Unexpected antibody against the high-prevalence P antigen before cardiac surgery.

P is a high-prevalence antigen present in 99.9 percent of the population and is fully developed at birth. P- individuals form naturally occurring antibodies against P, which are often of immunoglobulin (Ig)M and/or IgG type, very potent in complement activation, and able to cause serious intravascular hemolytic transfusion reactions. Some people with anti-P have the rare P1 k phenotype, which lacks P in the presence of P1 and Pk. Blood transfusion in patients with anti-P is challenging, as is described here. A male patient without a history of blood transfusion was admitted for a planned cardiac surgery. The preoperative ABO blood group could not be determined because of unexpected reactions in the reverse grouping, and all red blood cells (RBCs) in the antibody detection test were positive, except for the autocontrol. Further analysis of the patient's sample confirmed the presence of the P1 k phenotype, and anti-P was identified. If transfusion was needed, P- blood would be required, and the only P- RBCs available were at the national Sanquin Bank of Frozen Blood. These units are limited, expensive, and only available for 48 hours after thawing. In the case of massive blood loss, first ABO and Rh-compatible units should be transfused, followed by P- units after the bleeding stops. In our case, the surgery was conducted without transfusion. This case illustrates the importance of preoperative ABO blood group testing and antibody screening in cases where blood loss can be expected. In recent years, more focus has been put on patient blood management. A good collaboration between the local laboratory, surgery department, and dedicated blood transfusion laboratory is critical to prevent unnecessary incompatible blood transfusions with potentially serious outcomes.

Implementation of a new platelet pooling system for platelet concentrates led to a higher corrected count increment after transfusion: a comparative observational study of platelet concentrates before and after implementation.

OBJECTIVES: To study the effect of extended storage of platelet concentrates (PCs) and the implementation of a new platelet pooling system for PCs on corrected count increment (CCI) after transfusion. BACKGROUND: Due to new developments and changes in processes or procedures, one should remain alert for the effects of these changes. Besides in vitro studies and validation, in vivo studies are also important, as it has been shown that in vitro results do not always predict in vivo outcomes. METHODS/MATERIALS: After introduction of extended storage of PCs for 5-7 days prepared from five buffy coats and plasma, transfusion monitoring for transfusions of PCs in haemato-oncological patients was set up. After 9 months, a new pooling system for PCs was implemented, Composelect instead of Optipure PLT, and transfusion monitoring was continued for another 8 months. The CCI was used as primary outcome. RESULTS: In total, 93 patients were included and transfused with PCs prepared in the Optipure PLT system (262 transfusions) or in the Composelect system (127 transfusions). Extended storage of PCs for 7 days had no significant effect on CCI. Although the implementation of the Composelect system did not influence the CCI1 h (13.8 ± 6.0 vs. 13.0 ± 5.8; n.s.), it seemed to have a positive effect on CCI24 h (7.0 ± 4.9 vs. 4.7 ± 4.5; P < 0.05). CONCLUSION: Although the influence of confounders could not be excluded, it seemed that implementation of the Composelect system for PCs led to an improved CCI24 h and that extended storage of PCs did not influence the CCI.

A positive effect of immune suppression on corrected count increment after platelet transfusion at 1 but not at 24h.

The effects of patient characteristics on corrected count increment (CCI) in hemato-oncology patients were studied. CCI values were 13.6 ± 6.5 (1h, n=79) and 6.3 ± 5.3 (24h, n=69). With concomitant immune suppression CCI was higher at 1h (20.0 ± 7.9 versus 13.2 ± 6.3, p=0.023), but not at 24h (5.9 ± 6.7 versus 6.3 ± 5.2; p=0.88). The observed effect is short lived, potentially benefiting bleeding patients, but may not increase intervals between transfusions. Further, CCI1h was lower if fever was present (9.7 ± 3.6 versus 14.3 ± 6.7; p=0.002), and corresponding CCI24h values were 3.7 ± 6.3 versus 6.7 ± 5.0 (p=0.09). At 24h an effect for previous transfusions was observed, 6.7 ± 5.1 (with) versus 1.6 ± 5.4 (without p=0.02).

