Determination of 3-monoiodotyrosine and 3,5-diiodotyrosine in newborn urine and dried urine spots by isotope dilution tandem mass spectrometry.

High levels of 3-mono- and 3,5-diiodotyrosine (MIT and DIT, respectively) in urine have been related to iodotyrosine dehalogenase 1 deficiency, a type of congenital hypothyroidism. However, the determination of MIT and DIT in urine is not included in newborn screening programs performed in clinical laboratories to detect inborn errors of metabolism. We report here on the development of an analytical method for the determination of MIT and DIT in newborn urine and dried urine spots (DUS) by Liquid Chromatography Isotope Dilution tandem Mass Spectrometry (LC-IDMSMS). The development included the synthesis of 15N-monoiodotyrosine and 13C2-diiodotyrosine through the iodination of 15N-tyrosine and 13C2-tyrosine, respectively, using bis(pyridine)iodonium(I) tetrafluoroborate (IPy2BF4). Both labelled analogues were added at the beginning of the sample preparation procedure and used to develop both single- and double-spike LC-IDMS methods for the determination of MIT and DIT. The developed double spike methodology was able to quantify and correct possible MIT ↔ DIT interconversions throughout the sample preparation, which was observed for concentrated urine samples but not for DUS. Suppression matrix effects on the absolute signals of MIT and DIT were observed in urine samples but did not affect the IDMS results as recoveries on urine samples at different dilution factors could be considered quantitative. Method detection limits were 0.018 and 0.046 ng g-1 (limits of quantification 0.06 and 0.15 ng g-1) by single-spike IDMS, for MIT and DIT, respectively, in the analysis of urine samples and 0.07 and 0.05 ng g-1 (limits of quantification 0.23 and 0.17 ng g-1) for MIT and DIT, respectively, in the analysis of DUS. No significant differences were obtained for MIT concentrations in the analysis of the same newborn samples stored as liquid urine or DUS when the results were corrected for the creatinine content. Finally, 36 DUS samples from healthy newborns were analyzed and MIT was detected in all samples at low ng mg-1creatinine levels.

Effect of different cooking methods on the content and bioaccessibility of iodine components in abalone (Haliotis discus hannai).

The present study aimed to evaluate the changes in total iodine and iodine species (iodide, iodate, 3-iodo-l-tyrosine, and 3,5-diiodo-l-tyrosine) content in abalone after different treatments (raw, semi-drying, steaming, grilling, and boiling) and in-vitro digestion using high-performance liquid chromatography-inductively coupled plasma-mass spectroscopy (HPLC-ICP-MS). The highest reduction in iodine content was found in boiled abalone (64.95%), followed by steamed (32.40%) and grilled (32.11%) abalones. There is no significant difference between iodine content of raw and semi-dried abalone. Absorption efficiency was determined by an in vitro digestion procedure using simulated gastro/intestinal solutions. Unlike total iodine content after cooking, absorption efficiency increased after cooking. Absorption efficiency of semi-dried abalone is the highest (28.53%), followed by boiled (23.85%), grilled (22.62%), steamed (21.51%), and raw (12.20%) abalones. Iodide was the major form of iodine present in the abalone after cooking and in vitro digestion. No iodate was observed, and the organic iodine content was very low.

Proteins oxidation and autoantibodies' reactivity against hydrogen peroxide and malondialdehyde -oxidized thyroid antigens in patients' plasmas with Graves' disease and Hashimoto Thyroiditis.

