BACKGROUND

The effect of calcium carbonate on the absorption of levothyroxine has not been studied systematically. Such a potential drug interaction merits investigation because concurrent treatment with both drugs is common, particularly in postmenopausal women.

OBJECTIVE

To investigate the potential interference of calcium carbonate in the absorption of levothyroxine.

METHODS

Prospective cohort study conducted from November 1998 to June 1999, supplemented with an in vitro study of thyroxine (T(4)) binding to calcium carbonate.

METHODS

Veterans Affairs Medical Center in West Los Angeles, Calif.

METHODS

Twenty patients (age range, 27-78 years; n=11 men) with hypothyroidism who were taking a stable long-term regimen of levothyroxine were included in the study. All patients had serum free T(4) and thyrotropin values in the normal range before beginning the study.

METHODS

Subjects were instructed to take 1200 mg/d of elemental calcium as calcium carbonate, ingested with their levothyroxine, for 3 months.

METHODS

Levels of free T(4), total T(4), total triiodothyronine (T(3)), and thyrotropin, measured in all subjects at baseline (while taking levothyroxine alone), at 2 and 3 months (while taking calcium carbonate and levothyroxine), and 2 months after calcium carbonate discontinuation (while continuing to take levothyroxine).

RESULTS

Mean free T(4) and total T(4) levels were significantly reduced during the calcium period and increased after calcium discontinuation. Mean free T(4) levels were 17 pmol/L (1.3 ng/dL) at baseline, 15 pmol/L (1.2 ng/dL) during the calcium period, and 18 pmol/L (1.4 ng/dL) after calcium discontinuation (overall P<.001); mean total T(4) levels were 118 nmol/L (9.2 microg/dL) at baseline, 111 nmol/L (8.6 microg/dL) during the calcium period, and 120 nmol/L (9.3 microg/dL) after calcium discontinuation (overall P=.03). Mean thyrotropin levels increased significantly, from 1.6 mIU/L at baseline to 2.7 mIU/L during the calcium period, and decreased to 1. 4 mIU/L after calcium discontinuation (P=.008). Twenty percent of patients had serum thyrotropin levels higher than the normal range during the calcium period; the highest observed level was 7.8 mIU/L. Mean T(3) levels did not change during the calcium period. The in vitro study of T(4) binding to calcium showed that adsorption of T(4) to calcium carbonate occurs at acidic pH levels.

CONCLUSIONS

This study of 20 patients receiving long-term levothyroxine replacement therapy indicates that calcium carbonate reduces T(4) absorption and increases serum thyrotropin levels. Levothyroxine adsorbs to calcium carbonate in an acidic environment, which may reduce its bioavailability. JAMA. 2000;283:2822-2825

In women monitored for thyroid carcinoma, short-term stimulation with rhTSH induced an acute decrease in serum C-telopeptides of type-1 collagen and an increase in serum BALP levels without any effect on OPG production. The inhibitory effect of TSH on bone resorption occurred only in postmenopausal women who showed low BMD and a high bone turnover rate as an effect of L-thyroxine suppressive therapy.

BACKGROUND

It has been recently shown that thyrotropin (TSH) has an inhibitory activity on skeletal remodeling in in vitro conditions. Here, we have aimed at evaluating whether TSH has similar effects in vivo. For this purpose, we have evaluated the sequential profile of serum bone metabolism markers during acute stimulation with recombinant human TSH (rhTSH) in thyroidectomized women monitored for thyroid carcinoma.

METHODS

The study group included 66 thyroidectomized patients, of whom 38 were premenopausal and 28 postmenopausal, who underwent routine rhTSH-assisted whole body radioactive iodine scanning for differentiated thyroid carcinoma. The patients were sequentially evaluated for TSH, free triiodothyronine (FT3), free thyroxine (FT4), bone alkaline phosphatase (BALP), C-telopeptides of type-1 collagen (CrossLaps), and osteoprotegerin (OPG) levels during rhTSH stimulation. The samples were drawn just before and 2 and 7 days after the first administration of rhTSH. BMD was evaluated by ultrasonography at baseline. Seventy-one healthy women (41 premenopausal and 30 postmenopausal) acted as a control group.

CONCLUSIONS

At study entry, all patients had subclinical thyrotoxicosis as effect of L-thyroxine (L-T4) treatment. The patients had higher serum CrossLaps and OPG levels and lower BMD than healthy subjects. Postmenopausal patients showed comparable serum FT4 and FT3 concentrations with those found in premenopausal patients. However, postmenopausal patients showed higher serum CrossLaps (p < 0.001), OPG (p = 0.03), and BALP (p < 0.001) levels and lower BMD (p < 0.001) than those measured in premenopausal patients. Two days after the first administration of rhTSH, all patients had serum TSH values >100 mUI/liter. At this time, serum CrossLaps levels decreased significantly (p < 0.001) and BALP values increased (p = 0.001) with respect to the baseline values in postmenopausal but not in premenopausal patients. rhTSH did not induce any significant change in serum OPG values either in premenopausal or in postmenopausal patients. One week after the first rhTSH administration, serum CrossLaps values decreased again to values comparable with those measured at baseline, whereas serum BALP values remained high. This study shows that subclinical thyrotoxicosis is accompanied by high bone turnover rate with an increase in serum OPG levels compared with euthyroid healthy subjects. Acute increase in serum TSH levels is accompanied by a reversible inhibition of bone resorption. This effect is characterized by a decrease in serum CrossLaps and an increase in BALP levels without any evident effect on OPG production. The activity of TSH occurs specifically in postmenopausal women in whom the negative effects of L-T4 suppressive therapy on bone mass and metabolism are more marked compared with premenopausal women.

