Professional Documents
Culture Documents
We have designed a re-usable dialysis cell and a complex bound T4 before direct measurement of the free T4 by RIA
dialysis buffer, with which undiluted serum samples can be (22). Diluting the serum sample with simple laboratory
dialyzed with minimal changes in their serum matrix. Dialy- buffers, a procedure that has little effect on free-T4 concen-
sate thyroxin (free 14) is then measured by a sensitive RIA trations in serum from healthy euthyroid subjects, decreases
for T4. The range of reportability was 2-128 ng/L, the normal free-T4 concentrations in serum from some patients with
range was 8-27 ng/L, and the interassay CV was 7%. Free nonthyroidal illness (23). This suggests that methods in
T4 concentrations in various disorders were as follows: which free T4 is measured in undiluted sera will be more
hyperthyroidism, 32-478 ng/L; in both excess thyroxin-bind- relevant to clinical status than methods involving serum
ing globulin (TBG) and familial dysalbuminemic hyperthyrox- dilution (23).
We have been working with a sensitive RIA for measur-
inemia, 9-27 ng/L; primary hypothyroidism, <2-7 ng/L;
ing free T4 directly in dialysates of undiluted serum. For
central hypothyroidism, 4-6 ng/L; severe TBG deficiency, 9-
this, we have developed a convenient-to-use dialysis cell and
25 ng/L; hypothyroxinemias of nonthyroidal illness, 8-35
a dialysate buffer that provides a biochemical environment
ng/L. With this free-I4 assay, which is adaptable to clinical
similar to that in vivo. With this method, results for free T4
laboratory use, one can differentiate hyperthyroidism from
are high in hyperthyroidism but within the normal refer-
the major euthyroid hyperthyroxinemias and hypothyroidism
ence interval in cases of hyperthyroxinemia of severe TBG
from the major euthyroid hypothyroxinemias. excess and in familial dysalbuininemic hyperthyroxinemia.
Moreover, the free-T4 results are low in hypothyroidism,
Additional Keyphrases: thyroid disorders diagnostic aid normal in severe TBG deficiency, and normal or increased
in the hypothyroxinemias of nonthyroidal illness. This
Measurement of free thyroxin (T4) in serum in the clinical
method appears to be adaptable to routine clinical labora-
laboratory is still problematic.2 Free-T4 index values are
tory use, if made available as a kit procedure.
often abnormal and misleading in euthyroid individuals
with the more severe forms of thyroxin-binding globulin Methods and Materials
(TBG) excess (1, 2) or deficiency (3, 4). They are also
Dialysis Cell Design
misleading in euthyroid individuals whose blood has vari-
ant albumin molecules with increased affinity for T4, e.g., The new dialysis cell (Figure 1), designed for this assay,
familial dysalbuminemic hyperthyroxinemia (5, 6). Values has three major parts: a dialysate vial, a membrane cylin-
for the free-T4 index are high in the euthyroid hyperthyrox- der, and a cap. The dialysate vial is made of clear acrylic
inemias associated with transthyretin (prealbumin) excess plastic and the cap of polypropylene. The cap snaps onto the
(7) and with variant transthyretin molecules having in- top of the vial and contains an X-slit opening (not shown)
creased affinity for T4 (8). In addition, these values are through which a serum sample can be introduced with a
misleading in certain individuals who have variant TBG hand-held pipetter. The membrane cylinder consists of five
molecules with decreased affinity for T4 (9, 10) and in the parts: an inner cylinder of acrylic plastic with three grooves;
hypothyroxinemias associated with nonthyroidal illness an 0-ring of medical-grade silicone (RE.A.L. Seal Co., Inc.,
(11-13). In general, the labeled-analog RIA methods for free Escondido, CA 92025), which fits into a groove on the
T4 are unaffected by changes in TBG concentration (14, 15) bottom of the inner cylinder inside the dialysis membrane; a
but are unreliable for use in euthyroid patients who have piece of washed dialysis membrane; a second 0-ring, made
