Pro-atherogenic postprandial profile: meal-induced changes of lipoprotein sub-fractions and inflammation markers in obese boys.

BACKGROUND AND AIMS: Obesity is a pro-atherogenic condition and postprandial lipoprotein profile and circulating cytokines changes may contribute to promote the process. The aim of this study is to investigate postprandial metabolic response, lipoprotein oxidation and circulating cytokine levels, after the ingestion of two different meals with different fat/carbohydrate ratio. METHODS AND RESULTS: Ten prepubertal obese boys consumed two meals with the same energy and protein content but with a different carbohydrate to fat ratio: 1) moderate fat (MF): 61% carbohydrate, 27% fat; 2) high fat (HF): 37% carbohydrate, 52% fat. The AUC of glucose and insulin were significantly (p < 0.05) lower after the HF meal. HF meal was followed by a significant decrease in the cholesterol carried in the HDL fractions, while cholesterol in the small, dense LDL and in the VLDL particles increased, as compared to baseline (p < 0.05 for all). No differences were found in the cholesterol distribution after the MF meal. Moreover, HDL-C concentration was lower (p < 0.05) at 300 min after HF vs. MF meal. Oxidized LDL (ox-LDL) concentration increased after the HF meal but not after the MF meal [9.3(2.2) vs 1.8(2.2)% from baseline, P < 0.02)]. A positive association (r > 0.3, P < 0.05) was observed between the densest LDL particles and the ox-LDL plasma levels. A reduction of IL-6 was found at 120 min after the MF [-23.3(5.5) vs -8.4(3.8)% from baseline, P < 0.05)] compared with the HF meal. CONCLUSION: A simple change of ≈25% of energy load from fat to carbohydrate in a meal significantly improves postprandial pro-atherogenic factors in obese boys.

Serum lipoprotein profile and APOE genotype in Alzheimer's disease.

Alterations in cholesterol homeostasis are associated with Alzheimer's disease (AD). The role played by specific fractions of serum lipoproteins in modifying the risk of AD, and the interaction with APOE genotype has not yet been investigated. We studied serum lipoprotein profiles using a gradient-density ultracentrifugation method in a cohort of late-onset sporadic AD patients without cerebrovascular lesions and in healthy elderly subjects. In the AD group the lipoprotein cholesterol distribution showed an increase in LDL cholesterol, reaching a significant difference with respect to controls in the LDL sub-fractions representing the transition between small dense-LDL (fraction 11, p = 0.04) and normal-density LDL particles (fraction 12, p = 0.03). APOE genotype and LDL cholesterol were independently associated with AD. The mean concentration of LDL in fractions 11 and 12 increased the risk of developing AD (p = 0.01 and p = 0.025, respectively). These results confirm that an alteration of cholesterol homeostasis is associated with AD and that serum concentrations of LDL cholesterol are higher in AD patients without cerebrovascular pathology than in elderly normal subjects. The presence of the APOE epsilon4+ allele is a risk factor for AD independent of increased serum cholesterol or a modification of other vascular risk factors. Increased levels of specific sub-fractions of LDL cholesterol may be associated with increased risk of AD.

Relevance of hepatic lipase to the metabolism of triacylglycerol-rich lipoproteins.

HL (hepatic lipase) is a glycoprotein that is synthesized and secreted by the liver, and which binds to heparan sulphate proteoglycans on the surface of sinusoidal endothelial cells and on the external surface of parenchymal cells in the space of Disse. HL catalyses the hydrolysis of triacylglycerols and phospholipids in different lipoproteins, contributing to the remodelling of VLDL (very-low-density lipoprotein) remnants, as well as IDL, LDL and HDL (intermediate-, low- and high-density lipoprotein respectively). HL deficiency in humans is associated with diminished conversion of VLDL remnants into IDL and a near-complete absence of IDL-to-LDL conversion. Remnant lipoproteins and IDL are major determinants of coronary artery disease risk, and accumulation of these lipoproteins in the presence of low HL activity might lead to increased atherosclerosis. In addition to and independently of its lipolytic activity, HL participates as a ligand in promoting the hepatic uptake of remnants and IDL particles, and the latter may represent an additional mechanism linking low HL levels to plasma accumulation of these atherogenic lipoproteins. On the other hand, high HL activity may also result in an increased atherosclerotic risk by promoting the formation of atherogenic small, dense LDL particles. Finally, HL is also synthesized by human macrophages, suggesting that, at the arterial wall site, HL may also contribute locally to promote atherosclerosis by enhancing the formation and retention in the subendothelial space of the arterial wall of VLDL remnants, IDL and small, dense LDL. In conclusion, by interfering with the metabolism of apolipoprotein B100-containing lipoproteins, HL may have pro- as well as anti-atherogenic effects. The anti- or pro-atherogenic role of HL is likely to be modulated by the concurrent presence of other lipid abnormalities (i.e. LDL-cholesterol levels), as well as by the genetic regulation of other enzymes involved in lipoprotein metabolism.

