Gianfranco Tonnarini

Bone and Mineral Metabolism in Patients with Primary Aldosteronism

Primary aldosteronism represents major cause of secondary hypertension, strongly associated with high cardiovascular morbidity and mortality. Aldosterone excess may influence mineral homeostasis, through higher urinary calcium excretion inducing secondary increase of parathyroid hormone. Recently, in a cohort of PA patients a significant increase of primary hyperparathyroidism was found, suggesting a bidirectional functional link between the adrenal and parathyroid glands. The aim of this study was to evaluate the impact of aldosterone excess on mineral metabolism and bone mass density. In 73 PA patients we evaluated anthropometric and biochemical parameters, renin-angiotensin-aldosterone system, calcium-phosphorus metabolism, and bone mineral density; control groups were 73 essential hypertension (EH) subjects and 40 healthy subjects. Compared to HS and EH, PA subjects had significantly lower serum calcium levels and higher urinary calcium excretion. Moreover, PA patients showed higher plasma PTH, lower serum 25(OH)-vitamin D levels, higher prevalence of vitamin D deficiency (65% versus 25% and 25%; P < 0.001), and higher prevalence of osteopenia/osteoporosis (38.5 and 10.5%) than EH (28% and 4%) and NS (25% and 5%), respectively. This study supports the hypothesis that bone loss and fracture risk in PA patients are potentially the result of aldosterone mediated hypercalciuria and the consecutive secondary hyperparathyroidism.

Oxidative stress in patients affected by primary aldosteronism

Objective: Primary aldosteronism, an important form of secondary hypertension, is associated with significant increase of cardiovascular risk (ischaemic heart, cerebrovascular events, arrhythmias) (relative risk 4.6). The specific treatment of primary aldosteronism significantly reduces cardiovascular risk. In addition to high blood pressure values and direct action of aldosterone, new mechanisms such as increased oxidative stress are involved in the development of organ damage, metabolic, endothelial and coagulation complications. Methods: The aim of the study was to evaluate parameters of oxidative stress in 38 patients (21 men, 17 women, mean age 53.3 ± 4.7 years) with primary aldosteronism [11 aldosterone-producing adenoma (APA) (4 men, 7 women, mean age 50.2 ± 4.5 years) and 27 idiopathic adrenal hyperplasia (IHA) (17 men, 10 women, mean age 54.5 ± 5.3 years)] at diagnosis and after specific treatment (surgical or pharmacological), with respect to 50 patients with essential hypertension (26 men, 24 women, mean age 49 ± 7.4 years) and 50 healthy individuals (28 men, 22 women, mean age 48.7 ± 4.4 years). Results: Patients with primary aldosteronism showed significant increase of NADPH oxidase (Nox2-dp) plasma levels and urinary isoprostanes (34.9 ± 4.3 &mgr;g/dl and 216.3 ± 15.7 ng/mg, respectively; P < 0.05) than essential hypertensive patients (27.1 ± 3.7 &mgr;g/dl and 144.8 ± 9.4 ng/mg, respectively; P < 0.05). In APA patients undergoing adrenalectomy, we observed significant reduction of both circulating levels of Nox2-dp (29 ± 2.1  vs. 22,4 ± 1.7 &mgr;g/dl; P < 0.05) and urinary levels of isoprostanes (221.1 ± 10.5 vs. 132.6 ± 8.7 ng/mg; P < 0.05). Conclusions: This is the first study showing an increased oxidative stress in primary aldosteronism, characterized by increased serum levels of Nox2-dp and urinary excretion of isoprostanes. After APA removal with laparoscopic adrenalectomy, we found reduction of serum Nox2-dp and urinary isoprostanes.

ET-1 Plasma Levels During Cold Stress Test in Sclerodermic Patients

Endothelial cell injury in blood vessels of small arteries and capillaries is considered the primary event in the pathogenesis of systemic sclerosis (SSc). Because endothelin-1 (ET-1) is mainly released in the site of endothelial cell damage, thereby inducing a potent vasoconstriction, it was our intention to study ET-1 release in a group of SSc patients during a cold pressor test (CPT). Twelve SSc patients and a control group of 10 healthy subjects underwent CPT. Blood samples for ET-1 assay were collected at 90 and 180 seconds of exposure to cold stress. Heart rate and blood pressure were recorded at the same intervals. A capillaroscopic examination was performed in both groups before and after CPT. We observed significantly higher levels of plasma ET-1 in SSc patients compared with those of the control group at baseline (P=0.007) and at 90 (P=0.006) and 180 seconds (P=0.03) of CPT. During the test, the capillaroscopic examination showed a dramatic worsening of the vascular picture that was parallel to the increase in ET-1 plasma levels. This suggests that higher ET-1 plasma levels can have a part in the acute vascular reac tivity of SSc patients undergoing CPT.