other above-mentioned conditions (6-8, 15-17). The analog of buna rubber (R.E.A.L. Seal Co., Inc.), which fits into
assays have now been thoroughly criticized, on both practi- another groove outside the dialysis membrane; and an outer
cal and theoretical grounds (15-20). In addition, both the sleeve of acetyl resin, which is slipped over the membrane
free-T4 index and the labeled-analog RIA methods are and both 0-rings and snaps into the upper groove on the
subject to interference from endogenous thyroid autoanti- inner cylinder. Leaks are prevented by compression of the
bodies, when they bind the labeled thyroid hormones used in two 0-rings against the membrane. The placement of the
these assay procedures (15,21). assembled membrane cylinder within the dialysate vial is
Theoretically, the most reliable methods for measuring maintained by a seal extending from the lower portion of the
free T4 in serum should be the equilibrium dialysis and membrane cylinder to the wall of the dialysate vial and by
ultrafiltration methods that separate free T4 from protein- spacers extending from the upper portion of the membrane
cylinder. Because of a slightly conical shape of the interior of
Department of Pathology, White Memorial Medical Center, 1720 the dialysate vial, the seal and spacers hold the dialysis
Brooklyn Ave., Los Angeles, CA 90033. membrane at a constant distance from the bottom of the
Present address (and address for correspondence): Section of vial. The seal prevents evaporation of dialysate buffer
Endocrinology, Department of Internal Medicine, Loma Linda during storage. With the snap-fit cap in place, the seal also
University Medical Center, Loma Linda, CA 92354.
prevents dialysate buffer from leaving the dialysate com-
2Nonsdard abbreviations: T4, thyroxin; TBG, thyroxin-bind-
ing globulin; uapzs, 4-(2-hydroxyethyl)-1-piperazineethanesulfon- partment if the dialysis cell is accidentally inverted during
ate; T3, triiodothyronine; TSH, thyrotropin. storage.
Received January 19, 1988; accepted May 6, 1988. The disc of dialysis membrane, which separates the
Results DiscussIon
This direct equilibrium dialysis/radioimmunoassay meth-
Normal Controls
od separates free T4 from protein-bound T4 in a chemical
Free T4 concentrations in sera from the 120 normal milieu approaching that of unmodified serum. There is
controls (Figure 3) ranged from 8 to 30 ng/b. The single minimal dilution of the serum sample. Minimizing dilution
highest value was considered an outlier and the range of may be important because, as has been shown, diluting the
observed values in the remaining 119 controls-8 to 27 serum (by mixing with laboratory reagents) can cause an
ng/b-was used as the normal reference interval. The mean anomalous reduction in free concentrations when T4-bind-
free T4 in these controls was 14.5 (SD 4.2) ng/L and the ing inhibitors, which are themselves protein-bound ligands,
median was 14.0 ng/b. are present in the serum sample (23). A similar anomalous
fall in free-T4 concentrations occurs with dilution of serum
Hyperthyroxinemic Patients
from some patients who have the hypothyroxinemia of
The total T4 and free-T4 concentrations in the patients nonthyroidal illness, a phenomenon not seen in normal
with hyperthyroxinemia are shown in Figure 4. Free-T4 individuals or in those with hypothyroxinemia caused by
concentrations were high in clinically evident hyperthyroid- primary hypothyroidism (23). Concentrations of the major
ism and ranged between 32 and 478 ng/L. By contrast, free- serum electrolytes were maintained within the physiologi-
T4 concentrations were in the normal reference interval in cal range, except for serum bicarbonate, which was neutral-
severe TBG excess (9 to 27 ngfL) and in dysalbuminemic ized by uxzs acid. Nonphysiological anion concentrations
hyperthyroxinemia (9 to 27 ng/L). reportedly have variable effects on T4 binding in different
sera (30). Variations in chloride concentrations are particu-
Hypothyroxinemic Patients
larly important in sera with variant albumin molecules (31).
