Oxygen free radical injury of IEC-18 small intestinal epithelial cell monolayers.

Oxygen radicals can cause endothelial and epithelial permeability changes and mucosal injury of the small intestine. There is no clear consensus concerning the relative injurious potential of individual oxygen radicals. In this study, the small intestinal cell line IEC-18 was used as an in vitro model to study the relative injurious effects of reactive oxygen metabolites. By introducing different combinations of oxygen metabolite-producing enzymes, xanthine oxidase, superoxide dismutase, and catalase, and an iron chelator, deferoxamine, to the fully confluent monolayers and to proliferating IEC-18 cells, the differential injurious effects of the oxygen metabolites O2-, H2O2, and OH. could be evaluated. The extent of cellular injury was assessed using [3H]thymidine uptake, 51Cr release, and morphological evaluations. Our results suggest that OH. produced as a by-product of O2- and H2O2 via the Haber-Weiss reaction was the most injurious oxygen species involved in cellular injury of IEC-18 monolayers induced by xanthine oxidase. O2- produced by xanthine oxidase appeared to be only minimally injurious, and H2O2 produced by xanthine oxidase and as a result of conversion of O2- by superoxide dismutase was moderately injurious. Superoxide dismutase and deferoxamine at appropriate concentrations were protective against xanthine/xanthine oxidase-induced monolayer injury. H2O2 added directly or produced indirectly by glucose oxidase was very injurious to the intestinal monolayers, and this injury was mitigated by catalase.     

Brief Report: Lack of Effect of L-Methionine Ingestion on 14CO2 Excretion from L-Histidine (Imidazole-2-14C) in Folic Acid and Vitamin B12 Deficient Humans

Three human subjects with folic acid and one with vitamin B₁₂ deficiencyfailed to show any effect of ingestion of L-methionine (400-800 µg./kg. bodyweight) on the production of ¹⁴CO₂ from parenterally administered high specific activity L-histidine (imidazole-2-¹⁴C). It is suggested that the inhibitoryeffect of methionine on intestinal absorption and cell membrane transport ofhistidine can explain previously published effects of methionine on the oxidation of the imidazole-2 carbon atom of L-histidine and that a direct effect ofmethionine on histidine metabolism need not be invoked.

Submitted on February 17, 1969 Accepted on July 22, 1969     

Iron in the Duodenal Mucosa of Normal, Iron-loaded and Iron-deficient Rats

Iron absorption studies with oral ⁵⁹FeSO₄were performed on 13 iron-depleted, 11iron-loaded, and 10 control rats, and nonheme iron was determined in both isolated epithelial cells and defoliated mucosa obtained from the duodenum. Meanabsorption by the control animals was18.5% of the dose. Both iron-depleted andiron-loaded groups showed significant differences in iron absorption (54.5% and2.2% respectively). Compared with thenormal controls, iron was decreased inthe epithelial cells of the iron-deficientgroup, whereas higher concentrationswere observed in the defoliated mucosaof iron-loaded animals. The latter observation was confirmed by the presence ofiron-laden macrophages seen in sectionsof the lamina propria of the iron-loadedrats.

Submitted on February 20, 1973 Revised on June 7, 1973 Accepted on June 13, 1973     

The Effect of Chronic Iron Deficiency on Some Biochemical Functions of the Human Hemopoietic Tissue

In view of the erythroid hyperplasiafound in the bone marrow of subjectswith chronic iron deficiency anemia, thedelayed appearance of reticulocytosis following iron therapy is unexplained. Profound disturbances were observed insome biochemical functions of the bonemarrow cells isolated from such patients.There was a substantial decrease in thecellular nucleic acid content, associatedwith a marked drop in the rate of ³H-thymidine incorporation into DNA. Theutilization of ⁵⁹iron and of glycine-2-¹⁴Cfor heme production, and of the lattercompound for protein synthesis was alsoreduced, as compared to the findings inbone marrow cells from normal subjects.These metabolic alterations returned tothe normal pattern in the bone marrowcell suspensions obtained from the patients following recovery after irontherapy.

