Technical aspects of the estimation of triiodothyronine in human serum: Evidence of conversion of thyroxine to triiodothyronine during assay

Technical Aspects of the Estimation of Triiodothyronine in Human Serum : Evidence of Conversion of Thyroxine to Triiodothyrouine

During Assay

By P. R. LARSEN The method of Sterling et al. for measuring triiodothyronhre in human serum has been modified to provide greater sensitivity. The changes in technique include greater separation of triiodothyronine from thyroxine during paper chromatography, quantitation of triiodothyronine using a more dilute binding-protein solution, and the use of modifications which minimize the artifacts due to unknown substances in the chromatography paper. Complete recovery is obtained of known quantities of triiodothyronine added to human serum in amounts of 0.7 to 4.7 @ml. Mean tiodothyronhte concentrations in human serum using this method are 1.8 2 0.4 (SD) in normals, 6.7 +

3.3 (SD) in untreated hyperthyroid subjects, and 0.66 +- 0.39 (SD) in hypothyroid patients. These values are 18 to 30% lower than those previously reported. We have examined the influence of thyroxine on the determination of triiodothyronine using both labeled and unlabeled hormone. The results suggest that about 0.3 to 0.4% of the total thyroxine present in serum is deiodinated to triiodothyronine. This appears to occur during the paper chromatographic step. Because of these observations, it would appear that the modiied method, while quite satisfactory for clinical purposes, does not as yet provide the sensitivity and precision required for studies of triiodothyronine kinetics.

A

PRACTICAL METHOD for the measurement of triiodothyronine (T,) in human serum has recently been reported by Sterling, Bellabarba, Newman and Brenner.l This method consists of three major steps: (1) extraction of iodothyronines from serum by column chromatography; (2) separation of T3 from thyroxine (T,) using paper chromatography; (3) elution and quantitation of TR using dilute human serum and protein-binding displacement methodology. In the course of adaptation of this method for use in our laboratory, certain technical modifications have been introduced in an attempt to improve the sensitivity and precision of the method. We have carefully evaluated the possibility that Ts levels may be artifactually elevated due to the presence of relatively large amounts of T4 in human serum. In this report, we will describe our methodologica modifications, the results obtained using them, and the source and magnitude of the potential artifacts. From the Division of Endocrinology and Metabolism, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication August I, 1970. Supported by NIH Grant AM-14283 from the National Institute of Arthritis and Metabolic Diseases; the Health Research and Services Foundation of Pittsburgh Grant M-IO: the Dreyfus Charitable Fund: and General Clinical Research Center Grant FR-56 from the National Institutes of Health. P. R. LARSEN, M.D.: Assistant Professor of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pa. METABOLISM, VOL. 20, No. 6 (JUNE),

1971

609

610

P. R. LARSEN

Fig. l.-Radioactive scan of paper strip following chromatography. ments designated A, B, C, and D were each assayed for triiodothyronine.

Paper seg-

METHODSANDRESULTS Assay of T3 in Human Serum Extraction of iodothyronines from human serum. The extraction

of Ts and T4

from human serum was carried out as described by Sterling et al. on Dowex AG-50 WX 2 columns after addition of about 0.1 ng/ml of I251 T3 (Abbott Laboratories, Chicago, Ill.) for recovery calcu1ations.l The column eluate was dried under air at 65C. Approximately 80% of the T3 and 63% of the Ti present in the serum were extracted during this step as determined by tracer recoveries. Separation of Ts from T,. The dried column eluate was applied to the origin of chromatographic strips (2.5 X 58 cm) cut from Whatman No. 3 MM paper. Three 200-~1 washes of MeOH/2N NH40H (99/l, V/V) were used for transfer. Tracer T4 ( < 0.1 ng) was placed on the origin to document the migration of Td in the system. The strips were developed for 21 to 22 hr using the solvent system tertiary amyl alcohol/NHAOH( 2 N) /hexane (5/6/l ) described by Bellabarba, Peterson and Sterling.3 The T3 peak was located by scanning with a Vanguard radiochromatogram scanner (Fig. 1). Three or four 2.3- to 2.5-inch segments were then cut from each strip. One segment was located immediately behind and adjacent to the T3 peak (labeled A in Fig. 1) , the second consisted of the T3 peak (B), the third and, sometimes, a fourth strip (C and D) were located just ahead of the Ta peak. The paper segments were divided into smaller pieces and shaken for 30 min in 4 ml of MeOH/2N NHIOH in polystyrene counting tubes (Amersham-Searle) . The papers were then removed with wooden applicators and 5 ng of T3 in MeOH/2N NH,OH added to the extracts of paper segments A, C, and D (but not B). This was done to allow detection of even small amounts (about 1 ng) of TB-displacing material in the paper itself. The MeOH/2N NH,OH was then evaporated in a 50°C water bath under NZ and counted to determine recovery of 125I Ts. The purity of each tracer preparation was determined by chromatography and varied from 89 to 98%. Recovery was calculated using the following formula: Recovery = cpm extracted from paper/ (total counts in serum) (tracer purity). A major modification of this step is the 5 to 6 hours longer allowed for the separation of T3 and Ti. This gave average distances between these peaks of 18 to 22 cm as opposed to the 11 cm reported by Sterling et a1.l The recoveries of T3 varied from 35 to 5096, similar

to the range previously

reported.

