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Aim. Asymmetric and symmetric dimethylarginines (ADMA and SDMA, respectively) are protein breakdown markers; both compete with arginine for cellular transport and both are excreted in urine. Moreover, ADMA is a non-selective inhibitor of nitric oxide (NO) synthase that is metabolized by a specific hydrolase in which the activity during stress remains controversial. While an increase in ADMA is known to be associated with adverse events, little is known about SDMA. We investigated plasma ADMA and SDMA levels during ICU stay to reveal the time course of endogenous NO inhibition in patients with sepsis. Methods. A post hoc analysis from a prospective random controlled trial conducted in three ICUs was performed to study the pathophysiological pathways of sepsis. ADMA, SDMA, the ratio of ADMA/SDMA (a marker of ADMA catabolism), arginine, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and C reactive protein (CRP) were measured on days 1, 3, 6, 9, 12 and at discharge in 72 consecutive severely septic patients. Results. Fasting basal glycemia, creatinine, IL-6, TNF-α, CRP, DMA, and SDMA were higher than normal. The ADMA/SDMA ratio was decreased by 50%, and arginine levels were low. ADMA levels were related to the total Sequential Organ Failure Assessment (SOFA) scores and arginine levels, and inversely related to IL-6 and CRP levels. SDMA levels were related to Simplified Acute Physiologic Scores II (SAPS II), SOFA scores, blood urea, creatinine, and arginine levels. The ADMA/SDMA ratio was inversely related to IL-6 levels. In 58 ICU survivors, creatinine, IL-6, and CRP levels decreased over time; ADMA levels increased, SDMA levels remained stable, and the ADMA/SDMA ratio increased. In 14 non-survivors, creatinine, IL-6, TNF-α, CRP, and ADMA levels were stable, whereas the SDMA levels increased and the ADMA/SDMA ratio remained low. In both ICU survivors and non-survivors, the levels on the last ICU day confirmed the data trends. SDMA, but not ADMA, was associated with ICU mortality. Conclusion. ADMA catabolism appears to be activated by inflammation; its increase during the advanced septic phase in surviving patients may suggest an endogenous inhibition of NO synthesis during the full-blown septic phase. In severe sepsis, SDMA, but not ADMA, appears to be a marker of alterations in vital functions and mortality.

Despite research progresses, the chance to accurately predict the risk for diabetic nephropathy (DN) is still poor. So far, the first evidence of DN is micro-albuminuria, which is detected only 10–20 years after the onset of diabetes. Our goal is to develop new predictive tools of nephropathy starting from urine, which can be easily obtained using noninvasive procedures and it is directly related to kidney. Since it is reasonable to suppose that, in predisposed patients, the mechanisms leading to nephropathy start acting since the diabetes onset, urine from children with recent diagnosis of type 1 diabetes was subjected to proteomic analysis in comparison to age-matched controls. Targeted confirmation was performed on children with a longer history of diabetes using Western Blotting and applying a urinary lipidomic approach. To definitively understand whether the observed alterations could be related to diabetic nephropathy, urine from diabetic adults with or without albuminuria was also examined. For the first time, lipid metabolisms of prostaglandin and ceramide, which are significantly and specifically modified in association with DN, are shown to be already altered in children with a recent diabetes diagnosis. Future studies on larger cohorts are needed to improve the validity and generalizability of these findings. Data are available via ProteomeXchange with identifier PXD011183 Submission details: Project Name: Urinary proteomics by 2DE and LC-MS/MS. Project accession: PXD011183 Project DOI: https://doi.org/10.6019/PXD011183 Significance Nephropathy is a very common diabetic complication. Once established, its progression can only be slowed down but full control or remission is achieved in very few cases, thus posing a large burden on worldwide health. The first evidence of diabetic nephropathy (DN) is micro-albuminuria, but only 30% of patients with micro-albuminuria progress to proteinuria, while in some patients it spontaneously reverts to normo-albuminuria. Thus, there is clear need for biomarkers that can accurately predict the risk to develop DN. Herein, by applying proteomic and lipidomic approaches on urine samples, we show that alteration of prostaglandin and ceramide metabolisms specifically occurs in association with DN. Interestingly, we demonstrate that the modification of these metabolic pathways is an early event in diabetic patients, suggesting the identified changed proteins as possible predictive biomarkers of diabetes-induced renal function decline.

