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CLIN. CHEM.

34/9, 1737-1744 (1988)

Direct Determination of Free Thyroxin in Undiluted Serum by Equilibrium


Dialysis/Radioimmunoassay
Jerald C. Nelson1 and Ray 1. Tomel

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

CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988 1737


retentate compartment above from the dialysate compart- troit, MI 48232) and rabbit IgG, Cohn Fraction II (Sigma
ment below, is 1.3 cm in diameter. This small size did not Chemical Co., St. Louis, MO 63178). These proteins did not
prolong the time required to reach dialysis equilibrium for bind thyroid hormones but prevented adsorption of T4 to the
T4, compared with dialysis cells with larger membranes, in dialysis-cell components. We observed marked variation in
a retentate volume of 0.6 mL and a dialysate volume of 2.4 different lots of gelatin and rabbit IgG, and the lots used
mL, the volumes used in this study. The osmotic water gain were carefully screened for their suitability in this assay.
in this configuration averaged 48 L in 16 h at 37 #{176}C, for an Each liter of dialysate buffer contained sodium chloride,
average 8% dilution of serum proteins during dialysis.. 5.265 g; 60% syrup of rn.-lactic acid, as the sodium salt, 1.0
The dialysis membrane (Spectropor No. 2, molecular- mL; i..-glutamic acid as the monosodium salt, 561 mg;
mass cutoff 12 to 14 kDa; Van Water & Rogers, Norwalk, potassium chloride, 224 mg; monopotassium phosphate, 180
CA 90650) was washed by soaking in three changes of mg calcium chloride 2 H20, 275 mg; magnesium sulfate .7
distilled water with constant magnetic stirring for 2 h before H20, 246 mg; urea, 300 mg; tiaras sodium salt, 5.891 g;
cell assembly. After assembly of the membrane cylinder, we HEPES acid 7.190 g; penicillin, 100 000 USP units; strepto-
washed the cylinders, dialysate vials, and caps with tap mycin, 100 mg; amphotericin B, 0.25 mg; gentamicin, 100
water, then soaked them in 50 mLIL detergent wash solu- mg; sodium azide, 520 mg; gelatin, 500 mg; and rabbit IgG,
tion (Radiac Wash; Atomic Products Corp., Center Mor- 200 mg. Sodium chloride was obtained from Mallinckrodt,
iches, Long Island, NY) containing 0.5 g of sodium azide per Inc., Paris, KY 40361; potassium phosphate (monobasic,
liter. After soaking the parts in this for 1 h at room crystalline), calcium chloride (dihydrate, granular), and
temperature, we again rinsed them with tap water, then magnesium sulfate (crystalline) were obtained from J. T.
placed them in a pan of running tap water for 30 mm. Baker Chemical Co., Phillipsburg, NJ 08865. The other
During the washing, the membrane cylinders were held in a constituents were obtained from Sigma Chemical Co.
sandwich rack, which kept them from touching each other The sodium chloride, sodium lactate, sodium glutamate,
and prevented damage to the membranes. After a final rinse potassium chloride, potassium phosphate, magnesium sul-
with distilled water, the caps and the vials were allowed to fate, and urea were combined in 800 mL of distilled water
air-dry, but the dialysis cylinders were placed in clean and placed on a magnetic stirrer until completely dissolved.
dialysate vials containing 2.4 mL of fresh dialysate buffer to The calcium chloride was dissolved in 5 mL of distilled
keep the dialysis membranes from drying out. The assem- water and added to the mixture quantitatively. The mixture
bled dialysis cells could then be stored for up to one month at was then heated to 44#{176}C and the gelatin was added slowly,
room temperature before use. Dialysis was initiated when with constant stirring. The rabbit IgG was added after the
serum was introduced into the retentate compartment. The solution was cooled to room temperature. Then the HEPES
cells could be washed and re-used daily for at least four sodium salt, HEPES acid, penicillin, streptomycin, amphoteri-
months before there was evidence of deterioration. cin B, gentamicin, and sodium azide were all added, with
constant stirring until completely dissolved. The pH of the
Dialysate Buffer Design
mixture was adjusted to 7.35 at room temperature with
The dialysate buffer was designed to approximate the sodium hydroxide or hydrochloric acid as needed. The
composition of a protein-free ultrafiltrateof normal human solution was then diluted to 1000 mL with distilled water,
serum, at least for those compounds present in serum at a ifitered through glass wool, and stored at 4#{176}C until used.
concentration of 1 mmol/L or more, except that glucose was This reagent was stable indefinitely at 4#{176}C and for one
omitted and preservatives were included. Because the dialy- month at room temperature. This produced a buffer contain-
sate volume was greater than the retentate volume (2.4 mL ing, per liter, 131 mmol of sodium, 4.3 mmol of potassium,
vs 0.6 mL), the final ionic composition of different sera could 1.9 mmol of calcium, 1.0 mmol of magnesium, 98 mmol of
be normalized, even for sera from severely ill patients. The chloride, 1.3 mmol of phosphate, 1.3 mmol of sulfate, 5.4
dialysate contained a pH buffer, 4-(2-hydroxyethyl)-1-piper- mmol of lactate, 3.3 mmol of glutamate, and 8 mmol of urea.
azine ethanesulfonate (tiaras acid/sodium apas; Calbio- There was slight variation in the sodium and chloride
chem-Behring Corp., La Jolla, CA 92073), and a mixture of concentration, depending on the amount of sodium hydrox-
antimicrobials. It also contained gelatin (Difco Labs, De- ide or hydrochloric acid needed to adjust the pH.
With this dialysate buffer, values for serum analytes were
altered only slightly during equilibrium dialysis, as shown
for eight randomly chosen patients serum samples in Table
1. One aliquot of each sample was stored untreated while
another aliquot was dialyzed. Measurements of pH were
made at 37 #{176}C. The ionic composition of the sera was more
uniform after dialysis than without dialysis, as evidenced by

