User:Njmcdaniel/sandbox

Njmcdaniel/sandbox

Names
IUPAC name (2S)-2-Amino-3-[4-(4-hydroxy-3,5-diiodophenoxy)-3-iodophenyl]propanoic acid
Identifiers
CAS Number	5817-39-0 Y
3D model (JSmol)	Interactive image
MeSH	Reverse+triiodothyronine
PubChem CID	22069
UNII	8NZ4Y08T96 Y
SMILES C1=CC(=C(C=C1C[C@@H](C(=O)O)N)I)OC2=CC(=C(C(=C2)I)O)I
Properties
Chemical formula	C15H12I3NO4
Molar mass	650.974
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa). Y verify (what is YN ?) Infobox references

Tracking categories (test):

• SizeSet

Reverse triiodothyronine (3,3’,5’-triiodothyronine, reverse T₃, or rT₃) is an isomer of triiodothyronine (3,5,3’ triiodothyronine, T₃). Reverse T₃ is the third-most common iodothyronine the thyroid gland releases into the bloodstream, of which 0.9% is rT₃; tetraiodothyronine (levothyroxine, T₄) constitutes 90% and T₃ is 9%. However, 95% of rT₃ in human blood is made elsewhere in the body, as enzymes remove a particular iodine atom from T₄.

The production of hormone by the thyroid gland is controlled by the hypothalamus and pituitary gland. The physiological activity of thyroid hormone is regulated by a system of enzymes that activate, inactivate or simply discard the prohormone T₄ and in turn functionally modify T₃ and rT₃. These enzymes operate under complex direction of systems including neurotransmitters, hormones, markers of metabolism and immunological signals.

Once believed to be the only significant thyroid hormone, T₄ is now known to act primarily as a precursor, having also some non-genomic effects. Formerly considered to have no clinical relevance, T₃ is recognized as the active form of thyroid hormone. Until recently considered without function, reverse T₃ has been shown to have multiple non-genomic effects and to act as a competitive inhibitor of T₃ and its stimulatory effects. There is no doubt that at least one “anti-thyroid hormone” exists, though the exact relations of rT₃ to T1AM remain to be clarified.

Function

Reverse T₃ primarily inhibits thyroid signaling. It is commonly thought to have no biological activity, as early studies showed none of the stimulatory effects expected of thyroid hormone (TH). This belief became widely accepted despite contemporaneous work indicating rT₃ could inhibit the effects of T₄. Even after T₄ was discovered to be a prohormone for the potent T₃, the conversion of T₄ to rT₃ was considered simply a deactivation of excessive T₄.

This appraisal has been revised. Firstly, surplus T₄ is amply cleared by alternative pathways. More importantly, multiple functions for reverse T₃ have been revealed – inhibitory functions. Notably, there are several ways (genomic and non-genomic) in which rT₃ impedes the actions of thyroid hormone. These can be physiologically significant, depending on the relative proportion of T₃ to rT₃.

The conversion of T₄ to either T₃ or rT₃ is regulated. The balance of T₃ and rT₃ is crucial: A shift of the predominant hormone from rT₃ to T₃ triggers the transition from cellular proliferation to differentiation during embryogenesis and amphibian metamorphosis. Similar effects are apparent in wound healing and tissue repair and in some cancers. Reverse T₃ is importantly involved in reducing the metabolic rate during the stress response, which makes the body energy-efficient and prolongs survival in times of crisis.

However, this physiological adaptation, when protracted, can lead to a maladaptive condition called Euthyroid Sick Syndrome (or Non-Thyroidal Illness Syndrome) that is associated with significantly higher morbidity. Some physicians believe Chronic Fatigue Syndrome is a milder form of this illness. By extension, such an imbalance of T₃ with rT₃ could contribute to diverse other conditions whose apparent relation to thyroid dysfunction was thought to have been ruled-out by normal blood TSH and T₄ levels: depression; attention deficit and other cognitive disorders; infertility and more.

The possibility that reverse T₃ might have inhibitory effects was proposed in 1974. A review of accumulated research (following) supports this idea; data from many studies show that rT₃ is a significant element of thyroid hormone signaling, both genomic and nongenomic. The prohormone T₄ can be processed into either T₃ or rT₃, each an active hormone with opposing effects (a dichotomy analogous to the conversion of testosterone into either estradiol or dihydrotestosterone). The relative balance of T₃/ rT₃ indicates the physiological effects of thyroid hormone.

Biosynthesis, clearance, and regulation

Intra-thyroidal production, storage and release

About 5% of rT₃ in the human body is made in the thyroid gland; the rest is subsequently derived from T₄, the gland’s major hormone product. Thyroid gland hormone production is controlled by the hypothalamic-pituitary axis via the pituitary hormone TSH. Thyroid follicular cells increase every aspect of their many functions upon stimulation by TSH, ultimately directed to the manufacture and release of thyroid hormone.

The first step in thyroid hormone production is assembling the scaffolding upon which hormones will be constructed and stored. This is thyroglobulin (formerly called “colloid”), a long protein rich in the amino acid L-tyrosine; it is synthesized in the follicular cells and passes out (“exocytosis”) into a space between cells, the follicle. In this extracellular space, the enzyme thyroid peroxidase (TPO) – also made by the follicular cell – attaches iodine atoms to the aromatic rings virtually bristling from L-tyrosine molecules within the thyroglobulin.

Thyroid hormone synthesis.

Usually, two iodine atoms are attached to each ring, one apiece to the 3 and 5 carbon atoms. Therefore, the product is called diiodotyrosine (literally, “two-iodine tyrosine,” DIT). Remaining incorporated into thyroglobulin by peptide bonds, DIT is the major precursor of thyroid hormone. Occasionally, only a single iodine atom is attached to an L-tyrosine ring, producing monoiodotyrosine (MIT). MIT Also can be used to make thyroid hormones, the actions of which will be very different.

When thyroglobulin assumes its tertiary structure, appropriately-spaced iodinated tyrosyl rings – most of which are DIT – are brought into proximity. Two are joined (“conjugation”), again by the versatile TPO enzyme, to form one thyroid hormone molecule, still an intrinsic part of thyroglobulin. More than 90% of hormone in thyroglobulin comes from joining two of the abundant DITs; the product, with its four iodine atoms, is called T₄ (3,5,3',5'-tetraiodothyronine, levothyroxine).

Less than 10% of thyroid hormone is made by splicing MIT and DIT, creating either of two isomers with only three iodine atoms: rT₃ and T₃. Reverse T₃ has one iodine atom on the inner (tyrosyl) ring; T₃ features the single iodine on the outer (phenolic or “prime”) ring. In conjugating MIT and DIT, molecular conformation forces preferentially direct MIT to the outer ring position. is about ten-times more abundant in thyroglobulin than is rT₃.

Large amounts of thyroid hormones, a supply sufficient for 2-3 months, are thusly stored as integral parts of the long thyroglobulin molecule, within the capacious extracellular follicles (from Latin: “little bags”). Upon TSH signaling, follicular cells ingest thyroglobulin (“endocytosis”) as cytoplasmic vacuoles and lysosomal enzymes break thyroglobulin’s peptide bonds (“proteolysis”), freeing thyroid hormones into the cytosol. There, liberated hormones promptly encounter deiodinase (DI) enzymes and are altered before they exit the follicular cell – normally to increase T₃. In humans, the proportions of thyroid hormones entering the bloodstream are 90% T₄; 9% T₃, 0.9% rT₃ and approximately 0.1% T₂, depending on the availability of iodine, the rate of secretion and other variables.

