Continuing efforts to standardize measured serum growth hormone values in Japan.

Determination of serum growth hormone (GH) levels is mandatory for diagnosis of GH deficiency and excess. In the present study, we, the Study Committee for GH and Its Related Factors, The Foundation for Growth Science, Japan measured GH values in serum samples using all the commercially available kits in Japan. Significant discrepancies in the GH values were observed among the kits in spite of using the unified recombinant human GH-based standards. To deal with the discrepancies, we established a formula using a linear structural relationship model and were able to standardize the GH values. We propose to use the formula to diagnose GH deficiency and excess in Japan.

Thyroid-stimulating hormone-secreting ectopic pituitary adenoma of the nasopharynx.

Thyroid-stimulating hormone-secreting ectopic pituitary adenoma of the nasopharynx is highly unusual, with only three reported cases in the world literature. We describe the clinical presentation and radiologic findings in one patient with such rare lesions. A 46-year-old male with typical symptoms of Grave's disease was found to have a mass on magnetic resonance imaging. An otolaryngologic examination revealed a nasopharyngeal mass lesion, which was endoscopically resected. The results of immunohistochemical staining for thyroid-stimulating hormone were positive. After the resection, the patient's TSH was within normal limits. The clinical significance of the case and a brief literature review are presented.

Involvement of SIK3 in glucose and lipid homeostasis in mice.

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

Standardized centile curves and reference intervals of serum insulin-like growth factor-I (IGF-I) levels in a normal Japanese population using the LMS method.

Measurements of insulin-like growth factor-I (IGF-I) are useful not only for diagnosis and management of patients with growth hormone (GH)-related disorders but also for assessing nutritional status. We reported population-based references of serum IGF-I in 1996. However, they did not properly reflect data in the transition period from puberty to maturity. The aim of the present study was to re-establish a set of normative data for IGF-I for the Japanese population. The study included 1,685 healthy Japanese subjects (845 males, 840 females) from 0 to 83 years old. Subjects suffering from diseases that could affect IGF-I levels were excluded. Obese or extremely thin adult subjects were also excluded. IGF-I concentrations were determined by commercially available immunoradiometric assays. The reference intervals were calculated using the LMS method. Median IGF-I levels reached 310 ng/mL in males at the age of 14 years and 349 ng/mL in females at the age of 13 years, falling to 124 ng/mL and 103 ng/mL, respectively, by the age of 70 years. The mean pretreatment IGF-1 SD scores in patients with severe GH deficiency (GHD) obtained from the database of the Foundation for Growth Science and from clinical studies for adult GHD were -2.1±1.6 and -4.9±2.5, respectively. The present study established age- and gender-specific normative IGF-I data for the Japanese population and showed the utility of these references for screening patients with severe GHD.

Functional polymorphisms in TBX21 and HLX are associated with development and prognosis of Graves' disease.

BACKGROUND: It has been indicated that T-box 21 (TBX21) and H 2.0-like homeobox (HLX) are transcription factors related to the differentiation of T helper 1 cells, whereas GATA-binding protein 3 is the master transcription factor of T helper 2 cells. METHODS: We genotyped -1514T/C (rs17250932) and -1993T/C (rs4794067) polymorphisms of TBX21, - 742C/G polymorphism (rs2184658) of HLX and -1420G/A polymorphism (rs1269486) of GATA3 in genomic DNA samples from Japanese patients; 51 patients with severe Hashimoto's disease (HD), 39 with mild HD, 66 with intractable Graves' disease (GD), in whom remission was difficult to induce, 47 with GD in remission and 79 healthy volunteers. RESULTS: The T alleles of the TBX21-1514T/C and -1993T/C polymorphisms were more frequent in patients with intractable GD than in those with GD in remission. Among individuals with the TBX21-1993TT genotype, the G allele of HLX-742C/G polymorphism, which correlates with low HLX expression, was more frequent in patients with intractable GD than in those with GD in remission. CONCLUSIONS: Functional polymorphisms in TBX21 are associated with the development of autoimmune thyroid diseases and prognosis of GD, and a functional polymorphism in HLX in combination with the TBX21 polymorphism is also associated with the prognosis of GD.