TRIIODOTHYRONINE

IN HUMAN

SERUM

611

Quantitation of T, by binding-displacement assay. The binding-protein solution was prepared by the dilution of 3 to 4 ml of pooled human serum to 100 ml with glycine-acetate buffer (0.2 M glycine, 0.13 M Na acetate) pH 8.6. lz51 TS was added at a concentration of approximately 1 ng/ml. One milliliter of this solution was added to each tube containing the dried paper extract and to tubes containing standard amounts of TB. These were then mixed briefly and incubated at 4OC overnight. Each tube was then counted and 1 ml. of dextran-coated charcoal solution was added to each tube. The tubes were incubated for 45 min in an ice bath and then centrifuged for 20 min. The supernatant was decanted and 1 ml counted in an automatic well-counter (Packard). Sufficient counts were collected to assure statistical counting errors of less than 2.0%. The per cent tracer bound was calculated by reference to the total number of counts previously determined in each sample tube. Thus while the counts in tube B were higher than the others due to the recovered tracer, the per cent bound could be read from the same standard curve. This was verified in other studies by the use of 1311Ts as recovery tracer and lzaI Ta as the binding-protein tracer. The per cent bound of each isotope was identical. The nanograms of Tx recovered were determined by reference to the standard curve and corrected for losses. Results are expressed as ng T,/ml serum. Dextran-coated charcoal was prepared by suspending 12.5 g of Norit Neutral Decolorizing Charcoal (Fisher Scientific) in 500 ml of a solution containing 450 ml of 0.85% NaCl, 25 ml of 0.1 N NaOH and 25 ml glycine-acetate buffer, pH 8.6. Dextran T80 (Pharmacia, Uppsala, Sweden), 1.25 g was dissolved in 500 ml of a similar solution. Equal volumes of the charcoal and dextran solutions were combined and left for at least 12 hr at 4C prior to use. Preliminary standard curves were performed with each new binding protein solution used to determine the optimal concentration of dextran-coated charcoal to be used. The dilution (with glycine-acetate buffer) was generally l/8 to 3 /lO (dextran-coated charcoal/buffer).

Triiodothyronine standards were gravimetrically prepared at a T3 concentration of 20 pg/ml in 0.04N NaOH using sodium 3,5,3’ triiodothyronine (Mann Research Laboratories, New York, N.Y.) . The Ts content in this preparation was estimated precisely by determination of the absorption at 320 rnp in a Zeiss spectrophotometer using the molar extinction coefficient reported by Gemill. This concentration was in good agreement with the gravimetric calculation. This solution was then diluted l/100 with MeOH/2N NH40H, stored at -4C and used for up to 2 wk. In preliminary studies, we observed that in this assay system we were not able to obtain quantitative recovery of known amounts of T:< added to MeOH/2N NH+OH extracts of blank chromatography paper. When simultaneous standard curves were performed, one in tubes containing only 4 ml of MeOH/2N NHhOH and the other containing MeOH/2N NH,OH extracts of paper, the standard curve in the presence of paper extract was systematically displaced upwards over its entire range (Fig. 2). The variance was slightly increased, although not beyond tolerable limits. If the standard curve performed in the presence of dried paper extract was used for reference, quantitative recoveries

612

P. R. LARSEN

Triiodothyronine(nanograms) Fig. 2.-Standard curve using 4% human serum as binding protein. Solid line indicates curve obtained in presence of paper extract; broken line is control curve. Points are mean, and bars indicate SEM of triplicate determinations.

of T3 were obtained. Therefore, the standard curve was prepared by addition of various amounts of the T3 standard to 4 ml MeOH/2N NH40H extracts of blank chromatography paper which were of identical size and which had been developed simultaneously with the experimental strips. The standards generally used were 0,1,2,4,6,8,10 and 15 ng in triplicate. All tubes were then dried in a 50°C water bath under Nz along with the experimental tubes. Customarily, 2 to 10 ng of T3 were actually measured in the tube containing the extract of the B segment. This value is on the sensitive portion of the standard curve of Fig. 2. As little as 1 ng of T3 equivalent as background material in the paper could represent up to 50% of the total T3 measured. When TS was not added to the extracts of segments A, C, and D, the binding-displacement assay indicated 0 to 1.5 ng of T3 in these extracts. Due to the shallow slope in this part of the curve, we were not able to obtain precise quantitation of these amounts. To give greater confidence at these levels, 5 ng of TB were added to these extracts. The added TB was consistently recovered (+ 0.5 ng) from the extract of segment A located between the T3 and the Tq peaks. It was also recovered from segment D ahead of but separated from the T3 peak by about 2.5 inches. In segment C, immediately ahead of the T3 peak, and adjacent to it, we regularly found displacement of la51 T3 from TBG equivalent to from 1 to 2.5 ng of T1. Some (about 0.5 ng) of this activity oould be accounted for by the presence in this segment of approximately 5 to 10% of the original T3 which

TRIIODOTHYRONINE

IN HUMAN

Table l.-Recovery

613

SERUM

of Added Triiodothyronine From Pooled Human Serum Enrichment * 1.0 ng/ml 4.0 ng/ml

Experiment

A

Recovered t

ng Tdml

1

2.3

2 3

3.1

Mean 2 SEM

ng Tdml

2.6

5.5 5.6 5.6

2.7 k 0.2

5.6 rc 0.03

Observed

ng Ts/d

2.9

Enrichment * 4.1 ng/ml 0.7 ng/ml Exwriment

B

1 2 3 Mean -C SEM

Recovered 7 ng Tz/ml ne, Ts/ml

1.5 1.5 1.7 1.6 + 0.1

5.8 6.2 5.5 5.8 k 0.2

Observed ng Ta/ml

4.1

Differences T&eogra&l

3.0

Differences ~geo+;~~l

4.0

* Two serum pools, A and B, were enriched with exogenous T, in the amounts indicated. j Recovered T, is the total amount, endogenous plus exogenous, assayed in each of the triplicate specimens.

