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Department of Intensive Care Medicine (L.M., L.L., G.V.d.B.), Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and Department of Internal Medicine (T.J.V.), Erasmus University Medical Center, 3015 GE Rotterdam, The Netherlands
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.
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Abstract |
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Objective: Our objective was to study D2 expression and activity in skeletal muscle of acute and prolonged critically ill patients.
Design and Setting: We conducted a clinical observational study in acute and prolonged critical illness with comparison with healthy controls at a university hospital surgical intensive care unit.
Patients: Subjects included 63 prolonged critically ill patients who died in the intensive care unit, 21 acutely ill patients, and 38 controls matched for age, gender, and body mass index.
Results: Elevated expression of the D2 gene and D2 activity in skeletal muscle of prolonged, but not acute, critically ill patients were observed in the face of low circulating thyroid hormone levels.
Conclusions: Reduced D2 activity does not appear to play a role in the pathogenesis of the low T3 syndrome of critical illness.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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Although the pathogenesis of the low T3 syndrome remains poorly understood, altered peripheral metabolism of thyroid hormones plays a role. The peripheral conversion of thyroid hormones is regulated by three types of iodothyronine deiodinases. Type 1 deiodinase (D1) catalyzes conversion of T4 to T3 and breakdown of rT3, and this enzyme is considered to be the main source of circulating T3. Type 3 deiodinase (D3) inactivates T4 and T3 by conversion to rT3 and 3,3'diiodothyronine (T2), respectively (5). Prolonged critically ill patients have reduced D1 activity and increased D3 activity. This results in a diminished activation and an increased breakdown of thyroid hormone, thereby contributing to the low T3 syndrome (6).
Data on the role of type 2 deiodinase (D2) in the pathogenesis of the low T3 syndrome during critical illness are scarce. D2 converts T4 to T3 and is thus called an activating enzyme. It is expressed in the thyroid, skeletal muscle, pituitary, and brain where it is considered to be important for local thyroid hormone concentrations (7). Recently, it was shown that, in contrast to previous beliefs, D2 expressed in the human muscle may have a significant contribution to circulating T3, particularly in the hypothyroid state (8, 9). We therefore hypothesize that diminished D2 activity during critical illness contributes to the reduced T4 to T3 conversion in the low T3 syndrome.
Measuring D2 activity has been previously attempted in muscle from critically ill patients, but the method lacked sensitivity (6). We studied D2 expression and activity in muscle biopsies from prolonged critically ill patients as compared with acute surgical stressed patients and healthy volunteers.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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We studied prolonged critically ill patients who participated in a large randomized controlled study on the effects of intensive insulin treatment in intensive care unit patients (n = 1548), of whom the major clinical outcomes have been published in detail (10). From 63 of the 98 patients who had died in the intensive care unit, good quality RNA from postmortem biopsies was available. Postmortem biopsy samples of skeletal muscle (right musculus rectus abdominis) were taken within minutes after death. Tissue samples were snap-frozen in liquid nitrogen and stored at –80 C until further analysis. Blood samples were stored at –80 C.
For comparison, we studied skeletal muscle (right musculus rectus abdominis) biopsy samples harvested from 22 age-, gender-, and body mass index-matched patients during laparotomy for clinical indications (hemi- or segmentary hepatectomy or restorative rectal resection).
Thirty-eight matched healthy subjects were studied as controls. From 13 of these volunteers, muscle tissue (quadriceps) was sampled under local anesthesia. Because the amount of harvested tissue was limited, six samples were used for RNA isolation and five for activity measurements, and two samples could be used for both measurements.
Characteristics of prolonged ill and acute surgical stressed patients and healthy controls are given in Table 1
. All protocols were approved by the Institutional Review Board of the Leuven University. Written informed consent was obtained from all healthy volunteers and from the patients or, when the patient was unable to give consent, from the closest family member.
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Commercial RIAs were used to determine total serum T4, T3, TSH (Immunotech, Marseille, France), and rT3 (Biocode Hycel, Liege, Belgium) concentrations. The detection limits were, respectively, 13 nmol/liter, 0.1 nmol/liter, 0.025 mIU/liter, and 0.008 nmol/liter with an intraassay coefficient of variation (CV) of, respectively, 5.1, 3.3, 3.7, and 3%. All samples were assayed in duplicate. A highly specific commercial RIA was used to measure total cortisol (Immunotech) with a sensitivity of 10 nmol/liter and an intraassay CV of 5.8%. All samples were assayed in duplicate. Cortisol-binding globulin (CBG) levels were determined by radial immunodiffusion using an in-house polyclonal antibody raised against purified human CBG as previously described (11), with an interassay CV of 2.1%. Free cortisol was calculated from the total cortisol and CBG levels (12). The validity of applying the formula for critically ill patients was verified previously (13).
D2 activity
Muscle samples were homogenized on ice in 10 vol of PED10 buffer (0.1 M phosphate, 2 mM EDTA, 10 mM dithiothreitol, pH 7.2) using a Polytron (Kinematica AG, Lucerne, Switzerland). Homogenates were cooled on ice and immediately analyzed. Protein concentration was measured with the Bio-Rad protein assay using BSA as the standard following the manufacturer’s instructions. D2 activity was assayed as described previously (9) by duplicate incubation of 500 µg homogenate protein for 240 min at 37 C with 1 nM [3',5'-125I]T4 (100,000 cpm) in a final volume of 0.1 ml PED10 buffer. The incubations were carried out in the absence or presence of 0.5 µM unlabeled T4, which is sufficient to saturate D2. Reactions were stopped by addition of 0.1 ml 100% methanol on ice. After centrifugation, 0.1 ml of the supernatant was added to 0.1 ml 0.02 M ammonium acetate (pH 4.0), and 0.1 ml of the mixture was applied to a 4.6 x 250 mm Symmetry C18 column connected to an Alliance HPLC system (Waters, Etten-Leur, The Netherlands). The column was eluted with a linear gradient of acetonitrile (28–42% in 15 min) in 0.02 M ammonium acetate (pH 4.0) at a flow of 1.2 ml/min. The radioactivity in the eluate was measured online using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT). The difference in radioactivity in the T3 peak after incubation without and with excess unlabeled T4 was considered to mirror specific D2 activity.
