Karl B. Sluis

Protein Carbonyl Measurement by a Sensitive ELISA Method

We describe a new immunoassay for measuring protein carbonyls as an index of oxidative injury. Protein samples were reacted with dinitrophenylhydrazine then adsorbed to wells of an ELISA plate before probing with a commercial antibody raised against protein-conjugated dinitrophenylhydrazine. The biotin-conjugated primary antibody was then reacted with streptavidin-biotinylated horseradish peroxidase for quantification. The method was calibrated using oxidized albumin and results correlated well with the colorimetric carbonyl assay. The method required only 60 microg protein and was used to analyze the amount of protein carbonyls in plasma and lung aspirate samples. It was sensitive in the 0-2.5 nmol/mg protein range within which clinical samples fell and was linear up to 10 nmol/mg protein. The ELISA method for protein carbonyls is more sensitive and discriminatory than the colorimetric assay and should have wide application for analysing experimental and clinical samples, especially where concentrations are low and where only small amounts of sample are available.

Limiting light-induced lipid peroxidation and vitamin loss in infant parenteral nutrition by adding multivitamin preparations to Intralipid

Parenteral lipids are susceptible to light‐induced peroxidation, particularly under phototherapy. Ascorbic acid is protective. The aim of this study was to investigate whether dark delivery tubing and/or coadministration of multivitamin preparations could prevent peroxidation of Intralipid without undue vitamin loss. In experiments carried out on the benchtop, lipid peroxidation occurred in ambient light and was more extensive under phototherapy. Dark tubing decreased peroxide formation, but only by about 65%. In simulated clinical conditions in which solutions were pumped through standard clear or dark minibore plastic tubing, Intralipid accumulated lipid peroxides as measured by the FOX assay (280 μM) or as triglyceride hydroperoxides (52 μM). Multivitamin preparations (MVIP or Soluvit/Vitlipid) inhibited peroxide formation almost completely, and were fully protective when used with dark tubing. There was loss of riboflavin (65% from Soluvit and 35% from MVIP) in clear tubing but this was decreased to 18% and 11%, respectively, in dark tubing. Ascorbate loss was 20% (MVIP) and 50% (Soluvit) and only slightly less in dark tubing. Ascorbate loss was also seen in the absence of Intralipid and is due to riboflavin‐induced photo‐oxidation.

The correlation of elevated levels of an index of lipid peroxidation (MDA‐TBA) with adverse outcome in the very low birthweight infant

The objective of this study was to examine the relationship between malondialdehyde‐thiobarbituric acid (MDA‐TBA) levels, as a measure of lipid peroxidation, in very low birthweight (VLBW) infants and outcome measures. A prospective observational longitudinal study was carried out in two level III neonatal units in the South Island of New Zealand measuring MDA‐TBA levels in 61 VLBW infants in 1993. MDA‐TBA levels were measured in (i) maternal plasma within 48 h of parturition, (ii) cord plasma, and (iii) infants’plasma at 2, 7, 14 and 28 days of age, and correlated with antenatal and postnatal factors. Elevated levels of plasma MDA‐TBA at 7 days were associated with adverse respiratory and ophthalmological outcome in the VLBW infants. Elevated MDA‐TBA levels were measured at sample times close to the time of death in the infants who died. These results substantiate previously reported preliminary observations and support the hypothesis that oxidative injury, particularly within the first 7 days of life, is associated with the development of the long‐term complications of the pre‐term infant. MDA‐TBA levels appear to be a useful measure to continue to explore the role of free radical mediated disease in the VLBW infant.

Selenium and Glutathione Peroxidase Levels in Premature Infants in a Low Selenium Community (Christchurch, New Zealand)

ABSTRACT: By world standards, the selenium status of the adult population of Christchurch, New Zealand is low. To determine the status of infants undergoing neonatal intensive care, plasma and red cell selenium and glutathione peroxidase levels were measured in infants admitted to the regional neonatal unit. Plasma levels in all newborn infants were one third to one half those in adults. Premature infants had levels significantly lower than those in cord blood from term infants, but their levels were not different from those of term infants admitted to the unit. There were no differences between adult and infant red cell levels. The premature infants remaining in the neonatal unit showed dramatic decreases in plasma selenium and glutathione peroxidase with age, with many infants having selenium levels of <0.13 μmol/L (10 μg/L). Low levels were seen in infants fed orally as well as those on parenteral nutrition. Thus, the low selenium status of New Zealanders is associated with particularly low selenium levels in premature infants. Because these infants have a high risk for oxidative diseases such as bronchopulmonary dysplasia (chronic lung disease) and retinopathy of prematurity, the possibility that these conditions are more serious in the New Zealand population needs to be assessed and consideration given to dietary supplementation.

Proteinase-antiproteinase balance in tracheal aspirates from neonates

We wanted to identify the inhibitors of neutrophil elastase, quantify their activities in the upper airways of neonates, and relate these to the presence of active elastase and the likelihood of elastolytic injury occurring due to inhibitory capacity being overwhelmed. Activities of neutrophil elastase and its inhibitors were measured in tracheal aspirates from 17 infants, 10 of whom subsequently developed bronchopulmonary dysplasia. All aspirates contained immunologically detectable alpha 1-proteinase inhibitor (alpha 1-PI), but their inhibitory capacity against neutrophil elastase ranged from being undetectable to being in excess of the amount of alpha 1-PI detected immunologically. When the alpha 1-PI was removed from each of the aspirates, using a specific antibody, from 0-50% of the original activity remained, indicating the presence of another elastase inhibitor. Its properties were consistent with it being the low molecular mass, secretory leucoproteinase inhibitor (SLPI), also known as bronchial antileucoproteinase. The alpha 1-PI was from 0-100% active. Most of the inactive inhibitor was shown by western blotting to be complexed with elastase, with a small amount of cleaved material. There was no evidence of major oxidative inactivation. Free elastase was detected in only three of the aspirates; these had little or no detectable elastase inhibitory capacity, and most of their alpha 1-PI was complexed. Elastase load, comprising the sum of free and complexed elastase, correlated closely with myeloperoxidase activity, a recognized marker of inflammatory activity. Active SLPI levels showed a positive correlation with gestational age (r = 0.66). We conclude that most neutrophil elastase in the upper airways of ventilated infants is complexed.(ABSTRACT TRUNCATED AT 250 WORDS)

Endotracheal suctioning of the neonate: Comparison of two methods as a source of mucus material for research

Endotracheal suctioning in the neonatal intensive care setting is a routine procedure performed to maintain patency of the airway in ventilated infants. Harvested material can also be a source of mucus for research into neonatal respiratory disorders. We aimed to investigate whether the composition of material obtained by our clinically preferred technique of dry shallow suctioning differed significantly from that obtained with saline lavage and deep suctioning. Eleven pairs of dry and saline lavage aspiration samples were compared for neutrophil enzyme myeloperoxidase, total and active α1‐antitrypsin, α1‐antitrypsin complexed with elastase, and secretory leukoprotease inhibitor. Even though individual values of each analyte, expressed per gram of albumin, varied over at least a fivefold range, there was no difference between mean values of dry and lavage samples for any of the constituents. We conclude that the yield of material for research obtained by dry shallow suctioning is adequate and the quality at least as satisfactory as that provided by saline lavage. Pediatr. Pulmonol. 1997; 23:217–221

Selenium status of New Zealand infants fed either a selenium supplemented or a standard formula

Objective: New Zealand soils are deficient in the essential micronutrient, selenium. New Zealand infants have low selenium levels at birth and experience a further decline if fed cows milk based formula. This study examined the selenium status of infants fed with a new commercially available selenium supplemented formula.