from segment B. However, the major part of this material could best be correlated with the volume of serum used in the assay rather than its Ta (or T,) content. Thus, in hyperthyroid sera, when only a l-2-ml sample was used, no displacement was observed in the paper segment C extract whereas when 7-S-ml of hypothyroid sera were examined, there was more Ts in this area than in the area indicated by the radioactive TR peak. These mlodifications appeared to offer a number of advantages. The use of 3.5 to 4.0% serum enabled us to obtain a steep slope for the standard curve with a maximum decrease of about 30 to 40% in the absolute value for per cent bound over a range of 0 to 15 ng of T3 (Fig. 2). This curve is about three times more sensitive than that described in the original meth0d.l The use of the dextrancoated charcoal allowed optimal concentrations of the charcoal to be used for each binding-protein solution. The use of paper extracts as part of the standard curve enabled us to obtain complete recoveries of known quantities of Ta. The separation of TB from Tq during paper chromatography was verified by assaying the material extracted from paper segment A for Ts-displacing activity. Since Tq displaces lz51 T3 from the binding protein in this assay about three times as well as does TIl itself, this assay provides a very sensitive estimate of potential T4 contamination. Recovery of added T, from pooled human serum. The data obtained using these modifications in typical recovery studies is presented in Table 1. In these studies, each of two different serum pools was enriched with two concentrations of TS; one low and the other high. This allowed measurement of both the absolute Ta levels as well as the differences between the two totals. Triplicate samples of high and low specimens were assayed using three and 6-ml samples of serum, respectively. The T, concentrations given are the total amount recovered. The differences between the high and low enriched samples were in good agreement with the expected findings. overlapped

614

P. R. LARSEN

In other recovery studies performed periodically during our experience with this assay, the recoveries have ranged from a low of 87 to a high of 110%. The mean recovery was 100%. Influence of T, on the Measurement of TS in Human Serum Since the amounts of T4 in human serum are considerably greater than those of Ta, it is important to eliminate any effect of this hormone on the measurement of Ta. Theoretically, T, could cause an artifactual increase in T3 either through contamination of the T3 spot with T4 itself or via monodeiodination of Td to Ta during the assay. The former possibility appears to be adequately eliminated by the absence of significant T3 activity in the extract of paper segment A. The second possibility, that of deiodination of Tq to T3 during the assay procedure, is more difficult to evaluate. This deiodination could occur either during the column chromatography or the paper separation. Both of these possibilities were investigated. Eflect of column chromatography on conversion of lz51 T, to lz51 T,$. TO examine the possibility that Td could be deiodinated to TS during this procedure, the following studies were performed. Large amounts (2-3 X 10° counts/min) of lz51 T, and tracer amounts of 1311T3 were added to serum and then extracted by the above methods. The dried eluate was chromatographed as above. As controls, aliquots of the same I’751Ts and 1311T7 were applied to paper and run simultaneously to allow detection of any lzaI T, contamination in the ?l T1 preparation. The 1311TS was necessary for both localization and recovery calculations. The strip was divided into 0.5-inch segments and the five segments containing the highest lzlI counts constituted the T3 peak. This was similar in length to segment B of Fig. 1. The percentage of the original counts added to the paper for both isotopes found in this strip could then be calculated (Table 2, columns 1 and 2). Invariably, 1.3 to 1.6% of the lz51 initially added as T1 was present in the 13iI T3 peak. There were similar amounts of 1251in segments corresponding to both A and C of Fig. 1 (not shown). These I251 counts in the T, peak could represent laaI TR, either that present in the original tracer or generated during chromatography, 1251T1, or labeled debris present in relatively small amounts in all portions of the strip. Since roughly similar amounts of lz51 are found in the T3 segment of the three control samples in both studies, it is clear that this material is not generated during the column chromatography. In both studies, the mean lz51 in segment B in the serum specimens (1.52 and 1.44) is lower than that found in the controls (1.67 and 1.93) which had not been passed over the column. This could result from the fact that the proportion of the 1311T3 recovered in the serum T3 peak is also substantially lower than that found in the control specimens. The difference between the recovery of 1311T3 in the serum extract (65.5 and 67.9) and the control samples (78.4 and 80.0) may reflect either the results of damage to the 1311T3 during drying and/or a broadening of the T3 peak in the chromatographed serum extract due to the technical process involved in the transfer of the dried column eluate to the paper.

TRIIODOTHYRONINE

Table t.-Effect

IN HUMAN

of Column Chromatography on 125IThyroxine in Human Serum Radioytivity

Experiment

A

1 Serum 2 Serum 3 Serum 4 Serum

Mean 2 SEM Control Control Control

1 2 3

Mean i SEM Experiment Serum

Serum Serum Serum Serum Serum

IsI T3 After Rechromatography of Segment B in Paper Segn$ent B 4t 1aq Net ‘51 Tz as Fraction3 Ef Initial % of Total I=1 Ta % of Total 1311T3 IsI in Segment B

=I % of Total I=1 Ta

1.53 1.50 1.44 1.60

63.6 66.8 67.6 64.0

24.8 32.3 39.2 27.1

0.47 0.57 0.66 0.53

1.52 + 0.03

65.5 ? 1.0

30.9 -r- 3.2

0.56 _t 0.04

1.73 1.69 1.59

78.5 78.4 78.2

36.6 38.6 34.2

0.63 0.65 0.54

1.67 ? 0.04

78.4 ? 0.1

36.5 _t 1.3

0.61 + 0.03

1.57 1.59 1.31 1.38 1.32 1.44

59.5 69.5 71.5 66.5 74.4 65.7

44.4 53.5 51.4 41.7 40.9 35.8

0.94 0.98 0.75 0.69 0.58 0.63

1.44 ? 0.05

67.9 2 2.1

44.6 ? 2.7

0.76 t 0.07

1.98 1.89 1.92

79.8 80.2 80.1

48.9 35.1 45.5

0.97 0.66 0.87

B

1 2 $ 3 4 $ 5 6 1:

Mean C SEM Control Control Control

615

SERUM

1 2 3

Mean ? SEM 1.93 * 0.03 80.0 f 0.1 43.2 f 4.1 0.83 r 0.09 -___ * Figures in column 3 represent fraction of 1251 counts in column 1 that were again found in the T, peak after rechromatography. t Figures in column 4 are determined by multiplication of figures in column 1 by those in column 3 and correcting this for difference in 1311Ts yield between each individual serum sample and mean of control samples as given in column 2. Thus, for Experiment A, #l; 24.8 x 1.53 x 78.4163.6 = 0.47. $ KI was added at 2 x 10-s M to eluate after column chromatography to eliminate potential iodine exchange.

Further procedures were necessary to determine what fraction of the 1351in the T, peak was actually lzSI TB. The radioactivity in segment B was eluted from paper in MeOH/2N N&OH, dried under Nz at 50°C, and rechromatographed. The fraction of the lz51 counts in segment B still running with 1311T3 on this second chromatography was then determined (Table 2, column 3) and corrected for any deviation of the 1311T3 yield from 82%. This latter figure is the maximum yield of purified 1311 T3 in the 2.5-inch T3 segment B when chromatographed in this manner. The fraction of the 1251counts in the initial T3 peak due to actual lz51 T3 was then determined by multiplication of this fraction (Table 2, column 3) by the initial gross percentage of 1251in the B segment (Table 2, column 1) and correcting each to the mean T3 yield of the appropriate control segments (Table 2, column 2). This figure then represents the fraction

616

P. R. LARSEN

of the initial lz51 T4 counts which are apparently I”51 TB. In both experiments A and B there is good agreement between the mean per cent of lZ51T3 in the serum and that in the control specimens. This indicates that there is no significant net conversion of lz51 Tq to la51 TB during the extraction and drying procedure. Since the amounts of lZ51T3 found in these studies could be present as a contaminant in the original 1251Tq, one cannot assume it arose during the paper chromatography. It is somewhat disturbing to note that there is a significant variation of the calculated T3 fraction in the individual samples (from 0.47 to 0.98). This could reflect one of two possibilities: experimental variation or true differences in Tq to T8 conversion in different specimens either during column or paper chromatography. Evaluation of the effect of pupperchromatography on the generation of 1g51 TS from lz5Z T,,. A solution containing a large amount of 125I T*, about 0.5 pg of carrier Tq, and 1311Tq in tracer amounts was chromatographed in triplicate. The 2.5-inch Ts peak was localized using the 1311counts as in the above studies, and the 12jI counts appearing in this peak were expressed as a fraction of the Ia51 counts originally applied to the paper (Table 3, Run I,l-3, columns 1, 2, and 3). The la51 T* peak was also located and the tracer and carrier Td eluted in MeOH/2N NH,OH and dried under Nz at 50°C. A tracer amount of Ia11 T3 and more carrier T1 were added to each of the triplicate tubes containing the dried eluate, and the material was rechromatographed (Run II, l-3). The T3 peak was isolated as before, the fraction of 1251in this peak determined and the 125I TJ eluted again. The same 125I T4 was in this way carried through five successive chromatographic runs and the amount of 12sI appearing in the T3 peak determined for each run in the triplicate samples (Table 3, columns 1 and 2). As in the above studies, 1.3 bo 2.4% of the total 1251originally applied to the paper as lP51 T+ was present in T3 peak. To determine what fraction of the 125I present in the Ts peak was actually 125I T3 (as opposed to labeled debris or 12jI T4), the material in each of these peaks was eluted, dried and rechromatographed until a constant 1251/1311ratio was obtained in the 1311T, peak. This required three to four chromatographic runs for I-IV. With the triplicates from run V, the *?51 counts were too low relative to the 1311counts to obtain accurate estimates after the third run and the lZ51/1311ratio was still increasing.When a constant lZ51/1311ratio is attained, the 1251counts present are assumed to be pure T,. They were corrected for 1251 decay, the recoveries of 1311TR and the losses of lZ51during the repeated elutions from the chromatography paper. These corrected counts were expressed as a percentage of the original counts (Table 3, column 4) and this fraction multiplied by the initial percentage of 1251in the original Ta peak for all five runs (Table 3, column 2) to give an overall figure for generation of TA. 12jI T, appeared during each successive chromatographic run of the 1?51T* preparation. While there was an initial decrease in this fraction from 0.66 to 0.38%, relatively constant amounts of 0.3 to 0.4 % of initial 1251T., were found in the last four runs. The higher value in the first run presumably represents T.? present as contaminant in the tracer preparation. In the last column of Table 3, the fractions are multiplied by two, assuming that random lz51 labeling and deiodination of T_, in the 3’ and 5’ position occurs so that 21Z51Tq + 1251- + I-

617

TRIIODOTHYRONINE IN HUMAN SERUM

of Conversion of lz51 Thyroxine to 125Triiodothyronine During Paper Chromatography

Table 3.-Estimation Radioactivity

IsI

in Paper Segment B

Triiodothyronine

1

2*

3t

=2 CPM

Fraction of ‘=I Ta (%)

