Developing multianalyte assays.

Multianalyte 'binding' assays represent a major advance in microanalytical technology for the measurement of substances of biological importance. Their further development should facilitate sensitive and reproducible quantification of analytes in many areas that are currently problematic, including diagnostic medicine and the standardization of biologicals.

Effects of immunoglobulins upon the interaction of thyrotropin with receptor in human fat tissue.

TSH bound to human fat membranes is shown to be unusually sensitive to the addition of immunoglobulin G (IgG). Significant displacement was observed with 10 microgram/ml, and 50% displacement was obtained with 50-100 microgram/ml IgG prepared from control sera pooled from euthyroid subjects. It was not possible to demonstrate increased TSH displacement by IgG preparations from sera of thyrotoxic compared to that of euthyroid subjects using human fat membranes, as was demonstrable with human thyroid membranes. The increased sensitivity of fat membranes to IgG was not due to nonspecific protein interactions and is suggested to derive from interactions with Fc receptors associated with fat membranes.

Differences in nuclear triiodothyronine binding in rat brain cells suggest phylogenetic specialization of neuronal functions.

The central nervous system depends on thyroid hormones (TH) in regard to its development, maturation, and maintenance of normal functions. As there is much evidence to suggest that the effects of TH are mainly mediated through specific nuclear binding sites, we have studied the anatomical distribution of T3 nuclear receptors in different regions of adult rat brain, and the localization of receptors in the fractionated neuronal and glial nuclei of neocortex, paleocortex, and cerebellum. Purified nuclei from the various brain regions were prepared by ultracentrifugation in 2.2 M sucrose. Purified neuronal and glial fractions were obtained by discontinuous sucrose gradient centrifugation in 2.2 and 2.4 M sucrose. The washed nuclear fractions were used for T3 binding assay at 37 C for 30 min and the data analyzed by least squares nonlinear regression analysis. Nonfractionated nuclei from all regions studied were found to have similar dissociation constant (Kd) values (1.04-1.38 nM) and Eadie-Hofstee plots indicated the presence of an apparently ubiquitous single class of high affinity, low capacity binding sites. The increase in binding from cerebellum (54 +/- 24 fmol/mg DNA; mean +/- SE) to neocortex (666 +/- 89 fmol/mg DNA) showed a caudo-cranial pattern. In fractionated neuronal nuclei, the same trend was observed, only to a greater degree (1628 +/- 266, 994 +/- 76 and 212 +/- 29 fmol/mg DNA in neocortex, paleocortex, and cerebellum, respectively); the difference between corresponding values for glial nuclei of neocortex and paleocortex (357 +/- 139 and 250 +/- 92 fmol/mg DNA, respectively) was not statistically significant, and no specific T3 binding was found in cerebellar glial nuclei. These data suggest that TH may have an important role in neurons from phylogenetically newer regions, concerned with higher mental functions. The caudo-rostral distribution pattern may also indicate a gradient of TH actions in central nervous system regions.

Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators.

Selected clones of monoclonal antibodies from mice immunized with solubilized preparations of bovine TSH receptors have been characterized in a cytochemical bioassay (CBA) for thyroid stimulators. This assay is based upon quantification of changes in naphthylamidase activity of sections of guinea pig thyroids with use of a chromogenic substrate. Monoclonal 22A6 is a thyroid-stimulating antibody directed at a site within the TSH receptor. Thus, although it is a weak inhibitor of 125I-TSH binding to thyroid membranes, 22A6 is inhibited from binding to membranes by TSH, exhibits a more than additive agonist effect on adenylate cyclase activity when tested at low TSH concentrations in thyroid cells, and is a competitive antagonist of TSH enhancement of adenylate cyclase activity at high TSH concentrations. In the CBA, 22A6 is a stimulator whose maximal activity is obtained with 77 pg/ml (3-min exposure). Dose-response curves of a long acting thyroid stimulator (LATS)-B standard and 22A6 have slopes which are not significantly different; as anticipated, the response to LATS-B is inhibited by antihuman immunoglobulin G (IgG) and that due to 22A6 by antimouse IgG. In contrast to 22A6, monoclonal 11E8 is a relatively potent inhibitor of 125I-TSH binding as well as TSH stimulation of adenylate cyclase activity, while failing to act as a stimulator itself. 11E8 is itself inactive as a stimulator in the CBA over a wide dose range; it does, however, inhibit TSH stimulation in the CBA. This inhibition is abolished by antimouse IgG. The transient peak of response observed in time courses to TSH occurs later in the presence of 11E8. Unlike its effect on TSH 11E8 shows relatively low potency (greater than 10,000-fold lower) when inhibiting stimulation by the thyroid stimulating antibodies, 22A6 or LATS-B. Since this difference cannot be explained by quantitative differences in the ability of 22A6 or 11E8 to bind to thyroid membranes, the CBA data suggest that the stimulating antibodies, 22A6 and LATS-B, may interact with different determinants on TSH receptors then either TSH or the blocking antibody, 11E8. This also implies that in Graves' disease blocking antibodies may be incompletely expressed in the presence of stimulating antibodies, although they may be potent inhibitors of TSH binding, as measured in receptor assays.