The effects of prenatal oral administration of 0.2, 0.6, and 1.8 mg/kg body wt of 3,3',4,4'5,5'-hexachlorobiphenyl (HCB) on Day 1 of gestation and a combination of 1 mg/kg 3,3',4,4'-tetrachlorobiphenyl (TCB) from Day 2 to Day 18 with 0.6 mg HCB/kg body wt on Day 1 of gestation on thyroid hormone status and peripheral thyroid metabolism were studied in pregnant Wistar rats and their fetuses and offspring. Plasma total thyroxine and free thyroxine levels were reduced by HCB in a dose-dependent fashion in pregnant rats (Days 12 and 20 of gestation) and neonates (Day 21 postpartum), while only a combined dose of HCB and TCB was effective in decreasing fetal thyroid hormone levels by 65% on Day 20 of gestation. The activity of type II thyroxine 5'-deiodinase (5'D-II), the enzyme responsible for the deiodination of thyroxine (T4) to biologically active triiodothyronine in the brain, was examined in whole brain homogenates in fetuses and neonates. Decreases in plasma thyroid hormones were accompanied by significant increases, up to 100%, in 5'D-II activity in brain homogenates from fetuses (Day 20 of gestation) and neonates (Days 7 and 21 postpartum). The glucuronidation of 125I-T4 by hepatic microsomes was increased by at least 100% relative to control levels by all treatments in fetuses (Day 20 of gestation) and increased at least 40% in neonates (Days 7 and 21 postpartum) by a dose of 0.6 and 1.8 mg HCB/kg and the combined dose. These data indicate that prenatal HCB and/or TCB administration result in increased peripheral T4 metabolism. The increase in 5'D-II activity suggests that local hypothyroidism occurs in the brains of fetal and neonatal rats exposed to HCB and/or TCB. Since these effects occur during a period in which thyroid hormones play an important role in brain maturation, they may help explain the mechanism of developmental neurotoxicity induced by polychlorinated biphenyls.

OBJECTIVE

In this study, we explore the associations of decreased thyroid hormone levels with inflammation, wasting and survival in biochemically euthyroid patients with end-stage renal disease (ESRD).

METHODS

After exclusion of 23 patients with thyroid-stimulating hormone (TSH) values outside the normal range (0.1-4.5 mIU L(-1)), 187 clinically and biochemically euthyroid incident ESRD stage 5 patients starting dialysis were followed for a median of 20 (range 1-60) months. Measurements of total and free forms of thyroid hormones, s-albumin, hs-CRP, interleukin (IL)-6, vascular adhesion molecule (VCAM)-1 and insulin-like growth factor 1 (IGF-1) were performed at baseline.

RESULTS

In this population, 17 out of 210 patients (8%) were defined as subclinically hypothyroid. Multivariate analysis, according to receiver operating characteristic (ROC) curves, showed that mortality was best predicted by total triiodothyronine (T3). When using the cut-off levels derived from ROC, low T3 levels were associated with increased inflammation (higher hs-CRP, IL-6 and VCAM-1) and lower concentration of both s-albumin and IGF-1. Finally, low T3 but not low free triiodothyronine was associated with worse all-cause (Likelihood ratio = 45.4; P < 0.0001) and cardiovascular mortality (Likelihood ratio = 47.8; P < 0.0001) after adjustment for confounding factors.

CONCLUSIONS

This study showed that low T3 levels are independent predictors of all-cause and also cardiovascular disease mortality in biochemically euthyroid patients, perhaps due to an intimate association with inflammation. Based on these results, the use of T3 levels in studies assessing the relationship between thyroid dysfunction and mortality risk is recommended.

The aims of this study were to (a) determine the prevalence of patients without elevated thyroid hormone levels in Graves' ophthalmopathy (GO) using current generation free thyroid hormone assays, (b) measure the prevalence of thyrotropin receptor antibodies (TRAb) in these cases, and (c) identify possible predictors of hyperthyroidism. Over a 30-month period, 1020 cases of thyroid eye disease were evaluated, of which only 19 (1.9%) met the diagnostic criteria. Ten (1%) had subclinical thyrotoxicosis, 7 (0.7%) were euthyroid, and 2 (0.2%) were hypothyroid as determined by a third-generation thyrotropin (TSH) assay. TRAb levels were measured in 16 of these 19 patients. The prevalence of TRAb varied according to the assay used. Polyethylene glycol-extracted thyroid-stimulating immunoglobulin (PEG-TSI), unfractionated thyroid-stimulating immunoglobulin (uTSI), first-generation porcine TSH-binding inhibitory immunoglobulin (pTBII), and second-generation human TSH-binding inhibitory immunoglobulin (hTBII) assays were positive in 93.8%, 50%, 18.8%, and 81.3% of patients, respectively. TRAb was detected by at least one method in all patients. Patients were followed up for 15 to 45 months. Hyperthyroidism developed in 4 patients (25%). Suppressed TSH levels and elevated TBII were predictors of hyperthyroidism. When sensitive assays are used, the prevalence of GO patients without elevated thyroid hormone levels is extremely low. The sensitivities of assays for TRAb detection differ substantially in these cases. PEG extraction improves the detection rate of TSI (p = 0.02), and hTBII assays improve the detection of TBII in these patients (p = 0.002). The high prevalence of TRAb in such cases supports a role for these antibodies in the pathogenesis of thyroid-associated eye disease.

OBJECTIVE

To compare relative bioavailability of Synthroid, Levoxine (Levoxine has been renamed Levoxyl), and 2 generic levothyroxine sodium preparations.

METHODS

Single-blind (primary investigators blinded), randomized, 4-way crossover trial.

METHODS

Ambulatory care.

METHODS

Twenty-two women with hypothyroidism who were clinically and chemically euthyroid and were receiving levothyroxine sodium, 0.1 or 0.15 mg.

METHODS

All patients received each of the 4 levothyroxine products for 6-week periods in the same dosage as their prestudy regimen with no washout period. The order of the drug sequences was randomly determined before study initiation.

METHODS

Area under the curve, time to peak serum concentrations, and peak serum concentrations of thyroxine, triiodothyronine, and free thyroxine index for all 4 products.

RESULTS

All data analyses were completed prior to unblinding of the product codes. No significant differences between the 4 products were found in area under the curve or peak serum concentrations of total thyroxine, total triiodothyronine, or free thyroxine index. Although Synthroid produced a more rapid rise in total serum triiodothyronine concentration and a higher total peak serum triiodothyronine concentration than the other products, these differences were not statistically significant (P=.08). The Food and Drug Administration criterion for relative bioequivalence within 90% confidence intervals (0.8-1.25) was demonstrated (P<.05) for all pairs of products. Relative bioequivalence of 0.95 to 1.07 was demonstrated, tighter than the current bioequivalence criterion for oral formulations.