The possible implications of these findings are discussed in the light of available information.

Submitted on September 15, 1969 Revised on October 12, 1969 Accepted on March 12, 1970     

Brief Note: Effects of Prostaglandins, Epinephrine and NaF on Human Leukocyte, Platelet and Liver Adenyl Cyclase

Despite the limited scope of this study, the data show that homogenates ofhuman leukocytes, platelets and liver are capable of synthesizing cyclic AMP,and that such preparations are responsive to hormonal stimulation. Epinephrine stimulates cyclic AMP production in liver and leukocyte homogenates,and NaF shows a stimulatory effect in each tissue.^1,8 The data confirm theprevious reports^10,11 that prostaglandin E₁ stimulates platelet adenyl cyclase,but further shows a lack of stimulation by prostaglandin F/₁. Incubation ofleukocytes with the prostaglandins showed a similar effect.

It is suggested that further study may establish a relationship betweenadenyl cyclase stimulation and chemotaxis or phagocytic activity of circulatingleukocytes as has been reported in amebae.¹² Since systemic enzyme deficienciessuch as phosphorylase in Hers’ Disease¹³ and amylo-1.41.6-transglucosidasein Andersens’ Disease¹⁴ are evident in leukocytes, it is suggested that furtherstudy of circulating leukocytes may also detect an abnormal responsiveness ora deficiency of adenyl cyclase.

Submitted on August 8, 1969 Accepted on December 29, 1969     

Role of xanthine oxidase-derived oxidants and leukocytes in ethanol-induced jejunal mucosal injury

Previous reports indicate that intestinal intraluminal ethanol increases mucosal permeability (an index of mucosal injury) and histamine release by mast cells, and that the released histamine plays a role in mediating the increased permeability. In the present study, we investigated whether reactive oxygen metabolites and their major sources (xanthine oxidase and leukocytes) were involved in these ethanol effects. In rabbits, segments of the jejunum were perfused with a control solution or with 6% ethanol. In these segments, mucosal permeability was assessed by determining jejunal clearance of i.v. administered⁵¹Cr-ethylenediaminetetraacetate (⁵¹Cr-EDTA) and¹²⁵I-bovine serum albumin (¹²⁵I-BSA), and mast cell histamine release was estimated from the histamine concentration of the gut effluent. Ethanol increased⁵¹Cr-EDTA clearance,¹²⁵I-BSA clearance, and histamine release. These ethanol effects decreased when the animals were given superoxide dismutase plus catalase (scavenger of O^2– and H₂O₂, respectively), allopurinol, or oxypurinol (xanthine oxidase inhibitors). Administration of a monoclonal antibody (R15.7) against leukocyte adhesion molecule, CD18, inhibited completely the ethanol-induced increased⁵¹Cr-EDTA and¹²⁵I-BSA clearances and histamine release. These and supplementary data suggest that (a) ethanol-induced mucosal injury and mast cell histamine release are mediated primarily by leukocytes, and (b) oxy radicals, especially those generated by xanthine oxidase, mediate these ethanol effects mainly by promoting leukocyte infiltration.     

The Significance of Iron Turnover in the Control of Iron Absorption

Following acute blood loss, there is a 4- to 5-day lag before any demonstrable change in plasma iron kinetics. Then there is a shortened Fe⁵⁹T and transient fall in the plasma iron. Associated with this is a decreasein the intestinal content of iron and an increased absorption of iron. We suggest the iron content of the epithelial cell is important in the regulation ofits ability to absorb iron. The intestinal iron content is in turn closely controlled by the plasma iron turnover.

