- Email: [email protected]
Of newborn hearts and nucleic acids
1 0 7 2 Society for Pediatric Research
using labeled fatty acids. Brockerhoff demonstrated complete randomization of triglyceride fatty acids by the time they reach depot stores. This all bears on our data because it indicates that the composition of depot fat is not affected very much by changes in diet. DR. T~IOMAS K. OLIVER, JR., Department of
Pediatrics, University of Washington School of Medicine, Seattle, Wash. It has been shown in a number of infant species and more recently in adult humans that noradrenaline is perhaps more effective in releasing free fatty acids than is epinephrine. I wonder if you have studied the effects of this model. DR. HAESSLER, We have not studied specifically noradrenaline. We used Parke-Davis epinephrine which I believe is a mixture of adrenaline and noradrenaline. I think there is no reason to believe that the release of the fatty acids would be substantially different had we used purified noradrenaline rather than the mixed preparation, DR. ARLAN L. ROSENBLOOM, University Hospital, Madison, Wis. On a normal caloric intake, when feeding is limited to one meal a day, rats show a twenty-five fold increase in lipogenesls and a marked adaptive increase in enzyme synthesis in liver and adipose tissue. These observations are interesting in comparison with the frequently reported one-big-meaI-a-day eating pattern of the obese patient, the frequent inability to lose weight on severe calorie restriction, and the numerous metabolic alterations demonstrated in h u m a n obesity, including a subnormal response of plasma free fatty acids to epinephine, just as in Dr. Haessler's in vitro study. The disturbance in the metabolically obese individual is felt to involve hypertrophy of certain enzyme systems and this metabolic defect could also conceivably result from hyperphagia of hypothalamic origin. I would like to ask Dr. Haessler if there are any studies on rats made obese by an abnormal dietary pattern that have shown similar palmitic-linoleic ratios to those he has demonstrated in his "lesioned" animals ? DR. HAE,SSLER. I am not aware of any studies of fatty acid composition in animals that have been made obese because of dietary manipulation alone. We have some observations, however, on a type of animal which has been termed metabolically obese, that is, the obese hyperglycemic mouse. These animals also show the same sort of change in fatty acid composition and diminution in the release of fatty acids from tissue which we have observed in the "brain-lesioned" rat. DR. ROBERT E. GREENBERG, Department o[
Pediatrics, Stanford University, School of Medicine, Palo Alto, Catif. A very important point made in your presentation revolves around the discrepancy between the specific ratio you are utilizing and weight of the animal. Would this discrepancy perhaps be obviated if, instead of measuring weight per se, one attempted to measure the ratio as a function of the amount of fat? Then one possibly would not find differences between force-fed and experimental adiposity. DR. HAESSL~R. No. We have "brain-lesioned" animals that have been given restricted diets so
December 1964
they gained no weight. They have low ratios and normal total body fat content. We also have "brain-lesioned" animals that have been obese and brought back to normal weight by restricted feeding. They too have characteristically low ratios. Apparently the linoleic-palmitic ratio is not a function of the ~ize of the adipose organ, but a characteristic alteration due to the brain lesion. DR. BENJAMIN KRAMER, 4802 Tenth Ave., Brooklyn 19, N. Y. Did you study the influence of the age of the animal upon the rate of turnover of the fat? In the h u m a n adult the effect of change in diet is manifest after anywhere from six months to a year and a half, whereas the prematurely born can demonstrate such an effect as early as two or three weeks. DR. HAESSLER. We have studied animals in only a fairly small age range. O u r rats usually weighed around 200 grams when the lesions were made. We have made some lesions in animals as small as I00 grams and a few in larger animals. We have found no difference in the age within these relatively small limits in terms of the fatty acid composition after the lesion was made.