Thyroid hormones in the blood

In the bloodstream, these hormones strongly but reversibly bind to transport proteins. Binding serves multiple purposes: Free hormones are poorly water-soluble but joining to protein allows them to travel easily in the aqueous medium of blood. Protein-carriage also guards hormones against loss by renal clearance and liver breakdown. Finally, a large, inactive reserve of hormone is maintained, as bound hormone cannot enter cells. The equilibrium between bound and free (unattached) hormones leaves just a tiny amount free – in the picomolar range: 0.13% of rT₃ is unbound, comparable to T₃ (0.3% free) but contrasting with only 0.03% of T₄ being free in circulation.

The relative proportions of total (bound and free) thyroid hormones in the bloodstream can be determined. Among carefully-vetted donors, healthy humans have about 56 times more total T₄ (tT₄) than total T₃ (tT₃). Though this study didn’t measure reverse T₃, its tT₄ and tT₃ results validate the selection of healthy, normal patients by another group, who did: These investigators determined reference intervals for total rT₃ as well as tT₄ and tT₃. We see tT₃ is 7 to 10-times more abundant than rT₃ in healthy human serum, using their data to compare tT₃ and rT₃ at the upper limits of normal and then again at the lower.

Free values are quite different: The amount of biologically-available free T₄ is only about 3-times higher than free T₃ in healthy serum when measured directly, due to their greatly differing binding affinities noted above. There is no accurate test for free rT₃, as its lower normal values fall considerably below the sensitivity of radioimmunoassay (for comparison, so do women’s free estradiol and free testosterone). However, similar binding affinity values for T₃ and rT₃ noted above indicate the total values will satisfactorily reflect their proportionate free levels.

Extra-thyroidal production

Whilst 100% of total (bound and free) serum T₄ is produced by the thyroid gland, most rT₃ and T₃ are not, being derived from T₄ by enzymatic alterations carried out in other tissues. Approximately 95% of circulating rT₃ is made by selectively removing one iodine atom from the inner ring of the prohormone T₄ (“5-deiodination”). Similarly, 80% of T₃ in the blood comes from T₄, when one iodine atom is removed from the outer ring (5’-deiodination). These transformations are performed within the cytoplasm of cells by an elegant system of enzymes and are purposefully directed. This process is now recognized as the primary means of regulating the biological activity of thyroid hormone.

Actions of deiodinase enzymes

Only free thyroid hormone can enter cells to exert its major effects. Because of membrane-solubility challenges, various transport proteins exist to facilitate this entry. Equipage with specific transporter subtypes is important to the particular activities of individual cell types, tissues and organs. Although genetically defective transporters can cause functional hypothyroidism with severe developmental consequences, this system may play no more than a minor part in adaptively regulating the biological effect of thyroid hormones. That role primarily belongs to thyroid hormone-altering enzymes within the cells.

Arriving just inside the cell membrane, T₄ encounters the first of a variety of enzymes by which it can be modified. Some twenty percent of T₄ is deactivated by conjugation or other “alternative pathway” (e.g. deamination, decarboxylation, ether-link cleavage) and thusly is cleared away. Evidently, the thyroid gland normally produces an ample surplus of T₄. This system is also adaptive: Up to four-fold more T₄ can be inactivated by conjugation as necessary.

The remaining unaltered T₄ now becomes subject to the leading actors, a family of selenoprotein deiodinase enzymes (DI). There are three major isoforms of DI enzymes, each with different actions: Type-2 converts T₄ to T₃, making 50-70% of all T₃ in the blood. Type-3 changes T₄ into rT₃. Type-1 is most complex – the first one discovered but the last to be understood; it can act on T₄ to produce either T₃ or rT₃, depending on a variety of biological signals now being revealed by research. Many cell types studied contain DI enzymes, though the types and amounts vary greatly by organ, tissue and cell. Those cells able to convert T₄ to T₃ supply the blood with T₃ for those which cannot.

Clearance

Reverse T₃ is broken down to T₂ by 5’-deiodination, removal of an iodine atom from the outer, “prime” ring (Fig. 3). The same step converts T₄ to T₃. By these two results of the single reaction, types-1 and 2 deiodinase increase the metabolic rate (Fig. 3). People with a healthy metabolism demonstrate a higher ratio of T₃ to rT₃.

Conversely, 5-deiodination by DI types-1 and 3 removes iodine from the inner ring to deactivate T₃ into T₂ and convert T₄ into rT₃. In both ways, this reaction reduces the stimulatory effects of thyroid hormone. Starving, sick and otherwise significantly stressed people have a lower ratio of T₃ to rT₃. As these findings suggest, deiodinase activity is remarkably important and it is adaptively regulated.

Regulation of biosynthesis and degradation

Peripheral metabolism of T₄, T₃ and rT₃ by deiodination regulates the biological activity of thyroid hormones. Deiodinase enzymes, called the “gatekeepers to thyroid hormone action,” are encoded each in a different DNA location. The deiodination of thyroid hormones is closely controlled by various means, including: Up- or down-regulation of enzyme synthesis; significant variations in substrate preference; differential reaction mechanisms; variable sensitivity to chemical and neuroendocrine messengers and different inactivation mechanisms. These interact to maintain a circumstantially-appropriate level of thyroid hormone signaling.

Mechanisms of action

Genomic

The hormone T₃, largely produced from T₄ by outer ring (5’) deiodination, enters the nucleus of the cell and binds to a nuclear receptor (Thyroid Receptor, TR). Thyroid Receptor – and its effect – is also influenced by various other ligands. When T₃ joins with TR, the resulting complex is a transcription factor; it attaches to DNA at specialized thyroid response elements. In essence, the T₃+TR unit activates DNA programs that increase the metabolic rate and it blocks any DNA message that slows the metabolism. T₃ has few effects outside of the cell nucleus. This contrasts again with T₄ and rT₃, which act significantly upon mitochondria, membranes, ion pumps and more. Though not the only aspect of thyroid signaling, the “genomic” effects (those influencing DNA transcription) of T₃ are the most important.

The inhibitory genomic activity of rT₃ challenged investigators. Reverse T₃ was tested with the same measures used to evaluate the effects and mechanisms of T₃. Experimental designs that were, in retrospect, one-dimensional yielded valid but misleading data. Comparing the stimulatory potency of rT₃ to other iodothyronines – T₄, T₃ and T₂ and derivatives – revealed it had none: Reverse T₃ evoked no calorigenic effect, meaning it did not increase cellular metabolism; neither was it “anti-goiterogenic.” It did not block thyroid iodine uptake nor did it diminish the release of T₄ from the thyroid gland. The latter three observations indicate rT₃ exerts negligible feed-back inhibition on the hypothalamic-pituitary axis. This was later confirmed in finding T₃ reduces TRH-induced TSH secretion at least one hundred-times more potently than does reverse T₃. These results led researchers to conclude – and reaffirm for decades afterwards – that reverse T₃ had “little or no biological activity.”

The correct conclusion to be drawn from these data is: “Reverse T₃ has little or no positive thyroid-mimetic genomic activity.” Another biological effect had been overlooked: rT₃ can act as an inhibitor. Using an automotive metaphor, the researchers studied the brake pedal using every appropriate test for an accelerator. Finding it in no way increased the speed of the vehicle, they declared it useless.