CONCLUSIONS

The 4 generic and brand-name levothyroxine preparations studied are different but are bioequivalent by current Food and Drug Administration criteria and are interchangeable in the majority of patients receiving thyroxine replacement therapy. Further investigation is required to determine whether our results are equally applicable to all existing levothyroxine preparations.

1. Glycerol and dihydroxyacetone, both antiketogenic and readily metabolized, but differing in their effects on the redox state of the hepatic NAD couples, were given to starved rats and the contents of metabolites were measured in freezeclamped liver and in the blood. The object was to study the effects of changes in the redox state and of the availability of oxidizable substrates on the rate of ketone-body formation. 2. Intramuscular administration of dihydroxyacetone, glycerol or glucose to starved rats decreased the concentrations of acetoacetate and 3-hydroxybutyrate in the blood by 70-80% within 60min., whereas there was no major change in the free fatty acid concentration. 3. Dihydroxyacetone, but not glucose or glycerol, caused an immediate and sustained twofold increase in the blood lactate concentration. 4. Dihydroxyacetone and glycerol caused a rapid fall in the hepatic concentrations of ketone bodies, dihydroxyacetone being more effective. 5. This decrease was not accompanied by significant changes in the concentrations of acetyl-CoA, long-chain acyl-CoA or free CoA. 6. The hepatic glycerophosphate concentration rose about 40-fold on administration of glycerol, whereas with dihydroxyacetone the increase was only about 50%. The large increase in glycerophosphate concentration after administration of glycerol was completely prevented by pretreatment of the rats with tri-iodothyronine. Triiodothyronine-treated rats showed the same decrease in ketone-body concentrations after administration of glycerol as the untreated rats. 7. Glycerol and dihydroxyacetone caused an increase in the hepatic lactate concentration; the pyruvate concentration rose only after injection of dihydroxyacetone. 8. Both compounds increased liver glycogen. 9. Calculation of the [free NAD(+)]/[free NADH] ratios indicated that dihydroxyacetone increased the ratio in cytoplasm and mitochondria, whereas glycerol caused a prompt fall in both compartments, followed at 10min. by a slight rise in the mitochondrial compartment. 10. Dihydroxyacetone did not alter the hepatic content of ATP. 11. The findings suggest that the main reason for the antiketogenic effect of glycerol and dihydroxyacetone was a consequence of their ready metabolism and the provision of an increased supply of C(3) intermediates for conversion into oxaloacetate. Under the test conditions, neither the hepatic content of alpha-glycerophosphate nor the redox state of the NAD couples appeared to play a major role in the regulation of ketogenesis.

BACKGROUND

Free thyroxine (FT4) and free triiodothyronine (FT3) measurements are useful in the diagnosis and treatment of a variety of thyroid disorders. The IFCC Scientific Division established a Working Group to resolve issues of method performance to meet clinical requirements.

METHODS

We compared results for measurement of a panel of single donor sera using clinical laboratory procedures based on equilibrium dialysis-isotope dilution-mass spectrometry (ED-ID-MS) (2 for FT4, 1 for FT3) and immunoassays from 9 manufacturers (15 for FT4, 13 for FT3) to a candidate international conventional reference measurement procedure (cRMP) also based on ED-ID-MS.

RESULTS

For FT4 (FT3), the mean bias of 2 (4) assays was within 10% of the cRMP, whereas for 15 (9) assays, negative biases up to -42% (-30%) were seen; 1 FT3 assay was positively biased by +22%. Recalibration to the cRMP eliminated assay-specific biases; however, sample-related effects remained, as judged from difference plots with biologic total error limits. Correlation coefficients to the cRMPs ranged for FT4 (FT3) from 0.92 to 0.78 (0.88 to 0.30). Within-run and total imprecision ranged for FT4 (FT3) from 1.0% to 11.1% (1.8% to 9.4%) and 1.5% to 14.1% (2.4% to 10.0%), respectively. Approximately half of the manufacturers matched the internal QC targets within approximately 5%; however, within-run instability was observed.

CONCLUSIONS

The study showed that most assays had bias largely correctable by establishing calibration traceability to a cRMP and that the majority performed well. Some assays, however, would benefit from improved precision, within-run stability, and between-run consistency.

BACKGROUND

Perfluorooctanesulfonate (PFOS) is widely distributed and persistent in humans and wildlife. Prior toxicological studies have reported decreased total and free thyroid hormones in serum without a major compensatory rise in thyrotropin (TSH) or altered thyroid gland histology. Although these animals (rats, mice and monkeys) might have maintained an euthyroid state, the basis for hypothyroxinemia remained unclear. We undertook this study to investigate the causes for the PFOS-induced reduction of serum total thyroxine (TT4) in rats.

OBJECTIVE

We hypothesized that exposure to PFOS may increase free thyroxine (FT4) in the rat serum due to the ability of PFOS to compete with thyroxine for binding proteins. The increase in FT4 would increase the availability of the thyroid hormone to peripheral tissues for utilization, metabolic conversation, and excretion. We also hypothesized that PFOS does not directly interfere with the regulatory functions of the hypothalamic-pituitary-thyroid (HPT) axis in rats.

METHODS

Three experimental designs were employed to test these hypotheses. (1) Female Sprague-Dawley (SD) rats were given a single oral dose of 15 mg potassium PFOS/kg body weight. At intervals of 2, 6, and 24h thereafter, measurements were made for serum FT4, TT4, triiodothyronine (TT3), reverse triiodothyronine (rT3), thryrotropin (TSH), and PFOS concentrations, as well as liver PFOS concentrations, UDP-glucuronosyltransferase 1A (UGT1A) family mRNA transcripts, and malic enzyme (ME) mRNA transcripts and activity. (2) To provide evidence for increased uptake and metabolism of thyroxine (T4), 125 I-T4 was given to male and female SD rats by intravenous injection, followed in 2h by a single oral dose of 15 mg potassium PFOS/kg body weight. 125 I radioactivity was determined in urine and feces collected over a 24-h period and in serum and liver collected at 24h. (3) To assess the potentials effect of PFOS on the hypothalamic-pituitary-thyroid axis, over an 8-day period, groups of male SD rats were given PFOS (3mg/kg-d), propyl thiouracil (PTU, 10 microg/mL in water), or PTU and PFOS in combination, with controls receiving 0.5% Tween 20 vehicle. On days 1, 3, 7, and 8, TT4, TT3, and TSH were monitored. On day 8, pituitaries were removed and placed in static culture for assessment of thyrotropin releasing hormone (TRH)-mediated release of TSH.

