Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes.

We recently developed a novel keratin-derived protein (KDP) rich in cysteine, glycine, and arginine, with the potential to alter tissue redox status and insulin sensitivity. The KDP was tested in 35 human adults with type-2 diabetes mellitus (T2DM) in a 14-wk randomised controlled pilot trial comprising three 2×20 g supplemental protein/day arms: KDP-whey (KDPWHE), whey (WHEY), non-protein isocaloric control (CON), with standardised exercise. Outcomes were measured morning fasted and following insulin-stimulation (80 mU/m2/min hyperinsulinaemic-isoglycaemic clamp). With KDPWHE supplementation there was good and very-good evidence for moderate-sized increases in insulin-stimulated glucose clearance rate (GCR; 26%; 90% confidence limits, CL 2%, 49%) and skeletal-muscle microvascular blood flow (46%; 16%, 83%), respectively, and good evidence for increased insulin-stimulated sarcoplasmic GLUT4 translocation (18%; 0%, 39%) vs CON. In contrast, WHEY did not effect GCR (-2%; -25%, 21%) and attenuated HbA1c lowering (14%; 5%, 24%) vs CON. KDPWHE effects on basal glutathione in erythrocytes and skeletal muscle were unclear, but in muscle there was very-good evidence for large increases in oxidised peroxiredoxin isoform 2 (oxiPRX2) (19%; 2.2%, 35%) and good evidence for lower GPx1 concentrations (-40%; -4.3%, -63%) vs CON; insulin stimulation, however, attenuated the basal oxiPRX2 response (4%; -16%, 24%), and increased GPx1 (39%; -5%, 101%) and SOD1 (26%; -3%, 60%) protein expression. Effects of KDPWHE on oxiPRX3 and NRF2 content, phosphorylation of capillary eNOS and insulin-signalling proteins upstream of GLUT4 translocation AktSer437 and AS160Thr642 were inconclusive, but there was good evidence for increased IRSSer312 (41%; 3%, 95%), insulin-stimulated NFκB-DNA binding (46%; 3.4%, 105%), and basal PAK-1Thr423/2Thr402 phosphorylation (143%; 66%, 257%) vs WHEY. Our findings provide good evidence to suggest that dietary supplementation with a novel edible keratin protein in humans with T2DM may increase glucose clearance and modify skeletal-muscle tissue redox and insulin sensitivity within systems involving peroxiredoxins, antioxidant expression, and glucose uptake.

Elevated seminal plasma myeloperoxidase is associated with a decreased sperm concentration in young men.

Myeloperoxidase is a major neutrophil protein which generates oxidants that are highly reactive, and if present in seminal fluid, could be potentially damaging to spermatozoa. We recruited young males aged 18-35 years, unscreened for fertility status, for a pilot study measuring seminal plasma myeloperoxidase. On three occasions, over a 3-month period, we measured parameters of semen quality and correlated these with seminal myeloperoxidase protein and activity. After baseline measurement, participants were supplemented daily with 250 mg of vitamin C, a potent scavenger of reactive oxygen species with antiinflammatory activities. Seminal plasma from eight of the 12 participants had measurable concentrations of myeloperoxidase protein, across a broad range (15-250 ng/mL). Median myeloperoxidase protein concentrations were ~45-fold higher in semen samples with low vs. high sperm concentrations. Seminal plasma myeloperoxidase protein concentration was inversely correlated with the percentage of rapidly motile spermatozoa assessed by computer-assisted sperm analysis, and the total number of spermatozoa per ejaculate, but positively correlated with sperm maturity, measured by DNA staining ability. We measured an inverse correlation between semen vitamin C concentration and seminal plasma myeloperoxidase protein concentration, although vitamin C supplementation had no effect on semen quality. Our pilot data suggest that high concentrations of myeloperoxidase were present in the seminal plasma of many of our young participants, and that this may be associated with decreases in semen quality. A larger study is required to confirm these findings.

The role of oxidants and vitamin C on neutrophil apoptosis and clearance.

We have investigated the role of neutrophil oxidants in the surface changes that result in recognition and uptake of neutrophils by macrophages. We have shown that H2O2 produced by stimulated neutrophils is essential for the surface expression of phosphatidylserine. This does not occur in neutrophils from mice with chronic granulomatous disease and may explain the formation of granuloma in this condition. We have also investigated the role of intracellular vitamin C on neutrophil apoptosis. Cells from vitamin C-deficient mice were found to be less likely to undergo both spontaneous and oxidant-induced apoptosis, with eventual necrosis being the most probable outcome.

Regulation of apoptosis by vitamin C. Specific protection of the apoptotic machinery against exposure to chlorinated oxidants.