Table 1. Effect of Dialysis on the Serum Matrix


Wfthout Normal reference
dialysIs After dialysis Interval
Sodium, mmol/L 142 5 141 16 135-148
Potassium, mmol/L 4.6 0,3 4.5 0.1 3.5-5.3
Calcium, mg/L 918 913 81-107
Phosphate, mg/L 436 412 26-48
Chloride, mmol/L 102 6 105 3 96-109
Albumin, g/L 367 336 35-51
VIAL Membrane pH 8.11 0.20 7.44 0.02 7.35-7.45
Mean ( SD) of results for eight different sera,
Fig. 1. Design of the re-usable dialysis cell

1738 CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988


the lower standard deviation after dialysis, and serum From these experiments we concluded that the antibiotics
electrolyte concentrations remained within physiological and the buffer did not affect T4 binding during equilibrium
ranges. We examined the pH buffering capacity of the dialysis. We also concluded that the dialysis cells did not
dialysate by adding to normal sera unbuffered HEPES acid, introduce a blank effect in the sensitive T4 radioim-
up to 24 mmol/L, and unbuffered sodium bicarbonate, up to munoassay and that there was no adsorption of T4 to the
24 mmolfL. This resulted in serum pH values at 37 #{176}C dialysis cells during equilibrium dialysis.
ranging from 7.35 to 7.49 after dialysis, a variation in pH
Radioimmunoassay
that negligibly affects T4 binding to serum proteins (24).
We studied the effect of HEPES anion on T4 binding by The sensitive T4 radioimmunoassay for measuring free T4
measuring free T4 concentrations in aliquots of a normal in 500-tL aliquots of dialysate has previously been reported
serum poo1, using HEPEs-contaming dialysates and HEPES- (23). In the present studies, we used the first antibody at a
free dialysates. The other constituents of the dialysates were final dilution of 1:300 000 and used an RLA buffer similar to
unchanged. In this experiment, serum pH was controlled by the dialysis buffer described above, except that the HEPES
preliminary titration with i.e-lactic acid. This required con- acid was decreased to 6.046 g/L to give a final HEPES anion
centrations up to 30 mmol/L. Lactate anion, added as the concentration of 48 mmol/L (the concentration present in
hemi-calcium salt, did not alter T4 binding in concentrations dialysate buffer after dialysis against serum). The pH of the
up to 120 mmoWL (data not presented), confirming previous RIA buffer was adjusted to 7.40 at 37 #{176}C (i.e., pH 7.54 at
data that lactate anion does not alter T4 binding to serum room temperature). We used a first-antibody incubation of
proteins (25). Results of the 1-tEPES experiments showed that 60 mm at 37#{176}C, followed by 30 miii at 4#{176}C. This brings the
varying HEPES anion concentrations between 0 and 80 first antibody reaction to equilibrium. The second antibody
mmolIL had no effect on T4 binding. incubation was for 30 mm at 4#{176}C. The average total counts
The antibiotics and sodium a.zide did not, singly or in per minute were 60000, the average maximum binding was
combination, alter T4 binding to serum proteins at the 26%, the 50% B/B0 interpolated to an average free-T4
concentrations used. To show this, we compared the free-T4 concentration of 22 ng/L, the 2 ng/L standard averaged 90%
