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Diabetes is known to be associated with delayed lung development in humans and in experimental animals. This includes delayed expression of surfactant apoproteins. An important component of the metabolic abnormalities in diabetes is elevated levels of analogs of butyric acid, and the effects of diabetes on surfactant apoproteins can be reproduced by exposure of fetal rat lung explants to butyrate. Dexamethasone has the opposite effects on lung development. In humans, antenatal exposure to dexamethasone results in a lower incidence of RDS, whereas in experimental animals, dexamethasone increases the expression of surfactant apoproteins. A subset of Hox genes are expressed in developing lung, and their level of expression decreases with advancing gestation. We hypothesized that: 1) lungs of fetuses of rats with streptozotocin-induced diabetes would have altered levels of expression of Hox genes, 2) the effect would be mediated in part through elevated levels of butyrate, and 3) dexamethasone would reverse the effect. We tested our hypotheses in vivo using fetuses from streptozotocin-treated rats and in vitro by treating lung explants from normal rats with sodium butyrate. Streptozotocin treatment increased expression of Hoxb-5 at 18 d of gestation, but did not affect Hoxa-5 expression. This was associated with a 20-fold increase in alpha-aminobutyrate levels. Dexamethasone tended to reverse this effect. In contrast, butyrate treatment of explants decreased the expression of Hoxa-5 and Hoxb-5. We conclude that diabetes alters expression of Hox genes, but that the effect of butyrate on lung development, and in particular on surfactant apoprotein expression, is independent of its effects on Hox genes.

Cysteine availability is rate limiting for the synthesis of glutathione, an important antioxidant in the lung. We used rat alveolar epithelial type II cells to study the mechanism of cysteine and cystine uptake. Consistent with carrier-mediated transport, each uptake process was saturable with Michaelis-Menten kinetics and was inhibited at 4°C and by micromolar levels of amino acids or analogs known to be substrates for a specific transporter. A unique system XAGwas found that transports cysteine and cystine (as well as glutamate and aspartate, the only substrates previously described for system XAG). We also identified a second Na+-dependent cysteine transporter system, system ASC, and two Na+-independent transporter systems, system xcfor cystine and system L for cysteine. In the presence of glutathione at levels measured in rat plasma and alveolar lining fluid, cystine was reduced to cysteine and was transported on systems ASC and XAG, doubling the transport rate. Cysteinylglycine, released from glutathione at the cell surface by γ-glutamyl transpeptidase, also stimulated uptake after reduction of cystine. These findings suggest that, under physiological conditions, cysteine and cystine transport is influenced by the extracellular redox state.

Glucose transporter (GLUT) modulation can be an important mechanism that contributes to adaptation to hypoxic stress, but little is known about GLUT modulation in heart and skeletal muscle with prolonged hypoxia. In this work, the effect of chronic hypoxia on GLUT-4 and GLUT-1 mRNA and protein was studied in these two tissues in the adult and during development. Hypoxia (fractional inspired O2= 9 ± 0.5%) was administered to two groups, i.e., an immature group exposed from 3 to 30 days of age and an adult group exposed from 90 to 120 days of age. Rats were then killed and their heart and skeletal muscles were sampled for measurements of GLUT mRNA and protein with Northern and Western blots. In the adult, chronic hypoxia significantly decreased cardiac GLUT mRNA level by >25% of control ( P < 0.05), but had little effect on GLUT protein. A very different hypoxic effect was seen in the immature rat heart with a major increase in protein and no appreciable change in mRNA density. Adult skeletal muscle had no change in GLUT mRNA level but GLUT protein increased (15–20%, P < 0.05) while both GLUT mRNA and protein were significantly increased in the immature skeletal muscles (60–90% over control). We conclude that during chronic O2deprivation, GLUT-1 and GLUT-4 expressions show a similar pattern but greatly depend on tissue type and age. These differences in GLUT regulation may be due to different strategies for coping with prolonged O2deprivation in both immature and adult animals.

Tissues from fetuses and neonates of control and streptozotocin (STZ)-treated Sprague-Dawley rats were used to study the content and distribution of the hydrophobic surfactant protein B (SP-B) and the mRNAs for SP-B and SP-C using immunohistochemistry, RNA blotting, and tissue in situ hybridization. A dose of 50 mg/kg STZ was used to treat female rats before mating. The fetuses were sacrificed at fetal days 18 through 21 and neonates were obtained on neonatal days 1 and 2 (day of birth = end of day 22). At fetal day 18, SP-B was barely detectable by immunohistochemistry in control animals but the levels were progressively increased through gestation and easily detected by fetal day 21. At all fetal ages, SP-B was decreased in the STZ group compared with control animals. Both SP-B and SP-C mRNA were detectable at fetal day 18 in the control group and increased with advancing gestational age. In fetal lungs from the STZ group, SP-B and SP-C mRNA also showed an increase with advancing gestational age, but the levels were decreased compared with controls at fetal days 18, 20, and 21 (P less than 0.05). At fetal day 19, this difference did not achieve statistical significance. Differences between the two groups were no longer detected by neonatal days 1 and 2. The difference between the STZ and control groups, in both protein (SP-B) and mRNA (SP-B and SP-C), diminished with advancing fetal age but remained significant up to fetal day 21.(ABSTRACT TRUNCATED AT 250 WORDS)

