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.

Antibodies, Monoclonal↗

PubMedDate not supplied

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.

Animals↗

PubMedDate not supplied

Lipid peroxide and hydrogen peroxide formation in parenteral nutrition solutions containing multivitamins.

BACKGROUND: Infusion of parenteral solutions containing peroxides may be detrimental to premature infants. Intralipid frequently contains lipid peroxides and undergoes further peroxidation when exposed to light. Peroxidation is inhibited by ascorbate, and we have proposed that administration of peroxides could be minimized by mixing multivitamins with the Intralipid. In contrast, others have reported that multivitamins generate peroxides and have advised against mixing them with lipid. Our objective was to assess whether light-dependent reactions in parenteral solutions containing MVI Pediatric (MVIP) generate hydrogen peroxide and establish whether addition of multivitamins to Intralipid is beneficial or detrimental. RESULTS: We were unable to make accurate peroxide measurements in MVIP using the ferrous oxidation of xylenol orange (FOX) assay, even though others have used it for this purpose, because of interference by ascorbate. Therefore oxygen release on adding catalase was measured to assay for hydrogen peroxide. Freshly reconstituted solutions contained 250 to 500 micromol/L hydrogen peroxide, and this increased dramatically in ambient light. This is presumably due to light-dependent, riboflavin-catalyzed reduction of oxygen by ascorbic acid. The rate of peroxide generation was less for MVIP diluted in Intralipid than in dextrose solution. CONCLUSIONS: Taken together with our previous findings, we conclude that multivitamins protect Intralipid against lipid peroxidation, but light-dependent hydrogen peroxide production and ascorbate loss occur. These latter changes are less than for multivitamins in other total parenteral nutrition solutions, so there is an advantage in mixing multivitamins with Intralipid. However, prevention of ascorbate loss and hydrogen peroxide formation in any multivitamin solution requires protection of the delivery system from light.

Ascorbic Acid↗

PubMedDate not supplied

Pharmacologic levels of heparin do not destabilize neonatal parenteral nutrition.

BACKGROUND: Calcium and heparin are known to destabilize the lipid emulsion of total parenteral nutrition (TPN). However, these observations were made over long periods of time, using 5 to 10 times the amount of heparin used in a neonatal intensive care unit. We investigated the effects of lower heparin concentrations with lipid-to-nutrient ratios normally administered to premature infants. METHODS: Lipid emulsion stability was assessed over 30 minutes by measuring viscosity at 10 mmol/L calcium after the addition of 0, 0.5, 1, and 5 IU heparin/mL. This was done at a range of lipid-to-nutrient ratios in clinical use. The effect of varying calcium concentration and different multivitamin preparations on emulsion stability also was observed. RESULTS: Heparin caused an immediate increase in viscosity of pure Intralipid 20% (Intralipid; Kabi Pharmacia AB, Stockholm, Sweden), which eventually separated into two phases. Although changes in viscosity were observed at 1:1 lipid-to-nutrient ratios, no effect was seen at a 1:9 ratio. With the 1:1 ratio, the multivitamin preparations, MVI Pediatric (Rhone-Poulec Rorer, Montreal, Canada) and Vitlipid (Kabi Pharmacia AB), reduced the increase in viscosity. CONCLUSIONS: Heparin and calcium destabilize Intralipid. This is unlikely to cause problems for most infants receiving TPN, provided low heparin concentrations are used. In all cases, especially where the lipid ratio is high, the risk of the lipid phase separating out can be further minimized by (1) the addition of multivitamin preparations containing detergent or an emulsifying agent and (2) by having the shortest possible delivery tube between the point of mixing the lipid and amino acid solutions of TPN and entry into the infant.

Calcium↗

PubMedDate not supplied

Reactivity of the beta-93 SH groups in Hb Köln.

The beta-93 SH groups were found to be present in freshly purified Hb Köln, but over a period of several days storage they gradually oxidized to form inter-beta chain disulfide bonds. It is suggested that this gradual oxidation accounts for the low SH values which have been reported. The SH groups of Hb Köln were also found to react more readily than normal with H2O2 and O2-.

Blood Preservation↗

PubMedDate not supplied

Free radical inactivation of rabbit muscle creatinine kinase: catalysis by physiological and hydrolyzed ICRF-187 (ICRF-198) iron chelates.

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vulnerable to inactivation under conditions when it would be used as a diagnostic marker of tissue damage such as during cardiac ischemia/reperfusion or other oxidative tissue injury. Oxidative stress in tissues can induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatinine kinase inactivation in a heart reperfusion model has been documented, the mechanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen peroxide. Ferrous iron enhanced the inactivation. In addition, micromolar levels of iron and iron chelates that were reduced and recycled by superoxide or Adriamycin radicals were effective catalysts of creatinine kinase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological chelate of interest because of its putative role in alleviating Adriamycin-induced cardiotoxicity, also catalyzed the inactivation. Scavenger studies implicated hydroxyl radical as the oxidant involved in iron-dependent creatine kinase inactivation. Loss of protein thiols accompanied loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GSH depletion.

Animals↗

PubMedDate not supplied

The unstable hemoglobins.

The hemoglobin variants have a range of stabilities, a number being sufficiently unstable to cause a hemolytic anemia in the heterozygote. These unstable hemoglobins can be readily detected by standard stability tests. The abnormalities in the unstable hemoglobins result in an increased molecular flexibility that affects both the globin chain and the hemoglobin tetramer. The unstable globins are generally synthesized at a normal rate but their distorted structure leads to proteolytic destruction, primarily in the bone marrow. This explains why they are always present in much reduced amounts in the circulation. Evidence from the unstable hemoglobins is that overall globin synthesis at the cellular level continues until there is compensation for the loss of the abnormal chain. Increased flexibility or distortion of the hemoglobin molecule allows more ready oxidation to methemoglobin but, more importantly, there is also rapid conversion of methemoglobin to hemichrome with resultant denaturation and precipitation. The process is accompanied by the release of activated oxygen but there is no evidence that the amounts formed are sufficient to give cell damage. Hemolysis primarily occurs in the microcirculation due to the mechanical obstruction produced by the rigid Heinz bodies.

1-Propanol↗