- Email: [email protected]
SCREENING FOR PHENYLKETONURIA
522 The accumulation of
p-hydroxyisovaleric
acid
and
P-M.C.G. is most probably caused by an enzyme defect in the degradation of leucine. This normally proceeds as follows: after transamination, oxidative decarboxylation, and dehydrogenation, leucine is converted to p—methylcrotonyl-CoA. By biotin-dependent CO2-fixation, this acid is converted
p-methylglutaconyl-CoA, which is further metabolised to P-hydroxy-p-methylglutaryl-CoA. A block in the CO2fixation mechanism was most probably the cause of the metabolic defect in our patient. Such a block would primarily lead to intracellular accumulation of P-hydroxyisovaleric acid.! Conjugation of &bgr;-methy1crotonyl-CoA with glycine is also possible, in the same way as hippuric acid and other glycine conjugates are formed.2 The appearance of large amounts of P-hydroxyisovaleric acid and P-M.C.G. in our patient can thus be explained by a defect of methylcrotonyl-CoA carboxylase (E.C. 6.4.1.4). The probability of a failure in the leucine-degradation pathway prompted us to try dietary treatment. In order to minimise the leucine intake the child was given a diet containing the minimum requirements of the essential aminoacids (about 150 mg. per kg. body-weight of leucine). After introduction of this diet, the excretion of P-hydroxyisovaleric acid dropped rapidly from 400 mg. per day to about 50 mg. per day, whereas only a slight reduction of P-M.C.G. was seen. With the reduced excretion of these metabolites, there was a coincident drop in the urinary excretion of creatine, from 90 mg. to 30-40 mg. per day. There were, however, no signs of improvement. The child died three months later, of pneumonia. Permission for
to
necropsy was refused. We are convinced that our patient had a hitherto undescribed inborn error of metabolism. The error is most probably genetically determined, since both the parents and the two siblings excrete one of the abnormal meta-
bolites. They
are probably healthy heterozygotes, while the patient was a homozygote. Clinically as well as biochemically the metabolic error is different from those previously described in leucine metabolism. Although patients with
isovaleric acidemia
present disorder and isovaleric acidwmia are two different errors of metabolism. Most likely the errors are caused by failures in two neighbouring enzymes in the leucinedegradation nathwav. Institute of Clinical Biochemistry,
Rikshospitalet, University of Oslo. Department of Pædiatrics, Haukeland Hospital, University of Bergen, Norway.
2. 3. 4. 5.
From 1963 to 1968 in the Massachusetts screening programme for phenylketonuria, two blood filter-paper specimens were requested on each infant, one to be obtained at the time of discharge from the newborn nursery (usually at 3-4 days of age) and the other to be obtained at the time of the first well-baby visit (usually at 4-6 weeks of age). This screening laboratory received the first specimen on about 98% of all infants born in the State. The second specimen was received from about 60-70% of all infants during the initial years of the programme and from about 80-90% of all infants during the latter years of the programme. During this period of time, the total number of first specimens received was 571,387 and the number of second specimens received was 435,465. All infants who were diagnosed as having phenylketonuria (on the basis of careful follow-up studies), and all except three cases of " atypical phenylketonuria ", had a phenylalanine elevation greater than 2 mg. per 100 ml. in the first blood specimen. We stopped requesting the second blood specimen after 1968, when it became evident that this specimen was not disclosing additional cases of phenylketonuria. However, we are now receiving a urine filter-paper specimen from over 85% of all infants at the age of 3-4 weeks. This urine specimen, though it is less reliable than a blood specimen in detecting phenylketonuria, allows many more metabolic disorders to be diagnosed. The first blood specimen continues to form the basis of phenylketonuria detection in Massachusetts. INITIAL BLOOD-PHENYLALANINE CONCENTRATIONS (AS DETERMINED BY GUTHRIE BACTERIAL-INHIBITION ASSAY), 1962-70, IN INFANTS " ATYPICAL DETERMINED TO HAVE EITHER PHENYLKETONURIA OR PHENYLKETONURIA "*
3-5
also show urinary excretion of P-hydroxyisovaleric acid, these patients do not excrete P-M.C.G. Furthermore, our patient did not accumulate isovaleric acid in the blood. Clinically there were also pronounced differences between our patient and patients with isovaleric acidxmia, a disease which is dominated by episodes of metabolic acidosis and coma, accompanied by a body odour of isovaleric acid. Isovaleric acidaemia is also characterised by drowsiness, cerebellar ataxia, hyperreflexia, and mild psychomotor retardation.3 Our patient had no tendency to metabolic acidosis. The main symptoms were muscular hypotonia and atrophy, probably of spinal origin. The disease progressed gradually, despite the effect of the diet in reducing the excretion of abnormal metabolites. The question therefore remains open whether or not her clinical symptoms were caused by the error in the leucine metabolism. It seems quite clear, however, that the
1.
SCREENING FOR PHENYLKETONURIA SIR,-Professor Hsia and Mr. Dobson1 suggest that some females with phenylketonuria or hyperphenylalaninsemia are not being identified via newborn screening because they have a slower rise in blood-phenylalanine levels during the newborn period than do males.
LORENTZ ELDJARN EGIL JELLUM ODDVAR STOKKE.
HELENE PANDE PER ERIK WAALER.
Bachhawat, B. K., Robinson, W. G., Coon, M. J. J. biol. Chem. 1956, 219, 539. Schachter, D., Taggart, J. V. ibid. 1954, 208, 263. Budd, M. A., Tanaka, K., Holmes, L. B., Efron, M. L., Crawford, J. D., Isselbacher, K. J. New Engl. J. Med. 1967, 277, 321. Tanaka, K., Orr, J. C., Isselbacher, K. J. Biochim. biophys. Acta, 1968, 152, 638. Tanaka, K., Isselbacher, K. J. J. biol. Chem. 1967, 242, 2966.
*
Specimens obtained between 2 and 8 days in females.
the ages of 2 and 15
days in males and
The accompanying table shows the blood-phenylalanine concentrations in the first blood specimen on all infants who have been classified as having either phenylketonuria or
" atypical phenylketonuria ". These blood specimens obtained between the ages of 2 and 15 days (average
were
5-7
days) in males and between the ages of 2 and 8 days (average 4-2 days) in females. All the infants with initial blood-phenylalanine concentrations below 8 mg. per 100 ml. were subsequently found to have " atypical phenylketonuria ".
Most of the infants with initial blood-
phenylalanine concentrations of 20 mg. per 100 ml. or greater were subsequently classified as having phenylketonuria on the basis of follow-up studies. Professor Knox and Dr. Kang have lately given information regarding many of these infants.2 In every instance, except for three infants with " atypical phenvlketonuria 1. 2.
"
(and who
Hsia, D. Y.-Y., Dobson, J. Lancet, 1970, i, 905. Knox, W. E., Kang, E. S. ibid. p. 1390.
are not
523
receiving specific therapy), the level of phenylalanine in the first specimen was high enough for clear differentiation from the normal infant. Whatever the comparative rise in phenylalanine between males and females, it would appear that females with phenylketonuria are not missed in the Massachusetts newborn screening programme. Division of Diagnostic Laboratories, State Laboratory Institute, Massachusetts Department of Public Health, Boston, Massachusetts 02130.
HARVEY L. LEVY VIVIAN E. SHIH VALERIE KAROLKEWICZ ROBERT A. MACCREADY.
AN UNUSUAL CASE OF RABIES SIR,-During service in Burma in 1945, I had under my care a Burmese Army officer with rabies. He remembered being bitten by a mad mongoose fourteen years previously, but was unaware of any more recent contact. I do not know whether he had any prophylactic treatment at the time. He died in a most unpleasant way with convulsions, despite injection of 21/4 grains of morphia and 5 grains of phenobarbitone in the few hours before death. A further point of interest in this case was his desperate increase in libido; in retrospect this may be analogous with the condition in animals where mares are known to go into season and bitches to go on heat. The pathological basis for this is presumably the infection of the hippocampus by the virus; Negri bodies were indeed seen on section of this patient’s
was
the
patients.
extremely low IgA values in borderline-psychotic This is at present under investigation.
Psychochemistry Institute, Rigshospitalet, Copenhagen, and Protein Laboratory, University of Copenhagen, Denmark.
ELISABETH BOCK B. WEEKE O. J. RAFAELSEN.
LITHIUM DISTRIBUTION IN THE BRAINS OF TWO MANIC PATIENTS SiR,-Lithium levels in serum, liver, and brain have been measured in animals treated with lithium salts, but there is little published information about the distribution of lithium in the human brain. We have measured lithium concentrations in various parts of the brains of two patients treated with lithium for mania. Both patients had received lithium carbonate, 900 mg. daily in three divided doses. Both died the same morning -the first after 4 days’ treatment, the second after 3 days’ treatment, the causes of death being respectively asthma and cardiac disease (probably beriberi, because of a grossly dilated heart and very fatty liver). Necropsies were performed 30 and 29 hours after death. Slices were taken from BRAIN LITHIUM LEVELS
hippocampus. Newton Abbot Hospital, Devon.
A. M. N. GARDNER.
IMMUNOGLOBULINS IN SCHIZOPHRENIC PATIENTS SIR,-Dr. Strahilevitz and Dr. Davis (Aug. 15, p. 370) detected significantly raised IgA levels in schizophrenic patients, but unlike Solomon et all were unable to find increased IgM. We have measured 20 serum proteins in 32 psychiatric patients and in 32 healthy medical-staff members matched for age and sex.2 The immunoglobulins were determined by the method of Laurell.3 All blood-samples were drawn during the first two weeks after admission, and all patients had been off drugs for at least one month. As shown in the accompanying table, we found normal IgA and low IgM values in the schizophrenic patients. We agree that it is important that the patients tested should be newly admitted, to avoid the possible influence on serum proteins of infection and malnutrition, but we believe it is of similar importance to eliminate the influence of psychotropic drugs. Perhaps our most interesting finding 1.
2. 3.
Solomon, G. F., Allansmith, M., McClellan, B., Amkraut, A. Archs gen. Psychiat. 1969, 20, 272. Bock, E., Weeke, B., Rafaelsen, O. J. Unpublished. Laurell, C. B. Anal. Biochem. 1966, 15, 45.
the frontal lobe, the lateral aspect of the cerebellum, and through the pons (transverse section), and were frozen pending testing. For testing they were thawed enough to allow specimens of grey and white matter to be cut out. (Specimens from cerebellum and pons contained both grey and white matter.) Lithium content was determined by the method of Schou,l using a UnicamSP90’ atomicabsorption spectrophotometer and lithium sulphate as standard solution. Later, the sodium and potassium levels were measured in the same specimens; but the results of these estimations are less reliable because no special precautions were taken to prevent contamination. The results are shown in the accompanying table. Of particular interest are the lithium levels in the pons, which Since the release of lithium were higher than elsewhere. 1.
Schou,
M. Acta
IMMUNOGLOBULIN LEVELS IN PATIENTS AND CONTROLS
pharmac.
tox.
1958, 15, 115.
p-hydroxyisovaleric
acid
and
P-M.C.G. is most probably caused by an enzyme defect in the degradation of leucine. This normally proceeds as follows: after transamination, oxidative decarboxylation, and dehydrogenation, leucine is converted to p—methylcrotonyl-CoA. By biotin-dependent CO2-fixation, this acid is converted
p-methylglutaconyl-CoA, which is further metabolised to P-hydroxy-p-methylglutaryl-CoA. A block in the CO2fixation mechanism was most probably the cause of the metabolic defect in our patient. Such a block would primarily lead to intracellular accumulation of P-hydroxyisovaleric acid.! Conjugation of &bgr;-methy1crotonyl-CoA with glycine is also possible, in the same way as hippuric acid and other glycine conjugates are formed.2 The appearance of large amounts of P-hydroxyisovaleric acid and P-M.C.G. in our patient can thus be explained by a defect of methylcrotonyl-CoA carboxylase (E.C. 6.4.1.4). The probability of a failure in the leucine-degradation pathway prompted us to try dietary treatment. In order to minimise the leucine intake the child was given a diet containing the minimum requirements of the essential aminoacids (about 150 mg. per kg. body-weight of leucine). After introduction of this diet, the excretion of P-hydroxyisovaleric acid dropped rapidly from 400 mg. per day to about 50 mg. per day, whereas only a slight reduction of P-M.C.G. was seen. With the reduced excretion of these metabolites, there was a coincident drop in the urinary excretion of creatine, from 90 mg. to 30-40 mg. per day. There were, however, no signs of improvement. The child died three months later, of pneumonia. Permission for
to
necropsy was refused. We are convinced that our patient had a hitherto undescribed inborn error of metabolism. The error is most probably genetically determined, since both the parents and the two siblings excrete one of the abnormal meta-
bolites. They
are probably healthy heterozygotes, while the patient was a homozygote. Clinically as well as biochemically the metabolic error is different from those previously described in leucine metabolism. Although patients with
isovaleric acidemia
present disorder and isovaleric acidwmia are two different errors of metabolism. Most likely the errors are caused by failures in two neighbouring enzymes in the leucinedegradation nathwav. Institute of Clinical Biochemistry,
Rikshospitalet, University of Oslo. Department of Pædiatrics, Haukeland Hospital, University of Bergen, Norway.
2. 3. 4. 5.
