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The effect of low doses of x-ray (diagnostic procedures) on the chromosomes of human infants
1 04 0
American Pediatric Society
a disproportionate part of the otherwise normal urinary amino acids, but was not absolutely increased. Blood urea nitrogen and total urea excretion were normal on a normal protein diet, as was uric acid and creatine excretion. Liver chemistry determinations were normal. Following the oral administration of Nl~-glycine and NIS-ammonium chloride the rates of incorporation of this stable isotope into urinary ammonia and urea were determined. There was little incorporation of N ~5 into urea, and prolonged elevation of ammonia N aS, indicative of a defect in urea synthesis. N15-citrulline was rapidly converted to urea. Enzyme analysis of liver biopsy material revealed a reduction in carbamyl phosphate synthetase. The other urea cycle enzymes were normal. The in vivo demonstration of an incomplete block in citrulline synthesis was thus confirmed. Intermittent block in glucose utilization, elevation of blood glycine, and cyclic neutropenia remain unexplained. DISCUSSION DERRICK LOXSDALE, Cleveland Clinic, Cleveland, Ohio. I wondered whether you had DR.
an opportunity to test the use of arginine, glutamic acid, or neomycin in bringing down the level of the ammonia. DR. W. L. NVnAN, Department o[ Pediatrics,
University of Miami School of Medicine, Coral Gables, Fla. This is indeed a scholarly work-up of a new problem, and we are interested in the very thorough comparison given of the similarities between this condition and hyperglycinemia. As I listened to the presentation, I was particularly struck by the nature of the ketosis which, as you point out, is one of the most striking and devastating features of hyperglycinemia. I wonder, therefore, why you tend to exclude the possibility that the ketoses might result from the sequestration of alpha-ketoglutarate by the excess of ammonia. Certainly the most beautiful demonstration that I know of for the experimental production of ketosis is given by the experiments by Recknagel and Potter which indicate that one can regularly produce accumulations of acetoacetate simply by the addition of ammonium chloride in vitro, which will, by depleting the citric acid cycle, result in acetoacetate formation. I have privately thought that the most likely. simple explanation for the ketosis that one sees in hyperglycinemia is that this might be a confirmation for the cycle of Shemin in which deltaamino-levulinic acid is formed from succinyI-CoA and glycine. One might think of the production of ketosis in hyperglycinemia as being due to the drainage of succinyl-CoA simply by the metabolism of excess amounts of glycine. It seems to me, then, that there is the possibility now of considering the existence of two syndromes unrelated to carbohydrate metabolism, which would provide a better argument for ketosis produced in this manner. De. FREE~AN. In answer to Dr. Lonsdale's question, we did not have an opportunity to test
December 1964
the effects of arginine or glutamic acid on reduction of blood ammonia. In view of the defect in converting ornithine to citrulline, we would not have expected arginine to stimulate the conversion of ammonia to urea. Since we were able to reduce the blood ammonia by restriction of dietary protein, we did not attempt to sterilize the intestine with neomycin. This was a chronic situation, and we were wary of the effects of prolonged neomycin administration. Dr. Nyhan's question touches one of the central and least understood problems of this condition and of hyperglycinemia: the mechanism of the ketosis which was the cause of death in our patient. Although sequestration of alpha-ketoglutarate has been postulated as the mechanism of hepatic coma, there has been little in vivo experimental evidence to support this thesis. Ammonia intoxication in vivo is not associated with ketosis. In our patient the ketosis was not related to the level of blood ammonia, or to the duration of ammonia elevation. While there would appear to be a blockage of the citric acid cycle both in this condition and in hyperglycinemia, whether this bIockage is secondary to depletion of one of the cycle intermediates or to some inhibiting substance is at present unclear. However, as Dr. Nyhan stated, a major manifestation of both of these conditions involves interference with carbohydrate metabolism, although the primary defect is in a different system.