concentrations found by using dialysates containing the B/B0, and the 128 ng/L standard averaged 13% B/B0.
antibiotics with those observed by using dialysates contain-
Characteristics of the Direct Equilibrium
ing no preservatives. In these experiments a semi-sterile
Dialysis/Radioimmunoassay Method
technique was used after the dialysis cells had been steril-
ized by autoclaving. Autoclaving did not affect dialysis-cell Figure 2 depicts a representative result of experiments to
performance (data not presented). In five experiments, using determine the time to T4 binding equilibrium. Equilibrium
a normal serum pool, we found that free-T4 concentrations was achieved in 14 h, but assay incubations were routinely
in the presence of the antibiotics averaged 17.6 (SD 1.4) performed for 16 to 18 h, to ensure that we were measuring
ngfL; without antibiotics, the concentrations averaged 16.9 on the plateau of the time-response curve.
(SD 1.7) ng/L (P >0.9). To study intra-assay precision, we dialyzed a single
In studies of bacterial control, we found that a decline in normal serum pool in 57 cells. The mean free-T4 concentra-
glucose concentration was a sensitive early indicator of tion was 15 ng/L; the coefficient of variation (CV) was 5.3%.
bacterial growth. Periodically, over the two years during Interassay precision was similarly studied by using a second
which this assay has been in use, we have measured glucose normal serum pool run in one dialysis cell per day on 57
concentrations in the dialysate (using a glucose-enriched different days during four months. The mean free T4 in
serum pool) and cultured the retentate after dialysis, look- serum was 18 ngfL, with a CV of 6.9%. A pool of dialysates
ing for evidence of bacterial growth. Consistently, the was also run in these same 57 assays, the pool containing an
dialysates have remained crystal clear, glucose utilization average T4 concentration of 14 ng/L. The CV was 7.4%.
has not occurred during dialysis, and no bacterial growth Although the dialysate pool did not go through the dialysis
has been observed in cultures of the retentates. step of the assay, the variability was similar for both the
The possibility that the materials used in the dialysis cell serum pool and the dialysate pool. Thus the dialysis step
might introduce ifiA blank effects was studied by comparing apparently adds little to the variability in results.
the performance of radioimmunoassay buffer before and Serum sample stability was examined in two ways. First,
after incubation in dialysis cells. In five experiments the
mean B/B0 of buffer exposed to the dialysis cells was 99.1%
20
of the BIB0 of buffer that was not exposed to the dialysis
cells. Several other plastics and other 0-ring materials,
initially considered for use in this dialysis cell, proved
15
unacceptable because they produced blank effects in the
sensitive T4 assay.
The effectiveness of the dialysate proteins in preventing FREE T4
ngIL 10
T4 adsorption to the dialysis cells was studied by measuring
free-T4 concentrations in T4-containing buffers, with and
without proteins, both before and after incubation in the 5

