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Endocrinology, doi:10.1210/en.2007-1566
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Endocrinology Vol. 149, No. 8 4218-4228
Copyright © 2008 by The Endocrine Society

Effects of Substitution and High-Dose Thyroid Hormone Therapy on Deiodination, Sulfoconjugation, and Tissue Thyroid Hormone Levels in Prolonged Critically Ill Rabbits

Yves Debaveye1, Björn Ellger1, Liese Mebis, Theo J. Visser, Veerle M. Darras and Greet Van den Berghe

Department of Intensive Care Medicine (Y.D., B.E., L.M., G.V.d.B.), Laboratory of Comparative Endocrinology (V.M.D.), Catholic University of Leuven, B-3000 Leuven, Belgium; Department of Internal Medicine (T.J.V.), Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands; and Department of Anesthesiology and Intensive Care Medicine (B.E.), University Hospital of Muenster, D-48149 Muenster, Germany

Address all correspondence and requests for reprints to: Greet Van den Berghe, M.D., Ph.D., Department of Intensive Care Medicine, Catholic University of Leuven, B-3000 Leuven, Belgium. E-mail: greet.vandenberghe{at}med.kuleuven.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To delineate the metabolic fate of thyroid hormone in prolonged critically ill rabbits, we investigated the impact of two dose regimes of thyroid hormone on plasma 3,3'-diiodothyronine (T2) and T4S, deiodinase type 1 (D1) and D3 activity, and tissue iodothyronine levels in liver and kidney, as compared with saline and TRH. D2-expressing tissues were ignored. The regimens comprised either substitution dose or a 3- to 5- fold higher dose of T4 and T3, either alone or combined, targeted to achieve plasma thyroid hormone levels obtained by TRH. Compared with healthy animals, saline-treated ill rabbits revealed lower plasma T3 (P = 0.006), hepatic T3 (P = 0.02), and hepatic D1 activity (P = 0.01). Substitution-dosed thyroid hormone therapy did not affect these changes except a further decline in plasma (P = 0.0006) and tissue T4 (P = 0.04). High-dosed thyroid hormone therapy elevated plasma and tissue iodothyronine levels and hepatic D1 activity, as did TRH. Changes in iodothyronine tissue levels mimicked changes in plasma. Tissue T3 and tissue T3/reverse T3 ratio correlated with deiodinase activities. Neither substitution- nor high-dose treatment altered plasma T2. Plasma T4S was increased only by T4 in high dose. We conclude that in prolonged critically ill rabbits, low plasma T3 levels were associated with low liver and kidney T3 levels. Restoration of plasma and liver and kidney tissue iodothyronine levels was not achieved by thyroid hormone in substitution dose but instead required severalfold this dose. This indicates thyroid hormone hypermetabolism, which in this model of critical illness is not entirely explained by deiodination or by sulfoconjugation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROLONGED CRITICAL illness is invariably characterized by a suppressed thyroid axis as revealed by a reduced pulsatile TSH secretion, low plasma T4 and T3, and normal or only slightly elevated reverse T3 (rT3) concentrations, a constellation commonly known as low T3 syndrome (1, 2). The pathophysiology of this low T3 syndrome is complex and involves alterations in both the central nervous system and the peripheral tissues (3). Depressed hypothalamic TRH gene expression appears to play a role specifically in the chronic phase of critical illness (4). Infusion of TRH is able to reactivate TSH secretion in prolonged critical illness and to increase and maintain plasma T4 and T3 within the normal range (5, 6). Concomitantly, changes in peripheral metabolism of thyroid hormone occur (7, 8) that could explain why both in an animal model of prolonged critical illness (9) and in critically ill patients (10, 11) high doses of T4 and T3 appear to be required to normalize plasma T4 and T3 levels, as is obtained with TRH infusion (5, 6, 9, 12).

It is still controversial whether the reduction in plasma T3 is a beneficial adaptation resulting in a protection against catabolism or whether it is a maladaptation contributing to a worsening of the disease (2, 13). It has not been clearly demonstrated that substitution of critically ill patients with thyroid hormone reduces intensive care unit mortality (>20% worldwide) (2, 10, 11), and it is even unclear whether thyroid hormone is taken up and metabolized in tissues when T4 and/or T3 are administrated during critical illness.

In normal physiological conditions, the bulk of thyroid hormone is metabolized via sequential monodeiodination whereby T4 is converted to T3 via outer ring deiodination by iodothyronine deiodinase type 1 (D1) and type 2 (D2), whereas both T4 and T3 are inactivated by inner ring deiodination, catalyzed primarily by type 3 deiodinase (D3) (14). During prolonged critical illness, thyroid hormone metabolism is substantially altered. Suppressed hepatic D1 activity and increased hepatic D3 activity were recently documented in prolonged critically ill animals (12) and patients (15, 16). Moreover, elevated plasma levels of T4 sulfate (T4S) in patients who died in the intensive care unit (17) and increased plasma T3S/T3 ratios in patients with severe systemic illnesses (18) have been reported. Although these findings suggest that sulfoconjugation could also be involved in accelerated degradation of thyroid hormone during critical illness (17, 18), the precise role of this major alternate pathway of thyroid hormone metabolism (19) in this setting remains unknown.

