Olli Pitkänen

Generation of Free Radicals in Lipid Emulsion Used in Parenteral Nutrition

ABSTRACT: Lipid emulsions used in parenteral nutrition are prone to peroxidation that may be an important feature of oxygen-associated tissue damage. Incubation of lipid emulsion [Intralipid (IL)] with H2O2 and FeCl2 increased lipid peroxidation, measurable as increased production of pentane, from 0.39 ± 0.33 to 0.99 ± 0.18 μM (p < 0.0001). Malondialdehyde was increased from 0.010 ± 0.005 mM to 0.380 ± 0.025 mM (p < 0.001). Superoxide dismutase and catalase (each 100 U/mL) or vitamin C (10 mM) inhibited pentane and malondialdehyde production (p < 0.0001). Incubation of human erythrocytes in the presence of FeCl2 caused 11.0 ± 3.2% hemolysis (control 0.95 ± 0.14%). Addition of 0.44% IL increased hemolysis to 66.5 ± 3.4%, whereas further addition of vitamin E or C significantly inhibited hemolysis to 16.4 ± 8.1 and 38.9 ± 7.1%, respectively (p < 0.0001). IL was administered i.v. to eight preterm infants. It increased 3− to 28-fold (p < 0.001) the amount of pentane in expired breath. Partly, this increase was due to pentane dissolved in IL as a result of lipid peroxidation during storage. After discontinuing IL infusion, the elimination of pentane was nonexponential, consisting of a rapid and a slow component. According to our results, IL undergoes peroxidation causing free-radical-dependent damage to human cells. We propose that the adverse effects of parenteral IL are partially caused by free oxygen radicals generated by lipid peroxidation.

Correlation of free oxygen radical-induced lipid peroxidation with outcome in very low birth weight infants

Lipid peroxidation was measured in 19 very low birth weight infants with respiratory distress syndrome by quantitating ethane and pentane in expired air during the first 5 days postnatally. Despite high levels of inspiratory oxygen, the ethane and pentane output was low within the first 24 hours; thereafter it increased up to 100 and 30 fold, respectively. On days 1 to 3 there was no detectable correlation between lipid peroxidation and fractional inspiratory oxygen. However, on days 4 and 5, lipid peroxidation and fractional inspiratory oxygen showed a significant correlation. Maximal amounts of expired ethane and pentane were significantly higher for patients with a poor outcome (five deaths, six cases of bronchopulmonary dysplasia) than for those with good outcome (eight infants surviving intact) (p less than 0.01). The results imply a role for free oxygen radicals in the pathogenesis of life-threatening complications in the very low birth weight infant.

Immaturity-dependent free radical activity in premature infants.

ABSTRACT: To examine the role of immaturity in the free radical-mediated rate of lipid peroxidation in premature infants, we studied 27 infants [gestational age, 27.1 (SD 2.4) wk; birth weight, 970 (SD 330) g]. Ethane and pentane were quanti-tated in expired air during the first 18 d of life. During the first 2 postnatal d ethane [24.1 (SEM 7.8) pmol × kg−1 × min−1] and pentane [24.2 (SEM 4.1) pmol × kg−1 × min−1] were stable but increased during d 5 to maxima of 79.1 (15.8) pmol × kg−1 × min−1 and 62.1 (8.1) pmol × kg−1 × min−1, respectively. Maximum ethane and pentane correlated with gestational age (r = −0.42, p = 0.03 and r = −0.52, p = 0.005, respectively) and birth weight (r = −0.38, p = 0.05 and r = −0.59, p = 0.001, respectively). Infants with high maximum expired ethane and pentane (exceeding 40 pmol × kg−1 × min−1) had higher odds of dying or having bronchopulmonary dysplasia than those with low ethane and pentane (odds ratio, 6.5; 95% confidence interval, 1.1 to 38.5; p < 0.05 for ethane and odds ratio, 5.6; 95% confidence interval, 1.1 to 29.3; p < 0.05 for pentane). We conclude that degree of prematurity is the single most important factor explaining free radical-mediated lipid peroxidation in premature infants. A therapeutic intervention to limit the effects of free radicals should be started during the 1st postnatal d in premature infants to be effective.

