Plasma levels of C-reactive protein, leptin and glycosaminoglycans during spontaneous menstrual cycle: differences between ovulatory and anovulatory cycles.

PURPOSE: To assess the plasma levels of the inflammatory markers such as C-reactive protein (CRP), leptin, and glycosaminoglycans (GAGs) during the menstrual cycle. METHODS: Eighteen healthy volunteers were divided into two groups according to the presence of ovulatory or anovulatory menstrual cycles. Blood samples were collected at different time points: at the menstrual phase (days 2-3), periovulatory phase (days 12-13), and luteal phase (days 23-24). CRP and leptin concentrations were measured by enzyme immunoassay. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and quantified as hexuronate. The structural characterization of chondroitin sulfate (CS) isomers was performed by fluorophore-assisted carbohydrate electrophoresis (FACE). RESULTS: In the women with ovulatory cycles, plasma GAG levels differed significantly during menstrual cycle, with increased values at the periovulatory with respect to the menstrual phase. No significant differences in CRP and leptin concentrations were observed through the menstrual cycle in both the examined cycles, but inter-group analysis revealed significant differences of CRP and leptin levels between the ovulatory and anovulatory cycles with higher values at periovulatory phase in the ovulatory cycles. CONCLUSIONS: There are no fluctuations of both total GAG concentration and CS isomer content during menstrual cycle in the anovulatory cycles. A significant correlation between CRP and gonadotrophins was found. There is no significant difference in CRP across the menstrual cycle among ovulatory cycles, but there is a trend toward higher CRP at the periovulatory than the other phases, consistent with the significant difference in CRP between ovulatory and anovulatory cycles at the periovulatory phase. Both the trend and the significant result suggest an elevation in CRP with ovulation. These observations provide additional evidences to the hypothesis that the ovulation is an inflammatory-like phenomenon.

A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries.

OBJECTIVES: By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques. METHODS: The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable and 29 unstable plaques) and separated by two-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software. RESULTS: A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis. CONCLUSIONS: Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.

Glycosaminoglycan and transforming growth factor beta1 changes in human plasma and urine during the menstrual cycle, in vitro fertilization treatment, and pregnancy.

OBJECTIVE: To evaluate transforming growth factor beta1 (TGF-beta1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy. DESIGN: Prospective clinical study. SETTING: University hospital. PATIENT(S): Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3). INTERVENTION(S): We assayed plasma and urine concentrations of TGF-beta1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10-12, 22-24, and 30-32 and 1 month after delivery. MAIN OUTCOME MEASURE(S): Changes in TGF-beta1 and GAG content. RESULT(S): The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-beta1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-beta1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected. CONCLUSION(S): Transforming growth factor beta1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.

A novel LIF-CE method for the separation of hyaluronan- and chondroitin sulfate-derived disaccharides: Application to structural and quantitative analyses of human plasma low- and high-charged chondroitin sulfate isomers.

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.

Quali-quantitative analysis of urinary glycosaminoglycans for monitoring glomerular inflammatory activity.

OBJECTIVE: A 2-year follow-up study was carried out in patients with IgA nephropathy (IgAN) in order to verify the possible use of quali-quantitative analysis of urinary glycosaminoglycans (GAGs) as a prognostic index of disease and for drug treatment monitoring. MATERIAL AND METHODS: Ten patients with IgAN were evaluated at four time points: baseline, and 6, 9 and 24 months later. GAGs were isolated from 24-h urine using ion-exchange chromatography on diethylaminoethyl-Sephacel, and concentrations were expressed as milligrams of hexuronate per gram of creatinine. GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by means of densitometric scanning of Alcian Blue-stained strips. RESULTS: The relative content of total low-sulphated chondroitin sulphate species decreased significantly during the study period compared to baseline, whereas the relative percentages of heparan sulphate and chondroitin sulphate increased significantly. Moreover, a significant correlation was noted between the relative contents of urinary GAGs, renal function and inflammation indexes. CONCLUSIONS: It is likely that the excretion of various types of GAGs may be related to different glomerular pathophysiological conditions. Therefore, the determination of urinary GAG composition may represent a reliable indicator of disease activity.

Factors affecting S-homocysteinylation of LDL apoprotein B.

BACKGROUND: Hyperhomocysteinemia is an important risk factor for vascular disease and atherosclerosis, but the mechanisms by which homocysteine exerts its deleterious effects are not known. Because oxidation and/or homocysteinylation may increase atherogenicity of LDL, we investigated S-homocysteinylation of LDL as a possible contributor to atherosclerosis pathogenesis. METHODS: We used capillary electrophoresis to measure LDL-bound thiols [homocysteine, cysteine (Cys), cysteinylglycine, glutathione, and glutamylcysteine] in 104 healthy study participants We also assessed total plasma thiol concentrations and lipid profiles. RESULTS: Our data suggest that apoprotein B (apoB)-cysteinylglycine (CysGly), apoB-Hcy, and apoB-Cys concentrations are markedly higher in men than in women. The percentage of CysGly and glutathione on apoB was higher than that of the same thiols in plasma, whereas the other thiols were markedly less prevalent in lipoprotein than in plasma. Pearson correlation showed that among all thiols, only total plasma Hcy is related to apoB-Hcy concentrations. Multiple correlation analysis confirmed that total Hcy was the most important determinant of apoB-Hcy. Age and LDL cholesterol also showed positive associations, but Cys and, mainly, CysGly were negatively associated with apoB-Hcy concentrations. CONCLUSIONS: apoB-Hcy derivative formation is mainly dependent on total homocysteine concentration. Increased cholesterol concentrations are related to increased apoB-Hcy. CysGly seems to compete with Hcy for binding to LDL apoprotein, suggesting that CysGly may protect against atherosclerosis by decreasing the concentrations of Hcy transferred by LDL from plasma to endothelial and subendothelial spaces.

A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients.

BACKGROUND: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A(1c) (HbA(1c)) > or =8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA(1c) < 8.0% at baseline who maintained better metabolic control over time. METHODS: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA(1c) levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (+/-SD) follow-up of 6.8+/-1.1 years. RESULTS: All patients remained normoalbuminuric at follow-up, and had normal urinary alpha(1)-microglobulin levels. In patients with HbA(1c) <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA(1c) > or =8.0% and were significantly related to both HbA(1c) levels and the duration of poor glycemic control. CONCLUSIONS: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.

Urinary glycosaminoglycan composition in chronic glomerulonephritis.

BACKGROUND: Glycosaminoglycans (GAG) play an important role in regulating glomerular permeability, and a reduction in their plasmatic concentration or urinary loss has been implicated in the pathogenesis of diseases associated with increased albumin permeability. The purpose of this study was to evaluate GAG excretion in renal pathology by analyzing the composition of urinary GAG in antibody mediated glomerular injury, such as mesangial glomerulonephritis (IgAGN) and primitive membranous glomerulonephritis (MGN), to verify the effects of glomerular capillary wall lesion with and without mesangial cell injury. METHODS: Urinary GAG excretion was analyzed in 20 patients with IgAGN, 18 patients with MGN, and in 18 healthy subjects (controls). GAG were isolated from 24-hr urine using ion-exchange chromatography on DEAE-Sephacel, and the results are expressed as mg hexuronate/g creatinine (Cr). GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: We found total GAG levels significantly higher in the urine of patients with MGN in comparison with controls and patients with IgAGN. The electrophoretic pattern analysis demonstrated low sulfated chondroitin sulfate proteoglycan (LSC-PG) in all patients compared to 44% in controls (8/18), but also low sulfated chondroitin sulfate (LSC) in 18.4% of patients (7/38) and slow migrating LSC (SM-LSC) in 8% of patients (3/38), only in the MGN group. Moreover, in patients with MGN, the LSC-PG relative content was significantly higher when compared to that observed in controls. Finally, in IgAGN and MGN patients, a significant reduction in chondroitin sulfate (CS) relative content was observed. CONCLUSIONS: It seems likely that an increase in total GAG levels takes place when a reduction in renal function occurs, but the alteration of CS and herapan sulfate (HS) relative contents, and the presence of LSC-PG and free LSC also in the urine of IgAGN patients, allows us to suggest that the GAG distribution pattern becomes abnormal before an increase in total urine GAG excretion. In addition, the quali-quantitative determination of urinary GAG and GAGprotein complex could constitute an additional non-invasive marker to appraise the metabolism of renal connective tissue in some renal diseases.

Urinary transforming growth factor-beta 1 in various types of nephropathy.

