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Article Type: 3 Original Article - Australia, Japan, SE Asia

Macro TSH in patients with subclinical hypothyroidism A short title: Macro TSH in subclinical hypothyroidism

Naoki Hattori1, Takashi Ishihara2, Keiko Yamagami3 and Akira Shimatsu4

1) Department of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 2) Department of Endocrinology, Kobe City General Hospital, Kobe, 3) Internal medicine, Endocrinology, Osaka City General Hospital, Osaka, 4) Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

Correspondence: Naoki Hattori, Department of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu-city, Shiga 525-8577, JAPAN Tel: 81-77-561-2581, Fax: 81-77-561-2581, E-mail: [email protected] Key words: TSH, autoantibodies, subclinical hypothyroidism This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cen.12643 This article is protected by copyright. All rights reserved.

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Declaration of interest. We declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding. This work was supported by grants from the Ministry of Culture, Sports, Science, and Technology of Japan, from Ritsumeikan University, and from the Ministry of Health, Labour and Welfare of Japan.

Acknowledgments. We thank Dr. Yasuhiko Saiki for assisting in the laboratory work. We also thank Geoff Gillespie for his assistance in preparing this manuscript.

Abstract Objective TSH is a sensitive indicator of thyroid function. In subclinical hypothyroidism, however, serum TSH concentrations are elevated despite normal thyroid hormone levels, and macro TSH is one of the causes. This study aimed to clarify the prevalence and nature of macro TSH in patients with subclinical hypothyroidism.

Design We conducted a 2-year cross-sectional observational study. This article is protected by copyright. All rights reserved.

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Patients We included 681 patients with subclinical hypothyroidism and 38 patients with overt hypothyroidism (controls).

Measurements Macro TSH was screened by polyethylene glycol (PEG) method and analyzed by gel filtration chromatography and bioassays.

Results Among 681 serum samples, 117 exhibited PEG-precipitable TSH ratios greater than 75% (mean + 1.5 SD in controls) and were subjected to gel filtration chromatography. TSH was eluted at a position greater than 100 kDa in 11 patients with subclinical hypothyroidism (1.62%); these patients were diagnosed with macro TSH. The nature of macro TSH included eight anti-TSH autoantibodies of IgG class, two non-IgG-associated, and one human antimouse antibody (HAMA). Macro TSH showed low bioactivity.

Conclusions Macro TSH was heterogeneous, but it is mostly comprised of TSH and antiTSH autoantibodies. When PEG-precipitable TSH exceeds 90% in serum samples with TSH above 10 mU/l, clinicians should strongly suspect the presence of macro TSH and confirm it by gel chromatography. Because macro TSH exhibited low bioactivity, thyroid hormone

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from ng/ml to mU/l is 7.54). Based on a serum sample with a total TSH concentration of 4.5 mU/l, the intra- and inter-assay coefficients of variation were 3.5% and 5.2%, respectively. There was a good correlation (r=0.907) between the TSH values determined with our EIA system and the values determined with the Vitros TSH assay kit, which was used for TSH determinations in the hospital.

Gel filtration chromatography Gel filtration chromatography was performed with a 1 × 60 cm column of Ultrogel AcA 44 (IBF, La Garenne, France) equilibrated with 0.01 mol/l sodium phosphate buffer (pH 7.0), which contained 0.1 mol/l NaCl, 0.1% bovine serum albumin, and 0.01% NaN3. Samples (50-500 µl) from 117 patients with subclinical hypothyroidism that showed more than 75% PEG-precipitable TSH (mean + 1.5 SD in controls) and from 38 control patients were applied to the column, and 1-ml fractions were collected for TSH determinations. The column was calibrated with various molecular weight markers (Sigma, St. Louis, MO, USA).

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Macro TSH is a large molecular sized TSH on gel filtration chromatography and there are several case reports regarding it [10-16]. We hypothesized that the bioactivity of macro TSH might be low like macro PRL and that macro TSH may be accumulated in the circulation causing elevated serum TSH levels. Thus, macro TSH is likely to be found in patients with subclinical hypothyroidism. The diagnosis of macro TSH benefits clinical practice, because it may change the therapeutic strategy for patients with subclinical hypothyroidism. In this study, we tested our hypothesis by examining the prevalence of macro TSH in patients with subclinical hypothyroidism and by investigating the nature of macro TSH.

Subjects and methods Subjects From 2012 to 2014, 3047 serum samples were submitted to the laboratory of Kobe City General Hospital for thyroid function tests including serum concentrations of TSH and free T4. Blood was drawn in the morning, the serum was separated, thyroid function tests were performed, and the remaining serum samples were stored at -30 °C for further analysis. The serum concentrations of TSH and free T4 were assayed with the Vitros TSH and Vitros free T4 assay kits, respectively (Ortho-Clinical Diagnostics, Tokyo, Japan). Elevated TSH

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concentrations were found in 793 serum samples, including 112 that had low free T4 levels (overt hypothyroidism) and 681 that had normal free T4 levels (subclinical hypothyroidism). Samples from the 681 patients with subclinical hypothyroidism (385 women and 296 men, aged 64.5 ± 19.2 years) were screened for macro TSH. These patients were diagnosed with Hashimoto’s thyroiditis (n=342), post-operative or radioisotope-treated thyroid cancer (n=116), post-operative or radioisotope-treated Basedow’s disease (n=68), simple goiter (n=27), and painless thyroiditis (n=1). The other patients either had no known cause of hypothyroidism or no explanation was given in their medical records for why serum TSH levels were measured. A total of 401 patients with subclinical hypothyroidism had been receiving thyroid hormone replacement therapy at the time when the blood sample was collected. As a control group, we included 38 serum samples from patients with overt hypothyroidism (24 women and 14 men, aged 65.5 ± 16.2 years). This study was approved by the Clinical Research Review Board of Kobe City General Hospital.

Polyethylene glycol (PEG) precipitation and TSH assays To screen for macro TSH, we used the PEG precipitation method, which is typically used for macroprolactinemia screening [9]. In brief, serum samples (50 µl) were treated with 50µl of

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25% PEG (the final concentration of PEG; 12.5%) to precipitate γ-globulin fractions, and we measured the free TSH in the supernatant. These serum samples (50µl) were also diluted with 50µl of water (without PEG), and we measured the total TSH. The PEG-precipitable TSH (%), which may represent the amount of macro TSH was calculated as follows: (total TSH free TSH) / total TSH × 100. The mean PEG-precipitable TSH in controls was 58.0 ± 11.4%.

The enzyme immunoassay (EIA) system for human TSH was established according to the method used for EIA of human growth hormone [17]. In brief, polystyrene balls (3.5 mm in diameter, Precision Plastic Ball Co. Chicago, IL, USA) were coated with human TSH β monoclonal antibodies (10-T25C, Fitzgerald Industries International, Suite, MA, USA). These balls served as a solid phase. Human TSH β monoclonal antibodies (10-T25B, Fitzgerald) were conjugated to horseradish peroxidase (HRP, Wako Pure Chemical Industries, Ltd. Osaka, Japan) with the maleimide method. EIA for TSH was carried out as previously described for PRL [9]. Highly purified recombinant reference human TSH (hTSH RP-2SIAFP) and human pituitary TSH (NIDDK hTSH-SIAFPI-8) were provided by Dr. A. F. Parlow (Harbour-UCLA Medical Center, National Hormone and Peptide Program, Torrance, CA. USA; [email protected]). The limit of detection was 0.0075mU/l (conversion factor

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levels decreased, but did not fall below 100 mU/l. Discussion Diagnosis of macro TSH is clinically important, because it may change the therapeutic strategy for patients with subclinical hypothyroidism.

We found that the prevalence of macro TSH in patients with subclinical hypothyroidism was 1.62%, including one patient with HAMA. The present data should be carefully interpreted because this is not an epidemiological study but a retrospective study that examined the patients attending our hospital. Many of our patients had known thyroid pathology and were receiving thyroid hormone replacement therapy. Therefore, our data does not mean that 1.62% of patients with mild thyroid failure will have macro TSH. Recently, it was reported that 3 of 495 serum samples (0.6%) with serum TSH levels greater than 10 mU/l contained macro TSH [19]. Thus, macro TSH is not uncommon.

Macro TSH seems to be a heterogeneous entity. HAMA, a human IgG that binds to mouse IgG, bridges capture and detection antibodies without the ligand in sandwich type immunoassay systems, which results in false positives [2]. HAMA behaves like macro TSH,

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Measurement of IgG-bound TSH To determine the ratio of IgG-bound TSH, we used protein G Sepharose (GE Healthcare, Uppsala, Sweden), which characteristically binds IgG, as previously described [9]. The ratio of IgG-bound TSH (%) was calculated with the equation: TSH in the bound fraction / (TSH in the unbound fraction + TSH in the bound fraction) × 100. A small amount of TSH was bound to protein G in control sera, which may be due to non-specific binding (mean ± SD: 3.7 ± 3.8 %). We used the control mean + 1.96 × SD as a cutoff value for indicating the presence of IgG-bound TSH in patient samples; thus, a measurement greater than 11.1% indicated that the sample was positive for IgG-bound TSH.

Serial dilution study of TSH Serum samples containing macro TSH were subjected to the serial dilution. For all but one sample, the sera were diluted by 6, 12, 24, and 48; for one sample (case 1), the serum was diluted by 80, 160, 320, and 640. The TSH immunoreactivity was measured with EIA.

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macro TSH and confirm it by gel chromatography. In conclusion, macro TSH is not uncommon in patients with subclinical hypothyroidism. The etiology of macro TSH seems to be heterogeneous but it is mostly comprised of TSH and anti-TSH autoantibodies. Because macro TSH has low bioactivity, patients with macro TSH may not require thyroid hormone replacement therapy.

References 1. Biondi B. & Cooper D.S. (2008) The clinical significance of subclinical thyroid dysfunction. Endocrine Review, 29, 76-131. 2. Kricka L.J. (1999) Human anti-animal antibody interferences in immunological assays. Clinical Chemistry, 45, 942-956. 3. Fahie-Wilson M.N. & Soule S.G. (1997) Macroprolactinaemia: contribution to hyperprolactinaemia in a district general hospital and evaluation of a screening test based on precipitation with polyethylene glycol. Annals of Clinical Biochemistry, 34, 252-258. 4. Vieira J.G., Tachibana T.T., Obara L.H. & Maciel R.M. (1998) Extensive experience and validation

of

polyethylene

glycol

precipitation

macroprolactinemia. Clinical Chemistry, 44, 1758-1759.

This article is protected by copyright. All rights reserved.

as

a

screening

method

for

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23. Szkudlinski M.W., Fremont V., Ronin C. & Weintraub B.D. (2002) Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiological Review, 82, 473-502. 24. Freeman M.E., Kanyicska B., Lerant A. & Nagy G. (2000) Prolactin: structure, function, and regulation of secretion. Physiological Review, 80, 1523-1631. 25. Wei Y., Han C.S., Zhou J., Liu Y., Chen L. & He R.Q. (2012) D-ribose in glycation and protein aggregation. Biochimica et Biophysica Acta, 1820, 488-494.

Figure legends Figure 1. The distribution of PEG-precipitable TSH ratios in patients with subclinical hypothyroidism The γ-globulin fractions in sera were precipitated with 12.5% polyethylene glycol (PEG). The concentrations of TSH in the supernatant were measured (free TSH) and compared to the TSH in sera (total TSH). The PEG-precipitable TSH (%) was calculated as follows: (total TSH - free TSH) / total TSH × 100.

Figure 2. Gel filtration profiles of serum TSH in patients with macro TSH and one control patient with overt hypothyroidism This article is protected by copyright. All rights reserved.

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Molecular forms of TSH were determined by chromatography in sera from 11 patients with subclinical hypothyroidism that had high levels of PEG-precipitable TSH and one control patient (lower right). Two main peaks of TSH immunoreactivity appeared; one with a molecular mass of 28 kDa (arrow; centered in fraction 29) and the other with molecular mass greater than 100 kDa (macro TSH; centered in fractions 17-21).

Figure 3. Characterization of macro TSH (a) Serial dilution curves for TSH in patients with macro TSH. The serum samples were diluted 6, 12, 24, and 48-fold, except for case 1 (*), where serum was diluted 80, 160, 320, and 640-fold. (b) The mechanism that causes human anti-mouse antibodies (HAMA) to produce spuriously high TSH values is shown. This sandwich-type immunoassay used mouse monoclonal antibodies. (Top) The solid phase antibodies and HRP-labeled antibodies efficiently capture TSH; (bottom) HAMA can bind both mouse antibodies to produce a false positive fluorescent signal. (c) TSH levels (%) in 11 patients with macro TSH were measured in the absence (100%) and presence of three HAMA-blocking reagents, as follows: mouse whole serum (black bars), mouse IgG (grey bars), and mouse IgM (dotted bars). (d) Gel filtration profiles show macro TSH (arrow at 150 kDa) in the serum from case 1 (○); the

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(final concentration: 0, 1.0, 6.25, 12.5, 25, 50, and 100 mU/l) or 10 µl of a serum sample for another 4 days. Cell growth was examined with a Cell Counting kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. The assay was performed in quadruplicate, and the detection limit for TSH bioactivity was 10 mU/l. The intra- and interassay coefficients of variation were 10 and 14%, respectively.

The morphology of cells was evaluated with a IX71 inverted microscope (Olympus, Tokyo, Japan).

Statistical analysis All measurements are expressed as means ± SD and the value greater than mean + 1.96 SD was considered to be significantly elevated.

Results Screening for macro TSH with the PEG method The distribution of PEG-precipitable TSH ratios in sera from 681 patients with subclinical hypothyroidism showed a normal distribution, ranging from 21.4 to 100% (63.4 ± 11.5%;

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Fig.1). Macro TSH was examined by gel filtration chromatography in 117 patients that had PEG-precipitable TSH ratios greater than 75% (mean + 1.5 SD in controls).

Gel filtration chromatography In 38 control patients with primary hypothyroidism, gel filtration chromatography showed a main peak of TSH immunoreactivity with a molecular mass of 28 kDa, the same as TSH standard (Fig.2, right bottom). The ratios of TSH with a large molecular weight (greater than 100 kDa) ranged from 0 to 5.5% (1.0 ± 1.5%) in controls. Among the 117 patients with PEGprecipitable TSH ratios greater than 75%, gel filtration chromatography showed that, in 11 patients, the ratio of TSH with molecular mass greater than 100 kDa (macro TSH) was significantly higher than that of controls, ranging from 17.0% to 100% (56.6 ± 30.7%; Fig.2). The prevalence of macro TSH in all 681 patients with subclinical hypothyroidism was 1.62%.

Clinical and biochemical characteristics of macro TSH Table 1 shows clinical data of 11 patients with macro TSH. Five patients had chronic thyroiditis, and one patient had post-operative Basedow’s disease. Although total TSH levels in all 11 patients exceeded the normal range, the levels of free TSH were normal in 8 patients.

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Significantly high amounts of TSH (25.6 to 96.3%) were bound to the protein G column in 9 of the 11 patients; in control patients, only 3.7 ± 3.8% of TSH was non-specifically bound to the column. This finding suggested that macro TSH was comprised of IgG-bound TSH, in most cases. Six patients had been taking thyroid hormone replacement therapy.

The serial dilution study of the serum TSH from 11 patients with macro TSH showed that the slopes of serum TSH dilution curves were nearly identical to the slope of the TSH standard curve (Fig.3a). HAMA might produce spuriously high TSH values in sandwich type immunoassay systems employing two mouse monoclonal antibodies as shown in Fig.3b. HAMA could bridge the solid phase antibody and the HRP-conjugated antibody in the absence of TSH. We tested the effects of three HAMA blockers on TSH determinations in samples from the 11 patients with macro TSH (Fig. 3c). For one patient (case 2), all three HAMA blockers dramatically decreased TSH immunoreactivities. This result suggested that this patient had

HAMA in the serum, which resulted in a spuriously high serum TSH

measurement. Thus, acid treatment was performed in 10 patient samples other than case 2. Acidification of sera completely dissociated macro TSH to monomeric TSH. The addition of TSH standard to the sera from patients with macro TSH generated macro TSH (Fig. 3d).