[Arterial hypertension and oxidative stress induced by cyclosporin. Effect of carvedilol]. / Ipertensione arteriosa e stress ossidativo indotti dalla ciclosporina. Effetto del carvedilolo.

Cyclosporin-induced hypertension and endothelial dysfunction have been attributed to the effects of cyclosporin on factors controlling vasomotor tone. Endothelial nitric oxide (NO) regulates basal vasodilation, and an NO-mediated protective mechanism from cyclosporin-induced vasoconstriction has been proposed. In transplanted patients with cyclosporin-induced hypertension, we have recently demonstrated upregulation of the NO system and superoxide and free radical overproduction, which, by increasing NO metabolism, could induce hypertension, vascular remodeling and chronic rejection. In the present work, we have evaluated endothelial constitutive NO synthase (ecNOS), transforming growth factor beta and heme oxygenase-1 (protective against oxidative stress), mRNA production and plasma NO metabolites, peroxynitrite and antioxidant power in 15 kidney transplanted patients before and after 4 months of treatment with carvedilol alpha 1-beta-blocker and potent antioxidant. Our aim was to study the efficacy of the reduction of oxidative stress on complications such as endothelial dysfunction and fibrogenesis. Monocyte ecNOS and plasma NO metabolites remained higher versus those of control subjects and were unchanged by carvedilol, while antioxidant power and heme oxygenase-1 mRNA production increased. Peroxynitrite, as well as transforming growth factor beta mRNA, were reduced by carvedilol. It also normalized blood pressure. In conclusion, carvedilol reduces oxidative stress and normalizes blood pressure; ecNOS remains upregulated while mRNA for transforming growth factor beta, a key fibrogenic cytokine, is reduced by carvedilol, which seems to preserve protective mechanisms such as NO and heme oxygenase-1 against long-term complications of oxidative stress, e.g., endothelial dysfunction, fibrogenesis and chronic rejection.

Direct effect of chronic cyclosporine treatment on collagen III mRNA expression and deposition in rat kidneys.

BACKGROUND: It is hypothesized that in acute and chronic CsA nephrotoxicity, in vivo models CsA side-effects are mediated by Renin-Angiotensin II (RAS)-TGF-beta-1 pathway. However, to induce chronic nephrotoxicity, CsA administration has to be combined with a low salt diet, which causes hemodynamic changes and RAS up-regulation. MATERIALS AND METHODS: In order to define any direct correlation between CsA and nephrotoxicity, we studied in normal sodium fed rats, the chronic effects of CsA administration (group-1 treated with 12.5 mg/Kg/day of CsA subcutaneously; group 2 received daily placebo; group 3 interrupted CsA injection after 60 days), on renal TGF-beta-1 and collagen III expression, and on TGF-beta-1, collagen III and IV deposition. Sacrifices were performed after 2, 4, 8 and 12 weeks (wks) and kidneys were harvested for immunohistological studies and RT/PCR analysis. RESULTS: No difference of TGF-beta-1 expression and deposition was found among groups. Starting from the 2nd week of treatment, an increased collagen III deposition was evident in vessels and in outer medulla with subsequent extension at the 4th week to medullary rays and to cortex interstitium. The deposition paralleled the renal collagen III mRNA up-regulation: it was significantly higher in group 1 than in group 2 (p < 0.009 at 2nd wk; p < 0.016 at 4th wk). Collagen IV deposition did not differ between groups at any point. CONCLUSIONS: Our results suggest that chronic CsA administration can induce, in normal fed rats, the process of interstitial fibrogenesis through TGF-beta non-related mechanisms.

Molecular biology of diabetic glomerulosclerosis.