Nuclear envelope-related muscular dystrophies, in particular those referred to as laminopathies, are relatively novel and unclear diseases, also considering the increasing number of mutations identified so far in genes of the nuclear envelope. As regard LMNA gene, only tentative relations between phenotype, type and localization of the mutations have been established in striated muscle diseases, while laminopathies affecting adipose tissue, peripheral nerves or progerioid syndromes could be linked to specific genetic variants. This study describes the biochemical phenotype of neuromuscular laminopathies in samples derived from LMNA mutant patients. Since it has been reported that nuclear alterations, due to LMNA defects, are present also in fibroblasts from Emery–Dreifuss muscular dystrophy and familial partial lipodystrophy patients, we analyzed 2D-maps of skin fibroblasts of patients carrying 12 different LMNA mutations spread along the entire gene. To recognize distinctive proteins underlying affected biochemical pathways, we compared them with fibroblasts from healthy controls and, more importantly, fibroblasts from patients with non-lamin related neuromuscular disorders. We found less abundance of cytoskeletal/structural proteins, confirming a dominant role for Lamin A/C in structural support of nuclear architecture. Interestingly, we also established significant changes in the expression of proteins involved in cellular energy production and oxidative stress response. To our knowledge, this is the first report where proteomics was applied to characterize ex-vivo cells from LMNA patients, suggesting that this may represent a new approach to better understand the molecular mechanisms of these rare diseases and facilitate the development of novel therapeutic treatments.

Recent observations have shown subclinical intestinal abnormalities in human type 1 diabetes. Whether these are related to the pathogenetic process or secondary to the diabetes remains to be clarified. The aim of this study was to investigate this issue by examining intestinal permeability to sugars in subjects at different stages of type 1 diabetes: preclinical, new-onset and long-term established disease. Eighty-one subjects with islet autoimmunity (18 preclinical, 28 new-onset and 35 long-term type 1 diabetes) and 40 healthy control subjects were investigated by a lactulose-mannitol test, consisting of oral administration of the two sugars and measurement of their urinary excretion. All groups of subjects with islet autoimmunity showed an increase in intestinal permeability (p

OBJECTIVE: The purpose of this study was (a) to study whether a folate and vitamin B12 treatment, aimed at decreasing homocysteine levels, might ameliorate insulin resistance and endothelial dysfunction in patients with metabolic syndrome according to the National Cholesterol Education Program-Adult Treatment Panel-III criteria and (b) to evaluate whether, under these metabolic conditions, there is a relationship between hyperhomocysteinemia and insulin resistance. DESIGN AND METHODS: A double-blind, parallel, identical placebo-drug, randomized study was performed for 2 months in 50 patients. Patients were randomly allocated to two groups. In group 1, patients were treated with diet plus placebo for 2 months. In group 2, patients were treated with diet plus placebo for 1 month, followed by diet plus folic acid (5 mg/day) plus vitamin B12 (500 microg/day) for another month. RESULTS: In group 2, folate treatment significantly decreased homocysteine levels by 27.8% (12.2+/-1.2 vs 8.8+/-0.7 micromol/l; P

OBJECTIVE Islets after kidney transplantation have been shown to positively affect the quality of life of individuals with type 1 diabetes (T1D) by reducing the burden of diabetes complications, but fewer data are available for islet transplantation alone (ITA). The aim of this study was to assess whether ITA has a positive impact on hemostatic and cerebral abnormalities in individuals with T1D. RESEARCH DESIGN AND METHODS Prothrombotic factors, platelet function/ultrastructure, and cerebral morphology, metabolism, and function have been investigated over a 15-month follow-up period using ELISA/electron microscopy and magnetic resonance imaging, nuclear magnetic resonance spectroscopy, and neuropsychological evaluation (Profile of Mood States test and paced auditory serial addition test) in 22 individuals with T1D who underwent ITA (n = 12) or remained on the waiting list (n = 10). Patients were homogeneous with regard to metabolic criteria, hemostatic parameters, and cerebral morphology/metabolism/function at the time of enrollment on the waiting list. RESULTS At the 15-month follow-up, the group undergoing ITA, but not individuals with T1D who remained on the waiting list, showed 1) improved glucose metabolism; 2) near-normal platelet activation and prothrombotic factor levels; 3) near-normal cerebral metabolism and function; and 4) a near-normal neuropsychological test. CONCLUSIONS ITA, despite immunosuppressive therapy, is associated with a near-normalization of hemostatic and cerebral abnormalities.