3. Of newborn hearts and nucleic acids Louis Gluck, ~ Norman S. Talner, Thomas H. Gardner, ~ and Marie V. Kulovich, r
Yale University School of Medicine, New Haven, Conn. Selectively increased R N A concentration in the left ventricle as compared to the right followed banding of the aorta in puppies. Similar selective increase in right ventricular RNA occured with pulmonary artery banding. Because of these observations R N A concentrations in the two ventricles were compared in the fetus and followed after birth to determine whether hemodynamic changes with birth and subsequent normal enlargement of the left ventricle are also associated with RNA differences. Studies in the rabbit showed equal concentrations of R N A in the ventricles of the fetus and the just born rabbit. Following delivery, left ventricular R N A rose to a peak concentration at 16 hours while right ventricular R N A levels changed little. Grossly increased left ventricular muscle mass was noted by 3 days, and R N A levels in both ventricles were again equal at 5 days. However, in premature rabbits delivered by cesarean section, no selective increase in R N A concentration in the left ventricle was seen, and the RNA levels in both ventricles remained equal during the first 30 hours of life. These findings suggest either that failure of response of the premature left ventricle is associated with immaturity or, more likely, that differences in the systemic vascular resistance in term and premature rabbits which vary the hemodynamic load on the left ventricle are reflected biochemically by differences in ventricular R N A levels and left ventricular protein synthesls. T h e findings
"YBy invitation. Asterisk indicates the same throughout.
Volume 65
Number 6
Abstracts
Part 2
also suggest a basic observation--that distortion of the myocardial cell stimulates RNA production. DISCUSSION DR. SOLOMON A. KAPLAN, Childrens Hospital, Los Angeles, Cali[. This is a most interesting biochemical phenomenon, that a mechanical stimulus can result in increased RNA synthesis and presumably subsequent protein synthesis. It would be of great interest to know what type of R N A is being increased as a result of the mechanical stimulus, ribosomal, messenger, or transfer RNA. Techniques for isolating myocardial ribosomes capable of synthesizing protein in vitro are now available. It would seem to me that fractionation of R N A in your experiments would be an area worth exploring. DR. GLUCK. As a matter of fact, this is under investigation currently. It has been our hypothesis that during embryonic development the appearance of a particular R N A marks the beginning of differentiation--perhaps the differentiation itself. We are attempting to isolate the various kinds of template R N A and other R N A that appear. The bulk of the R N A increase following hemodynamic stress is ribosomal as it is in the R N A synthesis in the very early embryo.
4. Aldosterone secretion rates (A.S.R.) in congenital adrenal hyperplasla Avinoam Kowarski, r Jordan W. Finkelstein, ~" John S. Spaulding, ~ Gerald Holman, r and Claude J. Migeon, The Johns Hopkins University, Baltimore, Md. A.S.R. (in micrograms per 24 hours) were measured in 9 patients with congenital adrenal hyperplasia by a modification of the method of Kliman and Peterson using 1,2-HS-D-aldosterone. In two adult patients with the non-salt losing type on 64 and 120 mEq. Na per 24 hours, the A.S.R. were 306 to 350 (our normal range 44 to 92). There was some increase in A.S.R. (434 and 600) when Na intake was restricted to 9 mEq. A 14-day-old untreated infant taking 10 mEq. of Na per 24 hours had an A.S.R. of 64. The A.S.R. remained unchanged (61) on the second day of a 3 mEq. Na intake per 24 hours during obvious clinical and laboratory signs of salt loss. Two other untreated infants under I month of age were studied when clinically well (8 and 4) and again during obvious salt loss (9 and 11). A fourth infant, aged 6 months, had an A.S.R. of 53 while taking a regular diet and 29 when restricted to 1 mEq. Na per 24 hours; D O C A pellets implanted 5~2 months earlier may have influenced the results in this patient. A 5-year-old salt loser had an A.S.R. of 22 #g twenty days after cessation of all therapy and normal Na diet. A 12-year-old male patient under cortisol treatment for the hypertensive form of the disease had a low A.S.R. (I9) while on a regular diet which did not increase (11) during a 9 mEq. Na diet.