Salivary cortisol as an alternative for serum cortisol in the low-dose adrenocorticotropic hormone stimulation test?

BACKGROUND: Salivary cortisol is unaffected by cortisol binding globulin and reflects free serum cortisol as compared to total serum cortisol. AIM: The aim of the present study was to compare the salivary cortisol response with the serum cortisol response in a low-dose (1-microg) ACTH test in a clinical setting and to determine the optimal cut-off value of salivary cortisol as an alternative to serum cortisol. MATERIAL/SUBJECTS AND METHODS: We measured serum and salivary cortisol responses to iv administration of 1-microg ACTH in 51 patients (17 males) referred to the Department of Clinical Chemistry for ACTH-testing. Serum cortisol was assessed before, 20, and 30 min after ACTH-administration, and salivary cortisol was assessed before and 30 min after ACTH administration. RESULTS: Mean cortisol at baseline, 20, and 30 min were 0.44 micromol/l (SD: 0.22), 0.64 micromol/l (SD: 0.24), and 0.70 micromol/l (SD: 0.25), respectively. Median basal salivary cortisol was 8.4 nmol/l [interquartile range (IQR): 3.8-14.2]. Salivary cortisol at 30 min equaled 35.9 nmol/l (IQR: 21.1-46.2). Basal salivary cortisol was significantly correlated with salivary cortisol at 30 min (r=0.53; p<0.001). Salivary cortisol at 30 min of 23.5 nmol/l had a sensitivity and specificity of 78.1% and 70.0%, respectively as compared to the serum cortisol cut-off values of >0.50 micromol/l. CONCLUSIONS: The salivary low-dose ACTH-test yields more dynamic responses than serum cortisol. However, the sensitivity and specificity of salivary cortisol are too low to be adequate as an alternative to the serum cortisol measurements. In women on estrogen therapy, however, the use of salivary cortisol might be superior to serum cortisol.

Neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in The Netherlands: the importance of enzyme analysis to ascertain true MCAD deficiency.

The outcome was determined of population-wide neonatal screening for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency using tandem mass spectrometry (MS/MS) in The Netherlands, between October 2003 and September 2005. Prospective population-wide neonatal screening for MCAD deficiency was performed in the northern part of The Netherlands. In newborns with blood octanoylcarnitine (C(8:0)) concentrations > or =0.3 micromol/L, clinical and laboratory follow-up was initiated, including MCAD enzymatic measurements which played a decisive role. In a 2-year period, 66 216 newborns were investigated for MCAD deficiency and follow-up was initiated in 28 newborns. True-positives (n = 14) were identified based upon MCAD enzyme activity <50%, measured with hexanoyl-CoA as substrate. The observed prevalence of MCAD deficiency was 1/6600 (95% CI: 1/4100-1/17 400). In addition to an elevated C(8:0) concentration, a C(8:0)/C(10:0) molar ratio >5.0 turned out to differentiate between false-positives and true-positives. Measurement of MCAD activity using phenylpropionyl-CoA as a substrate further discriminated between newborns with MCAD deficiency and so-called mild MCAD deficiency. To summarize, neonatal screening for MCAD deficiency in the northern part of The Netherlands resulted in the predicted number of affected newborns. Measurement of MCAD activity in leukocytes or lymphocytes using phenylpropionyl-CoA as a substrate can be regarded as the gold standard to diagnose MCAD deficiency upon initial positive screening test results.

Occurrence and management of an aberrant free T4 in combination with a normal TSH.