Characterization of Thr-354 in the human sodium/iodide symporter (NIS) by site-directed mutagenesis.

Sodium/iodide symporter (NIS) is the key molecule concentrating iodide in the thyroid gland. The first-described human NIS (hNIS) mutation to cause a complete iodide transport defect was the T354P mutation. The Thr-354 lies in the midst of the putative ninth transmembrane segment which is well-conserved within the members of the SLC5A transporter family. Here we have investigated the molecular function of Thr-354 using site-directed mutagenesis and found that T354S and T354A mutations result in significantly decreased iodide transport activity, 50 % and 2 % of wild-type hNIS. Our findings indicate that whereas Thr-354 is indispensable for the complete NIS activity, the ß-hydroxyl group accounts for half, and the α-helical structure alone contributes for one-fiftieth of wild-type hNIS activity.

Congenital combined pituitary hormone deficiency attributable to a novel PROP1 mutation (467insT).

BACKGROUND: Combined pituitary hormone deficiency (CPHD) is an anterior pituitary disorder, commonly resulting in growth retardation. PROP1 gene mutations appear to be frequently responsible for CPHD, particularly in Middle and Eastern Europe and the Americas, but few cases have been reported in Japan. PATIENTS AND DESIGN: Two sisters (aged 8.4 and 4.3 years at presentation) exhibited proportional short stature from about 2 years of age. Genetic analysis determined the nature and location of mutations. RESULTS: Pituitary size by magnetic resonance imaging (MRI) indicated only slight hypoplasia, while hormone analysis revealed deficiencies in secretion of growth hormone (GH), thyroid stimulating hormone, prolactin and gonadotropins; adrenocortinotropin secretion appeared adequate. Genetic analysis revealed a novel familial inherited PROP1 mutation. A unique insertion mutation was found in codon 156 (467insT) located in the transcription-activating region of the PROP1 gene. The resulting PROP1 protein (191 amino acids) would lack the transcription activation domain and consequently be non-functional. CONCLUSION: Gene analysis suggested that the siblings had inherited a unique autosomal recessive PROP1 gene mutation resulting in severe GH deficiency and subsequent growth retardation.

A novel PROP1 gene mutation (157delA) in Japanese siblings with combined anterior pituitary hormone deficiency.

OBJECTIVE: The majority of cases of combined anterior pituitary hormone deficiency (CPHD) reported in Japanese patients have PIT1 abnormality. This study describes for the first time a homozygous mutation of the PROP1 gene in two Japanese siblings with CPHD born to consanguineous parents. PATIENTS: Two siblings were growth retarded at 3 years of age and developed hypothyroidism. Pituitary function tests showed combined deficiency of GH, TSH, PRL and gonadotrophins. The size of their pituitary glands decreased with age, as demonstrated by magnetic resonance imaging (MRI). RESULTS: The PROP1 gene was analysed by polymerase chain reaction (PCR) followed by direct sequencing. Both children were homozygous for a novel single base deletion at codon 53 (157delA), while their parents were heterozygous. This mutation, if translated, predicts the production of a protein lacking the paired-like homeodomain required for DNA binding, suggesting that the mutation was the direct cause of CPHD in these patients. CONCLUSIONS: 157delA is the first reported Japanese PROP1 gene mutation. In Japan, PROP1 abnormality appears to be a less frequent cause of CPHD than does PIT1 abnormality, whereas PROP1 abnormality predominates in CPHD patients of Caucasian and European origin.

[Differential diagnosis of hepatitis: development of new laboratory tests for autoimmune hepatitis and progress in pathophysiology].