10 7 3
An adult female with an atypical hypertensive form of the disease, while under cortisol treatment, had a low A.S.R. (24) on a regular diet which rose to 79 on the seventh day of 9 mEq. Na intake per 24 hours. We can conclude that the non-salt losing pa2 tients have high A.S.R. without clinical manifestations of hyperaldosteronism. The salt-losing and hypertensive patients had A.S.R. lower than those of the non-salt loser. DISCUSSION DR. LYTT I. GARDNER, State University o / N e w
York, Upstate Medical Center, Syracuse I0, N. Y. This is a very elegant study of a topic dear to the hearts of many of us who were pupils of Lawson Wilkins. Dr. Jos6 Cara, in our laboratory, has been much involved recently in a study of the juxtaglomerular apparatus and its relationship to virilizing adrenal hyperplasia and salt loss in this syndrome. He described hyperplasia and hypertrophy of the juxtaglomerular ceils of the kidney, and hypertrophy of the zone glomerulosa of the adrenals in autopsy specimens from a girl afflicted with the salt-losing form of this syndrome who died suddenly at age 4sA2 years, during the course of a febrile illness. It was hypothesized that the chronic state of salt loss caused changes in the intravascular compartment which led to compensatory activity of the juxtaglomerular cells with resultant stimulation of the zona glomerulosa of the adrenal, presumably through a reninangiotensin mechanism. This was in contrast with specimens obtained from autopsy of a 7-year-old boy with the hypertensive form of the disease who died suddenly during measles and who presented hypertrophy of the zona glomerulosa of the adrenals, without hypertrophy of the juxtaglomerular cells (Cara, J., and Gardner, L. I.: Pediatrics 32: 825, 1963). I wonder if you have had any opportunity to go into this aspect of your patients and make any observations along these lines. DR. KOWAI~SXL No, we did not study this question directly in these patients.
5. Immediate effects of phenylalanine-deficient diet in young in[ants D. Ingall, "~J. D. Sherman, ~ F. Coekburn, "~ and R. Klein, Boston City Hospital and Boston University School of Medicine, Boston, Mass. The effects of a phenylalanine-deficient diet on bone marrow and amino acid metabolism were studied in normal young infants of varying ages and maturity because the first two authors have reported the production of bone marrow changes similar to those seen in chloramphenicol toxicity in one anemic infant with phenylketonuria on a phenylalanine-deficient diet. Bone marrow exam-
using labeled fatty acids. Brockerhoff demonstrated complete randomization of triglyceride fatty acids by the time they reach depot stores. This all bears on our data because it indicates that the composition of depot fat is not affected very much by changes in diet. DR. T~IOMAS K. OLIVER, JR., Department of
Pediatrics, University of Washington School of Medicine, Seattle, Wash. It has been shown in a number of infant species and more recently in adult humans that noradrenaline is perhaps more effective in releasing free fatty acids than is epinephrine. I wonder if you have studied the effects of this model. DR. HAESSLER, We have not studied specifically noradrenaline. We used Parke-Davis epinephrine which I believe is a mixture of adrenaline and noradrenaline. I think there is no reason to believe that the release of the fatty acids would be substantially different had we used purified noradrenaline rather than the mixed preparation, DR. ARLAN L. ROSENBLOOM, University Hospital, Madison, Wis. On a normal caloric intake, when feeding is limited to one meal a day, rats show a twenty-five fold increase in lipogenesls and a marked adaptive increase in enzyme synthesis in liver and adipose tissue. These observations are interesting in comparison with the frequently reported one-big-meaI-a-day eating pattern of the obese patient, the frequent inability to lose weight on severe calorie restriction, and the numerous metabolic alterations demonstrated in h u m a n obesity, including a subnormal response of plasma free fatty acids to epinephine, just as in Dr. Haessler's in vitro study. The disturbance in the metabolically obese individual is felt to involve hypertrophy of certain enzyme systems and this metabolic defect could also conceivably result from hyperphagia of hypothalamic origin. I would like to ask Dr. Haessler if there are any studies on rats made obese by an abnormal dietary pattern that have shown similar palmitic-linoleic ratios to those he has demonstrated in his "lesioned" animals ? DR. HAE,SSLER. I am not aware of any studies of fatty acid composition in animals that have been made obese because of dietary manipulation alone. We have some observations, however, on a type of animal which has been termed metabolically obese, that is, the obese hyperglycemic mouse. These animals also show the same sort of change in fatty acid composition and diminution in the release of fatty acids from tissue which we have observed in the "brain-lesioned" rat. DR. ROBERT E. GREENBERG, Department o[