19. The e]]ect of low doses of x-ray (diagnostic procedures) on the chromosomes of human infants Barbara Ruben Migeon, M.D., ~ and Timothy Merz, Ph.D., ~ Departments of Pediatrics and Radiological Sciences, Johns Hopkins University, Baltimore, Md. Introduced by Barton Childs, M.D. To determine the in vivo radiosensitivity of the chromosomes of infants, we examined metaphase chromosomes from leukocyte cultures of 4 infants less than 3 months of age following diagnostic x-ray procedures and of 5 unirradiated newborn controls. Replicate specimens were examined whenever possible and studies were repeated on the same subject at various times following irradiation. Chromosome preparations were analyzed in respect to number of cells containing chromosomal aberrations and the type of aberration, chromosome or chrmnatid. Diagnostic procedures included chest films, intravenous pyelograin, cinecystourethrogram, upper gastrointestinal series, aortogram, and cineangiocardiogram. Frequencies of abnormal cells in the infant control group ranged from 1.6 to 8 per cent. The irradiated infants showed an aberration frequency of approximately 20 to 24 per cent. Two infants were examined before and after diagnostic doses
Volume 65
Number 6
Part 2
of x-ray and showed an approximate doubling of chromatid breaks following x-ray. The predominant aberration type was the chromatid deletion, an aberrant chromosomal configuration resulting from damage to the chromosome when it is functionally bipartite, subsequent to DNA replication. Previous studies had indicated that in adults the chromosomes from circulating lymphocytes have not yet synthesized their DNA and should not, therefore, exhibit chromatid type aberrations. Subsequent studies of lymphocytes from unirradiated adults also showed a high frequency (10 to 22 per cent) of chromatid aberrations. Studies with tritiated thymidine indicated that there was no significant population of lymphocytes whose D N A had been previously replicated (in infant or adult), leading to the conclusion that induction of apparent chromatid deletion by x-ray is unlikely. In vitro studies of the effect of x-ray on lymphocyte populations in the G1 and G~ stages of the mitotic cycle emphasized the artifactual nature of the previously observed chromatid deletions but underscored the well-documented observation that chromosome aberrations result from x-ray exposure. DISCUSSION DR. P. E. CONI~N,Hospital for Sick Children, Toronto, Ontario. I am pleased you point out this note of caution in interpreting chromatid deletions. I noticed that the three examples you showed were at the edge of the cell. This is by far the most common position in which to see them. I think at the edge of the cell, particularly when you are air-drying them, is the place of greatest stress. We had begun a study with children who had cardiac catheterization but were unable to complete the study because we had changed the x-ray technique. I think you do find fragments and exchanges, but these may be more frequent in mongols or children who have a carrier type abnormality. I wondered if you noticed, where there were chromosomal changes, if there were actual carrier type abnormalities. DR. GLEN CAYLER, Sutter Hospital, Sacramento, Cali[. Do you have data on the duration of the chromosomal changes which you observed? In the experimental animal, chromosomal changes are more marked when exposure to the teratogen occurs during fetal life. Do you have data on x-radiated h u m a n fetuses ? DR. AUDREY K. BROWN, Department o[ Pedi-
atrics, University o[ Virginia Hospital, Charlottesville, Va. Have you cultured leukocytes from bone marrow of these infants ? DR. MIOEON. Your question, Dr. Conen, as it is phrased is not entirely clear. The artificial chromatid deletions that we have observed are found in unirradiated lymphocyte populations with a wide range of frequencies and, therefore, should not be interpreted as evidence of x-ray damage in children whether their karyotypes are normal or
Abstracts
1 04 1
abnormal. These chromatid breaks are most likely technical in origin but not, in our experience, limited to specimens which have been air-dried; we have observed them with the same kind of frequency in squash preparations; they are not invariably on the edge of the cell. Dr. Cayler, these chromatid aberrations are not induced in vivo, so would be expected to show no constant relationship with time following in vivo irradiation. We have not studied irradiated fetuses. Dr. Brown, we have not examined bone marrow from infants in regard to these observations.
20. Neonatal respiratory distress syndrome in rats: An experimentally reproducible disease associated with neurohormonal imbalance Edward C. Defoe, Jr., M.D., ~ Barbara A. Burke, M.D., ~ and John A. Anderson, M.D., Ph.D., Department of Pediatrics, University of Minnesota, Minneapolis, Minn. This study is based upon certain clinical and histopathologic observations which suggest that the h u m a n neonatal respiratory distress syndrome may be secondary to transient limitation in the ability of affected neonates to mobilize or activate sympathetic neurohormones. Direct establishment of such a relationship has been hampered by the practical limitations imposed by the seriously ill h u m a n neonate and by the technical problems of amine measurement in this period where normal values are at the lower limits of procedural sensitivity. The study demonstrates that alteration of sympathetic neurohormone metabolism through drug administration to the pregnant rat results in a respiratory distress syndrome in the neonatal rat. To minimize catecholamine stores available for mobilization at birth, pregnant inbred Sprague-Dawley rats were administered hypoglycemia-producing doses of insulin daily for a 14 to 18 day period prior to parturition. Approximately one week before delivery a pharmacologically active inhibitor (alpha-methyl-3,4-dihydroxyphenylalanine) of the metabolic decarboxylation of 3,4-dihydroxyphenylalanine was administered daily in varying dosage. Neonates of mothers so treated demonstrated striking differences from those delivered of mothers receiving no treatment, partial treatment with one or the other drug, or treatment with other drug combinations. The stillbirth rate was high (35 per cent) and approximately one half of the liveborn animals exhibited varying degrees of respiratory distress. In a few the distress was transient; in most, however, it was progressive. Clinical symptoms paralleled those of the disease in humans (delayed respirations, hypoactivity, flaring nostrils, intercostal retraction, progressive
American Pediatric Society
a disproportionate part of the otherwise normal urinary amino acids, but was not absolutely increased. Blood urea nitrogen and total urea excretion were normal on a normal protein diet, as was uric acid and creatine excretion. Liver chemistry determinations were normal. Following the oral administration of Nl~-glycine and NIS-ammonium chloride the rates of incorporation of this stable isotope into urinary ammonia and urea were determined. There was little incorporation of N ~5 into urea, and prolonged elevation of ammonia N aS, indicative of a defect in urea synthesis. N15-citrulline was rapidly converted to urea. Enzyme analysis of liver biopsy material revealed a reduction in carbamyl phosphate synthetase. The other urea cycle enzymes were normal. The in vivo demonstration of an incomplete block in citrulline synthesis was thus confirmed. Intermittent block in glucose utilization, elevation of blood glycine, and cyclic neutropenia remain unexplained. DISCUSSION DERRICK LOXSDALE, Cleveland Clinic, Cleveland, Ohio. I wondered whether you had DR.