Submitted on October 7, 1963 Accepted on December 3, 1963     

Delivery of Co57B12 to Erythrocytes from agr and beta Globulin of Normal, B12-Deficient, and Chronic Myeloid Leukemia Serum

In a study of Co⁵⁷B₁₂ uptake by reticulocyte-rich erythrocytes, transfer fromchronic myeloid leukemia (CML) serum was less efficient than from normalserum. Decreased transfer of Co⁵⁷B₁₂ was not due to excessive transfer ofendogenous B₁₂ (which is normally elevated in CML) but was associatedwith an -globulin B₁₂-binder (isolated from "baby" DEAE columns) unableto normally deliver B₁₂ to erythrocytes. Delayed plasma clearance of intravenously injected B₁₂ in CML may be due to this phenomenon.

B₁₂-deficient serum delivered slightly more added Co⁵⁷B₁₂ to erythrocytesthan did normal serum, possibly due to less interference by endogenous B₁₂.- and -globulin B₁₂ binders from B₁₂-deficient serum transferred boundCo⁵⁷B₁₂ as efficiently as did normal serum - and -binders; the -binders delivered more than the -binders. CML -binder delivered as well as did - binder from pernicious anemia and normal serum. These findings suggest thatthe -binder of CML serum is physiologically and therefore chemically differentfrom the -binder in normal and pernicious anemia serum.

Reticulocyte-rich, B₁₂-deficient erythrocytes from pernicious anemia patientstook up Co⁵⁷B₁₂ less well than did B₁₂-sufficient reticulocyte-rich erythrocytes.This may partially explain delayed plasma clearance of B₁₂ in B₁₂ deficiency.

Rat liver homogenate uptake of Co⁵⁷B₁₂ was much greater when B₁₂ wastransferred from normal serum than from normal gastric juice or saline. Preliminary investigations showed that both - and -binders from normal serumtransferred B₁₂ to liver homogenate but CML -binder transferred poorly.

Submitted on September 9, 1966 Accepted on November 24, 1966     

Febuxostat Improves the Local and Remote Organ Changes Induced by Intestinal Ischemia/Reperfusion in Rats

Background

Xanthine oxidase has been implicated in the pathogenesis of a wide spectrum of diseases, and is thought to be the most important source of oxygen-free radicals and cell damage during re-oxygenation of hypoxic tissues.

Aims

The present study was undertaken to demonstrate whether febuxostat is superior to allopurinol in prevention of the local and remote harmful effects of small intestinal ischemia/reperfusion injury in rats.

Methods

Intestinal ischemia was induced by superior mesenteric artery ligation. The rats were assigned to five groups: the sham control; the intestinal ischemia/reperfusion; the allopurinol; and the febuxostat 5 and 10 mg/kg pretreated ischemia/reperfusion groups. Treatment was administered from 7 days before ischemia induction. After the reperfusion, the serum and tissues were obtained for biochemical, pharmacological, and histological studies.

Results

Intestinal reperfusion led to an elevation in the serum levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, malondialdehyde, and xanthine oxidase as well as intestinal myeloperoxidase, malonadialdehyde, and xanthine oxidase/xanthine dehydrogenase activity. Furthermore, the ischemia/reperfusion induced a reduction in the contractile responsiveness to acetylcholine. These changes were significantly regulated by the pretreatment with febuxostat compared to allopurinol. The degree of pathological impairment in the intestinal mucosa, liver, and lung tissues were lighter in the pretreated groups.

Conclusions

Febuxostat may offer advantages over allopurinol in lessening local intestinal injury as well as remote hepatic and lung injuries induced by small intestinal ischemia/reperfusion.     

Acute intestinal iron intoxication. I. Iron absorption,serum iron and autopsy findings

To simulate the fatal iron poisoning reported in children and to study themechanism of the iron toxicity, dissociable iron salts were given by stomach orduodenal tube or by enema to rabbits and dogs. In either route of administration, the lethal dose was found to be approximately 150 to 200 mg. Fe per kilogram body weight.

Dissociable iron salts in toxic doses were rapidly absorbed through the histologically intact mucosa both from the small and from the large bowel. In themajority of the animals, no histological changes were seen in the intestinalmucosa, but intestinal bleeding occurred in some instances by diapedesis fromthe greatly congested capillaries.

