Ameliorative Effect of Trigonella Foenum Graecum L. on Lipid Profile, Liver Histology and LDL-Receptor Gene Expression in High Cholesterol-Fed Hamsters.

BACKGROUND: Trigonella foenum graecum L. (Fenugreek, FG) is used in many countries as a medicinal plant. Evidence has suggested the hypolipidemic effect of Fenugreek; however, its actual mechanism has not been determined yet. OBJECTIVES: The purpose of our research was to investigate the effect of Fenugreek on lipid profile, liver histology and LDL receptor gene expression in male hamsters fed with high cholesterol diet. METHODS: These animals were given normal diet (ND), high cholesterol diet (HCD: 2% cholesterol and 0.5% cholic acid added to ND), HCD supplemented with 2g and 8g fenugreek per 100g ND (HCD+FG2 and HCD+FG8) respectively for four weeks. At the end of treatment, serum lipids, lipoproteins and liver enzymes were measured. Finally, LDL receptor (LDLR) gene expression was determined in the liver of the studied animals using Real Time-PCR method and liver histological changes were evaluated by H&E staining method. RESULTS: A significant reduction was observed in serum triglyceride (p<0.01), total cholesterol, low and very low density cholesterol, aspartate and alanine transaminases in HCD+FG8 group (p<0.05) compared with HCD group, while serum level of HDL-c (p<0.01) increased. In addition, LDLR gene expression in HCD+FG8 group increased 7.8 folds. The results confirm the protection effect of liver tissue in HCD+FG8 group against pathological changes. There was no significant change in LDLR gene expression in HCD+FG2 group. In conclusion, fenugreek ameliorated dyslipidemia by up-regulation of LDLR gene expression and can be used as a protective agent against hypercholesterolemia.

Expression of interleukins 7 & 8 in peripheral blood mononuclear cells from patients with metabolic syndrome: a preliminary study.

BACKGROUND & OBJECTIVES: Metabolic syndrome (MS) is a common but serious public health problem in developed countries. Chronic inflammation plays a key role in MS. Interleukins (IL)-7 and 8 are considered to have proinflammatory effects and may be involved in the pathogenesis of MS. Therefore, the aim of this study was to determine gene expression level of IL-7 and IL-8 in peripheral blood mononuclear cells (PBMCs) of patients with MS compared to healthy control subjects. METHODS: Using real-time RT-PCR, the relative amounts of IL-7 and IL-8 mRNA were determined in PBMCs from 20 female patients with MS and compared with those of 20 healthy control subjects. Biochemical and anthropometric parameters of MS were also assessed. RESULTS: Total cholesterol, triglyceride, and fasting blood sugar were significantly higher in MS patients compared to healthy subjects. There were no significant differences in HDLc and LDLc between the two groups. IL-8 expression in PBMC was significantly decreased in MS versus control subjects (fold of change was 0.395±0.1824), while no difference in the IL-7 expression was detected between them. IL-8 expression had negative correlation with MS components especially with triglyceride and total cholesterol (r=0.5, P<0.001). INTERPRETATION & CONCLUSIONS: In this preliminary study, no detectable differences were found in IL-7 expression and decreased expression of IL-8 in PBMCs of MS patients as compared to those of control subjects. Study on a larger population and investigating the mechanisms involved can reveal more details.

Total antioxidant capacity of saliva and dental caries.

OBJECTIVE: Dental caries is one of the most common infectious diseases worldwide. Saliva has many functions in the oral cavity and is the first line defense against dental caries. Oxidative stress can affect initiation and progression of many inflammatory and infectious diseases such as dental caries. Thus the aim of this study was to evaluate the relationship between total antioxidant capacity (TAC) of saliva and dental caries. STUDY DESIGN: 100 healthy high school students (50 female and 50 male) with age range of 15 -17 years were randomly selected, divided to four groups. Unstimulated whole saliva specimens were collected at the morning. TAC of saliva was evaluated by spectrophotometric assay. Statistical comparisons were performed using Student's t-test, by SPSS 13. RESULTS: The level of TAC was significantly higher in the saliva of caries active group relative to the caries free subjects. Statistical analysis for male and female groups showed a statistically significant reduction of TAC level in female group. CONCLUSION: TAC was higher in caries active group. Thus this result showed that total antioxidant capacity may influence in dental caries and activity can be measured by salivary factors and this may be helpful in preventive dentistry.

Increased receptor for advanced glycation end products in spermatozoa of diabetic men and its association with sperm nuclear DNA fragmentation.

Although the majority of patients with diabetes have disorders in sexual function, associations between diabetes mellitus and sperm function at the molecular level are largely unknown. As receptor for advanced glycation end products plays a key role in many diabetic complications, we hypothesised that it may be involved in sperm nuclear DNA fragmentation. RAGE levels were determined using ELISA and western blot analysis in sperm samples from 32 diabetic and 35 nondiabetic men. Sperm DNA fragmentation was assessed using TUNEL assay. Diabetic men had significantly higher mean levels of RAGE protein (P < 0.001) and DNA fragmentation (P < 0.001) in spermatozoa. Sperm RAGE was directly correlated to sperm DNA fragmentation in diabetic men (r = 0.81, P < 0.001). The high positive correlation between RAGE levels and nuclear DNA fragmentation in spermatozoa of diabetic men suggests a central role of RAGE in disturbances in sexual function of diabetic men.

Relationship between advanced glycation end products and increased lipid peroxidation in semen of diabetic men.

AIMS: Majority of diabetic male patients have disturbances in their reproductive systems. However, the mechanisms underlying these disturbances are largely unknown. Since advanced glycation end products (AGE) have a key role in oxidative stress and cell damage in diabetic complications, we hypothesize that AGEs may be involved sperm lipid peroxidation. METHODS: total AGEs in seminal plasma of 32 diabetic and 35 non-diabetic men was determined by spectrofluorimetric method and carboxy methyl lysine (CML) level was assayed using ELISA. Contents of lipid peroxidation in sperm and seminal plasma were determined by thiobarbituric acid reaction. Total antioxidant capacity (TAC) was measured by a colorimetric assay. RESULTS: total AGEs were found significantly higher in seminal plasma of diabetic men than non-diabetic group (p<0.001) whereas no significant differences in seminal plasma CML values between two groups was observed. Moreover, sperm and seminal plasma lipid peroxidation were significantly higher in diabetic subjects than non-diabetic men and a significantly lower TAC was detected in diabetic group compare to non-diabetics. CONCLUSIONS: these results showed an increment in AGEs in seminal plasma of diabetic subjects and may suggest a key role for glycation process and increased oxidative stress in reproductive system dysfunction.

The effect of increasing blood flow rate on dialysis adequacy in hemodialysis patients.

Inadequacy of dialysis is one of the determinants of morbidity and mortality in patients undergoing dialysis. The aim of this study was to assess the effect of increasing blood flow rate during hemodialysis (HD) on the adequacy of dialysis. In this study, 42 patients on maintenance HD were assessed. Body weight and blood urea nitrogen (BUN) levels before and after HD sessions were recorded on all the study patients. Volume of ultrafiltration, the duration of dialysis, percentage of filter clearance and flow rate of the dialysate were collected and documented in a checklist. Both Kt/V and urea reduction ratio (URR) were determined at two different blood pump speeds, i.e. 200 and 250 mL/min. During HD, hemodynamic status and vital signs of patients were monitored and controlled. After collecting the necessary data, efficiency of dialysis was calculated using the standard formula. Descriptive and analytical statistics was carried out to analyze the data. Using blood flow rate of 200 mL/min, 16.7% of patients had Kt/V higher than 1.3 and URR higher than 65. On the other hand, with flow rate of 250 mL/min, 26.2% of patients had Kt/V higher than 1.3 and 35.7% of subjects had URR higher than 65. Paired t-test with 95% confidence showed a significant difference in dialysis efficiency between two groups. Our data further confirm that increasing the blood flow rate by 25% is effective in increasing dialysis adequacy in HD patients.

Reproducible and sensitive determination of charged oligosaccharides from haptoglobin by PNGase F digestion and HPAEC/PAD analysis: glycan composition varies with disease.

Many studies have reported changes in the carbohydrate structure of serum glycoproteins in disease, but this information is often of limited value for understanding disease mechanisms because it is obtained with simple and/or indirect methodologies that determine only one structural feature. On the other hand, more detailed carbohydrate methodologies are time-consuming and require a lot of purified material. Using haptoglobin (Hp) as a model protein, a new procedure was devised that determined the oligosaccharide composition of very small amounts of Hp in a relatively short time. The Hp was purified by batch affinity-chromatography, oligosaccharides were removed with PNGase F, and the oligosaccharide composition of charged species was determined using HPAEC/PAD (Dionex carbohydrate analyser). The method was applied to the analysis of Hp from eight healthy individuals and 37 patients with different inflammatory diseases or cancers. Twenty-seven oligosaccharides were consistently detected, but the majority could not be identified. However, by calculating retention times relative to the sialylated biantennary peak (Neu5Ac(alpha)2-3/6Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-6(Neu 5Ac(alpha)2-3/6Gal(beta)1-4GlcNAc(beta)1-2Man(alpha)1-3)Man(beta)1-4G lcNAc(beta)1-4GlcNAc) it was possible to compare profiles quantitatively. Although no peak was identified as disease-specific, characteristic and reproducible profiles were obtained. Particularly striking were reductions in the major peaks in Crohn's disease, rheumatoid arthritis, stomach cancer, accompanied by increases in unidentified peaks. Previous studies suggested that many of the unknown peaks were due to increased sialylation and fucosylation. Only small changes in patterns were observed for breast and ovarian cancer. The new procedure will be very useful in the characterization of oligosaccharide composition of glycoproteins in clinical specimens.

