unbound hormone (Mendel 1989, 1992). This
means that all hormones with a significant degree of
protein binding will have a bioactive fraction much
less than 1. For these hormones, the measurement
of the total hormone concentration is not a reliable
estimate of the concentration of the bioactive
hormone; the concentration of unbound hormone
must be measured.
Unbound hormone concentration is measured by
separation of the protein-bound from the unbound
hormone and determination of the hormone concen-
tration in the sample portion containing the unbound
form. Methods used to separate bound from
unbound hormone include equilibrium dialysis,
ultrafiltration, kinetic radioimmunoassay, and
chromatography. Equilibrium dialysis remains the
reference technique for the measurement of unbound
analyte concentrations for all protein bound analytes,
but the technique is too tedious and time consuming
for routine laboratory use. The separation tech-
niques of ultrafiltration and kinetic radioimmunoas-
say are practicable and therefore are preferred.
In vivo
plasma ultrafiltration can be taken advan-
tage of in the assay of cortisol. The renal glomeru-
lar filtration of unbound cortisol results in urine
cortisol levels that can be readily and precisely
measured. Empirically, the daily renal excretion of
unbound cortisol correlates well with the mean daily
circulating levels of unbound cortisol. The other
hormones cannot be analyzed this way because their
greater protein binding and lesser plasma concentra-
tions result in urine hormone concentrations too
small for accurate detection. Another
in vivo
separa-
tory process occurs in the formation of saliva. The
unbound form of the steroid hormones enter saliva
from the salivary gland capillaries but not the bound
forms. As a result, salivary concentrations of
hormone are very similar to circulating unbound
hormone concentrations (Vining and McGinley
1986).
The concentration of unbound hormone can be
estimated by calculation of indices proportional to its
concentration. One such index is the ratio of the
concentration of hormone to the concentration of
specific hormone-binding protein. This index is
derived from the general equilibrium mass action
equation. If there is minimal competition from other
hormones for the binding sites on the binding protein
and if the fraction of hormone bound to albumin is
small compared to that bound to the specific binding
protein, the equation simplifies to,
[bound hormone] =
k
bp
[
binding sites
] [
unbound hormone
]
1
+
k
bp
[
unbound hormone
]
where
k
bp
is the association constant of the hormone
for the specific binding protein. Substituting the
concentration of total hormone for the concentration
of bound hormone, based upon an assumption of
extensive protein binding of the hormone, and divid-
ing through by the concentration of specific binding
protein yields,
[
hormone
]
[
binding sites
] =
k
bp
[
unbound hormone
]
1
+
k
bp
[
unbound hormone
]
The ratio of the concentration of hormone to the
concentration of specific binding protein is,
therefore, proportional to the concentration of
unbound hormone. Note, however, that the relation-
ship is nonlinear. In addition, it is based upon a
number of assumptions which limit its reliability: it
is valid only for highly protein-bound hormones that
are largely bound to their specific binding protein.
A hormone:hormone-binding protein ratio in
clinical use is the testosterone:SHBG ratio (also
called the androgen index). Testosterone is almost
completely protein bound in the plasma but a protein
other than sex-hormone binding globulin contributes
significantly to its binding; approximately 50 percent
is bound to albumin (Dunn 1981). As a result, the
testosterone:SHBG ratio has only an approximate
relationship to the unbound testosterone concen-
tration.
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Organ Function
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