The present study attempts to further characterize the metabolic fate of thyroid hormone during critical illness (monodeiodination and sulfoconjugation metabolic pathway and tissue levels) by studying the effect of treatment with two doses of thyroid hormone as compared with saline and with TRH. The two dose regimes for thyroid hormone treatment comprised either substitution dose or a higher dose of T4 and T3, either alone or combined, targeted to achieve at least those plasma hormone levels that can be obtained by TRH infusion. The impact of these interventions on plasma 3,3'-diiodothyronine (T2) and T4S levels, tissue deiodinase activities, and thyroid hormone levels in liver and kidney was documented.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study set-up and protocol
For the experiments, we used a validated, chronically instrumented in vivo animal model of prolonged critical illness that reveals several of the clinical, biochemical, and endocrine manifestations of the human counterpart (20). In this model, prolonged critical illness is brought about by a third-degree burn injury of 20% body surface area of adult, male New Zealand White rabbits of ±3 kg. All animals were parenterally fed from d 2 on to avoid starvation-induced endocrine alterations (21). In addition, blood glucose levels were kept less than 180 mg/dl by frequent blood glucose monitoring and titration of insulin infusion when necessary. The model has been described in detail previously (12, 20, 22). All animals were treated according to the Principles of Laboratory Animal Care formulated by the U.S. National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health. The study protocol was approved by the University of Leuven ethical review board for animal research (protocol P03052), and the animal experiments were supervised by an ethical review board veterinarian.

On the morning of d 4 of the illness, surviving rabbits were randomized by sealed envelopes to receive a 4-d treatment with saline 0.9% (placebo group), T4, T3, T4 plus T3, or TRH (60 µg/kg/h after an initial bolus of 60 µg/kg TRH) (Fig. 1Go). T4 and T3, alone or combined, were infused in two dose schemes. In the so-called substitution-dose experiment, rabbits received T4 (3 µg/kg/d), a continuous infusion of T3 (1 µg/kg/d), or the combination of both. Animals allocated to the so-called high-dose experiment were infused with 9 µg/kg/d T4, 5 µg/kg/d T3, or the combination of both. The doses used in the substitution-dose experiment approximated the replacement doses of T4 and T3 observed in rabbit iodothyronine kinetic studies (23, 24). Those used in the high-dose experiment attempted to bring plasma T4 and T3 levels at least into the range obtained by TRH infusion (9). We used this specific target because TRH infusion can restore physiological plasma T4 and T3 levels and can normalize D1 and D3 activities in critically ill rabbits (12, 25). The dose of TRH (UCB Pharma, Brussels, Belgium) was defined in previous studies (20, 22).


Figure 1
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  		FIG. 1. Flow chart of study design. Animals were randomized on d 4 after prevelation of a reference sample to receive saline, substitution doses (SD), and high doses (HD) of T4, T3, and T4 + T3 or TRH. After 4 d of intervention, animals were killed and tissue samples taken [saline, n = 8; T4 (SD), n = 8; T4 (HD), n = 9; T3 (SD), n = 9; T3 (HD), n = 9; T4 + T3 (SD), n = 8; T4+T3 (HD), n = 9; TRH, n = 8). In addition, four healthy animals were used as a control group. PN, Parenteral nutrition.

 
To control blood glucose levels, at least two arterial blood samples were taken daily. On d 4 before randomization and at 0900 h on d 5, 6, 7, and 8, an additional 4 ml blood was sampled in lithium citrate tubes (BD Biosciences, Temse, Belgium), immediately centrifuged, and then stored at –30 C until assay for T4, T3, rT3, and TSH.

On d 8, the animals were weighed and killed by sodium pentobarbital (Nembutal; Sanofi-Winthrop, New York, NY). Blood and tissue samples from four healthy animals were used as a control group. Tissue samples were taken from liver and kidney, snap-frozen in liquid nitrogen, and stored at –80 C until further analysis. The experiments were ended when a minimum of eight survivors on d 8 was obtained in all groups. To avoid bias from the inclusion of animals with terminal organ failure and to avoid unnecessary suffering, irresponsive or moribund animals were euthanized during the course of the experiment. These latter rabbits were excluded from hormonal analyses but included into the mortality calculation.

Determination of plasma thyroid hormone concentrations
Plasma total T4, T3, and rT3 concentrations were determined by RIA using 125I-labeled T4, T3, and rT3 (Amersham, Buckinghamshire, UK), antisera against T4 and T3 (Byk-Sangtec Diagnostica, Dietzenbach, Germany), and standard preparations of T4, T3, and rT3 in hormone-free human serum (26). The antiserum against rT3 was developed and provided by T. J. Visser (27). Measurement of total thyroid hormone levels was performed because catheters for blood sampling needed to be heparinized to prevent clotting, which induces artifactual results with free hormone determinations (28).