The aim of this study was to evaluate proteins oxidation in plasmas of two autoimmune thyroid diseases (AITD): Graves' disease (GD) and Hashimoto Thyroiditis (HT), and to determine whether oxidative modification of thyroid antigens (T.Ag) enhanced the reactivity of autoantibodies in plasmas of AITD patients compared with the reactivity towards native T.Ag. Carbonyl and thiol groups and MDA-protein adducts were assessed spectrophotometric methods in plasmas of 74 AITD patients and 65 healthy controls. The reactivities immunoglobulin (Ig)G autoantibodies towards malondialdéhyde (MDA)-modified T.Ag, hydrogen peroxide (H2O2)-modified T.Ag, native T.Ag and native derm were checked by enzyme-linked immunosorbent assay (ELISA). Evaluation of oxidized proteins exhibited high levels of MDA bound to proteins and carbonyl groups, as well as reduced thiol level in plasmas of AITD patients by comparison to healthy controls (p < 0.05). The ELISA test showed that AITD patients' plasmas' reactivity to native T.Ag was significantly increased to the reactivity towards native derm, whereas, no differences were found in the reactivity to native T.Ag and derm in controls plasmas. In addition, treatment of T.Ag by oxidants revealed enhanced reactivity of IgG circulating autoantibodies against H2O2-oxidized T.Ag compared to native ones (p < 0.001) in plasmas of both AITD. Also, reactivity's to MDA-oxidized T.Ag in GD plasmas decreased compared to native ones (p < 0.05) and no changes were noted for HT. Pearson correlation study resulted in positive correlation between reactivity's to H2O2-oxidized T.Ag and free triodotyronine level in GD patients (r = 0.42, p < 0.05) in one hand and thyroid stimulating hormone level in HT patients in the other (r = 0.65, p < 0.001). The data suggest that high production of H2O2 probably occurred during hormone synthesis could contribute to protein oxidation in AITD and to create neoepitopes responsible for autoantibody reactivity's to H2O2-oxidized T.Ag enhancement. These results provide support to the involvement of oxidative stress in AITD development and/or exacerbation.

Rapid Identification of Unknown Organic Iodine in Small-Volume Complex Biological Samples Based on Nanospray Mass Spectrometry Coupled with in-Tube Solid Phase Microextraction.

A new method for rapid screening of unknown organic iodine (OI) in small-volume complex biological samples was developed using in-tube solid phase microextraction (SPME) nanospray mass spectrometry (MS). The method proposed a new identification scheme for OI based on nanospray high-resolution mass spectrometry (HR-MS). The mass ranges of OI ions were confirmed using the t-MS2 scan mode first; then, the possible precursor ions of OI were selected and identified orderly in full MS/ddMS2 and t-MS2 scan modes. Besides, in-tube SPME was used for the pretreatment of small-volume biological samples, and it was the first time in-tube SPME combined with nanospray MS for OI identification. The whole analysis procedure took only 8 min and consumed 50 µL per sample. Using the new method, six kinds of OI added to urine and an unknown OI C12H23O11I in human milk were successfully identified. Moreover, the proposed identification scheme is also suitable for other ambient mass spectrometry (AMS) to determine unknown compounds with characteristic fragment ions.

[Method of determination organic iodine (iodotyrosines) in food].

It is important to control the substances of the synthesis of biologically active supplements, based on organic forms of iodine (iodotyrosines). But it is no less important to control the content of iodotyrosines in foods. The developed method is sensitive and selective and can determine iodotyrosines with a lower limit of detection (1 ppb). Iodotyrosines have been determined by HPLC-MS/MS. The article contains parameters for chromatographic separation of 3-iodo-L-tyrosine and 3.5-diiodo-L-tyrosine and parameters of the electrospray ionization (ESI) mass spectrometry, describes the methodology of sample preparation and solid phase extraction. The article substantiates the use of mass spectrometry as the most sensitive and selective method for determining the organic iodine as compared to HPLC with UV detection. The enzymatic hydrolysis with proteolytic enzymes has been used for sample preparation in iodothyronine analyses. Solid phase extraction was performed using C18 cartridge. For HPLC-MS/MS analysis iodothyronine derivatives were obtained with a mixture of butanol-acetyl chloride. Degree of iodotyrosine extraction from the matrix of the foodstuffs was not less than 85%, the correlation coefficient of the calibration curve in the concentration range of 1-2000 ng/mL was 0.999, reliable determination of iodine content in foods in the range from 10 to 20 000 mcg/kg.