We have investigated the ability of intracellular vitamin C to protect human umbilical vein endothelial cells from exposure to hypochlorous acid (HOCl) and a range of derived chloramines. Ascorbate provided minimal protection against the cytotoxicity induced by these oxidants, as measured by propidium iodide uptake. In contrast, there was a marked effect on apoptosis, monitored by caspase-3 activation and phosphatidylserine exposure. Extended incubation of the cells with glycine chloramine or histamine chloramine completely blocked apoptosis initiated in the cells by serum withdrawal. This effect was significantly abrogated by ascorbate. Inhibition of apoptosis required the oxidant to be present for an extended period after serum withdrawal and occurred prior to caspase-3 activation. General protection of thiols by ascorbate was not responsible for the protection of apoptosis, because intracellular oxidation by HOCl or chloramines was not prevented in supplemented cells. The results suggest a new role for vitamin C in the regulation of apoptosis. We propose that, by protection of an oxidant-sensitive step in the initiation phase, ascorbate allows apoptosis to proceed in endothelial cells under sustained oxidative stress.

Hypochlorous acid stimulation of the mitogen-activated protein kinase pathway enhances cell survival.

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 microM HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 microM and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 microM HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H2O2 and the lack of JNK activation contrasts with the activation frequently seen with H2O2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.

Fatty acid chlorohydrins and bromohydrins are cytotoxic to human endothelial cells.

Reaction of unsaturated lipids with the hypohalous acids (hypochlorous acid and hypobromous acid) results in the addition of the halide (X) across double bonds to form halohydrins (-CH2CH(OH)CH(X)CH2-). These modified lipids could be potentially destabilising to cell membranes due to their increased polarity. We have investigated the effect of pre-formed halohydrins on human umbilical vein endothelial cells (HUVEC) by incubating cultured cells with oleic acid micelles containing chlorohydrins or bromohydrins. Cell detachment and necrotic death were observed with increasing doses of halohydrins, whereas the cells were unaffected by equivalent doses of oleic acid. Bromohydrins caused more lysis than did chlorohydrins at equivalent doses. Complete lysis was seen with 200 microM fatty acid/chlorohydrin micelles and with 50 microM fatty acid/bromohydrin micelles. Chlorohydrin uptake was much less than the oleic acid control whereas bromohydrins were incorporated into the endothelial cells similarly to oleic acid. This difference or the bulkier nature of the bromohydrins could account for their increased toxicity. This study has demonstrated the potential toxicity of the halohydrins, and implications for their formation in inflammation are discussed.

Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide.

Treatment of cells with hypochlorous acid (HOCl) at sublethal doses causes a concentration-dependent loss in reduced glutathione (GSH) levels. We have investigated the products of the reaction of HOCl with GSH in human umbilical vein endothelial cells. Despite a complete loss of GSH, there were only very small increases in intracellular and extracellular glutathione disulfide and glutathione sulfonic acid after exposure to HOCl. (35)S labeling of the GSH pool showed only a minimal increase in protein-bound GSH, suggesting that S-thiolation was not a major contributor to HOCl-mediated loss of GSH in endothelial cells. Rather, the products of the reaction were mostly exported from cells and included a peak that co-eluted with the cyclic sulfonamide that is a product of the reaction of GSH with reagent HOCl. Evidence of this species in endothelial cell supernatants after HOCl treatment was also obtained using electrospray mass spectrometry. In conclusion, exposure to HOCl causes the irreversible loss of cellular GSH with the formation of novel products that are rapidly exported from the cell, and resynthesis of GSH will be required to restore levels. The loss of GSH would alter the redox state of the cell and compromise its defenses against further oxidative stress.

Living with a killer: the effects of hypochlorous acid on mammalian cells.

The production of hypochlorous acid (HOCl) by the myeloperoxidase-H2O2-Cl- system of phagocytes plays a vital role in the ability of these cells to kill a wide range of pathogens. However, the generation of a potent oxidant is not without risk to the host, and there is evidence that HOCl contributes to the tissue injury associated with inflammation. In this review, we discuss the biological reactivity of HOCl, and detail what is known of how it interacts with mammalian cells. The outcome of exposure is dependent on the dose of oxidant, with higher doses causing necrosis, and apoptosis or growth arrest occurring with lower amounts. Glutathione (GSH) and protein thiols are easily oxidized, and are preferred targets with low, sublethal amounts of HOCl. Thiol enzymes vary in their sensitivity to HOCl, with glyceraldehyde-3-phosphate dehydrogenase being most susceptible. Indeed, loss of activity occurred before GSH oxidation. The products of these reactions and the ability of cells to regenerate oxidized thiols are discussed. Recent reports have indicated that HOCl can activate cell signaling pathways, and these studies may provide important information on the role of this oxidant in inflammation.

Myeloperoxidase.

