Increased copeptin levels in metabolic syndrome from a Romanian population.

Rationale: Arginine vasopressin (AVP) is secreted under conditions of water deprivation. Since AVP has a low half-life in the plasma, the C-terminal fragment of AVP-precursor (copeptin) was used to estimate the AVP levels. High copeptin levels increase the risk for the development of diabetes mellitus. Aim: This study was aimed to measure copeptin levels in the metabolic syndrome (MetS) in Romanians using a competitive enzyme immunoassay. Methods and results: Patients prone to present MetS (n = 63) were compared to controls (n = 42). In the MetS group, the syndrome was confirmed in 93.6%. Affected patients displayed 85.7% obesity and insulin resistance (HOMAIR of 4.9 ± 0.4 versus 1.1 ± 0.8 in controls). Low HDL-cholesterol was less represented (47.5%). Copeptin levels were 0.6 ± 0.0 in MetS versus 0.42 ± 0.0 ng/ mL in controls (P < 0.004). Higher copeptin (0.79 to 1.83 ng/ mL) was associated with MetS, P < 0.0018, OR 20, 95%CI [3.03 - 131.7]. In ANOVA, high copeptin was equally explained by MetS or obesity (P < 0.05,α = 3.8). The best correlation was found with high triglyceride levels (P < 0.013,α = 6.3) while the correlation with HOMAIR remained not significant. Discussion: These data indicated a concordant correlation between increased copeptin and MetS or its components. In the light of epidemiological data, indicating that more than 50% of the European population has a lower daily water intake and a fraction of 25% displaying high copeptin, our data further sustained that copeptin may be a good biomarker for MetS and/ or obesity, which should be further investigated with other members of the osmoregulation pathway at both pathogenesis and genetic levels.

Quantifying subtle changes in cardiovascular mechanics in acromegaly: a Doppler myocardial imaging study.

AIM OF THE STUDY: To describe morphological and functional cardiovascular changes in acromegaly (ACM) patients, as well as to investigate the ability of Doppler-based myocardial deformation imaging (DMI) to characterize subtle dysfunction in ACM. METHODS: 69 patients (pts) with ACM (mean age 47 ± 10 years, 27 men) and 31 controls (mean age 43 ± 16 years, matched for age and gender) were recruited. Standard echocardiography and DMI data were obtained for all patients. Peak systolic longitudinal strain values (S) were determined for the left and right ventricles. Radial S was measured at the level of the mid inferolateral segment. Using a high-resolution echo-tracking system, the main indices of arterial stiffness were measured. RESULTS: Of the ACM subjects, 57 had active disease (group A), and 12 controlled ACM (group B). All pts with ACM presented structural changes: a higher LV indexed mass (112 ± 36, 118 ± 23 vs 74 ± 18 g/m(2), p < 0.001) and a higher relative wall thickness (0.45 ± 0.09, 0.50 ± 0.07 vs 0.40 ± 0.07, p = 0.003) compared to controls. Also, ACM pts had functional changes: reduced LV ejection fraction (57 ± 5, 55 ± 5 vs 64 ± 4%, p < 0.001) and altered diastolic function (E/A 1.0 ± 0.4, 1.1 ± 0.1 vs 1.3 ± 0.3, p = 0.005) compared to controls. Both longitudinal and radial LV S values were lower in ACM compared to controls: -16.5 ± 3.5, -16.8 ± 4.3 vs -21.5 ± 3.8%, p < 0.001 for longitudinal and 38.3 ± 12.3, 35.6 ± 11.8 vs 52.2 ± 11.7%, p = 0.002 for radial strain. CONCLUSIONS: ACM pts present LV concentric hypertrophy and LV systolic and diastolic dysfunction, even in controlled disease. Altered global LV systolic function appears to be due both to longitudinal and radial dysfunction.

Colocalization methods in pituitary tumorigenesis aged-related in MEN1 KO and wild type mice.

