Decreased VLDL-Apo B 100 fractional synthesis rate despite hypertriglyceridemia in subjects with type 2 diabetes and nephropathy

Subjects with Type 2 Diabetes Mellitus (T2DM) and diabetic nephropathy (DN) often exhibit hypertriglyceridemia. The mechanism(s) of such an increase are poorly known. OBJECTIVE: We investigated VLDL-Apo B 100 kinetics in T2DM subjects with and without DN, and in healthy controls. DESIGN: Stable isotope 13C-leucine infusion, and modelling analysis of tracer-to-tracee ratio dynamics in the protein product pool in the 6-8 hr period following tracer infusion, were employed. SETTING: Male subjects affected by T2DM, either with (n=9) or without (n=5) DN, and healthy male controls (n=6), were studied under spontaneous glycemic levels in the post-absorptive state. RESULTS: In the T2DM patients with DN, plasma triglyceride (TG) (2.2\ub10.8 mmol/L, Mean\ub1SD) and VLDL-Apo B 100 (17.4\ub110.4 mg/dl) concentrations, and VLDL-Apo B 100 pool (0.56\ub10.29 g), were 3e60-80% greater (p<0.05 or less) than those of the T2DM subjects without DN (TG: 1.4\ub10.5 mmol/L; VLDL-Apo B 100: 9.9\ub12.5 mg/dl; VLDL-Apo B 100 pool: 0.36\ub10.09 g), and 3e80-110% greater (p<0.04 or less) than those of nondiabetic controls (TG: 1.2\ub10.4 mmol/L; VLDL-Apo B 100: 8.2\ub11.7 mg/dl; VLDL-Apo B 100: 0.32\ub10.09 g). In sharp contrast however, in the subjects with T2DM and DN, VLDL-Apo B 100 FSR was 6550% lower (4.8\ub12.2 pools/day) than that of either the T2DM subjects without DN (9.9\ub14.3 pools/day, p<0.025) or the control subjects (12.5\ub19.1 pools/day, p<0.04). CONCLUSIONS: The hypertriglyceridemia of T2DM patients with DN is not due to hepatic VLDL-Apo B 100 overproduction, which is decreased, but it should be attributed to decreased apolipoprotein removal

Polar Electrophoresis: Shape of Two-Dimensional Maps Is as Important as Size

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of “isobaric” spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis

High Abundance Proteins Depletion vs Low Abundance Proteins Enrichment: Comparison of Methods to Reduce the Plasma Proteome Complexity

BACKGROUND: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a direct comparison of these two approaches. Following either approach, the plasma sample was further fractionated by SCX chromatography and analyzed by RP-LC-MS/MS with a Q-TOF mass spectrometer. The depletion of the 20 most abundant plasma proteins allowed the identification of about 25% more proteins than those detectable following low abundance proteins enrichment. The two datasets are partially overlapping and the identified proteins belong to the same order of magnitude in terms of plasma concentration. CONCLUSIONS/SIGNIFICANCE: Our results show that the two approaches give complementary results. However, the enrichment of low abundance proteins has the great advantage of obtaining much larger amount of material that can be used for further fractionations and analyses and emerges also as a cheaper and technically simpler approach. Collectively, these data indicate that the enrichment approach seems more suitable as the first stage of a complex multi-step fractionation protocol

Rapid, simple and effective technical procedure for the regeneration of IgG and HSA affinity columns for proteomic analysis.

In plasma and serum, the presence of high-abundance proteins can overwhelm the signals of low-abundance proteins, which then become undetectable either by two-dimensional gels or chromatographic techniques. Therefore, depletion of abundant proteins is a prerequisite to detect low-abundance components. Furthermore, the regeneration of pre-purification tools could be money-saving. We applied an affinity chromatography kit to remove albumin and the immunoglobulin chains from plasma and propose a simple and effective technical procedure for the regeneration of these affinity column

Middle age is not associated with altered fibrinogen concentration and production in males.

Whether ageing is associated with increased fibrinogen concentration and production remains unclear. We measured fibrinogen fractional (FSR) and absolute synthesis (ASR) rates in male volunteers, of either young (mean age: 28 years, range: 22-34) or middle age (mean age: 57 years, range: 38-72), using a leucine-tracer isotope dilution technique. In the middle-age group, neither fibrinogen FSR (20.8 +/- 1.6%/day) nor ASR (1.8 +/- 0.1 g/day), or concentration (274 +/- 15 mg/dl), were different from those of the younger group (FSR: 20.2 +/- 1.4; ASR: 1.7 +/- 0.2; concentration: 265 +/- 8, respectively). Leucine Ra, an index of endogenous proteolysis, was approximately 20% lower in the older than in the younger group (P < 0.02). Thus, middle age in males is not associated with increased fibrinogen concentration and turnover, whereas endogenous protein breakdown in decreased. Factor(s) different from age per se are likely to be involved in the dysfibrinogenemia possibly occurring with ageing. Protein turnover is already reduced in middle-age males

Effect of liver cirrhosis on phenylalanine and tyrosine metabolism

PURPOSE OF REVIEW: Phenylalanine conversion to tyrosine (i.e., 'hydroxylation') is the first irreversible step in phenylalanine catabolism and a source of circulating tyrosine. The purpose of the present review is both to examine hydroxylation from a biochemical standpoint and to report data measured in vivo under physiological conditions, as well as in liver and kidney disease. RECENT FINDINGS: The simultaneous infusion of phenylalanine and tyrosine tracers in humans allows us to determine the hydroxylation rate in vivo. Hydroxylation accounts for a minor ( approximately 10-20%) although significant portion of tyrosine flux. The liver and the kidney are the key organs accounting for virtually the whole-body hydroxylation rates. It is regulated by substrate availability, being acutely stimulated by mixed meal ingestion and by dietary adaptation to high phenylalanine intakes. Theoretically, it may be impaired in advanced liver and kidney disease. Nevertheless, in compensated liver cirrhosis, hydroxylation as well as tyrosine flux are not decreased but rather increased. Only in end stage liver disease hydroxylation may be impaired and is corrected by transplantation. Hydroxylation is also reduced in end stage renal disease. SUMMARY: Phenylalanine hydroxylation in vivo appears to represent a regulatory step of phenylalanine disposal and tyrosine production under acute and/or extreme conditions

Kinetics of albumin homocysteinylation measured with matrix-assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer.

Abstract: Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin. We have compared this method with a (35)S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The (35)S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximate to 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance

Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown

Proteome analysis of cultured fibroblasts from type 1 diabetic patients and normal subjects

CONTEXT: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. OBJECTIVE: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. DESIGN: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and alpha-actinin 4 and a decrease of tubulin-beta2 and splice isoform 2 of tropomyosin beta-chain were detected. CONCLUSIONS: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed