Bone metabolism in ochronotic patients.

We investigated skeletal involvement in five male and two female patients with ochronosis, aged 26-82 years. The main parameters of mineral metabolism, together with biochemical markers of bone resorption (urinary N-telopeptides of type I collagen) and formation (serum bone isoenzyme of alkaline phosphatase and serum osteocalcin) were evaluated. In the same subjects lumbar spine and femoral bone mineral density (BMD) were also measured by dual energy X-ray absorptiometry. All patients but the younger 26-year-old patient had lower than normal bone mass at femoral neck and total hip, showing marked osteopenia in three cases and osteoporosis in the remaining three cases. However, at lumbar spine BMD measurement provided spuriously overestimated results, because of intervertebral disc calcification and osteophyte formation. As far as biochemical markers of bone turnover are concerned, the most relevant finding was the increased N-telopeptides of type I collagen urinary excretion. Our results suggest that ochronosis may be associated with increased bone resorption rate leading to an accelerated bone loss. A role of the homogentisic acid polymer deposit in bone matrix and cells, possibly with osteocyte damage and interference in collagen metabolism, might be hypothesized.

The lungs and platelet production.

Several studies have suggested that thrombopoiesis may occur in the lungs. To investigate the role of the lungs in platelet production, we measured automated platelet parameters in blood from the pulmonary artery and the radial artery (n=125) or aorta (n=26) in patients undergoing aorto-coronary bypass. No significant differences were found between pulmonary and radial arterial blood with regard to platelet count (192.132 +/- 46.250 vs. 192.004 +/- 46.294 x 10(9)/l), mean platelet volume (11.03 +/- 1.04 vs. 11.03 +/- 1.03 fl), plateletcrit (0.212 +/- 0.051 vs. 0.212 +/- 0.051 x 10(-2)), platelet distribution width (14.48 +/- 2.16 vs. 14.47 +/- 2.08 fl) and platelet-large cell ratio (0.350 +/- 0.076 vs. 0.351 +/- 0.078). Similar results were obtained in comparisons between pulmonary arterial and aortic blood. A coefficient of linear correlation of 0.98 was found between the pulmonary and radial arterial and aortic platelet counts. These findings suggest that the platelet population entering the lungs was the same as the platelet population leaving them. Our results do not therefore support the theory of pulmonary platelet production.

HLA antigens in alkaptonuric patients.

BACKGROUND: In alkaptonuric patients a disabling ochronotic arthropathy develops, due to the deposit of a pigmented polymer of homogentisic acid. Since in inherited diseases the clinical expressions may be multifactorial, involving genetic and environmental factors, where the HLA system may play a role, we studied HLA antigens in ochronotic patients. METHODS: The study was carried out in 21 members of three families of six ochronotic patients and in two isolated ochronotic patients. The HLA typing has been done testing for antigens from loci A, B and C, by international standard microlymphocytotoxicity method, and for loci DR and DQ, by fluorescence method on immunologically isolated cells by means of antibody-coated microspheres. The chi square test was used for statistical analysis, with Yates correction due to the low number of observations. RESULTS: Despite the limited number of subjects, due to the rarity of the disease, a significantly higher prevalence of HLA-DR7 antigen was found in the alkaptonuric patients when compared to a general population (p<0.02), suggesting a possible association, while the prevalence of HLA A, B, C and DQ showed no significant differences. CONCLUSIONS: It might play a role in the pathophysiology and in the clinical expression of the disease.

Hormone metabolism in the pulmonary circulation.

We measured hormonal levels in blood samples from pulmonary and radial arteries in 117 patients undergoing aorto-coronary by-pass surgery with the aim of investigating the role of the pulmonary vessel endothelium in hormone metabolism. Insulin and glucagon concentrations were significantly higher in pulmonary artery blood with respect to radial artery blood (73 +/- 65 vs. 65 +/- 47 pmol/l, p < 0.005, and 80 +/- 49 vs. 73 +/- 51 ng/l, p < 0.01, respectively), while no difference was found for growth hormone, prolactin, C peptide, insulin-like growth factor I, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, parathyroid hormone, thyroglobulin, triiodothyronine, thyroxine, free triiodothyronine, and free thyroxine. Moreover, prolactin concentrations were more than twice the normal levels, this being an effect of propafol and the opiate fentanyl used for the general anesthesia. Assuming that the arteriovenous differences observed are a marker of peptide hormone degradation, our study has demonstrated that with similar kinetics insulin and glucagon secreted into portal circulation and escaping from hepatic extraction undergo further homeostatic removal of about 9-10 % in the pulmonary circulation before entering the general circulation.

