AMER. ZOOL., 15:881-903 (1975).
Radioimmunoassay and Radioreceptor Assays for Prolactin and Growth
Hormone: A Critical Appraisal
CHARLES S. NICOLL
Department of Physiology-Anatomy, University of Calif ornia, Berkeley, California 94720
SYNOPSIS. The promise and the problems associated with using radioimmunoassays (RIA's)
for mammalian prolactins (PRL's) and growth hormones (GH's) for the measurements of
these hormones in the blood of foreign species (mammalian and nonmammalian) are
considered. When crossreactivity is found with the plasma of a foreign species in heterologously applied RIA's for these hormones of mammalian origin, one can have little confidence about the nature of the crossreacting material that is being measured. Extensive
analysis is necessary to establish that a particular RIA system measures the PRL or GH in
a foreign species.
The question of whether RIA's for PRL and GH give physiologically meaningful measurements of the blood levels of these hormones is considered. In the case of PRL, analysis of
our own results and data in the literature raises serious questions about the physiological
validity of existing RIA's for mammalian PRL's. Evaluation of the available information on
RIA's for mammalian GH discloses that there is no basis for concluding that any of them give
measurements of the circulating levels of the hormone that are physiologically meaningful.
It is also apparent that the physiological significance of the radioreceptor assays for PRL and
GH remains to be established.
From analysis of discrepancies between bioassay and RIA estimates of PRL and GH levels
in adenohypophysial tissue, incubation medium, and plasma or serum, and of studies on the
metabolism of purified and secreted forms of both hormones, it is suggested that the major
intraglandular forms of PRL and GH are in fact prohormones.
surprising because with most "exotic"
species, it is difficult to obtain large quanRadioimmunoassays (RIA's) are now the tities of pituitary tissue from which hormost widely used methods for measuring mones can be extracted and purified for
various kinds of hormones. The popularity use in the development of RIA's. However,
of RIA's is understandable because they as will be discussed later, it is possible to
offer exceptionally high sensitivity and pre- develop homologous RIA's for prolactin
cision and they are much more convenient (PRL) and growth hormone (GH) with a
and less expensive than are most bioassays. relatively small number of pituitary glands.
Furthermore, when applied to homologous
Because of the unavailability of purified
species, RIA's are not complicated by non- preparations of PRL and GH from nonspecific reactions that may confound many mammalian species, some investigators
biological assays.
have attempted to measure these hormones
Although RIA procedures are currently in "lower" forms by applying existing RIA's
being used in numerous laboratories for mammalian PRL or GH. Such
throughout the world for measuring pep- heterologous1 applications of homologous
tide and protein hormones of mammalian
1
Homologous RIA's are those in which the same
species, attempts to apply these methods to
studies on endocrine physiology of non- hormone (e.g., ovine PRL) is used for generation of
the antiserum, as a label and as the standard. When
mammalian vertebrates are sparse. This lag such
an RIA is used to measure PRL in sheep blood the
on the part of comparative endocri- assay is homologously applied. If the same assay were
nologists in adopting RIA methods is not used to detect PRL in a foreign species, the procedure
INTRODUCTION
would represent an heterologous application of an
homologous RIA. Mixed heterologous RIA's can also
I am indebted to Dr. Sharon M. Russell for proof- be developed. These RIA's consist of antiserum to a
reading the manuscript and the proofs. Work in my hormone from one species (e.g., ovine PRL) used with
laboratory that is referred to in this chapter was sup- an iodinated label of the same hormone of another
ported by grants from theNIH (AM-13605) and from species (e.g., pig PRL). Ovine, pig, or other PRL's
could be used as a standard (see Midgley, 1972).
the NSF (GB-42687).
881
882
CHARLES S. NICOLL
or mixed heterologous RIA's for mammalian hormones have been discussed previously by Midgley et al. (1971) and by
Midgley (1972). They are used fairly commonly by investigators of mammalian
species (see Robyn et al., 1973; Greenwood
et al., 1973; Aubert et al., 1974).
I n this chapter the promise and problems
of heterologous application of RIA's for
mammalian PRL and GH to nonmammalian species will be evaluated. In
addition, the prospects of developing
homologous RIA's for these hormones of
non-mammalian species and the potential
value of the newer radioreceptor assays
(RRA's) will be discussed. Finally, the question of what the RIA's and RRA's for PRL
and GH are really measuring will be considered.
HETEROLOGOUS RADIOIMMUNOASSAY OF PROLACTIN AND GROWTH HORMONE
The feasibility of using RIA's for ovine
PRL for the detection of the hormone in
non-mammalian species was indicated by
the observation that fluorescein-conjugated antisera to the sheep hormone
localized in the presumptive lactotropes of
the adenohypophyses of a variety of vertebrates, including teleosts (see Schreibman
etal., 1973; Nagahama, 1973; Hansen and
Hansen, 1975). Thus, the PRL's of a
number of vertebrate species appear to
have antigenic determinants in common
with those of ovine PRL. Nicoll and Bryant
(1972) reported that with several homologous and mixed heterologous RIA's for
mammalian PRL's, cross-reacting material
could be detected in pituitary extracts of a
variety of vertebrate species. The occurrence and degree of the cross-reactions obtained by these investigators did not show
any phylogenetic consistency. Thus, extracts of pituitaries of some nonmammalian species (including teleosts and
chondrichthyeans) showed stronger crossreactions than did those of some mammalian species. Similar lack of phylogenetic
consistency is seen in the observation that
RIA's for rat prolactin are unsuitable for
measurement of the hormone in mice
(Sinha et al., 1972) but can be applied satis-
factorily to hamsters (Donofrio et al.,
1973/74).
These results of Nicoll and Bryant (1972)
with RIA's for mammalian PRL's are in
contrast with the findings of Hayashida
(1970), who reported that with an RIA for
rat GH, phylogenetically related crossreactions could be obtained with pituitary
extracts from representatives of all classes
of tetrapods. Despite these differences between the findings of Hayashida (1970) and
of Nicoll and Bryant (1972), both sets of
observations support the conclusion derived from the immunohistochemical
studies to the effect that existing RIA's for
mammalian PRL's and GH's could be
applied to the measurement of these hormones in foreign species.
Bryant and Nicoll (unpublished) investigated the possibility of using an RIA for
human PRL to measure PRL in the teleost
Tilapia mossambica. It was first established
that homogenates of whole Tilapia
pituitaries cross-reacted in the RIA for
human PRL. Since the prolactin cells of
Tilapia, as in other teleosts, are localized in
the rostral region of the pars distalis (see
Nagahama, 1973; Schreibman etal., 1973),
we next determined whether the crossreacting material in the homogenates of
whole pituitaries was sequestered in the region of" the prolactin cells. To test this possibility, the rostral half of adenohypophyses
from Tilapia was separated from the caudal
half and homogenates of the separate
halves were tested in the RIA for human
PRL. The results obtained are presented in
Figure 1.
The homogenates of both the rostral and
caudal halves of the Tilapia pituitaries inhibited binding of the 131I-labeled human
PRL to the antibody in a manner which
paralleled that obtained with the human
PRL standard. It was also evident that the
cross-reacting material was concentrated in
the rostral pars distalis inasmuch as the estimated amount of prolactin contained
therein (in terms of equivalents of the
human PRL standard) was more than 12
times the level found in the caudal region.
The cross-reacting material found in the
caudal region could have been PRL that
was present in the posterior portion of the
APPRAISAL OF RADIOASSAYS
883
adapted to sea water. The results obtained
from a test of this possibility are presented
A
in Figure 2. Pooled serum samples from
fish adapted to each habitat gave parallel,
\ \
dose-related slopes of inhibition which also
\
paralleled that obtained with the human
<A\
S
PRL standard. This parallel inhibition with
A Caudal
80\ \ (6.5jjg/mg)
the serum samples suggests (but does not
prove) that the cross-reacting material in
the
serum is the same as that found in the
\x \
pituitary homogenates. These assay results
Human
\
PRL STD "A
indicate that serum from the sea water60adapted Tilapia contained about 200 ng/ml
\
of PRL (in equivalents to the human stanRostral
,
(80)jg/mg)
dard), whereas the serum from the fresh
O.I
1.0
10
water-adapted fish contained 8,000 ng
equivalents per ml. Thus, with adaptation
ng of human PRL standard or ug of Tilopia
to fresh water the levels of circulating PRL
Pituitary extract
FIG. 1. Cross-reactivity of homogenates of the rostral (or of cross-reacting material) in Tilapia inand caudal regions of the pars distalis of the teleost, crease about 40-fold. This finding is conTilapia mossambica in a radioimmunoassay for human sistent with the available evidence on the
prolactin (PRL). The results indicate that the rostral role of PRL in fresh water adaptation in
and caudal regions contain, respectively, 80 and 6.5 Tilapia and other euryhaline teleosts. Ac/xg per mg wet weight of Tilapia "prolactin" in equivacordingly, these data from the pooled
lents of the human PRL standard (STD).
serum samples support the conclusion derived from the results with pituitary
gland as a result of incomplete separation homogenates that the RIA for human PRL
of the two regions. On the other hand, the could be used to measure Tilapia PRL.
possibility must be considered that Tilapia However, before it can be concluded that
growth hormone, which would be present
in the caudal half of the gland (see
1
Schreibman et al., 1973; Nagahama, 1973)
r
—r
may also cross-react in this RIA (see below).
