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.

Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases released from neutrophils.

Human neutrophils, when stimulated with phorbol myristate acetate or fMet-Leu-Phe in the presence or absence of cytochalasin B, released metalloproteinases that catalytically inactivated the plasma serine proteinase inhibitor, alpha 1-antitrypsin. Inactivation, measured as loss of elastase inhibitory capacity, was accompanied by cleavage of a Mr 4,000 peptide from the COOH-terminus. Cleavage of alpha 1-antitrypsin by cell supernatants was inhibited by EDTA, o-phenanthroline, and DTT, but not by inhibitors of serine or thiol proteinases. Gelatinase and collagenase were separated from the medium of stimulated neutrophils. Both preparations cleaved and inactivated alpha 1-antitrypsin, with cleavage occurring close to the reactive center, at the Phe-Leu bond between positions P7 and P6. Cleavage by purified gelatinase was very slow and could account for only a minor fraction of the activity of neutrophil supernatants. The collagenase preparation was more active. However, the unusual cleavage site, and the ability of fMet-Leu-Phe-stimulated neutrophils to cleave alpha 1-antitrypsin without releasing collagenase, suggests that collagenase is not responsible for cleavage by the cells, which, by implication, is due to an as yet uncharacterized metalloenzyme. Our results demonstrate that by releasing metalloproteinases, neutrophils could proteolytically inactivate alpha 1-antitrypsin at sites of inflammation. This provides an alternative to the previously documented mechanism of inactivation by neutrophil-derived oxidants.

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 effect of oxidants on neutrophil-mediated degradation of glomerular basement membrane collagen.

The contribution of activated oxygen species to neutrophil-mediated degradation of basement membrane collagen was investigated. In preliminary experiments, pre-exposure of either albumin or glomerular basement membrane to neutrophil myeloperoxidase with H2O2 and chloride increased their susceptibility to proteolysis 2-3-fold. In the basement membrane model, neutrophils are stimulated by trapped immune complexes to adhere, produce oxidants and degranulate. Degradation, measured as the amount of hydroxyproline solubilised, was due to neutral proteinases, particularly elastase, and depended on cell number and the amount of proteinase released. Experiments with oxidant scavengers and inhibitors and with neutrophils from donors with chronic granulomatous disease or myeloperoxidase deficiency showed that oxidants did not affect degradation of the basement membrane when this was measured on a per cell basis. However, oxidative inactivation of the released granule enzymes occurred. Activities of elastase, beta-glucuronidase and lysozyme were 1.5-2-times higher in the presence of catalase, but were unaffected by superoxide dismutase or hydroxyl radical scavengers. Inactivation did not occur with chronic granulomatous disease or myeloperoxidase deficient neutrophils. When related to the activity of released elastase, or to other degranulation markers, collagen degradation was decreased in the presence of catalase, or with chronic granulomatous disease or myeloperoxidase deficient cells. This implies that the basement membrane was made more digestible by myeloperoxidase-derived oxidants, as occurred in the cell-free experiments. Taken together, the results indicate that neutrophil oxidants have two opposing effects. They increase the susceptibility of the collagen to proteolysis and inactivate the proteinases responsible.

Changes in ascorbate levels on stimulation of human neutrophils.

Changes in ascorbate levels have been measured in human neutrophils stimulated with opsonized zymosan, phorbol myristate acetate and formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), in the presence and absence of cytochalasin B. After stimulation with opsonized zymosan or phorbol myristate acetate, there was no loss of total ascorbate, but 30-40% of the reduced ascorbate was oxidized to dehydroascorbate. Superoxide dismutase and catalase added to the cell suspension did not inhibit this oxidation. fMet-Leu-Phe, however, gave no net oxidation but about 20% of the total ascorbate was lost during 2 h incubation. These results imply that there is not a simple relationship between superoxide and hydrogen peroxide production and ascorbate oxidation, and that release of ascorbate into the phagolysosomes does not occur.

Degradation of glomerular basement membrane by human neutrophils in vitro.

The glomerular basement membrane is susceptible to immunologic injury when immune complexes or anti-basement-membrane antibodies become lodged in its network. We have studied the digestion of glomerular basement membrane prepared from normal human kidney by isolated neutrophils. In the absence of immunoglobulin aggregates or immune complexes, there was little evidence of neutrophil adherence to the membrane, of release of lysosomal enzymes, or of digestion. However, when the basement membrane contained immunoglobin G (IgG) aggregates generated in situ by heating the membrane impregnated with IgG to 63 degrees C, electron micrographs showed neutrophils adherent to the basement-membrane surface and phagocytosis of smaller fragments. Lysosomal enzymes were detectable in the extracellular medium, and measurements of either total protein or hydroxyproline solubilized showed digestion of 80 micrograms basement membrane/h per 10(7) cells. Hydroxyproline solubilization was almost totally inhibited by phenylmethylsulphonyl fluoride, indicating that the neutrophil serine proteinases, elastase and cathepsin G are responsible for degradation. These findings provide direct evidence for the digestion of extracellular matrix by neutrophils stimulated in situ by deposited immune complexes as a contributor to inflammatory tissue damage.