Serum iron levels of several milligrams per cent developed within sixty minutes after ingestion of the iron salts, and with the exception of 300 to 400 micrograms per cent, the serum iron was non-beta₁-globulin bound and in ferric state.

Although only a fraction of the total dose administered was found to be absorbed within nine hours, the observed serum iron rise was roughly proportionalto the dose ingested, and the survival time varied inversely with the dose.

Acute intestinal iron poisoning must therefore be considered as a true absorptive intoxication. Any possibly occurring mucosal damage in stomach andintestines is of secondary importance.

Submitted on March 22, 1954 Accepted on July 6, 1954     

In Vitro Effect of Erythropoietin on the Spleen of The Polycythemic Mouse: II. Radiosensitivity of Stem Cells

1. An in vitro method to observe radiosensitivity of stem cells was developedin the present study. In vivo and in vitro effect of ⁶⁰Co irradiation on the erythropoietin-induced stem cell differentiation into erythroblasts was observed,using a tissue culture method of polycythemic mouse spleen. Response to erythropoietin was demonstrated by an appearance of heme synthesis and erythroblasts in spleen fragments.

2. A significant correlation between the rate of appearance of erythroblastsand heme synthesis of the spleen fragments was observed.

3. After irradiation, marked impairment of both heme synthesis and production of erythroblasts was observed, yielding D₃₇ values in the vicinity of70 r in vivo and 120 r in vitro irradiation, respectively.

4. Marked recovery of erythropoietin-induced heme synthesis in the polycythemic mouse spleen was observed 9 days after 300 r irradiation, with an"overshooting" phenomenon on the 12th day.

Submitted on September 14, 1966 Accepted on December 12, 1966     

Brief Report: Hemoglobin-Coated Charcoal Radioassay for Serum Vitamin B12. A Simple Modification To Improve Intrinsic Factor Reliability

Reliable binding of vitamin B₁₂ by intrinsic factor (IF) is essential in thehemoglobin-coated charcoal radioassay for serum vitamin B₁₂ as described byLau et al.⁴ The fact that some IF preparations bind less B₁₂ in the saline IFcontrol tube than in those tubes containing serum can result in a falsely lowmeasurement of unknown sera. This may be avoided by the simple substitution of 0.5 ml. of 5 per cent human albumin solution for the saline in the IFcontrol tube.

Submitted on February 17, 1969 Accepted on May 8, 1969     

Intestinal mucosal alterations in rats with carbon tetrachloride-induced cirrhosis: changes in glycosylation and luminal bacteria

Spontaneous bacterial peritonitis is a major cause of mortality after liver cirrhosis. Altered permeability of the mucosa and deficiencies in host immune defenses through bacterial translocation from the intestine due to intestinal bacterial overgrowth have been implicated in the development of this complication. Molecular mechanisms underlying the process are not well known. In order to understand mechanisms involved in translocation of bacteria, this study explored the role of oxidative stress in mediating changes in intestinal mucosal glycosylation and luminal bacterial content during cirrhosis. CCl4-induced cirrhosis in rats led to prolonged oxidative stress in the intestine, accompanied by increased sugar content of both intestinal brush border and surfactant layers. This was accompanied by changes in bacterial flora in the gut, which showed increased hydrophobicity and adherence to the mucosa. Inhibition of xanthine oxidase using sodium tungstate or antioxidant supplementation using vitamin E reversed the oxidative stress, changes in brush border membrane sugar content, and bacterial adherence. In conclusion, oxidative stress in the intestine during cirrhosis alters mucosal glycosylation, accompanied by an increased hydrophobicity of luminal bacteria, enabling increased bacterial adherence onto epithelial cells. This might facilitate translocation across the mucosa, resulting in complications such as spontaneous bacterial peritonitis.     