Liver dysfunction has been found in 8.1% of postpartum women in the general population. This dysfunction was speculated to be developed by postpartum aggravation of subclinical autoimmune hepatitis. Therefore, we developed two methods for detection of autoantibodies to liver-specific antigens: an ELISA for anti-liver-specific arginase antibodies, and a highly sensitive radioligand assay for anti-CYP2D6 antibodies. Basic examinations of dilution curve, inhibition study and reproducibility were satisfactory for clinical application in both assays. Anti-arginase antibodies and anti-CYP2D6 antibodies were found in 28.6% and 42.6% of patients with autoimmune hepatitis, respectively. There was no correlation between the two autoantibodies and thus, combined use of these antibodies detects 55.3% of autoimmune hepatitis. Autoimmune hepatitis exists frequently when we include mild cases.

Development of a radiotransporter assay for determination of iodide: preliminary studies.

BACKGROUND: The principle of the radiotransporter assay (RATRA) is that the concentration of the substance to be assayed (analyte) is determined by the degree of its competitive inhibition of the binding of radioactive analyte with transporter. METHODS: To illustrate this approach, the iodide concentrations in urine samples were determined by means of RATRA using Na+/I- symporter (NIS). RESULTS: Iodide concentrations ranging from 9 x 10(-6) to 9 x 10(-4) mol/l could be measured without any significant interference of 0.85 mol/l NaCl. The mean recovery rate of added iodide to urine was 96.5%, serial dilutions of urine samples gave almost straight dose response lines passing near the zero point, the mean within assay coefficient of variation (CV) was 8.8% and between assay CV was 12.9%. Although urinary iodide concentrations determined by RATRA correlated with those using a chemical method (r = 0.97) and an electrode method (r = 0.85), there were discrepancies in absolute values particularly at the low level among these. CONCLUSIONS: The RATRA may have a limitation with respect to the specificity for determining analytes in the biological materials, but we suggest it has the ability to detect some factors influencing the transport.

No increase of blocking type anti-thyrotropin receptor antibodies during pregnancy in patients with Graves' disease.

Serial changes in serum levels of anti-TSH receptor antibodies were examined during and after pregnancy in six patients with Graves' disease receiving no or minimal maintenance doses of antithyroid drugs. During pregnancy, serum levels of TSH-binding inhibitory Igs (P < 0.001) and thyroid-stimulating antibodies (TSAbs) (P < 0.01) decreased gradually but increased after delivery in all patients. Activities of thyroid-stimulation blocking antibodies (TSBAbs) were lower than the cut-off value in early pregnancy, and values significantly decreased in four patients during pregnancy. The other two patients showed no significant change during pregnancy. In contrast, TSBAb levels increased significantly (P < 0.01) after delivery in all patients. We found that activities of TSH-binding inhibitory Igs, TSAb, and TSBAb decrease during pregnancy and increase after delivery, suggesting that amelioration of Graves' disease during pregnancy is induced by decrease of TSAb but not by the appearance of TSBAb.

Relation between post-partum liver dysfunction and anti-cytochrome 2D6 antibodies.

PROBLEM: Autoimmune thyroid disease frequently aggravates or develops after delivery through the immune rebound mechanism. However, little is known about the post-partum development of autoimmune hepatitis (AIH). METHOD OF STUDY: We examined 18 patients who developed liver dysfunction after delivery or abortion. Serial examinations were performed in 10 of these cases. Anti-cytochrome 2D6(CYP2D6) antibodies, which are liver-specific autoantibodies, were measured using a sensitive radioligand assay. RESULTS: Liver dysfunction developed between 1 and 5 months after delivery and was mild and transient except in one case. One patient developed liver dysfunction after abortion. Eight of 10 patients who underwent serial serologic examinations were positive for anti-nuclear antibodies, but anti-smooth muscle antibodies were positive in only three patients, and were present only at low titer. None of the patients had anti-mitochondrial antibodies. Nine of these 10 cases were diagnosed as definite or probable AIH according to the international scoring system of AIH. Nine of these 10 patients were positive for anti-CYP2D6 antibodies. The increase of anti-CYP2D6 antibodies was slightly delayed compared to increase of aminotransferase. Of the 18 patients who developed liver dysfunction, 15 cases (83.3%) were positive for anti-CYP2D6 antibodies. Of the 77 post-partum control subjects only three (3.9%) had positive antibodies. CONCLUSIONS: Autoimmune hepatitis developed after delivery, similarly to the development of post-partum autoimmune thyroid disease. Measurement of anti-CYP2D6 antibodies by a sensitive radioligand assay could provide information important for the detection of post-partum AIH.

