Peroxide Hemolysis Research Articles

Utility of Breath Ethane as a Noninvasive Biomarker of Vitamin E Status in Children

The purpose of our study was to determine if the ethane content of expired air could be a useful index of vitamin E status in children. Eight children with vitamin E deficiency secondary to chronic severe liver disease were studied: six of these children were treated with parenteral vitamin E (2-5 mg/kg/dose every 4-7 d). Measures of vitamin E status pre- and posttherapy were: serum vitamin E, 2 +/- 1 versus 7 +/- 1 micrograms/mL (p less than 0.001); serum vitamin E:total lipids, 0.3 +/- 0.1 versus 1.0 +/- 0.1 mg/g (p less than 0.001); and erythrocyte peroxide hemolysis test, 80 +/- 10 versus 6 +/- 12% (p less than 0.001). Fasting breath ethane in the patients pre- and posttherapy was 78 +/- 10 versus 31 +/- 11 pmol/kg/min (p less than 0.001). Breath ethane correlated negatively with serum vitamin E (p less than 0.042) and serum E:total lipids (p less than 0.004) and positively with the erythrocyte peroxide hemolysis test (p less than 0.003). Values for treated patients did not differ from those for fasted sibling controls (34 +/- 12 pmol/kg/min), postprandial sibling controls (31 +/- 12 pmol/kg/min), and healthy children sampled randomly, in the nonfasted state (21 +/- 14 pmol/kg/min). Breath ethane production in one patient (up to 168 pmol/kg/min) did not normalize after treatment of vitamin E deficiency until her selenium deficiency was corrected as well. We conclude that this noninvasive test can be useful as a screen for vitamin E deficiency in children and for ascertaining response to therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

VITAMIN E SUPPLEMENTATION REDUCES FREQUENCY OF PERIVENTRICULAR HAEMORRHAGE IN VERY PRETERM BABIES

231 babies, born at ≤32 weeks' gestation were enrolled in a randomised, controlled trial to assess the efficacy of vitamin E (dl-alpha-tocopherol acetate) in the prevention of periventricular haemorrhage. Daily supplementation with 20 mg/kg vitamin E intramuscularly during the first 3 days of life was associated with a rise in plasma vitamin E concentration and a reduction in hydrogen peroxide haemolysis of red blood cells in vitro. Among babies without haemorrhage on entry to the trial (n = 210), supplemented babies had a lower frequency of intraventricular haemorrhage than controls (8·8% v 34·3%; p< 0·005) and a lower combined frequency of intraventricular and parenchymal haemorrhage (10·8% v40·7%; p< 0·0001) on the final ultrasound brain scan. This protective effect was observed in both inborn and referred babies but was stronger in the former. Supplementation had no effect on mortality, but among survivors fewer supplemented babies than controls had intraventricular or parenchymal haemorrhage (10·7% v 32·6%; p<0·001). Possibly, vitamin E scavenges free radicals generated during ischaemic injury of the subependymal region and thereby limits tissue damage and the extent of periventricular haemorrhage on reperfusion.

Intramuscular Vitamin E Repletion in Children With Chronic Cholestasis

Progressive spinocerebellar degeneration was identified in six children with chronic cholestatic liver disease and attributed to severe vitamin E deficiency. In addition to areflexia, ataxia, dysmetria, and diminished vibratory and position sense, three patients had pigmentary retinopathy. Abnormalities were present on electromyography, nerve conduction studies, and electroretinography. Because the vitamin E deficiency was not corrected by oral administration of massive doses of vitamin E, vitamin E was administered by the intramuscular route. With doses of 50 to 100 mg of vitamin E every three to seven days, over a 32-month interval (range, 15 to 44 months), vitamin E deficiency and abnormal red blood cell peroxide hemolysis were corrected. Other than discomfort and occasional edema at the site of injection, there were no side effects of parenteral vitamin E therapy. In several other studies intramuscular vitamin E therapy has produced significant neurologic improvement in patients with similar characteristics. In this study clinical progression of spinocerebellar degeneration was arrested but improvement could not be demonstrated despite adequate vitamin E replacement.

Characterization of vitamin E status in cholestatic children by conventional laboratory standards and a new functional assay.

In the present study, the clinical application of a new functional test for vitamin E deficiency was evaluated. Erythrocytes from cholestatic children at risk for vitamin E deficiency and appropriate controls were incubated in vitro with hydrogen peroxide and the malondialdehyde generated and released into the supernatant quantitated. The results of these incubations were compared with fasting plasma vitamin E levels, the ratio of plasma vitamin E to plasma lipid levels, and, in some instances, hydrogen peroxide hemolysis tests. Malondialdehyde formation was less than 6% in controls and vitamin E-sufficient cholestatic children. However, cholestatic vitamin E-deficient children had a mean malondialdehyde formation of 41%. The results also suggest that for children less than 4 months of age, a ratio of plasma vitamin E to total plasma lipids less than 0.6 mg/g may be sufficient to provide protection from in vitro peroxidation. The authors suggest that this functional assay of vitamin E status be included in the evaluation of individuals with the potential for vitamin E deficiency.

Characterization of Vitamin E Status in Cholestatic Children by Conventional Laboratory Standards and a New Functional Assay

SummaryIn the present study, the clinical application of a new functional test for vitamin E deficiency was evaluated. Erythrocytes from cholestatic children at risk for vitamin E deficiency and appropriate controls were incubated in vitro with hydrogen peroxide and the malondialdehyde generated and released into the supernatant quantitated. The results of these incubations were compared with fasting plasma vitamin E levels, the ratio of plasma vitamin E to plasma lipid levels, and, in some instances, hydrogen peroxide hemolysis tests. Malondialdehyde formation was less than 6% in controls and vitamin E‐sufficient cholestatic children. However, cholestatic vitamin E‐deficient children had a mean malondialdehyde formation of 41%. The results also suggest that for children less than 4 months of age, a ratio of plasma vitamin E to total plasma lipids less than 0.6 mg/g may be sufficient to provide protection from in vitro peroxidation. The authors suggest that this functional assay of vitamin E status be included in the evaluation of individuals with the potential for vitamin E deficiency.

Vitamin E sufficiency in children with cholestasis: A comparison between erythrocyte peroxide hemolysis and serum α-tocopherol

Vitamin E sufficiency was assessed in 19 children with chronic cholestasis by determining both erythrocyte peroxide hemolysis (EPH) and serum α-tocopherol (α-T) levels. Eight had normal α-T (1.19 ± 0.67 mg/dl; normal > 0.5). The mean EPH in this group was 13.1 ± 14.3% (normal < 20%). The remaining 11 patients had low α-T levels (0.25 ± 0.15 mg/dl) and elevated EPH (83.9 ± 17.1%). Children found to be vitamin E deficient received either oral α-T (50–100 IU/kg per day) or parenteral α-tocopherol acetate in sesame oil every 2–4 wk (200–300 mg). This permitted serial monitoring of EPH and α-T. We found that normalization of the EPH was uniformly accompanied by normalization of the α-T level. However, normal α-T levels occurred with elevated EPH (between 20% and 80%) on 11 occasions. EPH > 80% correctly identified vitamin E deficiency in all cases. Hence, EPH is a satisfactory screening test of vitamin E sufficiently. When the EPH is < 20%, the patient is vitamin E sufficient. Conversely, when the EPH is > 80%, the patient is vitamin E deficient. Serum α-T measurements are needed to determine vitamin E sufficiency when the EPH is > 20% and < 80%.

The effect of vitamin E deficiency on some erythrocyte membrane properties.

The erythrocytes from the vitamin E-deficient rats showed no changes in comparison with the erythrocytes from the vitamin E-supplemented rats in lipid peroxides, membrane fluidity, lipid composition, and activities of defensive enzymes against the lipid peroxide formation. Once erythrocytes from vitamin E-deficient rats were exposed to H2O2, the erythrocyte membrane fluidity was significantly reduced, which was presumed to be accompanied by an increase in the hemolysis and lipid peroxide formation. Large decreases in arachidonic acid and phospholipid were also observed. These findings suggest that the reduced membrane fluidity is due to the loss of phospholipid from the erythrocytes, and raises the possibility that the reduced fluidity might contribute to the peroxidative hemolysis of the vitamin E-deficient rat erythrocytes.

Pulmonary Excretion Rate of Carbon Monoxide as an Index of Total Bilirubin Formation in Adult Male Wistar Rats with Common Bile Duct Ligation

Total bilirubin formation (TBF) in the rat after a short period of common bile duct ligation was studied by measuring the pulmonary excretion rate of CO (VECO). At postoperative day 3, the VECO of experimental animals was higher when compared with the pre‐operative VECO (p &lt; 0.005); whereas the VECO of control animals did not change. Also, on the 3rd postoperative day, the relative rate of early labeling of bilirubin following the administration of δ‐aminolevulinic acid‐5‐14C, a preferential hepatic heme synthesis precursor, was similar between the experimental and control animals; only the experimental animals had an abnormal peroxide hemolysis test. We conclude that common bile duct ligation in the rat is associated with elevations in the VECO, indicating significant increases in TBF, and the source of the increase is probably of erythropoietic origin. This finding may be relevant to the understanding of the pathophysiology of obstructive jaundice in human neonates.

Pulmonary excretion rate of carbon monoxide as an index of total bilirubin formation in adult male Wistar rats with common bile duct ligation.

Total bilirubin formation (TBF) in the rat after a short period of common bile duct ligation was studied by measuring the pulmonary excretion rate of CO (VECO). At postoperative day 3, the VECO of experimental animals was higher when compared with the preoperative VECO (p less than 0.005); whereas the VECO of control animals did not change. Also, on the 3rd postoperative day, the relative rate of early labeling of bilirubin following the administration of delta-aminolevulinic acid-5-14C, a preferential hepatic heme synthesis precursor, was similar between the experimental and control animals; only the experimental animals had an abnormal peroxide hemolysis test. We conclude that common bile duct ligation in the rat is associated with elevations in the VECO, indicating significant increases in TBF, and the source of the increase is probably of erythropoietic origin. This finding may be relevant to the understanding of the pathophysiology of obstructive jaundice in human neonates.

Mechanism Causing Vitamin E Deficiency During Chronic Childhood Cholestasis

In order to characterize the mechanism(s) causing vitamin E deficiency during chronic childhood cholestasis, we studied 6 vitamin E-deficient cholestatic children with clinical evidence of neurologic dysfunction (group A), 4 vitamin E-sufficient cholestatic children and young adults with normal neurologic status (group B), and 6 vitamin E-sufficient noncholestatic children (group C). Intestinal absorption of dl-alpha-tocopherol (assessed by an oral tolerance test) was markedly impaired (p less than 0.001) in group A compared with groups B and C, which did not differ from each other. Intraluminal total bile acid concentrations were markedly depressed in group A compared with age-matched controls (0.50 vs. 7.00 mM, p less than 0.001), whereas concentrations were low normal in group B. Intramuscular dl-alpha-tocopherol was well absorbed in 4 group A subjects and corrected abnormal hydrogen peroxide hemolysis. Our data suggest that low intraluminal bile acid concentrations result in malabsorption and deficiency of vitamin E in children with prolonged, severe cholestasis. Intact plasma transport and tissue uptake of vitamin E during cholestasis suggest that intramuscular vitamin E should be utilized for prevention and therapy of the neurologic abnormalities caused by vitamin E deficiency.

Vitamin E deficiency during chronic childhood cholestasis: Presence of sural nerve lesion prior to 2½ years of age

Vitamin E malabsorption and deficiency during chronic childhood cholestasis has been associated with a progressive ataxic neurologic syndrome. Hyporeflexia, the first sign of neurologic dysfunction, may begin prior to age 2 years, but severe symptoms do not develop until age 5 to 10 years. To establish the age of onset of neuropathologic lesions, we prospectively evaluated four young children with severe cholestasis. Malabsorption and deficiency of vitamin E were documented by low serum vitamin E concentrations, low serum vitamin E to total serum lipids ratios, elevated hydrogen peroxide hemolysis, and impaired absorption of a pharmacologic dose of alpha-tocopherol. Abnormal neurologic findings in two patients were limited to areflexia, ptosis, mild truncal ataxia, and hypotonia; two patients had minimal signs of neurologic dysfunction. Sural nerve histology at age 6 to 25 months revealed a degenerative axonopathy involving large-caliber myelinated fibers, but without quantitative axonal loss. Muscle histology and histochemistry tests yielded normal results. Our study suggests that neurologic injury may occur during the first two years of life in vitamin E-deficient children with cholestatic hepatobiliary disease, obligating aggressive attempts at correcting this deficiency state at a very young age.

Bioavailability of vitamin E in rats fed diets containing pectin

Weanling male Sprague-Dawley rats were fed: o (1) a basal diet without supplemental vitamin E (-E) (2) a basal diet supplemented with 39 IU vitamin E/kg of diet ( +E) or (3) an E supplemented basal diet to which 10 percent citrus pectin was added (+E+P) The animals were studied over a 56 day period. Growth rate as measured by body weight was noticeably slower in the +E+P group as compared to both the -E and the +E groups. The final average body weight of the +E+P group was 28 percent less (P<0.05) than the final average body weight of the +E group. No difference was observed in body weight between the +E and the -E groups. Plasma and red cell vitamin E levels in both the -E and the +E+P groups decreased rapidly after starting the dietary regimen. Parallel with this rapid decrease in red cell vitamin E level was a rapid increase in the in vitro peroxide hemolysis value in both the +E+P and the -E groups. At the end of the study, plasma vitamin E values (mg/dl) were 0.13±0. 1(-E), 0.04±0.2 (+E+P), and 1.08±0.17(+E) and red cell vitamin E values (ug/ml) were 0.44±0.10(-E), 3.49±0.30(+E+P), and 5.74±0.37(+E). Vitamin E contents of other tissues including liver, heart and lung were 3 to 4 fold lower in both the +E+P and the -E group compared to the +E group. These results indicate that the consumption of a diet high in pectin content reduces the bioavailability of vitamin E.