A high-performance capillary electrophoresis (HPCE) method based on laser-induced fluorescence detection is presented here. It enables the determination of sulfur-containing amino acids within 15 min. Fluorescence of sulfur-containing amino acids in plasma is linear over a range of 50-150 micromol/L for L-methionine, 5-100 micromol/L for L-homocysteine, and 50-200 micromol/L for L-cysteine. For homocysteine, we were able to detect 1 fmol injected, equivalent to a plasma concentration of 10 nmol/L. A similar sensitivity is present for cysteine, an even lower one being found for methionine. The intra- and interassay relative standard deviations are < 1%. High-performance liquid chromatography (HPLC) methods are commonly employed for quantifying blood concentrations of sulfur-containing amino acids. A comparative analysis of HPCE and HPLC quantitation of homocysteine has been carried out in 61 blood samples. Plasma concentrations measured by HPCE were in good agreement with those obtained employing an HPLC-based method, a satisfactory correlation being observed between the concentrations obtained by the two methods (r= 0.9972). Thus, the HPCE-based procedure presented here for the measurement of sulfur-containing amino acids in plasma is a simple, fast, accurate, and very sensitive method, suitable for routine determinations in clinical studies.

The aim of the study was to evaluate the effects of an acute increase in triglyceride levels induced by Intralipid (Kabivitrum, Stockholm, Sweden) infusion on forearm glucose uptake, glucose oxidative metabolism, and hepatic glucose production independent of circulating free fatty acid (FFA) levels in man. Six normal subjects underwent three different tests in random order. Each test consisted of a control period of 120 minutes followed by a euglycemic, hyperinsulinemic clamp lasting 120 minutes. In test 1, a high-dose intravenous Intralipid infusion was performed to increase triglyceride and FFA levels. In test 2, heparin (30 U/min) plus low-dose Intralipid infusions were performed to maintain triglyceride at normal levels and increase only FFA levels. Test 3 was performed as a control study. During the 120-minute control period, forearm glucose uptake and hepatic glucose production were not affected by increasing only FFA levels (test 2) or FFA and triglyceride levels (test 1) as compared with the control study. On the contrary, glucose oxidation was significantly decreased as compared with the control study during tests 1 and 2, without a further significant decrease during simultaneously increased FFA and triglyceride levels. Concomitantly, lipid oxidation was similar in tests 1 and 2, at values significantly greater than in test 3. During the euglycemic clamp, forearm glucose uptake and glucose oxidation were significantly lower during tests 1 and 2 than test 3. At variance with the control period, the increase of triglyceride levels during test 1 caused a significant 30% to 40% decrease of both parameters as compared with test 2. Opposite results were found for lipid oxidation, which remained unchanged during test 1 as compared with the control period but significantly decreased during tests 2 and 3. Hepatic glucose production was less inhibited during test 1 than during tests 2 and 3, and less so during test 2 than test 3. In conclusion, at least under these particular conditions, acute hypertriglyceridemia induced by Intralipid infusion seems to decrease forearm glucose uptake, glucose oxidation, and insulin-induced suppressibility of hepatic glucose production. Taking our results together, it seems that the triglyceride effect on carbohydrate metabolism occurs via triglyceride hydrolysis using the intracellular pathways of FFA without interference with the circulating FFA pool. © 1995.