Fra:Y I311 Ts (%1

4$ Fraction of Initial 1zI in s;g(m;yt 0

I.1 2 3

28,130 23,224 23,951

1.46 1.28 1.27

81.7 76.9 60.8

48.6 54.7 45.7

0.71 0.70 0.58 Mean 0.66

II 1 2 3

21,505 24,002 17,876

1.93 2.38 1.68

77.6 79.8 80.2

22.8 15.5

0.44 0.37 0.32 Mean 0.38

0.75

0.39 0.42 0.38 Mean 0.40

0.79

0.26 0.37 0.32 Mean 0.32

0.63

Chroma‘OK”a,pt;lc

III 1 2 3

IV 1 2 3

Vl

2 3

12,435 11,111 13,215

3,549 3,641 3,727

2,797 2,184 2,595

2.18 2.30 2.35

1.29 1.57 1.35

1.69 1.61 1.62

77.8 78.3 76.1

81.3 80.2 79.4

77.2 78.3 80.1

19.0

17.9 18.3 16.2

20.2 23.6 23.7

24.3 ** 20.5 21.6

55

6Q

Fraction of ‘=I T4 (%)

Net Deiodination of ISI T4 (%1

0.41 0.33 0.35 Mean 0.36

1.73 r 0.1 SEM

0.72 k 0.05 SEM

* Column 2 is computed by dividing 1251 counts of column 1 by total 1251 T, counts applied to paper. f Column 3 is computed by dividing 1311 counts in Segment B by total 1311 T3 counts applied to paper. New 1311 T, in tracer quantities was added to 1251 T, for each run. $ Figures in Column 4 represent fraction of 1251 counts in Column 1 that can be identified as 1251 Ts after repeated rechromatography of B Segment eluate to constant 125IJ1W. Counts were corrected for losses due to decay, incomplete elution from the paper and net 131T T, recovery. $ Column 5 is computed by multiplying figures in Column 4 by those in Column 2. ll Column 6 is Column 5 multiplied by 2 to correct for loss of 1261 from 50% of 12&IT,. 1) Roman numerals indicate five chromatographic runs. Arabic numerals are triplicate samples. lzaI T4 starting material for each run was obtained by elution of T, from strips of previous run. ** I251 counts were too low to obtain accurate yields after third rechromatography. Recovery of lzsI was then about 64%; recovery of 1311T, was 88%.

+ lz51 Ta + T.?. Therefore, in this system, it would appear that about 0.7% of the 9 T4 is deiodinated to T3 during paper chromatography. Effects of paper chromatography on L-thyroxine. Attempts were then made to verify this figure by the use of carrier T4 with actual assay of T7 generated during the paper chromatographic separation. Sodium L-thyroxine pentahydrate

618

P. R.

LARSEN

Table 4.-Generation of Triiodothyronine From Thyroxine During Paper Chromatography T3 Activity T4 Applied to Paper (ng)

1. 2. 3. 4. 5.

655 752 675 736 627

A

13.1 11.1 8.7 8.1 6.7

Recovered

(ng)

B

C

Net Ta Assayed (ng) B-(A+C)/Z

13.0 11.6 12.2 15.8 12.2

8.4 7.4 3.8 4.3 2.5

2.2 2.3 5.9 9.6 7.6

Corrected for Recovery and for Added 1311Ta

3.3 3.0 10.6 14.0 11.6

Mean + SEM

Gen&$d -

0.5 0.4 1.6 1.9 1.9

1.3 -C 0.3

* Measured T, was divided by amount of T4 applied to paper.

(Mann Research) was dissolved in 0.04 N NaOH at a concentration of about 20 pg/ml. The exact concentration was verified by determining the optical density at 325 rnp using the molar extinction coefficient.3 I351 Tq (about 0.5 ng) was added in tracer concentration and an aliquot taken for recovery calculations. Approximately 0.15 ml of this solution was chromatographed as above. The Tq was eluted from the 2.5 inches of paper in MeOH/2N NH40H, dried under Nz and rechromatographed two additional times. The first two chromatographic runs were for purposes of purification. During the third chromatography, I311Ts (0.5 ng) was added to localize and determine the recovery of Ts. The 2.5-inch segment containing the 1311T3 peak and segments of equal size from both sides of the peak (A and C of Fig. 1) were assayed for T3. Significant amounts of Ts were present in all three segments (Table 4). This T3 activity was consistently higher in the A segment than in the C segment suggesting that it might represent some Tq spilling over into this area. In an attempt to correct for this, we have assumed that the decrement in T4 will be linear with increasing distance from the TA peak. Therefore, we have subtracted a background activity from the B segment which is the average of that present in the A and C strips. After making this correction, there is still a significant amount of T,-displacing activity which cannot be accounted for by the presence of Tq and presumably represents Ts derived from deiodination of thyroxine. This was expressed as a fraction of the amount of TJ initially applied to the paper as determined from the lz51 counts. The estimated mean conversion rate by this method was 1.3%) approximately double that obtained with the tracer studies and there is considerable difference between the various individual samples. In other studies, the order of magnitude of the conversion estimate was the same, but there was considerable variation from sample to sample. Triiodothyronine

determinations in serum enriched with purified L-thyroxine.