The effect of 3,5,3'-triiodothyronine on leucine uptake and incorporation into protein in cultured neurons and subcellular fractions of rat central nervous system.

Previous studies have shown that nuclear thyroid hormone receptors in rat brain are preferentially localized within neurons. These cells also synthesize protein at a high rate, and the aim of the present study was to investigate any relationship between these two characteristics. In this paper we have shown that T3 stimulates leucine uptake and incorporation into protein in primary cell cultures of neurons. Stimulation was apparent with concentrations of hormone as low as 1.25 nM and increased in a dose-dependent manner up to 10 nM T3. However, the rapidity of the effect (evident at 25 min, and significant at 40 min) suggests that protein synthesis is stimulated at the level of translation, rather than transcription. More detailed study with 5 nM T3, revealed that incorporation into both soluble (cytoplasmic) and insoluble (membrane-associated) protein fractions was stimulated to similar degrees, and therefore the effect on protein synthesis was general. Furthermore, T3-mediated stimulation of leucine uptake into neurons was completely abolished in the presence of the protein synthesis inhibitors, actinomycin D and cycloheximide, and therefore the effect on leucine uptake was attributed to an increased requirement for the amino acid in protein synthesis (pleiotrophic effect). Parallel studies conducted with synaptosomes and mitochondria isolated from the central nervous system of adult euthyroid animals revealed that 5 nM T3 was without effect on leucine uptake and incorporation into protein. Possible reasons for this lack of effect are discussed.

Triiodothyronine binding in adult rat brain: compartmentation of receptor populations in purified neuronal and glial nuclei.

In order to investigate the localization of nuclear T3 receptors in adult rat brain, we have developed a technique for separating neuronal and glial nuclei, and compared nuclear T3 binding in both fractions. Glial and neuronal nuclear populations were prepared by homogenization of the whole brain except the cerebellum, followed by discontinuous density gradient centrifugation in 2.4 and 2.2 M sucrose. For purposes of comparison, liver nuclei were prepared by centrifugation in 2.4 M sucrose. Microscopic examination of the nuclei confirmed the purity of the two nuclear fractions to be above 90% with minimal extranuclear contamination. T3 binding studies were performed on the washed nuclear fractions using saturation analysis techniques. Maximum binding (Bmax) and dissociation constant (Kd) were obtained by nonlinear least-squares regression analysis. After 30 min incubation at 37 C, Bmax in neuronal nuclei was 1203 +/- 118 fmol/mg DNA (mean +/- SE) (n = 6), in comparison with 196 +/- 14 fmol/mg DNA in glial nuclei (n = 7). The corrected value for Bmax in glial nuclei, assuming 8% neuronal nuclear contamination, was 100 fmol/mg DNA. Bmax in liver nuclei was contamination, was 100 fmol/mg DNA. Bmax in liver nuclei was 299 +/- 38 fmol/mg DNA (n = 6). The Kd values were 0.38 +/- 0.04 nM, 0.34 +/- 0.05 nM, and 0.33 +/- 0.04 nM in neuronal, glial, and liver nuclei, respectively. The specificity of T3 binding to the nuclear receptor in both neuronal and glial nuclei was studied in competition studies, and revealed closely similar relative binding affinities (T3 greater than T4 greater than rT3) in both. We conclude that the higher T3 binding capacity of neuronal nuclei may be related to their higher protein synthetic ability, suggesting that T3 may be of importance in a rapid structural turnover in these cells. The effect of T3 on myelination in glial cells may be mediated through the nucleus.