Definite effects of rT₃ have been demonstrated – anti-thyroid effects: Quite early-on and in several reports, reverse T₃ was found to inhibit the action of T₄. Subsequent efforts exemplify the difficulties in designing experiments and interpreting their results: Several studies of rT₃ binding to nuclear receptors proved reverse T₃ incapable of significantly displacing T₃ from the Thyroid Receptor. These consistent findings were prematurely interpreted as proof rT₃ has but poor affinity to TR. By simply reversing the order of this experiment, the converse also was found to be true: T₃ cannot effectively displace Thyroid Receptor-bound rT₃ and reverse T₃ was indeed shown to have genomic effects– to impede thyroid signaling. When cells are infused with rT₃ prior to receiving T₃, the effects of T₃ are blocked Just as rT₃ cannot remove T₃ already bound to TR, neither can histamine-receptor antagonists like diphenhydramine (Benadryl®) displace histamine bound to its receptors. However, they have unquestioned antihistaminic action through prophylactically blocking open histamine receptors. A subsequent study of unbound nuclear receptors offered supporting evidence that antagonism between T₃ and rT₃ alters cellular function.

The presence of more than a single Thyroid Receptor became apparent. A number of groups reported having identified a specialized nuclear rT₃-receptor – and also that rT₃ had particular genomic effects. Much has been learned about the two groups of thyroid receptor subtypes since these studies were reported. Consequently, earlier conclusions about rT₃ receptors should be questioned. Importantly though, recent work has reaffirmed that rT₃ exerts significant genomic actions: Injected rT₃ blocked a number of known hepatic thyroid-response genes. These researchers wrote in 2007: “That rT₃ may function as an inhibitor of T₃ offers an interesting new regulatory pathway in the thyroid hormone signaling cascade.”

Nongenomic

Beyond activating the thyroid-responsive DNA programs, thyroid hormones influence various cell functions. These are “non-genomic” effects. As their receptors and mechanisms have been identified, the complexity of thyroid hormone signaling – including rT₃ activity – has been better understood. Nongenomic receptors include Integrin-αv/β3 on cell membrane surfaces, variants of nuclear TR lingering in the cytosol, mitochondrial and other receptors. Many of these bind T₄ and rT₃ preferentially, with only poor affinity for T₃.

Thyroid hormones can thusly exert multiple actions – sometimes without even entering the cell. Examples of nongenomic thyroid activities include influencing membrane ion pumps (sodium, calcium and protons) and altering the effects of other signaling pathways, such as the adrenergic system and the metabolic-sensing nuclear receptors (e.g. peroxisome proliferator-activated receptors). Also, plasma membrane and cytosolic targets of THs have been strongly, though circumstantially linked to mitogenic and anti-apoptotic signaling pathways.

An important nongenomic function of rT₃ – and a plausible explanation for many of its observed inhibitory effects – was identified upon confirming that reverse T₃ blocks type-2 deiodinase. This enzyme performs outer-ring (5’) deiodination, converting T₄ to the active T₃ and clearing rT₃ to T₂. Eighty percent of the T₃ required each day must be produced peripherally by T₄-deiodination; of that, type-2 DI provides 50-70%. The functional importance of blocking the greatest source of T₃ is obvious but evidence shows there are further significant effects of reverse T₃.

Non-genomic receptors can bind rT₃, influencing ion pumps and transporter function can be altered:

• The thyroid hormone binding site of apolipoprotein-E was found to accept rT₃ with affinity equal to T₄ and significantly more than for T₃.
• Reverse T₃ competitively inhibits – once again, when given as a pre-treatment – effects of T₄, T₃, 3,5-T₂, and DIT on sodium currents in muscle cells, apparently mediated by a non-genomic thyroid hormone receptor.
• The presence of rT₃ can influence carrier protein-mediated transport of other thyroid hormones in and out of cells.

Through non-genomic actions, synthetic reactions and enzyme systems are effected to influence metabolic energy and more:

• A study from Japan indicated that rT₃ inhibits the metabolism, reporting reduced ATP/ADP ratio in cultured cells under various circumstances – effects that were completely reversed when cells were incubated with T₃.
• Reverse T₃ may be a physiological modulator of the enzyme inosine monophosphate dehydrogenase with implications for cellular differentiation. This enzyme catalyzes the rate-limiting step in de-novo guanosine triphosphate (GTP) synthesis. GTP is a source of energy or an activator of substrates in metabolic reactions – in addition to being a substrate for the synthesis of DNA and RNA during replication and transcription.
• An rT₃-responsive post-translational protein synthesis-dependent pathway has been identified, by which pre-incubation with rT₃ increases the antiviral action of interferon-gamma – an effect shared by T₄ and T₃ but not by T₂ or other thyroid derivatives.
• Reverse T₃, along with T₄, exerts effects essential to normal embryonic central nervous system development. Their non-genomic effects to promote normal granule cell migration and neuronal process outgrowth by influencing actin content and polymerization were markedly attenuated by the presence of T₃.
• Other researchers studying the effect of rT₃ injections on lipaemia in response to stressors such as endotoxin, adrenaline and dexamethasone write: “Reverse triiodothyronine (rT₃) displays hypometabolic properties and antagonizes the hypermetabolic effect of 3,5,3'-triiodothyronine (T₃).”
• Reverse T₃ effects cancerous cells, stimulating the growth of some and inhibiting the proliferation of others. This may be relevant for normal physiology but could be due to mutated receptors commonly found in such tissues.

Clinical significance

Reverse T₃ has emerged as a valid indicator in the evaluation and management of patients. For some decades, rT₃ had been said to have “little or no biological activity.” It was considered an inactive byproduct of unwanted T₄ – a waste product offering no more than iodine atoms for recycling. Blood levels of rT₃ were believed to show only the extent to which T₄ was directed away from T₃ production, as perhaps a more sensitive marker than low T₃ itself. Now, the accumulated weight of evidence shows rT₃ is an important participant in thyroid signaling, primarily as an inhibitor. There are many clinical situations in which this can be significant.

The last barrier to perceiving rT₃ as an inhibitory hormone (besides cognitive habit and orthodoxy) may have fallen with the demonstration that 3-iodothyronamine (T1AM) rapidly exerts a potent anti-thyroid effect. Now, the existence of a thyroid hormone metabolite with effects opposed to thyroid-mimetic signaling is unquestionably proven. This may enhance our ability to conceive of rT₃ also as an active, if inhibitory iodothyronine – if only, as suggested by some, as a precursor of T1AM.

Measurement

Practical assays for rT₃ using radioimmunoassay (RIA) technique were reported in the mid-1970s, having been developed by Chopra and others. The range of normal hormone values is determined by testing carefully-selected healthy individuals, whose results analyzed statistically. The reference range (reference interval, RI) is defined as the span of values from -2 to +2 standard deviations – the 2.5th to 97.5th centiles; these include 95% of people expected to be healthy. However, it is difficult to determine the normal range for any thyroid hormone, particularly so for rT₃. The many challenges include a non-Gaussian distribution of thyroid values; the prevalence of thyroid problems in apparently healthy populations; variability of binding proteins with the use of hormones such as oral contraceptives and the rapidity with which rT₃ can rise in response to stress – in fact, as patients enter a surgical suite or simply with experiencing preoperative anxiety.

The normal range for reverse T₃ was found to be 9.1–22.1 ng/dL (0.14–0.34 mmol/L – the conversion factor is 65 for rT₃ and T₃ both) in a reliable study. Blood tests were taken from a well-selected group of 270 healthy human adults to determine the reference intervals for thyroid hormones by RIA. The similarity of this group’s RIs for TSH (0.4–4.3 μU/dl), tT₄ (4.51–9.95 μg/dl) and tT₃ (92.8–162.9 ng/dl) with other carefully-executed work, as noted above, supports the validity of these findings.

