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Urea biosynthesis during development of the mammal
950
Society for Pediatric Research
November 1965
Paulus Zee,* Laboratory of Nutrition and Metabolism, St. Jude Hospital and Departments of Physiology and Pediatrics, University of Tennessee, Memphis, Tenn. Introduced by Donald Pinkel Serum phospholipid levels and lecithin fatty acid compositions were determined in the cord blood of healthy newborns and venous blood of their mothers in order to establish and compare their normal values. Lipid extracts of the sera were fractionated on silicic acid columns and the fatty acid distributions of the lecithin fractions were determined by gas chromatography. The cord serum phospholipid concentrations were approximately one-half the maternal values. However, the distribution of the phospholipid fractions (cephalin, lecithin, sphyngomyelin, and lysolecithin) was similar to that reported for normal adults, but different from that of the mothers. The sera of the mothers had considerably lower lysolecithin and higher cephalin concentration than the sera of their newborn or of normal adults. The lecithin composition of maternal serum was consistently different from that of neonatal sera; in the mothers the lecithin contained more linoleate than oleate, while in the newborn the reverse was true. T h e arachidonate in the serum lecithin was lower in the mothers than in their newborn. These differences in phospholipid levels and compositions demonstrate the dissimilarity in lipid metabolism of mothers and their newborn.
cose. The fed normal infants received 30.8 calories per kilogram on day 1 and 37.7 calories per kilogram on day 2. The distressed infants received 26.6 calories per kilogram on the first day and 36.9 calories per kilogram on the second. BUN, serum Na, and K, total urinary nitrogen, urinary Na, and K were measured during the first 48 hours of life. Average values relevant to protein catabolism are presented in Table I and are interpreted to show a reduction of protein catabolism in the normal premature given calories. Catabolism of the distressed infant appears to have been reduced as well. Urinary sodium excretion was higher in both intravenous groups suggesting a possible disadvantage of intravenous 10 per cent glucose.
81, Urea biosynthesis during development o[ the mammal Niels C. R. R~iih~i* and Norman Kretch. mer, Stanford University School of Medicine, Palo Alto, Calif.
80. Effect o/ glucose on catabolism in the premature in[ant Peter A. M. Auld, Saroj Mehta,* and Pensri Bhangananda,* Cornell University Medical Center, New York, N. Y. A question exists whether catabolism in the premature in the days after birth is due to stress or starvation. A study was designed to determine the effect on catabolism of calories provided as 10 per cent glucose and water intravenously soon after birth. Normal prematures were alternately given glucose or starved and a number of infants with hyaline membrane disease were given glu-
These studies were designed to study the ability of the young animal to use protein for growth. Synthesis of urea reflects degradation of protein and consequently lack of use of protein for growth. The over-all capacity for synthesis of urea was studied in slices of liver incubated with optimal amounts of ammonia, ornithine, and HC140~ -. Following incubation, the samples were treated with urease, and the radioactive carbon dioxide was collected and measured. Development of capacity for urea biosynthesis was studied in slices of liver obtained from guinea pigs and rats. The amount of urea formed by samples taken from fetal animals was very low. The capacity for formation of urea, in the guinea pig, increased fivefold by the time the animal was 24 to 48 hours of age then decreased in the adult tissue to fetal values. In the newborn rat, the ureaforming activity was about twice that of the fetus and increased within 24 hours to the adult level which was about 10 times that of the fetus. When actinomycin D, 2.5 #g was administered as a single intraperitoneal injection immediately after birth, it had no effect upon urea-forming capacity: while puromycin, 0.25 mg. administered intraperitoneally every 3 hours from birth, resulted in 56 per cent inhibition of the increase in urea
Table I
Group NPO (9) IV (10) H M D (7)
Weight (grams) Day 1 Day 2 1,736 1,449 1,620
1,678 1,394 1,589
BUN (mg./lO0 ml.) Day 1 Day 2 59.6 25.3 36.9
57.9 19.8 35.4
Urine N~ Day 1 I Day 2 17.5 14.5 22.3
44.4 35.2 36.1
Nitrogen as product o[ protein catabolism mg./Kg./48 hours 312.3 22.5 211.0
Volume 67
Number 5
Abstracts 9 5 1
part 2
biosynthesis by 12 hours. These results suggest that new enzyme protein is synthesized from preformed mRNA. Schimke (1962) has shown that a marked elevation in activity of enzymes of ur=a biosynthesis can be produced in rat liver by fasting and by increasing dietary protein. In our experiments with newborn rats, a 24 hour fast produced no change in the normal increase in urea-forming capacity. Moreover, when animals were nursed in small and large litters, resulting in a twofold difference in weight at 10 days of age, there was no significant difference observed in the urea-forming capacity of the liver. In these rapidly growing animals there is no apparent adaptation of enzymes of the urea cycle despite marked differences in diet.
