Serum Thyroxine, Free T,, Triiodothyronine, and Reverse-T, in Diphenylhydantoin-Treated Patients Ralph R. Cavalieri, Laurence A. Gavin, Arthur Wallace, Margaret E. Hammond, and Kathleen Cruse In order to determine the effects of the administration of diphenylhydantoin (DPH) on various parameters of thyroid function, serum samples from 47 male adults receiving therapeutic doses of DPH and 45 euthyroid control subjects were analyzed for total thyroxine (T,) and an index of free T, concentration, using both a competitive protein-binding assay (CPBA) and a solid-phase radioimmunoassay (MA), total 3,5.3’-triiodothyronine (T,), 3.3’5’-triiodothyronine (reverse-T,. rT,), and TSH, each measured by specific MA. Mean total T, by both methods was depressed in the DPH group to 0.78 of the control level. Free T, Index by MA was decreased on the average in DPH-patients exactly in proportion to the depression in total T,. By the CPBA. the difference between two groups in Free T, Index was less marked but still significant (DPH/controls = 0.88, p < 0.01). The concentrations of total T, were virtually identical in the DPH and the control groups. The average TJT, ratio was significantly higher in the DPH patients than in the controls (0.0178 versus 0.0132, p < 0.001). Serum rT, was depressed by DPH-treatment in approximately the same proportion to the decrease in total T,. None of the DPHpatients had an elevated serum TSH. The above findings are interpreted as indirect evidence in support of the view that DPH stimulates T4 metabolism, particularly the conversion of T, to T,. The normal level of free T, may help to maintain a euthyroid state in spite of the decrease in free T4. The data also define the “euthyroid” ranges for total and free T, levels by these methods in patients receiving DPH.
1961, Oppenheimer INserum protein-bound
et al.’ reported that the iodine level was abnormally low in patients receiving therapeutic doses of diphenylhydantoin (DPH). This effect was assumed to result from the competitive inhibition by DPH of binding of thyroxine (T,,) to TBG, a phenomenon that is observable in vitro.* However, the in vivo effects of DPH were recognized to be more complex as a result of Chin and Schussler’? demonstration that the total serum T, is depressed, the free T, fraction (by ultrafiltration) is unchanged, and the serum free T, concentration is diminished. Larsen et al.4 reported that the free T, clearance is augmented during DPH-treatment. The latter effect was attributed to DPH-induced stimulation of hepatic metabolism of T,. Several groups have documented a depression in both total and free T, levels in DPH-treated Metabolism. Vol.28, No. 11, (November), 1979
patients;‘-* in three of these reportss-7 the free T, index was calculated using resin or charcoal T,-uptake values, and in the most recent study free hormone was measured directly.8 These groups have found that DPH treatment causes little or no alteration in total serum T,,5.7V8 but none of the groups has specifically examined the relationship between serum T, and T, in individual subjects. There is only one study containing data on serum reverse-T, (rT,), but individual ratios of rT, to T, were not given.* There is an important practical problem associated with the evaluation of thyroid functional status in patients receiving DPH, since the serum total and free T, levels are the primary thyroid function tests in most centers. Clearly, the “euthyroid” range for the general population would not apply to individuals on DPH therapy. The present study was performed in order to define the “euthyroid” range for serum T, and free T, in DPH-treated patients, using two different commercial kits. In addition, the effects of DPH therapy were examined on total serum T, and rT, levels. Indirect evidence was obtained indicating that DPH causes a shift in T, metabolism toward the formation of T,. MATERIALS AND METHODS Forty-seven male adults who were receiving therapeutic doses of DPH were studied. All were clinically euthyroid and none had evidence of severe systemic illness. Serum DPH levels (measured by the Clinical Laboratory of the San Francisco V.A. Medical Center) ranged from 2 to 42 pg/ml (mean, 12 &ml). As controls, two groups were studied: 16 healthy laboratory workers and 29 ambulatory patients who were not receiving DPH. In none of the 45 control subjects was there a history of thyroid dysfunction. Samples of serum from each subject were stored at - 20°C until analyzed. To avoid repeated freezing and thawing, each specimen was divided into several aliquots before storage.