00 A
Nevertheless, it seems reasonable to con\
A
X
clude from these results that the cross\
\
reacting material was the PRL of Tilapia
\
\
because it showed parallelism with the
\
\ o
human PRL standard and because it was
\
80
\
highly concentrated in the region of the
A \
\
\
gland where one would expect the horX \
S.W serum
mone to be localized.
o\
v
(0.2jjg/ml)
The possibility of using this RIA for
\
Human
human PRL to measure the hormone in the
F. W. serum
PRL STD
cr\
serum of Tilapia was also investigated.
bO
(S.Opg/ml)
i
1
1
I
Numerous studies indicate that PRL is
0.5 1.0
100
necessary for adaptation of Tilapia (Dharmamba and Maetz, 1972) and other
ng of human PRL or p\ of Tilapia serum
euryhaline teleosts to the freshwater FIG. 2. Cross-reactivity of serum from Tilapia in a
habitat (see Bern and Nicoll, 1968; Ensor radioimmunoassay for human prolactin. The fish
arid Ball, 1972; Johnson, 1973; Nicoll, were adapted to fresh water (F.W.) or to sea water
1974). Accordingly, one would anticipate (S.W.) prior to collection of blood. The results indicate
the serum for the S.W.- and F. W.-adapted fish
that the blood of fresh water-adapted that
contained 0.2 fig and 8.0 /ig/ml, respectively, of "proTilapia should contain substantially more lactin" in equivalents of the human PRL standard
PRL than would the blood of animals (STD).
100 -
I
1
c
t
884
CHARLES S. NICOLL
this RIA measuresTilapia PRL and that the
results obtained are physiologically meaningful, consideration must be given to a
quantitative peculiarity of these data.
In relation to other RIA data of PRL
levels in serum of several mammalian
species, the levels of immunoreactive material detected in serum of even the sea
water-adapted fish are extremely high.
Concentrations of plasma PRL of 200
ng/ml or higher are found in normal humans only in circumstances where massive
prolactin release has been induced (e.g., by
the suckling stimulus in lactating women or
by injections of TRH; see chapters in Pasteels and Robyn, 1973). The circulating
levels of the hormone are usually less than
50 ng/ml in normal unstimulated subjects.
Furthermore, levels of immunoreactive
human PRL in the microgram range are
encountered only in pathological conditions, such as in subjects with PRL-secreting
pituitary tumors (Frantz et al., 1972a,b;
Turkington, 1972; Friesen, 1973). Accordingly, if the cross-reacting material that was
detected in serum of Tilapia is in fact PRL,
one would have to conclude that this species
either has unusually high levels of the hormone in its circulation, or that the antiserum to human PRL must have an extraordinarily high affinity for the fish PRL.
In fact, the affinity of the antiserum for
Tilapia PRL would have to be substantially
higher than its affinity for the hormone that
was used to produce the antiserum. This
possibility seems remote in view of the
phylogenetic displacement of Tilapia and
Homo sapiens from common ancestors.
Thus, although these results with the RIA
for human PRL are qualitatively consistent
with expectations based on the available
physiological evidence on the role of PRL in
euryhaline teleosts, the quantitative aspects
of the data render dubious the conclusion
that the RIA measures Tilapia PRL.
The levels of prolactin detected in the
rostral region of the Tilapia pars distalis of
80 /u.g/mg wet weight might also be considered to be rather high. However, in the
adenohypophysis of lactating rats immunoreactive PRL in concentrations of 10
to 20 /Ag/mg wet weight have been reported
(Sinha et al., 1974; Nicoll et al., unpub-
lished). When one considers that the rostral
pars distalis of Tilapia is composed almost
entirely of lactotropes, the observed concentration of prolactin is not unreasonable.
In order to establish that the crossreacting material that was detected in
Tilapia pituitaries and serum by the RIA for
human PRL was in fact Tilapia PRL, additional analyses would have to be conducted.
Unfortunately, we were not able to carry
out tests requisite to demonstrate that the
RIA did measure Tilapia PRL because the
supply of that particular antiserum became
exhausted. The tests that should have been
performed to establish the physiological
validity of these RIA results are the same
for other homologous or heterologous applications of RIA's. These tests are enumerated below.
1) It would be essential to determine
whether the cross-reacting material was
present either in the organs or in the serum
of hypophysectomized Tilapia. If the
cross-reacting material were undetectable
in the serum of hypophysectomized fish
one could conclude that it was either of
pituitary origin or that its presense in
serum was pituitary dependent. If it were
also undetectable in tissues other than the
pituitary, the pituitary origin of the crossreacting material would be highly probable.
2) It would also be desirable to demonstrate that the antiserum to human PRL
could neutralize the biological activity of
the Tilapia prolactin in pituitary extracts
and in serum.
3) It would be important to demonstrate
that the other hormones of Tilpia
pituitaries do not cross-react significantly in
this RIA. The pituitary product of major
concern in this regard is growth hormone
(see below). This test of validity is impractical for most "exotic" species because of the
unavailability of purified preparations of
their pituitary hormones. However, progress is being made in the isolation and purification of Tilapia pituitary hormones
(Farmer et al., 1975). Obviously, it would
not suffice to test purified pituitary hormones from other species (such as ovine or
bovine hormones) since their degree of
cross-reactivity has no bearing on the
APPRAISAL OF RADIOASSAYS
cross-reactivity of Tilapia pituitary hormones in this RIA.
4) The demonstration that purified
Tilapia PRL would give slopes of inhibition
that parallel those obtained with the pituitary homogenates and with the serum samples would be reassuring.
5) Ultimate proof that the RIA for
human PRL measures physiologically active Tilapia PRL in blood can only be obtained by extensive correlation analysis between RIA and bioassay of the hormone in
serum or plasma samples. As is discussed
below in connection with bioassay and RIA
of rat PRL and GH, the demonstration that
the RIA shows good correspondence with
bioassay measurements on purified preparations of the hormone or with levels in
pituitary extracts does not prove that it is
valid for measurements on serum or
plasma. The forms of the hormone in blood
may be different from those in pituitary
homogenates or purified preparations.
Since none of these tests on the
physiological validity of the RIA has been
carried out, we are obliged to conclude
simply that the RIA for human PRL can
measure cross-reacting material in Tilapia
serum and this material might be Tilapia
PRL. On the other hand, because of the
quantitative peculiarities, the crossreacting material in the serum might be
something else which happens to give
slopes of inhibition of binding which parallel that of the standard in the RIA.
Leatherland and McKeown (1973) and
Peter and McKeown (1974) have reported
885
some results with an RIA for material that
was presumed to be pollack PRL. This pollack "prolactin" was purified by Emmert
and Wilhelmi(1968) and identified as "prolactin" on the basis of immunologic crossreactivity with antiserum to ovine PRL. The
antiserum to the pollack "prolactin" was
produced in rabbits by Emmert and Bates
(1968). However, the purified protein was
never identified as a PRL by any biological
assay. In fact, it was found to be inactive in
hypophysectomized killifish (Emmert and
Wilhelmi, 1968). Therefore, its identity as a
PRL has not been established. The RIA
derived from this pollack "prolactin" does
cross-react with material in the plasma of
goldfish (Leatherland and McKeown,
1973; Peter and McKeown, 1974).
Radioimmunoassays for ovine PRL and
bovine GH have been applied to the detection of cross-reacting material in plasma
and pituitary tissues of salmon (McKeown
and van Overbeeke, 1972; Leatherland and
McKeown, 1974; Leatherland etal., 1974),
a toad (McKeown, 1972), and band-tailed
pigeons (March and McKeown, 1973). The
data of these investigators on the slopes of
inhibition obtained with these heterologously applied RIA's are presented in
Table 1 along with some representative
data on the slopes of inhibition in
homologous RIA's. With an RIA for ovine
PRL, purified ovine PRL gives slopes of
inhibition ranging from —50 to —150. The
steepness of the slope depends on the
characteristics of the antibody. In our experience with homologous RIA's for PRL
TABLE 1. Slopes of inhibition of binding of labels in different homologous and heterologous applications of homologous
radioimmunoassays for prolactin (PRL) and growth hormone (GH).
Reference
1,2
1
1
1
3
2
1
1
2
RIA for:
Ovine PRL
Ovine PRL
Ovine PRL
Ovine PRL
Human PRL
Frog PRL
Bovine GH
Bovine GH
Frog GH
Material
tested
Ovine PRL
Pigeon plasma
Toad plasma
Salmon plasma
Tilapia Serum
Frog PRL or plasma
Toad plasma
Salmon plasma
Frog GH or plasma
Slope
-50 to -150
-30
-10
-10
-26
-70
-15
-10
-40
1) Data from McKeown (1972), McKeown and van Overbeeke (1972), March and McKeown (1973).
2) demons and Nicoll (1974).
3) Bryant and Nicoll (unpublished).
886
CHARLES S: NICOLL
and GH of several species (rat, sheep; human, bullfrog), slopes of inhibition of about
- 4 0 to - 7 0 are routinely obtained.
In the RIA for human PRL that was used
by Bryant and Nicoll (Figs. 1, 2), the Tilapia
serum and the human PRL standard gave
parallel slopes of inhibition of about —26.
Although this slope is somewhat flat, it is
still steep enough to give reasonably precise
estimates of the quantities of cross-reacting
material present in the samples. When pigeon plasma was tested in the RIA for ovine
prolactin, March and McKeown (1973) obtained a slope of inhibition of -30. This
slope would also provide a usable and
reasonably precise assay. However, it was
not demonstrated by these investigators
that the cross-reacting material in the pigeon plasma is pigeon PRL since none of
the tests that would be required to validate
the assay (see above) were performed.
The results obtained from the
heterologous RIA of plasmas of salmon
and the toad stand in marked contrast with
those discussed in the previous paragraph.
With the RIA for ovine PRL, the fish and
amphibian plasmas gave slopes of inhibition of about -10. With the RIA for bovine
GH, the salmon and toad plasmas gave
slopes of - 1 0 and —15, respectively. With
such poor slopes of inhibition it becomes
difficult to distinguish between non-specific
inhibition of binding of the labeled hormone and weak but specific inhibitions that
might be produced by an immunoreactive
protein that is related to the hormone
which was used to develop the RIA. However, even with such flat slopes, useful, albeit imprecise, RIA data could be obtained
if the cross-reacting material could be
shown to be the hormone of interest.
As with the results obtained with the
Tilapia serum in the RIA for human PRL
(Fig. 2), uncertainties exist about what is
being measured by these heterologous applications of the RIA's for ovine PRL and
bovine GH in the plasma of the toad, salmon, and the pigeon.
The kinds of specificity problems that
can be encountered when homologous or
mixed heterologous RIA's are applied to
foreign species are well illustrated by the
observations of several groups of inves-
tigators. Hayashida et al. (1973) reported
that electrophoretically purified GH's from
several amphibian species gave good
cross-reactions in an RIA for rat GH. However, these investigators also found that
similarly purified PRL's from several of
these amphibians also showed a high degree of cross-reactivity in the RIA for rat
GH. Accordingly, although this RIA for rat
GH would probably measure immunoreactive GH in the plasma of these amphibian
species, it might also detect their circulating
PRL.
Frantz et al. (1972ft) found that a particular antiserum to highly purified ovine PRL
could neutralize the mammotropic activity
of human growth hormone (HGH) in an
organ culture assay. If this particular antiserum to ovine PRL were used in an RIA
to measure PRL in blood of humans, it
would presumably give erroneous results
because of its cross-reactivity with HGH.
The same problem would probably exist if
this RIA were applied to other primate
species and to non-primates.
Another problem that arises with the
heterologous application of RIA's for
pituitary hormones is the possibility that
cross-reacting material which is not of
pituitary origin may be present in the
plasma of a foreign species. Some results
with an RIA for ovine LH provide a clear
illustration of this difficulty. An homologous RIA for ovine LH was shown to detect
cross-reacting material in the serum of
rhesus monkeys (Niswender et al., 1971).
The cross-reacting material appeared to be
rhesus LH since the plasma levels that were
measured in adult monkeys in different
physiological conditions showed the expected changes. When serum from infant
monkeys was tested in this RIA for ovine
LH, surprisingly high levels of crossreacting material were detected (Peckham
and Foster, 1975). Of even greater concern,
however, was the finding that serum from
some hypophysectomized monkeys also had
high levels of this cross-reacting material
(Peckham, personal communication).
Hence, the nature and even the pituitary
origin of this "LH" in monkey serum that
cross-reacts in the RIA for ovine LH is uncertain. It has been shown subsequently
APPRAISAL OF RADIOASSAYS
with an RIA for LH that involves iodinated
rhesus LH and antiserum to human
chorionic gonadotropin, that serum from
infant or hypophysectomized monkeys
shows little or no detectable cross-reacting
material (Peckham and Foster, 1975). Accordingly, these two RIA's for LH show
grossly divergent results even in the same
species.
Licht et al. (1974) have found that RIA's
for mammalian and chicken LH can detect
cross-reacting material in fractions of reptilian pituitaries but that some of these fractions have little or no LH bioactivity.
Hence, these heterologously applied RIA's
for LH can apparently detect the LH of
some reptilian species but they also detect
materials that cannot be considered to be
LH because they lack appropriate biological properties. These cross-reacting fractions that are devoid of LH bioactivity could
represent inactivated LH or precursor
forms of the hormone. On the other hand,
they may be something else, perhaps other
pituitary glycoproteins.
It should be clear from the foregoing that
when homologous or mixed heterologous
RIA's for pituitary hormones are applied to
a foreign species and cross-reactions are
obtained, one cannot be certain about the
nature of the cross-reacting material even
with species that are fairly closely related to
the ones which gave origin to the hormones
used to develop the RIA's. One cannot even
be certain that the cross-reacting material is
of pituitary origin unless it is demonstrated
to be present in pituitary extracts and absent from other tissues and from the serum
of hypophysectomized animals. The
placenta of one species may produce hormones that cross-react in an RIA for a
pituitary hormone of another species and
some tumors of non-pituitary origin can
produce hormones or hormone-like materials (Liddle et al., 1969; Roof and Gordan,
1975).
After it has been determined that the
cross-reacting material is of pituitary origin, uncertainties still remain about which
pituitary principle it represents. It has been
well established that the GH's and PRL's,
including placental lactogens (Li, 1972;
Nicoll, 1974), the pituitary and placental
887
glycoproteins (Sairam and Papkoff, 1973;
Pierce, 1974), and the MSH's, ACTH's, and
lipotropins (Ramachandran, 1973;
Hoffman, 1974), represent three families
of structurally related peptides. The members of each family probably have common
origins in ancestral peptides or proteins
(see Geschwind, 1969). These structural
similarities are accompanied by varying degrees of overlapping biological properties.
Thus, the PRL's and GH's of numerous
vertebrate species have varying degrees of
common biological activities (see Nicoll,
1974). Similar overlap exists among the
pituitary and placental glycoproteins (see
Fontaine, 1969; Licht, \973a,b; Licht and
Papkoff, 1973; Pierce, 1974) and the MSH
ACTH-lipotropin family (see Ramachandran, 1973; Hoffman, 1974). Hence, it is
not surprising that these pituitary peptides
also show immunochemical relatedness.
Therein lies the major uncertainty of
heterologous application of RIA's.
When an RIA for a particular pituitary
hormone is developed, considerable effort
is usually devoted to establishing that the
assay is specific for the hormone in that
species. When such an RIA is applied to a
foreign species and cross-reactions are obtained, the nature of the cross-reacting
material should be established and the specificity of the RIA should be determined
with equal certainty. Otherwise, the nature
of the material that is being detected is open
to question.
DEVELOPMENT OF HOMOLOGOUS RADIOIMMUNOASSAYS FOR PROLACTINS AND GROWTH
HORMONES OF NON-MAMMALIAN SPECIES
The recent identification of PRLandGH
as molecular entities that are electrophoretically separable from the adenohypophyses
of various tetrapod species (Nicoll and
Nichols, 1971; Nicoll and Licht, 1971) has
provided a simple means for developing
RIA's for these hormones of nonmammalian forms without requiring large
quantities of pituitary glands from which
the hormones must be extracted and purified. Clemons and Nicoll (1974) have developed satisfactory homologous RIA's for
the PRL and GH of the American bullfrog
(Rana catesbeiana) using pituitaries from
888
CHARLES S. NICOLL
about 100 animals. The hormones were
first separated from adenohypophysial
homogenates or from medium in which the
glands had been cultured by polyacrylamide gel electrophoresis using the
standard tris-glycine system of Ornstein
(1964) and Davis (1964). The gel regions
containing the PRL and the GH were then
injected into rabbits to produce antibodies.
Both hormones can be further purified to a
degree satisfactory for labeling with
radioactive iodine and for use as standards
by re-electrophoresis on polyacrylamide
gel in different buffer systems. With these
hormones, homologous RIA's for bullfrog
PRL and GH were developed. Minimal
cross-reactivity of the PRL was observed in
the RIA for GH and vice versa. These RIA's
for bullfrog PRL and GH are currently
being evaluated. Similar procedures are
being used to develop RIA's for the PRL
and GH of pigeons and teleosts (Buntin,
Asawaroengchai and Nicoll unpublished;
Russell and Nicoll, unpublished).
DO THE RIA's FOR MAMMALIAN PROLACTINS AND
GROWTH HORMONES GIVE MEASUREMENTS THAT
ARE PHYSIOLOGICALLY MEANINGFUL?
Although RIA's for the PRL's and GH's
of several mammalian species are now
widely applied in laboratories throughout
the world it is surprising that very little
work has been devoted to determining
whether they give measurements that are
of physiological significance. Data are now
accumulating which raise doubts about
the physiological validity of measurements
obtained by these RIA's. We became concerned about RIA's for rat PRL when we
observed that in the lactating rat, pituitary
prolactin levels, as measured by the pigeon
crop-sac bioassay (Nicoll, 1967), did not
correspond with amounts of the hormone
that were measured by the RIA (Nicoll et
al., 1973). Relationships between these two
assays were further analyzed using in vitro
incubation of rat adenohypophyses
(Asawaroengchai et al. 1974a). The in vitro
procedure allowed changes in PRL levels in
the pituitary tissue and in the incubation
medium to be followed concurrently by
both assays.
Six experiments were conducted using
pituitaries from different kinds of donor
rats and correlation and regression
analyses were performed on the estimates
of PRL levels in the pituitary tissue and in
the incubation medium. No consistent relationship was obtained between the bioassay
and the RIA estimates of PRL levels in the
pituitary tissue. However, a highly significant correlation was obtained between
the two assays from estimates of PRL levels
in the incubation medium. When purified
preparations of rat PRL were tested in both
assays, a highly significant correlation was
also obtained (r = 0.99) with a slope of 1.00.
Thus, RIA gives estimates which are in perfect agreement with the bioassay when purified (extracted) preparations of rat PRL
are tested.
These results suggest that secreted and
purified rat PRL resemble each other more
closely than they do the PRL in homogenates of rat pituitary tissue. However, the
difference in the regression coefficients between purified and secreted prolactin (1.00
and 4.9, respectively) clearly indicates that
these forms of the hormone differ in their
relative detectability in the two assays. With
the secreted PRL, the RIA detects, on the
average, only about 20% of the hormone
that is measured by the bioassay. If this
relationship between the two assays for secreted PRL were a constant one, there
would be no cause for concern about the
physiological significance of measurements
made by the RIA. However, in the individual medium samples used for these
analyses the bioassay:RIA activity ratios
ranged from less than 1 to about 20.
Further analysis disclosed that PRL exists
within and can be secreted by rat
adenohypophyses in electrophoretically
separable forms with widely different
bioassay:RIA activity ratios (Asawaroengchai et al. 19746). Moreover, in terms of
RIA detectability, the secreted PRL was
found to be much more susceptible to inactivation by liver, kidney, and mammary tissue than was the purified hormone
(Asawaroengchai and Nicoll, 1975). This
immunological inactivation can occur without loss of bioactivity. In fact, in some samples, immunoinactivation was accompanied
APPRAISAL OF RADIOASSAYS
by enhanced biological activity of the secreted PRL. Thus, the differences in the
relationships between the two assays
among purified, secreted and intraglandular forms of rat PRL are probably a result of
variable amounts of the different forms in
the pituitary homogenates and incubation
medium and of the high susceptibility of
the secreted hormone to immunological inactivation.
Our conclusions from analysis of relationships between bioassay and RIA of rat
PRL would seem to be at variance with the
findings of Niswender et al. (1969), Neill
and Reichert (1971), and Gala and Kuo
(1972) who reported that good relationships exist between the two assays. However, inspection of their data discloses that
in fact their results and ours are in remarkably good agreement. Our results, and our
analysis of the data of these other investigators are summarized in Table 2.
Correlation and regression analysis of
the combined data of Niswender et al.
(1969) and of Neill and Reichert (1971) disclosed an excellent 1:1 correspondence between the RIA and the bioassay of purified
preparations of rat prolactin. With such
preparations, our results are in perfect
agreement with theirs. With pituitary tissue
homogenates, similar regression coefficients were obtained by analysis of the data
of these investigators and from our experiments. However, a significant correlation
coefficient was obtained from their data on
pituitary tissue homogenates, whereas with
our data, no positive correlation was ob-
889
tained. This difference may result from the
fact that the pituitary glands used in our
studies were in a dynamic state of secretory
activity, whereas the glands used by Niswender et al. (1969) and by Neill and
Reichert (1971) were in steady state conditions of PRL secretion. Our data indicate
that the relationship between the two assays
for estimating pituitary PRL levels breaks
down completely in dynamic states of hormone secretion in vivo (Nicoll et al., 1972)
or in vitro (Asawaroengchai et al., 1974a).
With incubation medium containing secreted PRL, we obtained a significant correlation between the two assays, in agreement
with the data published by Gala and Kuo
(1972). The regression coefficient of 4.9
derived from our data on secreted PRL is in
good agreement with the value of 3.1 obtained from our analysis of the data of Gala
and Kuo (1972). Our data indicate that in
short term incubations the RIA detects,
overall, only about 20% of the prolactin
that is detected by bioassay. The data of
Gala and Kuo (1972) indicate that in
medium from organ culture of rat
pituitaries, the RIA detects, on the average,
only about 30% of the biologically active
prolactin.
These relationships between the two assays for PRL secreted in vitro obviously
raise concern about the physiological validity of the RIA for rat PRL when it is used to
measure levels of the hormone in serum or
plasma. However, the significance of these
relationships to RIA estimates of circulating PRL levels can be questioned. The reliability, precision and specificity of the pigeon crop-sac bioassay can be criticized.
TABLE 2. Relationship between bioassay and radioim- Furthermore, one can ask whether estimunoassay of different forms of rat prolactin.
mates of rat PRL in incubation medium,
obtained by a bioassay in pigeons, have any
Prolactin
Regression
Correlation
preparation
coefficient (r) coefficient (b) bearing on the physiological significance of
RIA estimates of rat PRL in the serum or
1.08
0.92
Purified"
plasma of the rat. Unfortunately, the availPurified"
0.99
1.00
able data on bioassay estimates of PRL
0.5
Pituitary homogenate"
0.95
0.6 (ns)d levels in rat blood do not clarify this probPituitary homogenate6
0.15 (ns)d
3.1
Incubation mediuml:
0.92
lem.
0.80
Incubation medium*
4.9
Attempts have been made to extract PRL
0
Data from Niswender et al. (1969) and from Neill from serum or plasma of rats and to meaand Reichert (1972).
6
sure the hormone by the local pigeon cropData from Asawaroengchai (1974).
c
sac bioassay (see Ben-David et al., 1970;
Data from Gala and Kuo (1972).
Ben-David and Chrambach, 1973; Forsyth
" ns = not significant.
890
CHARLES S. NICOLL
and Parke, 1973; Bates, 1974). In all of
these studies only one dose of extract was
administered. Therefore, the reactions obtained were not shown to be quantitatively
similar to the responses that are produced
by purified PRL standards. Furthermore,
inflammatory reactions are usually elicited
in the crop-sac by such plasma or serum
fractions (see Kurcz et al., 1969). Accordingly, the reliability of the estimates of
blood PRL levels obtained by these procedures is questionable.
To my knowledge the only bioassay estimate of blood PRL levels in the rat which is
possibly reliable comes from the studies of
Wolthuis (1963). He tested unprocessed rat
serum in an assay which measures the
luteotropic activity of PRL. The published
results of Wolthuis' bioassay (1963) indicate
that estrogen-treated rats have serum PRL
levels of about 1.75 /xg/ml (in equivalents of
the RP-1 rat PRL that is distributed by
N.I.H.). In similarly treated rats, Chen and
Meites (1970) obtained serum PRL levels of
only 0.25 /u.g equivalents of the RP-1 standard per ml by RIA. However, the bioassays conducted by Wolthuis (1963) involved
injection of only one dose of the plasma.
Accordingly, the validity of his estimate of
serum PRL is uncertain. It is evident, therefore, that at this juncture we are obliged to
conclude that the physiological validity of
the RIA for estimating circulating levels of
rat PRL remains to be demonstrated.
With regard to RIA's for the PRL of
other mammalian species, relationships between bioassay and RIA estimates on samples of physiological interest (i.e., plasma,
incubation medium, or pituitary tissue)
have been reported only for the bovine
(Raud and Odell, 1971), ovine (Forsyth,
1972), and human (Frantz et al., 1972a)
systems. Raud and Odell (1971) found poor
correspondence between bioassay and RIA
estimates of PRL in bovine pituitary extracts. They ascribed this lack of correspondence to problems with the systemic
pigeon crop-sac bioassay, and not with the
RIA.
The RIA for human prolactin has been
evaluated extensively by Frantz et al.
(1972a) for correspondence with bioassay
(mouse mammary gland organ culture as-
say) estimates of serum levels of the hormone. Their data indicate that, overall, a
good relationship exists between the two
assays in serum samples with PRL levels
ranging from 15 to about 500 ng/ml. However, it is evident from their data that in
samples with low normal levels of the hormone (i.e., less than 50 to 60 ng/ml) the
relationship between the two assays is poor.
Frantz et al. (1972a) did point out that the
RIA tends to underestimate the amount of
biologically active PRL present when the
hormone levels are low, whereas in samples
with high PRL levels, the RIA gives estimates that are higher than those obtained
with the bioassay. The problem of relating
RIA estimates of human PRL in serum
samples to the amount of biologically active
hormone in these samples can be more
clearly visualized by appropriate analysis of
the data of Frantz et al. (1972a). Thebioassay:RIA activity ratios of their published
data were calculated and are presented as a
frequency distribution (Fig. 3). If good correspondence existed between the two assays, the ratios should be more or less normally distributed about a value of 1 with
little spread. In fact, considering the variability of the bioassay, a range of ratios of 0.5
to 1.49 could be regarded as an acceptable
distribution consistent with close correspondence between the two assays. It is obvious from Figure 3 that the distribution of
30-
0.00 050 100 150 2.00 250 300 350 4.00 450
-0.49-099-1.49 499 -2.49 -2.99 -3.49-3.99 -4.49 -4.99
750 800
-799-a49
Bioassay RIA activity ratios in serum samples
FIG. 3. Distribution of bioassay: RIA activity ratios of
prolactin in 64 samples of human serum. Data from
tables in Frantz et al. (1972a).
APPRAISAL OF RADIOASSAYS
bioassay:RIA activity ratios of human
serum samples is markedly skewed toward
the side of high ratios. Only 58% of the 64
samples tested gave ratios between 0.5 and
1.49 and almost 30% of the ratios are greater than 2.0. Thus, a substantial number of
samples of human serum have a relatively
high level of biologically active PRL that is
not detected by the RIA.
Forsyth (1972) has made comparisons
between RIA and bioassay levels of PRL
activity in goat serum. She used an RIA for
ovine PRL and a bioassay that involves
organ culture of rabbit mammary tissue.
Although correspondence was obtained between the two assays in terms of their estimates of relative amounts of PRL in a majority of the serum samples tested, Forsyth
(1972) does emphasize that in some samples the bioassay estimates were much
higher than those obtained by the RIA.
The published data of Frantz et al.
(1972a) and of Forsyth (1972) relating RIA
and bioassay estimates of PRL levels in
human and goat serum, respectively, indicate that overall, a correspondence between
the two assays exists. However, because of
large variations in the bioassay:RIA ratios
among individual samples, one can have
little confidence that the RIA estimate gives
an accurate reflection of the amount of
biologically active hormone in individual
serum samples. With the RIA for human
PRL, this degree of confidence would be
about 60% (Fig. 3). It can be argued that the
bioassays used by Frantz et al. (1972a) and
by Forsyth (1972) are semiquantitative, subjective and subject to interfering factors.
Accordingly, we should not expect the correspondence between the RIA's and the
bioassays to be very good. This criticism has
validity. However, if the discrepancies that
exist between RIA's and bioassays in
human and in goat plasma are attributable
to inadequacies in the bioassays, we are
then obliged to arrive at the same conclusion which we reached from our consideration of the RIA for rat PRL. That is, validation of the RIA's for human and ovine PRL
for measurement of levels of physiologically active hormone in serum is far from
satisfactory.
Although RIA's for the GH of several
891
mammalian species are currently in use in
many laboratories, the physiological significance of these assays is even more uncertain than is the validity of the RIA's for
mammalian PRL. The RIA for rat GH is
particularly problematic in this regard.
Several groups of investigators have published data which supposedly established
that RIA's for rat GH give estimates of the
hormone that are physiologically meaningful. This evidence includes measuring the
GH levels in the adenohypophyses of rats
of different age and showing that levels of
the hormone change with certain experimental manipulations, such as induced
hypothyroidism (Schalch and Reichlin,
1966; Garcia and Geschwind, 1968;
Daughaday et al., 1968). It has also been
reported by several groups that RIA estimates of the levels of GH in the rat
adenohypophysis correlate well with estimates obtained by the rat tibia test
(Daughaday et al., 1968; Dickerman et al.,
1969; Muller et al., 1970). However, the
consistency of the relationship between bioassay and RIA of GH in the pituitary of
undisturbed rats completely disintegrates
when assays are performed on adenohypophyses of rats that are subjected to
various acute stimuli.
Numerous papers from several laboratories have reported that stresses and injection of hypothalamic extracts can induce
depletion of bioassayable GH from the rat
adenohypophysis (for reviews, see Garcia
and Geschwind, 1968; Reichlin and
Schalch, 1969; Daughaday et al., 1970;
Reichlin, 1974). In some cases the reported
depletions were massive (e.g., Muller and
Pecile, 1966). In no case have such depletions been detected by the commonly used
RIA's for rat GH. In fact, in one study, the
same pituitary glands were analyzed by
both the bioassay and the RIA (see Reichlin
and Schalch, 1969) after injection of a
hypothalamic "GRF" preparation. The
bioassay recorded a 25% depletion of
pituitary GH while the RIA registered a
nonsignificant increase. Similar discrepancies have been obtained from in vitro
studies. Schally et al. (1972) reported that
presumptive GRF preparations were
highly effective in promoting release of
892
CHARLES S. NICOLL
bioassayable GH by rat adenohypophyses
in vitro. Analysis of samples of the same
medium by RIA disclosed little or no effect
of the "GRF" on the release of immunoreactive GH. However, it should be
noted that the bioassay results in the reports of Reichlin and Schalch (1969) and of
Schally et al. (1972) were obtained by injection of only one dose of their test preparations.
The possibility of obtaining quantitative estimates of the GH levels in
adenohypophysial tissue with the rat tibia
test has been seriously questioned by other
investigators (see Rodger et al., 1969;
Daughaday et al., 1970; Reichlin, 1974).
These critics point out that the bulk of the
published data which supposedly demonstrate depletion of bioassayable GH from
the rat adenohypophysis was obtained
through the use of invalid assay procedures
(e.g., a single dose of pituitary extract was
frequently given without injection of concurrent standards). They also emphasize
that in their hands and in the hands of
others, depletion of bioassayable GH from
the rat adenohypophysis by stresses and
other treatments could not be demonstrated consistently.
