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CLIN.CHEM.38/7, 1289-1293 (1992)
The Free Hormone Hypothesis and Measurement of Free Hormones
Concentrations
of free hormones in serum are generally measured in the belief-enshrined
in the “free
hormone hypothesis”-that
only free hormones are
physiologically
active and that their concentrations
as
measured in vitro constitute reliable indicators of in
vivo hormonal effects. Concentrations
of binding proteins in serum change markedly in pregnancy and may
for other reasons, including genetic abnormality (e.g.,
1), be grossly disturbed. Physiological feedback mechanisms respond by modifying the concentrations of bound
and free hormone, the former changing in line with that
of the binding protein, the latter usually rempining
within normal limits [although modern methods suggest a decrease in the concentrations of free thyroid
hormones (THs) during the second and third trimesters
of pregnancy}.1 These homeostatic adjustments
are accompanied by maintenance
of apparently normal transport of the hormones to target tissues.
Subsequent to the development of simple immunoassay methods, routine measurement
of free hormones has
become increasingly
popular. Nevertheless,
doubt has
centered on the validity of some of these methods, and
they have been less widely adopted (particularly
in the
US) than might otherwise have been anticipated.
But
before considering this issue, let us exRmine the validity
of the free hormone hypothesis itself, this having also
emerged as a subject of controversy.
Observation of a broad correlation between endocrine
status and the free hormone concentrations
measured in
serum formed the basis of the free hormone hypothesis
(other evidence, such as the low permeation rates of
binding proteins across capillary membranes, has been
adduced in its support). A corollary of the hypothesis is
that the protein-bound concentration is physiologically
irrelevant,
a view supported
by substantial experimental evidence. For example, subjects lacking thyroxinebinding globulin (TBG) because of genetic abnormality
suffer no obvious physiological
disadvantage,
despite
possessing greatly decreased serum concentrations
of
TH.
Paradoxically,
the lack of correlation between hormonal effects and the concentration
of protein-bound
hormone has been interpreted in contradictory ways by
steroidologists
and thyroidologists.
According to the
‘latter,
generally following Robbins and Rail (2), the
dissociation rate constants of TH-binding proteins are
such that, as blood traverses target organs, the net rate
of bound hormone release greatly exceeds the net rate of
‘Nonstandard abbreviations: TI!, thyroid hormone; TBG, thyroxine-binding globulin; T4, thyroxine; and f1, free thyroxine.
hormone effiux from capillary blood to adjacent tissue;
also, the fractional depletion in bound hormone resulting from tissue uptake is assumed to be low. The
intracapillary
concentration of free hormone is thus
maintained
at its equilibrium value throughout capillary transit. If we assume that only free hormone
permeates capillary walls, it follows that (a) hormone
transport
to target cells is proportional to the free
hormone concentration and is uniform along the capillary length and (b) the capillary blood flow rate is
essentially irrelevant to hormone uptake.
Underlying this view is the assumption that dissociation of bound TH complexes is sufficiently rapid not to
exert significant rate-limiting
effects (causing depression of the intracapillary free TI! concentration) in the
face of hormone loss from the capillary (2). However,
although this assumption may arguably be valid for TH
bound to TBG, albumin, and thyroxine-binding
prealbumin-with
thyroxine (T4) dissociation half times of
-39, <1, and 7.4s, respectively
(2)-it is unlikely to
hold for hormone bound to slowly dissociating endogenous antihormone antibodies. Clinical chemists should
therefore note that, if the Robbins-Rall model is essentially valid [as claimed by, e.g., Mendel et al. (3,4)], the
free hormone hypothesis is likely to break down when
serum TH antibodies are present, implying that measured concentrations of free TH may not correlate with
thyroid status in these circumstances,
irrespective of
any artefactual effects that endogenous antibodies may
exert within the assay used.
In contrast, steroidologists have traditionally held
that specifically bound steroid hormones do not dissociate at all during capillary transit, only the free hormone
moiety per se being available for tissue uptake. Nevertheless, Tait and Burstein (5), the originators of this
view, postulated that-to account for the high hepatic
uptake of certain steroids-hormone
that is only loosely
bound to albumin must be considered free. Following
from this view, hormone delivery to target tissues is (a)
dependent on the blood flow rate and (b) nonuniform
along the length of tissue capillaries. These ideas also
imply that slowly dissociating antihormone antibodies
will neither influence hormone delivery to target organs
nor invalidate the free hormone hypothesis.
These contradictory concepts clearly stem from differing perceptions
of the rate limitations
on hormone
uptake under the “disequilibrium”
conditions existing
in vivo. Conflicting views on this issue underlie major
controversies regarding the free hormone hypothesis
during the past decade, with the experimental
studies
on hormone transport having been interpreted
differCLINICAL CHEMISTRY, Vol. 38, No. 7, 1992
1289
ently, depending on the model assumed to represent the
kinetics of hormone loss from intracapillary
blood. For
example, Pardridge et al. (6, 7), relying on observations
of labeled-hormone uptake from albumin-hormone
mixtures perfused through rat brain and liver, repeatedly
challenged the hypothesis,
initially postulating
that
albumin-hormone
complexes do not dissociate during
their passage through organs characterized
by short
capillary transit times (6). This led Pardridge (8) to
propose, among other things, a specific liver-targeting
role for binding proteins. After criticism of the mathematical basis for these ideas (9), Pardridge (10, 11)
reinterpreted
his experimental
data, using a revised
model based on the assumption that albumin-hormone
complexes dissociate “instantaneously”
(i.e., at infinitely high rates) in response to hormone loss from the
capillary. However, experimental data failed to conform
to this model unless binding proteins in serum were
postulated as possessing lower affinities in vivo than are
measured in vitro (7), leading Pardridge to posit that
“transient
conformational
changes about the ligand
binding site within the microcirculation”
enhanced hormone dissociation within certain organs. However, his
experimental
observations are more plausibly explained
by using equations that (albeit in a simplified way) take
into account the rate-limiting
effects of the dissociation
of protein-bound hormone (12, 13). Thus, although the
intracapilary
hormone-dissociation
mechanisms
hypothesized by Pardridge aroused much interest, they
stemmed from reliance on an oversimplified theoretical
model and for this and other reasons attracted considerable criticism (14-16).
Nevertheless, the observations of Pardridge and coworkers indicate that the rate-limiting
effects of protein-bound
hormone dissociation
may, in certain circumstances,
significantly
affect hormone uptake,
thereby invalidating
the free hormone hypothesis. A
more comprehensive analysis of the kinetics of hormone
loss from tissue capillaries, encompassing hormone dissociation and diffusion effects (which depend on anatomical differences in target organ microvasculature),
suggests that changes in the concentrations of binding
proteins may serve to redirect hormones to particular
organs in certain physiological circumstances,
e.g., in
pregnancy (9,16-18). This analysis concomitantly
predicts a decrease in the concentrations
of free T4 (fF) in
pregnancy, a prediction apparently confirmed by modera assay methods. Another recent suggestion by Tait
and Tait (19), based on their analysis of published data,
is that transport of certain steroid hormones to target
tissues depends on the concentration of albumin-bound
hormone per se.
Of what significance to clinical chemists are the
current debates among endocrinologists
on this topic?
Essentially, they highlight issues that should not be
overlooked in discussions of the validity and diagnostic
significance
of methods for assay of free hormone.
Briefly stated, the issues are as follows: (a) the free
hormone hypothesis is unproven and constitutes at best
an approximation,
(b) no comprehensive theoretical
1290 CLINICALCHEMISTRY,Vol.38, No. 7, 1992
model exists that adequately represents the kinetics of
the complex hormone-protein interactions and diffusion
processes involved in hormone migration from intracapillary blood to target cells, (c) major disagreement exists
among endocrinologists on the rate-limiting constraints
governing hormone transport to target tissues in vivo,
and (d) neither the physiological role (if any) of binding
proteins in serum, nor the significance of the changes in
their concentrations that occur in pregnancy, are at
present understood.
Regrettably,
the uncertainties
related to the free
hormone hypothesis have been largely ignored by manufacturers of assay kits for free hormones, who generally regard “normality” of estimates in clinically normal
subjects (in whom protein concentrations are disturbed)
as proof of a kit’s validity. This clearly involves circular
reasoning. Nonetheless,
clinical chemists may be persuaded by such arguments, being generally less interested in a kit’s analytical validity than in its ability to
reveal abnormalities
in endocrine status. In short, a
divergence in the objectives of clinical chemists and
endocrinologists
may emerge if present doubts regarding the free hormone hypothesis prove justified. Indeed,
the possibility of such divergence is implicit in the
observation that the if, concentration
is depressed in
pregnancy. If confirmed, this not only removes one of the
cornerstones
supporting the free hormone hypothesis
but also significantly diminishes the diagnostic utility
of measuring serum if4.
Indeed, the potentially differing needs of clinical
chemists and endocrinologists in this area may necessitate a specific nomenclature
to distinguish
the assay
kits or methods that (to the extent technically possible)
measure the actual concentrations
of free hormones
from those that merely are empirically
contrived
to
yield results indicative of endocrine
status in defined
categories of patients. The American Thyroid Association has implicitly moved towards this position by
distinguishing
between ‘if4 index” methods and genuine f’F4 assays (20), although the present basis for this
classification is somewhat illogical: some of the most
reliable if4 assay methods, such as the one developed by
Ross and Benraad (21), fall within the association’s
definition of an if4 index.
Whether or not further studies on hormone transport
ultimately reveal the necessity for a distinction of this
kind, it is imperative that the term “free hormone
assay” be more rigorously defined. Increasing confusion
has resulted from the use of kits that lack any legitimate physicochemical
claim to be so described, with
different kits (even from the same manufacturer)
often
yielding widely differing results in certain sera.
One factor that has contributed to this state of affairs
is that the majority of modern assays are “comparative”
(22); i.e., the standards used generally consist of human
serum to which hormone has been added, after which
the resulting concentrations of free hormone are determined by an “absolute” method. [An exception is the
dialysis/RIA
procedure-originally
developed in my
own laboratory (23)-marketed
as a kit by Nichols.]
Thus, irrespective of the inclusion of various equilibrium-disturbing additives (detergents, preservatives,
albumin “blockers,” proteins, diluents, buffer components,
etc.) in kit reagents, and irrespective of the validity of
the physicochemical
basis of the methodology, assay
results will inevitably
be broadly “correct” in normal
subjects. Furthermore,
by judicious tinkering with the
system, results for pregnant subjects can be contrived to
fall broadly within expected limits. Empirically
constructed kits of this kind may therefore give an appearance of genuinely measuring free hormone concentrations, with their fundamental
invalidity
being revealed
only in other circumstances
in which protein-binding
abnormalities
occur.
An interesting example of this phenomenon is found
in the recent papers by Ross and Benraad (24) and van
der Sluijs Veer et al. (25), the latter in the current issue
of this journal. Both report major errors in if4 estimates
for sera containing abnormal TBG values measured by
so-called two-step assays, as a result of conducting the
assays at room temperature.
The physicochemical
principles governing such assays are fundamentally
sound;
however, because the temperature
coefficients governing T4 binding to TBG and albumin differ, the relative
change in fT4 concentrations at 37#{176}C
and at room
temperature
will vary between standards and samples
that contain unusual relative amounts of TBG and
albumin, so that results for such samples will be severely biased. The fact that kit reagents contain additives capable of disturbing
the equilibrium between free
and bound T4 (as used in the Delfia kit) may result in
similar differential
effects on if4 concentrations
in standards and unusual samples,
even if assays are performed at 37#{176}C.
Temperature
effects of the kind reported by these authors (24,25) are thus exacerbated by
the use of such additives; similarly, these effects would
be further
increased were standards not made up in
“normal” hunin serum.
The problems these papers reveal thus stem not from
invalidity
of the physicochemical
basis of two-step assays per se, but from the violation of a cardinal rule
governing all valid immunoassays
of free hormones, i.e.,
that neither the reagents used nor the conditions under
which the assay is performed should significantly
disturb the pre-existing equilibrium in standards and serum samples [this rule underlies the use of very small
amounts of antibody in such assays (26)]. Any disturbance is likely to vary between samples, particularly
if
the concentrations
of binding protein or competitor in
the samples differ significantly.
