Ettore Bartoli

Hyperglycemia-Induced Thrombin Formation in Diabetes: The Possible Role of Oxidative Stress

Diabetes is characterized by the existence of a thrombosis-prone condition, possibly related to hyperglycemia. However, the mechanism linking hyperglycemia to the activation of the coagulation cascade is still unclear. It has been recently suggested that diabetes is accompanied by increased oxidative stress. In this work, the possibility that oxidative stress may be involved in the hyperglycemia-induced coagulation activation has been evaluated. Prothrombin fragment 1 + 2 (F1+2), which represents a reliable marker of the amount of thrombin released in the circulation, has been chosen for studying thrombin formation in vivo. In nine type II diabetic patients and in seven healthy control subjects, matched for age and body mass index, three different experiments were performed: oral glucose tolerance test (OGTT), intravenous antioxidant glutathione (GSH) administration for 2 h, and OGTT plus intravenous GSH administration. Samples were drawn at −15 min and every 30 min from 0 to 180 min. During the OGTT, F1+2 significantly increased in both diabetic and healthy subjects. GSH administration during OGTT normalized this phenomenon. GSH administered alone significantly decreased F1+2 in diabetic patients, while no effect was observed in the normal subjects. These data suggest that hyperglycemia may induce thrombin activation, possibly inducing an oxidative stress, and that antioxidant GSH may counterbalance this effect.

Total Radical-Trapping Antioxidant Parameter in NIDDM Patients

OBJECTIVE The existence of an oxidative stress in diabetes is still debated. This is largely due to the lack of good tools to assay the level of oxidative stress. The use of total radical-trapping antioxidant parameter (TRAP) has recently been proposed to explore the antioxidant property of a plasma sample. TRAP may be either directly measured by a fluorescence-based method (TRAPm) or calculated (TRAPc) by a mathematical formula, taking into account the serum levels of four natural antioxidants: protein-bound SH (thiol) groups, uric acid, vitamin E, and vitamin C. The difference between TRAPm and TRAPc is due to antioxidants, which are still unidentified, and to the possible synergism among the antioxidants. RESEARCH DESIGN AND METHODS In this study, we evaluated malondialdehyde (MDA), TRAPm, TRAPc, protein-bound SH groups, uric acid, vitamin E, and vitamin C in 40 NIDDM patients and 40 matched normal control subjects. RESULTS TRAPm and TRAPc were significantly lower in diabetic patients. A good correlation between TRAPm and TRAPc was found in both NIDDM patients (r = 0.68, P < 0.0001) and control subjects (r = 0.74, P < 0.0001). Protein-bound SH groups and uric acid were significantly lower in diabetic subjects, while MDA and vitamin E level were significantly higher. After correction for serum triglycerides (MDA) and cholesterol (vitamin E), MDA lost significance, while vitamin E did not. Vitamin C was not different in the two groups. CONCLUSIONS These data show decreased TRAP levels in NIDDM patients, suggesting the existence of lower antioxidant defenses in diabetes. The decrease appears to be due to various antioxidants, some of them not yet clearly defined. TRAP may represent a more reliable estimation of serum antioxidant capacity than the measurement of each known antioxidants. The correlation found between TRAPm and TRAPc values suggests that TRAPc, easier to measure than TRAPm, might be adequately reliable for routine assessment of oxidative stress in diabetic patients.

Renal Tubular Dysfunction in Chronic Alcohol Abuse -- Effects of Abstinence

BACKGROUND Alcohol abuse may be accompanied by a variety of disorders of electrolyte and acid-base metabolism. The role of the kidney in the pathogenesis of these disturbances is obscure. We sought to evaluate the alcohol-induced abnormalities of renal function and improvement during abstinence and to assess the relation between renal dysfunction and electrolyte and acid-base disorders. METHODS We measured biochemical constituents of blood and renal function before and after four weeks of abstinence in 61 patients with chronic alcoholism who had little or no liver disease. RESULTS On admission, 18 patients (30 percent) had hypophosphatemia and hypomagnesemia, 13 patients (21 percent) had hypocalcemia, and 8 patients (13 percent) had hypokalemia. Twenty-two patients (36 percent) had a variety of simple and mixed acid-base disorders. Twenty of these patients had metabolic acidosis, and among them, 80 percent had alcoholic acidosis. A wide range of defects in renal tubular function, with normal glomerular filtration rate, were detected in these patients. The defects included decreases in the threshold and maximal reabsorptive ability for glucose (38 percent of patients) and in the renal threshold for phosphate excretion (36 percent); increases in the fractional excretion of beta 2-microglobulin (38 percent), uric acid (12 percent), calcium (23 percent), and magnesium (21 percent); and aminoaciduria (38 percent). Seventeen patients (28 percent) had a defect in tubular acidification, and five an impairment in urinary concentrating ability. Urinary excretion of N-acetyl-beta-D-glucosaminidase and alanine aminopeptidase were increased in 41 and 34 percent of patients, respectively. The abnormalities of blood chemistry and renal tubular function disappeared after four weeks of abstinence. CONCLUSIONS Transient defects in renal tubular function are common in patients with chronic alcoholism and may contribute to their abnormalities of serum electrolyte and blood acid-base profiles.

Use of Testosterone To Prevent Cyclophosphamide-Induced Azoospermia

Several cytotoxic agents used to treat neoplastic or immunologic diseases may alter endocrine function in humans [1-5]. In particular, gonadal failure with azoospermia, amenorrhea, or anovulatory cycles has been reported in patients treated for various diseases [1-3]. Cyclophosphamide, an alkylating drug, is widely used as an antineoplastic or immunosuppressive agent. Severe gonadal failure with transient or permanent azoospermia is found in 50% to 90% of men treated with cyclophosphamide [6, 7]. Prepubertal patients who receive large doses of cyclophosphamide seem to recover gonadal function better than adults do; normal pubertal development and normal spermatogenesis have been reported in many of these patients [3, 8-10]. These findings may indicate that active germinal cells are more sensitive to cyclophosphamide because of their elevated mitotic activity. In designing our study, we assumed that the use of testosterone to inhibit germinal cell activity might reduce the sensitivity of these cells to the effects of cyclophosphamide. We administered cyclophosphamide as an immunosuppressive agent to manage glomerulonephritis, either orally or in an intravenous bolus [11, 12]. We therefore could evaluate the effects of the two methods of administration on germinal cell function. Methods We studied 15 men who had the nephrotic syndrome, (age range, 23 to 35 years). Each patient was told of the possible risks of treatment and gave informed consent. Patients were randomly divided into three groups. The five patients in group A (three with membranous nephritis, one with mesangial nephritis, and one with lupus nephritis) were given oral cyclophosphamide at an average dosage of 150 mg daily for 6 to 8 months; the total dose was 27 to 36 g. The five patients in group B (two with membranous nephritis, two with mesangial nephritis, and one with focal glomerulosclerosis) and the five patients in group C (two with membranous nephritis, one with mesangial nephritis, and two with lupus nephritis) received cyclophosphamide as a monthly intravenous bolus, 15 mg/kg of body weight, according to the protocol reported elsewhere [11, 12]. The average duration of treatment was 8 months; the total dose was 10.4 g. Patients in group C also received testosterone, 100 mg intramuscularly every 15 days, in addition to cyclophosphamide. Patients began receiving testosterone (a commercial blend of enanthate and propionate esthers of testosterone) 30 days before starting cyclophosphamide therapy, and they continued to receive it during the course of immunosuppressive therapy. Because ours was a pilot study, the testosterone dose that we used was lower than that currently used to treat hypogonadism; however, it proved to be effective in inhibiting gonadotropin secretion and spermatogenesis [13]. To ensure the possibility of future paternity, sperm samples from each patient were collected before the start of therapy and stored in liquid nitrogen. Additional sperm samples were collected from each patient before therapy, during the third and sixth months of cyclophosphamide therapy, and 3 and 6 months after the end of the therapy. All sperm samples were analyzed in the same laboratory by the same technician. Serum samples for luteinizing hormone and follicle-stimulating hormone assays were collected at the same intervals as the sperm samples. Hormone levels were measured in duplicate by specific immunoradiometric methods using commercial kits (Serono Diagnostic, Milan Italy). Both of the preceding assays are sensitive to about 0.15 IU/L. Levels are reported in IU/L in accordance with the First International Reference Preparation 68/40 and Second International Reference Preparation 78/549 guidelines for reporting luteinizing hormone levels and follicle-stimulating hormone levels, respectively. Statistical analysis was done using the Student t-test. All results are given as the mean SE. Results Under control conditions, all patients had normal sperm counts; the means were 43.70 6.56 106/mL in group A, 45.52 7.04 106/mL in group B, and 42.91 5.27 106/mL in group C. During immunosuppressive therapy, sperm counts abruptly decreased in all patients in groups A and B (Table 1), and all patients were azoospermic 6 months after starting immunosuppressive therapy. Three and 6 months after completing therapy, all patients in group A and four of the five patients in group B were azoospermic. During the third and sixth months of immunosuppressive therapy, three patients in group C had azoospermia or severe oligospermia; sperm counts were lower than 1 106/mL. After therapy was discontinued, sperm counts increased progressively in group C; mean counts were 28.64 3.47 106/mL 3 months after therapy ended and 45.78 3.89 106/mL 6 months after therapy ended. In these patients, mean percentages of sperm motility (88%; range, 63% to 92%) and sperm that showed no structural abnormalities (80%; range, 73% to 89%) were in the normal range. Table 1. Sperm Counts under Control Conditions and during and after Cyclophosphamide Therapy* In groups A and B, serum luteinizing hormone levels did not change throughout the observation period (Table 2) but follicle-stimulating hormone levels progressively increased. The following are the mean follicle-stimulating hormone levels: group A, 8.54 0.69 IU/L and 14.82 2.15 IU/L during the third and sixth months of cyclophosphamide therapy and 18.40 1.79 IU/L and 19.20 1.28 IU/L 3 and 6 months after discontinuation of therapy; group B, 9.30 0.87 IU/L and 18.48 2.81 IU/L during the third and sixth months of cyclophosphamide therapy and 16.80 2.15 IU/L and 16.04 2.22 IU/L 3 and 6 months after discontinuation of therapy. All of these values were significantly higher than those seen under control conditions (P = 0.002). Luteinizing hormone and follicle-stimulating hormone levels decreased in group C during cyclophosphamide therapy because of the inhibitory effect of testosterone (Table 2). In particular, luteinizing hormone levels during the third and sixth months of immunosuppressive therapy were 1.62 0.24 IU/L and 1.06 0.19 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 2.12 0.38 IU/L and 1.56 0.33 IU/L, respectively. The differences with respect to baseline values were statistically significant (P < 0.005). In these patients, luteinizing hormone levels measured 3 and 6 months after discontinuation of therapy were 6.44 0.87 IU/L and 4.80 0.40 IU/L, respectively; follicle-stimulating hormone levels at the same time points were 6.60 0.86 IU/L and 5.08 0.56 IU/L, respectively. Differences between the follicle-stimulating hormone levels in group C and those in groups A and B were statistically significant (P < 0.002). All 15 patients in the study had clinical remission of their underlying diseases. No side effects related to testosterone administration, such as liver enlargement or gynecomastia, were seen in group C. Table 2. Serum Follicle-Stimulating Hormone and Luteinizing Hormone Levels under Control Conditions and during and after Cyclophosphamide Therapy* Discussion Our findings confirm that cyclophosphamide therapy can affect germinal cell function in humans [1-36, 7]. All patients who received daily oral cyclophosphamide and four of five who received a monthly intravenous bolus of cyclophosphamide were azoospermic for as long as 6 months after the end of immunosuppressive therapy. Serum follicle-stimulating hormone levels were elevated in all patients who had become azoospermic, a finding that reflects a severe alteration of the germinal epithelium. In contrast, spermatogenesis returned to normal in the five patients who received testosterone before and during immunosuppressive therapy; follicle-stimulating hormone levels for all patients were within the normal range. This finding indicates that the use of testosterone to inhibit germinal cell activity may protect against the effects of cyclophosphamide. Leydig cell function was not affected in any study patient, as shown by normal levels of luteinizing hormone; previous reports have shown that Leydig cells are more resistant than germinal cells to the action of alkylating agents [6, 10]. Testosterone has been used as a contraceptive in men; it induces azoospermia or severe oligospermia by reducing levels of gonadotropins and intragonadal testosterone [13, 14]. In accordance with this observation, our data show a substantial reduction in gonadotropin levels during testosterone administration. Moreover, other studies [13, 14] have shown that testosterone can be used safely at high doses and for long periods. No data are available on the effects of testosterone in nephrotic patients receiving cyclophosphamide, and previous reports on gonadal protection during therapy with cytotoxic drugs are conflicting. Experimental studies in rats have shown that the use of triptorelin to suppress gonadal function may protect the testes from the adverse effects of cyclophosphamide therapy [15]. In contrast, nafarelin given to dogs was shown to potentiate the damaging effects of the alkylating agents [16]. Oral contraceptives and luteinizing hormone-releasing hormone agonists have also been used in humans in attempts to preserve gonadal function during chemotherapy. However, results of previous studies are conflicting and inconclusive [3, 17-20]. Our data were obtained from a small number of patients observed for only 6 months after the discontinuation of immunosuppressive therapy. However, our findings may indicate a safe way to preserve gonadal function in nephrotic patients who are treated with cyclophosphamide. Dr. Bartoli: Medicina Interna, University of Udine, Piazzale S. Maria della Misericordia 1, 33100 Udine, Italy.