The content and distribution of the 26-to 38-kDa surfactant protein (SP-A) and its mRNA were determined in fetuses of control and streptozotocin (STZ)-treated Sprague-Dawley rats using immunohistochemistry, RNA blotting, and in situ hybridization. Female rats were treated with 50 mg/kg STZ before mating, and the fetuses were killed at fetal days 18-21 or on neonatal days 1 and 2 (day of birth = end of day 22). SP-A was barely detectable on fetal day 18 in controls and easily detected by fetal day 21. In the STZ group, SP-A was decreased compared with controls at fetal days 18-21. However, by neonatal days 1–2, there were no significant differences in SP-A levels between groups. SP-A mRNA was detectable at fetal day 18 in controls, but it was decreased in the STZ group at day 18-21 (P less than 0.02) and differences were no longer detected by neonatal days 1–2. SP-A and SP-A mRNA accumulated with advancing gestational age in both groups until neonatal days 1–2. The differences in SP-A and SP-A mRNA levels in the two groups diminished with advancing age but remained significant at fetal day 21. These data suggest that STZ-induced diabetes interferes with normal expression of SP-A in the developing fetal lung.

Fetuses of streptozotocin-induced diabetic rats exhibited delayed lung maturation and a 40% reduction in the steady-state level of lung Na+,K(+)-ATPase alpha 1 subunit mRNA and Na+,K(+)-ATPase activity at 21 d of gestation. In in situ hybridization experiments the signal specific for Na(+)-pump alpha 1 subunit message was strongest above columnar epithelial cells of air-conducting structures. Strong labeling was also present above cuboidal cells lining the forming alveoli, but not above mesenchymal cells. Immunocytochemical localization of the protein paralleled the distribution of the mRNA. Mesenchymal cells were more abundant in fetal lungs of diabetic mothers, and thus the decreased overall levels of Na+,K(+)-ATPase may result from the observed morphological pulmonary immaturity. One day after birth there was no apparent difference in lung morphology at the light microscopic level, in the localization or the steady-state level of Na+,K(+)-ATPase alpha 1 isoform mRNA, or in enzyme activity. Na+,K(+)-ATPase has a likely role in the active phase of fluid absorption in the airways of newborns before the onset of breathing. Decreased fluid clearance and lack of thinning of the lung's connective tissue may contribute to the increased risk for respiratory distress in infants of diabetic mothers.

Newborn rats were injected with vitamin E or placebo daily until 6 days after birth. The effect of vitamin E pretreatment on in vitro surfactant phospholipid synthesis was examined in isolated type II cells exposed to oxygen or air form 24 h in vitro. Type II cells were also isolated from untreated 6-day-old rats and cultured for 24 h in oxygen or air with control medium or vitamin E supplemented medium. These cells were used to examine the effect of vitamin E exposure in vitro on type II cell phospholipid synthesis and ultrastructure. Phosphatidylcholine (PC) synthesis was reduced in cells cultured in oxygen as compared with air. This decrease was not prevented by in vivo pretreatment or in vitro supplementation with vitamin E. Vitamin E pretreatment increased the ratio of disaturated PC to total PC and increased phosphatidylglycerol synthesis. The volume density of lamellar bodies in type II cells was increased in cells maintained in oxygen. Vitamin E did not affect the volume density of lamellar bodies. We conclude that in vitro hyperoxia inhibits alveolar type II cell phosphatidylcholine synthesis without decreasing lamellar body volume density and that supplemental vitamin E does not prevent hyperoxia-induced decrease in phosphatidylcholine synthesis.

By participating in glutathione (GSH) synthesis, gamma-glutamyl transpeptidase (GGT) influences the GSH redox cycle, which is a major contributor in protecting against reactive oxygen metabolites. This study determined the effect of prolonged exposure of neonatal rats to > 98% oxygen on expression of GGT and on GSH metabolism. Lungs of neonatal rats chronically exposed to hyperoxia had increased expression of GGT mRNA, resulting in significantly higher GGT protein levels and enzyme activity than in lungs of animals raised in room air. Hyperoxia also upregulated glucose-6-phosphate dehydrogenase, but Na-K-ATPase activity was not changed. GGT mRNA, protein level, and enzyme activity returned to control levels after recovery in room air for 3 days. Levels of GSH, glutathione disulfide, and protein-bound GSH (S-glutathiolated protein) rose with hyperoxia and fell during recovery. S-glutathiolation is likely a mechanism for protection and a regulatory modification of protein sulfhydryl groups. Hyperoxia-induced upregulation of GGT and the concomitant increase in protein S-glutathiolation appear to be additional components fundamental in protecting the lung against oxidative injury.