Dietary vitamin E and polyunsaturated fatty acid (PUFA) in newborn babies with physiological jaundice

We studied 69 term babies aged 2–8 days who had physiological jaundice and who were fed formula A (Ostermilk Complete; vitamin E, 0.46 mg per 100 ml; polyunsaturated fatty acid (PUFA), − 0.08 g per 100 ml), or formula B (Cow and Gate Premium; vitamin E, 1.0 mg per 100 ml; PUFA, −0.55 g per 100 ml) or breast milk. Babies fed formula B, with the greatest vitamin E and PUFA content, had a significantly higher mean plasma vitamin E level compared with those fed formula A, even as early as the second and third day. Breast fed babies, 2–3 days old, had a lower mean plasma vitamin E level compared with formula B fed babies, thereafter vitamin E levels in breast fed babies rose. The RBCs of babies fed formula B and breast milk were significantly less susceptible to hydrogen peroxide (H 2O 2) haemolysis compared with the RBCs of those fed formula A. Reduced susceptibility to H 2O 2 haemolysis in formula B fed babies was observed in those as young as 2–3 days. Susceptibility to H 2O 2 haemolysis did not correlate with haemoglobin concentration, plasma bilirubin nor with the reticulocyte count in babies on different feeds. We conclude that in term newborn babies the vitamin E and PUFA contents of the milk feeds influence plasma vitamin E levels and susceptibility of RBCs to H 2O 2 haemolysis, but do not have an important bearing on the occurrence of physiological jaundice.

Plasma vitamin E levels and vitamin E/beta-lipoprotein relationships in small preterm infants during the early anemia of prematurity.

The vitamin E status of AGA preterm infants (birth weights≦1500 g) was studied during the first 10 weeks of life. The total polyunsaturated fatty acid content of the diet was 12%, and medicinal iron was given from 4 weeks of age. Plasma vitamin E concentrations correlated significantly with β-lipoprotein levels in the infants not supplemented with tocopherol. The low plasma vitamin E levels observed in these infants thus reflect the low transport capacity of the plasma, and do not necessarily signify vitamin E deficiency. Erythrocytes from the preterm infants showed increased hemolysis in the hydrogen peroxide hemolysis test, also when the plasma tocopherol levels were above 11.6 μmol/l; and for the same level of tocopherol, the degree of hemolysis varied considerably. This indicates that factors other than the tocopherol concentrations influence this test. When glucose was added to the cells during the test the hemolysis decreased. A group of infants supplemented with 7.5 IU water-soluble tocopherol/day showed satisfactory median levels of the vitamin, both when assessed as plasma vitamin E concentrations and as vitamin E/β-lipoprotein ratios. However, some infants had low plasma concentrations and ratios, particularly at 4 weeks of age. This indicates that this supplementary dose is insufficient when a water-soluble preparation is used. A very wide range of plasma vitamin E concentrations was observed in the supplemented infants. A word of caution against too vigorous supplementation in small preterm infants is therefore warranted.

Inhibition of Oxidative Hemolysis and Lipid Peroxidation by Mepacrine&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Mepacrine at 50 microM completely protected vitamin E-deficient rat erythrocytes from peroxidative hemolysis induced by dialuric acid or reduced glutathione under the standard experimental conditions. Malondialdehyde formation, which precedes the hemolysis, was also inhibited by mepacrine. These effects of mepacrine were observed when it was added after incubating the cells with dialuric acid before the malondialdehyde formation reached 50% of its maximal value. Mepacrine also inhibited NADPH-dependent lipid peroxidation in rat liver microsomes. The degree of inhibition by mepacrine of lipid peroxidation and hemolysis was dependent on the amount of red blood cells or microsomes in the reaction mixture.