dialysis cells. In five experiments the T4 in protein-contain-


ing buffer was 23.4 (SD 0.5) ngfL without incubation in the
dialysis cells and 22.8 (SD 0.4) ng/L after incubation in the
0 4 12 16 20 24
dialysis cells (P >0.05). Similarly constituted protein-free
HOURS
buffer gave a mean free-T4 concentration after incubation in
the cells of 16.3 (SD 0.5) ng/L (p <0.01), a 29% loss by Fig. 2. Effect of the duration of dialysis on free-T4 concentrations;
adsorption. equilibrium was achieved in 14 h at 37#{176}C

CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988 1739


the serum pool used in the interassay precision study was was 9.5 ng/L (normal 2.4-4.5 ng/L).
frozen and stored in separate aliquots. Each aliquot was We studied 27 patients with euthyroid hyperthyroxin-
thawed only once before use. Those aliquots frozen for more emias. None had clinical signs of thyroid disease. Thirteen
than three months gave free-T4 values of 18.5 (SD 0.11) were outpatients with severe TBG excess, defined as a TBG
ng/L, which were similar to the free-T4 values for those concentration exceeding twice the upper limit of the normal
samples stored less than one month, 18.2 (SD 1.2) ngfL; this reference interval. All were discovered by T4 testing. All
indicates that free T4 was stable in frozen serum for four had normal values for serum thyrotropin, ranging from 0.7
months or longer. Secondly, we performed a systematic to 2.0 miui-int. unitsfL. Their serum TBG concentrations
freeze-thaw study. Ten aliquots of a normal serum pool ranged from 72 to 193 mg/L, their total T4 from 128 to 538
were frozen for 11 days. One aliquot was thawed at room pgfL. In each, the T/FBG ratio was within or below the
temperature once, one aliquot twice, and so on, up to a normal reference interval and ranged from 0.11 to 0.22,
maximum of 10 freeze-thaw cycles. All samples were then characteristic of severe TBG excess (46). The other 14
analyzed in triplicate in a single assay. Values for free T4 patients had euthyroid hyperthyroxinemia resulting from
were not altered by the 10 freeze-thaw cycles. The aliquot familial dysalbununemic hyperthyroxinemia. These 14 indi-
thawed once had a value for free T4 of 19 ngfL and the viduals came from five kindreds, and one or more members
aliquot thawed 10 times also had a free T4 of 19 ng/L. All of each kindred were shown, by reverse-flow electrophoresis,
intermediate aliquots had free-T4 values of either 18 or 19 to have increased F4 binding to serum albumin (data not
ng/L, and there was no drift of values with increasing presented). All individuals were discovered by T4 testing.
numbers of freeze-thaw cycles. All were outpatients. Values for thyrotropin (TSH) in serum
All patients samples were dialyzed in duplicate and each ranged from 0.6 to 18.7 milli-int. units/b and were within
dialysate was immunoassayed in duplicate. The results normal limits in all but one. The one patient with an
reported are the mean of the quadruplicate RIft values. increased serum thyrotropin value was nongoitrous and
clinically euthyroid but had increased antimicrosomal anti-
Other Assays
bodies, indicating co-existing subclinical autoimmune thy-
Serum T4 and TBG were measured with radioim- roiditis. Serum T4 in this group ranged from 145 to 209
munoassay kits (Clinical Assays, Baxter-Travenol Diagnos- serum TBG ranged from 9 to 27 mg/b, within the
tics, Inc., Cambridge, MA 02139). Thyrotropin was mea- normal range in all but one individual, who had a decreased
sured with a sensitive immunoradiometric assay kit (Nich- TBG concentration of 9 mg/b. The T4/TBG ratio was in-
ols Institute Diagnostics, San Juan Capistrano, CA 92675). creased in all, ranging from 0.53 to 1.30 and overlapping the