Ultrasound-assisted enzymatic hydrolysis for iodinated amino acid extraction from edible seaweed before reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry.

The combination of reverse phase high performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICP-MS) was used for the determination of monoiodotyrosine (MIT) and diiodotyrosine (DIT) in edible seaweed. A sample pre-treatment based on ultrasound assisted enzymatic hydrolysis was optimized for the extraction of these iodinated amino acids. Pancreatin was selected as the most adequate type of enzyme, and parameters affecting the extraction efficiency (pH, temperature, mass of enzyme and extraction time) were evaluated by univariate approaches. In addition, extractable inorganic iodine (iodide) was also quantified by anion exchange high performance liquid chromatography (AE-HPLC) coupled with ICP-MS. The proposed procedure offered limits of detection of 1.1 and 4.3ngg(-1) for MIT and DIT, respectively. Total iodine contents in seaweed, as well as total iodine in enzymatic digests were measured by ICP-MS after microwave assisted alkaline digestion with tetramethylamonium hydroxide (TMAH) for total iodine assessment, and also by treating the pancreatin extracts (extractable total iodine assessment). The optimized procedure was successfully applied to five different types of edible seaweed. The highest total iodine content, and also the highest iodide levels, was found in the brown seaweed Kombu (6646±45µgg(-1)). Regarding iodinated amino acids, Nori (a red seaweed) was by far the one with the highest amount of both species (42±3 and 0.41±0.024µgg(-1) for MIT and DIT, respectively). In general, MIT concentrations were much higher than the amounts of DIT, which suggests that iodine from iodinated proteins in seaweed is most likely bound in the form of MIT residues.

Development of anion-exchange/reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry methods for the speciation of bio-available iodine and bromine from edible seaweed.

Anion exchange high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry has been novelly applied to assess inorganic (iodide and iodate) and organic (3-iodotyrosine - MIT, and 3,5-diiodotyrosine - DIT) iodine species in a single chromatographic run. The optimized operating conditions (Dionex IonPac AS7, gradient elution with 175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase and flow rates within the 0.5-1.5 mL min(-1) range) have also been used to perform inorganic bromine speciation analysis (bromide and bromate). The developed method has been applied for determining the bio-available contents of iodine and bromine species in dialyzates from edible seaweed. Reverse phase high performance liquid chromatography (Zorbax Eclipse XDB-C8, gradient elution with 0.2% (m/m) acetic acid, and 0.2% (m/m) acetic acid in methanol, as mobile phases, and a constant flow rate of 0.75 mL min(-1)) also hyphenated with inductively coupled plasma-mass spectrometry was used to confirm the presence of organic iodine species (MIT and DIT) in the dialyzates. The verification of the presence of iodinated amino acids (MIT and DIT) in the extracts was also performed by reverse phase high performance liquid chromatography-electrospray ionization-mass spectrometry (LTQ Orbitrap). The developed methods have provided good repeatability (RSD values lower than 10% for both anion exchange and reverse phase separations) and analytical recoveries within the 90-105% range for all cases. The in vitro bio-availability method consisted of a simulated gastric and an intestinal digestion/dialysis (10 kDa molecular weight cut-off - MWCO) two-stage procedure. Iodide and MIT were the main bio-available species quantified, whereas bromide was the major bromine species found in the extracts.

HPLC-MS analysis of iodotyrosines produced by sample hydrolysis: A simple method for monitoring iodinated casein in feed premixes.

A new and simple HPLC-MS method was developed for monitoring iodinated casein in feed premixes. In this method, feed premixes were hydrolyzed, and the iodotyrosines thus released were analyzed. Sample pretreatment included precipitation of transition metals ions with Na(2)S, hydrolysis with sodium hydroxide, and cleaning up with an Oasis SAX cartridge. Gradient elution was carried out on a C(18) column with water (containing 0.1% formic acid) and acetonitrile. Ion detection was performed using ESI positive SIM at m/z 262, 308, 388, and 434. Iodinated casein levels were monitored by qualitative analysis of the iodotyrosines released upon sample hydrolysis and by quantifying the 3,5-diiodotyrosine released. The validation data demonstrated that the method was selective and sensitive (

Development of a validated HPLC method for the determination of iodotyrosines and iodothyronines in pharmaceuticals and biological samples using solid phase extraction.