Plasma T2 concentrations were determined by RIA employing 125I-labeled T2, anti-T2-antibody nos4105 (dilution 1:150,000), buffer (0.06 M barbital buffer; 0.15 M NaCl; and 0.1% BSA, pH 8.6), and standard preparations of T2 (Henning GmbH, Berlin, Germany) in hormone-free human serum (29). After incubation overnight at room temperature, nos4105-antibody bound [125I]T2 was precipitated by a secondary antibody, supernatants aspirated, and the radioactivity in the precipitates quantified. [125I]T2 was obtained via the iodination of 3-T1 (gift from Dr. P. Block, Jr., University of Toledo, Toledo, OH) with 125I via the chloramine T method and purified by chromatography on Sephadex LH-20 before use (29).

T4S was measured on methanol extracts of plasma by RIA procedure using antibody Wu091 (30, 31). The final methanol concentrations in the assay were 11%. T4S, both labeled and unlabeled, was prepared by the method of Eelkman Rooda et al. (30). The RIA was carried out in disposable glass tubes using 200 µl buffer (0.06 M barbital buffer; 0.15 M NaCl; and 0.1% BSA, pH 8.6), 100 µl 11% methanol containing either the unknown or standard amounts of unlabeled T4S, and 100 µl Wu091 antiserum (dilution 1:300,000), and 100 µl [125I]T4S (20,000 cpm). The tubes were mixed thoroughly and incubated at 4 C overnight. A sufficient amount of tittered secondary antibody was then added. The tubes were mixed, incubated at 4 C overnight, and centrifuged at 2000 x g for 20 min. Supernatants were aspirated, and the radioactivity in the precipitates was quantified.

Plasma concentrations of rabbit TSH (rTSH) were measured by a specific RIA, making use of antiserum against rTSH raised in guinea pig. The RIA reagents were provided by Dr. A. F. Parlow (National Hormone and Peptide Program, Torrance, CA) (12, 20). Because frequent blood sampling for determination of rTSH concentration time series would inevitably evoke an intolerable amount of blood loss (32), rTSH was measured only once daily.

For all used RIA systems, rabbit plasma dilution and loading tests showed good parallelism with the standard curve. The detection limit of the RIAs were 0.3 nmol/liter for T4, 0.03 nmol/liter for T3, 0.05 nmol/liter for rT3, 24 pmol/liter for T4S, 3 nmol/liter for T2, and 1.2 mU/liter for rTSH, All samples were measured in a single assay run.

Determination of thyroid hormone concentrations in liver and kidney tissue
T4, T3, and rT3 were measured by RIA after extraction and purification of the iodothyronines from tissues, as described previously (33, 34). In brief, the samples were homogenized directly in methanol, and 2000 cpm of outer ring-labeled [131I]T4 and [125I]T3 were added to each sample as internal tracers for recovery. Appropriate volumes of chloroform were added to extract with chloroform/methanol (2:1). The iodothyronines were then back-extracted into an aqueous phase with 0.05% CaCl2 and purified by passing the supernatant through Bio-Rad AG 1 x 2 resin columns. After a pH gradient, the iodothyronines were eluted with 70% acetic acid, evaporated to dryness, and resuspended in RIA buffer. The extracts were counted to determine the recovery of [131I]T4 and [125I]T3 added to each sample. Average recovery was 49 and 51% for [131I]T4 in liver and kidney, respectively, and 68 and 71% for [125I]T3. Because previous experiments have shown that recovery of T4 and rT3 is similar, we also used [131I]T4 as recovery tracer for the determination of rT3. Concentrations were calculated using the amounts of T4 and T3 found in the respective RIAs, the individual recovery of [131I]T4 and [125I]T3 added to each sample, and the weight of the tissue sample submitted to extraction. No corrections for the amount of iodothyronines contributed by the blood trapped in the tissue aliquots were carried out because in liver and kidney, iodothyronines are predominantly located intracellularly (35) and the amount of trapped plasma is low (33).