Antimicrosomal autoantibodies were measured with a sen- range seen in hyperthyroidism (0.38-1.12).
sitive radioimmunoassay, and free triiodothyronune (T3) in Forty-two patients had primary hypothyroidism. All were
serum was measured by equilibrium dialysis at Nichols outpatients with clinically apparent and untreated hypothy-
Institute Reference Laboratory, San Juan Capistrano, CA roidism, who were otherwise selected without conscious
92675. The normal ranges for these assays in our laboratory bias. Their serum TSH ranged from 15 to 342 milli-int.
were 54-113 pg/b for serum T4, 16-36 mgfL for serum TBG, units/b, T4 from <5 to 69 pg/b, and T4/TBG ratios from
0.4-5.0 milli-int. units/L for serum thyrotropin, <25 kilo- <0.01 to 0.20. Interestingly, three had total T4 values
unita/L for antimicrosomal autoantibodies, and 2.4-4.5 ngfL within the normal reference interval (54-113 pg/L). Their
for free T3 in serum. We also determined a normal reference respective values for total T4 were 56, 60, and 69 pg/b; for
interval for the T/FBG ratio. If we used the value 777 for free T4 5, 6, and 7 ng/L; and for thyrotropin 35, 24, and 31
the relative molecular mass of T4 and 63 000 for the relative milli-int. units/b. All three had high TBG concentrations
molecular mass of TBG, the normal range for the T4IFBG associated with their primary hypothyroidism: 57, 40, and
ratio was 0.20-0.38. All samples were analyzed in duplicate. 44 mg/b, respectively. Their total T4 concentrations, al-
though within the normal reference interval, were inappro-
Normal Controls
priately low for their TBG concentrations, as evidenced by
One hundred and twenty nonpregnant adult hospital the decreased T4JTBG ratios: 0.08,0.12, and 0.13, respective-
employees served as normal controls. All prospective con- ly.
trols underwent a general physical examination, a chemis- We studied six patients with documented central hypo-
try panel, urinalysis, complete blood-cell count, thyrotropin thyroidism. All had independent evidence of organic pitu-
assay, and testing for antimicrosomal antibodies. Only itary or hypothalamic failure, in addition to clinical signs of
individuals with all results normal were accepted as normal hypothyroidism. Their serum TSH concentrations ranged
controls. from <0.1 to 3.6 and were in the normal range in five of the
six patients. Total T4 concentrations ranged from <5 to 42
Patients pg/b. Serum TBG values were 26 to 44 mg/L and their
A total of 147 patients with carefully defined thyroid- T/FBG ratios were 0.03 to 0.13 mollmol.
hormone abnormalities were studied. Thirty outpatients Forty-two patients with euthyroid hypothyroxinemias
with clinically overt and untreated hyperthyroidism were were studied. None had clinical signs of thyroid disease and
studied without further selection. All had undetectable all had concentrations of TSH within the normal reference
thyrotropun in serum (<0.1 milli-int. unitlL). Their values interval. Fifteen individuals had severe TBG deficiency,
for total T4 ranged from 102 to 324 pg/L and T4ITBG ratios which we defined as a TBG concentration less than one-half
from 0.36 to 1.12. Two patients had total T4 within the of the lower limit of the normal range. All were males and
normal range. One of these, with a total T4 of 102 pg/b, had all were outpatients discovered by T4 testing. Their TBG
a TBG of 19 mg/L and a T/FBG ratio of 0.43, which is above concentrations ranged from <1 to 7 mg/b. Their serum
normal; free T4 was 44 ngfL. The second patient, with a total thyrotropin concentrations were within the normal refer-
T4 of 103 pg/L, had a TBG of23 mg/Land a T/FBG ratio of ence interval and ranged from 0.9 to 4.5 milli-int. units/b.
0.36, which is normal; the free T4 was 32 ng/L and free T3 Their total T4 concentrations were between 17 and 38 pg/b,

