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Studier over referencemetoder til måling af stofskiftehormoner

Steen Strange Holm

The aim of the study is to review the literature on the potential reference methods for the quantification of free thyroxine (FT4) and to develop an adequately sensitive and specific reference method for determination of FT4 in serum.

The practical part of the study is divided into two parts.

1. The first part focuses on separation of the free fraction of T4 from the bound fraction with a minimal disturbance of the equilibrium. Three different separation techniques are evaluated: Equilibrium dialysis, ultrafiltration and ultracentrifugation.

2. The aim of the second part is to establish a simple non-immunological quantitative reference method for measuring total thyroxine (T4) and triiodothyronine (T3) in serum.

In order to achieve sufficiently sensitivity to quantify the free fraction of thyroxine, FT4.Two different techniques are tested; ICP/MS (Inductively Coupled Plasma/mass spectrometry) and LC/MS (Liquid Chromatography/Mass Spectrometry).

This thesis consists of two published papers and a report.

The first paper is a review of the literature on the methods for the detection of FT4. An overview of the most important errors and interferences of the potential reference methods for determination of FT4 (and free triiodothyronine, FT3) are presented. Furthermore, a short review of methods based on isotope dilution/mass spectrometry (ID/MS) to quantify thyroxine is provided.

The second paper describes investigations, performed during the Ph.D study. Three different potential separations techniques to separate the free fraction of T4 from protein bound fraction of T4 were studied to find a method that best fulfills the following conditions:

· Minimum adsorption of T4 to membranes or to any in vitro solid surfaces to which T4 might adsorb.

· Minimum leakage of binding proteins through membranes (ultrafiltration /dialysis) or remain in the supernatant (ultracentrifugation).

The thesis is divided into five sections. The first section gives a brief introduction of the physiology and the patophysiology of the thyroid gland and the synthesis and metabolism of the thyroid hormones.

The second section describes the analytical quality goals and the preanalytical variables in connection with measuring the thyroid hormones.

The third section presents descriptions of the theoretical considerations and calculations pertaining to the equilibrium between FT4 (free thyroxine) and the protein bound T4 (thyroxine).

The fourth section provides descriptions of the feasible techniques for separating the FT4 from the bound T4. The advantages and disadvantages of these techniques are described and discussed. In particular, the technical difficulties experienced in connection with separating the FT4 are studied and discussed.

The fifth section firstly covers a study of the sample preparation for a potential reference method for the measurement of total T4 and total T3. The method is compared to previously developed reference methods.

The second part of section five describes the preliminary experiments carried out employing the methods of ICP/MS (Inductively Coupled Plasma/mass spectrometry) and LC/MS (Liquid Chromatography/Mass Spectrometry) aiming to measure total T4 (thyroxine) and T3 (trijodthyronin), and to measure the free concentration of thyroxine, FT4. The advantages and disadvantages in applying these techniques are elucidated with a view to determining total T4, T3 and FT4.

Based upon the theoretical considerations made and the experiments performed, the following conclusions are drawn:

1. The most suitable techniques for separation of free fraction of T4 from the bound fraction, proved to be the equilibrium dialysis and the ultrafiltration procedures in spite of the fact that these methods contain some weaknesses such as interference and errors or inaccuracy related to the analysis.

The review of the literature revealed, that the major inaccuracies related to equilibrium dialysis are in vitro dilution of possible serum binding inhibitors, primarily drugs, (e.g.: phenytoin, carbamazepine and salicylate) and heparin induced free fatty acid.

The experimental investigations showed that in the process of ultrafiltration, the adsorption to membranes and the separation of the free fraction, without determination of some of the bound fraction of T4, presumably constitute the main error related to the analysis.

2. A potential sample preparation procedure has been developed and in comparison to previously developed methods applicable to LC/MS, this potential method has been improved, i.e. higher recovery and a method less laborious.
The developed sample preparation procedure was tested in connection with employing ICP/MS, or LC/MS bearing in mind the fact that the testing might result in changes in the sample preparation procedure.

In a preliminary test, it was found technical difficult to use ICP/MS. Consequently, it was determined that henceforward much effort be devoted to develop a method for the quantification of T4 and T3 by LC/MS. Establishing a method for the quantification of the total T3 without the possibility of employing MS/MS would be particularly difficult as the MS/MS is highly selective and sensitive in comparison with the MS.

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