This review covers recent advances in the biology of myeloperoxidase. Mechanisms of posttranslational processing and how these fail in some of the common deficiency mutants are discussed. We also review the enzymology that points to myeloperoxidase having a number of physiologic substrates in addition to chloride and the evidence that it produces hypochlorous acid in the neutrophil phagosome in sufficient quantities to be bactericidal. Evidence is accumulating that myeloperoxidase-derived oxidants modify biologic macromolecules and cell-regulatory pathways and that they play a role in atherosclerosis. Investigation of disease incidence in relation to a polymorphism in the promoter region of the gene has produced interesting associations. These links with inflammatory diseases can now be pursued further using specific biomarkers of myeloperoxidase activity.

Hypochlorous acid causes caspase activation and apoptosis or growth arrest in human endothelial cells.

We have investigated the effect of hypochlorous acid (HOCl) on cultured human umbilical-vein endothelial cells and shown that, whereas higher concentrations cause rapid necrosis, smaller amounts of this oxidant induce apoptosis or growth arrest. Exposure to 20-40 nmol of HOCl per 1.2x10(5) cells initiated apoptosis that was determined morphologically, by the identification of apoptotic nuclei with Hoechst 33342, and by detection of phosphatidylserine on the outer membrane. Degraded DNA was detected by flow cytometry. HOCl induced caspase activity; specific inhibition of caspases was shown to prevent apoptosis. No caspase activation could be detected with 50 nmol of HOCl per 1.2x10(5) cells, a dose that caused more extensive necrosis. Lower doses of HOCl, which did not cause cell death, resulted in a transient growth arrest that was apparent with as little as 5 nmol of HOCl per 1.2x10(5) cells. These results show that HOCl can modify cellular responses that are dependent on signal transduction pathways in a manner similar to that of other oxidants.

Loss of GSH and thiol enzymes in endothelial cells exposed to sublethal concentrations of hypochlorous acid.

We investigated the effect of sublethal concentrations of hypochlorous acid (HOCl) on intracellular thiol groups. Exposure of human umbilical vein endothelial cells to HOCl caused a decrease in cell viability, with concentrations of

Building a flexible protocol information system with ready for use' web-technology.

This paper describes how web technology, currently available, can be used to build a fast and easy flexible protocol information system. The interface design and functionalities of the system were based on experiences with a previous version of a protocol information system (ProtoVIEW). A wide range of diagnostic or therapeutic protocols could be retrieved and viewed with ProtoVIEW. The Web-based version contains all functionalities of the non web-based version plus several new functionalities. The web version contains an X-ray viewer and a great deal of interactivity such as validation of electronic patient data forms. The most important additional function is the context sensitive protocol support that may lead to improved protocol adherence. Finally, the web-based version can be accessed from any working place since patient data and protocols are stored centrally.

Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis.

Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.

Presenting treatment protocols with Web technology.

This paper describes a Web-based version of a protocol information system (ProtoVIEW) with which a wide range of diagnostic or therapeutic protocols can be retrieved and viewed. The Web-based version contains all functionalities of the non web-based version plus several new functionalities. The web version contains an X-ray viewer and a great deal of interactivity such as validation of electronic patient data forms. The most important additional function is the context sensitive protocol support which may lead to improved protocol adherence. Finally, the web-based version can be accessed from any working place since patient data and protocols are stored centrally.

Introduction of a computerised protocol in clinical practice: is there anything to gain?

OBJECTIVES: To assess the potential benefit of a protocol for the diagnostic work-up and management of patients with obstructive jaundice, by comparing its recommendations with the policies actually followed in patients and to compare local expertise with diagnostic and therapeutic procedures with that described in published reports. DESIGN: A retrospective analysis of patients' records. SETTING: University hospital, The Netherlands. SUBJECTS: 49 consecutive patients who presented to the departments of internal medicine and surgery between June 1990 and June 1992 with serum alkaline phosphatase activities > 125 mumol/L, and serum bilirubin concentrations > 17 mumol/L. MAIN OUTCOME MEASURES: The proportions of diagnostic and therapeutic decisions that deviated from the recommendations, and the success rates of diagnostic and therapeutic procedures. RESULTS: In patients with bile duct stones the treatment strategies did not deviate from those recommended in the protocol. In patients with cancer 38 (30%) of the 128 diagnostic decisions and 4 (11%) of the 37 therapeutic decisions deviated from the protocol. Success rates of all diagnostic investigations were comparable with those reported, and success rates of endoscopic biliary drainage tended to be lower than those reported. CONCLUSIONS: The introduction of a protocol for the diagnostic work-up of patients with obstructive jaundice may reduce unnecessary investigations and diagnostic omissions by half. Because local expertise of some of the procedures seems to be significantly less than reported elsewhere it may be necessary to modify the protocol to better fit local circumstances.

Hypochlorous acid disrupts the adhesive properties of subendothelial matrix.