Sodium L-Thyroxine pentahydrate plus tracer Ta was chromatographed two times as described above to remove contaminating TS. After the second chromatography, the Ta peak was eluted from the paper in 5- to 6-ml aliquots of a single serum pool. The T, content of control and each enriched serum sample was then determined by the method of Murphy and Pattee Table 5, columns 1 and 2). The T3 content of both the pooled serum and the unpooled enriched samples was then determined by the standard technique. During the assay for Ts in samples 1 to 8, a small amount of T3 displacing activity (1.5 to 2.1 ng) was

TRIIODOTHYRONINE

IN HUMAN

619

SERUM

Table 5.-Triiodothyronine Determinations in Serum Enriched With Purified Thyroxine 1

Total Td SamPIe (M/loo ml)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

54.3

69.1 73.9 73.0 68.0 74.2 77.8 64.1 17.7 18.1

2

ToL

EadoF (lJg/lOO ml)

7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 1.0 1.0

hvl@J

ml)

47.3 62.1 66.9 66.0 61.0 67.2 70.8 57.1 16.7 17.1

6 End&e-

Net Increase

&%I,)

~~ilz~

m/ml

2.7 3.4 2.4

1.3 1.3 1.3 1.3 1.3 1.3 1.3 1.3 0 0

1.4 2.1 1.1 2.0 2.0 2.3

3.3 3.3 3.6 2.9 1.2 0.7

1.6

2

-

Ta

% of T4 Added

0.30 0.34 0.16 0.30 0.33 0.34 1.6 0.23 0 0 0.7 0.42 1.6 0.93 Mean 0.34kO.08

SEM

again found in the A and C paper segments of the heavily T4-enriched sera. This activity was presumably due to the extremely high concentrations of Tq present in the sera which appeared in the Ts segment despite the separation of about 20 cm between the T3 and T4 peaks. The amounts present in the A and C segments were averaged and subtracted from that present in the B segment. This correction amounted to 25% or less of the T3 actually measured in segment B which varied from 4.1 to 8.7 ng for these specimens. The total amount of Ta present in the serum samples and that present in the native serum pool are shown in Table 5, columns 4 and 5. The net increase in T3 due to enrichment of the serum with purified Tq could then be determined (column 6) and expressed as a fraction of the net increase in purified T4 (column 7). As can be seen in Table 5, the mean percentage deiodination was 0.34 t 0.08 (SEM). While this value is lower than that calculated for T3 generation from tracer Tq, it must be kept in mind that only about 65% of the Tq present in serum is actually extracted and applied to the paper. Serum Triiodothyronine

in Thyroid States

The modifications described above resulted in a more sensitive method for T1 determination. While there was evidence of artifactual elevation of the T3 due to deiodination of Tq, this did not appear to be quantitatively significant for clinical purposes. The median amounts of thyroxine present in normal serum are about 75 ng/ml.” If 0.4% of this thyroxine were deiodinated to Ts, an artifactual increase of only 0.3 ng would be anticipated. Therefore, we next evaluated the usefulness of this method in subjects who were normal, hyperthyroid and hypothyroid. The values reported were not corrected for thyroxine deiodination. All sera were collected from patients seen at the Presbyterian-University or Oakland Veterans Administration Hospitals. The diagnosis was verified in each patient by both clinical examination and suitable laboratory studies. No patient whlose thyroid status was equivocal was included in this data. Specimens were stored at -20X! until assayed. All determinations were performed in duplicate on different days to avoid systematic errors. In practice, 6-ml serum specimens

620

P. a.

LARSEN

Table 6 Clinical

Status

Pool A Pool B Euthyroid Hyperthyroid Hypothyroid

Number

11 12 17 13 6

2.1 1.8 1.8 6.7 0.66

I!z 0.3 * z!I 0.2 * Z!I0.4 +- 3.3 z? 0.39

7.6 8.6 7.5 f 26.4 + 13.0 2.2 r 1.3

69 107 (42) 41 a 11.0 $ 31 z!I 13.5 $

* Enriched with 1 ng/ml T, so that endogenous Ta levels are 1.1 and 0.8 ng/ml pools A and B, respectively. t Median value for normal sera is estimated from data of Ekins et al.5 $ Mean ? SD of T4/T3 for individual specimens.

for

were used for normals, 6 to 8 ml for hypothyroid patients and 1.5 to 6 ml for hyperthyroid subjects. Pooled serum was obtained from the Clinical Chemistry laboratory at the Presbyterian-University Hospital. Results of the T3 determinations in various thyroid states are presented in Table 6. The values for pools A and B show the results of assays of quality control sera. Each of the eleven or twelve determinations was performed in a different run and, therefore, the percent standard deviation (13% ) reflects the between-assay variation in the method. The within-assay standard deviation at normal T3 concentrations for eight samples was approximately the same ( 12% ) . Pools A and B were enriched with 1 ngJm1 of TS to allow more precise determinatilon of the T3 levels. Therefore, the endogenous T3 concentration in these sera is 1.I and 0.8 ng/ml, respectively. This is appreciably below the mean of 1.8 value obtained in specimens from normal euthyroid subjects (primarily ambulatory, professional personnel). The mean T3 in untreated hyperthyroid subjects was 6.7 ng/ml and was 0.66 ng/ml in those with hypothyroidism. The mean difference between duplicates was 0.6 ng/ml in the sera from euthyroid subjects, 0.3 ng/ml in the hypothyroid patients, and 0.8 ng/ml in those with hyperthyroidism. The T, values are presented along with the mean of the Ti concentrations in the same samples in the hyper and hypothyroid subjects. The latter was measured by the Murphy-Pattee technique modified as previously described and results are given as total thyroxine.@ In the last column of Table 6, the T4/TS ratios are presented. The T4/TS ratio in the hyperthyroid specimens is about the same as that calculated for normal serum using an estimate of the median T4 found in a normal population. 5 In hypothyroid patients, the ratio is somewhat lower. It would be anticipated that the acute effect of complete inhibition of thyroid hormone secretion would be to increase the T4/T3 ratio since the Ts turnover rate is considerably more rapid than that of T4. In Table 7 this phenomenon was observed in subject H.J. following 1311treatment of a diffuse toxic goiter. Patient P.D., who received propylthiouracil, showed no substantial alteration in the T4/TR ratio during the early treatment period. One may speculate from the same considerations that synthesis of Ts in this patient was more severely impaired than that of TX during treatment with this drug.