Pediatrics, Stanford University, School of Medicine, Palo Alto, Catif. A very important point made in your presentation revolves around the discrepancy between the specific ratio you are utilizing and weight of the animal. Would this discrepancy perhaps be obviated if, instead of measuring weight per se, one attempted to measure the ratio as a function of the amount of fat? Then one possibly would not find differences between force-fed and experimental adiposity. DR. HAESSL~R. No. We have "brain-lesioned" animals that have been given restricted diets so
December 1964
they gained no weight. They have low ratios and normal total body fat content. We also have "brain-lesioned" animals that have been obese and brought back to normal weight by restricted feeding. They too have characteristically low ratios. Apparently the linoleic-palmitic ratio is not a function of the ~ize of the adipose organ, but a characteristic alteration due to the brain lesion. DR. BENJAMIN KRAMER, 4802 Tenth Ave., Brooklyn 19, N. Y. Did you study the influence of the age of the animal upon the rate of turnover of the fat? In the h u m a n adult the effect of change in diet is manifest after anywhere from six months to a year and a half, whereas the prematurely born can demonstrate such an effect as early as two or three weeks. DR. HAESSLER. We have studied animals in only a fairly small age range. O u r rats usually weighed around 200 grams when the lesions were made. We have made some lesions in animals as small as I00 grams and a few in larger animals. We have found no difference in the age within these relatively small limits in terms of the fatty acid composition after the lesion was made.
3. Of newborn hearts and nucleic acids Louis Gluck, ~ Norman S. Talner, Thomas H. Gardner, ~ and Marie V. Kulovich, r
Yale University School of Medicine, New Haven, Conn. Selectively increased R N A concentration in the left ventricle as compared to the right followed banding of the aorta in puppies. Similar selective increase in right ventricular RNA occured with pulmonary artery banding. Because of these observations R N A concentrations in the two ventricles were compared in the fetus and followed after birth to determine whether hemodynamic changes with birth and subsequent normal enlargement of the left ventricle are also associated with RNA differences. Studies in the rabbit showed equal concentrations of R N A in the ventricles of the fetus and the just born rabbit. Following delivery, left ventricular R N A rose to a peak concentration at 16 hours while right ventricular R N A levels changed little. Grossly increased left ventricular muscle mass was noted by 3 days, and R N A levels in both ventricles were again equal at 5 days. However, in premature rabbits delivered by cesarean section, no selective increase in R N A concentration in the left ventricle was seen, and the RNA levels in both ventricles remained equal during the first 30 hours of life. These findings suggest either that failure of response of the premature left ventricle is associated with immaturity or, more likely, that differences in the systemic vascular resistance in term and premature rabbits which vary the hemodynamic load on the left ventricle are reflected biochemically by differences in ventricular R N A levels and left ventricular protein synthesls. T h e findings
"YBy invitation. Asterisk indicates the same throughout.
Volume 65
Number 6
Abstracts
Part 2
also suggest a basic observation--that distortion of the myocardial cell stimulates RNA production. DISCUSSION DR. SOLOMON A. KAPLAN, Childrens Hospital, Los Angeles, Cali[. This is a most interesting biochemical phenomenon, that a mechanical stimulus can result in increased RNA synthesis and presumably subsequent protein synthesis. It would be of great interest to know what type of R N A is being increased as a result of the mechanical stimulus, ribosomal, messenger, or transfer RNA. Techniques for isolating myocardial ribosomes capable of synthesizing protein in vitro are now available. It would seem to me that fractionation of R N A in your experiments would be an area worth exploring. DR. GLUCK. As a matter of fact, this is under investigation currently. It has been our hypothesis that during embryonic development the appearance of a particular R N A marks the beginning of differentiation--perhaps the differentiation itself. We are attempting to isolate the various kinds of template R N A and other R N A that appear. The bulk of the R N A increase following hemodynamic stress is ribosomal as it is in the R N A synthesis in the very early embryo.