The total T4 and free-T4 concentrations in the four types of This method is a direct method in the sense that serum
hypothyroxinemia studied are shown in Figure 5. Free-T4 is dialyzed directly without additions or modifications and
concentrations were below the normal reference interval in the dialysate is analyzed directly without preparation or
clinically evident hypothyroidism. They were below the purification. With this method, free-T4 concentrations were
limit of detection of the assay (2 rig/b) in 20 of the 43 normal in individuals whose hyperthyroxinemia was caused
patients with primary hypothyroidism, and ranged from 2 to by increased T4 binding to serum proteins. This is in
7 ng/L in the other 23. In central hypothyroidism (which agreement with previous studies performed with other
includes secondary and tertiary hypothyroidism) free-T4 equilibrium dialysis methods (5-8,32) and with theoretical
300 S
120
250 t -j
100
S
C
2
200 I- 80 .
Ui
S
Ui
0 #{149}
i- 150 S S IL 60 $
#{149}
=
#{149}5
100 40
Ii
50 20
A
0
HYPERTHYROID TBO FDH HYPERTHYROID TBO FDH
EXCESS EXCESS
Fig. 4. Total T4 concentrations (left) and free-T4 concentrations (tight) in 30 hyperthyroid patients and 27 individuals with euthyroid hyperthyroxinE
mias [TBG excess in 13 and familial dysalbuminemic hyperthyroxinemia (FDH) in 141
predictions of free-T4 concentrations (33), but contrasts with patients with hypothyroxinemia of nonthyroidal illness
free thyroxin index values, which are frequently increased transiently increased concentrations of TSH have beei
in hyperthyroxinemia because of severe TBG excess and are reported (39-42), which makes the distinction betweei
uniformly increased in familial dysalbuininemic hyperthyr- primary hypothyroidism and hypothyroxinemia of nonthyr
oxinemia (1-3, 5, 6). It differs from labeled-analog free-T4 oidal illness unusually difficult. Thus, an accurate measure
methods, which give high free-T4 values for patients with ment of free T4 would be especially useful in such patients
familial dysalbuminemic hyperthyroxinemia (6, 16, 34). The direct equilibrium dialysisfRlA free-T4 measurement
Perhaps the most important difference between this free- separated the patients with hypothyroxinemia of nonthyroi
T4 method and current clinical laboratory methods for free dal illness or hypothyroxinemia of TBG deficiency fron
T4 is shown in the evaluation of patients with hypothyroxin- those with hypothyroidism, whether it was primary ant
emma. Hypothyroxinemia is frequent in adult inpatients central. Faber et al. (13) recently reported measurements o
(35-37), the large majority of whom have decreased T4 free T4 in undiluted serum in which they used an indirec
binding to serum proteins rather than hypothyroidism (11, ultrafiltration method with careful chromatographic purifi
12). Yet the free T4 index and labeled-analog free-T4 meth- cation of tracer T4 in the ultraflltrate. Measurement of frei
ods regularly give low values, underestimating free-T4 T4 by this method also separated patients with the hypo
concentration in this type of patient (11, 12, 17-38). Al- thyroxinemia of nonthyroidal illness from patients with flu
though TSH concentrations are usually normal in serum of hypothyroxinemia of hypothyroidism. Only free-T4 method
70 S
.
60 S
30
#{149} LOWER LIMIT OF NORMAL
* -.
50
-J
.1: #{149} S
#{149}
C
20
S.
S
.
S : #{149}
Ui
Ui
#{149}SS.
#{149}#{149}
#{149}
:#{149} *
U- S.
#{149}5
#{149} S.
6 #{163} S
S
#{149}5
#{149}T
#{149}
10
S55
#{149}
S
#{149}5
- 55
- .
4. LIMIT OF DETECTION! -
S. LIMIT OF DETECTION
Fig. 5. Total T4 concentrations (left) and free-T4 concentrations (light)in 42 patients with primary hypothyroidism, six patients with centt
hypothyroidism, 15 individuals with euthyroid hypothyroxinemla caused by TBG defIcIency, and 27 individuals with euthyrokl hypothyroxinemia
nonthyroldal illness