Sialyl Lewis(x) expression on IgG in rheumatoid arthritis and other arthritic conditions: a preliminary study.

Both infiltrating leukocytes and soluble immunoglobulin form aggregates in synovial fluid during the inflammatory process in rheumatoid arthritis (RA). Some of these changes are probably mediated by the adhesion molecule, E-selectin, which increases its expression with disease activity. As glycosylation changes in IgG in RA are well established, the current study was undertaken to measure the expression of the carbohydrate antigen sialyl Lewis x (sLe(x)), on IgG in RA. sLe(x) is a major ligand for E-selectin. Using a recently developed ELISA, sLe(x) expression was determined in IgG isolated from 8 healthy individuals, 20 RA sufferers (10 early and 10 with more long-standing disease) and 20 patients with other rheumatic conditions (osteoarthritis, ankylosing spondylitis, systemic lupus erythematosus). S Le(x) expression on IgG was elevated above the reference range in all but one of the RA patients and this change was highly significant (P < 0.0006). Expression of this antigen on IgG was also significantly different from normal in the other arthritic groups (P < 0.02), but the changes were much less than that observed for RA. In early RA, sLe(x) was inversely correlated with parameters used to measure disease activity. This was not observed with the established RA, where there was weak positive association. These preliminary results indicate that a change in sLe(x) expression on IgG is an early finding in the development of RA, which may be important in the development of the disease or for predicting its outcome.

Do synovial fluid acute phase proteins from patients with rheumatoid arthritis originate from serum?

This study was performed in order to gain insight into the occurrence, glycosylation and the possible origin of the acute-phase proteins alpha1-acid glycoprotein (AGP) and alpha1-protease inhibitor (PI) in sera and synovial fluid from patients with rheumatoid arthritis (RA). Therefore paired sera and synovial fluid samples from patients with RA, and paired synovial fluid samples from right and left knees of patients with varying degrees of arthritis were studied. Crossed affinity immunoelectrophoresis (CAIE) was used with concanavalin A and Aleuria aurantia lectin for the detection of the degree of branching and fucosylation, respectively, and the monoclonal CSLEX-1 for the detection of Sialyl Lewis(X) (SLe(X)) groups on AGP. For PI, not only CAIE, but also high-pressure-anion-exchange chromatography with pulsed amperometric detection was used to study the glycosylation. It was established that the concentrations of AGP and PI were increased in the serum of RA patients compared to normal healthy controls, but that the concentration of both proteins, as well as albumin, was significantly lower in synovial fluid than in serum. Furthermore, the type of glycosylation of both AGP and PI found in RA was significantly different from that found in normals, with increased fucosylation, but there were no major differences in the degree of branching of AGP- or PI-glycans in RA, compared to normals. No differences in glycosylation could be established between serum and synovial fluid in RA. For PI an increased fucosylation was found, both in serum and synovial fluid, using both methods of detection, and it could be established that only the alpha1-->3- and not the alpha1-->6-fucosylation of PI was affected by RA. The increased fucosylation of AGP resulted in an increased expression of SLe(X) on AGP-glycans. Since the alpha1-->3-fucosylation of AGP was significantly increased in both serum and synovial fluid from RA patients, and this correlated with systemic but not with local disease parameters, it can be suggested that acute phase proteins in synovial fluid are most probably of hepatic origin.

A lectin method for investigating the glycosylation of nanogram amounts of purified glycoprotein.

To unravel the complexities of the glycosylation of a protein is a substantial task, which requires considerable effort and resources. However, in many situations this is unnecessary, because only a limited amount of information is required. A new lectin-binding assay is described which is rapid, cheap and versatile. A purified glycoprotein is absorbed on to the plastic surface of a microtitre plate. After removing unbound protein by washing, uncoated sites on the plate are blocked and digoxigenin or biotin-labelled lectin is added. The degree of lectin binding is measured using either an anti-DIG antibody or streptavidin conjugated enzyme, which is subsequently used to develop a colour reaction. Using this method it is possible to screen multiple specimens with high sensitivity and excellent precision. In addition, very small amounts of lectin are used, background absorbances are low, and the procedure does not require a high degree of technical skill. Because very small amounts of glycoprotein are needed, a glycoprotein can often be rapidly purified by batch affinity chromatography. The method has been successfully applied to several purified proteins using the lectins, Con A, LCA, LTA, MAA, and SNA, and the information obtained agrees with that produced by more sophisticated approaches, eg Dionex Carbohydrate Analyser. Using a panel of lectins, a carbohydrate structural profile is quickly built-up, and subtle differences in glycosylation identified. This method should be particularly useful for screening glycosylation in multiple clinical specimens; in specimens where very small amounts of material are available, such as membrane molecules; and in the screening of recombinant proteins produced commercially.