Background: Urine, being an ultrafiltrate of plasma, is a rich source for biomarker discovery. Since potential new disease markers are often present in low concentrations, a prefractionation/enrichment step could be useful in the discovery process. To enhance the detection of low-abundance proteins, three immuno-affinity depletion approaches were evaluated. Methods: To remove the most abundant proteins from a human urine sample, GenWay™ Spin IgY-12 kit, HPLC Agilent Hu-PL7 and a home-made column vs. human serum albumin [immuno-affinity column (IAC)] were compared. Quantification of total proteins, 2-D gel electrophoresis (2-DE), Progenesis gel images analysis and mass spectrometric proteins identification were applied to evaluate these strategies. Results: Reproducibility of depletion columns, by estimating protein content of unbound fractions, were: 343±20.0 μg, 5.8%; 186.3±13.3 μg, 7.2%; 292±20.6 μg, 8.8% [mean± standard deviation (SD), CV%], for GenWay™, Agilent and IAC methods, respectively. To isolate urinary protein after depletion, ethanol precipitation provided the highest recovery (80%). Applying 2-DE and Progenesis analysis, the number of spots visualized on the gels was 468±21, 331±7, 368±22 and 304±7 (mean±SD) for GenWay™, Agilent, IAC, and the undepleted urine pool sample, respectively, with a significant difference p Conclusions: The sequential procedure of urine samples using multi-protein immuno-affinity depletion represents a valid tool for simplifying 2-DE analysis of the urine proteome. Particularly, the GenWay™ kit followed by ethanol precipitation was found to be the most efficient method for exploring the urine proteome. Clin Chem Lab Med 2010;48:531–5.

OBJECTIVE: We investigated whether preventing hyperglycemia in septic patients affected the plasma concentration of asymmetric-dimethylarginine and if this was associated with clinical benefit. DESIGN: A prospective, multicenter, randomized, controlled, clinical study. SETTING: Intensive care units (ICU) in three university hospitals. PATIENTS: A total of 72 patients admitted for severe sepsis or septic shock, who stayed at least 3 days in the ICU. At admission the patients were assigned to receive either tight or conventional glycemic control. INTERVENTIONS: Determination of circulating levels of asymmetric-dimethylarginine, arginine, interleukin-6, C-reactive-protein and tumor-necrosis-factor-alpha. MEASUREMENTS AND RESULTS: Blood was sampled at admission (no differences between groups), and on the 3rd, 6th, 9th, and 12th (T12) days. Sequential organ failure assessment was scored at each sampling time. All the data were analyzed on an intention-to-treat basis. The control and treatment groups received the same energy intake, glycemia (110.4 +/- 17.3 vs. 163.0 +/- 28.9 mg/dL, P < 0.001) and insulin (P = 0.02) supply differed. No differences were found in high plasma levels of asymmetric-dimethylarginine (P = 0.812) at any time during the ICU stay. The clinical course, as indicated by markers of inflammation, average and maximum organ failure score, ICU stay and ICU and 90-day mortality, was the same. CONCLUSIONS: Intensive insulin treatment, while achieving glucose control, did not reduce asymmetric-dimethylarginine in high-risk septic patients fed with no more than 25 kcal/kg per day to limit ventilatory demand and to simplify glucose control.