an opportunity to test the use of arginine, glutamic acid, or neomycin in bringing down the level of the ammonia. DR. W. L. NVnAN, Department o[ Pediatrics,
University of Miami School of Medicine, Coral Gables, Fla. This is indeed a scholarly work-up of a new problem, and we are interested in the very thorough comparison given of the similarities between this condition and hyperglycinemia. As I listened to the presentation, I was particularly struck by the nature of the ketosis which, as you point out, is one of the most striking and devastating features of hyperglycinemia. I wonder, therefore, why you tend to exclude the possibility that the ketoses might result from the sequestration of alpha-ketoglutarate by the excess of ammonia. Certainly the most beautiful demonstration that I know of for the experimental production of ketosis is given by the experiments by Recknagel and Potter which indicate that one can regularly produce accumulations of acetoacetate simply by the addition of ammonium chloride in vitro, which will, by depleting the citric acid cycle, result in acetoacetate formation. I have privately thought that the most likely. simple explanation for the ketosis that one sees in hyperglycinemia is that this might be a confirmation for the cycle of Shemin in which deltaamino-levulinic acid is formed from succinyI-CoA and glycine. One might think of the production of ketosis in hyperglycinemia as being due to the drainage of succinyl-CoA simply by the metabolism of excess amounts of glycine. It seems to me, then, that there is the possibility now of considering the existence of two syndromes unrelated to carbohydrate metabolism, which would provide a better argument for ketosis produced in this manner. De. FREE~AN. In answer to Dr. Lonsdale's question, we did not have an opportunity to test
December 1964
the effects of arginine or glutamic acid on reduction of blood ammonia. In view of the defect in converting ornithine to citrulline, we would not have expected arginine to stimulate the conversion of ammonia to urea. Since we were able to reduce the blood ammonia by restriction of dietary protein, we did not attempt to sterilize the intestine with neomycin. This was a chronic situation, and we were wary of the effects of prolonged neomycin administration. Dr. Nyhan's question touches one of the central and least understood problems of this condition and of hyperglycinemia: the mechanism of the ketosis which was the cause of death in our patient. Although sequestration of alpha-ketoglutarate has been postulated as the mechanism of hepatic coma, there has been little in vivo experimental evidence to support this thesis. Ammonia intoxication in vivo is not associated with ketosis. In our patient the ketosis was not related to the level of blood ammonia, or to the duration of ammonia elevation. While there would appear to be a blockage of the citric acid cycle both in this condition and in hyperglycinemia, whether this bIockage is secondary to depletion of one of the cycle intermediates or to some inhibiting substance is at present unclear. However, as Dr. Nyhan stated, a major manifestation of both of these conditions involves interference with carbohydrate metabolism, although the primary defect is in a different system.
19. The e]]ect of low doses of x-ray (diagnostic procedures) on the chromosomes of human infants Barbara Ruben Migeon, M.D., ~ and Timothy Merz, Ph.D., ~ Departments of Pediatrics and Radiological Sciences, Johns Hopkins University, Baltimore, Md. Introduced by Barton Childs, M.D. To determine the in vivo radiosensitivity of the chromosomes of infants, we examined metaphase chromosomes from leukocyte cultures of 4 infants less than 3 months of age following diagnostic x-ray procedures and of 5 unirradiated newborn controls. Replicate specimens were examined whenever possible and studies were repeated on the same subject at various times following irradiation. Chromosome preparations were analyzed in respect to number of cells containing chromosomal aberrations and the type of aberration, chromosome or chrmnatid. Diagnostic procedures included chest films, intravenous pyelograin, cinecystourethrogram, upper gastrointestinal series, aortogram, and cineangiocardiogram. Frequencies of abnormal cells in the infant control group ranged from 1.6 to 8 per cent. The irradiated infants showed an aberration frequency of approximately 20 to 24 per cent. Two infants were examined before and after diagnostic doses
Volume 65
Number 6
Part 2
of x-ray and showed an approximate doubling of chromatid breaks following x-ray. The predominant aberration type was the chromatid deletion, an aberrant chromosomal configuration resulting from damage to the chromosome when it is functionally bipartite, subsequent to DNA replication. Previous studies had indicated that in adults the chromosomes from circulating lymphocytes have not yet synthesized their DNA and should not, therefore, exhibit chromatid type aberrations. Subsequent studies of lymphocytes from unirradiated adults also showed a high frequency (10 to 22 per cent) of chromatid aberrations. Studies with tritiated thymidine indicated that there was no significant population of lymphocytes whose D N A had been previously replicated (in infant or adult), leading to the conclusion that induction of apparent chromatid deletion by x-ray is unlikely. In vitro studies of the effect of x-ray on lymphocyte populations in the G1 and G~ stages of the mitotic cycle emphasized the artifactual nature of the previously observed chromatid deletions but underscored the well-documented observation that chromosome aberrations result from x-ray exposure. DISCUSSION DR. P. E. CONI~N,Hospital for Sick Children, Toronto, Ontario. I am pleased you point out this note of caution in interpreting chromatid deletions. I noticed that the three examples you showed were at the edge of the cell. This is by far the most common position in which to see them. I think at the edge of the cell, particularly when you are air-drying them, is the place of greatest stress. We had begun a study with children who had cardiac catheterization but were unable to complete the study because we had changed the x-ray technique. I think you do find fragments and exchanges, but these may be more frequent in mongols or children who have a carrier type abnormality. I wondered if you noticed, where there were chromosomal changes, if there were actual carrier type abnormalities. DR. GLEN CAYLER, Sutter Hospital, Sacramento, Cali[. Do you have data on the duration of the chromosomal changes which you observed? In the experimental animal, chromosomal changes are more marked when exposure to the teratogen occurs during fetal life. Do you have data on x-radiated h u m a n fetuses ? DR. AUDREY K. BROWN, Department o[ Pedi-
atrics, University o[ Virginia Hospital, Charlottesville, Va. Have you cultured leukocytes from bone marrow of these infants ? DR. MIOEON. Your question, Dr. Conen, as it is phrased is not entirely clear. The artificial chromatid deletions that we have observed are found in unirradiated lymphocyte populations with a wide range of frequencies and, therefore, should not be interpreted as evidence of x-ray damage in children whether their karyotypes are normal or
Abstracts
1 04 1
abnormal. These chromatid breaks are most likely technical in origin but not, in our experience, limited to specimens which have been air-dried; we have observed them with the same kind of frequency in squash preparations; they are not invariably on the edge of the cell. Dr. Cayler, these chromatid aberrations are not induced in vivo, so would be expected to show no constant relationship with time following in vivo irradiation. We have not studied irradiated fetuses. Dr. Brown, we have not examined bone marrow from infants in regard to these observations.
20. Neonatal respiratory distress syndrome in rats: An experimentally reproducible disease associated with neurohormonal imbalance Edward C. Defoe, Jr., M.D., ~ Barbara A. Burke, M.D., ~ and John A. Anderson, M.D., Ph.D., Department of Pediatrics, University of Minnesota, Minneapolis, Minn. This study is based upon certain clinical and histopathologic observations which suggest that the h u m a n neonatal respiratory distress syndrome may be secondary to transient limitation in the ability of affected neonates to mobilize or activate sympathetic neurohormones. Direct establishment of such a relationship has been hampered by the practical limitations imposed by the seriously ill h u m a n neonate and by the technical problems of amine measurement in this period where normal values are at the lower limits of procedural sensitivity. The study demonstrates that alteration of sympathetic neurohormone metabolism through drug administration to the pregnant rat results in a respiratory distress syndrome in the neonatal rat. To minimize catecholamine stores available for mobilization at birth, pregnant inbred Sprague-Dawley rats were administered hypoglycemia-producing doses of insulin daily for a 14 to 18 day period prior to parturition. Approximately one week before delivery a pharmacologically active inhibitor (alpha-methyl-3,4-dihydroxyphenylalanine) of the metabolic decarboxylation of 3,4-dihydroxyphenylalanine was administered daily in varying dosage. Neonates of mothers so treated demonstrated striking differences from those delivered of mothers receiving no treatment, partial treatment with one or the other drug, or treatment with other drug combinations. The stillbirth rate was high (35 per cent) and approximately one half of the liveborn animals exhibited varying degrees of respiratory distress. In a few the distress was transient; in most, however, it was progressive. Clinical symptoms paralleled those of the disease in humans (delayed respirations, hypoactivity, flaring nostrils, intercostal retraction, progressive