Thyroid disease with monoclonal (immunoglobulin G lambda) antibody to triiodothyronine and thyroxine.

A man with previous Graves' disease spontaneously developed hypothyroidism. He became euthyroid with T4 therapy, but developed inappropriately elevated serum levels of T3 and, to a lesser extent, T4. Gel filtration analysis (Sephadex G-150) of serum trace-labeled with [125I]T3 revealed binding to a high molecular weight fraction, distinct from normal T3-binding proteins. This abnormal activity cochromatographed with serum immunoglobulin G (IgG) by DEAE-cellulose chromatography and gel filtration, and was retained by the F(ab)2 fragment of IgG, indicating its true antibody nature. By isoelectric focussing, there was restricted heterogeneity of the [125I]T3-antibody complex (pI 9.0-9.1), and the antibody was identified as an IgG (lambda) monoclonal Ig by immune precipitation. Antigenic cross-reactivity with T4 was demonstrated by inhibition of hapten binding. The affinity of the antibody for T3 was high (Ka = 0.9 x 10(9) liter mol-1), and the T3 binding capacity of the antibody in serum was estimated as 1132 ng/dl, equivalent to 1.39 mg T3-specific IgG/liter (0.014% of the total serum IgG). This binding capacity was similar to the serum T3 values (1100-1300 ng/dl) at which transition from hypothyroid to euthyroid states was observed, as judged by clinical examination and measurement of serum TSH levels.

A rapid standardized enzyme immunoassay for autoantibodies to thyroglobulin.

A rapid, specific, standardized, objective and sensitive quantitative enzyme immunoassay (ELISA) procedure has been developed for the detection of human autoantibodies to thyroglobulin. It involves three ten minute incubations, with the positive control serum adjusted to yield an absorbance of approximately 1.00. The results are reported as 'percentage of positive control' absorbance. The specificity of the reactions for thyroglobulin autoantibodies was supported by the good correlation found between the ELISA values obtained and the passive hemagglutination (PHA) titers of 348 sera from 333 patients suspected of having thyroid disturbances, as well as by antigen absorption experiments. Rheumatoid factor did not interfere with the assay. An appreciable proportion of passive hemagglutination-negative sera were confirmably positive for thyroglobulin autoantibodies by the ELISA method.

The pituitary-thyroid axis after hemithyroidectomy in euthyroid man.

The pathogenesis of euthyroid goiter is assumed to be TSH dependent, but most studies have not demonstrated elevation of serum TSH. To elucidate the early events involved in thyroid growth, we studied thyroid function in eight euthyroid patients subjected to hemithyroidectomy for solitary cold nodules preoperatively and 2, 15, 60, and 90 days and 36 months, postoperatively. Although within the euthyroid range, serum T4 decreased at 30 days and thereafter up to 90 days (P less than 0.01). Basal serum TSH rose significantly by the 30th day and remained elevated for the entire 90-day period (P less than 0.001). The serum TSH response to TRH was exaggerated from days 15-90 (P less than 0.001). Thirty-six months postoperatively, all parameters of thyroid function were similar to control values, with the exception of one patient who showed an exaggerated response of serum TSH to TRH. We conclude that 1) the dynamic phase of recuperation post hemithyroidectomy is TSH dependent, 2) the serum TSH increase occurs within normal limits and is a temporary event, and 3) 3 yr postoperatively, a new steady state is achieved with normal parameters of thyroid function. Thus, we believe that TSH plays a dominant role in the genesis of goiter.

Control of puberty in female rats: the effect of PTU-induced hypothyroidism and systematic undernutrition.