Respiratory changes in human red cells.

To investigate physiological respiratory changes in human red cells, we measured automated red cell parameters in samples from the pulmonary and radial arteries of 86 patients undergoing aorto-coronary bypass and from the pulmonary artery and the aorta in 23 patients. Our results showed higher mean corpuscular volume (88.53 +/- 5.06 fl vs. 88.12 +/- 4.94 fl, P < 0.000001), haematocrit (0.369 +/- 0.039 vs. 0.367 +/- 0.038, P < 0.0005), red cell distribution width (43.38 +/- 4.16 vs. 43.04 +/- 4.05 fl, P < 0.000001) and a lower mean corpuscular haemoglobin concentration (338.3 +/- 15.9 vs. 339.9 +/- 16.8 g/l, P < 0.005) in pulmonary arterial as compared to radial arterial blood. There were no differences with respect to haemoglobin concentration, red blood cell count, or mean corpuscular haemoglobin. Similar differences were observed between pulmonary arterial and aortic blood. Our results suggest cyclic respiratory modifications of red cell parameters attributable to the CO2 Jacobs-Stewart cycle.

The acute myocardial infarction in very elderly.

A total 325 patients were studied at admission for myocardial infarction, measuring plasma fibrinogen (FBG), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) and automatized hemocromocytometric parameters in order to contribute to explain the excess mortality reported in very elderly patients. It was found that age positively correlated with fibrinogen and LDH values and inversely with CPK, hemoglobin concentration and lymphocyte count. The unpaired comparison of the variables studied in age subgroups showed no differences between patients aged 65 or less than 65 years and patients aged 66-75 years. In patients aged over 75 years FBG, neutrophile count and LDH were significantly higher in respect to 65 or less and 66-75 years age subgroups and hemoglobin concentration, red blood cell count, hematocrit and lymphocyte count were lower. In the very elderly patients the study shows a biochemical feature suggesting delayed hospitalization for myocardial infarction, that may contribute to their poorer prognosis.

Osteocalcin metabolism in the pulmonary circulation.

The aim of this study was to evaluate the role of the pulmonary vessel endothelium in the removal of circulating osteocalcin, by measuring the osteocalcin levels in serum from pulmonary and radial artery blood from 39 patients undergoing aorto-coronary bypass. Because of the discrepancies between methods of measurement, two methods were used. Significant differences were observed in group A (n = 18), tested with heterologous radioimmunoassay (2.85 +/- 0.67 microg l-1 in the pulmonary versus 2.69 +/- 0.67 microg l-1 in the radial artery serum, P<0.001) and in group B (n = 21), tested with a two-site immunoradiometric assay (5.22 +/- 1.46 versus 4.93 +/- 1.36 microg l-1, P<0.01). The percentage differences were -5.54 +/- 4.76% (P<0.001) in group A and -4.99 +/- 8.13% (P<0.01) in group B; the comparison between the percentage differences was not significant. These different osteocalcin concentrations between the two vascular compartments were considered a marker of osteocalcin degradation. Therefore, the study seems to demonstrate that, as well as kidney, liver and bone, the lung is a relevant site of osteocalcin catabolism. The proteolytic activity of pulmonary vessel endothelium seems to involve about 5% of the circulating peptide.

Role of monocytes in the early phase of acute myocardial infarction.

BACKGROUND: In acute myocardial infarction peripheral leucocytosis occurs early and fibrinogen levels increase in response to the tissue injury, expressed by the enhanced enzyme plasma levels. The aim of the present study has been to investigate if a unifying link between these modifications might be found. METHODS: In 325 patients, 246 men and 79 women, 61.46 +/- 11.00 and 70.03 +/- 11.30 years mean age respectively, at admission for myocardial infarction and before treatment, we simultaneously measured plasma fibrinogen (FBG), hemochromocytometric parameters and plasma enzyme aspartate and alanine transaminase (AST and ALT), lactate dehydrogenase (LDH) and creatine phosphokinase (CPK). The statistical analysis was performed by using standard multiple regression for dependent variable FBG and for the variables white blood cells (WBC), monocyte number, large unstained cells (LUC) and CPK. RESULTS: The results showed that FBG was significantly correlated with monocyte (p < 0.001) and LUC (p < 0.05) counts; assumed as dependent variable, further on with FBG, monocyte number was correlated with WBC count, LUC and CPK; further on with monocyte number, WBC with neutrophile and lymphocyte counts, LUC with CPK, CPK with LDH. CONCLUSIONS: The study seems to show that monocyte modifications occur already in the early phase of myocardial infarction. These modifications are directly related to damage extension, as deducible from CPK levels, and seem to mainly modulate FBG and WBC, since the cytokines and hematopoietic growth factors production by activated monocytes.