Deiodinase activity
For determination of D1 and D3 activities, liver and kidney microsomal fractions were prepared and assayed as described by Darras et al. (36). Final incubations were in a total volume of 200 µl. For D1 activity, final incubation mixtures contained 1 µM rT3, 50,000 cpm/tube [3',5'-125I]rT3 (labeled using 17 Ci/mg carrier-free 125I from NEN Life Science Products, Boston, MA), 0.05 mg microsomal protein/ml, 2 mM EDTA, and 5 mM dithiothreitol and were incubated for 30 min at 37 C. For the D3 enzymatic assay, outer ring-labeled T3 was used, and the reaction products were separated and identified by reverse-phase HPLC on a C18 column. In addition to 1 nM T3, also 1 µM rT3 was added to the test solution. This saturating concentration of the preferred substrate for D1, together with 0.1 nM propylthiouracil, ensured that no inner ring deiodination of T3 by D1 occurred (12). Final incubations for D3 activity thus contained 1 nM T3, 200,000 cpm/tube [3'-125I]T3 (labeled as described above), 1 mg microsomal protein/ml, 1 µM rT3 and 0.1 mM propylthiouracil, 2 mM EDTA, and 50 mM dithiothreitol and were incubated for 120 min at 37 C. We abstained from analyzing D2-expressing tissues because D2 activity was in previous experiments in critically ill rabbits undetectable in skeletal muscle (12), which is in general considered as the major D2-expressing tissue (37).

D1 gene expression
Total RNA was isolated from rabbit liver and kidney using Qiazol lysis reagent (QIAGEN, Valencia, CA) and subsequently purified using the RNeasy mini RNA isolation kit (QIAGEN). Samples were treated with deoxyribonuclease to remove all contaminating genomic DNA. One microgram of total RNA was reverse-transcribed using random hexamers. Reactions lacking reverse transcriptase were also run as a control for genomic DNA contamination.

D1 mRNA (GenBank accession no. EF428024) levels were quantified by TaqMan real-time PCR using forward primer 5'-GCCAGAAGACCGGGATAGC-3', reverse primer 5'-GGTGCTGAAGAAGGTGGGAAT-3', and TaqMan probe 5'-CAGAACCCCAACTTCGCCCAGGA-3'. The 10-µl real-time reaction mixture contained 5 µl TaqMan Fast Universal PCR MasterMix (Applied Biosystems, Foster City, CA), 0.5 µl forward primer, 0.5 µl reverse primer, 0.5 µl TaqMan probe ([5']6-FAM [3']BHQ-1 labeled), 0.5 µl water, and 3 µl cDNA (7.5 ng). Final concentrations were 900 nM for the primers and 300 nM for the probes. Unknown samples were run in duplicate, and individual samples with a cycle threshold value SD greater than 0.3 were reanalyzed. Data were analyzed using the comparative cycle threshold method. mRNA levels are expressed relative to those of the hypoxanthine guanine phosphoribosyl transferase (HPRT) housekeeping gene.

Data analysis
Statistical analyses were performed with the StatView 5 for Windows Program (SAS Institute Inc., Cary, NC). Effects of interventions were analyzed using repeated-measures and factorial ANOVA with Fisher’s protected least significant difference (PLSD) post hoc testing, and square root or logarithmic transformation was performed when appropriate to convert nonnormally to normally distributed data. Saline-treated prolonged critically ill and healthy animals were compared using a Mann-Whitney U test. Correlation analysis was performed with linear, exponential, or logarithmic regression. The area under the curve, calculated by trapezoid rule, was used as a marker for the total amount of hormone present in plasma during the intervention. All data are expressed as the mean ± SEM unless specified otherwise. A two-sided value of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean body weight of rabbits surviving the eighth day of the experimental period was 2855 ± 36 g at the start and 2682 ± 38 g at the end of the study (P < 0.0001). In both experiments, starting body weight, changes in body weight, and mortality rate (31%, of which half was induced by euthanasia) were not different among groups. At baseline, mean blood glucose concentration was 112 ± 3 mg/dl and remained constant throughout both experiments. Four rabbits (saline, n = 2; high-dose T4, n = 1; TRH, n = 1) received insulin (10 ± 2.5 IU/rabbit). The baseline hemoglobin level was 11.7 ± 0.2 g/dl and decreased progressively to 8.4 ± 0.2 g/dl on d 8 (P < 0.0001 vs. baseline). Hemoglobin levels were not different among groups.

Effects of interventions on plasma T4, T3, rT3, and TSH levels (Fig. 2Go)
Saline-treated sick animals revealed lower plasma T3 levels than healthy animals. Infusion of TRH in sick animals increased plasma T4 and T3 levels as compared with saline.


Figure 2
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  		FIG. 2. Plasma iodothyronine levels (d 8) in the substitution-dose (SD) (left panel) and high-dose (HD) (right panel) thyroid hormone experiment, as compared with saline and TRH infusion. The two dotted horizontal lines represent the upper and lower limits of levels in healthy rabbits (n = 4). Effects of interventions were analyzed using factorial ANOVA with Fisher’s PLSD post hoc testing. #, P = 0.006 vs. healthy animals by Mann-Whitney U test. NS, Not significant.

 
In the substitution-dose experiment, plasma T4 levels were lowered in animals treated with T4, T3, and T4 + T3 as compared with saline-treated ill rabbits. Plasma T3 levels remained low in animals treated with T4, T3, and T4 + T3. rT3 levels also did not differ among groups.