Identification, separation and quantitation of iodoaminoacids, is essential for the biological research and the clinical diagnosis of thyroid gland disease. Under this aspect a reversed-phase high-performance liquid chromatographic method was developed for the determination of thyroid gland hormones and some of their primary metabolites, 3,3',5,5'-tetra-iodo-L-thyronine (L-thyroxine), 3,3',5-tri-iodo-L-thyronine, 3,5-di-iodo-L-thyronine, L-thyronine, 3,5-di-iodo-L-tyrosine, 3-iodo-L-tyrosine and l-tyrosine. Analysis was performed on an Inertsil C(18) column with photodiode-array detection, using a 25 min gradient scale program of a binary mobile phase consisted of 0.1% aqueous solution of trifluoroacetic acid at pH 3 as solvent A and acetonitrile as solvent B, at a flow rate of 1 mL/min. Quantitation was performed using were obtained using theophylline as internal standard. The method was applied to commercial pharmaceuticals and biological samples (serum, urine and tissue). Drug-free urine and serum samples were spiked with known concentrations of the analytes standards and pretreated by solid phase extraction to remove matrix interferences. C(18) cartridges were used, yielding recoveries ranging from 87.1% to 107.6% for serum samples and from 92.1% to 98.7% for urine samples. With regard to total-T(4) concentrations in serum samples, results are cross-validated with RIA and found to agree well.

Role of multimerized porcine thyroglobulin in iodine storage.

Thyroglobulin (Tg), the prothyroid hormone, is stored in the lumen of the thyroid follicles as soluble dimers and tetramers and insoluble multimers, Soluble Tg is well characterized with regards to structure and role, but insoluble Tg (i-Tg) is not. Here we show that i-Tg, multimerized through formation of disulfide and dityrosine bonds, has a higher iodine content than soluble Tg and no thyroid hormones. Furthermore, the size and the resistance of i-Tg to proteolytic enzymes implied a new mechanism by which thyrocytes may degrade this form of Tg. Using peroxidase and H2O2 generating system, we found that about 80% of i-Tg was degraded and 24% of its iodine content was released. Our data point to a role for i-Tg in iodine storage and the involvement of TPO in i-Tg degradation and iodide release.

A micromethod for quantitative determination of iodoamino acids in thyroglobulin.

We describe a new method for quantification of iodoamino acids after enzymatic hydrolysis of thyroglobulin. The procedure involves separation of monoiodotyrosine (MIT), di-iodotyrosine, tri-iodothyronine and thyroxine by reverse phase HPLC with a Vydac C18 stationary phase and a mobile phase of water-acetonitrile-acetic acid. The separation is monitored by sensitive spectrophotometric detection through a 96-well microplate system based on the catalytic Sandell-Kolthoff reaction of iodide on the oxidation of arsenic(III) by cerium(IV). This new microassay is particularly convenient because of its high sensitivity and its rapidity (less than 2 h). It can detect 1 pmol MIT and 0.5 pmol of the other three iodoamino acids with a recovery higher than 96%. Moreover, the 96-well microplate system allows many samples to be tested simultaneously and avoids the use of radiolabeled iodine.

Identification and quantitation of iodotyrosines and iodothyronines in proteins using high-performance liquid chromatography by photodiode-array ultraviolet-visible detection.

We describe a new method for the separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T0), 3,5-diiodo-L-thyronine (T2), 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine (rT3) and 3,3',5,5'-tetraiodo-L-thyronine (T4)]. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Nucleosil C8 column with photodiode-array UV-Vis detection. A clearly defined elution profile was obtained of each iodoamino acid (iodotyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA), phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determined, taking into account retention times and spectral characteristics. Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode-array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths continuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 280 nm. This method allowed identification and quantitation of iodoamino acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinated and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.