Sprague-Dawley rats were fed Purina Lab Chow with or without propylthiouracil (PTU), 0.001%, 0.01% or 0.1% PTU, ad libitum from weaning to vaginal opening. Mean values for all pubertal measurements are included in Tables 1 and 2. Growth rate (mean +/- S.E.) was significantly reduced (Neuman-Keuls test; P less than 0.05 level) in all PTU-fed rats (controls 4.9 +/- 0.1 g/day, 0.001% PTU 4.2 +/- 0.2 g/day, 0.01% PTU 3.4 +/- 0.2 g/day, 0.1% PTU 2.5 +/- 0.1 g/day), while age at vaginal opening in rats fed 0.001% PTU (35.8 +/- 0.6 days) or 0.01% PTU (36.1 +/- 0.9 days) was not significantly different from controls (36.0 +/- 0.6 days). Nevertheless, body weight at vaginal opening was lower in rats fed 0.1% PTU (87.6 +/- 4.7 g) than in controls (113.6 +/- 3.7 g). Pubertal body weight of rats fed 0.1% PTU was also reduced (88.6 +/- 3.7 g) but vaginal opening delayed (40.4 +/- 0.8 days). Proportions of body fat (6.1 - 5.1%), protein (15.0 - 14.1%), and water (72.4 - 71.3%) at vaginal opening were the same in control and PTU groups. Serum T4 was greatly diminished and similar in all 3 PTU groups, 0.2 - 0.3 microgram/100 ml, vs 4.8 +/- 0.2 microgram/100 ml in controls; in rats fed 0.1% and 0.01% PTU, T3 was 0.9 +/- 0.4 ng/100 ml and 0.9 +/- 0.6 ng/100 ml, respectively vs 72.6 +/- 5.6 ng/100 ml in controls, but not significantly reduced in the 0.001% PTU-fed group (60.7 +/- 7.9 ng/100 ml). In a second experiment, a group of weanling rats (pair-fed) was selected in which each member was fed the daily amount of control diet eaten by a corresponding age- and weight-matched 0.01% PTU-fed rat. During the experiment, both groups maintained the same body weight, growth rate, and food intake, however, only 45% (n = 11) of the pair-fed animals had vaginal opening by the time their 0.01% PTU-fed counterparts attained first estrus. Although one of the pair-fed (undernourished) rats attained first estrus, no eggs were found. Despite greatly reduced body weight (105.3 +/- 3.5 g vs controls 127.5 +/- 6.6 g), growth rate (3.5 +/- 0.2 g/day vs controls 5.5 +/- 0.1 g/day) and food intake (13.9 +/- 0.7 g/100g BWt/day vs controls 10.1 +/- 0.3 g/100g BWt/day), the 0.01% PTU-fed rats exhibited vaginal opening (36.9 +/- 0.8 days vs controls 35.6 +/- 1 days) and first estrus (39.6 +/- 0.9 days vs controls 36.4 +/- 1 days) at the usual age. In contrast, pair-fed rats had a lower % fat (4.5 +/- 0.1% vs PTU 6.8 +/- 0.4%) and higher % protein (16.5 +/- 0.3% vs PTU 14.3 +/- 0.3%) at the age when 0.01% PTU-fed rats attained first estrus. Serum prolactin levels at first estrus did not differ in rats fed control diet (26.5 +/- 12.4 ng/ml) or 0.01% PTU (8.8 +/- 1.9 ng/ml), or in pair-fed animals (8.8 +/- 4.5 ng/ml) at the age when PTU-fed rats reached first estrus.

Hemoglobin A2 in hyperthyroidism.

Erythrocyte hemoglobin A2 (Hb A2) was quantitated in 28 hyperthyroid patients prior to antithyroid therapy and at intervals during therapy. Fetal hemoglobin (Hb F) levels and red cell mean corpuscular volume (MCV) were also monitored. Before therapy, Hb A2 was significantly elevated (mean +/- SD, 3.3 +/- 0.5%: normals, 2.5 +/- 0.3%: p less than 0.001). Hb F levels exceeded 1.0% in 10 cases. A tendency to red cell microcytosis was also observed (mean MCV 81.1 fl). To-date 10 of these subjects have had adequate studies following appropriate and sustained responses to antithyroid therapy. All showed a fall in Hb A2, usually accompanied by a rise in red cell MCV. A hematologic profile for hyperthyroidism is proposed.

Similar serum concentrations of thyroid hormones in two geographically separate populations on disparate iodine intake.

Serum thyroid hormones were measured in Montreal, Canada (urinary iodine 446 +/- 164 micrograms/day) and Zagreb, Yugoslavia (urinary iodine 108 +/- 32 microgram/day). The serum concentrations of thyroxine and triiodothyronine in the two populations were almost identical. We conclude that dietary iodine, within accepted normal limits, is not a factor in determining serum thyroid hormone levels. The wide differences in reported serum triiodothyronine concentrations are related to methodological problems.

Metabolism of monoamines and diamines in hyperthyroid and hypothyroid rats.

The in vivo rates of catabolism of 14C-labelled pentylamine, ethylamine, putrescine, and cadaverine were studied in thyroidectomized rats and others made hyperthyroid by the daily administration of 0.2 mg of L-thyroxine per kilogram for 20--21 days. Hyperthyroid rats metabolized the monoamines at an accelerated rate; thyroidectomized animals oxidized pentylamine at a reduced rate. There was no effect of hypophysectomy on the rate of pentylamine oxidation. The in vitro monoamine oxidase (MAO) activity of liver was reduced in hyperthyroid rats and unchanged in those thyroidectomized; MAO activity in skeletal muscle was increased in the hyperthyroid rats and decreased in the hypothyroid rats. Because of the large mass of skeletal muscle compared with liver, it is considered that the changes in muscle MAO could play an important role in determining the rate of oxidation of pentylamine in vivo. The oxidation of the two diamines tested was not significantly affected by thyroidectomy; the rates were increased in the hyperthyroid rats, but the increase was significant only for cadaverine.