82. The effect o/ albumin in reversing bilirubin toxicity in tissue culture
Marilyn L. Cowger,* University of Washington School of Medicine, Seattle, Wash. Introduced by R. P. Igo Previous work from this laboratory using purified respiratory enzymes and intact mammalian cells grown in protein-free media in tissue culture demonstrated that the primary effect of bilirubin was to uncouple oxidative phosphorylation. As the concentration of bilirubin was raised, a second effect appeared, that of inhibition at a specific site in the terminal electron transport chain. This type of inhibition resembles that of the barbiturates in their effect on NADH~ oxidase, but bilirubin was unique in causing a primary uncoupling effect in intact ceils. Additional work has now shown that, unlike barbiturates, bilirubin inhibits the uptake and conversion of amino acids into cellular protein. Cell viability as estimated by erythrocin B decreased proportionally to bilirubin concentration and length of exposure time. The degree of toxicity was affected by the pH of the incubation medium. A pH more acidic than 7.6 enhanced the toxic effects on terminal oxidation and viability. The role of bovine albumin in modifying bilirubin toxicity in this system was studied. Albuminbound bilirubin was nontoxic to cultured cells. Bovine albumin was introduced into cell suspensions exposed to bilirubin for varying lengths of
time. Cell-bound bilirubin could be extracted back into the incubating medium, and it has been possible to determine the relationship between exposure time and degree of reversibility of bilirubin toxicity. Once again pH was a critical factor, this time in relation to the ability of albumin to extract cell-bound bilirubin. These results have clinical implications in treating hyperbilirubinemia in the newborn. Furthermore, some of the biochemical assays developed for this study may prove useful clinically for determining availability of albumin-binding sites in the serum of the newborn.
83. The use of THAM-buffered ACD blood in high risk in]ants who require exchange trans]usion Thomas K. Oliver, Jr., University of Washington School of Medicine, Seattle, Wash. In 1961 we observed that CO~ output promptly rose when small increments of ACD blood were given to term infants undergoing exchange transfusion. This was believed to be due to a ventilatory response to an acid load. It did not occur when heparinized blood was infused. Blood bank facilities for providing heparinized blood are unavailable in many communities including Seattle. It was reasoned that buffering ACD blood with T H A M prior to exchange transfusion of high risk infants who could not make compensatory adjustments should be given a trial. Six exchange transfusions have been performed in 5 premature infants ranging in birth weight from 920 to 2,520 grams (median 1,410). All had pCO2 ~ 50 mm. Hg. T H A M 20 mM. as 0.3M in 10 per cent dextrose was added the first time, increasing pH to 7.70; thereafter 15 mM. was added resulting in a range of pH 7.44 to 7.54. Mean values are shown in Table I. Acid-base balance improved in each patient during and following exchange. No posttransfusion alkalemia was observed. Serum electrolytes were unchanged; glucose rose during exchange. The procedure was performed in a neutral thermal environment, usmg a servocontrolled open-ended hood which provides radiant heat. It appears that T H A M can be safely added to ACD blood for exchange in high risk infants and may have wider application in other infants particularly if large volumes are to be used.
Table I Donor blood
pH pCCh mm. Hg Base excess mEq./L.