BACKGROUND: Thyroid function tests may show the combination of a normal concentration of serum thyroidstimulating hormone (TSH) and an increased or decreased level of free thyroxine (free T4). How often this occurs is unclear and not everyone is familiar with how it should be adressed. METHODS: We conducted a retrospective cohort study of all adult patients who presented at a non-academic general hospital in the Netherlands between 1 January 2010 and 31 December 2014 and yielded an increased or decreased free T4 in combination with a normal TSH. Exclusion criteria included the use of thyroid medication, pregnancy, a history of thyroid surgery and treatment with radioactive iodine. The medical records of the patients included were retrieved and evaluated. RESULTS: Of the 30,143 combined TSH and free T4 measurements in 23,199 individual patients, 1005 measurements (3.33%) in 775 patients (3.34%) yielded an aberrant free T4 in combination with a normal TSH. 398 patients (1.72%) had a persistent aberrant free T4, 349 (87.7%) of whom had a decreased free T4 and 49 (12.3%) an increased free T4. In 58 of the 398 patients (14.6%) with a persistent abberant free T4 a possible cause was established by the treating physician. However, upon re-examination of medical files a possible causative factor could be identified in 123 patients (30.9%). CONCLUSION: In our study population the prevalence of hyperthyroxinemia or hypothyroxinemia in combination with a normal TSH was 334 per 10.000 patients. When records were thoroughly searched, identification of potential causative factors increased substantially. Clinicians should be encouraged to check for underlying causes.

Validation of the 1 µg short synacthen test: an assessment of morning cortisol cut-off values and other predictors.

BACKGROUND: Clinical practice shows that many low-dose short synacthen tests (LD-SSTs) for diagnosing adrenal insufficiency in an outpatient setting have a normal outcome and could be considered superfluous. The objective of this study is to provide a guideline to safely reduce the number of unnecessarily performed LD-SSTs. METHODS: Data of LD-SSTs performed in outpatients were collected. Optimal morning cortisol cut-off values were determined using ROC analysis. Subsequently the predictive value of several variables was tested using univariable and multivariable logistic regression analyses. RESULTS: A morning cortisol lower cut-off value of 145 nmol/l (specificity 89.9%, positive predictive value 90.0%) and an upper cut-off value of 375 nmol/l (sensitivity 100.0%, negative predictive value 100.0%) were found. Chronic fatigue symptoms and symptoms of hypotension or orthostasis as the main reason for performing the test predict a normal outcome. The use of glucocorticosteroids predicts an abnormal outcome of the LD-SST. Oral, topical, nasal and inhaled glucocorticosteroids are each significant predictors when analysed specifically for predicting central adrenal insufficiency. CONCLUSION: By using morning cortisol cut-off values of 145 nmol/l and 375 nmol/l instead of the conventional cut-off values, the number of LD-SSTs performed in an outpatient setting can be reduced by 12%, while maintaining high sensitivity and specificity. Furthermore, the outcome of the LD-SST can be predicted by additional variables such as the indication for performing the test and the use of glucocorticosteroids. Different routes of administration of glucocorticosteroids such as inhalation or topical use should be taken into account when central insufficiency is suspected.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. IV. Evidence for paracrine and autocrine regulation.

Disulfiram (Dis), an inhibitor of peptidyl-glycine alpha-amidating monooxygenase, the enzyme responsible for the production of alpha-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated. Dis (8 microM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion. Dis significantly elevated prepro-TRH (25-50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.

Effects of hypothyroidism on hypothalamic release of thyrotropin-releasing hormone in rats.

