Comprehensive study of urinary cortisol metabolites in hyperthyroid and hypothyroid patients.

OBJECTIVE: To further analyse the significance and mutual relationship of thyroid function-linked alterations in cortisol metabolism that have been separately and variously reported. PATIENTS AND MEASUREMENTS: Twenty-four-hour urine samples from 21 patients with hyperthyroidism (Graves' disease), 16 patients with hypothyroidism (Hashimoto's thyroiditis), 21 healthy age- and sex-matched controls for hyperthyroidism, and 16 healthy age- and sex-matched controls for hypothyroidism were evaluated for 6beta-hydroxycortisol (6beta-OHF), tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-tetrahydrocortisol (allo-THF), urinary free cortisol (UFF), urinary free cortisone (UFE) and 17-hydroxycorticosteroid (17-OHCS). RESULTS: Urinary 17-OHCS, THE and allo-THF levels increased considerably in hyperthyroid patients compared to the controls, while UFF and THF showed no difference between the two groups. Urinary 6beta-OHF was significantly lower in the hyperthyroid patients than in the controls. Both the urinary allo-THF + THF/THE and the UFF/UFE ratios were significantly lower in the hyperthyroid patients than in the controls, whereas only the former was significantly higher in the hypothyroid patients than in the controls. The urinary allo-THF/THF ratio was significantly higher in the hyperthyroid patients and significantly lower in the hypothyroid patients than in the controls. In an analysis of pooled subjects including all groups (n = 64), free T4 levels correlated negatively (P < 0.0001) with the urinary allo-THF + THF/THE ratio but not with the UFF/UFE ratio. The serum levels of free T4 correlated positively (P < 0.0001) with the urinary allo-THF/THF ratio. CONCLUSION: The thyroid hormones seem to affect the total 11beta-HSD activity (allo-THF + THF/THE) more strongly than the renal 11beta-HSD2 activity (UFF/UFE). 5alpha-reductase activity (allo-THF/THF) is also enhanced in hyperthyroidism, while the reduction of urinary 6beta-OHF in hyperthyroidism might be a secondary effect of the altered activity of the total 11beta-HSD and 5alpha-reductase.

Antioxidants and methimazole in the treatment of Graves' disease: effect on urinary malondialdehyde levels.

BACKGROUND: We have postulated that metabolic oxidation could be the source of signs and symptoms of hyperthyroidism. The present study was designed to evaluate urinary malondialdehyde levels in Graves' disease and compare this oxidative stress biomarker with the clinical evolution of patients suffering this illness. METHODS: We evaluated the concentration of urinary and serum malondialdehyde (MDA) in 36 patients with Graves' disease. Patients were treated with the antithyroid drug methimazole (MMI; Group A) or antioxidant mixture (200 mg vitamin E, 3 mg beta-carotene, 250 mg vitamin C, 1 mg Cu, 7.5 mg Zn, 1.5 mg Mn, and 15 microg Se; Group B). RESULTS: MDA concentrations were higher in hyperthyroid patients compared to euthyroid controls, and a positive correlation was observed between serum and urinary MDA levels. Group A decreased urinary MDA to control values. There was a positive correlation between the clinical score and the heart rate of patients with urinary MDA before and during the treatment with MMI (Group A). Similar results were observed after treatment with the antioxidant mixture. CONCLUSIONS: Urinary MDA might be a good parameter in the follow-up of patients during MMI treatment. We proposed that oxidative stress correlates with signs and symptoms of hyperthyroidism.

Elevated urinary 8-isoprostaglandin F(2alpha) in females with Graves' hyperthyroidism.

In this study, we investigate how oxidative stress alters the urinary F2-isoprostanes' level and we examine the correlation between 8-isoPGF(2alpha) and thyroid hormones in female patients with Graves' hyperthyroidism. We quantitatively determined the concentrations of urinary F2-isoprostanes using gas chromatography-mass spectrometry in the selected ion-monitoring mode. We recruited individuals in the following three groups of subjects for this study: (1) female hyperthyroidism patients (n = 14, 21-71 years), (2) female hypothyroidism patients (n = 16, 16-66 years), and (3) female age-matched normal controls (n = 10, 20-61 years). The average concentration of 8-iso prostaglandin F(2alpha) (8-isoPGF(2alpha)) in hyperthyroidism patients was significantly higher than that in the healthy controls (P < 0.05). The concentration of 8-isoPGF(2alpha) in hypothyroidism patients was similar to that in normal controls. Although the level of 8-isoPGF(2alpha) in two of the patients was slightly elevated, the P value was not significant (NS). Our data suggest that the increased level of urinary 8-isoPGF(2alpha) may reflect possible oxidation injuries in hyperthyroidism patients due to the high level of thyroid hormones. Therefore, the elevated 8-isoPGF(2alpha) in patients could be used as an important biomarker for hyperthyroidism disease.