Pregnancy-induced changes in thyroid function: role of human chorionic gonadotropin as putative regulator of maternal thyroid.

Marked changes in maternal thyroid activity occur in pregnancy. It has been suggested that hCG may stimulate maternal T4 secretion, given its in vitro thyrotropic activity ascribed to a significant degree of structural homology with TSH. In a longitudinal study of 32 normal pregnant women, we attempted to clarify the functional activity of the thyroid in early and late pregnancy and the possibility of a nonpituitary control on the thyroid. Total T4 and T4-binding globulin levels were increased from the first trimester onward. Free T4 levels did not differ in the first trimester from postpartum values, but were significantly decreased in second and third trimesters (P less than 0.001). A decrease in TSH levels was observed in the first trimester (0.72 +/- 0.09 vs. 1.23 +/- 0.12 mU/L; P less than 0.001), while second and third trimester values did not differ from those postpartum. A significant negative correlation (P less than 0.05) was observed between hCG and TSH levels in the earliest weeks (8-10) of the first trimester. No correlation was found between hCG and total T4 or free T4 levels. A stimulation of I- uptake in FRTL-5 cells was induced by first trimester serum, which also showed a different behavior at chromatofocusing, with a higher proportion of hCG eluting at acidic pIs compared to second trimester samples. However, neither hCG levels nor the amount of acidic hCG correlated with the thyroid-stimulating activity measured in vitro. Some correlation was found with the percentage of basic hCG (eluting at pI greater than 4.6), although these isoforms were equally present in first and second trimesters. The differing patterns of circulating hCG at various stages of gestation suggest that distinct hCG isoforms may regulate maternal thyroid activity.

Time-related thyroid stimulation by thyrotropin and thyroid-stimulating antibodies, as measured by the cytochemical section bioassay.

The time course of response of thyroid sections in the cytochemical bioassay to either TSH or thyroid-stimulating antibodies is bell shaped. The maximal staining for lysosomal naphthylamidase activity achieved was found to be the same regardless of the dose of stimulator applied; however, the rate at which the maximum was attained was dose dependent. Sections exposed to 10(-1) mU/liter TSH showed a maximal response at 120 sec, and those exposed to 10(-3) mU/liter TSH showed a maximal response at 210 sec. A similar dose-time effect was seen with immunoglobulin G from a thyrotoxic patient. Thus, by selecting a specific exposure time, a dose-response curve to the stimulator was obtained. A dose-response curve to a range of concentrations from 10(-4)-10(-1) mU/liter TSH was obtained by exposing sections to the hormone for 90 sec. TSH (10(-3) mU/liter) produced a response significantly different (P less than 0.0025) from the control. However, 10(-5) mU/liter TSH produced a response significantly different (P less than 0.0025) from the control after an exposure time of 180 sec, and the range of the dose-response curve at this exposure time was 10(-6)-10(-3) mU/liter TSH. Each point on these two dose-response curves was determined in quintuplicate, and precision profiles were constructed. The assay performed at 90 sec had a lower relative precision of 30% at a dose of 10(-1) mU/liter TSH, and at 180 sec, the best lower relative precision achieved was 80%. Thus, the sensitivity of the assay was improved by increasing the exposure time of the sections to TSH, but with a resultant loss of relative precision.

Adenylate cyclase activity and the accumulation and release of adenosine 3',5'-monophosphate in normal human thyroid tissue slice preparations: responses to thyrotropin and thyroid-stimulating antibodies.

The cAMP response to TSH was investigated in slice preparations of normal human thyroid tissue maintained under in vitro conditions. In particular, dose-response relationships for tissue obtained from different thyroid glands were assessed, and the sensitivity and precision of this system were compared with the adenylate cyclase response to TSH in plasma membrane preparations derived from similar tissue. The effect of thyroid-stimulating antibodies (TSAb) on the thyroid slice cAMP response was investigated. After an incubation period of 120 min, 58% of a series of 43 immunoglobulin G (IgG) fractions prepared from the sera of Graves' disease patients significantly elevated the slice level of cAMP when assayed in triplicate at a level of 5 mg/ml. From a total of 22 preparations of IgG from untreated thyrotoxic patients, 11 (50%) stimulated the accumulation of cAMP in thyroid slices. Six out of a group of 8 patients with Graves' ophthalmopathy had demonstrable TSAb activity. After the initiation of antithyroid therapy with carbimazole, IgG preparations from 5 out of a group of 8 patients were without detectable TSAb activity. However, cAMP accumulation was stimulated by all 5 of a group of IgG preparations from previously thyrotoxic patients who had relapsed upon cessation of antithyroid therapy. The elevated cAMP levels in tissue slices in response to either TSH or TSAb were accompanied by an increase in cAMP in the incubation medium. The magnitude and significance of this extracellular cAMP accumulation were similar to those observed in the tissue and, accordingly, may form the basis of a simplified bioassay procedure for TSAb.