Patients with growth hormone deficiency (GHD) are known to have reduced life expectancy due to increased cardiovascular and cerebrovascular events. An increase in asymmetric dimethylarginine (ADMA) levels previously found in GHD patients could promote premature atherosclerosis. The aim of this study was to determine whether 6-month growth hormone (GH) replacement therapy was able to decrease ADMA levels and ameliorate endothelial dysfunction. Thirty-one GHD patients were studied before and after 6 months of GH (4 microg/[kg d], daily) replacement therapy. Reduced pretreatment levels of serum insulin-like growth factor (IGF) 1 were normalized during GH treatment (88.2 +/- 62.5 to 191.7 +/- 80.3 ng/mL, P < .0001). After 6 months of GH replacement, plasma cyclic guanosine monophosphate levels significantly increased (2.14 +/- 0.52 to 3.54 +/- 1.2 ng/mL, P < .0001), serum ADMA levels were significantly decreased (0.65 +/- 0.1 vs 0.59 +/- 0.11 mumol/L, P < .05), and arganine (Arg) to ADMA ratio was significantly higher (155 +/- 53 vs 193 +/- 61, P < .01). No changes were observed for plasma nitric oxide end products (nitrite and nitrate) levels after GH treatment (21.9 +/- 14.9 vs 24.1 +/- 19.0 mumol/L, not significant). Basal forearm blood flow remained unchanged, whereas reactive hyperemia increased from 7.30 +/- 5.31 mL/100 mL forearm per minute before GH therapy to 13.18 +/- 7.30 mL/100 mL forearm per minute after 6 months of therapy (P < .001). There was a positive correlation between IGF-1 and cyclic guanosine monophosphate (r = 0.73, P < .0001), IGF-1 and reactive hyperemia (r = 0.63, P < .0001), and IGF-1 and Arg/ADMA ratio (r = 0.44, P < .01). Conversely, a negative correlation was found between IGF-1 and ADMA levels (r = -0.41, P < .02). At the end of the study period, fat-free mass, plasma glucose, and hemoglobin A(1c) levels significantly increased, even if they were still in the reference range, suggesting moderate alteration of glucose metabolism. In conclusion, in GHD patients, GH replacement contributes to decreased, to some extent, cardiovascular risk, reducing ADMA levels and improving Arg/ADMA ratio and endothelial dysfunction.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene are associated with different types of macular degeneration, including Stargardt disease, cone-rod dystrophy, Fundus flavimaculatus, Retinitis pigmentosa and probably age-related macular degeneration. Methods: Screening for mutations in the ABCA4 gene was performed using denaturing high-performance liquid chromatography and direct sequencing. Results: We describe the identification of a new de novo 44-bp deletion in an Italian patient affected by cone-rod dystrophy. The mutation, located in intron 48 of the ABCA4 gene, is predicted to cause exon 49 skipping, resulting in loss of the C-terminus of the ABCA4 protein. Interestingly, exon 49 also codes for a highly conserved VFVNFA motif, which has been demonstrated to be essential for the activity of ABCA1, another gene of the ABC transporter family. The presence of CT repeats at the breakpoints might have facilitated the generation of the deletion through a slippage mispairing mechanism. Conclusions: The new 6730-16del44 deletion is the first de novo mutation associated with cone-rod dystrophy and may contribute to a better understanding of the role of ABCA4 mutations in macular dystrophies.

Aim of this study was to set up a method by capillary electrophoresis to detect lactulose and mannitol in urine after an oral load, and to estimate the intestinal permeability in controls and in type I diabetes patients. The underivatized carbohydrates were monitored by indirect UV detection using sorbate, cetyltrimethylammonium bromide and LiOH as background electrolyte. Urines were purified by solid phase extraction, shaken with cation exchange resin, filtered and analysed. Carbohydrates migrated in10 min in relation to their pK(a) and M(r). Controls (n = 33) and patients (n = 23) had an excretion ratio lactulose/mannitol 0.025 (0.018-0.051) and 0.067 (0.050-0.127), respectively (p0.01, median, interquartile range).

The protective role of folate in vascular disease has been related to antioxidant effects. In 45 patients with previous early-onset (at age

Ferroportin is encoded by the SLC40A1 gene and mediates iron export from cells by interacting with hepcidin. SLC40A1 gene mutations are associated with an autosomal type of genetic iron overload described as haemochromatosis type 4, or HFE4 (Online Mendelian Inheritance in Man number 606069), or ferroportin disease. We report three families with this condition caused by novel SLC40A1 mutations. Denaturing high-performance liquid chromatography was employed to scan for the SLC40A1 gene. A D181V (A846T) mutation in exon 6 of the ferroportin gene was detected in the affected members of an Italian family and shown to have a de novo origin in a maternal germinal line. This mutation was associated with both parenchymal and reticuloendothelial iron overload in the liver, and with reduced urinary hepcidin excretion. A G80V (G543T) mutation in exon 3 was found in the affected members of an Italian family with autosomal hyperferritinaemia,. Finally, a G267D (G1104A) mutation was identified in exon 7 in a family of Chinese descent whose members presented with isolated hyperferritinaemia. Ferroportin disease represents a protean genetic condition in which the different SLC40A1 mutations appear to be responsible for phenotypic variability. This condition should be considered not only in families with autosomal iron overload or hyperferritinaemia, but also in cases of unexplained hyperferritinaemia.