The aim of this study was to investigate whether the severity and duration of primary hypothyroidism influence hypothalamic TRH release. Hypothyroidism was induced in male Wistar rats by treatment with different thyrostatic drugs or by thyroidectomy. Serum TSH in rats treated for up to 3 weeks with methimazole (MMI; 0.05% in drinking water) increased 20-fold, but TRH release into hypophyseal portal blood (HPB) did not change. Treatment with propylthiouracil (PTU; 0.1% in drinking water), which inhibits thyroidal T4 production and peripheral conversion of T4 to T3, resulted in a more rapid reduction in serum T3 levels and increase in serum TSH than those in rats treated with 0.1% MMI. Although these differences were no longer observed after 3 weeks of treatment, TRH release into HPB of rats treated with PTU was 34-49% higher than that in MMI-treated rats. Combined treatment with MMI (0.05-0.1% in drinking water) and iopanoic acid (IOP; 4 mg/100 g BW.day, ip), an inhibitor of both peripheral and central T4 to T3 conversion, also tended to produce a more rapid decrease in serum T3 and increase in serum TSH. After 3 weeks of treatment, serum T4, T3, and TSH were not different in the two groups, but TRH release into HPB was 48-65% increased by MMI plus IOP vs. MMI alone. Three to 10 weeks after thyroidectomy, TRH release into HPB was 58-72% higher than that in untreated controls. In vitro incubation of hypothalami isolated from rats treated for 3 weeks with MMI, MMI plus IOP, or PTU, as described above, showed that basal and 56 mM K(+)-induced TRH release were not influenced by the different drugs. Also, the total hypothalamic TRH content was not changed by any of these treatments. However, in rats treated for 1 or 2 weeks with MMI or PTU, the TRH content of the median eminence was decreased by 17-25%. These findings indicate that, depending on severity and duration, experimental hypothyroidism may cause a significant increase in hypothalamic TRH release in rats. The magnitude of these changes compared with the much larger increases in serum TSH suggests that the feedback of thyroid hormone on TSH secretion is mainly exerted at the pituitary level.

Hypothalamo-hypophysial-thyroid axis in streptozotocin-induced diabetes.

Diabetes mellitus is frequently associated with reduced levels of TSH, PRL, GH, and gonadotropins. In this study we have wanted to determine whether chemically induced diabetes mellitus is associated with a decreased hypothalamic release of TRH. Male rats were made diabetic with streptozotocin (STZ; 65 mg/kg), whereas controls received vehicle. After 2 weeks, STZ diabetic rats had 25% lower body weights, 3.5-fold higher blood glucose, and 40-60% lower plasma TSH, T3, and T4 levels than controls. The plasma T4 dialyzable fraction had increased 2.5-fold in STZ diabetic rats, and the plasma free T4 concentration was similar to that in controls. Thus, treatment with STZ results in decreased plasma TSH and T4 levels, but does not reduce free T4 concentrations. The content of TRH in hypothalami of 2-week STZ diabetic rats was similar to that in controls, but in vitro these hypothalami released less TRH than those of control rats. In 2-week STZ diabetic rats, TRH in hypophysial stalk blood was 30% lower than that in control rats. The in vitro TRH secretion from hypothalami of untreated rats was dependent on the glucose concentrations in the incubation medium; increasing the glucose concentration from 10 to 30 mM did not alter TRH secretion, but basal TRH release increased in the absence of glucose. In conclusion, STZ-induced diabetes in the rat is associated with reduced hypothalamic secretion of TRH, which, in turn, may be responsible for the reduced plasma TSH and thyroid hormone levels. Furthermore, it is suggested that the inhibitory effect of STZ-induced diabetes on TRH secretion is probably not due to hyperglycemia.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. II. Stimulation by glucocorticoids.

The present studies were undertaken to determine whether glucocorticoids (GC) regulate TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 18 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with 1) fetal calf serum (FCS), 2) charcoal-treated FCS, 3) normal rat serum, or 4) serum from rats that were adrenalectomized, rendered hypothyroid, and gonadectomized (ATG rat serum). Dexamethasone (Dex) or corticosterone (Cort) was added to the culture medium at various concentrations with exposure times ranging from 4-18 days. TRH and prepro-TRH-(25-50) in cellular extracts and release media were measured by RIA, and pro-TRH mRNA was determined by Northern blot analysis and in situ hybridization. Dex substantially stimulated cellular TRH and prepro-TRH-(25-50) accumulation under all culture conditions investigated, i.e. in medium supplemented with any of the four sera. TRH gene expression did not occur in medium supplemented with charcoal-treated FCS or ATG rat serum. Pretreatment with 10(-8) M Dex caused a significant increase in basal as well as cAMP- or phorbol ester-stimulated release of the peptide. Steady state pro-TRH mRNA levels rose 6.8- and 4.2-fold (both P < 0.01) after treatment with 10(-8) M Dex for 4 and 12 days, respectively. In situ hybridization experiments revealed that this rise in pro-TRH mRNA levels was probably the result of an increase in the number of AP cells expressing pro-TRH. Both Dex and Cort caused a dose-dependent increase in TRH accumulation, but Cort was approximately 40 times less potent than Dex. These results indicate that GC stimulate TRH gene expression in cultured AP cells. The presence of GC in culture medium is a prerequisite for the occurrence of TRH gene expression in the AP. As GC have been reported to reduce pro-TRH mRNA levels in the hypothalamus in vivo, our results may provide an example of the tissue-specific effects of GC on TRH gene expression.