Transforming growth factor-beta1 (TGF-beta1) is a potent multifunctional polypeptide that is involved in normal renal function and in the development of glomerular sclerosis. It is also an important mediator of the immune and anti-inflammatory responses. The purpose of this study was to examine whether the measurement of urinary TGF-beta1 excretion in patients with different types of renal diseases and in newly diagnosed type 1 diabetes mellitus represents a non-invasive tool to evaluate disease activity and to monitor response to therapy. We studied the urinary excretion of TGF-beta1 in 57 nephropathic patients divided in different groups according to the underlying disease: 15 had mesangial glomerulonephritis (IgAGN), 9 membranous glomerulonephritis (MGN), 7 rapidly progressive glomerulonephritis (RPGN), 8 systemic lupus erythematosus (SLE), 9 interstitial nephritis (IN), 9 chronic renal failure (CRF). TGF-beta1 was also measured in 38 patients with type 1 (insulin-dependent) diabetes mellitus (12 with newly diagnosed diabetes, 26 long-standing diabetes) and 31 healthy controls. Total urinary TGF-beta1 concentration was assayed by enzyme-linked immunoassay (ELISA), and expressed as a ratio to urinary creatinine concentration. The urinary TGF-beta1 levels were compared with the findings of biopsy and clinical parameters. Urinary TGF-beta1 excretion was significantly increased in all groups except MGN, IN and CRF. In non-diabetic patients, urinary TGF-beta1 levels correlated with crescent formation, floccular adhesion and mesangial proliferation, but not with the degree of tubulo-interstitial fibrosis. Urinary TGF-beta1 levels did not correlate with indices of renal function (serum creatinine, glomerular filtration rate (GFR), albumin excretion rate [AER]). Among diabetic patients, HbA(1C) significantly correlated with TGF-beta1 urinary excretion. Urinary TGF-beta1 levels may represent a valid indicator of acute glomerular flogosis associated with mesangial proliferation in glomerulonephrities. In newly diagnosed diabetic patients, hyperglycaemia seems to represent the principal factor leading to TGF-beta1 overproduction. Follow-up studies of urinary TGF-beta1 levels measured during optimal glycaemic control are necessary to clarify the relationship between hyperglycaemia and TGF-beta1 excretion.

Evidence for a proinflammatory and proteolytic environment in plaques from endarterectomy segments of human carotid arteries.

OBJECTIVES: Based on previous observations on apolipoprotein(a), apo(a), in human unstable carotid plaques, we explored whether in the inflammatory environment of human atheroma, proteolytic events affect other hepatic and topically generated proteins in relation to the issue of plaque stability. METHODS AND RESULTS: Forty unstable and 24 stable plaques from endarterectomy segments of affected human carotid arteries were extracted with buffered saline (PBS) and then 6 mol/L guanidine-hydrochloride (GdHCl) to identify loosely and tightly bound products, respectively. The extracts were studied before and after ultracentrifugation at d 1.21 g/mL. In the extracts, the concentrations of interleukin (IL)-6, -8, and -18 were significantly higher in the unstable plaques and correlated to those of MMP-2 and MMP-9. By Western blots, both apoB and apo(a) were highly fragmented and mostly present in the d 1.21 bottom that also contained fragments of apoE (10 and 22 kDa), decorin, biglycan, and versican. Fragmentation was higher in the unstable plaques. In baseline plasmas, concentrations of lipids, lipoproteins, and ILs did not differ between patients with unstable and stable plaques. CONCLUSIONS: In unstable and to a lesser extent in stable plaques, there is a proinflammatory and proteolytic microenvironment with the generation of fragments with potential pathobiological significance that requires investigation.

Urinary glycosaminoglycan and proteoglycan excretion in normoalbuminuric patients with type 1 diabetes mellitus.

BACKGROUND: Diabetic nephropathy may be related to an abnormal metabolism of glycosaminoglycans (GAG) in the glomerular basement membrane (GBM). The first manifestation of nephropathy is microalbuminuria, whose appearance indicates a loss of GBM selectivity. The present study evaluated whether GAG excretion becomes abnormal in parallel with microalbuminuria, and whether such abnormalities are also present in normoalbuminuric diabetic patients. METHODS: We measured urinary GAG excretion in 60 patients with type 1 (insulin-dependent) diabetes mellitus and in 22 healthy subjects. GAG were isolated from 24-h urine using ion-exchange chromatography on DEAE Sephacel. GAG composition was determined by cellulose acetate electrophoresis and expressed as percentages by densitometric scanning of Alcian Blue stained strips. RESULTS: On subgrouping for albuminuric status and glyco-metabolic control, we found high urinary GAG concentrations in all except the normoalbuminuric patients with good glyco-metabolic control. The urinary GAG electrophoretic pattern showed alterations in chondroitin sulphate (CS) and heparan sulphate (HS) relative contents. A higher frequency of low sulphated chondroitin sulphate-proteoglycan (LSC-PG) was observed in all patients, including those with normoalbuminuria and good glyco-metabolic control. CONCLUSIONS: This urinary pattern may be indicative of an abnormal GBM metabolism. Since GAG play an important role in GBM permeability, these anomalies might consequently represent a first step towards selective changes of GBM in type 1 diabetes mellitus.