Enhanced thyroxine metabolism and high uptake goiters in rats after a single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Nine days after treatment of rats with a single dose (25 microgram/kg) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the 1 h biliary excretion of [125I]-thyroxine (T4) was increased 4-fold and the [125I]T4 bile:plasma ratio and the biliary clearance rate of plasma [125I]T4 were increased 10-fold. The proportion of biliary 125I present as T4-glucuronide was larger in TCDD-treated rats. Unexpectedly, TCDD did not influence the 30 min biliary excretion of [125I]triiodothyronine (T3), the [125I]T3 bile:plasma ratio, and the biliary clearance rate of plasma [125I]T3. TCDD also elevated serum thyrotropin concentrations, produced goiters and increased thyroid 131I uptake. Serum T4 concentrations were reduced to half of normal in TCDD-treated animals, but their serum T3 levels were elevated. Sephadex uptake of serum [125I]T3 was reduced in the TCDD group.

Elevated serum concentrations of thyroxine-binding globulin and caeruloplasmin in methadone-maintained patients.

Patients on a methadone-maintenance program had high serum concentrations of thyroxine-binding globulin. This resulted in elevated serum thyroxine and triiodothyronine levels and low triiodothyronine uptake values. Their free thyroxine index was normal, as was serum thyroxine and triiodothyronine when related to thyroxine-binding globulin concentration. Serum caeruloplasmin concentration was also increased in methadone-treated patients.

Goitres in rats fed polychlorinated biphenyls.

Rats fed a polychlorinated biphenyl (PCB) mixture in a high- or low-iodine diet (HID or LID respectively) for 15 days had thyroid enlargement, low serum thyroxine (T4), and high serum thyrotropin concentrations. Although binding of thyroid hormones to serum proteins was reduced in PCB-fed animals, the free T4 index (reflecting free T4 in serum) was less in these rats. Both serum triiodothyronine (T3) and the free T3 index were elevated in rats fed PCB in HID. LID-maintained rats elevated serum T3 concentrations but the free T3 index was similar to that in HID-fed rats, owing to enhanced binding of thyroid hormone to serum proteins. Addition of PCB to LID reduced serum T3 levels but did not alter the free T3 index because binding was less. In rats fed HID containing PCB, thyroid 131I uptake was increased.

Minimal effects of free fatty acids on the competitive protein-binding assay of serum thyroxine on reusable Sephadex columns.

In rats; following oral administration of cream and intravenous heparin, plasma free fatty acids and triiodothyronine Sephadex uptake were elevated. Simultaneously apparent serum thyroxine, measured by the Tetrasorb-E kit, was increased, but not when measured using reusable Sephadex columns. Addition of oleate to a human serum albumin solution or to human serum produced similar effects; appreciable rises in apparent thyroxine were noted in the kit assay but only minor changes occurred when Sephadex columns were used. In the latter procedure almost all [14C]oleic acid added to serum was removed in the first barbital buffer wash, before application of the thyroxine-binding globulin solution. In the serum thyroxine assay on reusable Sephadex columns, there is minimal interference by free fatty acids because they are removed by the first barbital buffer wash.

A simple ligand-binding assay for thyroxine-binding globulin on reusable Sephadex columns.

A method for the assay of thyroxine-binding globulin on reusable Sephadex G-25 (fine) columns is described. It depends upon elution by diluted iodothyronine-free serum of protein-bound [125 I]thyroxine from the columns under conditions where binding to thyroxine-binding prealbumin and albumin are abolished. It is simple, rapid and precise and permits determinations in large numbers of samples. Values (mg/l; mean +/- S.D.) were: normals 31.6 +/- 5.4, hyperthyroid 28.3 +/- 4.8, hypothyroid 40.6 +/- 7.5, oral contraceptives 40.1 +/- 6.8, pregnant 50.3 +/- 5.4, cirrhotics 20.7 +/- 4.3. Concentrations were reduced in serum heated at 56 degrees C, while the uptake of [125 I]triiodothyronine was increased. There was a significant negative correlation between thyroxine-binding globulin concentration and triiodothyronine uptake in the heated serum samples and in euthyroid subjects.

Alterations in thyroxine metabolism produced by cutaneous application of microscope immersion oil: effects due to polychlorinated biphenyls.