Human platelets mediate iron release from transferrin by adenine nucleotide-dependent and -independent mechanisms.

We assessed the ability of platelet sonicates and mediators secreted by unstimulated and thrombin-stimulated platelets to facilitate the release of iron from transferrin. Platelet sonicates and platelet conditioned media potentiated the release of iron from transferrin. The rate of release of iron was dependent on the pH of the reaction and amount of platelet sample added. Conditioned media from thrombin-stimulated platelets was more effective in mediating the release of iron from transferrin than was conditioned media from unstimulated cells. The rate of iron released from transferrin following addition of ATP and ADP in amounts equivalent to that present in platelet conditioned media was significantly less than the rate of iron released following the addition of conditioned media from platelets. Depletion of ATP and ADP in platelet conditioned media by incubation with apyrase only partially inhibited their ability to enhance the rate of iron release from transferrin. These observations indicate that platelets enhance the release of iron from transferrin by adenine nucleotide-dependent and -independent mechanisms. These observations are consistent with the hypothesis that platelets promote oxidant-induced tissue injury at sights of inflammation secondary to their ability to enhance the local release of iron from transferrin.

A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils.

We have developed a method that enables the rates of phagocytosis and killing of bacteria by neutrophils to be measured in a single assay. Neutrophils were incubated with bacteria, and at specific intervals were separated from uningested bacteria by low speed centrifugation. Rates of phagocytosis and killing were calculated from the decrease in number of extracellular bacteria and change in the number of intracellular bacteria. Both phagocytosis and killing were shown to follow first-order kinetics, and rate constants were calculated without having to separate the assay into two phases. In contrast to two-step methods, our method measures killing from the moment the neutrophils start ingesting the bacteria, and also eliminates the need to halt neutrophil activity temporarily and restart the assay after the extracellular bacteria have been removed. We obtained reproducible results for the phagocytosis and killing of Staphylococcus aureus (t1/2 = 9 min and 6 min respectively) and Escherichia coli (t1/2 = 10 min and 2 min respectively). We also were able to detect a 56% impairment in the rate of killing of S. aureus by neutrophils from an individual with a low level of myeloperoxidase.

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.

Inhibition of hypochlorous acid-mediated reactions by desferrioxamine. Implications for the mechanism of cellular injury by neutrophils.

Inhibition of free radical mechanisms by desferrioxamine, an iron chelator, is often thought to be a good indicator of iron-catalyzed hydroxyl radical (OH.) production. The specificity of desferrioxamine is critical for such identification. This study was undertaken to determine whether desferrioxamine could prevent the in vitro cytotoxic reactions of hypochlorous acid (HOCl), a major neutrophil-derived oxidant. Red blood cells were used as a target for HOCl, and cell lysis and haemoglobin oxidation were measured. Desferrioxamine, and its iron-chelated form, ferrioxamine, were shown to prevent both effects of HOCl. However, desferrioxamine was 6 to 8 times more efficient than either ferrioxamine or taurine, another amine which prevents HOCl-mediated cell lysis, in preventing both lysis and Hb oxidation. After reaction with HOCl, ferrioxamine and taurine retained almost all the oxidizing equivalents as long-lived chloramine. However, with desferrioxamine less than half the oxidizing equivalents were recovered as chloramines indicating that sites other than the terminal amine reacted with HOCl. The chloramines formed were able to oxidize molecules in solution, but being hydrophilic they were confined to the extracellular medium and cell lysis did not occur. The results indicate that scavenging of HOCl could be a factor in the inhibition by desferrioxamine of neutrophil-mediated cell lysis in vitro.

Membrane changes associated with lysis of red blood cells by hypochlorous acid.

This study was carried out to investigate HOCl-induced lysis of human erythrocytes. Using reagent HOCl with isolated red cells, we showed that the rate of lysis was dependent on the dose of HOCl per red cell rather than on the concentration of oxidant. The process was inhibited by scavengers such as methionine and taurine, but only if they were present at the time of addition of HOCl. Lysis was preceded by a decrease in cell density, a change in the deformability of the membrane as evidenced by ektacytometry, and an increase in K(+)-leak. Electron microscopy showed extensive disruption of the membrane. Increasing doses of HOCl caused progressive loss of membrane thiols, but complete thiol oxidation by N-ethylmaleimide did not result in an equivalent rate of lysis. Restoration of oxidised thiols by incubation with glucose did not significantly alter the pattern of lysis. Taken together, these results suggest that thiol oxidation was not responsible for HOCl-mediated lysis. There was evidence of increasing crosslinking of membrane proteins on electrophoresis, only some of which was due to the formation of disulfides. TLC of the membrane lipids indicated that there may be formation of chlorohydrins by reaction of HOCl with the fatty acid double bonds. This reaction results in the formation of a more polar species which, if formed, would be extremely disrupting to the lipid bilayer. The results indicate that HOCl-mediated damage to the membrane proteins or to the lipid bilayer comprises an initial damaging event that sets the cells on a path toward eventual lysis.