Thyrotropin-releasing hormone gene expression in the anterior pituitary. III. Stimulation by thyroid hormone: potentiation by glucocorticoids.

The present study was designed to investigate the effect of thyroid hormone on TRH gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male rats were cultured for up to 14 days in Dulbecco's Modified Eagle's Medium-L-15 medium supplemented with either fetal calf serum (FCS) or FCS devoid of thyroid hormones. T4 or T3 were added at various concentrations to the medium for a duration of 2-14 days. TRH and GH were measured by RIA, and pro-TRH mRNA levels were determined by semiquantitative in situ hybridization. Addition of both T3 and T4, but not the biologically inactive diiodothyronine, significantly stimulated TRH accumulation in AP cells. T3 increased TRH content in a time- and dose-dependent fashion and was much more potent than T4. Dexamethasone (Dex) also raised the content of TRH significantly. The combination of 10(-9) M T3 and 10(-8) M Dex dramatically potentiated the effect of either treatment alone (T3, 8.9-fold rise; Dex, 37.2-fold rise) and increased TRH accumulation 251.2-fold (all P < 0.01). Levels of pro-TRH mRNA mirrored TRH content data. T3, Dex, or the combination of both raised pro-TRH mRNA levels 1.9-, 2.7 (both P < 0.05)-, and 11.1 (P < 0.01)-fold, respectively. The visualization of pro-TRH mRNA by in situ hybridization revealed that the combination of T3 and Dex treatment caused a substantial increase in the number of cells expressing pro-TRH. The results presented here demonstrate that T3 increases pro-TRH gene expression in cultured AP cells and that glucocorticoids markedly potentiate this effect. As pro-TRH is expressed in a subpopulation of somatotrophs, our data suggest that the TRH gene in this location may be coordinately regulated with the GH gene.

Thyrotropin-releasing hormone (TRH) gene expression in the anterior pituitary. I. Presence of pro-TRH messenger ribonucleic acid and pro-TRH-derived peptide in a subpopulation of somatotrophs.

We have previously reported the presence of authentic pro-TRH-derived peptides in cultured anterior pituitary (AP) cells. The present studies were undertaken to determine whether pro-TRH mRNA could be demonstrated in the AP and to elucidate the cell type expressing pro-TRH. AP cells were cultured for up to 18 days, during which time the content of both TRH and prepro-TRH-(25-50) rose significantly (P < 0.01). In contrast, the cellular contents of LH, FSH, TSH, and ACTH fell significantly (P < 0.01), whereas that of GH increased by 45.9% (P < 0.05). Northern blot analysis revealed that the levels of pro-TRH mRNA extracted from AP cells (18 days in culture) were similar to those in hypothalamic tissue from adult male rats, indicating a high relative abundance of this mRNA in the AP. In situ hybridization experiments showed a dense accumulation of silver grains over a subpopulation of cultured AP cells. A combination of in situ hybridization for pro-TRH mRNA and immunocytochemistry for pituitary hormones revealed colocalization of pro-TRH mRNA and GH in a subpopulation of somatotrophs. No colocalization with LH-, TSH-, PRL-, or beta-endorphin-containing cells was observed. Immunocytochemistry at the electron microscopic level demonstrated that prepro-TRH-(25-50) was contained in a subpopulation of secretory granules in AP cells expressing this pro-TRH-derived sequence. These studies demonstrate that pro-TRH mRNA is present in cultured AP cells in high concentration and that the pro-TRH gene is expressed within a subpopulation of somatotrophs.