Pulmonary Fluid Balance in the Human Newborn Infant

At birth, the infant’s lungs must be cleared of fetal lung fluid. This process is mediated through the activation of airway epithelial sodium channels (ENaC). In animals, ENaC is considered crucial for postnatal pulmonary adaptation. In humans, postnatal ENaC expression is gestational age dependent and its activity, measured as nasal potential difference, correlates with lung compliance. It is therefore likely that in the human newborn infant ENaC is also important for physiologic postnatal adaptation. Low pulmonary expression or activity of ENaC in the perinatal period may cause delayed clearance of lung fluid and thereby contribute to development of respiratory distress in both term and preterm infants.

Determination of ethane and pentane in free oxygen radical-induced lipid peroxidation.

It has been proposed that ethane and pentane reflect free oxygen radical-induced lipid peroxidation. However, methodological difficulties limit the use of these gases for assessment of free oxygen radical activity. In the present report we describe an improved method for the accurate analysis of picomole quantities (≥1 pmol) of ethane and pentane. They are first quantitatively trapped into an adsorbent and then heat-desorbed directly into a capillary column for gas chromatographic quantitation. During oxidation of linolenic (n−3) and linoleic (n−6) acid, ethane and pentane were formed, respectively. Nonstimulated granulocytes formed pentane. Upon addition of phorbol 13-myristate 12-acetate, the generation of pentane was increased by 540%. Addition of superoxide dismutase plus catalase inhibited lipid peroxidation in both a cell-free system and in isolated cells.The present method is useful in the evaluation of free oxygen radical induced damage.

Free radical activity during development of insulin-dependent diabetes mellitus in the rat.

Free radical-induced lipid peroxidation was quantified by measuring expired pentane from diabetic prone BB Wistar rats of 45-90 d of age. Insulin-dependent diabetes mellitus was manifest at the age of 71 +/- 8 d. Expired pentane increased from 2.1 +/- 0.7 to 5.0 +/- 3.0 pmol/100g/min (p less than 0.01) at manifestation of the disease and remained high throughout the test period. In healthy age-matched control rats it persisted low. In rats made diabetic with streptozotocin, expired pentane remained low. The changes in expired pentane suggest that the development of endogenous insulin-dependent diabetes mellitus in BB rats is associated with increased free radical activity. This is not due to hyperglycemia or ketosis per se, and reflects a fundamental difference in the free radical activity between the spontaneously diabetic BB rats and the disease produced by streptozotocin. Development of spontaneous insulin-dependent diabetes in BB rats is associated with increased free radical activity that persists after the manifestation of the disease.

G protein‐coupled receptor for asthma susceptibility associates with respiratory distress syndrome

Background. Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) have some common features with asthma. Aim. To study whether G protein‐coupled receptor for asthma susceptibility (GPRA) contributes to RDS or BPD. Methods. A haplotype association study was performed in a case‐control setting of 521 Finnish infants (including 176 preterm neonates with RDS and 37 with BPD). Immunoreactivity of GPRA isoforms A and B was determined in pulmonary samples of fetuses, term infants and preterm infants with RDS or BPD. GPRA mRNA expression was determined by quantitative real‐time polymerase chain reaction (PCR) in samples from nasal respiratory epithelium of adults, term infants and preterm infants. Results. In infants with RDS born at 32–35 weeks of gestation, GPRA haplotype H1 was significantly underrepresented in RDS, whereas haplotype H4/H5 was associated with an increased risk. As in asthma, GPRA B isoform was induced in bronchial smooth muscle cells in RDS and BPD. In nasal respiratory epithelium, relative GPRA mRNA expression was strong in adults, weak in preterm and slightly higher in term samples. Conclusions. The results suggest that near‐term RDS and asthma share the same susceptibility and protective GPRA haplotypes. Altered GPRA expression may play a role in the pathogenesis of RDS and BPD in preterm infants.