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy.

OBJECTIVE: Immunosuppressive treatment of Graves' opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time-consuming and difficult for routine use. The aim of the present study is to develop practical and simple methods for uGAGs and sHA and compare them to the activity and severity of thyroid-associated ophthalmopathy. DESIGN, PATIENTS AND MEASUREMENTS: We developed a microelectrophoresis technique for uGAGs and a fluoroassay for sHA and assessed each in 152 patients with Graves' disease, 25 without GO and 127 with GO, classified according to the Clinical Activity Score (CAS). All patients had been euthyroid for > 2 months. RESULTS: Patients with inactive disease (CAS = 2, n = 100) had uGAGs (4.2 +/- 1.3 micro g/mg/creatinine) and sHA (11.1 +/- 7.2 micro g/l) that did not differ from normal subjects (3.1 +/- 1.1 micro g/mg/creatinine, n = 138 and 13.9 +/- 9.6 micro g/l, n = 395). In contrast, patients with active eye disease (CAS = 3, n = 27) had uGAGs (8.4 +/- 2.7 micro g/mg/creatinine) and sHA (32.3 +/- 17.8 micro g/l) 2-3 times higher than those patients with inactive eye disease. Using a cut-off of 6.1 micro g/mg creatinine for uGAGs and 20.7 micro g/l for sHA we found, respectively, 85% and 81% sensitivity and 93% and 91% specificity for each test. The positive and negative predictive values were 77% and 96% for uGAGs and 71% and 95% for sHA. CONCLUSION: Employing these two new methods we have established a significant relationship between the levels of uGAGs and/or sHA and the clinical activity of GO. Therefore, together with CAS, uGAGs determination, and, to a lesser degree, sHA, would be very useful in the discrimination from active and inactive ocular disease and aid in deciding on the best therapy for GO patients.

Bone and calcium metabolism in subclinical autoimmune hyperthyroidism and hypothyroidism.

Bone turnover is reported to increase in favour of resorption in overt hyperthyroidism and the rate of resorption is associated with the levels of thyroid hormones. Hypothyroidism, on the other hand, was shown to cause no disturbance of calcium kinetics and found to associate lower trabecular resorption surfaces and increased bone cortical thickness. Similar studies are very rare in subclinical thyroid disorders and consequently we aimed to examine calcium and bone metabolism in subclinical thyroid disorders. Thirteen patients with subclinical hyperthyroidism secondary to untreated Graves' disease, 20 patients with subclinical hypothyroidism and 10 healthy subjects participated in this survey. Briefly calcium, phosphorus, and creatinine (Cre), urinary deoxypyridinoline (U-DPD) and serum osteocalcin (OC) were measured as biochemical markers for calcium metabolism. Concerning serum Ca and phosphorus levels, there were no differences between three of the groups, but urinary Ca excretion was higher in subclinical hyperthyroid patients compared to control and hypothyroid subjects. Hypothyroid patients had similar U-DPD levels with control subjects (p = 0.218). Serum OC and U-DPD were higher in subclinical hyperthyroid compared to control subjects (p<0.001 and p<0.001 respectively). We demonstrated a higher bone turnover and greater calcium excretion in subclinical hyperthyroid patients. Additionally, we found that subclinical hypothyroidism is not associated with disturbed calcium metabolism. As persistent increase in bone turnover is responsible for accelerated bone loss, patients with Graves' disease may have increased risk for osteoporosis.

[The assessment of urinary glycosaminoglycans (GAG) excretion during therapy of patients with progressive Graves' ophthalmopathy]. / Ocena wydalania z moczem glikozaminoglikanów (GAG) w trakcie leczenia postepujacej oftalmopatii obrzekowo-naciekowej w przebiegu choroby Gravesa i Basedowa.