Because surfactant protein (SP) mRNA levels in rat fetuses are increased by maternal dexamethasone (dex) treatment and decreased in streptozotocin-induced diabetic (STZ-DB) pregnancy, we investigated the in vivo effects of dex on SP gene expression in STZ-DB pregnancy. The mRNA levels of SP (SP-A, SP-B, SP-C) were assessed in d 18 and 20 fetuses by Northern blot analysis, and nuclear run-on assays were performed with lung nuclei from d 20 fetuses (term = 22 d). Our findings indicate: 1) dex causes a greater increase in SP-A and SP-B mRNA levels in d 18 (12-16-fold) compared with day 20 (4-6-fold) fetuses (p < 0.05) in normal and STZ-DB pregnancy; 2) a 2-3-fold increase in SP-C mRNA levels was observed in response to dex in d 18 and 20 fetuses; 3) the increase in transcription of SP-A and SP-B in d 20 fetuses after dex is 68 and 60%, respectively, of the increase in their mRNA levels whereas in STZ-DB, the decrease in transcription compared with mRNA levels is 3.67-fold for SP-A and 2.42 fold SP-B; and 4) changes in SP-C transcription in either in vivo model, dex-treated or STZ-DB, correspond well with changes in mRNA levels. Together, these findings indicate that dex can enhance SP expression in STZ-DB immature lungs and support differential regulation of fetal SP genes in the models studied. (Pediatr Res 38: 870-877, 1995)

We have shown previously that chronic hypoxia can regulate the expression of membrane proteins. Since there are virtually no glucose stores in the brain and glucose transport can be rate-limiting during stress, the role of glucose transporters becomes crucial for cell survival under stress. In the present study, we asked whether mRNA levels for glucose transporter 1 (GT1), which is expressed in a variety of cells in the brain, especially in the microvessels for glucose transport from blood vessels to brain, change in response to chronic hypoxia. Because major developmental changes occur in the rat CNS in-utero and in the first few weeks postnatally, we studied brain GT1 mRNA using Northern blot analysis at different ages after exposure of fetuses (from embryonic day 10 to birth), developing rats (from birth to 30 day old) or adult rats (from 90 to 120 day old) to hypoxia (Fractional inspired O2 9%). Our data show that (i) GT1 mRNA level was much lower in the newborn than in the adult and increased with age; (ii) chronic hypoxia caused a decrease of approximately 65% in GT1 mRNA in adult brain but induced an increase in fetal (more than 50%) and developing (approximately 80%) rats and (iii) the response of housekeeping gene (glyceraldehyde 3-phosphate dehydrogenase) was not similar to that of GT1, suggesting that the changes of GT1 mRNA are specific to glucose transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal stress is associated with the induction of a specific set of proteins called heat shock proteins and with the induction of thermal tolerance. Heat stress has been shown to be capable of inducing at least partial tolerance to other stresses, including some oxidant stresses. Furthermore, these oxidant stresses are reported to be inducers of heat shock proteins. We hypothesized that hyperoxic stress would induce heat shock proteins and that factors induced by thermal stress, including heat shock proteins, would offer at least partial protection from hyperoxic exposure. We were particularly interested in a level of protection that would be relevant to clinical situations. Lung fibroblasts and live animals were exposed to thermal stress and/or hyperoxic stress and examined for induction of HSP70 (the most conserved of the heat shock proteins) and for induced tolerance as determined by the ability of cells to metabolize 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and by comparison of lung wet to dry weight ratios in live animals. Each stress induced tolerance to itself, but there was no evidence of heat stress inducing tolerance to hyperoxic stress. Furthermore, there was only minimal induction of HSP70 mRNA by hyperoxic exposure. We conclude that some overlap of mechanisms of induced tolerance by hyperoxic and thermal stress exists, but that differences far outweigh similarities.

Acute lung injury is a frequent and treatment-limiting consequence of therapy with hyperoxic gas mixtures. To determine if IL-11 is protective in oxygen toxicity, we compared the effects of 100% O 2 on transgenic mice that overexpress IL-11 in the lung and transgene ( 2 ) controls. IL-11 markedly enhanced survival in 100% O 2 with 100% of transgene ( 2 ) animals dying within 72–96 h and . 90% of transgene ( 1 ) animals surviving for more than 10 d. This protection was associated with markedly diminished alveolar-capillary protein leak, endothelial and epithelial membrane injury, lipid peroxidation, and pulmonary neutrophil recruitment. Significant differences in copper zinc superoxide dismutase and catalase activities were not noted and the levels of total, reduced and oxidized glutathione were similar in transgene ( 1 ) and ( 2 ) animals. Glutathione reductase, glutathione peroxidase, and manganese superoxide dismutase activities were slightly higher in transgene ( 1 ) as versus ( 2 ) mice after 100% O 2 exposure, and IL-11 diminished hyperoxiainduced expression of IL-1 and TNF. Hyperoxia also caused cell death with DNA fragmentation in the lungs of transgene ( 2 ) animals and IL-11 markedly diminished this cell death response. These studies demonstrate that IL-11 markedly diminishes hyperoxic lung injury. They also demonstrate this protection is associated with small changes in lung antioxidants, diminished hyperoxia-induced IL-1 and TNF production, and markedly suppressed hyperoxia-induced DNA fragmentation. ( J. Clin. Invest. 1998. 101:1970–1982.)

Demonstration of Multiple Transporters for Cyst(e)ine Uptake in Type II Pneumocytes. ▴ 2311

Twelve premature infants with primary apnea were treated with theophylline as an alternative to mechanical ventilation. There was a significant (P< .005) reduction in the mean daily number and the severity of apneic episodes after treatment. The only significant side effect noted was a rise in heart rate.