RESULTS

(1) PFOS transiently increased FT4 and decreased TSH within 6h, with values returning to control levels by 24h. TT4 was decreased by 55% over a 24-h period. TT3 and rT3 were decreased at 24h to a lesser extent than TT4. ME mRNA transcripts were increased at 2h and activity was increased at 24h. UGT1A mRNA transcripts were increased at 2 and 6h. (2) 125 I decreased in serum and liver relative to controls and consistent with a reduction in serum TT4. Concomitantly, 125 I activity was increased in urine and feces collected from PFOS-treated rats. (3) During the 8 days of dosing with PFOS, TSH was not elevated in male rats, while TT4 and TT3 were decreased. Pituitary response to TRH-mediated TSH release was not diminished after 8-daily oral doses of PFOS.

CONCLUSIONS

These findings suggest that oral dosing in rats with PFOS results in transiently increased tissue availability of the thyroid hormones and turnover of T4 with a resulting reduction in serum TT4. PFOS does not induce a classical hypothyroid state under dosing conditions employed nor does it alter HPT activities.

The aim of this prospective, follow-up study was to examine the influence of overt hypothyroidism (OHP) and subclinical (SHP), before and during thyroxine (T4) treatment, on lipoprotein(a) [Lp(a)], other lipoproteins, and apolipoproteins. Twenty-four patients (17 females, 7 males) with OHP, aged 54 +/- 11.1 years (group A) and 23 patients (females) with SHP aged 50.1 +/- 13.2 years (group B) were evaluated and compared to 34 and 38 controls, respectively. All patients received T4 therapy in a stepwise fashion until euthyroidism was reached. Thyrotropin (TSH), free thyroxine (FT4), and total triiodothyronine (TT3) levels were measured before T4 therapy and repeatedly every 4 weeks after the initiation of treatment until the euthyroid state was reached. Levels of Lp(a), total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDLc), high-density lipoprotein cholesterol (HDLc), apolipoprotein A1 (apoA1) and apolipoprotein B (apoB) were measured before and 4 months after the achievement of euthyroidism. Additionally, body mass index (BMI) was also evaluated. We found that in OHP patients, levels of TC, LDLc, and apoB were elevated before treatment and decreased significantly after the return to the euthyroid state. BMI and levels of triglycerides also decreased significantly; Lp(a) was higher in OHP patients in comparison with controls and decreased significantly by 14.56% (25.29% in men and 10.34% in women) during T4 treatment. In SHP patients, levels of all common lipoproteins, apolipoproteins, and Lp(a) did not differ significantly from controls before treatment and did not change after the euthyroid stage was reached. It is concluded that in overt hypothyroidism, Lp(a) levels and most of the lipoproteins were elevated before treatment and decreased significantly. In subclinical hypothyroidism, lipoproteins and Lp(a) levels were normal at baseline and did not change during treatment.

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone through induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control.

The Wistar Kyoto (WKY) rat is hyperreactive to stress and exhibits depressive-like behavior in several standard behavioral tests. Because patients with depressive disorders often exhibit disruptions in the circadian rhythm of activity, as well as altered secretory patterns of the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid hormones, we tested the hypothesis that these phenomena occur in the WKY rat. Plasma ACTH and corticosterone levels remained significantly higher after the diurnal peak for several hours in WKY rats relative to Wistar rats. Also, plasma levels of thyroid-stimulating hormone were significantly higher in WKY relative to Wistar rats across the 24-h period, despite normal or slightly higher levels of 3,5,3'-triiodothyronine. In addition, under constant darkness conditions, WKY rats exhibited a shorter free running period and a decreased response to a phase-delaying light pulse compared with Wistar rats. In several ways these results are similar to those seen in other animal models of depression as well as in depressed humans, suggesting that the WKY rat could be used to investigate the genetic basis for these abnormalities.

Cultured rat pituitary cells (GC) respond to thyroid and glucocorticoid hormones by increases in growth hormone production and growth hormone mRNA. When these cells are transferred from medium containing normal animal serum (with 1.8 mug of thyroxine per dl) to a medium containing serum from a thyroidectomized calf, "hypothyroid medium" (with no detectable thyroid hormone), growth hormone production decreases markedly. In cells maintained for 5 days in hypothyroid medium, triiodothyronine induces within 50 hr a 17-fold increase in growth hormone production whereas glucocorticoids, during the same time, produce a negligible (3-fold or less) stimulation. In combination, the two hormones promote a 45-fold stimulation. In all instances the changes in growth hormone production are paralleled by changes in the levels of growth hormone mRNA as measured by cell-free translation. The transfer to hypothyroid medium and the hormonal induction do not affect the relative activities of other mRNAs whose products are detectable on polyacrylamide gels. These studies indicate that thyroid hormone can be an activator of the expression of the growth hormone gene. The results also show that triiodothyronine controls the magnitude of the effect of glucocorticoids on growth hormone mRNA, and provide a model for "permissive" triiodothyronine action. The synergistic effect of these two classes of hormone suggests that they increase levels of growth hormone mRNA by different mechanisms.