Cholinergic Staining of Bronchus- Associated Lymphoid Tissue

The cholinergic staining of human bronchus-associated lymphoid tissue (BALT) was studied in humans. Morsels of the human lung (containing BALT) were harvested, after having obtained the appropriate approvals, during autopsies in 24 human subjects. The samples were stained by means of the enzymatic technique of acetylcholinesterase (AChE) and/or the monoclonal immunohistochemical method of choline acetyltransferase (ChAT). A morphometrical analysis was performed by means of quantitative analysis of images and statistical analyses of the data. AChE and proteins were also measured by biochemical assay. Our results demonstrate that both AChE and ChAT are localized in the BALT of young and old humans. These enzymes undergo age-related changes. The biochemical values of AChE are as follows: 22.3 ± 2.5 international units in young subjects and 78.5 ± 1.9 international units in old ones. The morphometrical values of AChE confirm the biochemical ones. The morphometrical data for ChAT are 31.6 ± 1.4 conventional units in young subjects and 71.2 ± 1.5 conventional units in old ones. Further results are needed to draw definite conclusions concerning the location and the distribution of these two enzymatic activities in BALT. In our opinion, the presence of AChE and ChAT in BALT can be both ‘non-neuronal’, with a role in general metabolism, and/or ‘neuronal’ with a role in neuroimmunomodulation.

Distribution of Catecholaminergic Neurotransmitters and Related Receptors in Human Bronchus-Associated Lymphoid Tissue

Background: The functions of the bronchus-associated lymphoid tissue (BALT) are under the control of the autonomic nervous system (sympathetic and parasympathetic nerve fibers). Objectives: The relationships between the adrenergic nerve fibers and β-adrenergic receptors were studied in the human BALT with the aim to demonstrate a probable neuromodulation. Methods: Morphological observations (staining with hematoxylin-eosin and scanning electron microscopy images) were carried out on samples of human BALT harvested during autopsies. Moreover, histochemical staining for norepinephrine (adrenaline = adrenergic nerve fibers) as well as for other catecholamines was performed. Finally, β-adrenergic receptors were stained by means of a β-blocking, radiolabeled drug (pindolol 125I). All our data were submitted to morphometric analysis (quantitative analysis of images and statistical analysis of data). Results: Our results provide direct evidence of the presence and distribution of catecholaminergic nerve fibers and related β-adrenergic receptors in BALT. β-Adrenergic receptors are present above all in the most richly innervated part of the BALT, and are, therefore, in close relationship with their related adrenergic nerve fibers. Conclusions: Studies on the distribution of adrenergicneurotransmitters and related β-adrenergic receptors in the human BALT are the first step for the demonstration of a probable neuromodulation of BALT.

Age-related changes in the human thymus studied with scanning electron microscopy

This investigation describes some morphological, age‐related changes in different compartments and cells of the human thymus. Scanning electron microscopic observations were done on human thymus samples obtained from four young and eight elderly patients during thoracic surgery and/or diagnostic biopsy of the thymus, after receiving authorization from the Ethical Committee of our university. The morphological data were submitted to quantitative image analysis so as to obtain quantitative results. Subsequently, the related values were used for statistical analysis. Our findings demonstrate that (1) all thymus compartments (subcapsular spaces, cortical, medullar, thymus microenvironment) contain numerous thymocytes even after the thymus has aged. (2) In older humans, residual thymus lymphoid islets contain, in addition to fatty cells and/or fibrous cells, also the same types of resident and nonresident cells (permanent and moving cells) that are found in young and adult subjects. (3) Endothelial cells of thymus microvessels contain numerous gaps. These gaps are tight in young subjects and become loose with age. (4) Thymocytes, in older subjects, are always found near these loose endothelial gaps of thymus microvessels. (5) While thymus cortical microvessels are provided with pericytes and/or periarteriolar spaces, microvessels of the thymus medullar are free of such spaces. Our morphological and quantitative results lead us to consider the possibility that the thymus fraction of resident and permanent cells (including thymocytes and reticular epithelial cells) is larger in younger subjects compared with older ones. The endothelial loose gaps of thymus microvessels, in older subjects, can allow the bidirectional transit of thymocytes through the wall of the said microvessels. Microsc. Res. Tech., 2008.