Diabetic nephropathy is one of the leading causes of renal failure in Western countries, where diabetic patients account for nearly half of all patients on haemodialysis. Progressive expansion of the mesangial matrix, and thickening of the glomerular and tubular basement membranes without signs of major cell proliferation are hallmarks of human and experimental diabetic nephropathy. These lesions eventually lead to glomerular fibrosis, a central pathological feature in many human acute and chronic kidney diseases, which progressively destroys the renal filtration unit, and may finally cause renal failure. Indeed, structure function relationship studies have shown that mesangial matrix expansion is strongly related to the clinical manifestation of diabetic nephropathy.

Intracellular processing of transforming growth factor-beta in mesangial cells.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulator of cell-growth, differentiation and extracellular matrix formation in several physiological conditions. It plays a crucial role in the process of glomerulosclerosis. Mature TGF-beta 1 is secreted as a latent form associated with the latency associated peptide (LAP), and its activation occurs through the LAP cleavage. The intracellular localization and the mechanisms of activation of TGF-beta 1 protein have not been elucidated in the mesangial cell. In the present report we examined the intracellular processing from TGF-beta 1 precursor to the latent-TGF-beta 1 in cultured mesangial cells by immunocytochemistry, using three rabbit polyclonal antibodies directed against different epitopes of human TGF-beta 1. The anti-LAP-TGF-beta 1 precursor Ab stained mesangial cells in the perinuclear region and in the cytoplasm in the area corresponding to the rough endoplasmic reticulum; the anti-COOH-terminal fragment of TGF-beta 1 Ab reacted in the same area, in vesicular structures located in the cytoplasm and furthermore, in the mesangial cell clusters, so-called hillocks, with an extracellular pattern; the anti-NH2-terminal fragment of TGF-beta 1 Ab stained only large exocytotic vesicles at the periphery of the cytoplasma. Our investigations suggest a conformational rearrangement of pro-TGF-beta 1 molecule occurring between the rough endoplasmic reticulum and the TGF-beta 1 secretion and support the idea that in mesangial cells the activation of TGF-beta 1 occurs during the secretion process. In conclusion, the processing of TGF-beta 1 in mesangial cells seems to be similar to that one observed in other mesenchymal cells.

Urinary NO2- and NO3- evaluation by an ion chromatography system.

We describe an ion chromatography system using a Dionex AS4A-SC column with carbonate-bicarbonate buffer (1.8-1.7 mM) as eluent for the evaluation of urinary NO2- and NO3-. This chromatographic system gives an accurate measurement of NO2- and NO3- in the urine as an index of NO production in vivo, making also possible to evaluate their relative proportion and providing useful tools to investigate the NO system.

Is the red cell morphology really useful to detect the source of hematuria?

Morphological analysis of urinary red blood cells by phase-contrast microscopy to identify the source of bleeding was, and still is, widely used also as a starting point for workup. To evaluate the reliability of this approach, we studied 129 outpatients presenting with persistent isolated microhematuria; 31 subjects also had mild proteinuria (1 g/day), while 21 had pathological albumin levels. All patients were followed for a period of 6 years. During this time, 6 patients underwent renal biopsy for the onset of macrohematuria episodes and proteinuria of 2-3 g/day. Glomerular bleeding was identified in only 14.7% of the patients, despite the persistent microhematuria and the presence of proteinuria or microalbuminuria. The renal origin of the urinary erythrocytes correlated with histological findings in only 2 of 6 patients with dysmorphic erythrocytes who developed proteinuria (exceeding 1 g/day), and none with isomorphic erythrocytes showed urological abnormalities. These results challenge the validity and reliability of morphological analysis to identify the source of bleeding along the urinary tract.

[7-year-follow up (1987-1994) of a population with asymptomatic persistent microhematuria and normal renal function]. / Follow-up di 7 anni (1987-1994) su una popolazione con microematuria persistente asintomatica e funzione renale integra.

A group od 129 patients with persistent asymptomatic microhematuria was studied for 7 years (1987-1994). At the beginning of the study, 31 patients showed mild proteinuria (less than 1 g/day) and in the rest of 98 patients, 21 showed microalbuminuria. At the end of the study none of the patients developed renal failure, urological disease, hypertension. Six patients out of 31 with mild proteinuria (less than 1 g/day), developed an increase of proteinuria over 2 and 3/day and underwent a renal biopsy while 2 out of 21 patients with altered microalbuminuria completely recovered after the follow-up period. The rest of 77 patients at the end of the study still showed isolated microhematuria. The results of this study support the hypothesis that in a population with age range between 16 and 28 years, the presence of persistent microhematuria, also associated with mild proteinuria, even for a long time, does not seem to lead to changes of renal function or to urological diseases.

