPRL in their
serum.4(}-42
Proteolytically eleaved forms and fragments
When circulating hPRL and hGH are subjected
to immunoelectrophoresis under reducing conditions several low MW forms are detected. It
has been suggested that the intact monomeric
form of each hormone may be subjected to
sequential attack by specific proteases to
generate biologically active fragments with different functions.i':" Modified forms of both hormones have been described in which the large
loop is opened; subsequent reduction of the
intrachain disulphide bonds generates low
molecular weight fragments.
Cleaved forms ofhPRL appear to be present in
tissues such as pituitary and ovary and have also
been demonstrated in serum from pregnant
women." It is not certain that low MW fragments are produced in vivo, but this idea has
generated considerable interest in view of evidence that the 16kD fragment of rat PRL is
biologically active despite reduced immunoactivity and may have important local effects. 43.4S
Immunoactive hGH variants of MW 12-30 kD
may contribute significantly to the total immunoactivity of hGH in normal sera in the basal
state;" their contribution to hGH bioactivity is
unknown. However, fragments of hGH
generated in vitro have been shown to have enhanced activity in some bioassays" and may act
through specific receptors. Their physiological
functions are unknown.
SERUM BINDING PROTEINS
Until recently it was thought that both hormones
existed free in the circulation. It has recently
become clear however that a specific, high-affinity, low-capacity hGH binding protein (BP)
exists in human plasma." Under physiological
conditions, about 50% of hGH m D and 30% of
hGH 20kD are bound. The rabbit GH BP has the
546
TABLE
Smith and Norman
3.
Significance of major circulating variants of hGH
Variant
Analytical importance
Physiological importance
hGH 20kD
Immunoactivity 10-30% that of hGH m D
Growth activity = hGH m D
?Insulin-like and receptor-binding activity
reduced"
Immunoactivity of aggregates usually
reduced.
Bioactivity considerably reduced in in
vitro bioassays
Binding to a minor serum
BP may reduce RIA activity by 10-15%
May have increased or decreased bioactivity
t 1/2 in serum may be increased 2 or 3fold compared with hGH m D and in vivo
bioactivity norma!']
'Big' and 'big big'
Fragments
Placental hGH
Not distinguishable from pituitary hGH
in routine assays
same amino-acid sequence as the amino-terminal
end of the GH receptor, suggesting that in this
species the BP is the free extra-cellular domain of
the receptor." This relationship between the
serum binding protein and the hGH receptor has
not been confirmed in man. As yet, no convincing evidence for a hPRL serum BP has been
obtained.
The effect of the hGH BP on the measurement
of hGH levels in blood is likely to be dependent
on the relative affinities for hGH of the antiserum
used in the assay and the BP. Preliminary data
from Baumann et a/. 37 suggest that the affinity of
antiserum for bound hGH is likely to exceed that
of the BP, so hGH will preferentially bind to the
antiserum. The complex of hGH with the major
BP is in fact fully immunoactive, although a
small proportion of hGH bound to a minor BP
may be underestimated by RIA. This is unlikely
to represent more than 10-15% of total plasma
hGH.
TABLE
4.
Proportion of large forms not altered by
pituitary stimulation
High serum levels of aggregates may be
associated with short stature (rare)
Effect of binding to BP on in vivo bioactivity not known
? Produced in vivo by the action of tissue
proteases,
May have specific actions
The major component of hGH in maternal serum in late pregnancy.
Physiological significance unknown
CLINICAL SIGNIFICANCE OF
MOLECULAR HETEROGENEITY
The full clinical significance of the variant forms
of the hormones is unclear. Tables 3 and 4 summarize what is known about the physiological
and analytical significance of variant forms of
both hormones in serum.
The properties of modified forms of the hormones may help us to understand situations
where there appear to be discrepancies between
the results of immunoassay measurements of the
ho~ones and the clinical picture. The preservation of normal ovarian function, with maintained
fertility, in women with very high immunoassayable hPRL levels is one such example/":" Such
women have been shown to have high serum
levels of high MW hPRL oligomers which may
be of reduced bioactivity." Current immunoassay methods do not allow the routine detection of
such patients, who may not require treatment,
Significance of major circulating variants of hP RL
Variant
Analytical significance
Physiological significance
G-hPRL
Activity reduced in Nb2 bioassay (by
50%) significantly underestimated
in most immunoassays (by 50-70%)
Activity reduced in both bioassays and
immunoassays
Unknown
? Specific function in pregnancy
'Big' and 'big big' hPRL
Fragments
Bioactivity much increased relative to immunoactivity in some fragments (in the
rat, no data in man)
Ovulatory hyper-prolactinaemia
may be associated with high levels
of large forms
? Produced in tissues
Specific actions unknown: may have
paracrine actions different from
parent hormone
Prolactin and growth hormone
although serial dilution of their sera may show
non-parallelism with monomeric hPRL standard."