These criticisms regarding the reliability
of the bioassays for rat GH are valid in most
cases. However, significant depletion of
GH from the rat adenohypophysis has been
recorded independently by several groups
of investigators who used valid and rigorous bioassay methods (e.g., Miiller and
Pecile, 1966; Parkhie and Johnson, 1971;
Grindeland and Ellis, personal communication). Accordingly, depletion of bioassayable GH is not peculiar to the work of a
single investigator or of an isolated group
of investigators.
Daughaday et al. (1970) also emphasized
that they were unable to detect depletion of
pituitary GH in response to stresses when
the hormone was separated by polyacrylamide gel electrophoresis and measured
by densitometry. However, we have regularly observed depletion of rat pituitary GH
in response to insulin-induced hypoglycemia using this bioassay (Vodian and
Nicoll, 1970).
Discrepancies exist among the results ob-
tained by these three assay methods for GH
in other circumstances. In the lactating rat
the suckling stimulus is reported to cause
depletion of bioassayable GH from the
adenohypophysis (Grosvenor et al., 1968;
Sar and Meites, 1969). We have found that
the suckling stimulus does not deplete GH
even though pituitary PRL levels are depleted, when both hormones are measured
by disc electrophoresis and densitometry
(Vodian and Nicoll, 1970). On the other
hand, when the rat adenohypophysis was
extracted at neutral pH, Sinha et al. (1974)
did observe that the suckling stimulus
caused a depletion of radioimmunoassayable GH. Alkaline (pH 11) extracts of the
adenohypophysis showed higher levels of
pituitary GH than were obtained with neutral extraction but no depletion of hormone
was recorded with the pH 11 extracts.
These results of Sinha et al. (1974) clearly
indicate that the pH at which the pituitary is
extracted influences whether or not GH
depletion is recorded by an RIA. However,
these investigators found that the suckling
stimulus causes a significant decrease in
blood levels of immunoreactive GH while
inducing depletion of the hormone from
the adenohypophyses that were extracted
at neutral pH. Conversely, Chen et al.
(1974) reported that suckling can induce
substantial increases in immunoreactive rat
GH in blood.
The discrepancies between bioassay and
RIA estimates of pituitary GH levels in the
rat take on new dimensions of complexity
because of recent studies by Ellis and
Grindeland (1974; personal communication). These investigators have an antiserum for rat GH which, when used in an
RIA, does detect depletion of pituitary GH
occurring in response to stress. Thus, their
RIA gives results which are consistent with
apparently reliable bioassay data, in contrast with the findings of other investigators
who have used RI A's with different antisera
to rat GH (see Garcia and Geschwind, 1968;
Schalch and Reichlin, 1969; Daughaday et
al., 1970; Reichlin, 1974). This RIA of Ellis
and Grindeland (1974) does not measure
elevations in plasma immunoreactive GH
in the rats which show depletion of RIAdetectable hormone from the pituitary.
APPRAISAL OF RADIOASSAYS
Hence, with regard to changes in plasma
levels, their RIA is consistent with the other
RIA's for rat GH. Ellis and Grindeland
(1974) have also found that rat GH in a
cytosol fraction of rat adenohypophysis has
a higher bioassay to RIA activity ratio than
does the GH of a fraction rich in secretion
granules. This observation is consistent
with the suggestion by Nicoll (1972) that
hormones exist in the adenohypophysis in
forms that are differentially detectable by
different assay methods. The result of Ellis
and Grindeland (1974) indicate that these
different forms may be localized in different compartments.
Since the RIA's for rat GH are used almost exclusively to estimate circulating
levels of the hormone, discussion of
whether they give estimates of pituitary GH
levels that correspond to those obtained
with the bioassay might be considered to be
pointless. It is possible that the RIA's do
give estimates of GH levels in serum or
plasma that are physiologically meaningful
even though their validity for measuring
the hormone in pituitary extracts is doubtful. However, inspection of the scant data
that pertain to this question is not reassuring with regard to the physiological validity
of RIA estimates of GH levels in blood.
Few investigators have attempted to
measure GH levels in serum or plasma by
bioassay and only some of these have conducted assays that can be considered to be
valid. Nevertheless, these few bioassay data
on plasma GH levels in the rat are significant in connection with the question of
the physiojogical relevance of the RIA for
rat GH. Sawano et al. (1968) and Muller et
al. (1971) reported that injections of
hypothalamic fractions that cause depletion of bioassayable GH from the
adenohypophysis of rats will also cause elevation of plasma GH from undetectable
levels to concentrations equivalent to 3 or 4
/xg/ml of biologically active hormone. Unfortunately, the bioassays conducted by
these investigators involved injections of
only a single dose of the plasma samples.
More rigorous bioassays of plasma GH
levels (involving at least two doses of standard and unknown) have been conducted
by other investigators. These more valid
893
bioassays also register plasma GH levels in
normal rats in the microgram per ml range
(Contopoulos and Simpson, 1957; Dickerman et al., 1969; Ellis and Grindeland,
1974). Numerous laboratories have reported that RIA estimates of plasma (or
serum) GH levels in the rat rarely exceed
about 100 ng/ml. Much lower plasma levels
of the hormone are more commonly encountered. Hence, there is also a substantial
discrepancy (about 10- to 100-fold) between bioassay and RIA estimates of
plasma GH levels in the rat.
This dichotomy between the two assays
has been systematically analyzed by Ellis
and Grindeland (1974). These investigators have measured GH levels in the
same pools of plasma from rats by RIA and
by valid bioassays. The bioassay registered
plasma levels of GH that were 50 times
higher than those measured by the RIA.
Furthermore, in response to stress the
plasma levels of RIA-detectable GH were
not changed, but the bioassay recorded
substantial increases of the hormone. Ellis
and Grindeland (1974) have found even
greater discrepancies (up to 200-fold) between RIA and bioassay estimates of GH in
human plasma. In addition, they have succeeded in separating material from human
and rat plasma that has substantial GH
bioactivity but which has very low detectability in the RIA (Ellis and Grindeland
1974; personal communication).
Our findings from comparisons of bioassay and RIA estimates of rat GH are consistent with those of Ellis and Grindeland
(1974) in showing that the secreted hormone differs from preparations that are
extracted and purified from pituitary
glands (Vodian and Nicoll, 1974; 1975).
When medium containing secreted GH was
simply combined with rat serum, more than
90% of the RIA-detectable GH activity was
lost. Addition of serum to medium containing equivalent amounts of purified rat GH
(by RIA estimate) did not result in any loss
of its RIA detectability. Incubation of
medium containing in vitro secreted or
purified rat GH with explants of several
tissues (kidney, liver, fat, etc.) showed a
similar difference in RIA stability. The secreted hormone was much more readily in-
894
CHARLES S. NICOLL
PRL levels in serum (Frantz et al., 1972a;
Forsyth, 1972). Given the shortcomings of
the bioassays used by these investigators, it
is perhaps unrealistic to expect a very good
correspondence between the two assays in
all samples. Furthermore, the data on
blood levels of PRL that have been obtained
by RIA of the hormone in rats, humans,
sheep, and other species seem to make
good physiological "sense". Immunoreactive PRL is essentially undetectable in
serum from hypophysectomized animals
and in intact ones the levels of the RIAdetectable hormone rise and fall in the expected fashion under different physiological conditions and in response to various
manipulations.
The physiological validity of the RIA for
human GH can be defended on similar
grounds. Changes in blood levels of immunoreactive HGH in various physiological conditions conform to our expectations
in most cases. In addition, subjects showing
growth abnormalities usually have high or
low levels of immunoreactive GH consistent with their conditions. However, data
obtained with the RIA for rat GH are not
consistent with results obtained in primates
with regard to changes in blood levels of the
hormone occurring in response to various
treatments. Although various treatments
can induce marked increases in the blood
levels of immunoreactive GH in primates,
similar manipulations cause either no
change or a decrease in the levels of RIAdetectable GH in the rat (see Garcia and
Geschwind, 1968; Reichlin and Schalch,
1969; Daughaday et al., 1970; Reichlin,
1974). Such inconsistencies between or
among different species have not been observed
with any of the RIA's for mammaTo my knowledge, none of the RIA's for
GH of other mammalian species has been lian prolactins. As discussed above, the
evaluated for relationships with bioassays available information on relationships between bioassay and RIA estimates of rat GH
in samples of physiological interest.