However, a quite separate and potentially more serious problem arises in single-step unbound-analog
immunoassays,
whether of labeled analog or (more recently) of labeled-antibody
design. The physicochemical
basis of such assays is likewise valid, provided the
labeled or solid-phase-bound
analog is genuinely not
bound to serum proteins, i.e., provided it binds by --10%
or less at the serum dilution used in the assay, in the
absence of exogenous antibody (27). It is not sufficient
at the analog merely not compete with hormone
bound to binding proteins in serum (27). On the latter
grounds, analogs binding with affinities
10% of those
of the native hormone were claimed as suitable for use
in assays of this type (28), implying,
e.g., that T4
analogs that are 99.8% bound in undiluted serum conform to the somewhat idiosyncratic
definition of unbound (27,29). The physicochemical
concepts underlying this definition have never been formally substantiated. Indeed, it is readily demonstrable that aT4 analog
that binds with affinities -10% of those of native hormone (or even an analog that binds to serum proteins
with affinities 1% of those of T4) would yield assay
results correlating almost exactly with total serum T4
(30).
In practice, however, most labeled-analog
kits have
relied on analogs binding predominantly
to albumin
(16, 27, 29). Protein-bound analogs distributing
between-serum proteins in this manner permit assay systems to be constructed in which analog binding remains
approximately
constant in both normal and pregnancy
serum, in which circumstance the results in pregnancy
will be approximately
correct (27, 31). However, kits
functioning in this way yield spurious results in many
other circumstances
in which the equilibrium between
free and bound hormone moieties is abnormal, e.g.,
when endogenous or exogenous binding competitors are
present (32) or in analbuminemic
sera (33). Such kits’
vulnerability
to variations in serum albumin inevitably
attracted the earliest and greatest attention (e.g., 3437) and is now well recognized; however, an abnormal
concentration in serum of any analog-binding
proteinincluding endogenous antibody (38)-also
distorts results. The more conspicuous artefactua.l effects characterizing such kits have been extensively reported in the
literature and are the basic reason for the disrepute into
which labeled-analog
methods (in their current form)
have fallen (39-41).
Errors generated in kits of this type thus arise in part
as an inevitable and predictable consequence of the use
of analogs that do not conform to the physicochemical
theory underlying the “unbound analog” assay. Such
errors are in addition to, and compound, the errors
caused by the inclusion of various additives in assay
reagents of the kind described above-although
errors
arising from these two sources may tend to counterbalance in some circumstances.
For example, certain kits,
including the recently described labeled-antibody
if4
kit (42), incorporate large amounts of albumin in the
reagents, thereby attenuating
some of the effects of
analog binding to endogenous serum proteins, but at the
cost of increasing
other errors in commonly encountered
clinical situations.
The situation in this field is unfortunately little short
of chaotic. Manufacturers
have relied on different analogs; different reagent additives, buffers, or blockers;
and different operating temperatures. Such kits may
arguably be diagnostically useful in well-defined clinical situations, but many lack the valid physicochemical
basis that would entitle them to be regarded as genuine
assays of free hormones, and they yield diagnostically
CLINICALCHEMISTRY,Vol.38, No. 7, 1992 1291
misleading results in more complicated circumstances
(e.g., nonthyroidal illness). Consequently, the endocrine
and clinical chemistry literature
is now littered with
unreliable reports of studies on the concentrations
of
free hormones
in various
pathophysiological
states,
in
which the effects reported may reflect nothing more
than assay artefacts.
What can be done to rectify the present situation?
First, manufacturers
should refresh their understanding of basic physicochemical
laws and redesign assay
kits for free hormones in this light. Second, formal
bodies such as the American
Thyroid Association and
the U.S. Food and Drug Administration
should carefully
reconsider
the criteria that an assay of free hormones
must meet. Finally, and perhaps most important, professional clinical chemists must maintain the high degree of vigilance that many (notably in the United
States) have demonstrated in the past in regard to the
assertions of kit manufacturers.
Regrettably, the experience of the last 10 years teaches that names on packs
and claims in package inserts cannot always be trusted,
particularly in the field of free hormone assays.
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Roger Ekins
Department
of Molecular Endocrinology
University College and Middlesex
School of Medicine
University College London
London, UK WIN 8AA
CLINICAL CHEMISTRY, Vol. 38, No. 7, 1992
1293