Soybean trypsin inhibitor attenuates ischemic injury to the feline small intestine

Recent evidence suggests that oxygen free radicals are largely responsible for the increased vascular permeability and early mucosal lesions associated with partial intestinal ischemia. It is postulated that oxygen radicals are produced by the reaction of the enzyme xanthine oxidase with hypoxanthine and molecular oxygen. In normal healthy cells, xanthine oxidase exists as a nicotinamide adenine dinucleotide-reducing dehydrogenase and not the oxygen radical-producing oxidase. In the intestine, dehydrogenase-to-oxidase conversion is nearly complete with less than 1 min of ischemia. Biochemical evidence from the intestine and liver indicate that ischemia-induced conversion of xanthine dehydrogenase to xanthine oxidase can be prevented by administration of protease inhibitors such as soybean trypsin inhibitor. In order to assess the role of proteases in oxygen radical-mediated ischemic injury to the small bowel, quantitative analyses of mucosal lesion development and vascular permeability were performed in autoperfused segments of cat ileum subjected to 1 or 3 h of ischemia and pretreated with 15 mg/kg (i.v.) soybean trypsin inhibitor. One hour of ischemia produced a significant increase in intestinal vascular permeability. The ischemia-induced increase in vascular permeability was significantly attenuated by soybean trypsin inhibitor pretreatment. Three hours of ischemia led to the development of mucosal lesions in untreated animals. Pretreatment with soybean trypsin inhibitor largely prevented the development of the mucosal lesions. The findings of our study are consistent with biochemical evidence that, during ischemia, proteases trigger the conversion of xanthine dehydrogenase to xanthine oxidase and thereby lead to oxygen radical production and subsequent tissue injury.     

The Heterogeneity of the High Molecular Weight B12 Binder in Serum

The binding of vitamin B₁₂ by serum proteins was studied by separatingCo⁵⁷B₁₂-enriched serum by Sephadex gel filtration, column chromatographywith DEAE-cellulose, and paper electrophoresis. Each method of separationyielded two discrete B₁₂-binding fractions. However, the analysis of each serumby all three separation technics indicated that one of the fractions was, ineach case, bipartite.

The "high" molecular weight B₁₂-binding fraction defined by Sephadex gelfiltration consisted of transcobalamin I and just part of the transcobalamin IIfraction. The remaining portion of transcobalamin II was eluted from Sephadexgel in a "low" molecular weight fraction. Thus, transcobalamin II, equivalentto the -globulin B₁₂-binder, consisted of both "high" and "low" molecularweight components.

This suggests that there are at least three serum proteins that can bindvitamin B₁₂: two -globulins, together comprising the transcobalamin II fraction and differing in molecular weight; and transcobalamin I.

Submitted on November 4, 1968 Accepted on January 28, 1969     

Determination of transferrin-like immunoreactivity in the mucosal homogenate of the duodenum,jejunum, and ileum of normal and iron deficient rats

Summary 1. In intestinal mucosal tissue a transferrin-like immunoreactivity (TLIR) was determined after loading with ⁵⁹Fe-(FeCl₃) in vivo. The scraped-off mucosal tissue was homogenized and centrifuged at 100 000×g. The supernatant was fractionated chromatographically. The TLIR was determined by the Mancini-test.2. Extrapolated to infinite dilutions of the homogenate the following contents of the TLIR were calculated per cm of the intestinal segment: 28, 20, and 13, g/cm in the duodenum, jejunum, and ileum of iron-deficient animals and 15, 14, and 12 µg/cm in the corresponding intestinal segments of normal rats, respectively.3. The content of TLIR was compared with the iron uptake of the mucosal tissue from the intestinal lumen during absorption. An iron turnover number of 1.6±0.2 iron atoms per min during the absorption in duodenal and jejunal segments and of 0.5±0.1 in ileal segments of either iron-deficient and normal animals was calculated for the mucosal transferrin represented by the TLIR.Supported by a grant of the Wissenschafts-Ministerium Nordrhein-Westfalen     