Although these considerations on the re- indicate poor correspondence between the
lationships between bioassay estimates on two assays, except in extracts of pituitaries
the one hand, and RIA measurements of from rats that are undisturbed. AccordPRL and GH on the other, raise questions ingly, the physiological validity of the rat
about the physiological validity of the GH RIA is indefensible.
RIA's, several arguments can be advanced
Although the RIA's for mammalian PRL
in their favor. Results of RIA's for human and for primate GH give results that apand ovine PRL were found to correspond pear to be physiologically "valid," one could
to some extent with bioassay estimates of also ask to what extent our view of
activated by incubation with tissues than
was the purified hormone (Vodian and
Nicoll, 1974). Furthermore, the in vivo
immunological half life of secreted rat GH
is much shorter than is that of the purified
form of the hormone (tj = 2.7 min and 6.5
min, respectively) (Vodian and Nicoll,
1974, 1975).
Further analysis disclosed that rat pituitary tissue and medium in which
adenohypophyses had been incubated contained several forms of GH that were electrophoretically separable. The bioassay:RIA activity ratios of these different
forms ranged from 0.8 to 109 (Vodian and
Nicoll, 1975). Analysis of the recovery of
bioassay and RIA detectable GH from the
medium disclosed that with electrophoresis
on polyacrylamide gel, about 95% of the
RIA-detectable hormone that was initially
present in the medium was lost. In contrast,
35% of the initial bioassay-detectable hormone in the medium was recovered in the
several fractions. These results clearly indicate that in terms of RIA detectability, secreted rat GH is highly unstable, whereas
the purified hormone is very stable. Thus,
the discrepancies between bioassay and
RIA estimates of GH levels in pituitary tissue and in plasma can be ascribed to the
existence of forms of the hormone that
have differential detectability in the two assays and by the high susceptibility of secreted GH to immunological inactivation.
This latter phenomenon indicates that
after secretion in vivo the RIA detectability
of GH would be rapidly lost. Accordingly, it
is not surprising that bioassay estimates of
plasma GH levels in the rat are 10 to 100
times higher than RIA measurements.
APPRAISAL OF RADIOASSAYS
physiological validity has been biased by the
RIA data. For example, we did not expect
the condition of pseudopregnancy in the
rat to be maintained by nocturnal discharges of immunoreactive PRL (Freeman
and Neill, 1972). The pre-RIA dogma held
that pseudopregnancy was maintained by
tonic secretion of high levels of prolactin,
rather than by phasic discharges of the
hormone (see Everett, 1964, Rothchild,
1965). Furthermore, there are clinical data
which are not reassuring with regard to the
physiological validity of RIA's for human
PRL and GH in all circumstances. Some
human subjects have unusually high
plasma levels of immunoreactive PRL yet
do not show symptoms of gynecomastia or
galactorrhea. Other subjects who do have
these symptoms have blood levels of RIAdetectable PRL in the normal range (see
Frantzetal., 1972a; Malarkey, 1975). Some
humans have very high levels of immunoreactive HGH in their blood but are dwarfed or show no evidence of accelerated
growth (see Laron et al., 1972; Laron,
1974). Of even greater concern, however,
are the subjects who show an unusually
high growth rate in the face of low or normal blood levels of RIA-detectable HGH
(see Ryan, 1967; Zimmerman et al., 1967;
Holmes et al., 1968; Frantz et al., 1972a).
These anomalous conditions can be, for
the most part, rationalized within a
framework that leaves intact the app irent
physiological validity of the RIA's.
Gynecomastia and galactorrhea depend on
ovarian and adrenal steroids as well as PRL.
Hence, one should not expect a simple relationship between breast development and
function and serum PRL levels. The subjects with high levels of immunoreactive
GH who are dwarfed or who are devoid of
symptoms of elevated GH may be secreting
an abnormal (physiologically inactive) form
of HGH. Alternately, these subjects may be
unresponsive to normal HGH because of
receptor inadequacy. However, the individuals who show accelerated growth without elevated blood levels of HGH are less
readily rationalized within a framework of
RIA validity. Van den Brande and Du Caju
(1973) report that these subjects have normal quantities of somatomedin activity in
895
their blood. Somatomedins are the factors
that are thought to mediate GH action (see
Daughaday et al., 1972; Van Wyk et al.,
1974). Thus, these subjects may be unusually responsive to normal levels of
somatomedins. However, as far as I am
aware, these factors called somatomedins
have never been shown to cause body
growth in any animal. In fact, there appear
to be no published data showing that they
can mimic the effects of GH in the rat tibia
test of Greenspan et al. (1949). Nevertheless, somatomedins do have some interesting metabolic effects on tissues in vitro that
may be related to growth or GH actions
(Kostyo et al., 1973; Van Wyk et al., 1973;
Phillips et al., 1973).
Preliminary data on radioreceptor assays
(RRA's) for PRL and GH have appeared
(Shiu et al., 1973; Lesniak et al., 1973;
Parke and Forsyth, 1975). One would anticipate that these RRA's should provide
measurements of these hormones that are
physiologically more meaningful than are
the estimates obtained by RIA's since
hormone-receptor interaction is apparently related to activation of the physiological
response of hormones. However, the available data indicate that the RRA's give estimates that are more or less in agreement
with those obtained by RIA's. Friesen et al.
(1973) report that their RRA for PRL in
human serum samples gives estimates that
are closely correlated with RIA measurements. However, inspection of their data
indicates that the correspondence between
the two assays is poor in samples containing
concentrations of human PRL in the low
normal range (i.e., less than 50 to 60 ng/ml).
The RRA of Shiu et al. (1973) for PRL
and that of Lesniak et al. (1973) for HGH
have apparently not been evaluated by direct comparison with a bioassay in samples
of physiological interest. However, using
purified preparations of hormones, both
groups report that their RRA's do give estimates that correspond with bioassay estimates of the bioactivity of these preparations of PRL and GH. However, as shown
above in connection with the RIA for rat
PRL, the relationship between RIA and
bioassay estimates of purified preparations
of rat PRL differs from the relationships
896
CHARLES S. NICOLL
that are obtained with samples of
physiological interest.
The RRA of Lesniak et al. (1973) for
human GH apparently detects "small"
(probably monomeric) GH molecules in
human plasma but it shows little crossreactivity with the "big" (high molecular
weight) form of the hormone (Gordon et
al., 1973, 1974). The RIA for HGH detects
both forms equally well. Hence, this RRA
gives estimates of plasma levels of HGH
that are generally lower than those obtained
by RIA. Inasmuch as the available reliable
data from bioassay estimates of GH
levels in human plasma indicate a bioassay:
RIA activity ratio of about 200 (see Ellis and
Grindeland, 1974), one must again raise
the question of physiological significance
with regard to these RRA's and, again, the
obvious answer is that it remains to be demonstrated.
Although the RIA's for PRL and GH in
some mammalian species do appear to
make good physiological sense (the RIA
for rat GH is excluded from this generalization), such evidence does not prove that
the RIA (or an RRA) estimate of hormone
levels in a given plasma sample gives an accurate measure of the quantity of biologically active hormone present. In fact, in
view of recent findings, one should not
expect very good correspondence among
different assay methods. Substantial evidence now indicates that several human
protein and polypeptide hormones exist in
forms with different molecular weights in
the glands that secrete them and in the circulation (see Balaetal., 1973; Yallow, 1974;
Reiss and Canterbury, 1974; Suh and
Frantz, 1974; Rogal and Rosen, 1974; and
papers in Rabinowitz and Roth, 1974).
These forms of hormone differ in their
relative activities in bioassays, RIA's and
RRA's. It remains to be determined how
the forms of human PRL and GH with
different molecular weights relate to the
electrophoretically separable forms of rat
PRL and GH that we have studied (Asawaroengchai et al., 19746; Vodian and
Nicoll, 1975) or to the GH fraction
from human plasma with a very high
bioassay:RIA activity ratio that was isolated
by Ellis and Grindeland (1974). Nevertheless, the existence of different forms of
hormone, including physiologically inactive fragments (see Reiss and Canterbury,
1974; Segre et al., 1974), with differential
detectability in bioassays, RIA's, and RRA's,
simply emphasizes that none of these assays
measures all of the forms of hormones that
are present in the circulation. Hence, we
should not lose sight of the fact that RIA's
and RRA's measure only those forms of
hormone that they can detect and a growing body of evidence indicates that these
assays do not register all of the physiologically active hormone that is present in
blood.