BACKGROUND: Colocalization analysis of confocal fluorescence and electron microscopy (EM) are important tools to detect the expression of multiple anterior pituitary hormones within the same cell. Heterozygous (Men1+/-) mice developed pituitary tumors, mostly reported somatolactotrophinomas and ACTH secreting pituitary adenomas but also nonfunctioning tumors. The aim of the study was to run immunohistochemistry protocols to study colocalization of pituitary hormones in newborn mice in tumoral and non-tumoral tissue in MEN1-KO and wild type control mice. METHODS: Pituitary samples from nine Men1+/- mice, 29-34 days old male mice (n=8) and one year old (n=1) and control group, four new born (1,5 days old) wild type (mus musculus) mice were analyzed by immunofluorescence immunohistochemistry (GH, PRL, gonadotrophs) to find hormonal colocalization in pituitary cell. Moreover, pituitaries were embedded in LRGold for immunogold labeling technique (GH, PRL, gonadotrophs and alpha-SU) also. RESULTS: Pituitary tumors, immunoreactive only for PRL were found in three MEN1 - KO mice. No sign of pituitary hyperplasia was found in MEN1-KO. MEN1-KO non-tumoral pituitary displayed similar immunoreactivity to wild type pituitary. Colocalization studies revealed individual cells PRL-FSH immunoreactive and GH-FSH immunoreactive in the non-tumoral tissue from MEN1-KO mice and in wild type pituitaries respectively but no colocalization in the tumoral tissue. In conclusion, colocalization is a feature of neonate mouse pituitary but not in adults. The MEN1-KO pituitary tumors were prolactinomas and unlike non-tumoral pituitary tissue of MEN1-KO, displayed no PRL-FSH colocalization.

Structural and functional changes of carotid wall properties in patients with acromegaly are not restored after 1 year of GH/IGF1 normalization.

UNLABELLED: Vascular changes are common in acromegaly (ACM). Current therapies can normalise the levels of both growth hormone (GH) and insulin-like growth factor (IGF1). OBJECTIVE: To establish whether the ACM vascular changes in patients with effectively managed disease are different from those in patients with an active condition. METHODS: 64 ACM patients were tested for serum GH (random and during an oral glucose tolerance test) and IGF1. Ultrasonography of the right common carotid (RCC) explored structural (the carotid diameter and intima-media thickness index (IMT)) and functional (the augmentation index (AIx), elastic modulus (Ep), and local pulse wave velocity (PWV)) arterial parameters in the ACM patients (groups A and B) and an age- and sex-matched control group of 21 patients without acromegaly (group C). RESULTS: The ACM patients were divided into 2 subgroups that had similar cardiovascular risk factor profiles: A (n=10, with controlled ACM), and B (n=54, with active ACM). The AIx was higher in groups A (27.7% [2.2-54.3]) and B (20.0% [ - 38.2-97.1]) than in group C (3.5% [ - 11.3-31.1]), p=0.01 and 0.002, respectively. The group B patients presented with poorer functional carotid wall parameters than the control subjects: Ep-95.5 [33-280] KPa vs. 77.5 [39-146] KPa, p=0.01; and PWV-6 [3.6-10.4] m/s vs. 5.4 [3.9-7.2] m/s, p=0.03.The ACM patients had greater RCC diameters (6.4 ± 0.6 mm vs. 5.7 ± 0.6 mm, p<0.001) and IMT values (0.72 ± 0.13 mm vs. 0.58 ± 0.08 mm, p<0.001) than the subjects in group C. CONCLUSIONS: Both the controlled and active ACM patients showed structural arterial changes. After 1 year of disease control, the patients with controlled ACM showed improvements in the functional, but not the structural, arterial parameters compared with the patients with an active condition.

Conventional examination versus immunohistochemistry in the prediction of hormone profile of pituitary adenomas. An analysis on 142 cases.

The large majority of neoplasms located in the sella turcica are benign pituitary adenomas derived from cells of the adenohypophysis. Pituitary adenomas represent the third most common primary intracranial tumor in neurosurgical practice, outnumbered only by gliomas and meningiomas. Their biology of pituitary adenomas is complex and they can cause a variety of endocrine syndromes and disorders, based on hormone profile secreted by proliferating cells. The aim of this study was to evaluate the routine conventional methods and highly specific immunohistochemical methods in order to accurately predict the type of hormone secretion. Our study was investigated 142 cases admitted with pituitary adenomas and treated by open surgery. Sections from each case were stained with routine Hematoxylin and Eosin method for histopathologic evaluation. Immunohistochemistry was performed on additional slides in order to detect specific pituitary adenomas. Based on the immunohistochemical profile of pituitary adenomas we found following results: 37 growth hormone (GH)-secreting adenomas, 23 prolactin (PRL)-secreting adenomas, 15 mixed GH-cell/PRL-cell adenomas, three mammosomatotroph cell adenomas, two adrenocorticotrophic hormone (ACTH)-secreting adenomas, one thyrotrophic cell adenoma, 18 gonadotroph adenomas, 30 null cell adenomas and 13 plurihormonal adenomas. No correlation was found between conventional features and the immunohistochemical profile. The immunohistochemical profile of functional pituitary adenomas is mandatory for a correct diagnosis. We revealed that staining characteristics of the tumor cells, such as acidophilic, basophilic or chromophobe are nowadays outdated as main principle of classification, because they not identify specific adenoma types.