Excessive accumulation of hydrophilic glycosaminoglycans (GAG) in the retrobulbar tissue leads to many of the clinical manifestations of Graves' ophthalmopathy (GO). We examined the quantitative urinary GAG excretion in 35 patients with GO. GAG were isolated from 24-h urine collections by precipitation with cetylpyridinum chloride and ethanol according to Bitter. Patients with progressive ophthalmopathy showed on the average a twofold increase in urinary GAG excretion in comparison to patients with stable ophthalmopathy. The elevated values of GAG decreased significantly under treatment and correlated with the clinical picture of GO, however no correlation was found between the urinary GAG values and actual severity of ophthalmopathy classified according to Werner and Donaldson. There was no significant difference in GAG excretion between group 1, treated with methylprednisolon and irradiation of retrobulbar tissue and group 2, under therapy with methylprednisolon. In conclusion, elevated urinary GAG excretion is characteristic for the clinical picture of the progressive GO. The remarkable decrease in GAG values during treatment correlated to the positive response to immunosuppressive therapy.

Medical therapy of Graves' disease: effect on remission rates of methimazole alone and in combination with triiodothyronine.

In a prospective, randomized study of 135 newly diagnosed patients with hyperthyroidism due to Graves' disease we compared the effect on remission rates of additional triiodothyronine (T3) with conventional antithyroid drug therapy. To this end 114 patients were followed for at least 12 months (15.7+/-4.9, mean+/-s.d.) after the discontinuation of any therapy. After return of thyroid function to normal (8.5+/-7.4 weeks, mean+/-s.d.) patients were maintained on antithyroid medication for 9.0+/-2.5 months. They were then randomly assigned to one of three groups: group 1 (n=44) stopped methimazole, groups 2 (n=39) and 3 (n=31) continued with exogenous T3 (not exceeding 75 microgram/day in any patient) for a further 6 months either with (group 2) or without (group 3) a fixed dose of 10mg methimazole daily. The T3 dose was kept variable to keep TSH suppressed (<0. 1mU/l), which could be achieved in 82% of patients on 100% of their monthly visits. No serious side-effect requiring the discontinuation of the study occurred in any patient. Total T3, TSH-receptor antibodies and some previously suggested potential predictors of relapse including thyroid size by ultrasound, 24h urinary iodine excretion, history of cigarette smoking and ophthalmopathy were determined at the outset of the study and subsequently every 6 months (and total T3 every 4 weeks). No significant difference (P>0.05, Chi square) was seen in relapse of hyperthyroidism after a mean follow-up of 16 months (range: 12-31 months; groups 1:52%, 2:44% and 3:42%) in an area of low-to-moderate iodine intake (prevalence of 24h urinary iodine excretion <100 microgram/24h: 17 and 25% at two different measurements respectively). Concomitantly, no predictor of recurrence of disease could be identified, irrespective of treatment modality.

Glycosaminoglycans in thyroid eye disease.

Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAG) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with thyroid eye disease (TED). In order to analyze whether the alteration of distribution pattern and biochemical composition of GAG and proteoglycans play a role in the development of the disease, a highly specific high-pressure liquid chromatography (HPLC) method was developed. The concentration of total GAG, chondroitin sulfate A (CA), dermatan sulfate (DS), and hyaluronic acid (HA) was determined in patients and controls, revealing marked differences in urinary concentration of total GAG and HA, as well as an elevation of CA in patients versus controls. Method sensitivity was 0.86 for patients with active disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation. Furthermore, distribution pattern of orbital extracellular matrix GAG exhibited a significant increase in the tissue fractions of CA and HA in patients with TED in comparison to controls. In conclusion, GAG polysaccharides not only play a major role in the pathogenesis of TED but have been successfully introduced as an activity marker of the disease.

Longitudinal changes of metacarpal cortical striation in Graves' disease.