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When macro TSH was subjected to repeated freeze and thaw cycles, it was partially dissociated to monomeric TSH (Fig. 3e).

Bioactivity of macro TSH Bioactivity of macro TSH was examined using FRTL-5 cells (rat thyroid cells) in 3 methods. First, we measured the concentrations of cAMP released into the culture medium in response to TSH. The limit of cAMP detection in the bioassay was at 1 mU/l TSH (Fig. 4a), and the patient’s serum was diluted 20-fold (10 µl serum in 200 µl medium); thus, serum samples with TSH levels greater than 20 mU/l could be evaluated in this bioassay. Because serum TSH levels were elevated in case 2 due to the interference by HAMA, we examined the bioactivities of serum TSH in cases 1, 3, and 4. In these serum samples, TSH bioactivity was below the detection limit (1 mU/l), which suggested that bioactive TSH levels were below 20 mU/l. Next, we measured cell proliferation in response to TSH. In this proliferation assay, a TSH-dependent increase in cell number could be detected at TSH concentrations above the 10 mU/l. Thus, only bioactivity in serum from case 1 could be examined in this system. The TSH levels in diluted sera were less than 10 mU/l; this suggested that the bioactive TSH level was less than 200 mU/l, clearly lower than the level of immunoreactive TSH (716 mU/l).

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Lastly, we examined whether cell morphology was affected (Fig. 4b). FRTL-5 cells were grown and differentiated in medium that contained TSH. Then, cells were cultured in medium without TSH, where they spread in a sheet-like fashion with unclear cell shapes. In the presence of 1 mU/l TSH, FRTL-5 cell morphology did not change remarkably; however, in the presence of 50 mU/l TSH, the cells became thick and round with spikes. The 20-fold diluted serum from case 1 (immunoreactive TSH: 36 mU/l) did not induce any remarkable morphological changes, suggesting that the bioactivity of macro TSH was lower than the immunoreactivity.

Clinical course in case 1 Fig. 5 shows the clinical course of the patient designated as case 1. Thyroid hormone replacement therapy was initiated on January 16, 2013, and the dose was increased from 25 µg to 50 µg on March 5, 2013. TSH levels decreased from 499.4 to 90.5 mU/l, in accordance with the increase in free T4 levels (1.1 to 1.6 ng/dl). After the diagnosis of macro TSH, the replacement therapy was temporarily discontinued. Serum TSH returned to very high levels again. Because the patient claimed that her physical condition had improved a little with the levothyroxine, thyroid hormone replacement therapy was resumed. Consequently, serum TSH

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levels decreased, but did not fall below 100 mU/l. Discussion Diagnosis of macro TSH is clinically important, because it may change the therapeutic strategy for patients with subclinical hypothyroidism.

We found that the prevalence of macro TSH in patients with subclinical hypothyroidism was 1.62%, including one patient with HAMA. The present data should be carefully interpreted because this is not an epidemiological study but a retrospective study that examined the patients attending our hospital. Many of our patients had known thyroid pathology and were receiving thyroid hormone replacement therapy. Therefore, our data does not mean that 1.62% of patients with mild thyroid failure will have macro TSH. Recently, it was reported that 3 of 495 serum samples (0.6%) with serum TSH levels greater than 10 mU/l contained macro TSH [19]. Thus, macro TSH is not uncommon.

Macro TSH seems to be a heterogeneous entity. HAMA, a human IgG that binds to mouse IgG, bridges capture and detection antibodies without the ligand in sandwich type immunoassay systems, which results in false positives [2]. HAMA behaves like macro TSH,

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because it binds to a protein G column, and it is eluted at greater than 100 kDa in gel filtration chromatography. HAMA should be excluded from macro TSH because it is merely the interference to immunoassay systems and TSH levels are not actually elevated.

Analogous to macro PRL [9], our results suggested that macro TSH is likely to be comprised of TSH and anti-TSH autoantibodies. This conclusion is based on (1) the binding component of macro TSH belonged to the IgG family (because it bound to protein G); (2) monomeric TSH was dissociated from the binding components by acidification and by repeated freeze and thaw cycles; and (3) macro TSH could be generated by incubating the patient’s serum with exogenous TSH. In addition, six patients with macro TSH had chronic thyroiditis or Basedow’s disease, which suggested that an autoimmune mechanism may be involved in the generation of macro TSH. The nature of the non-IgG-associated macro TSH remains to be elucidated, but it might be an aggregate of highly glycosylated TSH analogous to non-IgG-associated macro PRL [9].