Commercial laboratories in the United States also offer rT₃ tests by liquid chromatography/tandem mass spectrometry (LC/MS-MS), a powerful technique with very high sensitivity and selectivity. Indeed, Laboratory Corporation of America (LabCorp) in 2012 discontinued rT₃ RIA in favor of LC/MS-MS; increased demand for rT₃ tests had created shortages of reagents and delays in reporting results. Though the limitations of commercial reference intervals have been noted, those for LC/MS-MS are consistent with the best research findings: At LabCorp, the adult RI for rT₃ is 9.2–24.1 ng/dL, and at Quest Diagnostics, it is 8–25 ng/dL. However, their RIA reference intervals agree only poorly, using values of 9–35 ng/dL and 11–31 ng/dL. Presently, LabCorp makes a credible effort to present age-adjusted RIs that seem consistent with clinical experience (Table).

Table: Reverse T₃ Reference Intervals by Age

LabCorp USA, LC/MS-MS

Age	Range (ng/dL)
Premature (26-31w)	33.0-147.0
Premature (32-35w)	49.0-217.0
Full Term (2-7d)	33.0-206.0
8d-5m	13.0-107.0
6m-12m	8.1-52.8
1-15y	8.3-22.9
16y and older	9.2-24.1

Clinical utility

The clinical value of an assay for rT₃ is not fully established. Commercial laboratories providing rT₃ tests offer allowable suggestions: Quest states “the assay may be useful in the diagnosis of nonthyroidal illness (NTI). Patients with NTI have low T₃ concentrations and increased concentrations of rT₃. RT₃ may be useful in neonates to distinguish euthyroid sick syndrome from central hypothyroidism.” LabCorp has written that rT₃ is biologically inert and does not stimulate thyroid receptors. They then added, with proper references, that levels of rT₃ are elevated in Euthyroid Sick Syndrome and correlate with both the degree of myocardial function impairment in patients with heart failure and shorter survival in elderly people, regardless of other confounding factors.

As the measurement of rT₃ became feasible, its remarkably robust presence in healthy amniotic fluid was noted – in stark contrast to the (then) immeasurably-low T₃ and very low T₄. The use of this assay was suggested to improve the diagnosis of fetal thyroid dysfunction and to identify pregnancies of less than 30 weeks' gestation. Studies of human and animal amniotic fluid soon supported this idea. Although such an approach was subsequently judged unnecessary, many other investigations of fetal and neonatal rT₃ have followed. A search of Pub Med for “3,3’,5’-triiodothyronine AND fetus” today yields 168 citations addressing widely varied topics (June 18, 2013).

Reverse T₃ levels in human blood are lower than normal in untreated hypothyroidism and elevated in hyperthyroidism. The production of T₄ is impaired in hypothyroidism and 95% of rT₃ is derived from T₄. Also, lower rT₃ suggests conversion of T₄ preferentially to the active T₃, an obviously efficient step in such a shortage. Conversely, higher rT₃ in hyperthyroidism is clearly an adaptive response to excessive amounts of T₄, regardless of whether rT₃ is an inhibitor of high T₃ or simply a byproduct of increased T₄ deactivation.

Higher than normal levels of rT₃ are also found in patients treated with physiological doses of oral levothyroxine (T₄). This observation has also been reported in animals given T₄, including horses and rats. Given the inhibitory effects of rT₃, this finding could have great significance; further discussion follows.

This relative excess of rT₃ may be due to the liver’s first pass effect – T₄ absorbed from the gut is broken down by a gauntlet of hepatic enzymes before it gains the general circulation. A second possibility is suggested by this fact: The conversion of T₄ to rT₃ is increased when free T₄ levels are high. Whilst the thyroid gland in good health releases hormone at a steady rate and normal blood levels vary but little, once-daily oral doses produce transiently high (supra-physiological) peak free T₄ levels some three hours later. This T₄ peak may stimulate the excessive rT₃ production observed in levothyroxine therapy. An example of the response to a large dose is provided in a case-report: Having taken 20-times her daily T₄ dose, a suicidal woman’s blood had very high free and total T₄ and rT₃ but with well-regulated deiodination, the peak T₃ values never exceeded normal. It is easy to consider this an adaptive response to protect against elevated T₄, as noted in hyperthyroidism – and that it may be invoked by non-physiological, once-daily T₄ doses.

Sufficiently elevated rT₃ can be important and ominous. A significant cut-off value of 267 ng/dL (0.41 nmol/L) was determined at Stockholm’s Karolinska Institutet: Death occurred within the first week after myocardial infarction in 10% (16 of 165) of patients whose rT₃ levels were above 267 ng/dL; there were no deaths among the 166 patients with lower rT₃ levels (P <0.0004). The same elevated reverse T₃ levels >267 ng/dL also predicted a significantly increased risk of death within one year – independently of age, previous myocardial infarction, prior angina, heart failure, serum creatinine level and peak serum creatine kinase-MB fraction levels. Serum reverse T₃ assay has also been useful in other, less dramatic ways, for instance to predict glucose intolerance in uremia patients on hemodialysis and as an indicator of metabolic control in diabetes mellitus.

The question “what significant value does an rT₃ assay convey” invokes an important principle: Due to the complexity of thyroid signaling, the clinical application of any single test of thyroid function is imprecise. Assays for TSH and free T₄ are useful indicators of HP-T axis function. The ratio of total T₃ to total rT₃ additionally - and uniquely - demonstrates the status of peripheral deiodination and in the opinion of some researchers is a better indicator of thyroid function than TSH.

Ratio of T₃: rT₃

Whilst a value for rT₃ is shown to be useful, the ratio of total T₃ to reverse T₃ has greater worth (rT₃ is clinically available only as a “total” measure, so it must be compared to total T₃ –in the same units!). Shortly after the rT₃ assay became available, clinicians reported their patients had rising rT₃ with reciprocally falling T₃ during a variety of stressful events, such as operations; acute illness; calorie-restricted diet and myocardial infarction. These changes represent an adaptive response to stress. However, such responses become maladaptive if inappropriately prolonged. The persistence of elevated rT₃ with low T₃ has been referred to variously as Low T₃-Syndrome, Euthyroid Sick Syndrome and Non-thyroidal Illness Syndrome (to be discussed below). Researchers soon found the ratio of tT₃ to reverse T₃ a clinically useful indicator for these ill patients.

The tT₃/rT₃ ratio eliminates some common concerns in thyroid testing, particularly their non-Gaussian distribution and vagaries of binding proteins (by comparing total values of both). Since the T₄ which is not cleared by “alternative pathways” shall be converted into either T₃ or rT₃, this measure has proven a useful and sensitive measure of deiodination and thyroid signaling. An accounting metaphor seems relevant: If the prohormone T₄ represents a business’ billing (a potential for revenue), T₃ would be “collections” and rT₃ the “expenses” or “losses.” The tT₃/rT₃ ratio then represents the profit/loss statement, an essential measure of any corporation’s financial health – as is the tT₃/rT₃ ratio to quantify thyroid signaling.

The utility of this ratio compared to rT₃ alone was powerfully demonstrated in a study of 451 critically ill patients who received intensive care (ICU) for more than 5 days – and of whom 71 (16%) died. Comparing patients whose values fell into the highest vs. lowest quartiles, the tT₃/rT₃ ratio was strongly predictive of life vs. death, expressed as the odds ratio (OR) of survival = 2.9. In contrast, rT₃ alone was a weak indicator, OR = 0.3. Though tT₃ values generally rose over time, T₃/rT₃ increased only in survivors and remained unaltered or declined in the dying.

The clinical significance of the total T₃ to rT₃ ratio having been demonstrated, determining the range of normal values will be an important step in its clinical application. Although direct evidence among carefully-vetted people is currently lacking, there are several lines of evidence and useful older work. Firstly, T₃ and rT₃ are released into the bloodstream from the normal thyroid gland in the ratio of 9:1. The same 9:1 ratio is found in the secretions of benign thyroid adenomas. Within the cited studies’ margins of error, the “neutral” or resting-state tT₃/rT₃ value is 9.0.

