P. Biver

Evidence for an endogenous ouabain-like immunoreactive factor in human newborn plasma coeluted with ouabain on HPLC.

The identification in human plasma of ouabain as an endogenous digitalis-like factor (EDLF) claimed by Hamlyn et al. has recently been contradicted by two studies which failed to demonstrate endogenous ouabain-like immunoreactivity in HPLC fractions in which exogenous ouabain was eluted. In this paper we report the results obtained on the cross-reactivity with antiouabain antibodies of an EDLF purified by us from human newborn cord plasma. We found that this EDLF coeluted with ouabain on HPLC and cross-reacted both with rabbit anti-ouabain antiserum and with the purified antibodies, which excluded possible interferences due to antibodies directed against non-ouabain portions of the immunogen. Similar but not identical slopes of the ouabain and EDLF displacements curves were observed in all competition ELISA experiments. The inhibitory effect of EDLF on erythrocyte 86Rb uptake was reversed by antiouabain antiserum and antibodies. The concentration of EDLF in newborn plasma, in the four different purifications studied ranged from 30 to 380 pM ouabain equivalents (o.e.) by ELISA and from 100 to 300 pM o.e. by 86Rb uptake. Our data thus support the existence, in human newborn plasma, of a factor with both biological and immunological ouabain-like properties, although not necessarily identical to ouabain.

Endogenous Digitalis-Like Factor from Umbilical Cord and Ouabain: Comparison of Biochemical Properties

Digitalis substances (cardenolides and bufodienolides) are potent and specific inhibitors of the cell membrane Na,K-ATPase. Cardenolides are synthetized in a number of plants and bufodienolides are endogenous substances in certain species of amphibians (1,2). The demonstration of cardiotonic steroids in lower vertebrates and of specific receptors for digitalis on the surface of most mammalian cells have suggested the existence of endogenous digitalis-like factors (EDLF) also in mammalians (2,3). Several substances with endogenous digitalis-like activity have been purified from mammalian tissues and body fluids (2,3). Recently, Hamlyn et al. have isolated from human plasma and identified by mass spectroscopy a substance that is indistinguishable from ouabain, proposing that ouabain is an endogenous digitalis-like factor (4). However, this conclusion is still object of controversies (5).

CORRELATIONS BETWEEN DIGOXIN-LIKE IMMUNOREACTIVITY AND ELECTROLYTE VALUES IN URINARY SAMPLES OF NEWBORNS

Elevated levels of a group of substances with digoxin-like immunoreactivity (DLIS) are present in biological fluids of newborns. These endogenous substances could also have the biological properties of cardiac glycosides, such as natriuretic and vasoconstrictor activity. Therefore, DLIS could be involved in electrolyte and volume regulation in infants. We measured DLIS, electrolyte (Na+, K+, Cl-) and creatinine concentrations in 157 urine samples collected from full-term and preterm infants, with the aim to establish if a correlation exists between the urinary DLIS levels and the respective values of these electrolytes. Significant and positive correlations were found between the urinary concentrations of DLIS and electrolytes. In addition, electrolyte values correlated significantly between themselves. DLIS, K+ and Cl- values correlated positively with urinary creatinine concentrations (n = 142). A significant multiple correlation was found among DLIS concentrations and creatinine and Cl- values. We also studied the DLIS concentrations in serum samples collected at birth in peripheral blood of the mothers and in umbilical cord and gastric aspirate of 5 full-term infants. We observed that DLIS values in newborns are significantly higher than the respective values of the mothers. In conclusion, our data do not demonstrate a clear relationship between DLIS and electrolyte concentrations in urine samples of newborns. Further investigations are necessary to elucidate the biochemical and physiological properties of DLIS.

Chromatographic characterization of endogenous digoxin-like immunoreactive substances present in serum and urine of infants using gel filtration with Sephadex G 25

We assayed digoxin-like immunoreactivity in the serum of 19 healthy neonates (blood sample from umbilical cord), not receiving digoxin therapy, using a solid-phase radioimmunological method. In addition, urinary concentrations of a digoxin-like immunoreactive substance (DLIS) were assayed throughout the first 10 weeks of extrauterine life in 50 healthy infants. The mean (+/- SD) serum DLIS concentration obtained was 211 +/- 60 ng/l digoxin equivalents (d.e.) while, in the urinary samples of the first day of life, the mean concentration was 818 +/- 560 ng/l d.e. (n = 20). These DLIS values in infants were higher (about 5- to 10-fold) than those previously observed in adults. In addition, urinary DLIS values were very high in the first week of life and fell quite rapidly in the next weeks of life. We used serum and urine pools from neonates for gel filtration experiments using a Sephadex G 25 column. In the urine sample we found some immunoreactive peaks eluted at the end and after the region of salts, while the chromatographic profiles of the serum pool showed only a major peak eluted with the peak of proteins. After boiling, the same serum pool showed an elution pattern characterized by a smaller immunoreactive peak eluted with that of proteins and by other peaks similar to those observed for the urine sample. Our study demonstrated that a substance or more substances which cross-react with digoxin-specific antibodies are present in blood and urine of healthy infants. In addition, our chromatographic studies indicate that DLIS is probably a low molecular weight substance(s) which circulate(s) in blood bound to a higher molecular weight molecule.

Recovery of aEEG Patterns at 24 Hours of Hypothermia Predicts Good Neurodevelopmental Outcome

Background: The clinical use of amplitude integrated electroencephalogram (aEEG) in the neonatal intensive care unit has largely increased. This method has been reported to have a very good predictive value for neurodevelopmental outcome in term neonates after perinatal asphyxia. Aim: The aim of this study was to assess the recovery of aEEG patterns during hypothermic treatment in full term asphyxiated neonates. Our working hypothesis is that children with aEEG recovery within 24 h of therapeutic hypothermia will have a normal development outcome (i.e., no or mild neurological impairment). Study design: We performed an observational prospective study on a group of asphyxiated patients admitted to our Neonatal Intensive Care Unit from April 2009 to April 2012. Results: 24 patients with moderate to severe perinatal asphyxia had an aEEG recorded for at least 72 h during hypotermia (at the beginning of the registration 13 patients presented moderate aEEG abnormalities and 11 severe aEEG abnormalities). Respectively 11 neonates with moderate aEEG abnormalities and 1 neonate with severe abnormalities normalized the aEEG pattern during the treatment. At the follow up 3 patients died during neonatal age, 5 babies developed cerebral palsy, 4 babies developed dyskinetic cerebral palsy and 12 babies did not develop any disability (babies with good outcome were those with normal aEEG pattern at 24 h). Conclusion: Recovery to a normal aEEG background pattern within the first 24 h of hypothermia after perinatal asphyxia predicts a normal outcome. Abnormal aEEG pattern persisting after 24 h correlates with poor outcome (death or cerebral palsy).