Dopamine receptor subtypes in the human coronary vessels of healthy subjects

Objective: Dopamine D1–D5 receptors subtypes were studied in human coronary vessels of healthy subjects to assess their localization and their expression. Methods: Samples of intraparenchymal and extraparenchymal branches of human coronary arteries and veins were harvested from four normal native hearts explanted from four young brain dead heart donors in case of orthoptic transplant, not carried out for technical reasons. In all the samples morphological, biochemical, immunochemical, and morphometrical studies were performed including quantitative analysis of images and evaluation of data. Results: Microanatomical section showed healthy coronary vessels, which expressed all dopamine receptors (from D1 to D5) with a different pattern of distribution between the different layers, in the intra and in the extraparenchymal branches.D1 and D5 (with a prevalence D1 over D5) were distributed in the adventitia and to a lesser extent in the outer media but they were absent in arterioles, capillaries and venules. Endothelial and the middle layer showed D2, D3 and D4 receptors, with a greater expression of D2. Immunoblot analysis of dopamine monoclonal antibodies and dopamine receptors showed a different migration band for each receptor: D1 (45 KDa); D2 (43 KDa); D3 (42 kDa); D4 (40–42 KDa); D5 (38–40 KDa) Conclusion: These findings demonstrate the presence of all dopamine receptor subtypes in the wall of human coronary vessels of healthy subjects. Dopamine D1 and D2 receptor subtypes are the most expressed, suggesting their prominent role in the coronary vasoactivity.

Cholinergic Innervation of Balt (Bronchus Associated Lymphoid Tissue) in Rat

The presence and distribution of acetylcholinesterase (AChE) and cholineacetyl transferase activities (Chat) were examined in the bronchusassociated lymphoid tissue (BALT) of juvenile, adult and old rats. Histoenzymatic and immunochemical methods were used in association with quantitative analysis of images and statistical analysis of the data. Our results showed that both AChE and Chat activities were primarily confined to the BALT lymphoid cells. Only a low level of activity was observed in the sub-pleural parenchyma of the lung and in the wall of the bronchus. Moreover, both AChE and Chat activities in the BALT are specifically located in the lymphoid cells. Histoenzymatic staining and corresponding values of quantitative analysis of images confirmed morphological and immunochemical results. Finally, the intensity of histoenzymatic staining for AChE and of immunochemical staining for Chat in BALT of rats strongly decreases with age. On the basis of our results we hypothesize that both AChE and Chat activities may play an important role in BALT and both these enzymes undergo specific age-related changes.

PLASMA ATRIAL NATRIURETIC HORMONE IN HYPERTHYROIDISM

In 22 hyperthyroid patients, atrial natriuretic hormone (ANH) levels (71.91 +/- 21.03 pg/ml), measured during a 3-h-Holter, were found to be significantly higher (p < 0.001) than those in 20 age-matched normal subjects (37.22 +/- 8.73 pg/ml). We have demonstrated that the presence of tachiarrhythmias does not influence ANH release. The positive and significant correlation of FT3 with both ANH and heart rate confirms the hypothesis of a direct action of thyroid hormones on ANH release.

Progressive loss in circulating volume during haemodialysis can be monitored by time voltage integral area of QRS complex: pilot study

Introduction Time voltage area of QRS is a parameter that showed a close association with modifications in endoventricular volume. The aim of the study was to investigate the efficacy of this parameter in identifying progressive reduction in circulating blood volume (BV) during haemodialytic treatment (HT). Material and methods Thirteen uraemic patients were studied. XYX like leads were monitored before, during and after HT. Summation of areas of each QRS complex was named QRS total area (TA). Results Increase in QRS TA and decrease in BV were found after vs. before HT. Progressive increase in QRS TA is strongly linked to a progressive reduction of BV during HT. Conclusions These findings encourage use of ECG monitoring during HT with a dual purpose: rhythm and haemodynamic control. In fact, excessive or insufficient subtractions of water, with consequent hypotensive or cardiorespiratory crisis, are the most frequent complications in these patients.