In case 1, administration of thyroid hormone decreased serum TSH levels. This finding suggested that the negative feedback mechanism for thyroid hormone was intact.

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Nevertheless, TSH levels were not normalized with levothyroxine therapy; this suggested that the secretion of TSH and the formation, dissociation, and clearance of the TSH-anti-TSH autoantibody complexes were equilibrated, at the elevated levels of macro TSH and normal levels of free TSH. In most patients with macro TSH, free TSH levels were normal despite elevated total TSH levels. There were three patients with chronic thyroiditis who had elevated both macro and free TSH levels. These patients are most likely to have both macro TSH and primary hypothyroidism.

The present study implemented TSH bioassay systems that were not highly sensitive. Therefore, we could only confirm in three patients that macro TSH had low bioactivity. Analogous to macro PRL, we speculate that macro TSH probably has low bioactivity because the autoantibodies against TSH may compete with TSH for binding to TSH receptors [20].

In which patients should we suspect the presence of macro TSH and therefore take the diagnostic procedures? While considering a large number of patients with subclinical hypothyroidism (4-10% of adult population samples) [1], it is impractical in clinical practice to perform the screening of macro TSH in all patients with mild thyroid failure. Most patients

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with macro TSH in this study had serum TSH levels greater than 10 mU/l, which is the level at which most clinicians will initiate hormone replacement therapy. Therefore, patients with subclinical hypothyroidism who have serum TSH levels above 10 mU/l may be candidates for the screening of macro TSH, especially those who have extremely high levels of TSH as in case 1.

Which diagnostic procedures should we proceed with? Serial dilution study [21] was not helpful to discriminate elevated serum TSH levels due to macro TSH from primary hypothyroidism. The PEG method that is widely used for screening macroprolactinemia [4] is simple and practical. However, as we showed in this study, non-specific precipitation ratios are much higher in TSH (mean + 2SD = 81%) than PRL (mean + 2SD = 57%)[9] or insulin (mean + 2SD = 27.8%)[22]. The fact that TSH is a glycoprotein [23] and PRL is also partially glycosylated [24], suggests that glycosylation could facilitate the aggregation of hormones [25], which could lead to the increased precipitation with PEG. All patient samples with PEGprecipitable TSH ratios greater than 90% were found to contain macro TSH on gel chromatography. Therefore, we recommend that when PEG-precipitable TSH exceeds 90% in serum samples with TSH above 10 mU/l, clinicians should strongly suspect the presence of

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macro TSH and confirm it by gel chromatography. In conclusion, macro TSH is not uncommon in patients with subclinical hypothyroidism. The etiology of macro TSH seems to be heterogeneous but it is mostly comprised of TSH and anti-TSH autoantibodies. Because macro TSH has low bioactivity, patients with macro TSH may not require thyroid hormone replacement therapy.

References 1. Biondi B. & Cooper D.S. (2008) The clinical significance of subclinical thyroid dysfunction. Endocrine Review, 29, 76-131. 2. Kricka L.J. (1999) Human anti-animal antibody interferences in immunological assays. Clinical Chemistry, 45, 942-956. 3. Fahie-Wilson M.N. & Soule S.G. (1997) Macroprolactinaemia: contribution to hyperprolactinaemia in a district general hospital and evaluation of a screening test based on precipitation with polyethylene glycol. Annals of Clinical Biochemistry, 34, 252-258. 4. Vieira J.G., Tachibana T.T., Obara L.H. & Maciel R.M. (1998) Extensive experience and validation

of

polyethylene

glycol

precipitation

macroprolactinemia. Clinical Chemistry, 44, 1758-1759.