Association of seasonal allergic rhinitis is high in Graves' disease and low in painless thyroiditis.

Hashimoto's thyroiditis is thought to be a T-helper cell type 1 (TH1)-dependent disease, but it is not clear whether Graves' disease is T-helper cell type 2 (TH2)-predominant or not. TH1-predominant diseases are infrequently and TH2-predominant diseases are frequently associated with allergic diseases. We examined the prevalence of seasonal allergic rhinitis to Japanese cedar pollen, a typical TH2-associated disease, in patients with Graves' disease (n = 126), painless thyroiditis (n = 46) and Hashimoto's thyroiditis (n = 88), and compared them to healthy controls (n = 766). Gender and age distribution were not different among patient groups and healthy controls, except for the higher age of patients with Hashimoto's thyroiditis. The prevalence of seasonal allergic rhinitis was significantly high in patients with Graves' disease (42.9%, p < 0.05) and low in patients with painless thyroiditis (13.0%, p < 0.01) but was not different in patients with Hashimoto's thyroiditis (26.1%) compared to that of healthy controls (32.6%). When patients with painless thyroiditis were included in Hashimoto's thyroiditis group, the prevalence of seasonal allergic rhinitis was 21.6% and significantly different from that of healthy controls (p < 0.05). These data indicate that Graves' disease is TH2 predominant and painless thyroiditis is a TH1-predominant disease. Our findings suggest that the shift from TH2 toward TH1 immunogenesis may be important for achieving earlier remission of Graves' disease.

Possible induction of Graves' disease and painless thyroiditis by gonadotropin-releasing hormone analogues.

Prolonged administration of gonadotropin-releasing hormone (GnRH) analogues induce a decrease in serum estrogen level, which may aggravate subclinical or mild autoimmune thyroid disease. Two patients developed Graves' thyrotoxicosis in association with an increase in anti-thyrotropin (TSH) receptor antibody activities at 4 months after initiation of buserelin acetate. GnRH analogue therapy was discontinued at the time of diagnosis but it took more than 2 years of methimazole therapy to obtain remission of Graves' disease. Another patient developed painless thyroiditis in association with an increase in antithyroid microsomal antibodies at 4 months after initiation of leuprolide acetate. These results indicate that GnRH analogues possibly induce clinical onset of Graves' thyrotoxicosis or destruction-induced thyrotoxicosis. Clinicians should be aware of this phenomenon. All patients who are to receive GnRH analogue therapy should be examined for antithyroid antibodies and family history of autoimmune thyroid disease, and should be followed accordingly.

Autoantibodies against muscarinic cholinergic receptor in chronic fatigue syndrome.

The disturbance of the central nervous system and immunological abnormalities have been suggested in patients with chronic fatigue syndrome (CFS). We focused on immunological abnormalities against neurotransmitter receptors in CFS. Using a sensitive radioligand assay, we examined serum autoantibodies to recombinant human muscarinic cholinergic receptor 1 (CHRM1), mu-opioid receptor (OPRM1), 5-hydroxytryptamine receptor 1A (HTR1A), and dopamine receptor D2 (DRD2) in patients with CFS (n=60) and results were compared with those in patients with autoimmune disease (n=33) and in healthy controls (n=30). The mean anti-CHRM1 antibody index was significantly higher in patients with CFS (p<0.0001) and autoimmune disease (p<0.05) than that in healthy controls, and positive reaction was found in 53.3% of patients with CFS. Anti-OPRM1 antibodies, anti-HTR1A antibodies, and anti-DRD2 antibodies were found in 15.2, 1.7, and 5.0% of patients with CFS, respectively. Anti-nuclear antibodies were found in 56.7% (34/60) of patients with CFS, but anti-nuclear antibody titers did not correlate with the activities of the above four autoantibodies. The patients with positive autoantibodies to CHRM1 had a significantly higher mean score (1.81) of 'feeling of muscle weakness' than negative patients (1.18) among CFS patients (p<0.01). Higher scores on 'painful node', 'forgetfulness', and 'difficulty thinking' were also found in CFS patients with anti-CHRM1 antibodies but did not reach statistical significance. In conclusion, autoantibodies to CHRM1 were detected in a large number of CFS patients and were related to CFS symptoms. Our findings suggested that subgroups of CFS are associated with autoimmune abnormalities of CHRM1.