Contribution of immunological mechanism in a case of ochronotic arthropathy.

We have studied cell mediated and humoral response on the synovial fluid and peripheral blood of a 60-year-old man affected by ochronosis. Results showed raised percentages of CD3+, CD8+, HLA-DR+ and CD25+ T cells, presence of TNF, enhanced levels of immunoglobulins and low levels of C3 in the synovial fluid, and a higher rate of HLA-DR+ and CD25+ T lymphocytes in peripheral blood. These data suggest a possible role of immunological response the evolution of an ochronotic arthropathy.

Lactate dehydrogenase and its isoenzymes in the marrow and peripheral blood from haematologically normal subjects.

Lactate dehydrogenase (LDH) activity and its isoenzymatic fractions were measured in bone marrow blood and in peripheral venous blood from 16 haematologically normal subjects. Total LDH activity was significantly higher in marrow than in venous blood (428.8 +/- 98.4 vs 260.1 +/- 40.2 mU/l, p < 0.0001). The same was true for the absolute values of its isoenzymatic fractions. The percentage fractions LDH 1 and LDH 5 were similar in the two regions, while LDH 3 and LDH 4 were higher in medullary blood (p < 0.05) and LDH 2 was higher in peripheral blood (p < 0.05). The Spearman test showed a limited correlation between marrow and peripheral total LDH activity values (p < 0.05). This seems to be at least in part sustained by the highly significant correlations existing in LDH 3 and LDH 4 values, reported to be pre-eminent isoforms in maturing haematopoietic cells (p < 0.005 and p < 0.001, respectively). These findings could be attributed to an apoptotic regulation of marrow cell production.

Heat inactivable fraction of alkaline phosphatase in marrow and peripheral blood.

We measured the total alkaline phosphatase activity and their heat-inactivable and heat-stable fractions both in marrow and in peripheral blood, collected from 16 patients undergoing bone marrow aspiration for diagnostic purposes. Total enzymatic activity and the heat-inactivable fraction, of bone source, were significantly higher in serum from medullary blood (p < 0.001). Although the heat-stable fraction, of non-bone source, was higher in serum from peripheral blood (p < 0.05), the inactivable fraction showed there the greater component (51.56 +/- 11.91%). Results seem to indicate that the quantification of the enzymatic fractions and of the ratios between them in comparison with marrow and peripheral blood may be useful in cases that pose diagnostic difficulties.

Platelet and hemocoagulative changes in elderly atherosclerotic patients after treatment with the antiaggregating drug picotamide.

The effects of the antiaggregating drug picotamide on platelet and hemocoagulative parameters were evaluated in a group of 28 elderly atherosclerotic patients. After 1 month of treatment, consisting of one 300 mg tablet three times per day, a significant inhibition of platelet aggregability in response to ADP, adrenaline, arachidonic acid and collagen and a reduction of the mean platelet volume (-4.62+/-7.46%, P<0.003) was observed. Fibrinogen plasma levels, unchanged at the 15th day, were significantly reduced at the 30th day, from 445.6+/-145.95 to 382.72+/-110.56 mg/dl (P<0.01), while no changes were observed in antithrombin III, factor VII, D-dimer plasma levels and in plasmatic and urinary fibrin(ogen) degradation products values. The results seem to indicate a reduction after antiaggregating treatment of the interaction between the platelets and the vessel wall, that may be responsible for the changes observed.

Differences in platelet count and size between marrow and peripheral blood.

Automatized platelet parameters were evaluated in 23 unselected patients undergoing a bone marrow examination for diagnostic purposes, both in medullary and in peripheral blood, with the aim of investigating the biological significance of platelet volume heterogeneity. In the marrow blood the platelet count was 10.07 +/- 21.25% (p < 0.05) lower and the platelet volume 7.93 +/- 6.88% (p < 0.001) higher compared to peripheral venous blood: 275,173 +/- 108,079 versus 296,652 +/- 104,814/mm3 (p < 0.05) and 8.93 +/- 1.46 versus 8.23 +/- 1.57 fl (p < 0.001), respectively. The same behavior was observed between the marrow and the arterial blood collected from 12 out of the 23 patients, while no significant difference was observed between arterial and venous blood. The differences observed might be attributable to a platelet population replacement under homeostatical regulation and seem to support the concept that the large platelets are younger elements.