RNA isolation and real-time PCR
Total RNA was isolated from skeletal muscle tissue using Qiazol lysis reagent (QIAGEN, Venlo, The Netherlands) and subsequently purified using the RNeasy mini RNA isolation kit (QIAGEN). Samples were treated with DNase to remove all contaminating genomic DNA, and 1 µg total RNA was reverse-transcribed using random hexamers.
D2 mRNA levels were quantified in real time with the ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA) which uses TaqMan chemistry for highly accurate quantitation of mRNA levels. We designed primers and probes for D2 (forward, 5'-GCCACATGCCACCTTCTTG-3'; reverse, 5'-GCTGAGCCAAAGTTGACCAC-3'; and probe, 5'-CTTTGCCAGCCCTGAGCGCC-3'). 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 (forward, 5'-TGTAGATTTTATCAGACTGAAGAGCTATTGT-3'; reverse, 5'-AAGGAAAGCAAAGTCTGCATTGTT-3'; and probe, 5'-TTTCCAGTTAAAGTTGAGAGATCATCTCCACCAAT-3').
Statistical analysis
Statistical analyses were done using StatView software (SAS Institute Inc., Cary, NC). Data were analyzed using one-way ANOVA tests with a post hoc Fisher’s least significant difference test for multiple comparisons and Mann-Whitney U tests when appropriate. Data are presented as means ± SEM or medians and interquartile ranges when appropriate. Statistical significance was assumed for a two-sided P value < 0.05.
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Results |
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Circulating total T3 levels were lower in the acute surgical stressed patients as compared with healthy controls, and these levels decreased further in the chronically ill population (Fig. 1). Plasma total T4, rT3, and TSH concentrations were comparable in healthy controls and acute surgical stressed patients. In prolonged critical illness, a significant rise in rT3 and a decrease in total T4 and TSH were observed (Fig. 1).
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). Deiodinase expression and activity in skeletal muscle
Skeletal muscle D2 mRNA levels were similar in acute surgical stressed patients and healthy controls. Prolonged critically ill patients revealed 2-fold higher muscle D2 mRNA levels than healthy controls (Fig. 1). Changes in enzyme activity mimicked that of gene expression. D2 activity in skeletal muscle of prolonged critically ill patients was 3-fold higher than in acute surgical stressed patients and healthy controls (Fig. 1).
Relationship between D2 expression/activity and levels of thyroid hormones and cortisol during critical illness
Circulating T3 levels showed a weak inverse correlation with skeletal muscle D2 gene expression (r = –0.18; P = 0.09) and activity (r = –0.35; P = 0.009). Circulating T4 levels correlated with D2 activity (r = 0.35; P = 0.01) but not mRNA expression levels.
Free cortisol levels showed a weak but significant correlation with D2 mRNA expression (r = 0.3; P = 0.004) levels but not with D2 activity.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The role of D2 in local T3 production has been well accepted, particularly in the brain where it is the only thyroid hormone-activating enzyme (7). Recent data by Maia et al. (8) suggest that expression of D2 in skeletal muscle may significantly contribute to circulating T3, particularly in the hypothyroid state (9). These data generated the hypothesis that a decrease in D2-dependent conversion of T4 into T3 in skeletal muscle could contribute to the low T3 syndrome in critical illness. In contrast to this hypothesis, however, we found increased levels of D2 gene expression and activity in the context of the low T3 syndrome of prolonged but not acute critical illness. The data suggest that at least in the prolonged phase of critical illness, D2 adapts appropriately to the low T3 levels and likely does not contribute to the low T3 syndrome in this condition.
Our observation of increased D2 gene expression and activity in skeletal muscle of chronically ill patients is in contrast to a previous observation. Peeters et al. (6) reported no detectable D2 activity in skeletal muscle of prolonged critically ill patients. The results obtained from the activity measurements in that study may be explained by lack of control samples and suboptimal assay for D2 activity in skeletal muscle based on the release of radioiodide from [3',5'-125I]T4. In our study, we have extended the incubation time and a HPLC system was used for quantitation of [125I]T3 production. In these analyses, the production of [125I]rT3 from [125I]T4 was in good agreement with previous measurements of D3 activity using [125I]T3 as the substrate.
In conclusion, during prolonged but not acute critical illness, D2 gene expression and activity are increased in the face of low circulating thyroid hormone levels. These data indicate that reduced D2 activity does not play a role in the pathogenesis of the low T3 syndrome of prolonged critical illness.
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Acknowledgments |
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Footnotes |
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First Published Online May 15, 2007
Abbreviations: CBG, Cortisol-binding globulin; CV, coefficient of variation; D1, type 1 deiodinase; D2, type 2 deiodinase; D3, type 3 deiodinase.
Received March 6, 2007.
Accepted May 7, 2007.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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in nonthyroidal illness syndrome induced by abdominal surgery. J Clin Endocrinol Metab 86:4198–4205This article has been cited by other articles:
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  J Kwakkel, O Chassande, H C van Beeren, W M Wiersinga, and A Boelen Lacking thyroid hormone receptor {beta} gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness J. Endocrinol., April 1, 2008; 197(1): 151 - 158. [Abstract] [Full Text] [PDF]
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