Rapid purification of human peripheral blood monocytes by centrifugation through Ficoll-Hypaque and Sepracell-MN.

We have developed a rapid and simple method for isolating human peripheral blood monocytes in suspension. The procedure combines two separation media and involves isolation of the mononuclear cells by centrifugation through Ficoll-Hypaque followed by purification of the monocytes using Sepracell-MN, a colloidal silica-based medium. The final cell population contained approximately 90% monocytes with good functional ability. The contaminating cells were lymphocytes. Viability was always greater than or equal to 99% with 90% recovery of the monocytes from the mononuclear cells.

Inhibition of neutrophil degranulation and superoxide production by sulfasalazine. Comparison with 5-aminosalicylic acid, sulfapyridine and olsalazine.

Sulfasalazine is a potent inhibitor of superoxide production and granule enzyme release by stimulated neutrophils, and modulation of these responses may contribute to its anti-inflammatory properties. It is a composite drug consisting of 5-aminosalicylic acid and sulfapyridine joined through an azo linkage. To investigate which functional groups on the molecule are active against neutrophil responses, 5-aminosalicylic acid, sulfapyridine and olsalazine were added to cells stimulated with fMet-Leu-Phe or immune complexes. The inhibitory effects of sulfasalazine on superoxide production, degranulation and neutrophil-mediated collagen degradation were closely mimicked by olsalazine, with the other two compounds having little effect on either function. Thus the azo link appears to be the important structural feature of sulfasalazine that affects neutrophil responses. This suggests that sulfasalazine could be anti-inflammatory in its own right rather than just acting as a source of 5-aminosalicylic acid. Our findings are also a favourable indication for olsalazine (Dipentum), which is currently under trial as an anti-inflammatory agent.

Inhibition by nonsteroidal anti-inflammatory drugs of superoxide production and granule enzyme release by polymorphonuclear leukocytes stimulated with immune complexes or formyl-methionyl-leucyl-phenylalanine.

The effects of nonsteroidal anti-inflammatory agents on superoxide production and granule enzyme release by human polymorphonuclear leukocytes stimulated with either formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe] or immune complexes were investigated. Cytochrome c reduction and the release of lysozyme, beta-glucuronidase, myeloperoxidase and gelatinase were measured. Auranofin, phenylbutazone, sulfasalazine and the phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited these responses in fMet-Leu-Phe stimulated cells, at concentrations below 50 microM. Indomethacin, piroxicam, mefenamic acid, primaquine and quinacrine at 50-250 microM were inhibitory. Up to 1 mM ibuprofen and chloroquine inhibited superoxide production but had little effect on degranulation. With cells stimulated by IgG aggregates (immune complexes), up to 1 mM ibuprofen, mefenamic acid and piroxicam did not inhibit either response. Indomethacin, phenylbutazone, sulfasalazine and primaquine inhibited, but considerably higher concentrations were required than with fMet-Leu-Phe. Quinacrine inhibited superoxide production equally well with both stimuli but inhibited enzyme release only with fMet-Leu-Phe. Only auranofin, 4-bromophenacyl bromide, and the weakly effective chloroquine exerted approximately the same effect with both stimuli. D-Penicillamine did not affect enzyme release with either stimulus and interfered in the superoxide assay. Gelatinase release induced by fMet-Leu-Phe was affected to the same extent, or slightly more, than release of the other granule enzymes. With immune complexes, there was only modest inhibition of gelatinase release by any of the drugs at 250-1000 microM. Our results reinforce previous observations that many anti-inflammatory drugs affect neutrophil functions, but their effects vary with stimulus. The relative insensitivity of immune complex-induced responses to most of the drugs must be taken into account when considering their mode of action.

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.

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.

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.

Protocol processing system (ProtoVIEW) to support residents at the emergency ward.

This paper describes the design, implementation and evaluation of a prototype protocol processing system (ProtoVIEW). ProtoVIEW provides protocol information for diagnostic and therapeutic purposes. The developed system described here is used as a research instrument to investigate the impact of automated protocols on the medical decision making of physicians. A first evaluation indicates that residents like to use the system and that usage of the system leads to a more uniform working strategy.

Development, implementation and a first evaluation of a protocol processing system (ProtoVIEW).

In this contribution the protocol information system ProtoVIEW is presented. The system provides the necessary information to physicians about diagnostic procedures and therapies. It is implemented as a stand alone system. The design criteria are discussed and the results of a first evaluation presented. It appears that interns can easily find the required information with the help of the system. The time that they need for accessing the relevant information is relatively short (about 1 min). The users expressed the opinion that the system is easy to use and does support them in the management of their patients. On the basis of this evaluation and evaluations reported elsewhere it is concluded (a) that stand-alone protocol systems can support daily patient management in a positive way and (b) that the design criteria for a protocol information system as presented in this paper are useful for prospective protocol information system developers.

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.