Characterization of monoclonal antibodies derived from lymphocytes from Graves' disease patients in a cytochemical bioassay for thyroid stimulators.

Monoclonal antibodies 208F7 and 307H6, derived from Graves' lymphocytes, were previously shown to stimulate thyroid function. We characterized these antibodies in the ultrasensitive cytochemical bioassay for thyroid stimulators. Bell-shaped dose-response curves were obtained for both antibodies, confirming their actions as thyroid stimulators; 307H6 was 10(7) times more potent than 208F7, and the ascending limb of the response curve to 307H6 was not significantly different from that of a reference preparation of thyroid-stimulating antibodies, namely LATS-B. Stimulation by both 208F7 and 307H6 was inhibited by antihuman, but not antimouse, immunoglobulin. Stimulation by 208F7, but not 307H6, was inhibited by 11E8 (a monoclonal antibody raised against the TSH receptor), which is a relatively potent inhibitor of TSH, but not thyroid-stimulating antibodies. These findings together with previous observations on the interactions of 208F7 and 307H6 with thyroid cells in both the presence and absence of TSH and of 208F7 and 307H6 with solubilized thyroid membrane components are summarized in a model relating the appropriate epitopic regions of the TSH receptor.

Theoretical limitations on immunoassay sensitivity. Current practice and potential advantages of fluorescent Eu3+ chelates as non-radioisotopic tracers.

The sensitivity of any immunoassay is a complex function of the underlying physico-chemical basis of the technique and the size and source of 'experimental' errors. Analysis of this relationship emphasizes the distinction between competitive labelled analyte methods, e.g., RIA, and the non-competitive labelled antibody techniques, e.g., IRMA, implying that, in practice, non-competitive methods may display sensitivities which are orders of magnitude greater than similar competitive assays. The achievement of the potential sensitivity of the non-competitive methods depends to a great extent on the application of very highly detectable labels. The long fluorescent lifetime and large Stoke's shift of certain europium (Eu3+) chelates permits such sensitive detection in a commercially available time-resolved fluorimeter. We briefly outline the current application of Eu3+ labels in immunometric assay, compare the application of Eu3+ labels in the immunofluorometric assay of human TSH with conventional RIA and discuss the future potential of this technique.

Nuclear tri-iodothyronine (T3) binding in neonatal rat brain suggests a direct glial requirement for T3 during development.

Tri-iodothyronine (T3) binding studies were performed on neuronal and glial nuclei prepared from developing rats brain by discontinuous sucrose gradient centrifugation. Maximum binding capacities (MBC) and dissociation constants (Kd) were obtained from Eadie-Hofstee plots of transformed data. An ontogenic study on nuclei prepared from whole brain revealed that on day 5 after birth, glial nuclear MBC was 1774 +/- 201 (S.E.M.) fmol/mg DNA compared with 974 +/- 117 fmol/mg DNA for the neurones (P less than 0.01). Although diminishing to 667 +/- 112 fmol/mg DNA by day 21, alterations in neuronal MBC over the neonatal period were not statistically significant, whereas glial MBC diminished steadily to 557 +/- 133 fmol/mg DNA in glial nuclei (P less than 0.05). Over the same period, a significant reduction in Kd was noted only in the glia, from 3.17 +/- 0.40 to 1.83 +/- 0.34 nmol/l (P less than 0.03). Ligand specificity of the receptor in both nuclear types on day 21 was tri-iodoacetic acid greater than T3 greater than thyroxine greater than 3,3',5'-T3, but this was less clearly demonstrated at day 5. Regional studies on days 15 and 21 demonstrated that for both neuronal and glial nuclei, receptors are concentrated in the cerebral cortex and diminish in a cranio-caudal direction. Cerebral glial MBC on day 21 was 2215 +/- 147 fmol/mg DNA, at this stage still exceeding the cerebral neuronal capacity of 1111 +/- 207 fmol/mg DNA. The results indicate that neonatal glia may respond directly to thyroid hormones via nuclear receptor binding, and that receptors are predominantly located in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of maternal hypothyroxinaemia in the rat on brain biochemistry in adult progeny.