Overall, the results discussed above on
evaluation of relationships between the
bioassay and RIA of PRL's and GH's in
general and of rat PRL and GH in particular have much more significance than simply raising questions about the physiological meaningfulness of RIA estimates of the
levels of these hormones in tissue, blood, or
incubation medium. As stated previously,
electrophoretically separable forms of both
hormones with different bioassay:RIA activity ratios are present in and can be secreted by the rat adenohypophysis
(Asawaroengchai et al., 19746; Vodian and
Nicoll, 1975). The results of Ellis and
Grindeland (1974) also indicate that GH
exists in plasma of rats and man in forms
that are separable by chromatography and
electrophoresis and these forms have differential detectability in the two assays. The
existence of these different forms of the
two hormones may provide an explanation
for the discrepancies between BA and RIA
estimates of changes in pituitary PRL and
GH levels that occur in response to certain
stimuli (see above) and the dichotomies that
have been observed in incubation medium
and in plasma.
It seems likely that the adenohypophysis
can contain variable amounts of these different forms of the hormones and that they
can be secreted differentially. Thus, discharge of a quantity of the forms of either
prolactin or GH with high bioassay:RIA activity ratios would be registered by the
bioassay as depletion of hormone from the
APPRAISAL OF RADIOASSAYS
adenohypophysis. A concomitant increase
in the bioassay-detectable hormone would
presumably be found in blood or incubation medium. The RIA would not detect
either the depletion or the release of the
forms of these hormones with high bioassay:RIA activity ratios since our data indicate that such forms arise mainly from loss
of RIA detectability (Asawaroengchai and
Nicoll, 1975; Vodian and Nicoll, 1975;
Farmer et al., 1975; Russell and Nicoll, unpublished).
There is no doubt that the rat
adenohypophysis can secrete significant
amounts of RI A-detectable PRL and GH in
response to appropriate stimuli. Our data
on the metabolism of secreted rat GH and
PRL in vivo and in vitro give clues to the
probable fate of the endogenously secreted
forms of these hormones that are detectable by the RIA. It seems likely that these
forms rapidly lose much of their RIA detectability in the circulation. This immunological inactivation is probably not accompanied by loss of bioactivity in view of the
high levels of bioassayable GH found in rat
plasma by Ellis and Grindeland (1974) and
our finding that while the clearance rate
of RI A-detectable exogenous rat GH from
the plasma of hypophysectomized rats is
relatively fast, the biological half-life of the
hormone is very long (Vodian and Nicoll,
unpublished).
The observed discrepancies between
bioassay and RIA of GH and PRL and the
existence of forms of these hormones with
different molecular weights and variable
detectability in the two assays, call into question one of the fundamental and long
standing assumptions of endocrinology.
That assumption holds that hormones that
are extracted and purified from the
adenohypophysis are essentially identical
with those contained within the gland and
that the intraglandular and purified forms
of the hormones are similar to the forms
that are secreted by it. Thus the circulating
forms of pituitary hormones are regarded
as being more or less the same as the forms
that are purified after extraction. The data
discussed above indicate that this assumption probably is invalid for PRL and GH.
897
Recent work clearly indicates that this assumption is also invalid for another
peptide-secreting gland, namely the
parathyroid (see Segre et al., 1974; Reiss
and Canterbury, 1974). Although the
parathyroid gland apparently secretes predominantly a single form of its hormonal
product, the secreted parathormone
(PTH) is rapidly metabolized in the circulation to yield fragments. The C-terminal
fragment possesses the biological activity
and it is undetectable by most RIA's for
PTH.
The existing RIA's and RRA's for mammalian PRL and GH have been developed
using purified homogeneous preparations
of these hormones that were extracted
from pituitary glands. Application of radio
assays to the measurement of these hormones in blood also operated on the assumption that the circulating forms of PRL
and GH are more or less the same as the
purified forms of the hormones. Thus, the
RIA's and RRA's should allow blood levels
of these hormones to be measured in
physiologically meaningful terms. Since the
available evidence indicates that the secreted and circulating forms of PRL and GH
(and other peptide hormones) are probably
different from the forms that are extracted
and purified from glandular tissue, it is not
surprising that correspondence between
radioassay and bioassay estimates of PRL
and GH in samples of physiological interest
is less than perfect. In fact, the conversion
of PRL and GH in the circulation to humors
that differ from the purified forms of these
hormones, in terms of detectability by
bioassay and RIA, suggests that the purified preparations of PRL and GH may actually be prohormones. One could speculate that conversion of the prohormone
forms to the fully active principles may
occur at the receptor sites that form the
basis for the RRA's. It is noteworthy in this
connection that liver tissue provides one of
the best sources for PRL receptors (Parke,
1973; Posner et al., 1974). Thus, the liver
may be a major site of conversion of prohormone PRL to the active form. If this
speculation is correct, then the RIA's and
RRA's for PRL and GH (and possibly other
898
CHARLES S. NICOLL
peptide hormones) may actually be assays physiologically active form of the correfor the prohormone forms of these princi- sponding hormone in a foreign species
ples.
than it does with the circulating form in
the homologous species.
As discussed previously, evidence indiCONCLUSIONS
cates that the circulating physiologically acThe physiological validity of RIA and tive forms of some peptide hormones (inRRA systems for PRL and GH of mamma- cluding PRL and GH) arise from converlian species is poorly verified in most cases sion of the secreted forms of these horand highly doubtful in some. The uncer- mones to entities that are poorly detectable
tain physiological significance of these by the homologous RIA's. It is possible,
radioassays might have its basis in the fact therefore, that conversion in vivo of the
that they were developed using prepara- secreted form of the hormone in a foreign
tions of these hormones that may actually species to its physiologically active form
be prohormones. Radioassays for PRL and may be accompanied by no loss of or even
GH that are physiologically meaningful enhanced cross-reactivity to the antibody
could undoubtedly be developed if an- that was produced against the purified
tibodies to purified preparations of the hormone of the first species. This possibilhormones or receptor preparations were ity may represent the most significant pofound that reacted with the biologically ac- tential value of heterologous application of
tive forms of the hormones that are in the the RIA's that are available for mammalian
circulation. Alternatively, physiologically PRL's and GH's.
valid radioassays could be developed if the
active forms of the hormones could be isolated from the circulation. In either case,
ADDENDUM
extensive evaluation would be required to
establish the physiological validity of such
Since the manuscript of this chapter was
radioassays.
first submitted we have tested several
In the few cases where comparisons be- mixed heterologous RIA's for mammalian
tween bioassay and RIA estimates of PRL PRL's for possible utility in measuring the
and GH levels have been made on the se- hormone in blood of teleosts (Schreibman,
creted (in vitro) or circulating forms of Buntin, Russell, and Nicoll, unpublished)
unpubthese hormones, the bioassays detected and pigeons (Buntin and Nicoll,
much higher levels of hormone than did lished). A combination of 125 I-labeled
the RIA in many samples. This discrepancy human PRL and an antiserum to ovine PRL
suggests that the RIA detects only a frac- was particularly useful since serum or
tion of the biologically active hormone that plasma from three teleost species (Tilapia
is present in these samples. In this connec- mossambica, Platichthys stellatus, and Giltion it is worthwhile to recall our experi- lichthys mirabilis) and from pigeons showed
ence with the RIA for human PRL that was excellent cross-reactivity. However, serum
applied to the measurement of cross-react- or plasma from hypophysectomized indiing material in the serum of Tilapia. We viduals of the three teleostean species and
concluded that the serum levels of the of hypophysectomized pigeons showed
Tilapia "PRL" were 10 to 100 times higher cross-reactivities that were as good as those
than would be expected. In retrospect, obtained with samples from intact animals.
however, perhaps this RIA for human PRL One plasma sample from an hypophysecgave a more accurate measure of the blood tomized pigeon that was found to have no
levels of physiologically active PRL in Tilapia detectable prolactin bioactivity in an assay
than such RIA's give for human PRL when which uses rat mammary explants in organ
they are applied to human blood samples. culture, showed very high cross-reactivity
It is conceivable that an antiserum to puri- in the mixed heterologous RIA. These refied PRL or GH of one species may show sults reemphasize that when such crossbetter cross-reactivity with the circulating reactivity is found in the blood of a foreign
APPRAISAL OF RADIOASSAYS
species one cannot conclude that the hormone of interest is being measured.
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