However, deiodinase enzymes continually metabolize T₄ into either T₃ or rT₃. What resulting ratio is then enjoyed by healthy people? To determine this, a group of significant number should be properly selected; then, tT₃ and rT₃ are tested to calculate each individual’s tT₃/rT₃ ratio. Statistical analysis sets the normal range for these ratios to include 95% (+/- 2 SD, as above). There is some data on people in control groups for various research studies: “Normal” serum tT₃: rT₃ values – expressed in the same units, of course – were 11.03 in 30 healthy males aged 23-40 years and in two studies by the same authors, 12.2 +/- 0.6 and 12.5 +/- 0.6 Not-really healthy controls matched against people with insulin resistance (IR) by sex, age, body mass index and TSH had tT₃/rT₃ = 7.33 ± 0.33, while those with IR averaged 8.78 ± 0.47.

We may indirectly infer a “normal” range of tT₃/rT₃ using the reference intervals determined for tT₃ and rT₃ separately by Peeters et al. Compare the values for tT₃ and rT₃ at their lowest limits and again at the upper end: The ranges (given above) suggest tT₃/rT₃ normally varies from 7 to 10. The lower value of this derived interval is somewhat below the “starting” ratio of 9 upon secretion from the thyroid gland. This result is plausible yet not primary data. Some clinicians’ experience suggests most patients feel best with their tT₃/rT₃ ratio in the range of 10–14, whilst all thyroid hormone levels are adequate. This impression is gained from including the tT₃/rT₃ ratio as a data point in following patients’ response to thyroid hormone replacement. The medical literature lacks data regarding such an "optimal" ratio. A recent publication reviewed reports comparing T₄, T₃ or combinations of both for the treatment of hypothyroidism. As the value of the tT₃/rT₃ ratio was only recently appreciated, none of the cited studies incorporated this powerful measure into their design.

Drug interactions

As should be expected of a system controlled by the up- and down-regulation of enzymes, blood levels of rT₃ and the tT₃/rT₃ ratio can be altered by pharmaceuticals including drugs and hormones. Medications causing elevated reverse T₃ levels with low tT₃/rT₃ balance include the iodine-rich amiodarone; the antithyroid agent propylthiouracil – but not methimazole; the iodinated contrast agent ipodate (iopanoic acid); perhaps tobacco smoking and agents acting on receptors involved in the stress response can raise rT₃, below.

Naturally-occurring substances also can increase rT₃ levels. Endogenous factors (made within the body) include excessive levels of T₄, which can direct the deiodination of T₄ to rT₃, as above – an appropriate physiological response. A human study showed type-3 DI can be up-regulated by prolonged treatment with T₄ in dosses sufficient to suppress TSH; this treatment is usually associated also with elevated levels of T₄. Cytokines including tumor necrosis factor alpha, interferon-alpha, NF-κB and interleukin-6 and free (non-esterified) fatty acids increase rT₃ by blocking outer-ring (5’) deiodination. Exogenous bacterial endotoxin (lipopolysaccharide) increases rT₃ to a greater extent than can be attributed to the cytokines it also provokes.

A few drugs have been observed to lower circulating rT₃, including lithium carbonate; phenytoin and carbamazepine. Dithiothreitol reduces serum rT₃ by accelerating its deiodination to T₂. The antibiotic rifampicin, commonly used to treat tuberculosis, seems also to increase T₃ and reduce rT₃ by a similar action. Bexarotene, a retinoid specifically-selective for the retinoid X receptor (as opposed to the retinoic acid receptor) can decrease RT₃ and increase T₃. Agents modifying the stress-response also can lower rT₃ levels; see below.

Reverse T₃ is reduced by several other signals and stimuli. These include exogenously administered growth hormone and oral – but not intravenous – glucose and carbohydrate feeding.

From the first application of the physics term “stress” to biology, the central role of the adrenal gland in stress responses has been appreciated. The importance of thyroid hormone could not be understood until research had shown deiodination of T₄ is regulated, not a random process. Stress hormones, including cortisol and catecholamines, enhance the conversion of T₄ preferentially to rT₃ instead of T₃ during the acute response to stress. Dexamethasone and therapeutic corticosteroids block 5’-deiodination of T₄ to T₃ and raise rT₃ levels. The systemic administration of adrenaline (epinephrine) also increases rT₃, though in some tissues, it can increase 5’ (outer ring) deiodination to reduce rT₃, acting at least partly at the pre-translational level – chiefly by its beta-agonist effects. Predictably then, while the beta-agonist drug isoproterenol lowers rT₃, beta-blockers, especially propranolol inhibit T₃ production and increase rT₃. This, along with their anti-adrenergic effects, make beta-blockers useful for treating acute symptoms of thyrotoxicosis, though perhaps a concern for hypothyroid patients taking T₄.

Cancer

Cancer (carcinoma) cells, being undifferentiated and rapidly dividing, resemble embryonic or fetal cells. Cancer, like fetal tissue, has long been associated with high circulating levels of rT₃ and low T₃. This cannot be explained solely by the stress of severe illness (Euthyroid Sick Syndrome). For example, people with hepatitis C virus (HCV)-related cirrhosis of the liver and hepatocellular carcinoma (liver cancer) were found to have significantly higher rT₃ than well-matched HCV-cirrhosis patients without carcinoma. Tumor cells themselves abnormally produce deiodinase enzymes. The excessive production of type-3 DI occurs particularly in some brain tumors; basal cell carcinomas; liver cancers and neonatal hemangiomas, the endothelium of which make fetal-levels of 3-DI sufficient to produce “consumptive hypothyroidism.”

These aberrations may create conditions favorable to tumor growth, locally and even systemically. In normal development, T₃ stimulates differentiation of fetal cells into their mature adult forms. Research has begun to address the question as to whether conversely, a low T₃/rT₃ balance supports the proliferation of undifferentiated cells, including cancers: Added reverse T₃ increased the growth of several human sarcoma cell lines, leading the investigators to suggest a specific rT₃-receptor exists in neoplastic cells. However, when added to cell cultures, reverse T₃ inhibits proliferation of certain lines of breast and ovarian cancer cells. If a specific rT₃-receptor is responsible, it could be non-genomic – known for mutagenic and anti-apoptotic effects – or a genomic mutated variant.

Mutated thyroid hormone receptors are strongly associated with the onset and growth of cancers, particularly non-T₃ binding receptors such as TRα-2 (v-erbA) and mutated TRβ. The DNA programs for these receptors are considered oncogenes and were first identified in birds infected with tumor-inducing retroviruses.Mutated TRβ has been identified in liver, kidney, thyroid and other cancers. Malfunctioning mutated TRβ also is likely to be involved in the process of carcinogenesis and increases cells’ malignant and metastatic potential. A study of clear cell renal carcinoma (ccRCC) was summarized thusly: “…reduced TRβ1 expression (91% less) and tissue hypothyroidism (58% less T₃, due to lack of type-1 DI) in ccRCC tumors is likely to be involved in the process of carcinogenesis or in maintaining a proliferative advantage.” Nongenomic actions of thyroid hormone are also observed to activate complex signaling leading to angiogenesis and tumor cell proliferation. It seems further study of rT₃ and what effects, if any, it may exert on mutated receptors may be a relevant aspect of cancer research.

AIDS

Strong evidence that rT₃ has biological activity – contributing a tonic inhibition of the metabolic rate – comes from HIV-infected young men who have lost this effect, a situation described as “unique.” Their (total) reverse T₃ was observed to decline as their HIV infection advanced, even though their thyroxine-binding globulin became elevated. Whilst low T₃ levels correlated with poor survival in advanced AIDS, as can be seen in other severe illnesses, the authors noted the persistence of normal T₃ (with falling rT₃) may contribute to weight loss.