[Insulinoma of the pancreas: analysis of a clinical series of 30 cases]. / Insulinomul pancreatic--analiza unei serii clinice de 30 de cazuri.

UNLABELLED: Insulinoma is the most frequent neuroendocrine pancreatic tumor and is the main cause for hypoglicemia due to endogenous hyperinsulinism. We performed an analysis of a clinical series in order to study the clinical and biological spectrum of presentation, the preoperatory imagistic diagnosis and results of the surgical approach. Between 1986-2009, 30 patients with symptoms suggesting an insulinoma were hospitalized in our department. Preoperatory localization of insulinomas was possible in 16 patients. The most sensitive imagistic methods were ecoendoscopy and magnetic resonance. Intraoperatory ultrasound was performed in 16 patients and its sensitivity in detection of insulinomas was 93%; the combination between intraoperative ultrasound and manual exploration of pancreas by the surgeon reached a 100% sensitivity. Before the intraoperatory ultrasound was used the tumor excision was predominantly done by extensive pancreatic resection, while after this was available in our centre more conservative (enucleo-resection) procedures were chosen. In 1 patient the resection was done by laparoscopy, and in 1 patient by robotic surgery. The dimensions of the tumor were less than 2 cm in most of the patients; 2 had nesidioblastosis and 2 had multiple insulinomas; all 28 patients proved to have benign insulinomas at histological specimens. Following surgery, the symptoms disappear in all patients. The most common complication following extensive pancreatic resections was acute pancreatitis, while after enucleation pancreatic fistula occurred more frequently. CONCLUSIONS: Due to small dimensions, the preoperative diagnosis of insulinomas is usually difficult, ecoendoscopy being the most sensitive method. Intraoperative ultrasound is essential for insulinoma localization and for chosing the optimal type of excision. Enucleation is the resection method to be chosen whenever this it is technical possible. In benign insulinomas the prognosis is excellent, surgical resection being curative in all cases.

Desmopressin increases IGF-binding protein-1 in humans.

CONTEXT: IGF binding protein-1 (IGFBP-1) is essential for IGF-I bioavailability. High levels of IGFBP-1 are encountered in critically ill patients and are a good predictor marker in acute myocardial infarction. The mechanisms responsible for the elevated IGFBP-1 levels in these conditions are still unclear. Interestingly, high levels of vasopressin have been reported in the above-mentioned conditions. OBJECTIVE: To study the effect of vasopressin on IGFBP-1 in humans. DESIGN: Placebo-controlled cross-over study in patients with central diabetes insipidus (CDI) in whom potential interference from endogenous vasopressin secretion is minimized. After a 3-day desmopressin washout period, each patient received i.v. saline on day 1 and desmopressin (3 mug) on day 2. Blood samples were taken after administration, every 2 h during the whole night, starting at 2000 h. PATIENTS AND SETTING: Fourteen inpatients with CDI in an endocrinology department of a university hospital. RESULTS: Serum IGFBP-1 increased within 4 h after 1-desamino-8-d-arginine vasopressin (DDAVP) by 375+/-73%, compared with a spontaneous fasting increase by 252+/-46% following placebo administration (P<0.05). No changes were registered in the levels of either classically regulators of IGFBP-1 (insulin, glucagon, and cortisol) or of IGF-I and glucose. The decrease in plasma osmolarity induced by DDAVP did not precede the increase in IGFBP-1. CONCLUSIONS: DDAVP increases serum levels of IGFBP-1. Further investigation is essential to unravel the clinical potential of this interaction in conditions associated with high IGFBP-1 levels.