The changes in metabolic efficiency, body composition, and nutrient partitioning induced by high-fat feeding were evaluated in adult rats (90 d of age). The alterations in serum free triiodothyronine, insulin, and leptin levels, as well as in hepatic and skeletal muscle metabolism, were also assessed. Rats were fed either a low- or a high-fat diet for 2 weeks. Relative to the low-fat feeding, energy intake and expenditure, as well as body-energy gain, lipid gain, and energetic efficiency, were increased by the high-fat feeding. Increased serum leptin levels accompanied these variations. A positive correlation between serum leptin levels and percentage of body fat was found in the rats fed the low- or high-fat diet, with a significant divergence between the slope of the regression lines. Furthermore, a negative correlation between serum leptin level and energy intake was found in the rats fed the low-fat diet, while a positive correlation was found in the rats fed the high-fat diet. Finally, the high-fat feeding decreased the hepatic and skeletal muscle mitochondrial oxidative capacity. It is concluded that, in adult rats, a nutritional factor such as a high level of fat in the diet induces obesity, leptin resistance, and impairment of mitochondrial capacity, all phenomena typical of unrestrained aged rats.

In view of the increasing number of in-vitro tests of thyroid function, rationalization of the biochemical assessment of patients with suspected thyroid disease was attempted. In addition to clinical examination of 285 consecutive new referrals to a thyroid clinic, measurements were made of serum total and free triiodothyronine (T3) and thyroxine (T4) and of thyrotropin (TSH) by radioimmunoassay before and 20 min after thyrotropin-releasing hormone (TRH) and basal TSH by immunoradiometric assay (IRMA). Analysis of these results demonstrated that: (i) a detectable and normal TSH (IRMA) result indicates that the patient is euthyroid and obviates the need for measurement of thyroid hormones and (ii) a raised or undetectable TSH (IRMA) level should be followed by measurement of free T4 (and rarely also free T3) to distinguish between subclinical and overt hypothyroidism and hyperthyroidism. This policy would considerably reduce the number of in-vitro thyroid function tests without resulting in either a delay in diagnosis or a reduction in its accuracy.

OBJECTIVE

This study was designed to improve our previously developed tandem mass spectrometry (MS/MS) method for free thyroxine (FT4) by enhancing sensitivity and permitting simultaneous measurements of both free triidothyronine (FT3) and FT4 using a smaller plasma/serum sample.

METHODS

An API-5,000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standard, T4-d(5). Four hundred microliters of human plasma/serum was filtered through a Centrifree YM-30 ultrafiltration device by centrifugation, and 450 microL of internal standard in methanol was then added to 150 microL of ultrafiltrate for deproteinization. After centrifugation, 500 microL of supernatant was diluted with 400 microL of distilled de-ionized water and a 650 microL aliquot was injected onto a C-18 column. After washing, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode.

RESULTS

The within-day and between-day coefficients of variation (CVs) were <or=9% for FT3 and <or=7% for FT4 at all concentrations tested. Accuracy ranged between 95% and 105%. The 2.5th-97.5th percentile for FT3 and FT4 was 0.09-0.4 ng/dL (1.4-6.2 pmol/L) and 0.8-2.1 ng/dL (10-26 pmol/L), respectively. The results correlated only moderately well with the immunoassays.

CONCLUSIONS

We describe an improved simple, accurate and fast isotope dilution tandem mass spectrometry method for the simultaneous determination of FT3 and FT4 in human serum/plasma samples.

We determined approximately 15,000 laboratory values in 236 individuals between the ages of 60 and 90 y, 22 individuals between 90 and 99 y, and 69 individuals greater than or equal to 100 y, and compared these with values in young adults. We tested 47 different analytes in the 60-90-y group and 93 analytes in the greater than or equal to 90-y group. Na, K, Cl, and CO2 values were either identical or showed minimal change with age; pH decreased slightly. Differences in Ca values were only minor, but ionized Ca increased slightly. Phosphate decreased in men, but changed only minimally in women; parathyroid hormone increased with age. Increases with age were also observed for glucose, insulin, and C-peptide. Among the enzymes, alkaline phosphatase increased in women, but in men only greater than 90 y; gamma-glutamyltransferase increased in both sexes. Creatine kinase (CK) decreased slightly in individuals greater than 70 y and markedly in those greater than 90 y of age, whereas CK-MB decreased markedly greater than 70 y, reaching the detection limit in individuals greater than 90 y. Lactate dehydrogenase isoenzyme 5 decreased slightly with age. Urea nitrogen increased gradually with age, but creatinine increased only in individuals greater than or equal to 90 y. The increase in urea is not paralleled by a loss of protein in urine, suggesting that the possible cause of azotemia may not always be renal pathology. Urate increased in women but not in men. Liver function, as measured by total bilirubin and liver enzymes, was exceedingly well maintained. Concentrations of most proteins show little change, except for slight decreases in prealbumin, albumin, and transferrin, proteins used as an index of nutritional status. IgA values increased, IgG ranges were wider, IgM and IgD decreased, and the range for IgE was narrower than in young adults. Cholesterol, high-density lipoprotein cholesterol, and triglyceride values increased with age, but decreased in individuals greater than or equal to 90 y. Among the trace elements, magnesium changed little, zinc and lead decreased, and copper values increased with age. Total triiodothyronine and thyroxine decreased, with concomitant increases in thyroid-stimulating hormone. More individuals had increased microsomal antibodies and thyroglobulin titers in the aging population than in the young. In men, the free, percent free, bioactive, and total testosterone values decreased, but luteinizing hormone (LH) and follicle-stimulating hormone (FSH) values increased. In women, estrone and estradiol values decreased, with concomitant increases in LH and FSH. Androstenedione and progesterone decreased in both sexes.(ABSTRACT TRUNCATED AT 400 WORDS)

The human colon, intratumoral subpopulations HCT 116 and HCT 116a were established in chemically defined medium supplemented with transferrin, insulin, epidermal growth factor (EGF), triiodothyronine, hydrocortisone, and sodium selenite. The responsiveness of the adapted cell lines to these growth factors was compared in anchorage-dependent and -independent assays. HCT 116 cells maintained in serum-free conditions were further adapted to growth factor deprivation, and the effects of these polypeptides were determined in anchorage-independent assays. In monolayer, HCT 116 cells adapted to grow in serum-free medium responded to transferrin but not to EGF or insulin. Similarly adapted HCT 116a cells were, however, insensitive to transferrin addition but manifested a 300 and 500% increase in growth rates with EGF and insulin, respectively. Optimal growth of HCT 116 cells was seen in the presence of insulin and transferrin, while maximum proliferation of HCT 116a cells depended on combined insulin, transferrin, and EGF. In soft agarose, both HCT 116 and HCT 116a subpopulations showed a stringent requirement for transferrin. No combination of growth factors without transferrin supported colony formation. These data suggest that (a) these colon tumor subpopulations may be subject to separate growth controls, and (b) there may be an important role for transferrin in anchorage-independent growth and possibly in the maintenance of malignant characteristics.