The finding of low rT₃ in HIV patients was soon corroborated and the important association of low rT₃ with increased metabolic rate and weight loss was recently validated. Patients with untreated stage-A HIV infections were studied. Their findings confirmed that low rT₃ and normal T₃ are associated with a hypermetabolic state, featuring increased resting energy expenditure, protein breakdown and 22% greater protein synthesis. Simultaneous findings of low urinary C-peptide and elevated IL-6 and TNF-α were coincidental, not causally-linked and there was no evidence of intestinal malabsorption. These patients apparently prevented Wasting Syndrome (cachexia) by increasing their caloric intake.

Nongenomic explanations for these metabolic effects of low rT₃ can be suggested. A Japanese report cited above indicated that rT₃ inhibits the metabolism, reducing the ATP/ADP ratio in cultured cells. We’ve also previously noted rT₃ blocks type-2 deiodinase: As euthyroid (normal) rT₃ levels suppress type-2 DI activity in the brain by 20-30%, this provides a degree of tonic inhibition. The high metabolic rate associated with low rT₃ may be due to the loss of these physiological restraints.

This effect also could be genomic: A highly-conserved (suggesting an important function) alternatively-spliced version of Thyroid Receptor alpha exists, called TRα-2; it is known as a “non-T₃ binding receptor.” The effects of TRα-2 clearly inhibit T₃. If not T₃, could this receptor bind rT₃? A Pub Med search for “thyroid receptor alpha 2 AND rT₃” reveals just 4 articles, none of which actually addresses the question. A review of publications investigating thyroid receptors is similarly unfruitful. Could the “binding protein for reverse T₃ (NrT₃BP) in the rat liver nuclear extract” described decades ago represent non-T₃ binding receptor variants?

Stress and the Euthyroid Sick Syndrome

The stress response, as noted, is a complex adaptation to improve survival chances during life-threatening circumstances. Euthyroid Sick Syndrome (ESS) is its maladaptive prolongation, particularly of altered peripheral deiodination to reduce T₃ and increase rT₃. There are several associated perturbations: The hypothalamic–pituitary–thyroid axis develops a degree of dysfunction; there is evidence of at least compensatory membrane transporter alterations and cytokine levels are elevated. Euthyroid Sick Syndrome is described primarily among critical care patients, though as above, it has been noted in a variety of other settings associated with severe situational and physiological stress. These patients have normal levels of TSH, so are biochemically “euthyroid.” No intrinsic thyroid disease being involved, EES alternatively may be called “non-thyroidal illness syndrome.” Whereas “low-T₃ syndrome” was an early designation for the condition, the term is now little-used because many patients actually have “normal” T₃. Most research in this area has disregarded the contribution of rT₃, considering it a functionless marker of inappropriate deiodination.

ESS Patients become physiologically hypothyroid as they produce less T₃ and more rT₃ – both, as above being active and in effect, counter-balancing hormones. This response has a positive survival value at the onset. A report of meningococcal sepsis showed rapidly-fatal cases had paradoxically higher tT₃/rT₃ than survivors; the authors felt the victims had insufficient time to adapt their thyroid metabolism. However, ESS becomes maladaptive when it is inappropriately continued.

The prolonged condition of ESS is clearly associated with significantly higher death rates and in post-operative patients, prolonged recovery. Ill-effects arise from reverse T₃, not just low T₃: Well-designed animal research has also shown that infusing rT₃ during shock caused significantly higher death rates. Dysfunction of the hypothalamus – itself a target of altered hormone signaling, drugs and cytokines in protracted critical illness – may worsen, causing falling T₄ levels. There is an ultimate dichotomy: Improvement in T₃/rT₃ heralds survival, whilst deterioration predicts a fatal outcome.

ESS is the most severe manifestation of “second level of thyroid control” dysfunction. The role of intervention to supplement T₃ (and by some, T₄) is not clear. Such efforts have been convincingly advocated by leading experts, while others state “there is no persuasive evidence it is beneficial” for these critically-ill people, though acknowledging thyroid hormone treatment causes no harm. Along with general supportive measures, the use of N-acetyl cysteine has been suggested to prevent ESS by protecting 5’-deiodination against blockade by IL-6 (cytokine). Supplementing the dysfunctional hypothalamus with stimuli to produce TSH and growth hormone (hGH) has shown good results, with the added benefit that hGH allows increased T₄ and T₃ with no rise in rT₃. Euthyroid Sick Syndrome has provided the impetus for much research into the deiodination of T₄. To date, though, little of it has esteemed rT₃ a functioning hormone.

In thyroid disorders

Altered T₄ metabolism can be seen with a variety of thyroidal disorders. Hyperthyroidism has been associated with reduced tT₃/rT₃ balance in numerous studies. This may be understood as a successful adaptive response to maintain physiological euthyroidism despite elevated thyroid levels. Support for this assertion may be found in reports citing hyperthyroid patients’ normal T₃ and lack of clinical symptoms despite the presence of high T₄.

Conversely, an increased tT₃/rT₃ balance could be appropriate when patients lack sufficient thyroid hormone. When tested, this finding is indeed reported in most studies of hypothyroidism. The “second level of thyroid control” adapts to maintain T₃ availability should T₄ production become insufficient – an effect to which elevated TSH contributes. Deiodination is also adjusted to the supply of iodine, a nutrient required to make thyroid hormone: People living in iodine-deficient areas increase their conversion of T₄ to T₃. Research has proven frank iodine deficiency increases 5’-deiodination of T₄ to T₃, particularly effecting enzymes within the thyroid gland itself, whilst iodine excess conversely reduces that action.

Contrary to these expectations, though, an older study reported seventeen hypothyroid patients having significantly lower tT₃/rT₃ ratios than normal people This study’s authors considered the finding maladaptive and likened it to Euthyroid Sick Syndrome. The probable explanation for these valid comments is impaired deiodination from autoimmune inflammation and this leads to several important associations:

Hypothyroidism is usually caused by autoimmune thyroiditis (AIT). Thyroid glands of patients with Hashimoto’s AIT are infiltrated by lymphocytes, which produce within the gland significantly high levels of inflammatory cytokines (notably TNF-α, IFN-γ, IL-2 and IL-6). These cytokines are known to block outer ring (5’)-deiodination and can increase type-3 DI production, to further reduce T₃ and raise rT₃. Intra-thyroidal 5’-deiodination significantly compensates for low thyroid production – a consequence of AIT – so its inhibition by locally high concentrations of cytokines can be important. People with autoimmune thyroiditis, as would be expected, have significantly elevated rT₃ levels (p<0.00002) and they respond abnormally to TSH, with impaired fT₃ and fT₄ release and increased rT₃ production.

Evidence shows autoimmune thyroiditis patients can have symptoms of hypothyroidism even with normal TSH values: Among women with euthyroid AIT – having diseased glands still capable of functioning normally – higher antithyroperoxidase antibody levels (thusly, more intra-thyroidal cytokines) correlate significantly with a greater symptom load and decreased quality of life. Also, the prevalence of euthyroid AIT is reportedly increased among chronically fatigued patients.

Patients with “subclinical hypothyroidism,” more advanced AIT requiring high TSH to produce T₄ normally, have greater rates of dysfunction and death. However, they show little benefit from treatment with T₄ alone, so intervention is not encouraged until the elevated TSH exceeds 10 μIU/mL. The classical definition and treatment of subclinical hypothyroidism, it should be noted, do not consider rT₃ or even account for T₃.