Expression of the Epithelial Sodium Channel in Airway Epithelium of Newborn Infants Depends on Gestational Age

OBJECTIVE. In the newborn infant, removal of fetal lung liquid from the airways depends on ion transport through the airway epithelium. The epithelial sodium channel is considered rate limiting for the postnatal clearance of lung liquid, but it is unknown whether during the early postnatal period the expression of epithelial sodium channel is associated with maturity. Our objective was to study the relationship between gestational age and epithelial sodium channel expression in airway epithelium. METHODS. In 90 newborn infants (preterm [gestational age < 37]: n = 29; term [gestational age ≥ 37]: n = 61), we measured the expression of epithelial sodium channel (reported as attomoles of subunit expression normalized to femtomoles of expression of cytokeratin 18) in nasal epithelium at 1 to 5 and 22 to 28 hours after birth. RESULTS. At 1 to 5 hours postnatally, airway expression of α-, β-, and γ-subunits of epithelial sodium channel was lower in preterm than in term infants. At this time point, significant correlations existed between gestational age and airway expression of α- and β-epithelial sodium channel. By 22 to 28 hours after birth, only the expression of β-epithelial sodium channel had decreased significantly in the preterm infants, whereas the expression of all epithelial sodium channel subunits had decreased significantly in the term infants. At this time point, no difference in expression of any of the subunits was found between preterm and term infants. CONCLUSIONS. Airway expression of epithelial sodium channel at 1 to 5 hours of age is significantly lower in preterm than in term infants. Low postnatal expression of α-, β-, and γ-epithelial sodium channel subunits in the airway epithelium may contribute to the development of respiratory distress in the preterm infant.

Attenuated lipid peroxidation in preterm infants during subsequent doses of intravenous lipids.

The aim of this study was to determine whether the administration of a lipid emulsion containing less polyunsaturated fatty acids but rich in monounsaturated fatty acids causes less in vivo lipid peroxidation in preterm infants. The prospective intervention study included 13 infants with birth weights and gestational ages ranging between 1,100 and 2,660 g and from 28.4 to 32.9 weeks. All were in a stable condition and randomly allocated for a 3-hour infusion (0.16 g/kg/h) of an olive oil-based and a soybean oil + medium chain fatty acid (MCT) emulsion on 2 consecutive days. Expired pentane and plasma triglycerides (TGs) were measured before, during, and after the 3-hour infusion. Basal exhaled pentane averaged 9.4 ± 7.0 pmol/kg/min (mean ± SD). During the olive oil-based emulsion, exhaled pentane increased to 95.2 ± 56.7, and during soybean oil + MCT it increased to 110 ± 93.9 pmol/kg/min (p < 0.05 both from basal, n.s. between preparations). One hour after discontinuation of the infusion, exhaled pentane returned to 21.1 ± 12.6 pmol/kg/min (p < 0.05 vs. basal). Combined data on expired pentane measurements demonstrated that on day 1 pentane peaked at 124 ± 87.0 pmol/kg/min which was significantly attenuated to 57.5 ± 24.4 pmol/kg/min after an identical dose of lipid on day 2 (p < 0.05). No difference in peak TGs was detected between the two preparations or the study days. Infusion of a constant dose of intravenous lipids on 2 subsequent days to the newborn infants is associated with a reduction in lipid peroxidation. This finding may be dependent on normal postnatal maturation or may represent an appropriate adaptive response aiming at a reduction in oxidative stress. Peroxidation of soybean oil + MCT and olive oil-based lipid emulsions was similar in the newborn infants.

Significance of ion transport during lung development and in respiratory disease of the newborn

Active ion transport plays a critical role in the liquid movement across the fetal and perinatal lung epithelium. The fetal lung liquid production is coupled with active secretion of Cl- into the luminal space. The potential for fluid absorbing mechanisms related to active Na+ transport from the apical to the basolateral side of the epithelium appears near the end of gestation. At birth there is a dramatic change of environment with commencement of air-breathing, sudden increase in oxygen partial pressure (PO2) and profound changes in the pulmonary circulation. A concurrent switch from fluid secretion to maintenance of low amounts of alveolar fluid is another major physiological adjustment taking place in the perinatal distal lung epithelium. The fluid-absorbing mechanism is a result of a well-synchronized co-operation between the basolateral membrane Na-K-ATPase and the apical membrane Na+ channels and it promotes salt and water movement from the airspace. Inability of the fetal lung epithelium to switch from fluid secretion to Na+ transport-dependent absorption seems to be an important factor adversely contributing to the respiratory distress of the newborn premature infant.