In the high-dose experiment, plasma T4 levels were increased in animals receiving T4 or T4 + T3 vs. saline, and lowered with T3 treatment. Plasma T3 levels were increased with T4, T3, and T4 + T3. Plasma rT3 was increased only in T4- and T4 + T3-treated animals. The highest levels of T4 were found in animals treated with T4 alone, highest levels of T3 with T4 + T3 treatment, and highest levels of rT3 with T4 treatment alone.

rTSH, measured once daily (overall 3.69 ± 0.13 mU/ml on d 8), remained stable during the experiment without differences among groups, both in substitution- and high-dose rabbits (data not shown).

Within the intervention groups, thyroid hormone plasma concentrations were not significantly altered by infusion of insulin.

Effects of interventions on tissue thyroid hormone levels (Fig. 3Go)
As compared with healthy rabbits, saline-treated critically ill rabbits revealed lower liver T3 and kidney rT3 levels. Infusion of TRH increased liver and kidney T4 and T3 levels.


Figure 3
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  		FIG. 3. Tissue iodothyronine levels (d 8) in liver (left panel) and kidney (right panel) in substitution-dose (SD) and high-dose (HD) rabbits, as compared with saline and TRH infusion. The two dotted horizontal lines represent the upper and lower limits of levels in healthy rabbits (n = 4). Effects of interventions were analyzed using factorial ANOVA with Fisher’s PLSD post hoc testing. {ddagger}, P = 0.02; {dagger}, P = 0.04 vs. healthy animals by Mann-Whitney U test. NS, Not significant.

 
In substitution-dose rabbits, liver and kidney T4 levels were lowered with the administration of T4, T3, and T4+T3 as compared with infusion of saline. No differences in tissue T3 and rT3 were found with these doses of T4 and/or T3.

In high-dose rabbits, liver and kidney T4 decreased with T3 and increased with T4 and T4 + T3 as compared with saline. Both liver and kidney T3 increased with T4, T3, and T4 + T3. No differences in liver rT3 were found among groups. Kidney rT3 was increased only with infusion of T4 but was not different from saline in the other intervention groups. The highest tissue levels of T4 were found with T4 alone, the highest tissue T3 levels were found with T4 + T3, and the highest tissue rT3 levels were measured in animals receiving T4 alone.

Within the intervention groups, tissue thyroid hormone concentrations were not significantly altered by infusion of insulin.

Effects of interventions on deiodinase activity and gene expression
Compared with healthy animals, saline-treated sick animals exhibited lowered hepatic D1 (Fig. 4Go) and increased renal D3 activities (0.45 ± 0.09 vs. 0.08 ± 0.04 fmol/mg/min, P = 0.02). TRH infusion increased hepatic D1 activity (Fig. 4Go).


Figure 4
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  		FIG. 4. Liver (left panel) and kidney (right panel) deiodinase activities in the substitution-dose (SD) and high-dose (HD) experiment. The two dotted horizontal lines represent the upper and lower limits of levels in healthy rabbits (n = 4). Effects of interventions were analyzed using factorial ANOVA with Fisher’s PLSD post hoc testing. *, P = 0.01 vs. healthy animals by Mann-Whitney U test.

 
In substitution-dose animals (Fig. 4Go, upper panels), no differences in hepatic and kidney D1 activity were found among groups. In high-dose rabbits (Fig. 4Go, lower panels), enhanced D1 activities were observed in the liver of animals infused with T4 alone and with T3 alone, and in the kidney of animals receiving T4 alone or T3 alone, as compared with saline. Coinfusion of T3 with T4 appeared to additionally increase hepatic D1 activity. No differences in hepatic D3 (1.55 ± 0.15 fmol/mg/min) and renal D3 activities (0.51 ± 0.06 fmol/mg/min) were found among groups, either in substitution- or in high-dose rabbits.

Compared with healthy animals, saline-treated sick animals exhibited lower D1 gene expression in liver [1.25 ± 0.37 vs. 2.81 ± 0.89 arbitrary units (a.u.), P = 0.09] and in kidney (0.64 ± 0.17 vs. 1.44 ± 0.22 a.u., P = 0.04). In the whole group, both liver (4.65 ± 0.75 a.u.) and kidney (2.01 ± 0.45 a.u.) expression of the D1 gene were positively correlated with plasma T3 (r = 0.62, P < 0.0001 in liver; r = 0.68, P < 0.0001 in kidney), area under the curve for T3 (r = 0.60, P < 0.0001 in liver; r = 0.65, P < 0.0001 in kidney) and its corresponding D1 activity (r = 0.63, P < 0.0001 in liver; r = 0.78, P < 0.0001 in kidney).

Within the intervention groups, neither deiodinase mRNA nor activity levels were significantly altered by infusion of insulin.