Insulin resistance and insulin secretion in non-diabetic acromegalic patients.

UNLABELLED: Insulin resistance (IR) can be induced by high amounts of growth hormone (GH). AIM: To set up, in acromegaly without diabetes mellitus, a correlation between the disease activity in GH-secreting adenoma (AA) - assessed by minimum GH serum level during an oral glucose tolerance test (OGTT) - and severity of insulin resistance (IR), assessed by HOMA-IR index. METHODS: 75 out of 88 consecutive patients with acromegaly hospitalized in our department were included in this study. 13 patients proved to have diabetes mellitus and were excluded. Serum glucose, GH and insulin levels were measured by immunoradiometricassay basal and at 30, 60 and 120 minutes after a 75 g OGTT in 88 patients with active or cured acromegaly. IR was assessed using HOMA-IR index (Homa-IR=basal serum glucose (mg/dl) x basal serum insulin (mU/L)/22.5 x 18). A value over 2.5 was considered indicating IR. RESULTS: Out of 75 patients without diabetes mellitus, 36 subjects (48%) were presenting with IR (34 with active disease, 2 cured). We found a significant positive correlation (r=0.56, p<0.001) between AA and HOMA-IR. The GH minimal level corresponding to the intersection of the exponential regression curve with the HOMA-IR level of 2.5 was 8.8 ng/mL, a cut-off point indicating IR with 82% specificity and 78% sensitivity. The odds ratio for developing IR becomes significant at a minimum GH level during OGTT of 2 ng/mL (odds ratio 7.6, 95% confidence interval 2-29). CONCLUSIONS: The severity of IR revealed by acromegaly correlates with GH production. A GH serum level higher than 2 ng/mL during OGTT indicates an increased risk for developing IR. This cut-off level of GH can be used as one of criteria of cured disease, regarding the lack of metabolic effects.

[Double localization of pancreatic insulinoma. Diagnostic and therapeutic difficulties]. / Insulinom pancreatic cu dubla localizare. Dificultati de diagnostic si atitudine terapeutica.

Insulinomas are the most common cause of hypoglycemia resulting from endogenous hyperinsulinism. Because most of insulinomas are less than 2 cm in size and rarely they not may be visible by CT scan or transabdominal ultrasonography. Intraoperative ultrasonography may be a solution. Although as surgical method is preferred enucleation because operative time is shorter and easier and the low frequency postoperative complications, pancreaticoduodenectomy Whipple is indicated in selected cases. We report a case of double insulinoma located in the head of the pancreas in which the diagnosis and surgical treatment presented difficulties which determined a particular clinical evolution.

Enhanced protein kinase B/Akt signalling in pituitary tumours.

Pituitary tumours have previously been shown to harbour several abnormalities that cause deregulation of the cell cycle, particularly down-regulation of expression of the cyclin-dependent kinase inhibitor p27. However, it has been unclear whether these are the primary initiating events, or are secondary to other more proximate alterations in signalling pathways. In other cellular systems the Akt signalling pathway has been associated with downstream modulation of cell-cycle control. The aim of the present study was to test the hypothesis that Akt signalling is enhanced in pituitary tumours, and to see if changes in Akt expression are related to previous findings on low expression levels of the nuclear cell-cycle inhibitor p27 in pituitary tumours. We examined normal and adenomatous human pituitary tissue for mRNA and protein expression of Akt1, Akt2 and p27, and the activation of Akt, as well the phosphatase involved in the inactivation of Akt, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). In pituitary adenomas Akt1 and Akt2 mRNA were found to be over-expressed compared with normal pituitary, while PTEN transcripts showed similar levels between the two tissue types. Immunohistochemical expression of phospho-Akt was found to be higher in the tumours than normal pituitaries, while the protein expression of nuclear p27 and PTEN was lower in the adenomas. However, the expression of p27 and Akt were not directly correlated. PTEN sequencing revealed no mutation in the coding region of the gene in pituitary adenomas, and thus we did not locate a cause for the increased phosphorylation of Akt. In summary, we have shown over-expression and activation of the Akt pathway in pituitary tumours, and we speculate that cell-cycle changes observed in such tumours are secondary to these more proximate alterations. Since Akt is a major downstream signalling molecule of growth factor-liganded tyrosine kinase receptors, our data are most compatible with an abnormality at this level as the primary driver of pituitary tumorigenesis.