RATIONALE AND OBJECTIVES: The authors verify whether metacarpal cortical striation can be used to assess longitudinal changes in bone turnover in Graves' disease. METHODS: Eight patients with untreated Graves' disease (5 men, 3 women; age 36 +/- 12 years) and six patients with the disease in remission (1 man, 5 women; age 38 +/- 12) were studied. Posteroanterior radiographs of the bilateral hands were obtained using fine-grain mammography film and direct magnification. Three observers independently determined the grade of cortical striation (striation index; SI) of the second and third metacarpals in randomly presented radiographs. RESULTS: The SI determined by all observers decreased significantly after the beginning of antihyperthyroid therapy compared with the SI before treatment (P < 0.05). Significant correlation was found between the observers' assessments (r = 0.74, P < 0.001). The average kappa value and percent agreement were 0.27 and 51.8%, respectively. Intraobserver variability provided relatively good kappa statistics (kappa: 0.56; percent agreement: 79.9%). Longitudinal decrease in the SI was in accordance with a decline in urinary pyridinoline cross-link excretion, a decline in serum osteocalcin, and an increase in bone mineral density of lumbar spine. CONCLUSIONS: The change in the SI can be used to detect increased and decreased bone turnover. Thus, the SI can be used as one index of bone turnover in patients with Graves' disease.

HPLC glycosaminoglycan analysis in patients with Graves' disease.

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.

The effect of somatostatin versus corticosteroid in the treatment of Graves' ophthalmopathy.

Uncontrolled study has demonstrated the usefulness of somatostatin in the treatment of mild Graves' ophthalmopathy (GO). We performed a prospective study to evaluate the usefulness of somatostatin as compared to corticosteroid in the treatment of moderately severe GO. All patients were rendered euthyroid and observed for 3 months to exclude spontaneous improvement without active treatment. They were randomized to receive either somatostatin (SS, octreotide 200 micrograms q8h subcutaneously, n = 8) or corticosteroid (CS, prednisone 1 mg/kg/day in decreasing doses, n = 10). Assessments of soft tissue inflammation, exophthalmos, palpebral aperture, intraocular pressure, diplopia, cornea, and visual acuity were made every 4 weeks for 3 months. MRI of the orbit was performed before and after treatment. Both SS and CS therapy decreased the palpebral aperture and activity score after 3 months (p < 0.05), but those treated with CS had a lower activity score after treatment when compared to SS [2.5 (1-7) v.s. 3.5 (0-4), median (range), p < 0.05]. Only CS, but not SS, was able to reduce intraocular pressure and muscle size as documented by MRI, but no significant reduction in proptosis was observed in either group. Also, patients' self-assessments of the eye changes after treatment were similar between the two groups. Both groups showed significant elevation of urinary glycosaminoglycan (GAG) excretion before therapy (SS 24.6 +/- 10.8; CS 27.8 +/- 11.4 mg/24 h), which was reduced after treatment (SS 12.5 +/- 7.3; CS 10.8 +/- 6.3 mg/24 h, p < 0.05). However, no significant correlation could be observed between the degree of GAG reduction and the clinical outcome of the patients. In conclusion, the long acting SS octreotide was effective in reducing soft tissue inflammation and providing symptomatic relief in GO but not as effective as corticosteroid in reducing muscle size. In view of the minimal side-effects and similar efficacy as compared to corticosteroid in patients with minimal extraocular muscle enlargement, it is suggested that a trial of SS may be considered in selected patients with GO.

Urinary glycosaminoglycans excretion in Graves' disease.

Urinary excretion of glycosaminoglycans was measured in 10 patients with pretibial myxoedema, 7 of whom also had thyroid-associated ophthalmopathy, and in 3 additional patients with ophthalmopathy but no skin changes. Total uronic acid excretion was raised above control levels in only 2 patients, who had both eye and skin disease of recent onset. In these patients excretion was initially three times the control level but declined sharply in subsequent months. This decline was in the absence of effective treatment or spontaneous improvement and would appear to reflect the natural history of the disease. These data show that although glycosaminoglycans excretion may be disturbed in Graves' disease, it provides an unreliable reflection of clinical status and of the effectiveness of treatment.

Response to methimazole in Graves' disease. The European Multicenter Study Group.