4. Aldosterone secretion rates (A.S.R.) in congenital adrenal hyperplasla Avinoam Kowarski, r Jordan W. Finkelstein, ~" John S. Spaulding, ~ Gerald Holman, r and Claude J. Migeon, The Johns Hopkins University, Baltimore, Md. A.S.R. (in micrograms per 24 hours) were measured in 9 patients with congenital adrenal hyperplasia by a modification of the method of Kliman and Peterson using 1,2-HS-D-aldosterone. In two adult patients with the non-salt losing type on 64 and 120 mEq. Na per 24 hours, the A.S.R. were 306 to 350 (our normal range 44 to 92). There was some increase in A.S.R. (434 and 600) when Na intake was restricted to 9 mEq. A 14-day-old untreated infant taking 10 mEq. of Na per 24 hours had an A.S.R. of 64. The A.S.R. remained unchanged (61) on the second day of a 3 mEq. Na intake per 24 hours during obvious clinical and laboratory signs of salt loss. Two other untreated infants under I month of age were studied when clinically well (8 and 4) and again during obvious salt loss (9 and 11). A fourth infant, aged 6 months, had an A.S.R. of 53 while taking a regular diet and 29 when restricted to 1 mEq. Na per 24 hours; D O C A pellets implanted 5~2 months earlier may have influenced the results in this patient. A 5-year-old salt loser had an A.S.R. of 22 #g twenty days after cessation of all therapy and normal Na diet. A 12-year-old male patient under cortisol treatment for the hypertensive form of the disease had a low A.S.R. (I9) while on a regular diet which did not increase (11) during a 9 mEq. Na diet.
10 7 3
An adult female with an atypical hypertensive form of the disease, while under cortisol treatment, had a low A.S.R. (24) on a regular diet which rose to 79 on the seventh day of 9 mEq. Na intake per 24 hours. We can conclude that the non-salt losing pa2 tients have high A.S.R. without clinical manifestations of hyperaldosteronism. The salt-losing and hypertensive patients had A.S.R. lower than those of the non-salt loser. DISCUSSION DR. LYTT I. GARDNER, State University o / N e w
York, Upstate Medical Center, Syracuse I0, N. Y. This is a very elegant study of a topic dear to the hearts of many of us who were pupils of Lawson Wilkins. Dr. Jos6 Cara, in our laboratory, has been much involved recently in a study of the juxtaglomerular apparatus and its relationship to virilizing adrenal hyperplasia and salt loss in this syndrome. He described hyperplasia and hypertrophy of the juxtaglomerular ceils of the kidney, and hypertrophy of the zone glomerulosa of the adrenals in autopsy specimens from a girl afflicted with the salt-losing form of this syndrome who died suddenly at age 4sA2 years, during the course of a febrile illness. It was hypothesized that the chronic state of salt loss caused changes in the intravascular compartment which led to compensatory activity of the juxtaglomerular cells with resultant stimulation of the zona glomerulosa of the adrenal, presumably through a reninangiotensin mechanism. This was in contrast with specimens obtained from autopsy of a 7-year-old boy with the hypertensive form of the disease who died suddenly during measles and who presented hypertrophy of the zona glomerulosa of the adrenals, without hypertrophy of the juxtaglomerular cells (Cara, J., and Gardner, L. I.: Pediatrics 32: 825, 1963). I wonder if you have had any opportunity to go into this aspect of your patients and make any observations along these lines. DR. KOWAI~SXL No, we did not study this question directly in these patients.
5. Immediate effects of phenylalanine-deficient diet in young in[ants D. Ingall, "~J. D. Sherman, ~ F. Coekburn, "~ and R. Klein, Boston City Hospital and Boston University School of Medicine, Boston, Mass. The effects of a phenylalanine-deficient diet on bone marrow and amino acid metabolism were studied in normal young infants of varying ages and maturity because the first two authors have reported the production of bone marrow changes similar to those seen in chloramphenicol toxicity in one anemic infant with phenylketonuria on a phenylalanine-deficient diet. Bone marrow exam-