Activation of thyrotropin-releasing hormone gene expression in cultured fetal diencephalic neurons by differentiating agents.

The present studies were undertaken to investigate the effect of 5-bromo-2'-deoxyuridine (BrdU; 50 microM) or forskolin/3-isobutyl-1-methylxanthine (F/I; 10/500 microM) on TRH gene expression in cultured fetal diencephalic cells. BrdU as well as drugs such as F/I that raise intracellular cAMP levels had been previously termed differentiating agents because they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalons may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracellular TRH and pro-TRH messenger RNA concentrations in cultured diencephalic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to cause maintenance of high TRH levels after withdrawal of the drug, F/I had to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expression in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I exposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulation of somatostatin and arginine vasopressin, which were determined in parallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these and possibly other neuropeptides in vitro.

Effect of thyroid status and paraventricular area lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood.

TRH is a potent stimulator of pituitary TSH release, but its function in the physiological regulation of thyroid activity is still controversial. The purpose of the present study was to investigate TRH and catecholamine secretion into hypophysial portal blood of hypothyroid and hyperthyroid rats, and in rats bearing paraventricular area lesions. Male rats were made hypothyroid with methimazole (0.05% in drinking water) or hyperthyroid by daily injections with T4 (10 micrograms/100 g BW). Untreated male rats served as euthyroid controls. On day 8 of treatment they were anesthetized to collect peripheral and hypophysial stalk blood. In euthyroid, hypothyroid and hyperthyroid rats plasma T3 was 1.21 +/- 0.04, 0.60 +/- 0.04, and 7.54 +/- 0.33 nmol/liter, plasma T4 50 +/- 3, 16 +/- 2, and 609 +/- 74 nmol/liter, and plasma TSH 1.58 +/- 0.29, 8.79 +/- 1.30, and 0.44 +/- 0.03 ng RP-2/ml, respectively. Compared with controls, hyperthyroidism reduced hypothalamic TRH release (0.8 +/- 0.1 vs. 1.5 +/- 0.2 ng/h) but was without effect on catecholamine release. Hypothyroidism did not alter TRH release, but the release of dopamine increased 2-fold and that of noradrenaline decreased by 20%. Hypothalamic TRH content was not affected by the thyroid status, but dopamine content in the hypothalamus decreased by 25% in hypothyroid rats. Twelve days after placement of bilateral electrolytic lesions in the paraventricular area plasma thyroid hormones and TSH levels were lower than in control rats (T3: 0.82 +/- 0.05 vs. 1.49 +/- 0.07 nmol/liter; T4: 32 +/- 4 vs. 66 +/- 3 nmol/liter; TSH: 1.08 +/- 0.17 vs. 3.31 +/- 0.82 ng/ml). TRH release in stalk blood in rats with lesions was 15% of that of controls, whereas dopamine and adrenaline release had increased by 50% and 40%, respectively. These results suggest that part of the feedback action of thyroid hormones is exerted at the level of the hypothalamus. Furthermore, TRH seems an important drive for normal TSH secretion by the anterior pituitary gland, and thyroid hormones seem to affect the hypothalamic release of catecholamines.

In vivo hypothalamic release of thyrotropin-releasing hormone after electrical stimulation of the paraventricular area: comparison between push-pull perfusion technique and collection of hypophysial portal blood.