Effects of Aspirin and Related Drugs in Vitamin E-Deficient Rats

Evidence from the literature indicates that in vitamin E-deficient animals prostaglandin (PG) synthesis in platelets is enhanced while it is decreased in the muscle and testis. In the present study the effects of aspirin, a known inhibitor of PG biosynthesis, on vitamin E deficiency signs in the rat were investigated. Administration of aspirin to vitamin E-deficient rats had no protective effect on fetal mortality, incisor depigmentation, body weight gain or red blood cell peroxidative hemolysis. Aspirin prevented the anemia and thrombocythemia observed in vitamin E-deficient rats. Aspirin, salicylic acid and a carbazole prostaglandin inhibitor exacerbated testis degeneration in vitamin E-deficient animals. Addition of aspirin to the diet more than doubled the vitamin E requirement for reversal of necrotizing myopathy.

Studies on the Normal Range of α-Tocopherol Levels in Plasma and RBC Membranes in Childhood, as Related to the Hydrogen Peroxide Hemolysis

Studies on the Normal Range of α-Tocopherol Levels in Plasma and RBC Membranes in Childhood, as Related to the Hydrogen Peroxide Hemolysis

Vitamin E and Neonatal Bilirubinemia

A study was designed to determine the effect of vitamin E on bilirubinemia in the preterm infant. Twenty infants with birth weight between 1,000 and 1,500 gm and 20 infants with birth weights between 1,501 and 2,000 gm were studied. Half the infants in each birth weight group received vitamin E administered intramuscularly in a total dose of 50 mg/kg during days 1 to 3 of life; the remaining infants served as controls. The administration of vitamin E produced significantly increased plasma tocopherol concentrations and normal hydrogen peroxide hemolysis tests by the end of the first week of life. Infants with birthweights less than or equal to 1500 gm who received vitamin E demonstrated a significant decrease in serum bilirubin on day 3 of life (6.5 +/- 2.2 vs 8.8 +/- 2.2 mg/dl) as well as a significant decrease in peak serum bilirubin during the first week of life (8.3 +/- 2.2 vs 10.6 +/- 2.6 mg/dl). The duration of phototherapy also was significantly less in the vitamin E-supplemented group (48 +/- 18 vs 107 +/- 31 hours). These differences were less pronounced in infants with birth weights more than 1,500 gm.

Influence of middle molecules on the anemia of uremic patients.

To evaluate their toxicity at the cellular level, middle molecules from uremic serum were incubated with erythrocytes from healthy subjects and the activity of the enzyme Delta-aminolevulinic acid dehydrase (D-ALA-D) and peroxidative hemolysis were investigated. Uremic middle molecules caused a significant decrease of the D-ALA-D activity of normal erythrocytes which was not due to differences in the concentrations of Pb, Cd or Zn. The decreased enzyme activity could be restored by adding reduced glutathione (GSH; 5 mmol/L) together with the middle molecules to the assay system. Uremic middle molecules caused a significant increase of peroxidative hemolysis in normal erythrocytes. Uremic middle molecules contribute to the anemia of uremic patients by impeding hemoglobin synthesis and by increasing peroxidative hemolysis, possibly by affecting SH-groups. H2O2-producing compounds should be avoided in uremic patients.

465 HEMATOLOGICAL AND BIOCHEMICAL CHANGES IN PREMATURE INFANTS ON 3 COMMERCIAL INFANT FORMULAS

Thirty healthy premature infants with birth weights between 1000g and 1600g were randomly assigned at 1 wk of age to 3 groups to determine the effect of 3 commercial infant formulas (Similac 20, Similac 20 with Iron, Iscmil - each containing 15 I.U. vit. E/liter) on various hematological, biochemical and growth parameters. During their hospital stay weekly determinations were made of hemoglobin (Hb); hematocrit (Hct); reticulocyte count; hydrogen peroxide hemolysis; red cell glutathione peroxidase (GSH-Px) and selenium (Se); plasma GSH-Px and Se; and serum Ca, P, alkaline phosphatase, protein, cholesterol and vit. E levels.Mean weekly rates of declines in Hb and in Hct, respectively, did not differ significantly in the 3 groups of infants. Among the infants with the highest rates of declines in Hct and Hb were some with persistently low serum vit. E levels; these were noted in all groups. Red cell and plasma GSH-Px and Se levels did not correlate with the rate of decline in Hb or Hct levels, nor was there a direct and constant correlation between hydrogen peroxide hemolysis and the rate of Hb decline. A decline in serum phosphorus levels was noted in all 3 groups, but was significantly greater (p<.01) in the Isomil group. Under the conditions of our study, iron at the concentration used in Similac with Iron (12 mg/L) did not appear to accelerate hemolysis. Individual variability in the hematological responses was noted irrespective of the formula used.