Finally, many frankly hypothyroid patients whose levothyroxine (T₄) treatment appears adequate upon testing the HP-T axis with TSH and T₄ levels according to prevailing practice standards nevertheless complain they feel no better. Their dissatisfaction – for which they may be offered psychoactive medication – is stated in numerous books in the popular press, some written in quite an angry tone.

These situations are linked by the common thread of autoimmune thyroiditis. As above, AIT effects deiodination to reduce the tT₃/rT₃ balance and rT₃ exerts inhibitory actions. Harvard researchers state such altered deiodination can cause physiological hypothyroidism – “disruption of thyroid hormone signaling” – whilst even T₃ levels remain within the normal range. They describe a new thyroid paradigm, hypothyroidism at the second level of regulation and its laboratory hallmark is neither high TSH nor low T₄ or T₃ but a low tT₃/rT₃ balance. The condition is loosely analogous to type-2 diabetes, in which abundant hormone nevertheless has inadequate effect. It has been suggested that the Integrative Medicine term “type-2 hypothyroidism” might appropriately be applied to designate this disorder. The clinical importance of the condition, in its prevalence and severity, is not delineated.

Such a state of low thyroid function, undetectable by the recommended tests for TSH and fT₄, has long been postulated. Barnes, Wilson and others noted patients’ symptoms and signs – and response to thyroid treatment incorporating T₃ – may not match those test results. Furthermore, studies report that up to sixteen percent of patients do not respond optimally to treatment with T₄ alone. Efforts to explain these discrepancies by determining patients’ resting metabolic rate; measuring their body temperature< or by testing the response to TRH stimulation have been, for various reasons, ultimately disappointing. Other mechanisms have been proposed, from “endocrine-disruption” by chemicals, of which the existence is well-supported but the clinical significance is uncertain, to the effects of electromagnetic fields, which can provoke changes characteristic of the stress response and may effect the thyroid.

There are several validated forms of “resistance to thyroid hormone”: The first is Refetoff Syndrome, caused by abnormal Thyroid Receptors and the second is due to defective trans-membrane thyroid hormone transport These, however, are uncommon hereditary conditions, for which the body compensates with greater thyroid production, elevated blood hormones and often an enlarged thyroid – all inconsistent with the observed clinical problem. The simplest explanation for a common condition of thyroid dysfunction characterized by normal blood TSH and fT₄ and poor response to levothyroxine (T₄) treatment is low thyroid signaling due to the inappropriate deiodination of T₄.

This less severe, “ambulatory” version of ESS can be observed in patients with AIT, subclinical and mild hypothyroidism and those taking levothyroxine, as above. However, just as ESS occurs in critical care patients without co-existing thyroid disease, it has been suggested that chronic dysfunctional deiodination can occur in stressed people with healthy thyroid glands. Despite the disapproval of symptom questionnaires expressed in current guidelines, such patients are said to be identified by their clinical symptoms. The diagnosis is then supported upon demonstrating a low T₃/rT₃ ratio – regarded by some a better marker of thyroid signaling than TSH – and may be confirmed by their response to treatment.

The best choice of treatment for hypothyroidism has been debated since synthetic-origin levothyroxine (T₄) was marketed to compete with desiccated animal thyroid. While T₃ constitutes 20% of the latter, the former contains none and its effectiveness depends wholly upon deiodination. A leading textbook states: “A primary advantage of levothyroxine therapy is that the peripheral deiodination mechanisms can continue to produce the amount of T₃ required in tissues under the normal physiologic control.” However, this may not always be possible: Some patients taking sufficient T₄ to become euthyroid (normal TSH) nevertheless have low T₃. In this study, larger doses of T₄ returned T₃ levels to normal but that tactic, as above, can result also in higher rT₃. While current consensus opinion endorses the use of T₄-only as a precursor, conservative and Integrative physicians alike advocate treatment with a “natural” mixture of both T₄ and T₃. They postulate treatment combining T₃ with a reduced amount of T₄ (as pharmaceutical liothyronine or “natural” desiccated thyroid) can improve patients’ clinical response. Their ideas are physiologically sound, as 95% of inhibitory rT₃ arises from T₄ – and are supported by data from an older clinical trial demonstrating added T₃ increased the tT₃/rT₃ ratio and improved outcomes. Renewed interest in combined T₃+T₄ treatment is indicated by the increased number of recent publications.

Studies comparing treatment with T₄ against T₃ or both combined yield varied results and despite flaws, the preponderance indicate physiological replacement with both T₄ and T₃ is superior to T₄-only. Basic physiological and design problems can be seen in some reports, including the use of once-daily dosing, particularly an issue with the “brief duration” of T₃; the failure to individualize doses – or even adjust them upon causing frank hyperthyroidism with mood impairment (making one study primarily a test of the investigators’ procrustean dosing hypothesis); monitoring therapy only with TSH and finally, neglecting the non-genomic requirements for T₄ when providing T₃-only treatment. Further support for T₃-added treatment – and a genomic rationale – is found in a study of a common (16% prevalence) mutation of the gene (DIO2) coding type-2 deiodinase. People with the mutated gene had impaired deiodination and they fared significantly better taking T₄+T₃ treatment compared to T₄-alone. In its summation, a recent review of combination T₃ and T₄ treatment concluded: “The outcome of our analysis suggests that it may be time to consider a personalized regimen of thyroid hormone replacement therapy in hypothyroid patients.”

T₃-only (liothyronine) treatment has been accepted for use in particular circumstances and is recommended by some physicians for ESS. In humans, treatment with sub-physiological doses of T₃ leads to reduced T₄ and rT₃ in the blood. Since T₃ therapy maintains or improves T₃ levels, the tT₃/rT₃ ratio thusly can be increased. Despite thoughtful opposition, treatment with T₃ has been suggested for selected symptomatic euthyroid patients and studies old and recent have shown its benefit. A number of T₃ intervention trials have been successful also in heart disease patients and Multiple sclerosis research.

Effects on reproduction

Thyroid hormone in appropriate amounts is essential for healthy reproduction. Virtually every aspect depends on thyroid signaling: Male fertility; ovulation; implantation; embryonic organogenesis and differentiation (particularly the central nervous system); intrauterine growth and development and carriage of the pregnancy to term. Of course, thyroid hormone continues to be essential through parturition and into the neonatal phase, when its role in non-shivering thermogenesis may be significant to humans and congenital hypothyroidism will cause tragic consequences if treatment is delayed.

Successful progression from conception through infancy involves changing expressions of deiodinase enzymes and also of Thyroid Receptors. Large shifts in fetal thyroid hormone balance occur first in local tissues and later systemically, particularly so through delivery and early neonatal life. Whilst the precise amount and timing of T₃-signaling is known to be absolutely essential to proper fetal development, the exact role of rT₃ in this process is uncertain. Reverse T₃ is abundantly present in fetal circulation, some 15-times more than found in adult sera. Indeed, it is preponderant and has even been called “fetal thyroid hormone.” That rT₃ in the adult can act as an inhibitor is particularly evocative, since ontogeny requires withholding the effects of T₃ until a precise moment. The significance of rT₃ in reproduction, then, could be great and if so, is likely complex.

The adult uterine lining is one of the few adult tissues to produce type-3 deiodinase, which acts on the inner ring. This suggests the local regulation of thyroid hormones – and a low T₃/ rT₃ balance – is important to normal uterine function

Following conception, the placenta must invade the maternal uterus to secure the fetal blood supply, an activity facilitated by T₃. Promptly, however and afterwards throughout the pregnancy, the placenta produces large amounts of type-3 DI. The resulting placental conversion of T₄ into rT₃ and deactivation of T₃ into T₂ greatly reduces TH transfer from mother to fetus, evidently a key regulatory step.