Unilateral electrical stimulation for 15 min of the paraventricular area of anesthetized rats induced a 2- to 3- fold increase in plasma TSH levels and caused an increased release of TRH into hypophysial stalk blood from 217 +/- 25 to 530 +/- 90 pg/15 min (n = 6). This experimental model was then used to determine the in vivo hypothalamic release of TRH by push-pull perfusion of either the mediobasal hypothalamus (MBH) or anterior pituitary (AP). Before stimulation, TRH release per 15 min was 4.2 +/- 0.7 pg from the MBH (n = 18) and 3.5 +/- 0.3 pg from the AP (n = 13). Unilateral electrical stimulation of the paraventricular area led to higher plasma TSH levels in 27 of 31 rats, and levels during stimulation increased from 0.89 +/- 0.04 to 1.86 +/- 0.10 ng/ml (n = 31). No significant increase in TRH in the perfusates was observed when push-pull perfusion was done in the MBH contralateral to the site of stimulation (n = 6). However, TRH release increased 2- to 3-fold during the perfusion of the MBH ipsilateral to the site of stimulation (15.4 +/- 4.3 pg/15 min; n = 13). In conclusion, push-pull perfusion of the MBH or AP can be used to estimate hypothalamic TRH release. However, the output of TRH by push-pull perfusion is low and varies considerably between individual rats. Thus, the practical value of push-pull perfusion for measurement of in vivo TRH release seems limited.

Hypothyroidism may account for reduced prolactin secretion in lactating rats bearing paraventricular area lesions.

Lesions in the paraventricular area (PVA) of lactating rats have been found to inhibit PRL release. We have examined whether this reduced PRL release is due to hypothyroidism resulting from destruction of the PVA. Rats were made hypothyroid by thyroidectomy on day 15 of pregnancy or by methimazole treatment from the day of parturition. Electrolytic lesions were placed bilaterally in the PVA on day 15 of pregnancy. The following variables were studied: weight gain of the pups, nursing behavior, thyroid status, and release of PRL. The treatments did not affect the time the mothers spent with the pups but reduced the daily weight gain of the pups. Rats with PVA lesions had reduced PRL and TSH levels during lactation compared with controls. Suckling-induced PRL release after 6 h of separation of mothers and pups was less in PVA-lesioned rats than in controls, but T4-treatment did overcome this blunted response in rats with lesions. Levels of T3 and T4 in PVA-lesioned rats were lower than those in controls. In rats made hypothyroid by thyroidectomy or treatment with methimazole, PRL levels were lower and TSH levels higher than those in euthyroid mothers on days 8, 15, and 22 of lactation. Suckling after 6 h of separation of pups and mothers raised PRL levels both in control and methimazole-treated rats, but in the latter animals the response was blunted. It is suggested that the reduced PRL release in lactating rats with PVA lesions could be due to hypothyroidism resulting from these lesions.

Regulation of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in the rat anterior pituitary gland.

TRH-like peptides share the N- and C-terminal amino acids with TRH (pGlu-His-Pro-NH2) but differ in the middle amino acid residue. One of them, pGlu-Glu-Pro-NH2 (< EEP-NH2; EEP) is present in the rat pituitary gland, but its biological significance is unknown. We investigated the localization and regulation of this tripeptide in the rat pituitary gland. To distinguish between TRH and EEP two antisera were used for RIA: specificity of antiserum 4319 for the TRH-like peptides pGlu-Phe-Pro-NH2 and EEP was equal to or greater than that for TRH, whereas antiserum 8880 is TRH-specific. Our RIA data showed the presence of a TRH-like peptide in the anterior pituitary gland (AP) and of TRH in the posterior pituitary gland (PP). The TRH-like peptide in the AP was identified on anion-exchange chromatography and subsequent HPLC as EEP. Pathophysiological conditions such as altered thyroid and adrenal status and suckling did not affect pituitary gland levels of EEP. In general, however, there is a clear sex difference: levels of EEP are higher in male than in female rats. In both sexes gonadectomy leads to a substantial two- to threefold rise in EEP levels, abolishing the sex difference. Testosterone administration to gonadectomized male rats normalizes levels of EEP again. Disulfiram, an inhibitor of the enzyme peptidylglycine alpha-amidating monooxygenase, reduced levels of EEP in the AP by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the regulation, localization and function of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in the rat anterior pituitary gland.