Hepatic changes and serum ferritin in pancreatic cancer and other gastrointestinal diseases: the role of cholestasis.

Serum ferritin, prealbumin, pseudocholinesterase, alpha-1-antitrypsin and caeruloplasmin were determined in control subjects and patients with pancreatic cancer, chronic pancreatitis or extra-pancreatic disease mainly of gastrointestinal origin, in order to investigate the different hepatic changes which influence serum ferritin in chronic pancreatic and other digestive diseases. Increased circulating ferritin was found in pancreatic cancer and extra-pancreatic disease when compared to controls. Correlations were detected between ferritin and the other proteins investigated and between ferritin and total bilirubin, alkaline phosphatase and alanine aminotransferase. Multiple regression analysis demonstrated that cholestasis accounts for 45% of circulating ferritin, the acute-phase response accounted for 18% and decreased liver function accounted for 11%. We conclude that the increase in serum ferritin in chronic pancreatic and other gastrointestinal diseases largely depends on liver changes, with cholestasis probably playing a primary role.

Role of local and systemic factors in increasing serum glycoprotein markers of pancreatic cancer.

This study was performed in order to evaluate the role of various local and systemic alterations in influencing serum glycoproteic markers in patients with pancreatic cancer, and in healthy and diseased controls. Cancer antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA), and ferritin were determined in the sera of 23 control subjects, 30 patients with pancreatic cancer, 27 with chronic pancreatitis, and 27 with extra-pancreatic diseases mainly of gastrointestinal origin. A number of acute-phase proteins and indices of liver function and cholestasis were also assayed. The three antigens increased only in patients with pancreatic cancer. Higher CA 19-9 and CEA, but not ferritin, levels were found only in patients with hepatic metastases. Acute-phase proteins and synthetic functional indices were found to be higher and lower, respectively, in patients with pancreatic malignancy when compared with controls. Multiple regression analysis documented the dependence of circulating ferritin, but not of CA 19-9 and CEA, on the systemic indices. Canonical correlation showed a similar trend for CA 19-9 and CEA, which differed from that of ferritin. Ferritin was found to depend on the presence of systemic and hepatic alterations, especially of cholestasis. We can conclude that the variations of serum glycoprotein markers in patients with pancreatic cancer depend on various regional and systemic factors. CA 19-9 and CEA are related mainly to the extent of the neoplasia. The influence of a decreased liver function capacity associated or not to cholestasis and the interrelation with the acute-phase response may also be suggested. Ferritin, on the other hand, is related to a higher degree than CA 19-9 and CEA to hepatic dysfunction and also behaves similar to an acute-phase protein.

Acute phase proteins in chronic pancreatic disease.

In order to evaluate the behaviour of some acute phase proteins in chronic pancreatic disease and to correlate these reactants with different factors, C-reactive protein, ceruloplasmin and alpha-1-antitrypsin were assayed in the sera of 24 control subjects, 26 patients with pancreatic cancer, 22 patients with chronic pancreatitis and 22 patients with a variety of diseases not of pancreatic origin. Alpha-1-antitrypsin, C-reactive protein and ceruloplasmin concentrations were found to be increased in 63%, 50% and 42% of patients with chronic pancreatic disease, respectively. In patients with pancreatic cancer no difference was found between the values of each protein considering the presence or otherwise the absence of liver metastases. Patients with chronic pancreatitis had higher C-reactive protein or alpha-1-antitrypsin values when increased serum amylase or pseudocysts were present. Significant correlations were found between the three acute-phase proteins considering the subjects as a whole; however in the single subjects they were not found to be concomitantly abnormal. Correlations were detected between these proteins and liver function test values. Alpha-1-antitrypsin is probably the most sensitive index in chronic pancreatic disease, while C-reactive protein seems better to reflect the stage of the disease. The variations of the levels of these proteins seem to be, at least in part, independent of each other; they are all partially influenced by the presence of liver damage.

C reactive protein in pancreatic cancer and chronic pancreatitis.