Portal vein thrombosis in hepatocellular carcinoma: age and sex distribution in an autopsy study

Abstract The purpose of this work was to investigate the prevalence, associated features and effect on survival of portal vein thrombosis (PVT) complicating hepatocellular carcinoma (HCC). The autopsy data of a series of 72 consecutive patients (57 male, 15 female) with HCC were reviewed. PVT was found in 32/72 patients (44%), and tended to be more common in female patients (10/15 versus 22/57, P = 0.052). Stratifying the data according to gender, it appeared that the mean age of patients with PVT compared to those without was greater in woman (71.9 ± 5.9 versus 63.2 ± 6.9 years, P = 0.024) and younger in men (58.8 ± 8.9 versus 66.0 ± 9.9 years, P = 0.007). When PVT was present, it was more likely that a definite diagnosis of HCC had been obtained before autopsy (P = 0.0001) and that death had been caused by bleeding complications (P = 0.007). Median survival times were similar, irrespective of the presence of PVT. During the natural history of HCC, PVT occurs in a substantial proportion of patients. Hormonal factors may have a permissive role in thrombus formation or neoplastic vascular invasion. Although in the presence of PVT a diagnosis of HCC is rarely missed and bleeding complications are likely to occur, patient survival does not seem to be significantly affected.

Relief of pruritus and decreases in plasma histamine concentrations during erythropoietin therapy in patients with uremia

BACKGROUND The pathophysiologic aspects of pruritus in patients with chronic renal insufficiency are poorly understood, and there is no universally effective treatment. The improvement of pruritus in several patients receiving erythropoietin therapy raised the possibility that erythropoietin affects uremic pruritus directly. METHODS We undertook a 10-week placebo-controlled, double-blind, crossover study in a group of patients receiving hemodialysis who had severe pruritus, to investigate the effects of recombinant human erythropoietin on their pruritus and plasma histamine levels. Twenty patients with uremia, of whom 10 had severe pruritus and 10 did not, received erythropoietin (36 units per kilogram of body weight three times weekly) and placebo in random order, each for five weeks. The severity of pruritus was scored weekly, and plasma histamine levels were measured at the beginning and end of each five-week period. RESULTS Eight of the 10 patients with pruritus had marked reductions in their pruritus scores during erythropoietin therapy. The mean (+/- SE) pruritus score decreased from 25 +/- 3 to 6 +/- 1 in these patients. The pruritus returned within one week after the discontinuation of therapy. The improvement was not related to the change in hemoglobin level. These eight patients were successfully treated again with low doses of erythropoietin (18 units per kilogram three times weekly), and the effect has persisted for six months. The patients with pruritus had elevated plasma histamine concentrations (20.7 +/- 2.7 nmol per liter), as compared with the patients without pruritus (4.2 +/- 0.6 nmol per liter; P less than 0.001) and normal subjects (2.1 +/- 0.2 nmol per liter; P less than 0.001). Therapy with erythropoietin induced a decrease in plasma histamine concentrations in both groups of patients with uremia, and recurrences of pruritus after the discontinuation of erythropoietin were accompanied by increases in plasma histamine concentrations. CONCLUSIONS Erythropoietin therapy lowers plasma histamine concentrations in patients with uremia and can result in marked improvement of pruritus.

Chronic lymphocytic sialoadenitis in HCV‐related chronic liver disease: comparison with Sjögren's syndrome

With the aim of morphologically characterizing chronic sialoadenitis in patients with hepatitis C virus (HCV) chronic liver disease, labial salivary gland biopsies from 22 chronic HCV liver disease and from 10 primary Sjögrens syndrome patients were compared. Only focus score (number of aggregates with more than 50 lymphocytes per 4 mm2 of glandular tissue) and grading of inflammation were able to discriminate significantly between the two patient groups. Duct ectasia, acinar depletion, presence of lymphoid aggregates with less than 50 lymphocytes and of lymphoid infiltration within intralobular salivary duct epithelium were evident in both disease groups and appeared to be non‐specific, mostly age‐related changes. In both patient groups plasma cell and lymphocyte typing showed similar features: T lymphocytes represented most of the lymphoid population, B lymphocytes were few unless follicles were present. Higher focus score values were associated with a plasma cell switch from an IgA to an IgM and/or IgG predominance. A greater morphological similarity was seen between biopsies of the primary Sjögrens syndrome group and those of female rather than male chronic HCV liver disease patients. Salivary gland tissue in HCV patients responds to damage in a fashion similar to primary Sjögrens syndrome, the only difference being a lesser degree of inflammation.