We have investigated whether the cell adhesion-promoting properties of the subendothelial matrix are affected by exposure to neutrophil-derived oxidants. Native subendothelial matrix was exposed to increasing doses of H2O2 in the presence of myeloperoxidase and Cl- or to reagent hypochlorous acid (HOCl). Increasing doses of either oxidant system resulted in progressive loss in the adhesive properties of the matrix, and phase contrast microscopy showed that the cells failed to attach to and spread on the oxidant-treated surface. When cells were replated on the treated matrix in the presence of 20% serum, they did attach, but showed abnormal spreading and morphology in longer-term culture. In a modified ELISA system, binding of antibodies specific to fibronectin, thrombospondin and laminin was also disrupted by prior exposure of the matrix to HOCl. Of these components, the cell-binding region of fibronectin was most affected by HOCl, thrombospondin and laminin were less sensitive, and the collagen-binding region of fibronectin was the most resistant. SDS-PAGE of 35S-labelled subendothelial matrix proteins indicated that there was no major irreversible crosslink formation or fragmentation after exposure to HOCl or the myeloperoxidase system, although formation of disulfides is quite likely.

Modification of red cell membrane lipids by hypochlorous acid and haemolysis by preformed lipid chlorohydrins.

Hypochlorous acid (HOCl), a strong oxidant generated by the myeloperoxidase system of neutrophils and monocytes, has been implicated in inflammatory tissue damage by these cells. Reaction of HOCl with the double bonds of unsaturated lipids produces alpha, beta-chlorohydrin isomers. We have exposed red cell membranes to HOCl and used thin layer chromatography (TLC) of the extracted lipids and enzyme-linked immunosorbent assay (ELISA), using an antichlorohydrin monoclonal antibody, to show that fatty acyl chlorohydrins are formed. The ELISA was approximately 25 fold more sensitive than TLC, and chlorohydrins were detected when membranes from 10(6) cells were treated with > or = 0.16 nmoles HOCl. Lipid chlorohydrins are more polar and bulky than their parent lipids and as such could affect membrane stability and function. To determine the effect of incorporation of lipid chlorohydrins into cell membranes, preformed fatty acid and cholesterol chlorohydrins were incubated with red cells. Lysis was measured as release of haemoglobin and incorporation of lipids was determined by 14C scintillation counting. Addition of HOCl-treated oleic acid to red cells resulted in rapid lysis of a fraction of the cells in a concentration dependent manner. HOCl-treated cholesterol also caused a small amount of cell lysis that was predominantly due to chlorohydrin 3, one of the three major cholesterol chlorohydrin products. Chlorohydrin 3, which has a decreased planarity and polarity, was also primarily responsible for altering the critical micelle concentration of HOCl-treated cholesterol-containing liposomes.

Evaluating the MACRO solution. Multimedia Application for Clinical Research in Oncology.

This paper describes the evaluation approach of the MACRO project. This approach is based on earlier work in the European 3rd Framework Programme (AIM). We describe how user requirements and expectations play a role in the design of the various assessment studies in MACRO.

Impact of a protocol processing system (ProtoVIEW) on clinical behaviour of residents and treatment.

A protocol processing system (ProtoVIEW), containing therapeutic trauma protocols, was used in the Accident and Emergency (A and E) department for a period of 7 months to investigate the impact of automated protocols on firstly, medical decision making of physicians and secondly, on quality of treatments eventually received by the patients. A randomized controlled trial showed that mandatory use of the system led to a more uniform working strategy while fracture treatment only seemed to improve in a subgroup of patient for whom residents established a correct diagnosis.

Effects of a supportive protocol processing system (ProtoVIEW) on clinical behaviour of residents in the accident and emergency department.

A randomized two period crossover trial was performed at the Accident and Emergency (A & E) department of the University Hospital in Nijmegen (The Netherlands). We assessed what the impact was of (mandatory) consultation of a protocol for the management of isolated traumas on treatment decisions of residents. All eight surgical residents who regularly worked in the A & E department participated in the trial. All patients who entered the A & E department between October 13, 1992 and June 9, 1993, of age 16 years or older with an isolated fracture without concomitant lesions were admitted to the study. During the experimental periods, the management protocol was available on computer (using ProtoVIEW) and during the control periods on paper. Main measurements were treatment adjustments made by residents (after consulting different information sources), and their opinion about ProtoVIEW as an information source assessed by means of a questionnaire. When protocol consultation was mandatory, residents changed their treatments almost four times more often towards the protocol than during the control periods (P = 0.01 Chi-square test). Most residents found ProtoVIEW easy to use, liked it as a useful training source while half of them said they would use the system in daily clinical practice. We conclude that mandatory protocol consultation using ProtoVIEW influenced protocol adherence positively.