An improved ELISA for the determination of sialyl Lewis(x) structures on purified glycoconjugates.

The membrane carbohydrate antigen, sialyl Lewis x (sLe(x)), is involved in cellular adhesive interactions in many diseases, such as cancer, inflammation and thrombosis. This antigen is also found on soluble macromolecules, such as serum glycoproteins, but the precise role of soluble sLe(x) in modifying disease processes, or reflecting the pathological changes is still unclear. Although methods were previously reported for the measurement of soluble sLe(x), many of these were not well characterised, measurements were mainly made on mixtures of molecules, and the anti-sLe(x) antibodies were used at concentrations that made the assay expensive. In this study an ELISA has been devised that detects sLe(x) in purified soluble glycoconjugates using the anti-sLe(x) antibody, CSLEX I. Commercially-available haptoglobin (Hp) and synthetic complexes of Lewis antigens with polyacrylamide were used as model substances in developing the procedure. Key steps were washing the antibody/antigen complex with ten times diluted salt solution to prevent dissociation of the complex and the use of bovine serum albumin for blocking non-specific interactions. The assay was shown to be very specific, its precision was in the range 6-12%, and it could detect less than a pmol of sLe(x). It could also distinguish between different densities of sLe(x) on the same amount of glycoconjugate. Determination of sLe(x) in Hp isolated from small groups of healthy individuals, cancer patients, and rheumatoid arthritis sufferers suggested that the antigen expression is increased in disease. This method, which is an improvement on those previously described will be useful for determining sLe(x) in many different types of soluble glycoconjugate, and used in combination with synthetic carbohydrate polyacrylamide complexes, will help to standardize measurements of soluble sLe(x) in the future.

Glycosylation of alpha-1-proteinase inhibitor and haptoglobin in ovarian cancer: evidence for two different mechanisms.

The change in glycosylation of the two acute-phase proteins, alpha-1-proteinase inhibitor (API) and haptoglobin (Hp), in progressive ovarian cancer is different. This has been shown by monosaccharide analysis and lectin-binding studies of proteins purified from serum. In the glycan chains of API, there is decreased branching (more biantennary chains), less branches ending in alpha 2-3 sialic acid, more branches ending in alpha 2-6 sialic acid and more fucose, probably linked alpha 1-6 to the core region. On the other hand, Hp shows increased branching (more triantennary chains), more branches ending in alpha 2-3 sialic acid, less branches ending in alpha 2-6 sialic acid, and more fucose, probably in the alpha 1-3 linkage at the end of the chains. This is surprising because API and Hp are thought to be glycosylated by a common pathway in the liver. We have also shown that the fucose-specific lectin, lotus tetragonolobus, extracts abnormal forms of both Hp and API in ovarian cancer, but the expression of this Hp is related to tumour burden and the expression of this API is related to lack of response to therapy. It is suggested that this difference in the behaviour of API and Hp in ovarian cancer may be associated with the different changes in their glycosylation. Of the many mechanisms that could explain these findings, a likely one is that a pathological process is removing API with triantennary chains from the circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased branching, increased fucosylation and changed sialylation of alpha-1-proteinase inhibitor in breast and ovarian cancer.

Proteolytic enzymes could be very important in spread of cancer, but the role of the body's natural inhibitors of these enzymes in this process is unknown. One such inhibitor is the serum glycoprotein, alpha-1-proteinase inhibitor (API). In previous studies we showed that the fucose-specific lectin, lotus tetragonolobus, extracted high amounts of API in cancer when patients were unresponsive to treatment. The aim of this study was to determine whether the carbohydrate structure of API is altered in cancer. API was isolated from the sera of healthy women and women with breast or ovarian cancer. By means of high-performance anion-exchange chromatography, cancer API was shown to contain more fucose and less N-acetylglucosamine than healthy API. Further investigation of the purified specimens using a lectin-binding assay suggested that the cancer API was less branched and contained more alpha 2-6 and less alpha 2-3 sialic acid. Observations from both methods were consistent with an increase in bi-antennary chains terminating in alpha 2-6 sialic acid and possibly more alpha 1-6 fucose in the core of the unit. These distinctive changes could have important consequences for the function of API in cancer and may help to develop more precise markers for monitoring pathological progression in this disease.