We describe a simple and reliable method to determine optimal airway pressure in infants with idiopathic respiratory distress syndrome who require continuous positive airway pressure treatment. Esophageal pressure was monitored in ten infants with IRDS during initial application of CPAP. As the level of CPAP was increased in 2 cm H2O increments, changes in Pes were compared with changes in PaO2. Below optimal airway pressure, Pes as well as PaO2 increased insignificantly. When optimal airway pressure (8.1 +/- 0.8 cm H2O) was applied, there was a marked increase in Pes (3.6 +/- 0.8 cm H2O. p less than 0.001) and PaO2 (39.0 +/- 10.0 mm Hg, p less than 0.01). Further increase in CPAP did not result in any subsequent appreciable increase in Pes while PaO2 decreased slightly and PaCO2 increased. Less than optimal CPAP increases F102 requirements and may increase the associated risk of bronchopulmonary dysplasia, while excessive levels of CPAP may increase the risk of pneumothorax. We suggest that esophageal pressure be monitored routinely to determine the optimal level of CPAP for each infant during the initial application of therapy.

We examined the effect of hyperinsulinemia in the absence of hyperglycemia on parameters of lung surfactant production in the fetal rhesus monkey at 134–148 days gestation. Hyperinsulinemia was achieved by infusion of insulin for 21 d from an Alzet minipump implanted in the fetal hind leg. Blood insulin levels were elevated 80-fold in the treated group. These fetuses were also slightly hypoglycemic and their body weights were 25% greater than expected. There were no differences, however, between the insulin-treated and control groups in the phospholipid content and composition of lung lavage or lavaged lung tissue or in the rate of choline incorporation into disaturated phosphatidylcholine in lung slices. These data suggest that hyperinsulinemiaper se does not inhibit surfactant production in the fetal primate at least up to 148 d gestation. Although an inhibitory effect of insulin later in gestation is not ruled out, it is also possible that hyperglycemia rather than hyperinsulinemia is responsible for the deficiency in surfactant often observed in human infants of diabetic mothers.

Intrauterine growth retardation is caused by a variety of genetic and environmental influences, recognition of which is important both for prevention and for therapeutic and prognostic reasons.

Administration of cortisol to fetal rabbits resulted in a 42% inhibition of pulmonary de novo fatty acid synthesis from acetyl coenzyme A (CoA) (P = less than 0.025). This was associated with inhibition of acetyl-CoA carboxylase (EC. 6.4.1.2.) activity (P = less than 0.01) and a tendency towards decreased activity of fatty acid synthetase. There was no effect on pulmonary microsomal fatty acid elongation activity. Light and electron microscopic examination of the apex of the right lung of control and cortisol-treated animals revealed changes consistent with accelerated lung maturation in the treated animals. The in vitro activities of acetyl-CoA carboxylase and fatty acid synthetase were similar in rabbit lung and thus acetyl-CoA carboxylase activity does not appear to be rate limiting for de novo fatty acid synthesis in lung. No significant change in the activity of enzymes associated with de novo fatty acid synthesis of microsomal fatty acid elongation was found in fetal brain after cortisol exposure. However, in a parallel study on fatty acid synthesis in fetal liver, cortisol administration resulted in a 30% increase in fatty acid synthetase activity (P less than 0.025). The finding of cortisol-induced inhibition of de novo fatty acid synthesis in fetal rabbit lung may be related to the known inhibitory effect of cortisol on lung growth in the fetus.

[ 3 H]norepinephrine was shown to bind to specific sites on isolated fat cells. A Scatchard plot of norepinephrine binding showed two apparent K a of 1.9 · 10 6 and 1.2 · 10 5 LM − . 1.4 · 10 −4 M Norepinephrine covalently-linked to agarose beads reduced [ 3 H]norepinephrine binding by over 50%. Several structurally related drugs were compared as inhibitors of [ 3 H]norepinephrine binding and as stimulators of lipolysis in preparations of similarly prepared cells. Dose-response curves for norepinephrine, epinephrine and isoproterenol showed the affinities for binding inhibition and for stimulation of lipolysis to be in the same range of 6 · 10 −7 -2 · 10 −6 M. Dopamine and dopa were potent inhibitors of [ 3 H]norepinephrine binding at 8.5 · 10 −7 M and 2.0 · 10 −6 M respectively, but did not stimulate lipolysis even at 10 −4 M. Propranolol, a β-adrenergic antagonist, had no effect on [ 3 H]norepinephrine binding at 10 −4 M but completely inhibited catecholamine-stimulated lipolysis at 10 −5 M. Phentolamine, an α-adrenergic antagonist, did not inhibit binding or catecholamine-stimulated lipolysis at 10 −4 M. Ephedrine, metaraminol, phenylephrine and normetanephrine were also ineffective both as [ 3 H]norepinephrine binding inhibitors and as stimulators of lipolysis. The results suggested the catechol ring of catecholamines is more important than the ethanolamine side chain as a requirement for binding, while both an intact catechol moiety and ethanolamine function appear necessary for physiological effect.

The records of 340 infants of 36 weeks' gestational age or less were reviewed to study the relationship between premature rupture of the membranes (PRM) and the development of the respiratory distress syndrome (RDS). Twins and infants of diabetic mothers were excluded from the data analysis. PRM of 16 hours or more was associated with a statistically significant reduction in the incidence of RDS in infants of 31 weeks' gestational age and older. The association between PRM in excess of 16 hours and survival, however, was only statistically significant for infants of 33 weeks' gestational age and older. The implications of these results and a proposed plan of management for premature infants with PRM are discussed.