Recent evidence shows that thyrotrophin-releasing hormone (TRH) immunoreactivity in the rat anterior pituitary gland is accounted for by the TRH-like tripeptide pyroglutamyl-glutamyl-prolineamide (pGlu-Glu-ProNH2, < EEP-NH2). The present study was undertaken to investigate further the regulation, localization and possible intrapituitary function of < EEP-NH2. Anterior pituitary levels of < EEP-NH2 were determined between days 5 and 35 of life, during the oestrous cycle and after treatment with the luteinizing hormone-releasing hormone (LHRH) antagonist Org 30276. Treatment of adult males with the LHRH antagonist either for 1 day (500 micrograms/100 g body weight) or for 5 days (50 micrograms/100 g body weight) reduced anterior pituitary < EEP-NH2 levels by 25-30% (P < 0.05 versus saline-treated controls). Anterior pituitary < EEP-NH2 increased between days 5 and 35 of life. In females, these levels were 2- to 3-fold higher (P < 0.05) than in males between days 15 and 25 after birth; these changes corresponded with the higher plasma follicle-stimulating hormone (FSH) levels in the female rats. After day 25, < EEP-NH2 levels in female rats decreased in parallel with a decrease in plasma FSH. Injections with the LHRH antagonist (500 micrograms/100 g body weight), starting on day 22 of life, led to reduced contents of < EEP-NH2 in the anterior pituitary gland of female rats on days 26 and 30 (55 and 35% decrease respectively). Levels of < EEP-NH2 in the anterior pituitary gland did not change significantly during the oestrous cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Starvation-induced changes in the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA and the hypothalamic release of proTRH-derived peptides: role of the adrenal gland.

The purpose of this study was to investigate the mechanisms involved in the reduced thyroid function in starved, young female rats. Food deprivation for 3 days reduced the hypothalamic content of prothyrotrophin-releasing hormone (proTRH) mRNA, the amount of proTRH-derived peptides (TRH and proTRH160-169) in the paraventricular nucleus, the release of proTRH-derived peptides into hypophysial portal blood and the pituitary levels of TSH beta mRNA. Plasma TSH was either not affected or slightly reduced by starvation, but food deprivation induced marked increases in plasma corticosterone and decreases in plasma thyroid hormones. Refeeding after starvation normalized these parameters. Since the molar ratio of TRH and proTRH160-169 in hypophysial portal blood was not affected by food deprivation, it seems unlikely that proTRH processing is altered by starvation. The median eminence content of pGlu-His-Pro-Gly (TRH-Gly, a presumed immediate precursor of TRH), proTRH160-169 or TRH were not affected by food deprivation. Since median eminence TRH-Gly levels were very low compared with other proTRH-derived peptides it is unlikely that alpha-amidation is a rate-limiting step in hypothalamic TRH synthesis. Possible negative effects of the increased corticosterone levels during starvation on proTRH and TSH synthesis were studied in adrenalectomized rats which were treated with corticosterone in their drinking water (0.2 mg/ml). In this way, the starvation-induced increase in plasma corticosterone could be prevented. Although plasma levels of thyroid hormones remained reduced, food deprivation no longer had negative effects on hypothalamic proTRH mRNA, pituitary TSH beta mRNA and plasma TSH in starved adrenalectomized rats. Thus, high levels of corticosteroids seem to exert negative effects on the synthesis and release of proTRH and TSH. This conclusion is corroborated by the observation that TRH release into hypophysial portal blood became reduced after administration of the synthetic glucocorticosteroid dexamethasone. On the basis of these results, it is suggested that the reduced thyroid function during starvation is due to a reduced synthesis and release of TRH and TSH. Furthermore, the reduced TRH and TSH synthesis during food deprivation are probably caused by the starvation-induced enhanced adrenal secretion of corticosterone.