From the Nuclear Medicine Service, Veterans Administration Medical Center, and Depariments of Medicine and Radiology, University of California, San Francisco, Calif Received for publication January 29, I979. Supported by the Veterans Administration. Address reprint requests to R. R. Cavalieri, M.D., Nuclear Medicine Service (I 15). V.A. Medical Center, 4150 Clement Street, San Francisco, Calif 94121. 0 I979 by Grune & Stratton, Inc. 0026%0495/79/2811&0013$0I.00/0 1161
1162
CAVALIER1
Fig. 1.
TOTAL THYROXINE (METHOD
I )
0
;
CONTROL
. .
DILANTIN
pg/lOO (METHOD
I
II)
8 0
CONTROL
*
ET
AL.
Serum total thyroxine concentration, in
ml, in control subjects and in Dilantin (DPH)
treated patients. IAl Results obtained by method 1 (solid-phase radioimmunoassay): (B) results by method 2 (competitive protein-binding assay). Brackets indicate *2 SEM. The difference between the groups is significant by both methods (p < 0.001).
DILANTIN
Total serum T4 and Free T, Index (FTJ) were determined in duplicate by two methods. Method 1 was a solid-phase radioimmunoassay (Corning Medical, Medfield, Mass.) that employs two sets of tubes. Tube A contained 25 rliter of test serum (or standard), ?-T, as tracer, and immobilized anti-T, antibody (Ab). Tube B contained all of the above plus thimerosal, which releases T4 from serum proteins. The amount of tracer taken up by Ab in each tube was determined at the end of 30 min. The ratio of Ab-bound tracer in tube A to that in tube B was proportional to the free T, concentration of the serum. From the A/B ratio obtained in a set of standard sera containing known concentrations of total and free T,, the FT,I was estimated for each test serum. From the amount of Ab-bound tracer in tube B, total T, concentration in the test serum was determined, using as reference the same set of standards. Sensitivity of the assay for free T, was 0.25 ng/dl. The coeficient of variation was 1.5% within assay and 3.7% between assays. Method 2 was a competitive protein-binding assay that employs small disposable Sephadex columns as adsorbing medium. The procedure was done in two stages, one providing total T, concentration, and the other a Free T4 Index.9 In the first stage, the test serum (or standard) containing “‘I-T4 as tracer was applied to the column at pH 11. At this pH, virtually all of the T, in serum is released from its binding proteins and is adsorbed to the Sephadex. The proteins were washed through the column, which was then adjusted to a lower pH, 8.4. A solution containing TBG was passed through the column and a fraction of the adsorbed T, (including tracer) was bound by the TBG. The amount of labeled T, retained by the column was proportional to the amount of T, originally applied in the test serum. (Columns were counted in a well-counter.) In the second stage, a small aliquot (20 pliter) of the test serum was applied to the same column used for the total T, determination. Following a wash with pH 8.6 buffer, the column was counted again. The amount of labeled T, retained after the final step was proportional to the free T, concentration. The IT41 result was expressed as a ratio of the final column counts for the test serum over the final column counts for a normal reference serum (pool of normal sera). Although neither method directly measures free T4, the “index” is based on T,-binding power of test serum using
labeled T, rather than TP; therefore, it is more closely related to the free T, concentration than the conventional FTJ, which is calculated from a T,-resin uptake value. Serum TSH and total T, were measured by solid-phase radioimmunoassays (Corning). Serum 3,3’,5’-triiodothyronine (rT,) was determined by a radioimmunoassay development in our laboratory.‘a RESULTS
Serum Thyroxine Both methods yielded results that agreed quite closely (Fig. 1). By method 1, the mean total serum T4 in the DPH-treated group (5.93 + 1.81 pg/lOO ml [mean + SD]) was decreased to 0.78 of that in the controls (7.58 + 1.18), a highly significant difference. By method 2, the proportional decrease was the same, 5.78 ? 1.63 versus 7.39 * 1.35. Nearly one-third of the DPH group had total T, values below the lower limit of the control range. The mean free T, concentration in the DPH cases was similarly depressed (Fig. 2). By the solid phase method (method l), the free T4 value in the DPH patients (1.17 + 0.32 ng/ 100 ml) was 0.78 of the control mean (1.50 f 0.28), exactly in proportion to the difference in the total T4 concentration, indicating that the proportion of free T, was unaffected by DPH treatment. By method 2, the difference in free T, index between the groups was less marked (DPH/controls = 0.86). Nevertheless, by this method, 30% of the DPH cases fell below the lower limit of the control range. Within the control group, there was no significant difference between the healthy laboratory workers and the euthyroid patients in either total or free T4 values. In the DPH group, there was no signifi-
DIPHENYLHYDANTOIN
AND THYROID
1163
FUNCTION
FREE T, INDEX
to the mean total T, levels, which were similar in the two groups, the ratio of total T, to total T, was significantly higher in the DPH-treated patients (Fig. 3). The mean Tj/TI ratio in the control group of patients (0.0131) was nearly identical to that in the healthy controls (0.0135), so the higher ratio noted in the DPH group (mean 0.0178 + 0.0056) represented a true increase and was not merely due to a lower T,/T, due to nonthyroidal illness in the control patients.”