Similarly, immunoactive but bioinactive hGH
has been suggested as a cause of short stature.
Subnormal ratios of hGH radioreceptor or
bioassay activity to immunoactivity have been
found in a smalI number of children, who
respond weli to treatment with hGH. 48- 1O One
such case of growth failure due to bioinactive
hOH was shown to be caused by the presence in
serum of large amounts ofhOH oligomers which
showed decreased binding to the hGH receptor
of human IM9 lymphocytes. 51
ANALYTICAL SIGNIFICANCE OF
MOLECULAR HETEROGENEITY
Immunoassays depend on the binding of one or
more antibodies to binding sites on the target
hormone; such sites may be distant from the
binding sites for physiologicalIy-relevant ligands.
Immunoassays may thus detect biologicalIy inactive hormone fragments or aggregates, or fail to
detect bioactive fragments where the assay antibody binding site is absent or obscured.
Antibodies for use in immunoassays are
usualIy raised against the unmodified monomeric
form of the hormone, and may not detect
modified but bioactive forms. Reference preparations for use in immunoassay also consist of
purified monomeric hormone and so may not
reflect the composition of the hormone in the
serum being assayed.
In view of the marked fluctuations in the proportions of hOH and hPRL variants with different MWs and immunoactivities in serum,
there is a strong case for continuing to report
results of assays for these hormones in international units rather than mass units.
The molecular heterogeneity of the hormones
raises the question of the significance of possible
in vitro interconversions between forms, particularly mass variants. Variable conversion of
high to low molecular weight forms of both hormones is known to occur with treatment with
reducing agents, or multiple freeze-thaw cycles.
Under usual assay conditions this does not
appear to cause problems, but there is some evidence that heat treatment of serum, as may be
used for HIV positive samples, can significantly
alter apparent immunoactivity in at least one
RIA. 52
Comparisons between different immunoassays
Systematic comparisons between assays using
547
different antisera are carried out as part of the
validation of new methods but there are few
published reports in the UK literature. In the
case of hPRL, some antisera recognize high MW
variants less welI than others":" and this may
explain the non-paralIelism sometimes found on
dilution of samples with very high hPRL concentrations.
Immunoradiometric
assays
(IRMAs) using monoclonal antibodies, usualIy
directed at two non-overlapping epitopes to form
a 'sandwich', tend to give lower estimates of
hPRL levels than do radioimmunoassays (RIAs)
or bioassays'v" and may not recognize some
forms of the hormone.
Comparisons of hOH results by different immunoassays provide evidence of considerable
variation; up to three-fold differences in absolute
results, both between RIA and IRMA and
between different IRMAs have been reported.":"
A least one commercial IRMA does not detect
hOH 2okD at alI.57 Such data re-emphasize the importance of establishing familiarity with one
assay method and appropriate local reference
and 'action' ranges. Similar problems have been
experienced with IRMAs for gonadotropins. 58
Comparison between immunoassay and bioassay
results
The recent development of a relatively simple
and convenient bioassay for lactogenic hormones
has facilitated comparative studies of their
biological and immunoactivity. The Nb2 celI
assay" uses a celI line derived from a lymphoma
which developed in an oestrogen-treated male
rat. The celIs proliferate in response to both hOH
and hPRL: the assay can be made specific by
using monoclonal antibodies or antisera to block
one hormone.