Correlation analysis of tissue iodothyronine levels with plasma iodothyronine levels and deiodinases
Liver T4, T3, and rT3 concentrations on d 8 showed a positive correlation with the respective hormone concentrations in plasma (r = 0.90, P < 0.0001; r = 0.92, P < 0.0001; r = 0.30, P = 0.02, respectively). Similarly, kidney iodothyronine concentrations were positively correlated with d-8 plasma iodothyronine concentrations (r = 0.85, P < 0.0001 for T4; r = 0.91, P < 0.0001 for T3; r = 0.59, P < 0.0001 for rT3) (Table 1Go and Fig. 5Go).


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  		TABLE 1. Correlation analysis of plasma and tissue (liver and kidney) iodothyronine levels

 

Figure 5
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  		FIG. 5. Correlation analysis of liver (left panel) and kidney (right panel) T4, T3, and rT3 levels with the respective hormone concentrations in plasma.

 
Both in liver and kidney, D1 activity correlated positively with tissue T3 (r = 0.86, P < 0.0001 in liver; r = 0.73, P < 0.0001 in kidney) and the tissue T3/rT3 ratio (r = 0.69, P < 0.0001 in liver; r = 0.47, P = 0.02 in kidney). Liver D3 activity was negatively correlated with plasma d-8 T3 (r = –0.53; P < 0.0001) and the d-8 plasma T3/rT3 ratio (r = –0.70; P < 0.0001). Similarly, D1 mRNA levels also correlated positively with tissue T3 levels in liver and kidney (r = 0.61, P < 0.0001 in liver; r = 0.70, P < 0.0001 in kidney) and the T3/rT3 ratio (r = 0.55, P < 0.0001 in liver; r = 0.98, P < 0.0001 in kidney).

Effects of interventions on plasma T2 and T4S levels
Plasma T2 was not altered by any of the interventions (Fig. 6Go).


Figure 6
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  		FIG. 6. Effects of interventions on plasma T2 over time (left panel) and on d 8 (right panel) in substitution-dose (SD) and high-dose (HD) rabbits. The bold P values indicate the overall significance level between the plasma T2 levels induced by the different interventions.

 
Plasma T4S levels were increased by TRH. In substitution-dose rabbits, plasma T4S remained stable during the experiment. In high-dose rabbits, plasma T4S declined over time with T3 treatment and increased with administration of T4 and T4 + T3. Compared with high-dose T4 alone, the combined infusion of T4 and T3 resulted in significantly lower T4S levels over time (Fig. 7Go). Plasma T4S on d 8 correlated exponentially with plasma T4 (r = 0.75; P < 0.0001).


Figure 7
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  		FIG. 7. Effects of interventions on plasma T4S over time (left panel) and on d 8 (right panel) in substitution-dose (SD) and high-dose (HD) rabbits. The bold P values indicate the overall significance level between the plasma T4S levels induced by the different interventions.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study on the metabolic fate of thyroid hormone in a low T3 syndrome model of prolonged critical illness showed that a substitution dose of T4 alone, T3 alone, or the combination of both did not affect any of the illness-associated changes in the thyroid axis except a further decline in plasma and tissue T4 levels. A 3- to 5-fold higher dose, chosen to target plasma T4 and T3 at least within the range obtained by TRH infusion, also elevated tissue thyroid hormone levels and D1 activity as did TRH. Changes in iodothyronine tissue levels mimicked the changes in plasma. Tissue T3 levels and the ratio of active over inactive thyroid hormone (T3/rT3) in the tissues correlated with tissue deiodinase activities. Neither the substitution dose nor the high dose of thyroid hormones altered plasma T2 levels. Only administration of T4 in high dose increased plasma T4S levels.

The present study shows that there is an enhanced degradation and/or excretion of thyroid hormone during prolonged critical illness. Indeed, the infusion of substitution doses of both T4 and T3 failed to increase plasma and tissue iodothyronine levels, and high doses of T4 and T3, severalfold the substitution dose were required to bring plasma levels within the physiological range as obtained with TRH. Combined with the results of iodothyronine kinetic studies in patients, showing that reduction in thyroidal T4 (7, 38) and T3 secretion (21) account for a only minor part of the low plasma T3 concentrations, and previous observations in this animal model (9, 12, 20, 22), our data suggest that the low T3 state of critical illness is at least in part explained by an increased clearance of T4 and T3 besides an impaired T4 to T3 conversion. Similar results were previously obtained with T4 (10) and T3 administration to critically ill patients (11). Coincidentally, the T3 dose that was chosen in those studies to maintain normal plasma T3 levels, five times the replacement dose (11), was identical to the one we used in the high-dose rabbit experiment.

In normal physiological conditions, the principle route of T4 disposal occurs by either inner or outer ring deiodination forming rT3 and T3, respectively. It is estimated that the combined production of T3 and rT3 accounts for 80% of total T4 metabolism in euthyroid man, whereas alternate pathways are responsible for the remaining 20% (14). However, there are strong arguments indicating that this pattern of T4 degradation may be substantially altered in the low T3 state of critical illness. The rather unexpected finding of a further reduction of plasma and tissue T4 levels in animals treated with a substitution dose of T4, without concomitant changes in T3 and rT3 parameters and in D1 and D3 activity, is consistent with this. Moreover, the absence of differences in plasma T2 levels among all intervention groups, even with those receiving a high-dose T4, further confirms that during critical illness, a considerable amount of T4 disposal is routed to non-deiodinative pathways (19).