(METHOD n i
(METHOD 11
2.0-
7 8
c F
l.O-
i.
: P
1961, Oppenheimer INserum protein-bound
et al.’ reported that the iodine level was abnormally low in patients receiving therapeutic doses of diphenylhydantoin (DPH). This effect was assumed to result from the competitive inhibition by DPH of binding of thyroxine (T,,) to TBG, a phenomenon that is observable in vitro.* However, the in vivo effects of DPH were recognized to be more complex as a result of Chin and Schussler’? demonstration that the total serum T, is depressed, the free T, fraction (by ultrafiltration) is unchanged, and the serum free T, concentration is diminished. Larsen et al.4 reported that the free T, clearance is augmented during DPH-treatment. The latter effect was attributed to DPH-induced stimulation of hepatic metabolism of T,. Several groups have documented a depression in both total and free T, levels in DPH-treated Metabolism. Vol.28, No. 11, (November), 1979
patients;‘-* in three of these reportss-7 the free T, index was calculated using resin or charcoal T,-uptake values, and in the most recent study free hormone was measured directly.8 These groups have found that DPH treatment causes little or no alteration in total serum T,,5.7V8 but none of the groups has specifically examined the relationship between serum T, and T, in individual subjects. There is only one study containing data on serum reverse-T, (rT,), but individual ratios of rT, to T, were not given.* There is an important practical problem associated with the evaluation of thyroid functional status in patients receiving DPH, since the serum total and free T, levels are the primary thyroid function tests in most centers. Clearly, the “euthyroid” range for the general population would not apply to individuals on DPH therapy. The present study was performed in order to define the “euthyroid” range for serum T, and free T, in DPH-treated patients, using two different commercial kits. In addition, the effects of DPH therapy were examined on total serum T, and rT, levels. Indirect evidence was obtained indicating that DPH causes a shift in T, metabolism toward the formation of T,. MATERIALS AND METHODS Forty-seven male adults who were receiving therapeutic doses of DPH were studied. All were clinically euthyroid and none had evidence of severe systemic illness. Serum DPH levels (measured by the Clinical Laboratory of the San Francisco V.A. Medical Center) ranged from 2 to 42 pg/ml (mean, 12 &ml). As controls, two groups were studied: 16 healthy laboratory workers and 29 ambulatory patients who were not receiving DPH. In none of the 45 control subjects was there a history of thyroid dysfunction. Samples of serum from each subject were stored at - 20°C until analyzed. To avoid repeated freezing and thawing, each specimen was divided into several aliquots before storage.
From the Nuclear Medicine Service, Veterans Administration Medical Center, and Depariments of Medicine and Radiology, University of California, San Francisco, Calif Received for publication January 29, I979. Supported by the Veterans Administration. Address reprint requests to R. R. Cavalieri, M.D., Nuclear Medicine Service (I 15). V.A. Medical Center, 4150 Clement Street, San Francisco, Calif 94121. 0 I979 by Grune & Stratton, Inc. 0026%0495/79/2811&0013$0I.00/0 1161
1162
CAVALIER1
Fig. 1.
TOTAL THYROXINE (METHOD
I )
0
;
CONTROL
. .
DILANTIN
pg/lOO (METHOD
I
II)
8 0
CONTROL
*
ET
AL.
Serum total thyroxine concentration, in
ml, in control subjects and in Dilantin (DPH)
treated patients. IAl Results obtained by method 1 (solid-phase radioimmunoassay): (B) results by method 2 (competitive protein-binding assay). Brackets indicate *2 SEM. The difference between the groups is significant by both methods (p < 0.001).