Dityrosine bridge formation and thyroid hormone synthesis are tightly linked and are both dependent on N-glycans.

Formation of dityrosine bridges is a ubiquitous process mainly attributed to oxidative stress leading to protein degradation and cellular damages. Here we show that dityrosine formation is involved in a physiological process, thyroid hormone synthesis, and is strictly dependent on structural characteristics, namely N-glycans, presented by the protein acting as the prothyroid hormone. We used two isoforms of the N-terminal thyroid hormone forming domain (NTD) of human thyroglobulin: one without N-glycan (19 kDa isoform) and the other with high mannose type structures (25 kDa isoform). Both isoforms were able to form iodotyrosines after in vitro iodination. However, iodotyrosine coupling to form thyroxine did not occur with the unglycosylated 19 kDa NTD. In contrast, the 25 kDa isoform formed thyroxine. Strikingly, thyroxine synthesis was accompanied by dimerization of the 25 kDa isoform and formation of a dityrosine bridge; none of this was observed with the 19 kDa isoform. Taken as a whole, our results indicate that dimerization through dityrosine bridging accompanies and could have a role in thyroid hormone synthesis.

Kinetics and mechanism of the peroxidase-catalyzed iodination of tyrosine.

The kinetics of iodination of tyrosine by hydrogen peroxide and iodide, catalyzed by both horseradish peroxidase (HRP) and lactoperoxidase (LPO), were studied. The initial rates of formation of both molecular I2 and monoiodotyrosine (MIT) were measured with stopped flow techniques. The following reactions occur in both systems. Enzymatic: FeIII + H2O2-->Fev = O + H2O; Fev = O + I(-)-->FeIII-O-I-; FeIII-O-I- + H(+)-->FeIII + HOI; FeIII-O-I- + I- + H(+)-->FeIII + I2 + HO-. Iodine equilibria: I2 + I-<-->I3-; I2 + H2O<-->HOI + I- + H+. Nonenzymatic iodination, one or both of the following: Tyr + HOI-->MIT + H2O; Tyr + I2-->MIT + I- + H+, where FeIII is native peroxidase, Fev = O is compound I and Tyr is tyrosine. The big difference in the two systems is that the following reaction also occurs with LPO: FeIII-O-I- + Tyr-->MIT + FeIII + HO-, which is the dominant mechanism of iodination for the mammalian enzyme. The overall rate of formation of MIT is about 10 times faster for LPO compared to HRP under comparable conditions. A small decrease in rate occurs when D-tyrosine is substituted for L-tyrosine in the LPO reaction. Thus LPO has a tyrosine binding site near the heme. A kinetically controlled maximum is observed in I3- concentration. Once equilibrium is established, I2 is the dominant form of inorganic iodine in solution. However, hypoiodous acid may be the inorganic iodination reagent.

Preparation of 125I-labeled human growth hormone of high quality binding properties endowed with long-term stability.

125I-labeled human growth hormone (125I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20 degrees C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20 degrees C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.

Evidence for a preferential iodination site within the thyroglobulin molecule.

Mouse 330 kDa thyroglobulin labeled in vivo was analyzed using a tryptic peptide mapping technique and high performance liquid chromatography (HPLC). 30 min after Na125I injection, one peptide spot (spot 7) on a silica gel plate was the only prominent labeled peptide, followed by other labeled peptide spots after 1 h. HPLC showed that spot 7 was rich in monoiodotyrosine. The ratios between the iodoamino acids were strictly maintained from 1 to 6 h after Na125I injection. Spot 7 was again the first spot that appeared from the samples of iodine-deficient mice. These data indicate that there is some preferential iodination site(s) within the thyroglobulin molecule and also that their iodoamino acid composition is predetermined.

Intermediate peptides of insulin degradation in liver and cultured hepatocytes of rats.