Bayley developmental assessments were performed at 12 months corrected age on 11 preterm infants, all of whom had undergone 133-Xenon cerebral blood flow measurements (CBF) on the 1st day of life and neonatal computerized tomography scans for the detection of intraventricular hemorrhage. The Bayley mental index was found to vary as a quadratic function of CBF to the left hemisphere, with moderate CBF values associated with more favorable outcomes than either low or high CBF values.

To investigate the presence of subtle narcotic depression following maternal narcotic analgesia, we have evaluated the effects of naloxone versus placebo in a double-blind parallel group study in 43 normal term newborn infants whose mothers had received routine narcotic analgesia within six hours prior to delivery. Infants were given either an intramuscular injection of 20 microgram/kg naloxone or 0.20 ml/kg placebo after determination of the one-minute Apgar score, and the following measurements were compared: Apgar scores at one and five minutes, capillary blood gas values at one, 60, 120, and 240 minutes, and neurobehavioral assessments at one, 4, and 24 hours. No adverse effects from naloxone were observed. Neither Apgar scores nor capillary blood gas determinations differed significantly between the two groups. Response to sound was significantly higher in the naloxone group at 24 hours. The alertness score was significantly higher for the naloxone group at one and four hours; the general assessment score for the naloxone group was significantly higher at four and 24 hours. Average scores of naloxone and placebo groups were also different at four and 24 hours of age. These data demonstrate that maternal narcotic analgesia may produce subtle changes in alertness and general behavior not reflected by Apgar scores or respiratory status, potentially reversible by administration of naloxone shortly following delivery.

SPR Presidential Address: Micro- and Macro-Environments in Academic Pediatrics. Annual Meeting, Washington, D.C. 1982

Previously we have shown ( E. D. Adamson, M. J. Deller, and J. B. Warshaw, 1981 , Nature (London) 291, 656–659) that epidermal growth factor (EGF) binds specifically to the cells and stimulates the incorporation of tritiated thymidine into DNA of several tissues of mouse embryos in a dose-dependent fashion when tested in vitro. However, in vivo a different response is obtained; exogenous EGF causes reduced incorporation of radiolabeled thymidine compared to buffer-injected control embryos. Several possible explanations are being explored. Here we present evidence that one of the responses of embryonic tissues in vivo to exogenous EGF is “down-regulation” of its receptors.

This discussion reviews cellular mechanisms underlying the development of oxygen toxicity and the antioxidant defenses that are present to counter the toxic effects of oxygen. Clinical manifestations of pulmonary oxygen toxicity in the newborn infant are presented and suggestions are offered for its prevention.

Extract: Fatty acids are important components of pulmonary surface-active lipids and lung membranes. We have investigated fatty acid synthesis in developing rabbit lung by measuring the rate of incorporation of (14C)acetyl or (14C)malonyl-CoA into pentane-extractable fatty acids. Because the lung can incorporate exogenously supplied as well as endogenously synthesized fatty acid into phospholipid, parallel studies of hepatic fatty acid synthesis were carried out. Fatty acids can be synthesized in fetal rabbit lung by the de novo pathway and to a lesser extent by chain elongation. The activity of enzymes related to de novo fatty acid synthesis is at adult levels by 23 days of fetal life and remains fairly constant thereafter. Enzymes associated with hepatic de novo fatty acid synthesis reach very ‘high levels of activity during fetal life but this decreases by 50% after birth, in association with the onset of suckling. Activity of the enzymes of de novo pulmonary synthesis appears to be unaffected by birth and onset of nutritional intake. Pulmonary and hepatic fatty acid elongation do not appear to be major routes of fatty acid synthesis in the fetal rabbit. Microsomal elongation activity is low in the fetus and on the first day of life, adult activity being fourfold greater. Pulmonary mitochondrial elongation becomes progressively more active during fetal life, but quantitatively appears less important than de novo synthesis. Gas-liquid chromatography of the fatty acids synthesized by fatty acid synthetase in the supernatant fraction of lung homogenates revealed that the main product was palmitic acid, a major component of dipalmityl lecithin which is an important surface-active lipid. The maior products of pulmonary mitochondrial and microsomal fatty acid elongation were stearic acid and 20− and 22-carbon unsaturated fatty acids. The early maturation of the enzymes of the de novo pathway ensures a source of fatty acid for lung membrane growth and for surfactant formation when the pathways for lecithin synthesis mature. Speculation: Inasmuch as the enzymes associated with de novo fatty acid synthesis are active before the pathway for lecithin synthesis matures, fatty acid synthesis is probably not the rate-limiting factor in surfactant production in the rabbit fetus. The role of other lecithin precursors such as choline remains to be investigated. Early dietary intake does not appear to influence pulmonary fatty acid synthesis, but may play an important role in overall pulmonary lipid metabolism and the regulation of lung surface activity.