DIOXIN TOXIC EQUIVALENCY FACTOR EVALUATION OVERVIEW: Polyhalogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have the ability to bind to and activate the ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Structurally related compounds that bind to the AhR and exhibit biological actions similar to TCDD are commonly referred to as "dioxin-like compounds" (DLCs). Ambient human exposure to DLCs occurs through the ingestion of foods containing residues of DLCs that bioconcentrate through the food chain. Due to their lipophilicity and persistence, once internalized they accumulate in adipose tissue resulting in chronic lifetime human exposure. Since human exposure to DLCs always occurs as a complex mixture, the Toxic Equivalency Factor (TEF) methodology has been developed as a mathematical tool to assess the health risk posed by complex mixtures of these compounds. The TEF methodology is a relative potency scheme that ranks the dioxin-like activity of a compound relative to TCDD that is the most potent congener. This allows for the estimation of the potential dioxin-like activity of a mixture of chemicals, based on a common mechanism of action involving an initial binding of DLCs to the AhR. The toxic equivalency of DLCs was nominated for evaluation, because of the widespread human exposure to DLCs and the lack of data on the adequacy of the TEF methodology for predicting relative potency for cancer risk. To address this, the National Toxicology Program conducted a series of 2-year bioassays in female Harlan Sprague-Dawley rats to evaluate the chronic toxicity and carcinogenicity of DLCs and structurally-related polychlorinated biphenyls (PCBs) and mixtures of these compounds. 3,3',4,4',5-Pentachlorobiphenyl (PCB 126) was produced commercially before 1977 for the electric industry as a dielectric insulating fluid for transformers and capacitors. Manufacture and use of the chemical was stopped because of increased PCB residues in the environment, but it continues to be released into the environment through the use and disposal of products containing PCBs, as by-products during the manufacture of certain organic chemicals, and during combustion of some waste materials. Bioaccumulation of PCB 126 results in persistent levels in animal and human tissues and the biological responses to PCB 126 are similar to those of TCDD, a known human carcinogen. PCB 126 was selected for study by the National Toxicology Program as a part of the dioxin TEF evaluation to assess the cancer risk posed by complex mixtures of polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and PCBs. The dioxin TEF evaluation includes conducting multiple 2-year rat bioassays to evaluate the relative chronic toxicity and carcinogenicity of DLCs, structurally related PCBs, and mixtures of these compounds. PCB 126 was included since this is the most potent coplanar PCB that has dioxin-like activities. While one of the aims of the dioxin TEF evaluation was a comparative analysis across studies, in this Technical Report only the results of the PCB 126 study are presented and discussed. Female Harlan Sprague-Dawley rats were administered PCB 126 (99% pure) in corn oil with acetone by gavage for 14, 31, or 53 weeks or 2 years. 2-YEAR STUDY: Groups of 81 female rats were administered 30, 100, 175, 300, 550, or 1,000 ng PCB 126/kg body weight in corn oil:acetone (99:1) by gavage, 5 days per week, for up to 104 weeks; a group of 81 vehicle control female rats received the corn oil/acetone vehicle alone. A group of 28 rats received 10 ng/kg for up to 53 weeks only. Up to 10 rats per group were evaluated at 14, 31, or 53 weeks. A stop-exposure group of 50 female rats was administered 1,000 ng/kg PCB 126 in corn oil:acetone (99:1) by gavage for 30 weeks then the vehicle for the remainder of the study. Mean body weights of 30 and 100 ng/kg rats were similar to those of the vehicle controls during most of the study, mean body weights of 175 and 300 ng/kg rats were less than those of the vehicle controls during year 2 of the study, and mean body weights of 550 ng/kg, 1,000 ng/kg core study, and 1,000 ng/kg stop-exposure rats were less than those of the vehicle controls after week 17. THYROID HORMONE CONCENTRATIONS: Alterations in serum thyroid hormone levels were evaluated at the 14-, 31- and 53- week interim evaluations. In the 550 and 1,000 ng/kg rats, total thyroxine (T4) and free T4 were significantly lower than vehicle controls and serum triiodothyronine (T3) and thyroid stimulating hormone (TSH) levels were significantly higher than vehicle controls at the 14-week interim evaluation. Serum T3 was also significantly higher in the 300 ng/kg rats compared to vehicle controls at 14 weeks. At 31 weeks, T3 was significantly higher at doses of 100 ng/kg or greater compared to vehicle controls. TSH levels were higher in 550 and 1,000 ng/kg rats than in vehicle controls. At 53 weeks, significantly lower serum concentrations of total T4 and free T4 were observed compared to vehicle controls in groups administered 175 ng/kg or greater and 30 ng/kg or greater, respectively. Serum T3 levels were significantly higher at doses of 175 ng/kg or greater compared to vehicle controls. No changes in TSH were observed between vehicle controls and dosed rats at 53 weeks. HEPATIC CELL PROLIFERATION DATA: To evaluate hepatocyte replication, analysis of labeling of replicating hepatocytes with 5-bromo-2'-deoxyuridine was conducted at the 14-, 31-, and 53-week interim evaluations. The hepatocellular labeling index was significantly higher at doses of 300 ng/kg or greater at 14 weeks and 175 ng/kg or greater at 31 weeks compared to vehicle controls. No statistically significant differences were observed between vehicle controls and PCB 126 dosed rats at 53 weeks. However at 53 weeks, a 5.8-fold increase above the vehicle controls was observed in the 1,000 ng/kg group. CYTOCHROME P450 ENZYME ACTIVITIES: To evaluate the expression of known dioxin-responsive genes, CYP1A1 associated 7-ethoxyresorufin-O-deethylase (EROD) activity and CYP1A2-associated acetanilide 4-hydroxylase (A-4-H) activity were evaluated at the 14-, 31-, and 53-week interim evaluations. In addition, CYP2B associated pentoxyresorufin-O-deethylase (PROD) activity was also analysed. Hepatic PROD (CYP2B1) and hepatic and pulmonary EROD (CYP1A1) activity were significantly greater in all dosed groups than in vehicle controls at weeks 14, 31, and 53. Hepatic A-4-H (CYP1A2) activity was significantly greater in the 30, 100, 175, 300, 550, and 1,000 ng/kg groups compared to vehicle controls at weeks 14, 31, and 53. DETERMINATIONS of PCB 126 CONCENTRATIONS IN TISSUES: The tissue disposition of PCB 126 was analyzed in the liver, lung, fat, and blood of all rats in vehicle controls and all dosed groups at the 14-, 31-, and 53-week interim evaluations and in 10 rats per group including vehicle controls at the end of the 2-year study (104 weeks). Detectable concentrations of PCB 126 were observed in the liver, fat, lung, and blood. Measurable concentrations of PCB 126 were present in the liver and fat at weeks 31, 53, and 104. Hepatic and fat concentrations increased with increasing doses of PCB 126. Measurable concentrations of PCB 126 were present in vehicle control lung tissue at 53 and 104 weeks. No PCB 126 was observed in the blood from the vehicle control rats. Lung and blood concentrations tended to increase with increasing doses of PCB 126, with a few exceptions. In the stop-exposure group, PCB 126 concentrations in liver and fat were lower than the levels observed in the 30 ng/kg group. In the stop-exposure group, lung tissue PCB 126 concentrations were equivalent to the levels observed in the 30 ng/kg group. In blood from the stop-exposure group, PCB 126 concentrations were equivalent to the levels observed in the 100 ng/kg group. PATHOLOGY AND STATISTICAL ANALYSES: Absolute and relative liver weights were significantly increased at all time points and correlated with increased incidences of hepatocellular hypertrophy. At 2 years, there were significant treatment-related increases in the incidences of cholangiocarcinoma and hepatocellular adenoma. Three hepatocholangiomas were seen in the 1,000 ng/kg core study group and a single incidence of cholangioma each occurred in the 550 and 1,000 ng/kg core study groups. At 2 years, a significant dose-related increase in hepatic toxicity was observed and was characterized by increased incidences of numerous lesions including hepatocyte hypertrophy, multinucleated hepatocytes, diffuse fatty change, bile duct hyperplasia, bile duct cyst, oval cell hyperplasia, necrosis, pigmentation, inflammation, nodular hyperplasia, portal fibrosis, cholangiofibrosis, and toxic hepatopathy. The incidences of these lesions were generally decreased in the 1,000 ng/kg stop-exposure group compared to the 1,000 ng/kg core study group. The lung weights of 1,000 ng/kg rats were generally significantly increased at weeks 14, 31, and 53. At 2 years, treatment related increases in the incidences of cystic keratinizing epithelioma and squamous cell carcinomas were observed. In addition, dose-related increases in the incidences of bronchiolar metaplasia of the alveolar epithelium and squamous metaplasia were also observed. The incidence of gingival squamous cell carcinoma of the oral mucosa was significantly increased in the 1,000 ng/kg core study group at 2 years. Gingival squamous cell carcinoma, although reduced in incidence as compared to the 1,000 ng/kg core study group, was still present in the 1,000 ng/kg stop-exposure group. At 2 years, adenomas and/or carcinomas were present in the adrenal cortex of most core study groups and in the 1,000 ng/kg stop-exposure group. Dose-related effects on the incidences of adrenal cortex atrophy and cytoplasmic vacuolization were also seen. (ABSTRACT TRUNCATED)