Patient
PreTHAM
After THAM
Pre-exchan~e
End exchange
Postexchange (14 to 15 hours)
6.70 ~ 150 ~-22
7.50 27 -5.3
7.32 74 +2.7
7.38 58 +6.0
7.39 51 +4.7
Society for Pediatric Research
November 1965
Paulus Zee,* Laboratory of Nutrition and Metabolism, St. Jude Hospital and Departments of Physiology and Pediatrics, University of Tennessee, Memphis, Tenn. Introduced by Donald Pinkel Serum phospholipid levels and lecithin fatty acid compositions were determined in the cord blood of healthy newborns and venous blood of their mothers in order to establish and compare their normal values. Lipid extracts of the sera were fractionated on silicic acid columns and the fatty acid distributions of the lecithin fractions were determined by gas chromatography. The cord serum phospholipid concentrations were approximately one-half the maternal values. However, the distribution of the phospholipid fractions (cephalin, lecithin, sphyngomyelin, and lysolecithin) was similar to that reported for normal adults, but different from that of the mothers. The sera of the mothers had considerably lower lysolecithin and higher cephalin concentration than the sera of their newborn or of normal adults. The lecithin composition of maternal serum was consistently different from that of neonatal sera; in the mothers the lecithin contained more linoleate than oleate, while in the newborn the reverse was true. T h e arachidonate in the serum lecithin was lower in the mothers than in their newborn. These differences in phospholipid levels and compositions demonstrate the dissimilarity in lipid metabolism of mothers and their newborn.
cose. The fed normal infants received 30.8 calories per kilogram on day 1 and 37.7 calories per kilogram on day 2. The distressed infants received 26.6 calories per kilogram on the first day and 36.9 calories per kilogram on the second. BUN, serum Na, and K, total urinary nitrogen, urinary Na, and K were measured during the first 48 hours of life. Average values relevant to protein catabolism are presented in Table I and are interpreted to show a reduction of protein catabolism in the normal premature given calories. Catabolism of the distressed infant appears to have been reduced as well. Urinary sodium excretion was higher in both intravenous groups suggesting a possible disadvantage of intravenous 10 per cent glucose.
81, Urea biosynthesis during development o[ the mammal Niels C. R. R~iih~i* and Norman Kretch. mer, Stanford University School of Medicine, Palo Alto, Calif.
80. Effect o/ glucose on catabolism in the premature in[ant Peter A. M. Auld, Saroj Mehta,* and Pensri Bhangananda,* Cornell University Medical Center, New York, N. Y. A question exists whether catabolism in the premature in the days after birth is due to stress or starvation. A study was designed to determine the effect on catabolism of calories provided as 10 per cent glucose and water intravenously soon after birth. Normal prematures were alternately given glucose or starved and a number of infants with hyaline membrane disease were given glu-
These studies were designed to study the ability of the young animal to use protein for growth. Synthesis of urea reflects degradation of protein and consequently lack of use of protein for growth. The over-all capacity for synthesis of urea was studied in slices of liver incubated with optimal amounts of ammonia, ornithine, and HC140~ -. Following incubation, the samples were treated with urease, and the radioactive carbon dioxide was collected and measured. Development of capacity for urea biosynthesis was studied in slices of liver obtained from guinea pigs and rats. The amount of urea formed by samples taken from fetal animals was very low. The capacity for formation of urea, in the guinea pig, increased fivefold by the time the animal was 24 to 48 hours of age then decreased in the adult tissue to fetal values. In the newborn rat, the ureaforming activity was about twice that of the fetus and increased within 24 hours to the adult level which was about 10 times that of the fetus. When actinomycin D, 2.5 #g was administered as a single intraperitoneal injection immediately after birth, it had no effect upon urea-forming capacity: while puromycin, 0.25 mg. administered intraperitoneally every 3 hours from birth, resulted in 56 per cent inhibition of the increase in urea
Table I
Group NPO (9) IV (10) H M D (7)
Weight (grams) Day 1 Day 2 1,736 1,449 1,620
1,678 1,394 1,589
BUN (mg./lO0 ml.) Day 1 Day 2 59.6 25.3 36.9
57.9 19.8 35.4
Urine N~ Day 1 I Day 2 17.5 14.5 22.3
44.4 35.2 36.1
Nitrogen as product o[ protein catabolism mg./Kg./48 hours 312.3 22.5 211.0
Volume 67
Number 5
Abstracts 9 5 1
part 2
biosynthesis by 12 hours. These results suggest that new enzyme protein is synthesized from preformed mRNA. Schimke (1962) has shown that a marked elevation in activity of enzymes of ur=a biosynthesis can be produced in rat liver by fasting and by increasing dietary protein. In our experiments with newborn rats, a 24 hour fast produced no change in the normal increase in urea-forming capacity. Moreover, when animals were nursed in small and large litters, resulting in a twofold difference in weight at 10 days of age, there was no significant difference observed in the urea-forming capacity of the liver. In these rapidly growing animals there is no apparent adaptation of enzymes of the urea cycle despite marked differences in diet.