Prior to 1972, radiation used to treat neonatal hyperbilirubinemia was based upon the photometric unit, the foot-candle, a measure of light illumination. Measurements in terms of microwatts per square centimeter for selective wavelengths is more precise. We compared the effectiveness of phototherapy provided by overhead phototherapy units in intensive care modules vs. conventional phototherapy units. Forty-two infants were studied over a six-month period and divided into three groups based upon radiant flux measurements as follows: Group 1 (No. = 6), 1.0µw to 1.9µw/sq cm/nm; group 2 (No. = 15), 2.0µw to 3.9µw/sq cm/nm; group 3 (No. = 21), 4.0µw to 6.0µw/sq cm/nm. All flux determinations were made within the 400- to 500-nm range. All infants in group 1 were treated with overhead phototherapy units in the intensive care modules. Because of multiple factors known to increase the risk of kernicterus, evaluation of effectiveness of phototherapy at low radiant flux was limited in group 1. Significant changes in bilirubin were noted by 48 hours when comparing group 3 with groups 1 and 2. A minimum of 4.0µw/sq cm/nm appears necessary for effective phototherapy. As designed, phototherapy units in intensive care modules are ineffective in delivering this therapeutic level of radiant flux.

Alveolar type II cells from 6-day-old rat pups were obtained at approximately 90% purity, as judged by light and electron microscopy. Within 48-72 h in culture, the cells formed a monolayer with tight junctions typical of epithelial cells. Type II cells aggregated into raised domelike areas, which when sectioned showed alveolarlike structures comprised of type II cells. After 3 days in culture these structures showed abundant lamellar body secretion. Phospholipid profiles were characteristic of type II cells, with disaturated phosphatidylcholine (DSPC) the predominant secreted phospholipid. The cells also released DSPC in response to beta-adrenergic stimulation. This release was inhibited by the specific beta-antagonist propranolol. The results show that morphologic differentiation and biochemical properties of neonatal type II cells can be studied using an isolated cell system in tissue culture.

Although the development of sophisticated neonatal intensive care has permitted the increasing survival of many small and critically ill preterm neonates, the incidence of major neurologic and developmental problems in this population has not significantly changed during the past decade,1-4 and much attention has been directed to the early identification of infants at risk. Motor handicaps and the delayed acquisition of milestones have been noted in the survivors of neonatal intensive care during the first several years of life.5,6 Learning difficulties and behavior problems may be later sequelae.7 Inasmuch as patients who have experienced intraventricular hemorrhage (IVH) have been shown to have a greater risk for neurodevelopmental handicaps than their peers who have not experienced hemorrhage,8-11 we report the first year post-term data from a prospective study of 67 consecutive preterm neonates of ≤1,250 gm birth weight who were admitted to our Newborn Special Care Unit and survived >36 postnatal hours; data include computed tomographic (CT) and/or postmortem evaluations for the determination of the incidence of IVH and neurodevelopmental followup of surviving infants.

THE HUMAN NEWBORN INFANT undergoes major metabolic changes during the transition from fetal to extrauterine life. Although glucose appears to be the major energy fuel during fetal development and an important precursor for glycogen and lipid depots, the newborn infant rapidly develops the capacity to oxidize fatty acids and ketone bodies as fuels alternative to glucose. Fatty acids derived from the high fat milk diet and endogenous fat stores become the preferred fuel of heart and other tissues with high-energy demands, tn the human neonate, there is a dramatic increase in serum levels of free fatty acids during the first hours of life." This is followed by increased concentrations of ketone bodies in the serum, Animal studies have shown that tissues of neonates, including brain, can actively utilize fatty acids and ketone bodies as oxidative substrates? ImPortant regulatory influences may include fatty acid availability, increased oxygen supply, and increases in the availability Of carnitine. Carnitine serves as an essential carrier for acyl groups across the mitochondrial membrane to sites of oxidation, and therefore has a central role in the mitochondrial oxidation of fatty acids. In the present study we have examined relationships between breast and formula feeding and serum levels of carnitine

The effects of parenteral vitamin E treatment on aspects of the pulmonary biochemical and morphologic response to 100% oxygen were studied in newborn rabbits manifesting chemical evidence of vitamin E deficiency. Pups treated with 2 mg/100 g body weight increased serum vitamin E levels from 0.39 to 2.17 mg/dl by 72 hr and lung tissue vitamin E content from 3.52 to 17 mg/mg wet weight of lung. In vitro lipid peroxidation in lung homoginates of animals in 100% oxygen for 72 hr was inhibited by approximately 80% in animals receiving 100% oxygen plus vitamin E. Hyperoxia-induced increases in the pulmonary antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase were diminished by vitamin E administration. Lungs from vitamin E-treated animals did not show the early lung epithelial injury seen in animals exposed to 100% oxygen but not treated with vitamin E. Mophometric analysis of lungs of animals in room air for 72 hr showed 81.6% of lung to be normal as compared with 43.3% normal lung in the group maintained in 100% oxygen for 72 hr. In the group treated with oxygen plus vitamin E, the lungs were similar to room air controls (82.6% normal). This study thus provides further evidence for a direct antioxident affect of vitamin E in lung.

We have compared fluorescent and nonfluorescent light sources for phototherapy for newborn infants with hyperbilirubinemia. Phototherapy was provided by a tungsten halogen lamp and conventional fluorescent lights with identical radiant flux of 6 µW/sq cm. For 22 infants treated with the nonfluorescent lamp the mean duration of phototherapy was 33.77 hours and the mean reduction of bilirubin was 3.84 mg/100 ml/day. This did not differ significantly from infants treated with conventional fluorescent lights. The nonfluorescent light can be utilized for infants in incubators or on radiant warmers. These results provide additional support for the relationship between radiant flux as a practical measure of phototherapy dose and the clinical response of a reduction in serum bilirubin.