This article is protected by copyright. All rights reserved.

as

a

screening

method

for

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5. Leslie H., Courtney C.H., Bell P.M. et al. (2001) Laboratory and clinical experience in 55 patients with macroprolactinemia identified by a simple polyethylene glycol precipitation method. Journal of Clinical Endocrinology and Metabolism, 86, 2743-2746. 6. Vallette-Kasic S., Morange-Ramos I., Selim A. et al. (2002) Macroprolactinemia revisited: a study on 106 patients. Journal of Clinical Endocrinology and Metabolism, 87, 581-588. 7. Smith T.P., Suliman A.M., Fahie-Wilson M.N. & McKenna T.J. (2002) Gross variability in the detection of prolactin in sera containing big big prolactin (macroprolactin) by commercial immunoassays. Journal of Clinical Endocrinology and Metabolism, 87, 5410-5415. 8. McKenna T.J. (2009) Should macroprolactin be measured in all hyperprolactinaemic sera? Clinical Endocrinology (Oxf), 71, 466-469. 9. Hattori N., Ishihara T. & Saiki Y. (2009) Macroprolactinaemia: prevalence and aetiologies in a large group of hospital workers. Clinical Endocrinology (Oxf), 71, 702-708. 10. Newman J.D., Bergman P.B., Doery J.C. & Balazs N.D. (2006) Factitious increase in thyrotropin in a neonate caused by a maternally transmitted interfering substance. Clinical Chemistry, 52, 541-542.

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11. Halsall D.J., Fahie-Wilson M.N., Hall S.K. et al. (2006) Macro thyrotropin-IgG complex causes factitious increases in thyroid-stimulating hormone screening tests in a neonate and mother. Clinical Chemistry, 52, 1968-1969. 12. Mendoza H., Connacher A. & Srivastava R. (2009) Unexplained high thyroid stimulating hormone: a "BIG" problem. BMJ Case Reports. doi:10.1136/bcr.01.

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13. Verhoye E., Van den Bruel A., Delanghe J.R., Debruyne E. & Langlois M.R. (2009) Spuriously high thyrotropin values due to anti-thyrotropin antibodies in adult patients. Clinical Chemistry and Laboratory Medicine, 47, 604-606. 14. Sakai H., Fukuda G., Suzuki N., Watanabe C. & Odawara M. (2009) Falsely elevated thyroid-stimulating hormone (TSH) level due to macro-TSH. Endocrine Journal, 56, 435440. 15. Rix M., Laurberg P., Porzig C. & Kristensen S.R. (2011) Elevated thyroid-stimulating hormone level in a euthyroid neonate caused by macro thyrotropin-IgG complex. Acta Paediatrica, 100, e135-e137. 16. Loh T.P., Kao S.L., Halsall D.J. et al. (2012) Macro-thyrotropin: a case report and review of literature. Journal of Clinical Endocrinology and Metabolism, 97, 1823-1828. 17. Hattori N., Kato Y., Murakami Y. et al. (1988) Urinary growth hormone levels measured

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by ultrasensitive enzyme immunoassay in patients with renal insufficiency. Journal of Clinical Endocrinology and Metabolism, 66, 727-732. 18. Tokuda Y., Kasagi K., Iida Y. et al. (1988) Sensitive, practical bioassay of thyrotropin, with use of FRTL-5 thyroid cells and magnetizable solid-phase-bound antibodies. Clinical Chemistry, 34, 2360-2364. 19. Mills F., Jeffery J., Mackenzie P., Cranfield A. & Ayling R.M. (2013) An immunoglobulin G complexed form of thyroid-stimulating hormone (macro thyroid-stimulating hormone) is a cause of elevated serum thyroid-stimulating hormone concentration. Annals of Clinical Biochemistry, 50, 416-420. 20. Hattori N., Nakayama Y., Kitagawa K., Ishihara T., Saiki Y. & Inagaki C. (2008) Antiprolactin

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23. Szkudlinski M.W., Fremont V., Ronin C. & Weintraub B.D. (2002) Thyroid-stimulating hormone and thyroid-stimulating hormone receptor structure-function relationships. Physiological Review, 82, 473-502. 24. Freeman M.E., Kanyicska B., Lerant A. & Nagy G. (2000) Prolactin: structure, function, and regulation of secretion. Physiological Review, 80, 1523-1631. 25. Wei Y., Han C.S., Zhou J., Liu Y., Chen L. & He R.Q. (2012) D-ribose in glycation and protein aggregation. Biochimica et Biophysica Acta, 1820, 488-494.