OBJECTIVE: A variety of regimens continue to be used in the treatment of Graves' disease with antithyroid drugs. We have investigated the factors which determine the initial response to methimazole (time until euthyroidism is achieved) in Graves' disease. PATIENTS: Five hundred and nine patients with Graves' disease in different European countries with normal and subnormal iodine supply. Patients were randomized to treatment with either 10 or 40 mg of methimazole per day for one year, with levothyroxine supplementation as required to maintain euthyroidism. Investigations were carried out before treatment and at 3 and 6 weeks and 3, 6, 9 and 12 months. MEASUREMENTS: Response was assessed by serial measurements of serum thyroid hormones. TSH receptor antibodies, thyroid autoantibodies and urinary iodide excretion were measured centrally. Twenty-minute thyroid uptake was measured by standard techniques. Data were collected and analysed centrally. Standard techniques as well as a stepwise logistic regression model were used to examine the relations between methimazole dose, age, goitre size, presence of endocrine eye signs, thyroid hormone levels, urinary iodide excretion, thyroid uptake, index of disease severity (Crooks), presence of TSH receptor antibodies and duration of the hyperthyroid phase. RESULTS: Within 3 weeks, 40.2% of patients responded to 10 mg of methimazole and 77.5% responded within 6 weeks. The corresponding figures for 40 mg of methimazole were 64.6 and 92.6%. Significant associations were found between duration of hyperthyroidism and the following variables: goitre size, urinary iodide excretion, methimazole dose, presence of TSH receptor antibodies (TBIAb), index of disease severity (Crooks) and pretreatment thyroid hormone levels. Response to methimazole was delayed in patients with large goitres, iodine excretion of > or = 100 micrograms/g creatinine, high pretreatment thyroid hormone levels, elevated levels of TBIAb and treatment with only 10 mg of methimazole. In the 10-mg group, 46% of patients were euthyroid within 3 weeks when urinary iodide was < 50 microgram/g of creatinine, and only 27% when iodide was above 100 micrograms/g. By stepwise logistic regression, the main factors for the response to methimazole were daily dose, pretreatment T3 levels, and goitre size. CONCLUSION: Methimazole dose, pretreatment serum T3 levels, and goitre size are the main determinants of the therapeutic response to methimazole in Graves' disease, at least in areas comprising low, subnormal and normal iodine supply.

Improved measurement of anti-thyroglobulin IgG in urine of patients with autoimmune thyroid diseases by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay.

Previously, antithyroglobulin IgG was assayed in dialyzed urine from patients with autoimmune thyroid diseases by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay), and most of the assay results were useful as a diagnostic aid for autoimmune thyroid diseases. However, dialysis of urine was laborious and time-consuming, and some results were less reliable due to low levels of anti-thyroglobulin IgG in urine. This paper describes some improvements of the assay. Useful assay results could be obtained for most of urine samples without dialysis, although some interfering substance(s) was suggested to be present in some urine samples before dialysis. Accurate assay results with no interference could be obtained after gel filtration by only two min centrifugation in place of dialysis. More reliable assay results for urine samples containing low levels of antithyroglobulin IgG were obtained after concentration using a molecular sieve.

Urinary glycosaminoglycans in Graves' ophthalmopathy.

An increased accumulation of glycosaminoglycans (GAG) in retrobulbar tissues has been reported in patients with thyroid eye disease. We examined the quantitative urinary GAG excretion in 101 patients with Graves' ophthalmopathy of different classes, 36 patients with Graves' hyperthyroidism without ophthalmopathy, 14 patients with toxic nodular goitre and 103 control subjects. Glycosaminoglycans were isolated from 24-h urine collections by precipitation with cetylpyridinium chloride and ethanol followed by photometrical quantification of hexuronic acids after reaction with carbazole. In comparison with the control group (15.8, 10.4, 21.6 mg/24 h; median, 25th, 75th percentile) a significant (P less than 0.005) elevation of urinary GAG excretion was found in patients with ophthalmopathy (19.2, 12.2, 28.7 mg/24 h), whereas patients with Graves' hyperthyroidism and no ophthalmopathy (16.2, 11.9, 21.7 mg/24 h) and patients with toxic nodular goitre (15.8, 11.5, 21.2 mg/24 h) exhibited no markedly increased values. Especially, patients with active, untreated ophthalmopathy showed on average a twofold increase (36.7, 28.1, 48.4 mg/24 h) in urinary GAG excretion. In contrast, high values were not found in patients with inactive ophthalmopathy and elevated values decreased under treatment, which correlated with clinical findings. Further, relapses were also accompanied by high GAG excretion. Thus, using a simple laboratory method, quantitative determination of urinary GAG excretion appears to present an effective parameter for the activity of Graves' ophthalmopathy.