The sulfoconjugation pathway is such an alternate pathway, and it has been suggested that enhanced sulfation of T4 may account for a substantial part of the so-called T4 disposal gap observed during critical illness (17, 18, 19). Equally, the failure of a replacement dose of T3 to increase plasma T3 levels might be explained by an enhanced degradation of T3 via the sulfoconjugation pathway (18). The observation that T4S did not differ among groups in the substitution-dose experiment, however, argues against a major role for sulfoconjugation in the enhanced degradation of thyroid hormone in critically ill rabbits. The rise in T4S observed in sick rabbits receiving TRH or higher doses of T4 may, on the other hand, be explained by an increased production and/or a decrease in its clearance by an overall reduction in D1 activity. Although sulfation of T4 via sulfotransferases is a substrate-dependent process (39), the latter is presumed to be the principle cause of the observed accumulation of T4S (40), especially because critical illness is characterized by decreased sulfotransferase activities (17). Indeed, in normal physiological conditions, plasma concentrations of T4S are low, even under suppressive T4 therapy (31), due to a rapid and irreversible degradation of T4S by D1 (40, 41). Although the literature suggests that also an increased urinary and biliary excretion of thyrosulfoconjugates may be implicated in the maintenance of the low T4S plasma concentrations (31, 42), we were unable to support or refute such a mechanism in critically ill rabbits because no such fluids or feces were collected.

Consequently, the failure to explain the observed T4 disposal gap by changes in iodothyronine deiodination or sulfation strongly suggest the involvement of distinct routes of thyroid hormone degradation. Other known non-deiodinative pathways of thyroid hormone metabolism include glucuronide conjugation, conversion to acetic acid analogs, and ether cleavage products (19). Apart from the knowledge that glucuronidation plays an important role in drug-accelerated T4 disposal (43), very little is known about these alternate routes of thyroid hormone metabolism, making further basic research into this field warranted (43).

Alternatively, several of the observed effects could also be explained by changes in thyroidal hormone secretion. The decrease in plasma and tissue T4 in high-dose T3-treated rabbits most likely resulted from a suppressed thyroidal T4 secretion due to feedback inhibition. Likewise, the decrease in plasma T4 observed in the substitution-dose T4 rabbits could also partly be explained by this mechanism. The bolus administration of a substitution dose of T4 may theoretically have resulted in a temporary surge of plasma T4 leading to a decline in pituitary TSH release. Although plasma TSH remained stable during the experiment without differences among groups, this does not allow excluding such an effect because a once-daily TSH measurement provides only limited information during critical illness (11, 44). However, repetitive administration of exogenous T4 is known to obliterate the TSH response to transient increments in plasma thyroid hormone levels markedly, making such an effect unlikely (45).

Our data also confirm the presence of low liver T3 concentrations in prolonged critical illness (46, 47) and demonstrate that thyroid hormone therapy in a substitution dose is unable to increase tissue thyroid hormone and D1 activity levels. This inability of thyroid hormone in substitution dose to affect D1 activity combined with the clear parallelism between the changes in plasma and in tissue T3 levels induced by the different interventions makes it very tempting to postulate that most of the liver T3 during critical illness is plasma derived. Indeed, such a mechanism would be consistent with the results from tracer kinetic studies in rats, showing that during low T3 states, the relative contribution of locally produced T3 in liver and kidney is minor (48, 49, 50, 51) due to a decreased local conversion of T4 to T3 by D1 (48, 50). Our data do not allow us to conclude this, because no labeling techniques were performed to differentiate between plasma-derived and locally produced T3. Moreover, the low D1 activity observed in critical illness might also be a secondary change, besides a possible cause, of the low liver T3 content (52). The decreased expression of liver D1 supports this (15, 53), because D1 is a very sensitive marker of tissue thyroid hormone status (54). Consequently, addition of T3 to T4 infusion in the high-dose experiment resulted in an enhanced T3 liver content, and both hepatic D1 expression and activity were positively correlated with tissue T3 and T3/rT3 ratio in liver. The observation of similar, albeit slightly different, changes in kidney D1 activity and kidney T3 levels further supports this premise but also indicates the presence of tissue-specific differences in iodothyronine deiodinases and tissue thyroid hormone regulation (14, 48, 52, 53, 55).

Another factor that could explain the low hepatic T3 content is the level of thyroid hormone uptake by the liver (56). However, the observation that our prolonged critically ill rabbits exhibited similar T4 liver levels as healthy animals argues against a major role for an impaired intrahepatic T4 availability in the pathogenesis of the low hepatic T3 levels (33, 57). Furthermore, expression analysis in liver samples of critically ill patients suggests that monocarboxylate transporter 8 (MCT8), a specific and very active thyroid hormone transporter, is not crucial for the regulation of hepatic iodothyronine levels (53).