Figure legends Figure 1. The distribution of PEG-precipitable TSH ratios in patients with subclinical hypothyroidism The γ-globulin fractions in sera were precipitated with 12.5% polyethylene glycol (PEG). The concentrations of TSH in the supernatant were measured (free TSH) and compared to the TSH in sera (total TSH). The PEG-precipitable TSH (%) was calculated as follows: (total TSH - free TSH) / total TSH × 100.

Figure 2. Gel filtration profiles of serum TSH in patients with macro TSH and one control patient with overt hypothyroidism This article is protected by copyright. All rights reserved.

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Molecular forms of TSH were determined by chromatography in sera from 11 patients with subclinical hypothyroidism that had high levels of PEG-precipitable TSH and one control patient (lower right). Two main peaks of TSH immunoreactivity appeared; one with a molecular mass of 28 kDa (arrow; centered in fraction 29) and the other with molecular mass greater than 100 kDa (macro TSH; centered in fractions 17-21).

Figure 3. Characterization of macro TSH (a) Serial dilution curves for TSH in patients with macro TSH. The serum samples were diluted 6, 12, 24, and 48-fold, except for case 1 (*), where serum was diluted 80, 160, 320, and 640-fold. (b) The mechanism that causes human anti-mouse antibodies (HAMA) to produce spuriously high TSH values is shown. This sandwich-type immunoassay used mouse monoclonal antibodies. (Top) The solid phase antibodies and HRP-labeled antibodies efficiently capture TSH; (bottom) HAMA can bind both mouse antibodies to produce a false positive fluorescent signal. (c) TSH levels (%) in 11 patients with macro TSH were measured in the absence (100%) and presence of three HAMA-blocking reagents, as follows: mouse whole serum (black bars), mouse IgG (grey bars), and mouse IgM (dotted bars). (d) Gel filtration profiles show macro TSH (arrow at 150 kDa) in the serum from case 1 (○); the

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Accepted Article

amount increased after the same serum sample was incubated with different concentrations of a TSH standard; ●: 8 ng (60 µU); ▲: 16 ng (120 µU). (e) Gel filtration profile of TSH in case 1, after macro TSH fractions (No18, 19, and 20 as shown by dotted area) were subjected to 10 freeze and thaw cycles.

Figure 4. Biological activities of TSH Bioactivities of macro TSH were examined in the FRTL-5 rat thyroid cell line. (a) Standard curve of TSH in the cAMP-based bioassay. (b) Microscopic images (×200) of FRTL-5 cells cultured in the presence of 1 mU/l TSH (upper panel), 50 mU/l TSH (lower right panel), and 20-fold diluted serum from case 1 (lower left panel).

Figure 5. Clinical course in case 1 Changes in serum TSH (filled circles) and free T4 (fT4; filled triangles) levels in case 1 are shown during intermittent thyroid hormone replacement therapy (doses and time periods indicated in boxes at top) over 20 month. The conversion factor of free T4 concentrations from ng/dl to pmol/l is 12.87.

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Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article

Article Type: 3 Original Article - Australia, Japan, SE Asia

Macro TSH in patients with subclinical hypothyroidism A short title: Macro TSH in subclinical hypothyroidism

Naoki Hattori1, Takashi Ishihara2, Keiko Yamagami3 and Akira Shimatsu4

1) Department of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 2) Department of Endocrinology, Kobe City General Hospital, Kobe, 3) Internal medicine, Endocrinology, Osaka City General Hospital, Osaka, 4) Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto, Japan.

Correspondence: Naoki Hattori, Department of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu-city, Shiga 525-8577, JAPAN Tel: 81-77-561-2581, Fax: 81-77-561-2581, E-mail: [email protected] Key words: TSH, autoantibodies, subclinical hypothyroidism This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cen.12643 This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Accepted Article This article is protected by copyright. All rights reserved.

Macro TSH in patients with subclinical hypothyroidism.

TSH is a sensitive indicator of thyroid function. In subclinical hypothyroidism, however, serum TSH concentrations are elevated despite normal thyroid...
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