1740 CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988


and the T/FBG ratios were above normal in 14 of the 15 30
individuals. The one individual with a normal T4ITBG ratio
e
had a TBG of 7 mg/b, a total T4 of 27 pg/b, and a T4ITBG
ratio of 0.31. There was no overlap between the T4/TBG 25 .
ratios in primary hypothryoidism and those in severe TBG .
deficiency. #{149}...
We studied 27 patients with the unique hypothyroxin- 20 ......
C......
emias associated with nonthyroidal illnesses. Patients re- #{149}...
ceiving dopamine or glucocorticoids were not included be- FREET, .......
......
cause these drugs suppress TSH secretion. Occasionally ngIL 15 .........
patients with nonthyroidal illnesses have hypothyroxine- #{149}1.#{149}#{149}#{149}...#{149}#{149}.#{149}#{149}
mia from unrecognized coexisting primary hypothyroidism. ...........
.........
In these patients, the concentrations of TSH in serum are 10 .............
increased. More often, patients with nonthyroidal illness .......
44- -

have hypothyroxinemia caused by TBG deficiency (3, 26,


27). These patients can be identified by their serum TBG
5
concentrations and their T/FBG ratios (28,29). If TBG is
reduced without a decrease in either albumin or prealbu- LIMIT OF DETECTION

mm, the T/1BG ratio will increase. If TBG, albumin, and


0
prealbumin are all decreased in parallel, the T/FBG ratio
will remain normal. The unique hypothyroxinemias of Fig. 3. Free-T4 concentrations in 120 healthy adults
nonthyroidal illness can be distinguished from primary The mean was 14.5 (SD 4.2) pg/I, the median 14.0 ng/L. and the observed range
hypothyroidism by the absence of clinical evidence of thy- (one outlier deleted) was 0.8 to 2.7 ng/L
roid disease and by normal concentrations of serum TSH;
they can be distinguished from TBG deficiency by a do- concentrations ranged from 4 to 6 ng/L and were detectable
creased T4iTBG ratio (a serum T4 inappropriately low for in all. Free-T4 concentrations clearly separated patients
the prevailing TBG concentration). In these 27 patients with the hypothyroxinemia of hypothyroidism from individ-
studied, hypothyroxinemia was discovered by T4 testing. uals with euthyroid hypothyroxinemia. Free-T4 concentra-
Thyrotropin was normal (0.5 to 4.0 miui-int. units/L), and tions were within the normal reference interval in all
total T4 was low, ranging from <5 to 48 pg/b. The T/FBG patients with severe TBG deficiency, 9 to 25 ng/L. They
was low and ranged from <0.03 to 0.15. There was no were either normal or high in the patients with hypothyrox-
overlap with the T/FBG values seen in TBG deficiency (i.e., inemia caused by nonthyroidal illness. In this group of
0.31 to >2.03), but there was complete overlap with the patients, free T4 values were 8 to 35 ng/L; the highest value
T4/FBG values seen in primary hypothyroidism (<0.01 to overlapped the free-T4 concentrations seen in hyperthyroid-
0.20). ism.