1. Bestatin, a microbial aminopeptidase inhibitor, induced accumulation of low-molecular weight intermediate peptides of insulin degradation in liver of rats in vivo and in primary cultured rat hepatocytes. However, bestatin did not affect the association and internalization of the hormone into hepatic cells. 2. Results of the HPLC analyses showed that the intermediate peptides of insulin degradation are small ones and specifically accumulate only in the presence of bestatin. 3. The above results, together with those employing other protease inhibitors, show that cytosolic bestatin-sensitive protease(s), trypsin-like protease(s) and thiol protease(s) play an important role in the intracellular degradation process of insulin.

Characterization and purification of iodinated bovine thyrotropin by high performance liquid chromatography.

Reversed-phase (RP) HPLC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel permeation chromatography have been used to study the incorporation of 125I into bovine (b) TSH by the lactoperoxidase-catalyzed radioiodination procedure. Two preparations of [125I]bTSH were studied, being freshly iodinated bTSH and tracer purified from this preparation by the method of receptor adsorption. It is demonstrated with these methods that both the alpha- and beta-subunits of bTSH are labeled with 125I, and that the tracer purified by receptor adsorption retains this incorporation pattern. However, the implied theoretical specific activity of (at least) 2 I atoms per TSH molecule (or approximately 140 muCi/micrograms) suggested by this result was not achieved, with observed tracer specific activity being 30-60 muCi/micrograms, indicating that hormone molecules with varying extents of labeling must exist. Evidence to support this was provided by comparison of the MIT/DIT ratios for the 2 tracer preparations. Receptor adsorption decreased the MIT/DIT ratio from 75:25 in the freshly iodinated bTSH to 93:7, indicating the selection of particular iodinated species. Tryptic mapping by RP-HPLC was used to study both tracer preparations, and it is shown that at least 14 iodine-containing tryptic peptides may be resolved for each preparation, which is greater than the theoretical maximum of 13 peptides if every tyrosine was labeled and tryptic cleavage occurred at all possible lysine and arginine residues. Tracer heterogeneity was also studied by purification using RP-HPLC. Selection of peak fractions demonstrated that intact [125I]bTSH may be recovered from RP-HPLC which in TSH radioreceptor assay exhibit increased assay sensitivity, increased saturable binding, and decreased nonsaturable binding.

Radioiodination of tyrosine residue(s) of ox testis and of wheat germ calmodulins.

Radioiodination of the two tyrosine residues (Tyr-99 and Tyr-138) of ox testis calmodulin was performed using several methods, and studied through the specific activity, and the [125I]iodoamino acid analysis of the radiolabeled calmodulins. Hydrolysis by thrombin of 125I-calmodulin labeled by the lactoperoxidase method and subsequent isolation of peptides TM1 and TM2 by gel electrophoresis showed preferential labeling by 125I of Tyr-99 (TM1) over Tyr-138 (TM2). Analysis of [125I]iodoamino acids of radiolabeled TM1, TM2 and calmodulin demonstrated that [125I]monoiodotyrosine was predominant, the remainder being [125I]diiodotyrosine. Radioiodination of wheat germ calmodulin, which contains a single tyrosine residue (Tyr-139), showed that only TM2 was labeled by 125I on the Tyr-139 residue and also on the His-108 residue (radiolabeled monoiodotyrosine, diiodotyrosine and monoiodohistidine being present).

Preparation and characterization of radioactive monoiodotyrosine and diiodotyrosine derivatives of parathyroid hormone.

Highly purified native parathyroid hormone was iodinated by the enzymatic method and separated from unlabeled hormone by isocratic HPLC. The separation system used also resolved iodohistidine, monoiodotyrosine, and diiodotyrosine forms of the hormone from one another. A simplified procedure for direct bioassay of the carrier-free, high specific activity, mono- and diiodinated parathyroid hormone (PTH) by the renal membrane adenylyl cyclase method was also developed. Both labeled forms of the hormone are very potent in this assay, but the iodinated forms appeared to give a lower Vmax than the native hormone. The methods for iodination, separation and biological characterization of this PTH tracer are exceptionally facile, inexpensive, and convenient.