BACKGROUND

The purpose of this study is to validate the generalizability of the efficacy and safety of radiofrequency (RF) ablation for treating autonomously functioning thyroid nodules (AFTN) in a large population multicenter study.

METHODS

This study included 44 patients from 5 institutions who refused or were not suitable for surgery or radioiodine therapy. Twenty-three patients were affected by a toxic nodule and 21 by a pretoxic nodule. RF ablation was performed using an 18-gauge, internally cooled electrode. Nodule volume, thyroid function, scintigraphy, symptom/cosmetic scores, and complications were evaluated before treatment and during each follow-up.

RESULTS

The mean follow-up period was 19.9±12.6 months. The mean nodule volume was initially 18.5±30.1 mL and significantly decreased after treatment at 1 month (11.8±26.9 mL, p<0.001) and the last month (4.5±9.8 mL, p<0.001). Significant improvement of triiodothyronine, free thyroxine, and thyrotropin was observed at the last follow-up. Regarding scintigraphy, 35 hot nodules became cold or were normal when scanned and 9 decreased uptake, although they remained hot nodules. The mean symptom and cosmetic scores were significantly reduced at the last follow-up. No major complications were encountered.

CONCLUSIONS

This multicenter study validated the efficacy and safety of RF ablation for treating AFTN; RF ablation can be considered an alternative to surgery or radioiodine therapy.

OBJECTIVE

To reinvestigate the relationship between circulating TSH levels and adiposity in a cohort of obese people, who have normal thyroid function.

METHODS

Retrospective cross-sectional analysis was carried out on 226 euthyroid obese or overweight female patients. Thirty-nine female lean and euthyroid subjects (BMI<25 kg/m2) were included in the study group. TSH, free thyroxine (FT4), free triiodothyronine (FT3), fasting plasma levels of insulin and glucose, homeostasis model assessment (HOMA) for insulin resistance (HOMA-IR) and insulin secretion (HOMA-b cell), body weight, height, body mass index (BMI) and waist circumference were assessed.