82. The effect o/ albumin in reversing bilirubin toxicity in tissue culture
Marilyn L. Cowger,* University of Washington School of Medicine, Seattle, Wash. Introduced by R. P. Igo Previous work from this laboratory using purified respiratory enzymes and intact mammalian cells grown in protein-free media in tissue culture demonstrated that the primary effect of bilirubin was to uncouple oxidative phosphorylation. As the concentration of bilirubin was raised, a second effect appeared, that of inhibition at a specific site in the terminal electron transport chain. This type of inhibition resembles that of the barbiturates in their effect on NADH~ oxidase, but bilirubin was unique in causing a primary uncoupling effect in intact ceils. Additional work has now shown that, unlike barbiturates, bilirubin inhibits the uptake and conversion of amino acids into cellular protein. Cell viability as estimated by erythrocin B decreased proportionally to bilirubin concentration and length of exposure time. The degree of toxicity was affected by the pH of the incubation medium. A pH more acidic than 7.6 enhanced the toxic effects on terminal oxidation and viability. The role of bovine albumin in modifying bilirubin toxicity in this system was studied. Albuminbound bilirubin was nontoxic to cultured cells. Bovine albumin was introduced into cell suspensions exposed to bilirubin for varying lengths of
time. Cell-bound bilirubin could be extracted back into the incubating medium, and it has been possible to determine the relationship between exposure time and degree of reversibility of bilirubin toxicity. Once again pH was a critical factor, this time in relation to the ability of albumin to extract cell-bound bilirubin. These results have clinical implications in treating hyperbilirubinemia in the newborn. Furthermore, some of the biochemical assays developed for this study may prove useful clinically for determining availability of albumin-binding sites in the serum of the newborn.
83. The use of THAM-buffered ACD blood in high risk in]ants who require exchange trans]usion Thomas K. Oliver, Jr., University of Washington School of Medicine, Seattle, Wash. In 1961 we observed that CO~ output promptly rose when small increments of ACD blood were given to term infants undergoing exchange transfusion. This was believed to be due to a ventilatory response to an acid load. It did not occur when heparinized blood was infused. Blood bank facilities for providing heparinized blood are unavailable in many communities including Seattle. It was reasoned that buffering ACD blood with T H A M prior to exchange transfusion of high risk infants who could not make compensatory adjustments should be given a trial. Six exchange transfusions have been performed in 5 premature infants ranging in birth weight from 920 to 2,520 grams (median 1,410). All had pCO2 ~ 50 mm. Hg. T H A M 20 mM. as 0.3M in 10 per cent dextrose was added the first time, increasing pH to 7.70; thereafter 15 mM. was added resulting in a range of pH 7.44 to 7.54. Mean values are shown in Table I. Acid-base balance improved in each patient during and following exchange. No posttransfusion alkalemia was observed. Serum electrolytes were unchanged; glucose rose during exchange. The procedure was performed in a neutral thermal environment, usmg a servocontrolled open-ended hood which provides radiant heat. It appears that T H A M can be safely added to ACD blood for exchange in high risk infants and may have wider application in other infants particularly if large volumes are to be used.
Table I Donor blood
pH pCCh mm. Hg Base excess mEq./L.
Patient
PreTHAM
After THAM
Pre-exchan~e
End exchange
Postexchange (14 to 15 hours)
6.70 ~ 150 ~-22
7.50 27 -5.3
7.32 74 +2.7
7.38 58 +6.0
7.39 51 +4.7