Increased Circulating Intercellular Adhesion Molecule-1 Levels in Type II Diabetic Patients: The Possible Role of Metabolic Control and Oxidative Stress

Blood levels of the circulating form of the integrin intercellular adhesion molecule-1 (ICAM-1), malondialdehyde (MDA), and hemoglobin A1c (HbA1c) were studied at baseline and 3 months after improved metabolic control in 25 type II diabetic patients without signs of macroangiopathy, and were compared with those in 15 matched healthy normal controls. Circulating ICAM-1 and MDA levels were increased in diabetic patients, both at baseline and 3 months later. However, with improving metabolic control HbA1c, circulating ICAM-1, and MDA significantly decreased. A significant correlation between circulating ICAM-1, HbA1c, and MDA was found in diabetic patients at each time. Multiple regression analysis considering circulating ICAM-1 as the dependent variable and HbA1c and MDA as independent variables, showed a significant correlation between the three variable at each time. Similar correlations were found in control subjects. These data show increased levels of circulating ICAM-1 in type II diabetic patients, independent of the presence of macroangiopathy. Moreover, these results suggest that oxidative stress and metabolic control might participate in determining increased circulating ICAM-1 levels in both type II diabetic patients and normal subjects.

Renal antioxidant enzyme mRNA levels are increased in rats with experimental diabetes mellitus

Summary Exposure to high glucose concentrations increases the mRNA levels of oxygen radical scavenging enzymes in cultured endothelial cells, suggesting a compensatory response to increased free radical production. To test the hypothesis that this response also occurs in vivo, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase mRNA levels, were measured in the kidneys of Sprague-Dawley rats 17 days after intravenous injection of streptozotocin (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens (2, 3–8, 6–10 IU/day) in two different experiments that were designed to achieve varying degrees of metabolic control. Cu,Zn-SOD and catalase mRNA levels were measured by Northern blot hybridization and standardized by 28S ribosomal RNA determination. Renal Cu,Zn-SOD and catalase mRNA levels were significantly greater in untreated diabetic and in low-dose (2 IU/day) insulin-treated rats than in controls. Treatment with a moderate dose (3–8 IU/day) of insulin normalized catalase but not Cu,Zn-SOD mRNA levels. The highest insulin regimen (6–10 IU/day), in addition to achieving complete metabolic control as evidenced by normal growth and plasma glucose levels, normalized both catalase and Cu,Zn-SOD mRNA levels. Thus, in rats with streptozotocin-induced diabetes Cu,Zn-SOD and catalase renal mRNA levels are greater than in normal rats. This difference is prevented by sufficient insulin dosage to normalize plasma glucose and might be due to an increased production of free radicals. [Diabetologia (1997) 40: 23–29]

Fibrinogen Plasma Levels as a Marker of Thrombin Activation in Diabetes

This study attempted to verify the existence of a correlation between fibrinogen, a major cardiovascular risk factor in diabetes, and indexes of thrombin generation and action, prothrombin fragment 1 + 2 (F1 + 2), and D-dimer (D-D), in a group of diabetic subjects compared with a matched control group. Forty insulin-dependent diabetes mellitus patients and 30 matched healthy control subjects participated in this study. The subjects were tested for the following parameters: fibrinogen, prothrombin F1 + 2, D-D, fasting glycemia, and HbA1c. In addition, 5 diabetic subjects who maintained stable fibrinogen plasma levels > 300 mg/dl for at least 6 months before the study were treated with 12,500 U/day subcutaneous heparin for 7 days. Diabetic subjects showed increased levels of fibrinogen, prothrombin F1 + 2, and D-D plasma levels. Simple linear regression analysis detected a positive correlation between fibrinogen and prothrombin F1 + 2, D-D, and glycosylated HbA1c. In the five diabetic subjects treated with heparin fibrinogen, prothrombin F1 + 2 and D-D levels decreased at the end of the treatment. All these parameters returned to baseline after 7 days of washout. These data indicate that fibrinogen plasma levels are correlated to parameters of thrombin activation in plasma in diabetic patients and suggest that high fibrinogen plasma levels might be a risk marker for cardiovascular disease in diabetes because it is an expression of an existing thrombophilia.