Background: Mutations in the retina-specific ABC transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes scanning of the ABCA4 gene cumbersome, we describe here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions. Results: DHPLC profiling not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene.

Denaturing HPLC (DHPLC) is a useful technique for the fast screening of known and unknown heterozygous gene mutations. Most DNA mutations causing genetic disorders consist of nucleotide substitutions, but insertions and deletions occur, albeit less frequently. The heteroduplexes with insertions/deletions have gaps that may affect molecular stability differently from the mismatches caused by substitutions. Therefore, gaps and mismatches may be distinguished by DHPLC analysis, which is based on the differential thermal stability of amplicons with different characteristics. To verify this hypothesis, we examined 12 DNA samples containing insertions and deletions of different sizes (one to 29 residues) from four different genes (ABCA4, CFTR, FTL, and SLC11A3). We found that all of them were detected by DHPLC runs at 50°C, which is considered a non-denaturing temperature, as well as by runs at the temperature optimized for mismatch recognition. The finding confirms that gaps reduce heteroduplex stability more than mismatches, and indicates that DHPLC analysis at low temperature may be applied to distinguish DNA deletions/insertions from substitutions. Hum Mutat 22:98–102, 2003. © 2003 Wiley-Liss, Inc.

Background— We tested the hypothesis that asymmetric dimethylarginine (ADMA) levels could be elevated and influence endothelin-1 and nitric oxide release and action in patients with cardiac syndrome X (CSX). In addition, we evaluated whether an intravenous infusion of l -arginine would improve endothelial function in these subjects. Methods and Results— Nine patients with CSX and 14 control subjects underwent a continuous infusion of l -arginine (0.125 g/min) or saline for 120 minutes. Sixty minutes after l -arginine or saline infusions, an intravenous insulin bolus (0.1 U/kg) combined with a euglycemic clamp was performed. Basal ADMA and endothelin-1 levels were higher in patients with CSX than in controls. At the end of the first hour of infusion, compared with saline, l -arginine infusion increased basal forearm blood flow, nitrite and nitrate (NOx), and forearm cGMP release and decreased endothelin-1. After insulin bolus, during saline, insulin-induced NOx, endothelin-1, and forearm cGMP release was almost abolished. Conversely, l -arginine restored a physiological profile of all endothelial variables compared with control subjects. In control subjects, compared with saline infusion, l -arginine infusion did not modify any parameter. ADMA levels were positively correlated with basal endothelin-1 levels and negatively correlated with insulin-induced incremental levels of NOx and forearm cGMP release. Conclusions— Plasma ADMA levels are increased in patients with CSX, and they are correlated with increases in endothelin-1 and reductions in insulin-induced increments in plasma NOx and cGMP, effects that are reversed by intravenous l -arginine. These data suggest that increased ADMA levels play a role in the abnormal vascular reactivity that is observed in patients with CSX.

The aim of the study was to investigate the effects of two hypocaloric (800-kcal) diets on body weight reduction and composition, insulin sensitivity, and proteolysis in 25 normal glucose-tolerant obese women. The two diets had the following composition: 45% protein, 35% carbohydrate (CHO), and 20% fat (HP diet, 10 subjects), and 60% CHO, 20% protein, and 20% fat (HC diet, 15 subjects); both lasted 21 days. A euglycemic hyperinsulinemic (25 mU/kg/h) clamp lasting 150 minutes combined with indirect calorimetry was performed before and after the diet. Both diets induced a similar decrease in body weight and fat mass (FM), whereas fat-free mass (FFM) decreased only after the HC diet. 3-Methylhistidine (3-CH3-HIS) excretion was reduced by 48% after the HP diet and remained unchanged after the HC diet (P < .05). A significant correlation was found between the changes in FFM and in 3-CH3-HIS excretion after the diet (rs = .50, P < .02). Blood glucose remained unchanged, while insulin decreased in both diets. Free fatty acids (FFA) significantly increased only after the HC diet (P < .05). During the clamp period, glucose disposal and glucose oxidation significantly increased after the HP diet and significantly decreased after the HC diet. Opposite results were found when measuring lipid oxidation. In conclusion, our experience suggests that (1) a hypocaloric diet providing a high percentage of natural protein can improve insulin sensitivity; and (2) conversely, a hypocaloric high-polysaccharide-CHO diet decreases insulin sensitivity and is unable to spare muscle tissue.