There are now several studies reporting altered
hPRL bioactivity:immunoactivity (B:I) ratios in
situations where hPRL molecular heterogeneity
is known to be altered. Using the Nb2 cell bioassay, we,bO and others," have found bioactivity to
be reduced compared with immunoactivity in
some patients with prolactinomas. This may
relate to the reduction in the proportion of serum
hPRL which is glycosylated in sera from these
patients, since glycosylation has significant
effects on both the bioactivity and immunoactivity of the molecule.F:" In uraemic or
myxoedematous patients we/ 5•M like Rowe et
al.,65 have been unable to demonstrate major
differences between hPRL activity in the Nb2
assay and in immunoassays, although other
workers have reported changes. 66.67
There are fewer studies of B:I ratios ofhOH in
548
Smith and Norman
serum. The insensitivity of even the Nb2 assay
for hGH makes measurement of unstimulated
hGH levels or confirmation of reduced bioactivity difficult in children with short stature, and
results are conflicting.v?" Using the Nb2 assay,
we and others have found no change in B:I ratios
in sera from acromegalic patients after stimulation of the pituitary.":"
Discrepancies between studies of B:I ratios
may relate to the immunoassay used for comparison with the bioassay, and definitive answers
await the isolation of individual variants such as
G-hPRL in sufficient amounts for their use as
standards and to raise specific antibodies. Since
changes in the molecular heterogeneity of the
hormone, such as glycosylation, affect both immunological and biological activities, determination of B:l ratios may conceal important changes
in the proportions of hormone variants.
CONCLUSIONS
I. hGH and hPRL do not circulate as single
molecular species but as 'families' of related
forms.
2. In a few patients, the molecular heterogeneity
of the hormones may help to explain clinical
paradoxes.
3. The development of new immunoassay
methods,
including
those
involving
monoclonal antibodies, is leading to recognition of variability between immunoassays;
the importance of establishing reference
ranges is emphasized.
4. Despite the heterogeneity of hPRL and hGH,
immunoassay results generally correlate well
with those of bioassays and will remain the
standard method of measurement of these
hormones in routine practice. In future assays
for variants such as G-hPRL may provide
additional information of clinical importance.
5. In view of the heterogeneity of the hormones,
there is a good case for using international
rather than mass units to express immunoassay results.
Acknowledgements
We thank Mr Barry Pike for his help with the
preparation of the figures, and Miss Joan Butler
for critical review of the manuscript and helpful
comments. During the writing of this review,
Caroline Smith was a Wellcome Trust Research
Training Fellow.
REFERENCES
I Pierce JG, Parsons TF. Glycoprotein hormones:
structure and function. Ann Rev Biochem 1981; 50:
465-95
2 Dufau ML, Veldhuis JD. Pathophysiological
relationships between the biological and immunological activities of luteinizing hormone. In:
Burger HG, ed. Reproductive Endocrinology. Bailliere's Clinical Endocrinology and Metabolism, Vol.
I (Feb). \987; pp 153-76
3 Hutchinson JSM. The interpretation of pituitary
gonadotrophin assays-a continuing challenge. J
Endocrinol 1988; 118: 169-7\
4 Baenziger JU, Green ED. Pituitary glycoprotein
hormone oligosaccharides: structure, synthesis and
function of the asparagine-linked oligo saccharides
on lutropin, follitropin and thyrotropin. Biochim
Biophys Acta 1988; 947: 287-306
5 Wallis M. The molecular evolution of pituitary
growth hormone, prolactin and placental lactogen:
a protein family showing variable rates of
evolution. J Mol EI'01198\; 17: 10-8
6 Owerbach D, Rutter WJ, Cooke NE, Martial JA,
Shows TB. The prolactin gene is located on
chromosome 6 in humans. Science 1981; 212: 8\5-6
7 Ower bach D, Rutter WJ, Martial JA, Baxter JD.
Shows TB. Genes for growth hormone, chronic
sommatomammotrophin and growth hormone-like
gene on chromosome \7 in humans. Science 1980;
209: 289-92
8 Seeburg PH. The human growth hormone gene
family: nucleotide sequences show recent divergence and predict a new polypeptide hormone.
DNA 1982; 1: 239-9
9 Hirt H, Kime1man J, Birnbaum MJ, et al. The
human growth hormone gene locus: structure,
evolution and allelic variations. DNA 1987; 6: 59-70
10 Frankenne F, Rentier-Delrue F, Scippo ML,
Martial J. Hennen G. Expression of the growth
hormone variant gene in human placenta. J Clin
Endocrinol Metab 1987; 64: 635-7
1\ Shome B, Parlow AF. Human pituitary prolactin:
the entire linear amino acid sequence. J Clin
Endocrinol Metab \977; 45: 1112-5
12 Cooke NE, Coit D, Shine J, Baxter JD, Martial JA.
Human prolactin: cDNA structural analysis and
evolutionary comparisons. J BioI Chem \981; 256:
4007-16
13 Li CH, Dixon JS. Human pituitary growth
hormone. The primary structure of the hormone:
revision. Arch Biochem Biophys 1971; 146: 233-6
14 Abdel-Meguid SS, Shieh H-S, Smith WW, Dayringer HE, Violand BN, Bentle LA. Three-dimensional structure of a genetically-engineered variant
of porcine growth hormone. Proc Natl Acad Sci
USA 1987; 84: 6434-7
15 Cunningham BC, Jhurani P, Ng P, Wells JA.
Receptor and antibody epitopes in human growth
hormone
identified
by
homolog-scanning
mutagenesis. Science 1989; 243: \330-6
16 Cunningham BC, Wells JA. High-resolution
epitope mapping of hGH-receptor interactions by
alanine-scanning mutagenesis. Science 1989; 244:
1081-5
Prolactin and growth hormone
17 Frankenne F, Closset J, Gomez F, Scippo ML,
Smal J, Hennen G. The physiology of growth hormones in pregnant women and partial characterization of the placental GH variant. J Clin Endocrinol
Metab 1988; 66: 1171-80
18 Cooke NE, Ray J, Watson MA, Estes PA, Kuo BA,
Liebhaber SA. Human growth hormone gene and
the highly homologous growth hormone variant
gene display different splicing patterns. J Clin Invest
1988; 82: 170-5
19 Cooke NE, Ray J, Emery JG, Liebhaber SA. Two
distinct species of human growth hormone-variant
mRNA in the human placenta predict the expression of novel growth hormone proteins. J Bioi
Chem 1988; 263: 9001-6
20 DeNoto FM, Moore DD, Goodman HM. Human
growth hormone DNA sequence and mRNA structure: possible alternative splicing. Nucleic Acids Res
1981; 9: 3719-30
21 Lewis UJ, Singh RNP, Tutwiler GF, Sigel MB,
VanderLaan EF, VanderLaan WP. Human growth
hormone: a complex of proteins. Recent Prog Horm
Res 1980; 36: 477-508
22 Lecomte CM, Renard A, Martial J. A new natural
hGH variant-17'5kD-produced by alternative
splicing. Nucleic Acids Res 1987; 15: 6331-8
23 Lewis UJ, Singh RNP, Sinha YN, VanderLaan
WP. Glycosylated human prolactin. Endocrinology
1985; 116: 359-63
24 Markoff E, Lee DW. Glycosylated prolactin is a
major circulating variant in human serum. J Clin
Endocrinol Metab 1987; 65: 1102-6
25 Hashim lA, Aston R, Butler J, McGregor AM,
Smith CR, Norman MR. The proportion of
glycosylated prolactin in serum is decreased in
hyperprolactinaemic states. J Clin Endocrinol
Metab 1990; 71: 111-15
26 Lewis UJ, Singh RNP, Lewis LJ. Two forms of
glycosylated human prolactin have different pigeon
crop-sac stimulating activities. Endocrinology 1989;
124: 1558-63
27 Heffner LJ, Iddenden DA,
Lyttle CR.
Electrophoretic analyses of secreted human endometrial proteins: identification and characterisation of luteal-phase prolactin. J Clin Endocrinol
Metab 1986; 62: 1288-95
28 Lee DW, Markoff E. Synthesis and release of
glycosylated prolactin by human decidua in vitro. J
Clin Endocrinol Metab 1986; 62: 990-4
29 Ray J, Jones BK, Liebhaber SA, Cooke NE.
Glycosylated human growth hormone variant.
Endocrinology 1989; 125: 566-8
30 Hennen G, Frankenne F, Scippo M-L, et al.
Hormone de croissance placentaire. Signification
par rapport aux hormones de croissance et lactogene. Reprod Nutr Dev 1988; 28:1699-706
31 Baumann G, MacCart JG, Amburn K. The
molecular nature of circulating growth hormone in
normal and acromegalic man: evidence for a principal and minor monomeric forms. J Clin
Endocrinol Metab 1983; 56: 946-52
32 Stolar MW, Amburn K, Baumann G. Plasma 'big'
and 'big big' growth hormone in man: an oligomeric series composed of structurally diverse growth
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
549
hormone monomers. J Clin Endocrinol Metab 1984;
59: 212-8
Suh HK, Frantz AG. Size heterogeneity of human
prolactin in plasma and pituitary extracts. J Clin
Endocrinol Metab 1974; 39: 928-35
Whitaker MD, Klee G, Kao PC, Randall RV,
Heser DW. Demonstration of biological activity of
prolactin molecular weight variants in serum. J Clin
Endocrinol Metab 1984; 58: 826-30
Garnier PE, Aubert ML, Kaplan SL, Grumbach
MM. Heterogeneity of pituitary and plasma prolactin in man: decreased affinity of 'big' prolactin in a
radioreceptor assay and evidence for its secretion. J
Clin Endocrinol Metab 1978; 47: 1273-81
Shoupe D, Montz FJ, Kletzky OA, diZerega GS.
Prolactin molecular heterogeneity. Response to
thyrotropin-releasing hormone stimulation of concanavalin A bound and unbound immunoassayable
prolactin during human pregnancy. Am J Obstet
Gynecol 1983; 147: 482-7
Baumann G, Amburn K, Shaw MA. The circulating growth hormone binding-protein complex: a
major constituent of plasma GH in man.
Endocrinology 1988; 122: 976-84
Benveniste R, Helman JD, Orth DN, McKenna rr,
Nicholson WE, Rabinowitz D. Circulating big
human prolactin: conversion to small human prolactin by reduction of disulphide bonds. J Clin
Endocrinol Metab 1979; 48: 883-6
Gordon P, Lesniak MA, Eastman R, Hendricks
CM, Roth J. Evidence for higher proportion of
'little' growth hormone with increased radioreceptor assay activity in acromegalic plasma. J Clin
Endocrinol Metab 1976; 43: 364-73
Jackson RD, Wortsman J, Malarkey WB. Characterization of a large molecular weight prolactin in
women with idiopathic hyper-prolactinaemia and·
normal menses. J Clin Endocrinol Metab 1985; 61:
258-63
Whittaker PG, Wilcox T, Lind T. Maintained fertility in a patient with hyperprolactinaemia due to big,
big prolactin. J Clin Endocrinol Metab 1981; 53:
863-6
Andersen AN, Pedersen H, Djursing H, Andersen
BN, Friesen HG. Bioactivity of prolactin in a
woman with an excess of large molecular size prolactin, persistent hyperprolactinaemia and spontaneous conception. Fertit Steril 1982; 38: 625-8
Mittra I. Somatomedins and proteolytic bioactivation of prolactin and growth hormone. Cell 1984;
38: 347-8
Sinha YN, Gilligan TA, Lee DW, Hollingsworth D,
Markoff E. Cleaved prolactin: evidence for its occurrence in human pituitary gland and plasma. J
Clin Endocrinol Metab 1985; 60: 239-43
Clapp C, Sears PS, Russell DH, Richards J, LevayYoung BK, Nicoll CS. Biological and immunological characterization of cleaved and 16K forms of
rat prolactin. Endocrinology 1988; 122: 2892-8
Baumann G, Stolar MW, Amburn K. Molecular
forms of circulating growth hormone during spontaneous secretory episodes and in the basal state. J
Clin Endocrinol Metab 1985; 60: 1216-20
Leung DW, Spencer SA, Cachianes G, et al.
550
Smith and Norman
Growth hormone receptor and serum binding
protein: purification, cloning and expression.
Nature 1987; 330: 537--43
48 Rudman D, Kutner MH, Blackston RD, Cushman
RA, Bain RP, Patterson JH. Children with normalvariant short stature: treatment with human growth
hormone for six months. N Engl J Med 1981, 305:
123-31
49 Kowarski AA, Schneider J, Ben-Galim E, Weldon
VV, Daughaday WHo Growth failure with normal
serum RIA-GH and low somatomedin activity:
somatomedin restoration and growth acceleration
after exogenous GH. J Clin Endocrinol Metab 1978;
47: 461--4
50 Frazer TE, Gavin JR, Daughaday WH, Hillman
RE, Weldon VV. Growth hormone-dependent
growth failure. Pediatr Res 1980; 14: 478 (abstract)
51 Valenta LJ, Siegel MB, Lesniak MA, et al. Pituitary
dwarfism in a patient with circulating abnormal
growth hormone polymers. N Engl J Med 1985;
312: 214-7
52 Hardy MJ, Ragbeer SS. Effect of heat treatment for
inactivation of HTLV III virus on serum immunoreactive prolactin. Ann Clin Biochem 1987; 24:
322--4
53 Jeffcoate SL, Bacon RRA, Beastall GH, Diver MJ,
Franks S, Seth J. Assays for prolactin: guidelines
for the provision of a clinical biochemistry service.