The influence of vasopressin deficiency and acute desmopressin administration on melatonin secretion in patients with central diabetes insipidus.

Melatonin secretion is modulated by the light-dark schedule, mainly through a sympathetic input to the pineal gland. Besides this, arginine vasopressin (AVP) has been found in the pineal glands of several animal species and there is experimental evidence that AVP modulates melatonin secretion in animals. However, the interaction between vasopressin and melatonin secretion in humans has not been systematically investigated. We proposed to study the nocturnal melatonin pattern in patients with central diabetes insipidus (CDI) who lack endogenous secretion of AVP, and the effect on their melatonin secretion of the agonist for V2 type receptors: desmopressin (1-Desamino [8-D Arginine] vasopressin). Plasma melatonin levels were measured in 14 patients with CDI, every 2 h starting from 22:00 h until 06:00 h, following iv injection of saline (day 1) and 3 microg desmopressin (day 2) at 20:00 h. The lights were turned off at 22:30 h and the samples were taken in a dim light. The plasma melatonin secretion pattern was normal in patients with CDI. Desmopressin at a dose 3 times higher than the antidiuretic one did not modify the melatonin levels or the time of the peak secretion. In conclusion melatonin secretion is not modulated by AVP in humans.

Diagnostic traps in porphyria: case report and literature review.

Porphyrias are metabolic disorders of heme biosynthesis, which encompass a broad range of symptoms and signs, neurologic, cutaneous or mixed. Because of lack of specificity and polymorphous clinical picture, porphyrias can mimic either neuropsychiatric, dermatologic, or gastrointestinal diseases. We present the case of a 58 years old man to whom clinical presentation suspicious of Addison's disease (melanoderma, fatigue, weight loss, intermittent abdominal pain) was the disguise of porphyria cutanea tarda. A general background of porphyrias and differential diagnosis with other forms of hepatic porphyria, as well as other causes of hyperpigmentation, are given. The clinician should be aware of the protean manifestations of porphyrias and include them in clinical judgment in various situations.

Amiodarone-induced thyroid dysfunction in cardiac patients from areas with iodine deficiency.

UNLABELLED: Amiodarone via iodine excess can determine thyroid dysfunction. AIM: to assess thyroid dysfunction in patients treated with amiodarone, according to previous daily iodine intake. MATERIAL AND METHODS: 63 patients treated with amiodarone were assessed. 11 of 63 were resident in a moderate iodine deficient area. Thyroid stimulating hormone (TSH), free and total thyroxine (T4) and total triiodothyronine (T3) were measured. Thyroid ultrasonography, color flow Doppler sonography (CFDS), radioiodine uptake (RAIU) at 2 and 24 hours were also performed. RESULTS: Amiodarone-induced thyrotoxicosis developed in 31 patients (49.2%); 17 patients (27%) remained euthyroid. Patients from iodine deficient areas developed more frequent hyperthyroidism (91% vs. 40.4%), at significant lower cumulative doses of amiodarone, and never hypothyroidism. Overt hyperthyroidism prevails (29/31 patients). Frequency of amiodarone-induced thyrotoxicosis type I was 19% (12/63), type II 12.7% (8/63), and of mixed forms 17.5% (11/63), without significant differences between the two geographical areas. There are no peculiar features of amiodarone-induced thyrotoxicosis versus common hyperthyroidism, but there is a tendency at recurrence of tachyarrhythmias at lower levels of T3 than in common hyperthyroidism. Amiodarone-induced hypothyroidism developed in 15 patients (23.8%) and none was from iodine deficient areas, but almost half show high levels of antithyroid peroxidase antibodies (ATPO). Subclinical hypothyroidism prevails (11/15 patients). CONCLUSION: a predictor for amiodarone-induced thyroid dysfunction is iodine deficiency in nutrition.

Somatotroph to thyrotroph cell transdifferentiation during experimental hypothyroidism - a light and electron-microscopy study.