Hydrolysis of cyclosporin A (CsA) was studied in order to clarify the still undefined point of attack of the acidic degradation. Among ether extractable and water-soluble products formed from CsA in HCl, two open-chain peptides were isolated by high-performance liquid chromatography which were identified as the deca- and nonapeptides deriving from CsA through the hydrolytic cleavage of amino acid residue 11 and both residues 11 and 10, respectively. Identification was carried out by fast atom bombardment tandem mass spectrometry.

A precolumn derivatization method was optimized for rapid and specific analysis of total urinary hydroxyproline by HPLC. After an overnight hydrolysis, urine samples dried and reconstituted with the internal standard cysteic acid (in sodium hydrogen carbonate, pH 9.3) were derivatized with N,N-diethyl-2,4-dinitro-5-fluoroaniline (FDNDEA) at 100 degrees C for 20 min. The DNDEA-hydroxyproline adduct was separated on an Ultrasphere ODS column with a mobile phase of acetate buffer (containing triethylamine, 6 mL/L, pH 4.3) and acetonitrile (80/20, by vol), and was detected at 360 nm. A single run took 18 min with a hydroxyproline retention time of 7.3 min. The assay showed a linear response to hydroxyproline concentrations from 5 to 100 mg/L with a detection limit of 0.8 ng injected, corresponding to 2 mg/L in urine. Mean (SD) analytical recovery was 94.2 (13)% and 104 (9)% at 10 and 50 mg/L, respectively. Within-run and between-run CVs (n = 10) were 3.74% and 4.33%, respectively, for 25 mg/L. Results for samples (n = 50) analyzed by HPLC (y) vs ion-exchange chromatography with postcolumn ninhydrin reaction (x) correlated well: y = 0.98x + 1.02 (r = 0.985, Sxy = 3.13). In another comparison, involving 173 samples, a colorimetric procedure (Hypronosticon, x) gave slightly higher values than the HPLC method (y): y = 0.83x + 2.21 (r = 0.937, Sxy = 4.6).

Investigations into the properties of haemoglobin often require the isolation of the valence intermediates (alpha o2 beta+)2 and (alpha+ beta o2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 micron) in an HR5/20 column at various temperatures (10-25 degrees C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (alpha o2 beta+)2, (alpha+ beta o2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods.

In this work we report data on the hydrolysis of CsA, allowing interpretation on the formation of previously identified open chain oligopeptides from CsA. © Munksgaard 1997.

Ferritin consists of two subunit types, H and L, which assemble in different proportions in a 24-mer protein.1 The H-subunit has ferroxidase activity and is mainly found in cell cytoplasm, where it has the major function of sequestering and detoxifying unwanted iron. The L-subunit has no catalytic activity on its own, but assists the functionality of the H-subunit2 and is also found in minor amounts in serum.3 Two types of genetic disorder are associated with mutations of the L-ferritin gene ( FTL ), both with autosomal dominant transmission. The first, hereditary hyperferritinaemia cataract syndrome (HHCS), is caused by mutations in the regulatory iron responsive element (IRE) in the 5′UTR of the transcript that reduce binding affinity to the iron regulatory proteins (IRPs) and lead to constitutive upregulation of the protein in tissue and serum.4–8 Subjects with the mutations show high levels of serum ferritin (500–2000 μg/l) and often early-onset bilateral cataracts6 likely caused by protein aggregation in the lens,9 but do not present alterations in iron metabolism. The disorder has been extensively studied and more than 21 different causative mutations have been identified.9,10 The second type of genetic disorder, neuroferritinopathy, is rare and few families have been identified. It is associated with an adenine insertion at position 460–461 in the coding region (exon 4) of the gene that causes a frame shift alteration of the C-terminus of the L-ferritin polypeptide.11 Affected subjects show late-onset movement disorders, iron deposition in the brain basal ganglia, and low serum ferritin levels.12,13 It is unclear whether the iron deposition in the brain is caused by a quantitative defect of L-ferritin or by an abnormal functionality caused by …