621

TRIIODOTHYRONINE IN HUMAN SERUM

Table 7.-Effect of 1311and PropyIthiouracil Treatment of Hyperthyroidism on ThyroxinelTriiodothyronine Ratios (T4/T3) in Serum

H.J.*

4/13 4/18 4/20 4/22

24.6 23.6 22.2 17.8

6.3 4.9 2.3 2.4

P.D.f

5/26 5/29 6/l

59 56 44.5 39.4 31.4

> 15 15 9.7 11.8 10.3

6/2 6/4

39 48 97 74

37 46 33 30

* 1311, 6 mCi, 4/16. f Propylthiouracil, 200 mg q-6-h, begun on S/27/70.

DISCUSSION

The modifications in the method of Sterling et al. described in this report were made in an attempt to improve the sensitivity and precision of the method. These include increased separation of T3 and Ta during paper chromatography, increased sensitivity of the binding-displacement assay, elimination of any possible T4 contamination by assay of paper between the T3 and T4 peaks and the use of paper extract in the standard curve to minimize artifacts from this source. During preliminary studies, we found that the limiting factor in the development of an even more sensitive assay was the unpredictable artifact contributed by this extract. Sterling and his colleagues have reported values of 220 ng/lOO ml in normals, 865 ng/lOO ml in untreated thyrotoxics and 98 ng/lOO ml in patients with spontaneous hypothyr0idism.l The values given in TabIe 6 are 18 to 30% lower than these. The recovery data for known amount of T3 added to serum pools would eliminate incomplete recovery as a possible explanation for the lower values determined in our laboratory. Several other explanations are possible. Since the separation we obtain between T3 and T4 is somewhat greater than that reported by Sterling (about 19 cm versus about 10 cm), the possibility of contamination of the T,? spot with T4 would appear less likely under the conditions used in our study. Since T4 has two to three times the affinity for TBG as does Ts in the binding-protein assay, it is possible that the presence of small amounts of T4 ( < 1 ng) could artifactually increase the T3 estimate. Sterling et al. have reported finding less than 0.5% of T4 label in the T3 area.l If this were actually T4, it could contribute as much as 0.5 to 1.0 ng. to the T3 estimate for the above reasons. The absence of Tq in segment A appears to provide reasonable assurance that this is not occurring during our assay. A second possibility is that the Ts-displacing material often found in the extract of paper segment C may not be sufficiently separated fern T3 during a shorter migration from the origin. This would also result in an apparent increase in the TB levels measured. A third possible reason for the difference is that the

622

P. R. LARSEN

Tq to T3 conversion rates are smaller in our laboratory due to unknown differences in technique. Despite the improvements described here, the per cent standard deviation in the quality control samples is rather large ( 11 to 14% ) . The mean differences between the duplicates in the euthyroid subjects is 30% of the mean, 46% of the mean for hypothyroid patients, and in serum from subjects with hyperthyroidism, 11% . It is clear from these data that the method is much better at higher Ts levels. No comparable data have been published by other workers in this field so it is not possible to compare this aspect of the assay. Some of this variability may be due to the differences in conversion of Tq to T3 during the paper chromatographic step. If conversion of T4 to T3 occurred at twice the rate in some samples as opposed to others, the measured T3 values would be substantially different. The other disadvantage of the method at this time, in addition to the difficulty with an accurate detection of the lower T3 concentrations, is that up to 15 to 20 ml of serum may be required for duplicate determinations in hypothyroid patients and even 10 to 12 ml in normals. The problems of measuring small amounts of Ta in the presence of relatively large amounts of T4 need no reemphasis. Certainly the most disturbing aspect of our studies is the demonstration of conversion of Tq to T3 during the paper chromatographic separation. If 0.73% of Ti were converted to TS during this step, as suggested by the tracer studies, the net deiodination of T4 to T3 during the assay would be on the order of 0.44 to 0.47% since only about 65% of the Tq in the serum is actually applied to the paper. This agrees fairly well with the observed value of 0.34% using sera enriched with purified Tq. The interpretation of the higher values for deiodination obtained during paper chromatography of thyroxine alone is complicated by the apparently greater overlap of Tq in the T3 area during these studies. A net Tq deiodination rate of 0.45% would add about 0.3 ng/ml or 20% to the endogenous T3 concentration in the sera of normal subjects. In hyperthyroid sera, a corresponding increase of about 1.l ng/ml would be predicted, while in the sera from hypothyroid subjects there would be a negligible contribution from Tq. Thus, the corrected mean values would be 1.5 ng/ml for euthyroid and 5.6 ng/ml for hyperthyroid subjects. There is a rather broad range in the calculated conversion rates in Table 5 (O-0.92%) that may reflect a variation either in the deiodination rate itself or its estimation. If the actual deiodination rate does vary from sample to sample, it is difficult to apply a constant conversion rate for all studies. Thyroxine deiodination of even a greater magnitude than reported here has been observed by Fisher and Dussault during the assay of T3 using the method of Sterling.7 In earlier reports, both Fisher and Taurog have demonstrated deiodination of 6 to 20% of labeled T4 following application to filter paper.8vg In the latter study, it was shown that either carrier Tq or human serum proteins could inhibit the deiodination although the effective amounts of carrier were greater than that present in 5 to 10 ml of human serum. In addition, while the percentage of 1311- increased after application of 1311Tq to paper, there was no increase in 1311T3 or other 1311labeled degradation products suggesting the deiodination always involved the labeled iodine atom.