Ann Clin Biochem 1986; 23: 638-51
54 Rose DP, Berke B, Cohen LA. Serum prolactin and
growth hormone determined by radioimmunoassay
and a two-site immunoradiometric assay: comparison with the Nb2 cell bioassay. Horm Metab
Res 1988; 20: 49-53
55 Smith CR, Butler J, Iggo N, Norman MR. Serum
prolactin in uraemia: correlations between bioactivity and activity in two immunoassays. Acta
Endocrinol (Copenh) 1989; 120: 295-300
56 Reiter EO, Morris AH, MacGillivray MH, Weber
D. Variable estimates of serum growth hormone
concentrations by different radioassay systems. J
Clin Endocrinol Metab 1988; 66: 68-71
57 Celniker AC, Chen AB, Wert RM, Sherman BM.
Variability in the quantitation of circulating growth
hormone using commercial immunoassays. J Clin
Endocrinol Metab 1989; 68: 469-76
58 Wheeler MJ, D'Souza A, Horn AN. Evaluation of
test kits for gonadotropins. Lancet 1989; ii: 616-7
59 Tanaka T, Shiu RPC, Gout PW, Beer CT, Noble
RL, Friesen HG. A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin
Endocrinol Metab 1980; 51: 1058-63
60 Smith CR, Butler J, Hashin lA, Norman MR.
Serum prolactin bioactivity and immunoactivity in
hyperprolactinaemic states. Ann Clin Biochem 1990;
27: 3-8
61 Subramanian MG, Spirtos NJ, Moghissi KS,
Magyar DM, Hayes MF, Gala R. Correlation and
62
63
64
65
66
67
68
69
70
71
72
73
comparison of NB2 lymphoma cell bioassay with
radioimmunoassay for human prolactin. Fertil
Steril 1984; 42: 870--4
Markoff E, Sigel MB, Lacour N, Seavey BK,
Friesen HG, Lewis UJ. Glycosylation selectively
alters the biological activity of prolactin.
Endocrinology 1988; 123: 1303-6
Pellegrini I, Gunz G, Ronin C, et al. Polymorphism
of prolactin secreted by human prolactinoma cells:
immunological, receptor binding and biological
properties of the glycosylated and non-glycosylated
forms. Endocrinology 1988; 122: 2667-74
Smith CR, Butler J, Norman MR. Biological activity of serum prolactin in patients with primary
hypothyroidism. Ann Clin Biochem 1988; 25: 540-5
Rowe RC, Cowden EA, Fairnan C, Friesen HG.
Correlation of Nb2 bioassay and radioimmunoassay values for human serum prolactin. J Clin
Endocrinol Metab 1983; 57: 942-6
Mooradian AD, Morley JE, Korchik WP, Ma KW,
Hartel MA, Parsons JA. Comparison between
bioactivity and immunoreactivity of serum prolactin in uraemia. Clin Endocrinol 1985; 22: 241-7
Silverman A Y, Schwartz SL, Steger RW. A quantitative difference between immunologically and
biologically active prolactin in hypothyroid
patients. J Clin Endocrinol Metab 1982; 55: 272-5
Tokuhiro E, Dean H, Friesen HG, Rudman D.
Comparative study of serum human growth
hormone measurement with Nb2 lymphoma cell
bioassay, IM9 receptor modulation assay and
radioimmunoassay in children with disorders of
growth. J Clin Endocrinol Metab 1984; 58: 549-54
Sneid D, Jacobs L, Weldon V, Trivedi B, Daughaday W. Radioreceptor-inactive growth hormone
associated with stimulated secretion in normal
subjects. J Clin Endocrinol Metab 1975; 41: 471--4
Gamier P, Job J-c. Correlative study of
radioreceptor assay and radioimmunoassay of
serum growth hormone in children: normal children and hGH-treated pituitary dwarfs.
Acta
Endocrinol ( Copenh ) 1977; 86: 50-9
Baldwin AL, Aston R, Butler J, Buchanan C,
Norman MR. Bioactivity and immunoactivity of
growth hormone during dynamic testing of patients
with acromegaly. Ann Clin Biochem 1988; 119: 32632
Tanaka T, Shishiba Y, Gout P, Beer C, Noble R,
Friesen HG. Radioimmunoassay and bioassay of
human growth hormone and human prolactin. J
Clin Endocrinol Metab 1983; 56: 18-20
Baumann G, Stolar MW, Buchanan TA. Slow
metabolic clearance rate of the 20,OOO-Dalton
variant of human growth hormone: implications
for biological activity. Endocrinology 1985; 117:
1309-13
Accepted for publication 23 May 1990