Thyroxine conjugation

The EMMIA system was developed by Ngo and Lenhoff (N3, N4). In this assay, enzyme activity is modulated by an enzyme modulator which is coupled to antigen (free form) but not by the complex of enzyme modulator-antigen and antibody (bound form). As shown in Fig. 2 and Table 6, in an enzyme inhibitor immunoassay, an enzyme inhibitor is used as a negative modulator. For example, the reaction mixture for measuring thyroxine consists of acetylcholine inhibitor-thyroxine conjugate [I-Ag], acetylcholinesterase [E], unlabeled thyroxine [Ag], and antithyroxine antibody [Ab]. When the amount of unlabeled thyroxine, which binds to antibody [Ab Ag], is increased, the free form of acetylcholine inhibitor-thyroxine conjugate [I-Ag] increases, and the enzyme activity decreases. Therefore, the enzyme activity is inversely proportional to the concentration of unlabeled thyroxine. A tetrazyme kit (Abbott) is now available for measuring thyroxine. 

As a special case, the fluorescence intensity of the fluorophor-thyroxine conjugate [F-Ag] is increased when antibody to thyroxine is bound to it [F-Ag Ab]. When unlabeled thyroxine [Ag] is added, the fluorescence enhancement is decreased (S6). 

The functional form of thyroxine (T3) is generated by the deiodination of T4, and PCBs can influence the tissue levels of this form by disturbing metabolism, as well as by reducing the binding of T4. PCBs have been shown to inhibit the sulfation of thyroid hormones and the deiodination of T4 to T3. They can also induce the glucuronyl transferase that conjugates T4 (Brouwer et al. 1998). 

Figure 19.1 Dose-dependent response curve for thyroxine (T4) assay using E5-Ab and double-antibody immune complex. Solutions (20 juL) of the reference samples containing 0.0 to 25.0 /ig/dL T4 were mixed with 180 fiL of the releasing solution. Fractions (132 juL) of this mixture were combined with 38 //Lof an E5-Ab solution. After standard incubation, 76 A. of this mixture was spotted on a blank Stratus tab. Incubation times and reagent volumes used for the rest of the reagents, such as the conjugate and the substrate wash, were identical to those defined for an existing T4 assay on Stratus ...

Figure 14.9. Overlap between the emission spectrum of a BPE-thyroxine donor conjugate and the absorption spectrum of the acceptor conjugate CY5-anti-thyroxine IgG Donor emission through a 580-nm interference filter is shown below the full emission spectrum. (From Ref. 34 with permission.)...

Figure 14.15. Multifrequency phase-modulation curves of 4 nM BPE-thyroxine donor conjugate ( ) of donor in the presence of CY5-anti-thyroxine IgG acceptor conjugate (a) and of both donor and acceptor in the presence of a nearly saturating amount of thyroxine ( ). (From Ref. 34 with permission).

Hypothyroidism developed within 2 weeks of rifampicin therapy in these patients and resolved when it was withdrawn. Rifampicin increases thyroxine clearance, possibly by enhancing hepatic thyroxine metabolism and the biliary excretion of iodothyronine conjugates. In healthy volunteers rifampicin reduces circulating thyroid hormone concentrations without affecting thyrotropin, suggesting that rifampicin directly reduces thyroid hormone concentrations. 

Substrates MTX, thyroxines, digoxin (Oatp2), bile salts, peptides, eicosanoids, statins, GSH, glucuronide and sulfate conjugates of hormones... 

The degradation of thyroxine in liver occurs primarily by conjugation with glucuronic acid. In this assay, [12SI]thyroxine glucuronide was quantitated by on-line radiochemical detection. 

Disposition in the Body. Incompletely and variably absorbed after oral administration. It is metabolised by de-iodination to liothyronine (tri-iodothyronine) which is the principal active form of thyroxine further de-iodination to thyroacetic acid (4-/7-hydroxyphenoxyphenylacetic acid), and conjugation with glucuronic acid and sulphate also occur. About 30 to 55% of a dose is excreted in the urine and 20 to 40% is eliminated in the faeces of the urinary material about 40% is thyroacetic acid and 20% is liothyronine. 

C, D. Conjugated proteins. Prosthetic group. Hormones epinephrine, thyroxin, insulin, cortisone, ACTH. Chemotherapy. Arsphenamine, sulfanilamide and other sulfa drugs, penicillin, streptomycin, chloramphenicol, aiireomycin. Bacteriostatic action of sulfa drugs through competition with a bacterial growth substance, para-aminobenzoic acid. 

The mechanism of action of o-thyroxine appears to be stimulation of oxidative cataboli.sm of cholesterol in the liver through stimulation of 7-a-cholcstcrol hydroxylase, the rate-limiting enzyme in the conversion of cholesterol to bile acids. The bile acids arc conjugated with glycine or taurine and excreted by the biliary route into the feces. Although thyroxine docs not inhibit cholesterol bio.synthesis. it increases the number of LDL receptors, enhancing removal of LDL from plasma. 

A second type of EMIT has been developed using the enzyme malate dehydrogenase as the enzymatic label. Research has shown that thyroxine competitively inhibits malate dehydrogenase. A conjugate prepared with thyroxine covalently bound close to the enzyme s active site shows very low specific activity that can be restored by binding of the thyroxine to arcP -thyroxine antibody. In this very specific assay for thyroxine, enzyme activity increases upon antibody binding, so that in a competitive assay for free thyroxine, activity decreases with increasing free thyroxine concentration. 

Fig. 1. Structure of thyroxine (T4) showing the outer and inner aromatic rings and the d -OH involved in sulfate or glucuronide conjugation. T3 is formed by loss of the 5 iodine and rT3 by loss of the 5 iodine.

Thyroxine-MDase conjugates are peculiar in that they are enzymatically inactive but become activated by the binding of anti-thyroxine antibodies (Ullman et al., 1975). It seems that the conjugated thyroxine inhibits the enzyme by hindering its active site, whereas the antibody reactivates the enzyme by pulling the thyroxine away from the active site. Although this mechanism is not clearly understood, inhibition appears to be due to an increase in the (Van Lente and Galen, 1980). Many of these assays are marketed under the trade name EMIT by Syva Co. Enzyme activities are measured as discussed in Chapter 10. 

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