The effects of maternal hypothyroxinaemia during pregnancy on subsequent brain biochemistry in progeny was studied. Normal and partially thyroidectomized rat dams were mated and progeny allowed to grow to adulthood. Brain regions (cerebellum, medulla, midbrain, cerebral cortex and paleocortex) were dissected out and the activities of various cell marker enzymes were determined, along with cholesterol contents. Maternal hypothyroxinaemia was without effect on body weight, brain weight or thyroid status of adult progeny. Oligodendroglial marker enzyme activities were altered in progeny from thyroidectomized dams. 2',3'-Cyclic nucleotide 3'-phosphohydrolase was decreased in the medulla (by 37%) and midbrain (by 32%). 5'-Nucleotidase was also diminished in the same brain regions, by 33% in the medulla and by 35% in the midbrain. In contrast, oleate esterase was increased (by 39%) in the paleocortex. Although these enzymes are putatively involved in myelin metabolism, no changes were observed in the concentration of a major myelin lipid (cholesterol). The activity of beta-D-glucuronidase (a general neuronal marker) was decreased (by 30%) in the paleocortex, whereas N-acetyl-beta-D-galactosaminidase (a general glial marker) was unchanged in all brain regions. In summary, maternal hypothyroxinaemia has irreversible effects on brain biochemistry in adult progeny. The damage is parameter-selective and brain region-specific, analogous to the pattern of neurological damage seen in offspring born to hypothyroxinaemic women in iodine-deficient endemias.

The influence of the maternal thyroid hormone environment during pregnancy on the ontogenesis of brain and placental ornithine decarboxylase activity in the rat.

The influence of maternal hypothyroxinaemia on early brain and placental development was examined in a partially thyroidectomized (parathyroid-spared; TX) rat dam model. Ornithine decarboxylase (ODC) specific activity, along with more general indices of cell growth, were determined in prenatal whole brain (at 15, 19 and 22 days of gestation), postnatal brain regions (at 5, 10 and 14 days) and placenta. Maternal hypothyroxinaemia resulted in reductions in fetal body weight, brain weight, brain DNA content and brain total protein content at 15 days of gestation; the latter effect persisting until 19 days of gestation. Further changes in brain cell growth were observed near term, when an increase in the DNA concentration was accompanied by a decrease in the total protein:DNA ratio. Growth of the postnatal brain regions appeared normal, with the exception of an isolated increase in the protein content of the cerebellum at postnatal day 5. Determination of the specific activity of brain ODC revealed a complex pattern of change in the progeny of TX dams, superimposed upon the normal ontogenetic decline. In the fetal brain, activity was initially deficient at 15 days of gestation but was increased at 22 days of gestation relative to controls. The compromise extended into the postnatal period; ODC specific activity being transiently reduced in the brainstem, the subcortex and the cerebral cortex. Placental development was less consistently affected; wet weight, gross indices of cell growth (DNA content, DNA concentration, total protein:DNA ratio) and ODC specific activity were all normal in the TX dam. However, cytosolic and total protein concentrations were reduced at 15 and 19 days of gestation respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition by normal immunoglobulins of thyrotrophin-stimulated production of cyclic AMP in slices of normal human thyroid.

Slice preparations of normal human thyroid tissue have been used to investigate the effect of normal immunoglobulin G (IgG) on thyrotrophin (TSH)-induced accumulation of cyclic AMP. Incubation of slices in the presence of both TSH and normal IgG for 20 min reduced the stimulation of cyclic AMP accumulation elicited by TSH alone by approximately 30%. However, preincubation of slices with IgG for 100 min before addition of TSH virtually abolished the response to TSH. The latter effect of normal IgG was reversible, and removal of IgG before exposure to TSH allowed an unimpaired cyclic AMP response to TSH. The implications of these observations with respect to the application of this system to the functional bio-detection of thyroid-stimulating antibodies in IgG fractions from thyrotoxic sera are discussed.