During the perinatal period metabolic adaptations take place which insure that substrates for energy production and growth are available in the immediate postnatal period. The fetus receives a constant infusion of glucose, fatty acids and protein by the placenta. Late in gestation the accumulation of storage fuels such as glycogen and adipose tissue anticipate the abrupt cessation of substrate supply from the placenta at the time of birth. Postnatally an increase in endogenous production of glucose in the form of glycogenolysis and gluconeogenesis become important for maintenance of glucose homeostasis. Increase utilization of fatty acids in ketone bodies as energy substrates also become important in the postnatal period. These metabolic adaptations are mediated by the development of new enzyme activities as well as by changes in hormonal millieu and substrate availability.

The developmental profile of pulmonary glycogen was investigated in fetuses of rats made diabetic before conception with the injection of 40 mg/kg streptozotocin. Lungs of control litters showed increasing pulmonary glycogen concentration from day 16-20, followed by significant decline by term (= 22 days). In contrast, the diabetic litters, which had pulmonary glycogen concentration equal to controls until day 20, showed significantly higher glycogen values (P less than 0.01) on days 21 and 22, consistent with a delay in glycogen degradation. This coincided with the finding of decreased amounts of fetal pulmonary phosphatidylcholine and disaturated phosphatidylcholine on day 21 of the diabetic gestation (P less than 0.05), but not before that time. Pulmonary glycogen phosphorylase A activity was significantly decreased in the diabetic litters on the final days of gestation, at the same time that the delay in glycogen breakdown became evident. Pulmonary glycogen synthase activity did not differ in the control and diabetic fetuses. The delay in pulmonary glycogen degradation seen in the fetus of the diabetic gestation is thus temporally related to the delay in lung maturation seen in this model and may be secondary to a decrease in the activity of the glycogenolytic enzyme phosphorylase A. Decreased availability of pulmonary glycogen stores for surfactant synthesis may be important in elucidating the mechanism of the delayed pulmonic maturation seen in fetuses of diabetic pregnancies.

Delayed fetal lung development is a feature of the diabetic pregnancy. Since fetal glucocorticoids are important in the regulation of lung maturation, we measured corticosterone and corticosteroid-binding globulin binding capacity in streptozotocin-diabetic pregnant rats and their fetuses. Previous studies have demonstrated delayed fetal lung maturation in this animal model. In control fetuses, total corticosterone concentration increased through day 20 of gestation, then declined until day 22 (term). The unbound steroid, which accounted for 5-10% of the total, increased approximately 3-fold from day 18 to term. Corticosteroid-binding globulin binding capacity peaked on day 19 after which it decreased. Maternal total and unbound corticosterone levels and corticosteroid-binding globulin binding capacity remained relatively constant throughout the final week of normal gestation. When compared to controls, fetuses from diabetic pregnancies had significantly lower total corticosterone from day 19 through 22. Corticosteroid-binding globulin binding capacity was also significantly decreased in these fetuses for the last 4 days of gestation. Similar differences were noted in maternal samples. However, no significant differences in unbound, biologically active, corticosterone were seen when diabetic and control groups were compared. Thus, delayed fetal lung maturation observed in fetuses of streptozotocin-diabetic rats is associated with a decrease in total circulating corticosteroid levels late in gestation. However, since unbound corticosteroid levels were similar in fetuses of control and diabetic animals, it is likely that other mechanisms may be responsible for the observed delay in lung development in fetuses of diabetic pregnancies.

Glucose, a major metabolic substrate for the mammalian fetus, probably makes significant contributions to surface active phospholipid synthesis in adult lung. We examined the developmental patterns of glycogen content, glycogen synthase activity, glycogen phosphorylase activity and glucose oxidation in fetal and newborn rat lung. These patterns were correlated with the development of phosphatidylcholine synthesis, content and the activities of enzymes involved in phosphatidylcholine synthesis. Fetal lung glycogen concentration increased until day 20 of gestation (term is 22 days) after which it declined to low levels. Activity of both glycogen synthase I and total glycogen synthase (I + D) in fetal lung increased late in gestation. Increased lung glycogen concentration preceded changes in enzyme activity. Glycogen phosphorylase a and total glycogen phosphorylase (a + b) activity in fetal lung increased during the period of prenatal glycogen depletion. The activity of the pentose phosphate pathway, as measured by the ratio of CO2 derived from oxidation of C1 and C6 of glucose, declined after birth. Fetal lung total phospholipid, phosphatidycholine and disaturated phosphatidylcholine content increased by 60, 90 and 180%, respectively, between day 19 of gestation and the first postnatal day. Incorporation of choline into phosphatidylcholine and disaturated phosphatidylcholine increased 10-fold during this time. No changes in phosphatidylcholine enzyme activities were noted during gestation, but both choline phosphate cytidylyltransferase and phosphatidate phosphatase activity increased after birth. The possible contributions of carbohydrate derived from fetal lung glycogen to phospholipid synthesis are discussed.