TRIIODOTHYRONINE

IN HUMAN

SERUM

623

Our efforts have been directed towards a different aspect of this problem: the quantities of T3 arising from T4 during the assay. Our inability to detect substantial amounts of labeled T3 arising from Tq is oonsistent with Taurog’s previous observations. When Tq is added to serum and the change in T3 concentration estimated, the difference, though significant for our purposes, is negligible when compared with the deiodination rates of 6 to 20% referred to above. It would appear either that T3 is not a major by-product of this deiodination reaction or that the presence of unknown substances in the serum extract inhibits the production of substantial amounts of T3 from T,. As a solution to the problem of deiodination, Dussault, Lam and Fisher have used a column chromatographic method for the separation of T3 from TA which alllows correction of the measured T3 for the increase due to T4 deiodination in each specimenlO The normal values obtained using this technique appear to be slightly lower than those presented in Table 6 even after correction of our values for deiodinated T,. An individual correction for each sample is not possible in the paper system. As can be seen in Table 2 and 3, 1.2 to 2.5% of the T, label is usually found in the T3 segment even with the wide separation of T3 from T, that we customarily obtain. Of this, only a small fraction (15 to 25% ) can be identified as T3 after repeated chromatography (Table 3). The procedures necessary to quantitate the fraction of T4 tracer which is actually T3 would obviate the assay for T3 levels in this segment. It is obvious that these artifacts represent only a relatively small fraction of even our lower T3 estimates and one may question their significance. We have had no difficulty distinguishing hyperthyroid patients from normals and have observed 2 of some 40 to 50 patients with hyperthyroidism who had normal Tq and elevated T3 levels. The data for patient H.J. in Table 7 demonstrate that a substantial change in T, can be measured in the absence of significant alteration in T* levels. However, the method does sot appear to have the necessary precision for use in kinetic studies. In these, the rapid turnover rate of TX and the large distribution volume considerably magnify any errors in the Ts concentration itself. In comparing the physiologic importance of T3 with Tq, there is a further multiplication by a factor of 3 to 4 to account for the greater biological potency of Ts. It is even more critical to eliminate the possibility of either T, deiodination or contamination in studies dealing with the question of in vivo T4 to T3 conversion. Braverman, Ingbar and Sterling have reported no deiodination of either tracer T4 or of carrier added to serum at concentrations of about 12 pg/lOO ml during paper chromatography. I1 Their inability to detect deiodination could be related to the use of smaller amounts of tracer or carrier T, in these studies. The estimated in vivo Ta deiodination rate would, of course, have to be corrected for any in vitro deiodination. While it would appear that this method is quite suitable for clinical purposes, we have not been able to attain sufficient freedom from artifact or sufficiently satisfactory precision to justify its use in physiologic studies. The primary problems in our hands arise during the paper chromatographic separation of Tq and Ts and in the quantitation of the isolated T,. Deiodination of T4 appears to occur only during paper chromatography and the presence of unknown substances

624

I’. R. LARSEN

in the MeOH/2N NHdOH extract of chromatography paper limits the development of suitable sensitivity. This may contribute as well to the difficulties with precision. The production of an antibody with relative specificity for Ts as opposed to Tq which has been reported by Brown, Ekins, Ellis and Reith suggests a possible solution to these prob1ems.l” This app roach is now being pursued in our laboratory. ACKNOWLEDGMENTS The author would like to thank Dr. Kenneth Sterling for providing details of his method prior to its publication. The careful technical assistance of Mrs. C. Livstone, Miss .I. Dockalova and Mrs. D. Sipula is gratefully acknowledged. The review of this manuscript by Dr. W. Tong was greatly appreciated. REFERENCES 1. Sterling, K., Bellabarba, D., Newman, E. S., and Brermer, M. A.: Determination of triiodothyronine concentration in human serum. J. Clin. Invest. 48: 1150, 1969. 2. Bellabarba, D., Peterson, R., and Sterling, K.: An improved method for chromatography of iodothyronines. J. Clin. Endocr. 28:305, 1968. 3. Gemell, C. L.: The apparent ionization constants of the phenolic hydroxyl groups of thyroxine and related compounds. Arch. Biochem. Biophys. 54: 359, 1955. 4. Murphy, B. P.: The determination of thyroxine by competitive protein-binding analysis employing an anion exchange resin and radiothyroxine. J. Lab. Clin. Med. 66: 161, 1965. 5. Ekins, R. P., Williams, E. S., and Ellis, S.: The sensitive and precise measurement of serum thyroxine by saturation analysis (competitive protein-binding assay). Clin. Biochem. 2:253, 1969. 6. Larsen, P. R., Atkinson, A. J., Jr., Wellman, H. N., and Goldsmith, R. E.: The effect of diphenylhydantoin on thy-

roxine metabolism in man. J. Clin. Invest. 49: 1266, 1970. 7. Fisher, D. A., and Dussault, J. H.: Contribution of methodologic artifacts to the measurement of T, concentration in serum. J. Clin. Endocr. (in press). 8. Fisher, D. A.: Artifactual deiodination during cellulose-starch thin layer chromatography. J. Clin. Endocr. 28:717, 1968. 9. Taurog, A.: Spontaneous deiodination of 1slI-labeled thyroxine and related iodophenols on filter paper. Endocrinology 73: 45, 1963. 10. Dussault, J. H., Lam, R., and Fisher, D. A.: The measurement of serum triiodothyronine by double column chromatography. Unpublished. 11. Braverman, L. E., Ingbar, S. H., and Sterling, K.: Conversion of thyroxine (T4) to triiodothyronine (T3) in athyreotic human subjects. J. Clin. Invest. 49:855, 1970. 12. Brown, B. L., Ekins, R. P., Ellis, S. M., and Reith, W. S.: Specific antibodies to triiodothyronine hormone. Nature 226: 359, 1970.