Characterization of thyroid-stimulating immunoglobulin-induced cyclic AMP accumulation in the rat thyroid cell strain FRTL-5: potentiation by forskolin and calibration against reference preparations of thyrotrophin.

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0.75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1.25 mu./ml) was 20.5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0.1 mumol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0.1 mumol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.

Hypothyroidism in the adult rat causes brain region-specific biochemical dysfunction.

The influence of hypothyroidism in the adult rat on brain biochemistry was investigated. Hypothyroidism was induced in 6-month-old male rats by partial thyroidectomy coupled with the administration of 6-n-propyl-2-thiouracil (0.005%, w/v) in the drinking water. Age-matched euthyroid males served as the controls. Hypothyroidism resulted in brain region-specific changes in certain catabolic enzyme activities. Acid phosphatase activity was reduced in the cerebellum (by 34%) and the medulla (by 38%), whereas alkaline phosphatase activity was decreased in the midbrain (by 37%) and the subcortex (by 49%). A differential response was also observed in the case of aryl sulphatase activity: aryl sulphatase A (myelin-degradative activity) was diminished in the cerebellum (by 56%), whereas aryl sulphatase B remained unchanged in all regions. Acetylcholine esterase activity was reduced in the cerebellum (by 45%), the medulla (by 34%) and the subcortex (by 45%), whereas monoamine oxidase activity was affected in only one region, the cerebellum, where it was increased by (61%). The compromise of myelin and neurotransmitter degradative enzyme activities may place severe restrictions on normal brain function. The vulnerability of the adult rat cerebellum to the effects of thyroidectomy is commensurate with the known clinical signs of cerebellar dysfunction in adult hypothyroid man. These findings raise the possibility of an important role for the thyroid hormones in the mature brain.

A rat granulosa cell plasminogen activator bioassay for FSH in human serum.

A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3.5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0.3-15 IU/l, with an assay sensitivity of 0.3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 degrees C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 microliters) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0.745, n = 15 and P less than 0.05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples.

Effect of maternal hypothyroxinaemia during fetal life on the calmodulin-regulated phosphatase activity in the brain of the adult progeny in the rat.

Calmodulin-regulated phosphatase activity was measured in the brain of 2-month-old rats born from hypothyroid and normal dams, using a fluorometric enzyme assay developed for this purpose. Calmodulin content was measured in the same brain regions by radioimmunoassay. Significant differences between groups in weight and protein content, basal phosphatase and calmodulin-regulated phosphatase activity were found. The brain region most affected was the cerebellum, where basal and calmodulin-regulated phosphatase activities, and protein content were increased. The data point towards a lasting effect of maternal hypothyroxinaemia on the brain function of the progeny.

Influence of maternal hyperthyroidism in the rat on the expression of neuronal and astrocytic cytoskeletal proteins in fetal brain.

Maternal hypothyroidism during pregnancy impairs brain function in human and rat offspring, but little is known regarding the influence of maternal hyperthyroidism on neurodevelopment. We have previously shown that the expression of neuronal and glial differentiation markers in fetal brain is compromised in hypothyroid rat dam pregnancies and have now therefore extended this investigation to hyperthyroid rat dams. Study groups comprised partially thyroidectomised dams, implanted with osmotic pumps infusing either vehicle (TX dams) or a supraphysiological dose of thyroxine (T4) (HYPER dams), and euthyroid dams infused with vehicle (N dams). Cytoskeletal protein abundance was determined in fetal brain at 21 days of gestation by immunoblot analysis. Relative to N dams, circulating total T4 levels were reduced to around one-third in TX dams but were doubled in HYPER dams. Fetal brain weight was increased in HYPER dams, whereas litter size and fetal body weight were reduced in TX dams. Glial fibrillary acidic protein expression was similar in HYPER and TX dams, being reduced in both cases relative to N dams. alpha-Internexin (INX) abundance was reduced in HYPER dams and increased in TX dams, whereas neurofilament 68 (NF68) exhibited increased abundance in HYPER dams. Furthermore, INX was inversely related to - and NF68 directly related to - maternal serum total T4 levels, independently of fetal brain weight. In conclusion, maternal hyperthyroidism compromises the expression of neuronal cytoskeletal proteins in late fetal brain, suggestive of a pattern of accelerated neuronal differentiation.