Serum C reactive protein was determined in 30 control subjects, 32 patients with pancreatic cancer, 28 with chronic pancreatitis and 23 with extra-pancreatic diseases of the upper gastrointestinal tract. The aim was to ascertain possible alterations of this index in chronic pancreatic disease and to speculate on some influencing factors. Higher C reactive protein levels were found in pancreatic cancer as compared to controls. Pancreatic cancer patients with systemic metastases had higher levels of this index compared to those with non-metastatic disease. Raised concentrations of C reactive protein were detected in 7/28 subjects with chronic pancreatitis. In this group these higher levels were found in patients in a relapsing phase of the disease; no association was observed with pancreatic pseudocysts. Among all subjects a correlation was found, between C reactive protein and age; patients with abnormal fasting blood glucose levels or increased white blood cell count had higher levels of this protein as compared to the remaining patients. We may conclude that C reactive protein increases in pancreatic cancer, specially in relation to tumour extent; in chronic pancreatitis it reflects the inflammatory status of the gland. While acting in the context of the acute phase response, this test may provide an adjunct in evaluating patients with a chronic pancreatic disease.

Ribonucleases and deoxyribonucleases in pancreatic cancer: clinical value and pathophysiological interrelationships.

In this study we evaluated some pathophysiological aspects of pancreatic and liver ribonucleases and alkaline deoxyribonuclease and their clinical usefulness in diagnosing pancreatic cancer. Pancreatic RNase was found to be a sensitive index of pancreatic malignancy; however it was not specific in distinguishing pancreatic malignancy from chronic pancreatitis or other pathologies. Liver RNase and alkaline DNase did not provide better results than pancreatic RNase. These three enzymes were found to be age-dependent and related to each other. Therefore serum nucleases are not useful for clinical purposes since they are influenced, at least in part, by different non-specific factors.

Serum deoxyribonuclease and ribonuclease in pancreatic cancer and chronic pancreatitis.

Serum ribonuclease (RNase) and deoxyribonuclease (DNase) were investigated in 18 control subjects, and in 22 patients with pancreatic cancer, 13 with chronic pancreatitis and 29 with extrapancreatic diseases in order to assess their clinical usefulness in pancreatic cancer diagnosis and to evaluate whether modifications were consensual and/or age-related. Increased DNase and RNase values were found not only in a notable proportion of pancreatic cancer, but also in chronic pancreatitis and extra-pancreatic diseases. Thus the clinical value of both enzymes in pancreatic cancer diagnosis is negligible. DNase does not seem to be strictly age-dependent, whereas serum RNase does. Elevated levels of the two enzymes, when present, were consensual, suggesting that factors involved in such an increase were partially common to both.

A kinetic assay for human urinary kallikrein determination.

A simple kinetic method for human urinary kallikrein determination is proposed. In this assay, the release of p-nitroaniline from the chromogenic substrate S-2266 at 37 degrees C and pH 8.2 is followed spectrophotometrically at 405 nm. The delta A/5 min (0-5 min) interval was chosen. This assay was shown to have good sensitivity since enzyme concentrations as low as 0.00125 KU/ml could be measured. The use of dialyzed urines minimizes the interferences associated with high urinary salt concentration. Because of its precision and reproducibility, this kinetic assay may be proposed in clinical investigation.

Study of retinol-binding protein in pancreatic cancer.

Serum RBP, prealbumin, and zinc were evaluated in normal subjects and patients with pancreatic cancer and chronic pancreatitis. A significant decrease of RPB was found in pancreatic cancer patients compared with controls. A concomitant reduction of prealbumin and zinc was also observed. Multiple regression analysis suggested that the modification of RBP serum levels might be accounted for mainly by diminished prealbumin levels, while the direct role of zinc is negligible.

Urinary ribonuclease excretion in pancreatic disease.

Urinary ribonuclease output and indices of renal tubular integrity were evaluated in control subjects and patients with pancreatic cancer, chronic pancreatitis and extrapancreatic diseases. The aim of the study was to ascertain the contribution to such diagnoses of ribonuclease determination in urine, and the possible influence of tubular damage on the extent of ribonuclease excretion. Information from the ribonuclease assay in urine offered no advantage over that obtained by the same determination in serum; tubular damage may contribution in some cases to an elevated ribonuclease excretion.