We have carried out studies using rat lung slices showing that the developing lung utilizes both glucose and fatty acids as oxidative substrates. Glucose oxidation to CO2 decreased at birth but showed higher activity after weaning. The activity of the pentose phosphate pathway also decreased postnatally. In contrast to glucose, the oxidation of palmitate and caprate to CO2 showed an increase in the immediate postnatal period. Cytochrome oxidase and carnitine palmitoyltransferase showed a parallel postnatal increase following the increase in fatty acid oxidation. Cytochrome oxidase activity in adult lung was approximately 30% of peak newborn values. Palmitate incorporation into total lipids was greatest at 18 to 19 days of fetal development, at which time the lung content of nonesterified fatty acids was highest.

In 9% of a series of 160 thyroid tumors (130 malignant and 30 benign), the only or the predominant cell type was the oxyphilic Hurthle cell. Three carcinomas (follicular or papillary) and two adenomas were evaluated by histochemical, electron microscopic and biochemical techniques. The characteristic cytological features were: cytoplasm loaded with mitochondria of relatively normal structural architectonics, and an increased number of cytoplasmic dense bodies which possessed acid phosphatase and nonspecific esterase activities and therefore could be classified as lysosomes. The mitochondria isolated from Hurthle cell tumors contained higher than normal activity of succinoxidase and cytochrome oxidase and exhibited normal difference spectrum. One follicular carcinoma and one follicular adenoma contained considerable amounts of a poorly iodinated thyroglobulin. When studied, the peroxidase and iodination activities were present but lower than normal. The subcellular distribution of perioxidase and ...

Intrauterine growth retardation can result from a variety of environmental and genetic influences on fetal growth. The sequelae of intrauterine growth retardation resulting from impairment of nutrient flow include low birth weight with sparing of brain growth, polycythemia, and hypoglycemia resulting from decreased storage fuels and defective gluconeogenesis. Available data suggest that the vast majority of nutritionally growthretarded infants have normal postnatal development without significant difference in IQ or neurologic scores from normal infants. The outcome of infants in whom there is decreased growth potential relates to the condition underlying growth retardation and may, of course, result in later severe handicap. Infants with nutritional intrauterine growth retardation may exhibit decreased fetal size and sparing of brain growth as an adaptive stage in the presence of lower oxygen and substrate availability. Under such conditions, a smaller size may decrease substrate and oxygen needs and improve outcome. All infants with intrauterine growth retardation, however, require follow-up and careful developmental evaluation.

Cultured chick embryo cardiac myoblasts specifically bind [ 3 H]nonrepinephrine. The binding is rapid and reversible. Bound [ 3 H]nonrepinephrine, dissociated by 1 M HCl, can be rebound to fresh cells. β-Adrenergic catecholamines were most potent in displacing [ 3 H]nonrepinephrine from the cellular bindign sites. The binding reaction did not show stereospecificity. α-Adrenergic amines were much less potent. Propranolol, but no phentolamine, competed for the sites. Approximately 2.5 · 10 6 specific binding sites are present per myocardial cell. The sites appear to be present predominantly at the cell surface in that nonrepinephrine linked to agarose beads competes for th sites. Similarly, the sites were degraded by either trypsin or trypsin bound to agarose. Two different binding constants, K = 2 · 10 6 and 1 · 10 5 , were observed. Proteolytic enzymes decreased binding whereas certain hospholipases led to an increase in specific binding. Divalent cations at concentrations > 1 mM diminished binding as did chelating agents.

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with 3H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8-10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48-60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7-8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of 3H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.

Acute nutritional deprivation results in significant reduction in the activities of acetyl-CoA carboxylase, fatty acid synthetase, microsomal fatty acid elongation and choline phosphotransferase in rat lung. This data establishes the enzymatic basis for the known inhibition of pulmonary surfactant production by acute starvation.

Extract Quantitative studies of the ultrastructure of heart muscle in iron- and copper-depleted rats show an increased mitochondrial area that contributes to cardiac hypertrophy in both conditions. The mean ratios of mitochondrial/myofibrillar areas are 1.73 and 1.69, respectively, in the deficient groups compared with 0.70 in control animals. The markedly enlarged mitochondria appear to displace and distort the myofibrils. After iron-deficient rats are provided with iron, the reversal of the abnormal mitochondrial/myofibrillar ratio and of cardiac hypertrophy requires about 16 days or approximately twice as long as the complete repair of anemia. In heart muscle from iron-deficient animals, the mitochondrial cytochromes, which all contain iron, remain essentially normal in concentration. In the copper-deficient rats, in contrast, cytochrome a+a3, which contains copper, is depressed to less than one-half the normal concentration. Isolated mitochondria from heart and liver of all animals deficient in iron and copper function normally with respect to respiration and phosphorylation. Thus, a correlation between abnormality of mitochondrial structure, composition, and function is not as yet apparent. The mitochondrial contribution to the cardiac hypertrophy of iron and copper deficiency cannot be attributed entirely to increased work load secondary to anemia, particularly in copper-deficient rats whose cardiac enlargement precedes the development of anemia. The morphologic changes are distinct from those observed in experimental work hypertrophy and can represent a response to the lack of essential precursors required for the cytochromes or other mitochondrial constituents. Speculation: Cardiac hypertrophy in iron and copper deficiency is in part attributable to enlargement of the mitochondrial compartment. This results from the lack of trace metals required for the production of cytochromes or other mitochondrial constituents.