A 30% solution of a polychlorinated biphenyl (PCB) mixture or a microscope immersion oil containing 34% PCB, when applied to the skin of rats, led to substantial increases in the biliary excretion of intravenously injected [125I]thyroxine (T4) in bile: plasma 125I ratios, in the biliary clearance rate of plasma [125I]T4, and in bile flow. Both PCB preparations also elevated liver weight, thyroid 125I uptake, and Sephadex uptake of [125I]triiodothyronine (T3), and depressed serum T4 concentrations; serum T3 levels were unaltered by the PCB solution or by the immersion oil containing PCB. PCB, either in mineral or immersion oil, reduced the free T4 index (serum T4 X fraction Sephadex T3 uptake), indicating a probable reduction in the concentration of free T4 in serum; the free T3 index, on the other hand, was elevated in PCB-treated rats. The same type of immersion oil, in which the PCB was replaced by a hydrogenated terphenyl, was without effect on any of the indices studied. Thus, the effects of microscope immersion oil on T4 metabolism were due to its PCB content. In thyroidectomized, T4-maintained rats, PCB in mineral oil again increased Sephadex uptake of [125I]T3, greatly reduced serum T4, and moderately reduced serum T3 levels; the free T4 index was substantially reduced and the free T3 index moderately lowered in treated animals. These data indicate that in PCB-treated rats both the peripheral conversion of T4 to T3 and thyroid T3 secretion were enhanced. The metabolic impact of thyroid hormone in PCB-treated animals was unchanged, as shown by normal activity of hepatic mitochondrial L-alpha-glycerophosphate dehydrogenase.

Assay of serum thyroxine by competitive protein binding: reuse of disposable sephadex columns.

A method for the assay of total serum thyroxine on reusable Sephadex G-25 fine columns, obtained from the Ames Tetralute kit, and competitive protein binding with TBG is described. The columns are regenerated with diluted plasma and may be used for at least 3 months. The procedure is rapid and simple and requires no extraction, centrifugation or evaporation. Recovery of added thyroxine from serum is essentially complete. The between-assay and within-assay coefficients of variation are about 5.5%. Correlation with values obtained using the Tetralute kit was good (r = 0.988). Values in 75 euthyroid subjects were normally distributed and therefore the normal range was taken as the mean +/- 2 SD or 4.6-10.4 mug/100 ml.

Polychlorinated biphenyl-induced modification of lymphocyte response to plant mitogens in rats.

In PCB-treated rats total white cell and differential counts and lymphocyte proliferative response to PWM were unaltered. The lymphocyte response to PHA was increased in treated animals.

Enchanced in vitro hepatic glucuronidation of thyroxine in rats following cutaneous application or ingestion of polychlorinated biphenyls.

In rats four daily skin application of a 30% solution of a polychlorinated biphenyl (PCB) mixture in mineral oil or of a microscope immersion oil, containing 34% PCB, led to increases in liver weight, protein concentration of the 10 000 X g supernatant fluid of liver homogenates and the in vitro glucuronidation of thyroxine (T4) by the supernatant fluid, whether related to liver weight or to protein concentration in the reaction mixture. Similar effects occurred after feeding 250 ppm (mg/kg) of PCB in either Purina chow or a low-iodine diet for 11 days. It is concluded that increased hepatic T4 glucuronidation contributes to the enhanced biliary excretion of T4 previously observed in PCB-treated rats.

Ehanced biliary thyroxine excretion in rats treated with pregnenolone-16alpha-carbonitrile.

Normal rats were treated with pregnenolone- 16alpha - carbonitrile (PCN) 10 mg/100 g by stomach tube twice daily for 3 days. In these animals the biliary excretion of intravenously injected 125I-thyroxine (T4) was enhanced and the bile: plasma 125I ratio (B/P ratio) and the biliary clearance rate of plasma 125I-T4 was increased. Normal rats were treated with PCN for 3 days and homozygous Gunn rats for 13 days. In both groups PCN enhanced the bile flow and elevated the B/P ratios and the biliary clearance rate of plasma T4 following ip injection of 125I-T4 17 h previously. PCN-treatment had no effect on the fractions of biliary 125I present as T4-glucuronide, T4 and I- in either the normal or Gunn rats. Treatment with PCN for 10 days produced goitres in normal and Gunn rats and in normal rats elevated the serum TSH (bioassay) levels and the 17 h thyroid 131I uptake as well as the serum PB125I concentrations, without affecting stable PBI concentrations. These data indicated increased pituitary TSH release in response to increased peripheral metabolism of thyroid hormone; enhanced hormonal release from the thyroid kept pace with the accelerated peripheral loss.

On the mechanism of polychlorinated biphenyl-induced hypobilirubinaemia.

Treatment of normal rats with a polychlorinated biphenyl mixture of DDT increased liver microsomal protein concentration. Hepatic microsomal bilirubin UDP-glucuronyltransferase activity was elevated in the DDT-treated, but not in the polychlorinated biphenyl-treated rats. In contrast polychlorinated biphenyl, but not DDT, reduced serum bilirubin concentrations in jaundiced Gunn rats, as did sulphadiazine; sulphonamides compete with bilirubin for binding sites on plasma albumin. Polychlorinated biphenyls appear to lower serum bilirubin concentration by reducing the binding of bilirubin to plasma protein.