RESULTS

Serum TSH levels were higher in the obese than in the lean subjects. In the study group (lean and obese subjects), there was a significant positive correlation between serum TSH and body weight (r=0.231, p<0.001), BMI (r=0.270, p<0.001), waist circumference (r=0.219, p=0.001), fasting insulin (r=0.201, p=0.002) and HOMA-IR (r=0.201, p=0.002); there was no correlation between serum FT4 and any of the parameters. A multivariate linear regression analysis revealed that only BMI (p=0.012, 95% CI=0.01-0.08) contributed significantly to the variance of TSH.

CONCLUSIONS

This study strongly supports existing, but contradictory evidence that serum TSH levels are positively correlated with the degree of obesity and some of its metabolic consequences in overweight people with normal thyroid function.

This study was designed to assess the changes in nonlinear properties of heart rate (HR) variability (HRV) during the menstrual cycle by means of complexity measures, including sample entropy (SampEn) and correlation dimension (CD), and explore probable physiological interpretations for them. In 16 healthy women (mean age: 23.8 +/- 2.7 yr), complexity measures along with the spectral components of HRV (sympathovagal markers) were analyzed over 1,500 R-R intervals recorded during both the follicular phase (day 11.9 +/- 1.4) and the luteal phase (day 22.0 +/- 1.4) of each woman's menstrual cycle. Simultaneously, serum ovarian hormone (estradiol-17 and progesterone) and thyroid-related hormone [free triiodothyronine, free thyroxine (T(4)), and thyroid-stimulating hormone] concentrations were measured. With regard to HRV measures, SampEn, CD, and high-frequency (HF) components decreased from the follicular phase to the luteal phase, whereas normalized low-frequency (LF) components and the LF-to-HF ratio as well as resting HR increased. In regard to hormone levels, whereas progesterone was increased, the other hormone concentrations were unchanged. Furthermore, across the menstrual cycle, both SampEn and CD were well correlated with the spectral indexes and free T(4) concentrations, and SampEn also showed significant correlations with the ratio of estradiol-17 to progesterone concentrations. These results suggest that the nonlinear properties in HRV are altered during the regular menstrual cycle and that the autonomic nervous system, ovarian hormone balance, and free T(4) may be involved in nonlinear HR control in healthy women. All of these factors may enrich the physiological meanings of complexity measures.

BACKGROUND

Female sexual dysfunction (FSD) is characterized by reduced sexual appetite and altered psychologic and physiologic response to sexual intercourse; it is reported to be frequent in diabetes mellitus, but no data have been reported in thyroid disorders.

OBJECTIVE

To compare the prevalence of FSD in diabetic, in obese, and in hypothyroid women vs. healthy women, and to correlate FSD with endocrine and metabolic profiles.

METHODS

We evaluated, through a questionnaire (Female Sexual Function Index [FSFI]), the prevalence of FSD in 91 women affected by diabetes mellitus, obesity, or hypothyroidism, and in 36 healthy women, all aged 22-51 years and in premenopausal state.

METHODS

FSFI score, endocrine and metabolic parameters (triglycerides, high-density lipoprotein [HDL] and low-density lipoprotein [LDL] cholesterol, free-triiodothyronine (FT3), free-thyroxine (FT4), thyroid stimulating hormone [TSH], 17-beta-estradiol, testosterone, glycated hemoglobin 1c (HbA1c), thyroid autoantibodies, E-selectin, P-selectin, intercellular adhesion molecule-1 [ICAM-1], plasminogen-activator inhibitor-1 [PAI-1]), and anthropometric parameters (body mass index, waist, blood pressure [BP]).

RESULTS

A reduced FSFI score was more frequent in diabetic, obese, and hypothyroid women vs. healthy women (P < 0.01). In the different groups of women, FSFI score was inversely correlated (pairwise correlation) with at least one of the following: HbA1c, TSH, LDL-cholesterol, PAI-1, diastolic BP, presence of thyroid Ab, and directly correlated with HDL-cholesterol (always P < 0.05 or less). At stepwise regression analysis, HDL-cholesterol (protective) and HbA1c, LDL-cholesterol, PAI-1, and diastolic BP (negatively) predicted reduced FSFI score.

CONCLUSIONS

These data indicate an increased prevalence of sexual dysfunction in diabetic, in obese, and in hypothyroid women, associated with markers of cardiovascular risk.

The relation between functional properties of the contractile apparatus, such as shortening velocity and ATPase activity, and myosin isoenzyme composition was studied in ventricular myocardium of adult (60-90-day-old) rats and of newborn (3-day-old) and young (10- and 20-day-old) rats. In adult animals, variations of isomyosin pattern were produced by reducing food intake and by changing the thyroid state. Hyperthyroidism was induced with triiodothyronine daily injection for 15 days; hypothyroidism was induced with iodine-free diet and KClO4 in drinking water for 50-60 days. The following parameters were studied: 1) calcium-magnesium-activated and magnesium-activated ATPase activity of washed and purified myofibrils, 2) calcium-activated ATPase activity of purified myosin, 3) isomyosin composition and relative content of alpha-myosin heavy chains (alpha-MHCs), and 4) force-velocity curve of left and right ventricle papillary muscles. To take into account the difference in excitation-contraction coupling between newborn and adult myocardium, the determination of the force-velocity curve was repeated in Krebs' solution with normal [CaCl2] (2.5 mM) and in Krebs' solution with high [CaCl2] (10 mM). During postnatal growth, the relative content of alpha-MHC increased and reached a maximum at about 20 days. Pronounced increases of myofibrillar and myosin ATPase activity and in shortening velocity occurred during the same period. In adult hyperthyroid rats, alpha-MHC content as well as enzymatic activity and shortening velocity were higher than in control adult animals. Hypothyroidism and food deprivation caused a decrease of alpha-MHC content and a reduction of both enzymatic activities and shortening velocity. The study of the relations between alpha-MHC relative content and functional parameters showed that 1) in ventricular myocardium of adult rats a linear relation existed between alpha-MHC content and myosin and myofibrillar ATPase activity and shortening velocity, and 2) in newborn and young rat ventricular myocardium, both enzymatic activities and shortening velocity were lower than would have been expected on the basis of the linear relation described above. This latter observation could be accounted for by a variation in specific activity of myosin during postnatal development or by the presence of peculiar isomyosins that cannot be detected with usual electrophoretic techniques.