Many or most fetal tissues also produce type-3 DI abundantly. In contrast, little type-2 DI is produced until required for organ-specific differentiation – and type-1 DI only appears after 30 weeks. This sequence of types of deiodination is the reason fetal blood contains mostly rT₃, at an order of magnitude greater than normal adult levels; has much conjugated T₃ (T₃-S) and but very small amounts of T₄ and T₃. This ensures the proper delivery of the correct type of thyroid signaling when necessary for normal development.

Such great differences from the maternal blood levels have long been understood to allow the fetal thyroid and hypothalamic-pituitary axis to develop and function without suppression by maternal TH. The formation of the brain and neutrally-derived structures, the eyes and ears in particular, has been carefully studied. Their development requires the proliferation of cells to a critical number, during which time T₃ must be very low – assured by plentiful type-3 DI production. Then, cells must assume their adult forms, a transition for which T₃ is essential. Local synthesis of “prophylactic” type-3 DI ceases and that of type-2 DI begins, which converts available T₄ into T₃ sufficient to activate cell differentiation.

Organs deprived of T₃ fail to differentiate properly. This was learned long ago, when scientists found tadpoles made hypothyroid could not metamorphose into frogs. Conversely, thyroid hormone stimulation in excess – or at the wrong time – causes rushed or imperfect development in amphibians and humans. In particular, disordered neuronal differentiation and skeletal abnormalities are seen when high hormone levels from hyperthyroid mothers overwhelm the protective ability of placental and fetal deiodinases.

The role of nongenomic effects in embryonic organ and tissue development is crucial and reverse T₃ has significant nongenomic effects, which must be considered. One such sequence is required for normal cellular migration and brain development. Noting that Down syndrome children have significantly low rT₃ values, a causal linkage of low rT₃ with their brain and other developmental abnormalities has been suggested. Other nongenomic effects of rT₃ noted above may be important. A yet broader view suggests rT₃ may substitute for T₄ in-utero: Some nongenomic receptors bind rT₃ as readily as T₄; possibly rT₃ maintains their effects during fetal development without the “risk” of prematurely producing T₃. Finally, understanding that proper embryogenesis requires orchestrated cellular proliferation and apoptosis (the removal of particular cells – the embryonic webs between fingers, for example), the statement above that nongenomic thyroid effects are characteristically “mitogenic and anti-apoptotic” is provocative.

Effects of rT₃ on the embryonic Thyroid Receptors and genome have not been defined. The roles of Thyroid Receptors in embryogenesis are crucial. Thyroid Receptor-alpha (TRα) is most important early in development, when fetal T₃ is scarce. If not bound to T₃, TRα covers the thyroid response element and actually reduces the transcription of T₃-mediated DNA programs. The production of TRα and Thyroid Receptor-beta (TR-β) varies with location (brain, limbs, etc.) and by phase of development. The functions of receptor variants, like non T₃-binding TRα-2 and TRα-3 are not yet clarified. Does rT₃, which several reports have found to compete with T₃ when given as a “pre-treatment,” play a role at the fetal TR? Perhaps it might bind TRα to produce a null-state. Could the “non T₃-binding” variants instead have affinity for rT₃? The TR is known to be influenced by various co-repressors, including retinoids (vitamin A derivatives); hormones such as estradiol; and other nuclear receptors like the PPARs. A reassessment of possible rT₃ participation using modern methods might be useful. This issue also has bearing upon the development and proliferation of cancer.

History

In terms of human scientific endeavor, Endocrinology is rather recent. The word “hormone” was not coined until 1905, when Starling had proven the intuitively evident: The mysterious lumps of tissue called glands – seemingly anatomically isolated – release potent chemicals into the bloodstream to influence the entire body. This work was, in various ways, propelled by Brown-Séquard’s interest in the adrenal glands and gonads and his well-publicized belief that the glands produced and stored that which came to be called hormones.

The role of the thyroid gland began to be revealed in the 1870s, when surgical science and Theodor Kocher’s skill had advanced sufficiently to allow his complete removal of large, functionally crippling goiters (the cause of which, iodine deficiency, was then unknown). His post-op patients could again easily breathe and swallow but they had become surgically hypothyroid, the unfortunate consequences of which had been prematurely dismissed. The condition was especially obvious in the children, mimicking the syndrome of congenital hypothyroidism and by 1883 was called “artificial Cretinism.” The post-op condition also confirmed that thyroid gland failure caused Gull’s disease or “myxedema,” the contemporary terms for hypothyroidism. Drawing on Brown-Séquard’s work, these patients were injected with extracts of sheep thyroid glands ca. 1890 and restored to health. Kocher’s studies of the thyroid gland were awarded the 1909 Nobel Prize in medicine. Over more than a century, research has further clarified the control of thyroid gland secretion and the roles of its hormones.

It took about 50 years to delineate the HP-T axis. Pioneering neurosurgeon Harvey Cushing appreciated the regulatory function of the pituitary by 1912; after having removed his patients’ glands because of large tumors, he saw them slip into a state similar to hibernation. By 1930, the importance of the pituitary-thyroid axis had been generally acknowledged. Despite obvious anatomical connections between the hypothalamus and the pituitary gland, both direct nerve-linkage and a portal venous system, the hypothalamic-pituitary axis could not be proven until more than 20 years later, in the 1950s. These efforts merited the 1977 Nobel Prize.

Concurrently, of course, thyroid hormones themselves were studied. The most abundant hormone, T₄ was isolated by 1915 and its structure was correctly reported in 1927. For some years, researchers did not understand why hormone extracted from the thyroid was more potent than an equal amount of T₄. The answer came in 1952, when T₃ was identified in humans and its power (to suppress the H-P axis) was found to be some five-times greater than T₄. By 1955, T₃ was acknowledged as the most active form of thyroid hormone.

The conversion of T₄ to T₃ had been described in 1951 and again in 1955 but this was retracted – incorrectly – in 1958. The validity and clinical importance of T₄ deiodination was not appreciated until 1970. Researchers were then able to accept Gross’ 1953 suggestion that T₄ is a prohormone, with little metabolic activity other than that arising from its transformation to T₃. Ingbar reiterated this proposal in the 1974 Harrison's Principles of Internal Medicine. Further research was needed to later define non-genomic effects of T₄.

The identification and synthesis of reverse T₃ was reported in 1955. By 1959, it had been found to inhibit the effects of T₄. However, reports both slightly earlier and later stated rT₃ had none of the stimulating effects expected of thyroid hormone. Reinforcing perceptions of irrelevance, difficulty measuring rT₃ led to the belief it was evanescent and did not linger in normal human serum. Chopra disproved this, demonstrating that deiodination of T₄ in humans produces significant amounts of rT₃ as well as T₃, writing also: “…rT₃ may not be just an inactivation product of T₄ but that it may be involved in physiological regulation of metabolism and biological action of T₄.” Whilst other writers suggested T₄ deiodination to T₃ might be a “random” process, Chopra asked whether the deiodination of T₄ could instead be a physiologically-regulated phenomenon. Time and meticulous research have validated his insights.

Recognition of the proper role of T₃ took 18 years, from 1952 to 1970 – at which time the major purpose for T₄ was also defined. In 1974, the earliest evidence that the peripheral metabolism of thyroid hormones could be modulated by physiological or physiopathological events was published: Calorie-deprived humans showed decreased circulating T₃ relative to T₄ with higher rT₃ concentrations. The importance of this “second level” of control was stated as fact only around 2005. The inhibitory role of rT₃ has also taken decades to define and has yet to be universally acknowledged.