Endogenous Production of 14CO: A Method for Calculation of RBC Life-span In Vivo

A kinetic model is presented for estimation of the degradation rate of labeledheme by measurement of appearance of¹⁴CO in the breath following injection ofglycine-2-¹⁴C. This method does not require sampling of blood or other bodyfluids, is absolutely independent of circulating blood volume and relatively independent of erythropoietic rate, andestimates the relative contribution to¹⁴CO production from destruction of circulating RBC hemoglobin heme and thatarising from other heme sources. Forcirculating RBC, rate of random hemolysis, mean potential lifespan and spreadof lifespans about this mean can be calculated. Mean overall RBC lifespan andthe fraction of RBC dying of senescencecan be derived from these calculations.In normal male buffalo rats, the average value for random hemolysis was0.67 per cent per day, corrected meanpotential lifespan 66.2 days and standard deviation about this mean of 7.6days. For normal female LAF₁ mice, thecorresponding average values were 0.60,51.8 and 9.1, respectively. Results intwo other inbred mouse strains weresimilar save for a shorter mean potential lifespan of 47.1 days in SEC/1Remice and a longer mean potential lifespan of 57.3 days in WC-B6 mice.Following splenectomy in the rat, an isolated significant increase in mean potential lifespan was seen. An isolateddecrease in mean potential lifespan wasseen in three rats recovering fromphenylhydrazine-induced anemia. Anexample of markedly increased randomhemolysis, together with shortened meanpotential life-span, was seen in a gastrectomized rat with a severe hypochromicanemia. The present method is shownto simultaneously determine the majorparameters defining RBC survival, andas such, should be quite useful in thestudy of red blood cell disorders in animals and man.

Submitted on December 19, 1969 Revised on May 13, 1970 Accepted on May 28, 1970     

Hemoglobin Andrew-Minneapolis agrA2beta144 Lys rarr Asn2: A New High-Oxygen-Affinity Mutant Human Hemoglobin

The functional properties and primarystructure of a new -chain mutant ofhuman hemoglobin are described. Themutant was transmitted as an autosomaldominant characteristic. Affected members of the kindred exhibited markederythrocytosis due to the high oxygenaffinity of the resultant hemoglobin. Theabnormality is associated with a substitution of an asparaginyl residue for lysinein the 144 position of the -chain,^A₂^144LysAsn₂, presumably due to anAAA/G to AAA/U transversion. The mutant hemoglobin displayed a profound increase in oxygen affinity, with a P₅₀ of thefresh whole blood of 14 mm Hg. The isolated mutant hemoglobin exhibited nearnormal heme—heme interaction, a half-normal Bohr effect, and normal reactivitywith 2,3-diphosphoglycerate.

Submitted on January 17, 1974 Accepted on April 15, 1974     

Porphyrin Synthesis and Heme Synthetase Activity in Pyridoxine-Responsive Anemia

Previous in vitro studies had shown that reticulocytes from a patient withpyridoxine-responsive anemia (PRA) incorporated glycine-2-¹⁴C into hemeat 11-25 per cent of the normal rate. Incorporation of the intermediate compound, -aminolevulinic acid-4-¹⁴C (ALA), varied from 46 to 81 per centof normal. These findings suggested two defects in the synthesis of heme: amajor one prior to formation of ALA, probably ALA synthetase, and a minorone somewhere between ALA and heme. This minor defect was investigatedin a patient with PRA during a period of repeated phlebotomies resultingin a 30 gram reduction in iron stores. Porphyrin synthesis from ALA wasmeasured in vitro and found to be normal. Heme synthetase activity in particulate fractions of reticulocytes measured by radioiron incorporation intoheme was 50 per cent less than normal when the iron stores were high andsignificantly improved when the iron stores were reduced to normal. Repeatstudies showed that incorporation of ALA-4-¹⁴C into heme by intact reticulocytes equaled the normal rate. Glycine incorporation was still reduced.Concomitant with the improvement in heme synthetase activity was an improvement in anemia. These results suggest that excess iron is a significantcause of the reduced heme synthetase activity observed in this patient withpyridoxine-responsive anemia.

Submitted on March 5, 1968 Accepted on June 10, 1968     

Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 51Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model.