Somatotroph and thyrotroph pituitary cells share a common precursor cell expressing the transcription factor Pit1 in ontogeny. Cells expressing both thyrotropin (TSH) and growth-hormone (GH) are found in adult rat pituitary and in human pituitary adenomas in acromegaly, and these tumors contain both thyrotropin-releasing hormone (TRH) and the TRH receptors (TRHR). It has been shown that stimulation of TSH expression in primary hypothyroidism promotes changes suggestive of somatotroph to thyrotroph cell transdifferentiation. We tested this hypothesis and the role of TRH in experimental primary hypothyroidism in rats. Adult female Long-Evans rats, 6 months old, were administered the antithyroid drug methimazole (0.1% w/v) in the drinking water for 42 days. Animals were sacrificed by perfusion fixation under anaesthesia at weekly intervals and pituitary tissue processed in acrylic resin for immunofluorescence and immuno-electronmicroscopy for TSH, GH and TRHR. In the hypothyroid rat pituitary immunofluorescent somatotrophs were greatly reduced in number and gradually replaced by thyrotrophs during methimazole administration. Colocalization of GH and TSH in the same cell was noted. Immunoelectronmicroscopy demonstrated the development of enlarged thyrotrophs with dilated rough endoplasmic reticulum containing an electron-dense material and intracisternal granules, both of which are immunoreactive for TSH ('thyroidectomy cells'). The somatotrophs showed reduced GH immunoreactivity and also the presence of TSH-type, small-size secretory granules. This suggests that the greatly increased number of TSH-cells in methimazole-induced-hypothyroidism is due, at least partially, to the transdifferentiation of somatotroph into thyrotroph cells. TRHR immunofluorescence was expressed in many somatotrophs in normal rat pituitary and unlike immunoreactive GH, its expression was enhanced during hypothyroidism. The number of TRHR-immunoreactive cells increased in parallel with the number of TSH-immunoreactive cells. This indicates a role for TRH stimulation in the transdifferentiation process. Taken together, these data suggest that, in addition to the cell mutation mechanism involving an early totipotential progenitor cell, transdifferentiation of existing somatotroph cells also plays a part in the pathogenesis of multihormonal GH-secreting adenomas.

Presence of oxytocinergic neuronal-like cells in the bovine pineal gland: an immunocytochemical and in situ hybridization study.

In the last decade, there is more and more evidence showing the role of the central innervation of the pineal gland, but there are controversies around the intra or extrapineal origin of oxytocin found within the pineal tissue. In order to check the amount and the site of synthesis of oxytocin in the bovine pineal gland, we performed a morphological and chromatographic study. The anatomical distribution of the pineal oxytocin was explored by immunohistochemistry and in situ hybridization for the corresponding mRNA. The results confirm the presence of oxytocinergic fibres in the bovine pineal, some of them endowed with big varicosities. Immunohistochemistry also displayed neuronal-like cells in the pineal body. The in situ hybridization for the mRNA encoding pre-pro/oxytocin-NFZ I used a mixture of three oligonucleotide probes labelled with (35)S. This allowed identification of positive cells in the bovine pineal. The content in oxytocin was evaluated by radioimmunoassay during 5 months, from July to November, and the peptidic extract revealed an increase of pineal oxytocin immunoreactivity in September as compared with July or November. The significance of intrinsic oxytocin innervation of the bovine pineal gland, as well as the threefold increase of the oxytocin content in the pineal in September, remains to be elucidated.

TRH synthesis in "mute" thyrotropinomas: cause-effect or coincidence?

In the pathogenesis of thyrotropin (TSH) immunopositive pituitary adenomas, trigger mutagenetic events are well recognized. However, the way towards a clinical significant tumor is followed under the pressure of growth factors, among which the intrapituitary synthesis of releasing factors could bring a significant contribution. In this study, the production of thyrotropin releasing hormone (TRH) and beta TSH chain was evaluated at the mRNA level by in situ hybridization and end product level by immunohistochemistry, in 18 patients submitted to neurosurgery for pituitary macroadenomas. The hormonal sampling showed abnormal secretion for FSH in 5 and TSH in 4 patients. Seven cases were immunopositive for TSH, and expressed TSH beta mRNA. All but one out of these expressed also TRH mRNA. FSH immunoreactivity was documented in 12/ 18, only one of these being negative for TRH mRNA. Paracrine TRH could contribute to the pathogenesis of these "mute" adenomas.

A chemical method to isolate hypothalamic nonapeptides by coupling cyst(e)in with bimane.

Vasopressin, oxytocin as well as other active nonapeptides (vasotocin, etc) are difficult to isolate from tissues. Traditionally they were identified using cumbersome biological assays or immunoassays, commercially unavailable, and with some cross reactivity. Based on the fact that all these peptides have two Cysteines in their molecules we developed a simple, sensitive and specific method to detect them by HPLC after pre-column fluorescent derivatization with monobromobimane (mBBr). The peptides were separated on a Vydac C18 column after reduction with Tris (2-carboxyethyl) phosphine (TCEP) and derivatization with mBBr for 5 minutes in dark. Using this method we were able to detect specific peaks for arginine-, lysine-vasopressin, and vasotocin at levels as low as 10 pmol. The method can be used to detect other active peptides with cyst(e)ins in their molecule, as well.

Melatonin shortens the survival rate of Ehrlich ascites-inoculated mice.

UNLABELLED: OBJECTIVES Pineal gland may have a role in organism's protection against cancer. Melatonin as well as still unidentified low-weight molecular pineal substance(s) have been reported to have growth inhibitory effect on different tumor cells. We tested the influence of melatonin and of a bovine pineal extract on the survival rate of AKR mice inoculated with Ehrlich ascites. The tumor is known to have an accelerated development after pinealectomy. MATERIAL AND METHODS: Male AKR mice, kept under a 14/10 hours - Light /Dark cycle, were inoculated intraperitoneally (i.p.) with 1.5x 10(6) Ehrlich ascites cells. On day three after inoculation the animals were divided in three groups (n=10). Each animal received i.p. daily (20.00H), until their death, 250 microl of solution containing melatonin (250 microg), pineal extract (equivalent of 1 bovine pineal gland) or saline. RESULTS: The average survival rate of the animals treated with melatonin was shorter (14.8+/-2.23 days) compared to control animals (21.9+/-2.21 days)(p=0.01). The animals treated with the pineal extract had a longer survival rate (22.6 +/- 1.8 days) but not statistically significant. The pineal extract was not available for testing at higher doses. CONCLUSION: In our model, melatonin had a deleterious effect on the survival rate raising the question whether it is correct to assume that the hormone shows lack of adverse reactions.

A cytotoxic, apoptotic, low-molecular weight factor from pineal gland.

Previous studies suggest that the pineal gland may play a role in tumour growth inhibition. In this respect, melatonin, as the major hormone of this gland, has been extensively studied. However, there is growing evidence for the existence of other yet unknown pineal factors that may have tumour growth inhibiting properties. Here we describe the partial purification of a highly cytotoxic low molecular weight (<400 Da) hydrophilic fraction (designated F2M3R), starting from a porcine pineal extract (PE), via methanol precipitation followed by reverse-phase HPLC. F2M3R is cytotoxic for a highly apoptosis-resistant human erythroleukemia cell line (K562) at a concentration as low as 30 microg/ml. The viability of the cells was not influenced by an identical prepared porcine pituitary extract or by melatonin. PE induces apoptosis in K562 cells as indicated by three different criteria: morphology, in situ TUNEL assay and bi-parametric FACS analysis with annexin V and propidium iodide, but does not influence the viability of stimulated peripheral blood mononuclear cells. These observations warrant further purification and validation of the cytotoxicity in a panel of different human tumour and non-malignant cells.

Blood-brain barrier for human growth hormone and insulin-like growth factor-I.

There is a blood-brain barrier (BBB) for GH. A certain, unknown amount of GH passes the BBB, acts on the neuronal GH receptors and directly influences the brain mechanisms serving the feedback and ultradian secretion of GH. The high density of GH receptors in the choroid plexus suggests a possible receptor-mediated transcytosis transport. The effects of GH on brain development, neuronal plasticity and neuroprotection seem to be mediated by IGFs. GH and IGFs are also synthesized in the brain. The relative contributions to brain functions of GHs produced inside and outside the BBB are unknown. The cerebrospinal fluid (CSF) space is the compartment inside the barrier accessible to clinicians. High GH levels in CSF were reported in acromegaly and also a small increase was reported after chronic administration of hGH in GH-deficiency syndromes. For the practitioner it is necessary to determine the normal range of hGH levels in CSF.