Cardiovascular disease and the development of coronary artery disease play a pivotal role in increasing mortality in patients with type 1 diabetes. The aim of our study was to evaluate the effects of pancreas transplantation on atherosclerotic risk factors, endothelial-dependent dilation (EDD), and progression of intima media thickness (IMT) in patients with uremia and type 1 diabetes after kidney-alone (KA) or kidney-pancreas (KP) transplantation. A cross-sectional study comparing two groups of patients with type 1 diabetes was performed. Sixty patients underwent KP transplantation and 30 patients underwent KA transplantation. Age and cardiovascular risk profile were comparable in patients before transplantation. In all patients, atherosclerotic risks factors (lipid profile, fasting and post-methionine load plasma homocysteine, von Willebrand factor levels, D-dimer fragments, and fibrinogen) were assessed and Doppler echographic evaluation of IMT and endothelial function with flow-mediated and nitrate dilation of the brachial artery was performed. Twenty healthy subjects were chosen as controls (C) for EDD. Compared with patients undergoing KA transplantation, patients undergoing KP transplantation showed lower values for HbA1c (KP = 6.2 +/- 0.1% vs. KA = 8.4 +/- 0.5%; P < 0.01), fasting homocysteine (KP = 14.0 +/- 0.7 mcromol/l vs. KA = 19.0 +/- 2.0 micromol/l; P = 0.02), von Willebrand factor levels (KP = 157.9 +/- 8.6% vs. KA = 212.5 +/- 16.2%; P < 0.01), D-dimer fragments (KP = 0.29 +/- 0.02 microg/ml vs. KA = 0.73 +/- 0.11 microg/ml;P < 0.01), fibrinogen (KP = 363.0 +/- 11.1 mg/dl vs. KA = 397.6 +/- 19.4 mg/dl; NS), triglycerides (KP = 122.7 +/- 8.6 mg/dl vs. KA = 187.0 +/- 30.1 mg/dl; P = 0.01), and urinary albumin excretion rate (KP = 13.5 +/- 1.9 mg/24 h vs. KA = 57.3 +/- 26.3 mg/24 h; P < 0.01). Patients undergoing KP transplantation showed a normal EDD (KP = 6.21 +/- 2.42%, KA = 0.65 +/- 2.74%, C = 8.1 +/- 2.1%; P < 0.01), whereas no differences were observed in nitrate-dependent dilation. Moreover, IMT was lower in patients undergoing KP transplantation than in patients undergoing KA transplantation (KP = 0.74 +/- 0.03 mm vs. KA = 0.86 +/- 0.09 mm; P = 0.04). Our study showed that patients with type 1 diabetes have a lower atherosclerotic risk profile after KP transplantation than after KA transplantation. These differences are tightly correlated with metabolic control, fasting homocysteine levels, lower D-dimer fragments, and lower von Willebrand factor levels. Normal endothelial function and reduction of IMT was observed only in patients undergoing KP transplantation.

A new method for the separation and quantification of primary and secondary amino acids by reversed-phase high-performance liquid chromatography and spectrophotometric detection is described. The use of the novel derivatizing reagent N,N-diethyl-2,4-dinitro-5-fluoroaniline yields derivatives that are more stable to light and heat in solution than the traditional ones. A simple gradient between acidic acetate buffer and acetonitrile allows the complete separation of 21 amino acids in 80 min at room temperature. The good reproducibility of peak areas and retention times allows the application of this simple and low-priced method to the determination of amino acids in the range 50–500 pmol per residue.