Soy protein diet improves endothelial dysfunction in renal transplant patients.

BACKGROUND: Since it has been demonstrated that soy diet can improve endothelial function, in the present study we evaluated the effect of dietary substitution of 25 g of animal proteins with soy proteins on endothelial dysfunction in renal transplant patients. METHODS: In 20 renal transplant patients (55 +/- 11 years, serum creatinine 1.7 +/- 0.6 mg/dl), brachial artery flow mediated dilation (FMD) and endothelium-independent vasodilation (sublingual nitroglycerine, 25 microg) were measured at baseline, after 5 weeks of a soy diet and finally after 5 weeks of soy wash-out. Changes in plasma lipids, markers of oxidative stress (lipid peroxides, LOOH) and inflammation (C-reactive protein), isoflavones (genistein and daidzein), asymmetric dimethyl arginine (ADMA) and L-arginine were also evaluated. RESULTS: At baseline, patients showed a significantly lower FMD as compared with age-matched healthy subjects (3.2 +/- 1.8 vs 6.3 +/- 1.9, respectively; P < 0.001), while response to nitroglycerine was similar. After soy diet, actual protein intake was not changed, cholesterol and lipid peroxides were significantly reduced, and isoflavones were detectable in plasma. Soy diet was associated with a significant improvement in FMD (4.4 +/- 2.0; P = 0.003 vs baseline), while response to nitroglycerine was unchanged. Improvement in FMD was related to L-arginine/ADMA ratio changes, but no significant relation was found to changes in cholesterol, lipid peroxides or genistein and daidzein plasma concentrations. After 5 weeks of soy diet discontinuation, FMD (3.3 +/- 1.7%) returned to baseline values and isoflavones were no longer detectable in plasma. CONCLUSIONS: A soy protein diet for 5 weeks improves endothelial function in renal transplant patients. This effect seems to be strictly dependent on soy intake as it disappears after soy withdrawal and is mediated by an increase in the L-arginine/ADMA ratio, independently of change in lipid profile, oxidative stress or isoflavones.

Preliminary approaches for the development of a high-performance liquid chromatography/electrospray ionization tandem mass spectrometry method for the detection and label-free semi-quantitation of the main storage proteins of Lupinus albus in foods.

Since recent literature has indicated that white lupin (Lupinus albus) may be a useful source of hypocholesterolemic proteins to be used in functional food formulation, our final goal is the development of a fast and automated high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the detection and the label-free semi-quantitation of the main lupin globulins in lupin foods and food ingredients. We present here some preliminary results in this direction. As a first step a total protein extract (TPE-WF) from lupin flakes was pre-fractionated by anion-exchange chromatography and each fraction was digested with trypsin and analyzed by HPLC/ESI-MS/MS. Subsequently, the tryptic digest of TPE-WF was directly analyzed by HPLC/ESI-MS/MS without any pre-fractionation. Eventually, in order to test the applicability of the method to real samples, a lupin beverage and two lupin protein isolates were analyzed. Both Mascot and Spectrum Mill MS Proteomics Workbench software were used to identify the protein composition in these samples and Spectrum Mill was used also to test the possibility of developing a label-free semi-quantitation method based on peptide hits. Encouraging results were obtained especially in the detection of the hypocholesterolemic component beta-conglutin.

Characterization and quantification of soy isoflavone metabolites in serum of renal transplanted patients by high-performance liquid chromatography/electrospray ionization mass spectrometry.

During a dietary intervention study on 16 renal transplanted patients, in which 25 g/day of animal proteins were replaced with 25 g of soy proteins, the metabolic profile of soy isoflavones in serum was characterized. This paper describes a reliable and fast liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) method, in negative ion mode, allowing the characterization and simultaneous quantification of several soy isoflavone metabolites. Six metabolites were identified and quantified: daidzein ([M-H](-) at m/z 252.8), dihydrodaidzein (DHD, [M-H](-) at m/z 254.8), equol ([M-H](-) at m/z 240.9), O-desmethylangolensin (O-DMA, [M-H](-) at m/z 256.8), genistein ([M-H](-) at m/z 268.8), and dihydrogenistein (DHG, ([M-H(+)](-) at m/z 270.8). Quantification was assessed using two deuterated internal standards, D(3)-daidzein and D(4)-genistein. This method permitted a limit of quantification (LOQ, S/N = 10) and a limit of detection (LOD, S/N = 3) of 0.05 microM and 0.005 microM for all analytes, except for genistein, where the LOQ and LOD were 0.005 microM and 0.001 microM, respectively. The linearity ranges were from 0.005 to 1.5 microM for genistein, from 0.05 to 1.5 microM for DHG, and from 0.05 to 0.7 microM for the other metabolites. The relative standard deviations (RSDs) were between 0.19% and 13.9% at the LOQ concentration for all metabolites, and between 0.6% and 4.8% at the maximum concentration. On the basis of the results obtained in the dietary intervention study, it was possible to split the patients into five groups characterized by different metabolic pathways.

Analysis of Lupinus albus storage proteins by two-dimensional electrophoresis and mass spectrometry.

A laboratory-prepared total protein extract (TPE) and a lupin protein isolate (LPI-E) produced in a pilot plant were submitted to a detailed two-dimensional (2DE) proteomic investigation. Recent findings have indicated that in an established rodent model of hyperlipidemia, moderate daily intakes of LPI-Es lead to a reduction of total and low-density lipoprotein cholesterol levels, and the knowledge of the actual composition of the protein sample used in that study is at the basis of further structure/action investigations. The experimental results indicate that the semi-industrial procedure used for the production of LPI-E damages only marginally the proteins. It does, however, cleave some disulfide bridges and induce mild proteolysis, as confirmed by the higher number of resolved protein spots in the low Mr and acidic pI region of the 2DE map. Out of 72 spots submitted to mass spectrometry and compared with available protein databases, 42 correspond to fragments of beta-conglutin, the 7S globulin of lupin, spanning between positions 37 and 495 of the protein sequence. Using the bioinformatic tool BlastP, these peptides were compared to the alpha'-subunit of beta-conglycinin, the 7S globulin of soybean, this being the most active hypocholesterolemic component of soybean protein, as shown by in vitro and in vivo experiments. At least 18 peptides derived from beta-conglutin, having a percentage identity higher than 50% and a similarity percentage higher than 70% vs the alpha'-subunit of beta-conglycinin, are likely candidates to be the biologically active components of lupin protein.

A simple method for the characterization and quantification of soy isoflavone metabolites in the serum of MMTV-Neu mice using high-performance liquid chromatography/electrospray ionization mass spectrometry with multiple reaction monitoring.

In a study aimed at elucidation of the possible role of dietary phytoestrogens in the growth of breast cancer, it was necessary to develop a convenient, accurate, and reproducible method for the characterization and quantification of isoflavone metabolites in the serum of MMTV mice that were fed diets containing different amounts of these polyphenols. The analytical method is based on liquid chromatography with mass spectrometry in multiple reaction monitoring mode, using deuterated genistein as internal standard. The identified metabolites were genistein and dihydrodaidzein (DHD); their average concentrations in serum were 0.71 and 0.21 microM, respectively, for animals fed 1.5 mg/day of genistein and 0.7 mg/day of daidzein. This method assured a limit of quantification of 0.04 microM for genistein and 0.08 microM for DHD, and a limit of detection of 0.018 microM for genistein and 0.035 microM for DHD. The coefficient of variation was 8.9% for genistein and lower than 15% for DHD. This study provides the first data indicating the presence of dihydrodaidzein in serum of mice fed diets containing soy proteins.

Proteins of white lupin seed, a naturally isoflavone-poor legume, reduce cholesterolemia in rats and increase LDL receptor activity in HepG2 cells.

White lupin (Lupinus albus, L.), a widely cultivated crop that has been consumed for many years in Western Europe, may provide a useful alternative for individuals wishing to substitute animal with plant proteins for cardiovascular disease prevention. Lupin seeds have a very low content of isoflavones, and lupin protein isolates are essentially isoflavone free. In rats fed a casein-based cholesterol + cholic acid diet, a relatively low daily intake (50 mg/d by gavage for 2 wk) of total lupin protein extract reduced plasma total and VLDL + LDL cholesterol concentrations by 21 and 30%, respectively (both P<0.001). In an attempt to elucidate the lipid-lowering mechanism, LDL receptor activity was evaluated in a human hepatoma cell line (HepG2). In this model, the lupin total protein extract was essentially inactive, whereas one purified minor protein component, conglutin gamma, had a remarkable upregulatory effect, with maximal increases of 53 and 21% (both P<0.05) for LDL uptake and degradation, respectively. This initial study indicates that lupin, although isoflavone free, has hypocholesterolemic activity similar to that of other leguminous proteins in an established animal model. Further, the cholesterol reduction appears to be associated with stimulation of LDL receptors by a well-defined protein component of the lupin seeds as demonstrated by in vitro studies.