DILANTIN
Total serum T4 and Free T, Index (FTJ) were determined in duplicate by two methods. Method 1 was a solid-phase radioimmunoassay (Corning Medical, Medfield, Mass.) that employs two sets of tubes. Tube A contained 25 rliter of test serum (or standard), ?-T, as tracer, and immobilized anti-T, antibody (Ab). Tube B contained all of the above plus thimerosal, which releases T4 from serum proteins. The amount of tracer taken up by Ab in each tube was determined at the end of 30 min. The ratio of Ab-bound tracer in tube A to that in tube B was proportional to the free T, concentration of the serum. From the A/B ratio obtained in a set of standard sera containing known concentrations of total and free T,, the FT,I was estimated for each test serum. From the amount of Ab-bound tracer in tube B, total T, concentration in the test serum was determined, using as reference the same set of standards. Sensitivity of the assay for free T, was 0.25 ng/dl. The coeficient of variation was 1.5% within assay and 3.7% between assays. Method 2 was a competitive protein-binding assay that employs small disposable Sephadex columns as adsorbing medium. The procedure was done in two stages, one providing total T, concentration, and the other a Free T4 Index.9 In the first stage, the test serum (or standard) containing “‘I-T4 as tracer was applied to the column at pH 11. At this pH, virtually all of the T, in serum is released from its binding proteins and is adsorbed to the Sephadex. The proteins were washed through the column, which was then adjusted to a lower pH, 8.4. A solution containing TBG was passed through the column and a fraction of the adsorbed T, (including tracer) was bound by the TBG. The amount of labeled T, retained by the column was proportional to the amount of T, originally applied in the test serum. (Columns were counted in a well-counter.) In the second stage, a small aliquot (20 pliter) of the test serum was applied to the same column used for the total T, determination. Following a wash with pH 8.6 buffer, the column was counted again. The amount of labeled T, retained after the final step was proportional to the free T, concentration. The IT41 result was expressed as a ratio of the final column counts for the test serum over the final column counts for a normal reference serum (pool of normal sera). Although neither method directly measures free T4, the “index” is based on T,-binding power of test serum using
labeled T, rather than TP; therefore, it is more closely related to the free T, concentration than the conventional FTJ, which is calculated from a T,-resin uptake value. Serum TSH and total T, were measured by solid-phase radioimmunoassays (Corning). Serum 3,3’,5’-triiodothyronine (rT,) was determined by a radioimmunoassay development in our laboratory.‘a RESULTS
Serum Thyroxine Both methods yielded results that agreed quite closely (Fig. 1). By method 1, the mean total serum T4 in the DPH-treated group (5.93 + 1.81 pg/lOO ml [mean + SD]) was decreased to 0.78 of that in the controls (7.58 + 1.18), a highly significant difference. By method 2, the proportional decrease was the same, 5.78 ? 1.63 versus 7.39 * 1.35. Nearly one-third of the DPH group had total T, values below the lower limit of the control range. The mean free T, concentration in the DPH cases was similarly depressed (Fig. 2). By the solid phase method (method l), the free T4 value in the DPH patients (1.17 + 0.32 ng/ 100 ml) was 0.78 of the control mean (1.50 f 0.28), exactly in proportion to the difference in the total T4 concentration, indicating that the proportion of free T, was unaffected by DPH treatment. By method 2, the difference in free T, index between the groups was less marked (DPH/controls = 0.86). Nevertheless, by this method, 30% of the DPH cases fell below the lower limit of the control range. Within the control group, there was no significant difference between the healthy laboratory workers and the euthyroid patients in either total or free T4 values. In the DPH group, there was no signifi-
DIPHENYLHYDANTOIN
AND THYROID
1163
FUNCTION
FREE T, INDEX
to the mean total T, levels, which were similar in the two groups, the ratio of total T, to total T, was significantly higher in the DPH-treated patients (Fig. 3). The mean Tj/TI ratio in the control group of patients (0.0131) was nearly identical to that in the healthy controls (0.0135), so the higher ratio noted in the DPH group (mean 0.0178 + 0.0056) represented a true increase and was not merely due to a lower T,/T, due to nonthyroidal illness in the control patients.”
(METHOD n i
(METHOD 11
2.0-
7 8
c F
l.O-
i.
: P