Practical treatment with minimum maintenance dose of anti-thyroid drugs for prediction of remission in Graves' disease.

Although many researchers have reported clinical and laboratory parameters for prediction of remission in Graves' disease during or after anti-thyroid drug therapy, there is no reliable one to assure the complete remission. We prospectively examined a practical therapy with minimum maintenance dose of anti-thyroid drugs for prediction of remission in Graves' disease. Fifty-seven patients with Graves' disease were treated with anti-thyroid drugs at the initial dose of 30 mg/day of methimazole (MMI) or 300 mg/day of propylthiouracil (PTU). Then, doses were gradually decreased, and finally discontinued when the patients were able to maintain euthyroid (normal FT4 and TSH) for at least 6 months with the minimum maintenance dose (MMI 5 mg every other day or PTU 50 mg every other day). After discontinuation of drugs, FT4, FT3, TSH and TSH-binding inhibitory immunoglobulin (TBII) were measured every one to two months for the first 6 months and every 3-4 months for the next 18 months to confirm continuous remission. After 2 years of drug cessation, 46 (81%) of 57 patients were in remission and the other 11 patients had relapsed into thyrotoxicosis. At the time of drug discontinuation, the serum concentration of FT4, FT3 and TSH, titers of anti-thyroglobulin antibodies and anti-thyroid microsomal antibodies, goiter size were not different between the remission and relapse groups. At the time of drug cessation, the activities of TBII and thyroid-stimulating antibodies (TSAb) overlapped between the two groups, although they were significantly lower in the remission group than in the relapse group (p<0.01). Forty percent (4/10) of TBII positive patients and 71% (23/32) of TSAb positive patients continued to be in remission. On the other hand, thyrotoxicosis relapsed in 5 (11%) of 47 TBII negative and 2 (8%) of 25 TSAb negative patients. These data indicate that minimum maintenance therapy to keep euthyroid (normal FT4 and TSH) for 6 months is a practical measure for 81% prediction of remission in Graves' disease. The measurement of TBII or TSAb gave little additional information for predicting remission.

Increased serum concentration of eosinophil-derived neurotoxin in patients with Graves' disease.

Eosinophil-derived neurotoxin (EDN) is released after activation and stimulation of eosinophils in allergic disease, which is a T(H)2-predominant condition. We previously reported that Graves' thyrotoxicosis develops or relapses after an attack of allergic rhinitis. In this study, to confirm the relation between Graves' disease and the allergic condition, we determined the serum level of EDN in 30 untreated patients with Graves' disease, 50 patients with Hashimoto's thyroiditis, and 39 normal controls. Compared to the serum level in normal subjects (30.1 +/- 15.6 ng/mL), EDN was increased in untreated patients with Graves' disease (52.4 +/- 27.6 ng/mL), but not in patients with Hashimoto's thyroiditis (thyrotoxic, 30.9 +/- 13.4 ng/mL; euthyroid, 30.0 +/- 11.9 ng/mL; hypothyroid, 23.4 +/- 10.2 ng/mL). A significant correlation was observed between the EDN level and the serum activity of thyrotropin (TSH) receptor antibody (r = 0.541, p < 0.0001). These data suggest that the allergic condition is closely related to Graves' disease and that a T(H)2-type immune response is crucial in the pathogenesis of Graves' disease.