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

CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988 1741


500j a 500 1
6
1 50J_

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

HYPOTHYROID LOW NONTHYROIDAL HYPOTHYROID LOW NONTHYROIDAL


PRIMARY CENTRAL TBO ILLNESS PRIMARY CENTRAL TBG ILLNESS

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

1742 CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988


involving membrane separation techniques have been 12. Kaptein EM, Grieb DA, Spencer CA, Wheeler WS, NicoloffJT.
shown to be capable of distinguishing these two forms of Thyroxine metabolism in the low thyroxine state of critical non-
hypothyroxinemia (13). thyroidal illnesses. J Clin Endocrinol Metab 1981;53:764-71.
Equilibrium dialysis methods for measuring free T4 have 13. Faber J, Kirkegaard C, Rasmussen B, Westh H, Busch-Soren-
sen M, Jensen 1W. Pituitary-thyroid axis in critical illness. J Clin
generally been thought to be reliable but too cumbersome
Endocrinol Metab 1987;65:315-20.
and time-consuming for clinical laboratory use. Some of the
14. Ashwell K, Hopton MB., Harrop JS. Serum free thyroxine
problems with previous equilibrium dialysis methods have
concentrations in subjects with high circulating levels of thyroxine-
been overcome with the current method. A direct assay binding globulin.Ann Clin Biochem 1983;20:285-8.
design eliminates the need for adding radiolabeled tracer to 15. Byfield PGH, Lalloz MRA, Pearce CJ, Himsworth RL. Free
the serum sample before dialysis, thereby eliminating the thyroid hormone concentrations in subjects with various abnormali-
need for tracer purification steps. The dialysis cell and its ties of binding proteins: experience with Amerlex free-T4 and free-
membrane can be washed and re-used, eliminating the need T3 assays. Clin Endocrinol 1983;19:277-83.
to replace the membrane after each assay. The sensitive T4 16. Stockigt JR, Stevens V, White EL, Barlow JW. Unbound
RJA used is similar to any standard double-antibody ifiA analog radioimmunoassays for free thyroxin measure the albwnin-
bound hormone fraction. Clin Chem 198329:1408-10.
except for the high affinity of the T4 antiserum, and is no
more difficult to perform than an RIA of total T4. 17. Ordonez-Llanos J, Rodriquez-Espinosa J, Gomez-Gerique JA,
Solans-Barri MD, Ruiz-Minguez MA. Effect of isolated decreases in
The modest increase in time and effort needed to perform
albumin and prealbumin on radioimniunoassay results for free
the preliminary dialysis fractionation of the serum sample thyroxin in nonthyroidal illnesses [Letter]. Clin Chem
before T4 quantification has, in our experience, been more 1984;30:496-8.
than justified by the improved diagnostic reliability of the 18. Weasel KW. Thyroxine analogue assay kits in clinical medi-
direct equilibrium dialysis/radioimmunoassay method, in cine [Letter]. Lancet 1985i:781-2.
comparison with free-T4 index and labeled-analog free-T4 19. Ekins R, Jackson T. Thyroxine analogue assay kits in clinical
methods, in our patient population. medicine [Letter]. Lancet 1985;i:782.
20. Alexander NM. Free thyroxin in serum: labeled thyroxin-
We are grateful to Nichols Institute for producing the plastic analog methods fall short of their mark [Editorial]. Chin Chem
dialysis cells to our specifications and for the Institutes gift of the 1986;32:417.
cells used in this study. We thank Dr. Elaine M. Kaptein for 21. Sakata S, Nakamura S, Miura K. Autoantibodies against
performing reverse-flow electrophoresis to document familial dysal- thyroid hormones or iodothyronine. Ann Intern Med 1985;103:579-
buminemic hyperthyroxinemia. We also thank Esther Carlthn and 89.
Drs. Richard L. Eddy, Elaine M. Kaptein, Kenneth E. W. Melvin,
22. Ekins RP. Methods for the measurement of free thyroid hor-
Robert Rosenquist, and Juan Sotos for helping us obtain serum
from patients with familial dysalbuminemic hyperthyroxinemia mones. In: International symposium on free thyroid hormones,
Venice. Excerpta Medica, 1978:72-92.
and congenital TBG abnormalities.
23. Nelson JC, Weiss RM. The effects of serum dilution on free
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1744 CLINICAL CHEMISTRY, Vol. 34, No. 9, 1988

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