It remains hitherto unclear whether the low T3 syndrome is a beneficial adaptation resulting in a protection or whether it is a maladaptation that contributes to a worsening of the disease (2, 13). The classical assumption that low thyroid levels reflect an adaptive, protective mechanism against catabolism in prolonged critical illness was recently challenged by showing that thyroid hormone levels are inversely correlated with urea production and markers of bone degradation in prolonged critically ill patients (6, 58). Furthermore, restoration of physiological levels of thyroid hormone by continuous infusion of TRH, in combination with a GH secretagogue, was able to reduce these biochemical markers of catabolism (6, 58). The current study further advocates that thyroid hormone therapy in prolonged critical illness, in a fed condition (59), may be not as harmful as previously proclaimed, because no change in body weight and mortality rate were not different among the intervention groups. Nevertheless, large-scale clinical studies examining the effect of thyroid hormone therapy on the outcome of critical illness should however be awaited before the use outside research protocols can be advised.

Several limitations of our study need to be highlighted. First, is it important to note that we explored thyroid hormone levels only in liver and kidney and that other organs such as muscle, heart, brain, and skin were ignored. Although liver and kidney has classically been regarded as the major sources of circulating T3 (38), several studies have questioned this premise (60) and even argue that D2-expressing tissues, in particularly skeletal muscle, are an important source of plasma T3 production (14, 37). Furthermore, D2-expressing tissues appear to be quite independent of plasma for tissue T3 due to intracellular activation of T4 into T3 (14, 50). Second, four rabbits received insulin to keep blood glucose levels less than 180 mg/dl. However, although an effect of insulin on tissue iodothyronine content (57, 61) and deiodinase activity (52, 57) has been suggested, both in this study and in prolonged critically ill patients (15, 16, 53), insulin did not alter plasma and tissue thyroid hormone levels or tissue deiodinase activities. Third, no corrections for the amount of iodothyronines contributed by the blood trapped in the tissue aliquots were carried out. However, because liver and kidney iodothyronines seem to be predominantly located intracellularly (35), the possible contribution of thyroid hormone located within the plasma present in liver and kidney is expected to be minor (33). Fourth, because no urine, bile, or feces samples were collected, we were unable to address the potential role of urinary and biliary excretion of sulfate and glucuronide thyroconjugates. Finally, the extrapolation of our data to human physiology should be done with great caution because substantial differences in thyroid hormone metabolism exist among mammals (14). Moreover, it should be realized that the deiodinase activities measured in vitro under saturating conditions may not represent the actual deiodination taking place in vivo.

We conclude that in prolonged critically ill rabbits, low plasma T3 levels were associated with low liver and kidney T3 levels. Restoration of plasma and tissue thyroid hormone levels could not be achieved by a substitution dose of thyroid hormone but instead required severalfold this dose, which suggests thyroid hormone hypermetabolism. Thyroid hormone hypermetabolism in this model of critical illness is not entirely explained by deiodination or by sulfoconjugation. Unraveling the exact pathophysiology requires further investigation.


   Acknowledgments
 
We acknowledge the generosity of U.C.B. Pharma Belgium, Baxter Belgium, Fresenius-Kabi Belgium, Eddy Vanonckelen (Alaris Medical Systems, Belgium), and Wouter Diddens (Tyco Healthcare, Belgium) in providing, respectively, TRH, Clinomel, TPN bags, infusion pumps, and arterial catheters for the experiments. We greatly appreciate the technical support of Sarah Vander Perre and Eric-Jan Ververs (Laboratory Intensive Care Medicine); Willy Coopmans and Erik Van Herck (Laboratory for Experimental Medicine and Endocrinology); Willy Van Ham, Francine Voets, and Lut Noterdaeme (Laboratory of Comparative Endocrinology); and Mark De Turcq and Wilfried Frooninckx (Technical Department University Hospital Leuven).


   Footnotes
 
This work was supported by the Fund for Scientific Research-Flanders, Belgium (Ph.D. Scholarship, Aspirantenmandaat to Y.D. and G.0144.00, G.0278.03 to G.V.d.B.); a grant from Innovative Medizinische Forschung (EL 610304) and B. Braun Stiftung, Germany, to B.E.; and the Research Council of the Catholic University of Leuven (GOA 2007/14) to G.V.d.B.

Disclosure Statement: None of the authors has a conflict of interest to disclose.

First Published Online May 1, 2008

1 Y.D. and B.E. contributed equally. Back

Abbreviations: a.u., Arbitrary units; D1, deiodinase type 1; PLSD, protected least significant difference; rT3, reverse T3; rTSH, rabbit TSH; T2, 3'-diiodothyronine; T4S, T4 sulfate.

Received November 13, 2007.

Accepted for publication April 16, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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