From 1963 to 1968 in the Massachusetts screening programme for phenylketonuria, two blood filter-paper specimens were requested on each infant, one to be obtained at the time of discharge from the newborn nursery (usually at 3-4 days of age) and the other to be obtained at the time of the first well-baby visit (usually at 4-6 weeks of age). This screening laboratory received the first specimen on about 98% of all infants born in the State. The second specimen was received from about 60-70% of all infants during the initial years of the programme and from about 80-90% of all infants during the latter years of the programme. During this period of time, the total number of first specimens received was 571,387 and the number of second specimens received was 435,465. All infants who were diagnosed as having phenylketonuria (on the basis of careful follow-up studies), and all except three cases of " atypical phenylketonuria ", had a phenylalanine elevation greater than 2 mg. per 100 ml. in the first blood specimen. We stopped requesting the second blood specimen after 1968, when it became evident that this specimen was not disclosing additional cases of phenylketonuria. However, we are now receiving a urine filter-paper specimen from over 85% of all infants at the age of 3-4 weeks. This urine specimen, though it is less reliable than a blood specimen in detecting phenylketonuria, allows many more metabolic disorders to be diagnosed. The first blood specimen continues to form the basis of phenylketonuria detection in Massachusetts. INITIAL BLOOD-PHENYLALANINE CONCENTRATIONS (AS DETERMINED BY GUTHRIE BACTERIAL-INHIBITION ASSAY), 1962-70, IN INFANTS " ATYPICAL DETERMINED TO HAVE EITHER PHENYLKETONURIA OR PHENYLKETONURIA "*
3-5
also show urinary excretion of P-hydroxyisovaleric acid, these patients do not excrete P-M.C.G. Furthermore, our patient did not accumulate isovaleric acid in the blood. Clinically there were also pronounced differences between our patient and patients with isovaleric acidxmia, a disease which is dominated by episodes of metabolic acidosis and coma, accompanied by a body odour of isovaleric acid. Isovaleric acidaemia is also characterised by drowsiness, cerebellar ataxia, hyperreflexia, and mild psychomotor retardation.3 Our patient had no tendency to metabolic acidosis. The main symptoms were muscular hypotonia and atrophy, probably of spinal origin. The disease progressed gradually, despite the effect of the diet in reducing the excretion of abnormal metabolites. The question therefore remains open whether or not her clinical symptoms were caused by the error in the leucine metabolism. It seems quite clear, however, that the
1.
SCREENING FOR PHENYLKETONURIA SIR,-Professor Hsia and Mr. Dobson1 suggest that some females with phenylketonuria or hyperphenylalaninsemia are not being identified via newborn screening because they have a slower rise in blood-phenylalanine levels during the newborn period than do males.
LORENTZ ELDJARN EGIL JELLUM ODDVAR STOKKE.
HELENE PANDE PER ERIK WAALER.
Bachhawat, B. K., Robinson, W. G., Coon, M. J. J. biol. Chem. 1956, 219, 539. Schachter, D., Taggart, J. V. ibid. 1954, 208, 263. Budd, M. A., Tanaka, K., Holmes, L. B., Efron, M. L., Crawford, J. D., Isselbacher, K. J. New Engl. J. Med. 1967, 277, 321. Tanaka, K., Orr, J. C., Isselbacher, K. J. Biochim. biophys. Acta, 1968, 152, 638. Tanaka, K., Isselbacher, K. J. J. biol. Chem. 1967, 242, 2966.
*
Specimens obtained between 2 and 8 days in females.
the ages of 2 and 15
days in males and
The accompanying table shows the blood-phenylalanine concentrations in the first blood specimen on all infants who have been classified as having either phenylketonuria or
" atypical phenylketonuria ". These blood specimens obtained between the ages of 2 and 15 days (average
were
5-7
days) in males and between the ages of 2 and 8 days (average 4-2 days) in females. All the infants with initial blood-phenylalanine concentrations below 8 mg. per 100 ml. were subsequently found to have " atypical phenylketonuria ".
Most of the infants with initial blood-
phenylalanine concentrations of 20 mg. per 100 ml. or greater were subsequently classified as having phenylketonuria on the basis of follow-up studies. Professor Knox and Dr. Kang have lately given information regarding many of these infants.2 In every instance, except for three infants with " atypical phenvlketonuria 1. 2.
"
(and who
Hsia, D. Y.-Y., Dobson, J. Lancet, 1970, i, 905. Knox, W. E., Kang, E. S. ibid. p. 1390.
are not
523
receiving specific therapy), the level of phenylalanine in the first specimen was high enough for clear differentiation from the normal infant. Whatever the comparative rise in phenylalanine between males and females, it would appear that females with phenylketonuria are not missed in the Massachusetts newborn screening programme. Division of Diagnostic Laboratories, State Laboratory Institute, Massachusetts Department of Public Health, Boston, Massachusetts 02130.
HARVEY L. LEVY VIVIAN E. SHIH VALERIE KAROLKEWICZ ROBERT A. MACCREADY.
AN UNUSUAL CASE OF RABIES SIR,-During service in Burma in 1945, I had under my care a Burmese Army officer with rabies. He remembered being bitten by a mad mongoose fourteen years previously, but was unaware of any more recent contact. I do not know whether he had any prophylactic treatment at the time. He died in a most unpleasant way with convulsions, despite injection of 21/4 grains of morphia and 5 grains of phenobarbitone in the few hours before death. A further point of interest in this case was his desperate increase in libido; in retrospect this may be analogous with the condition in animals where mares are known to go into season and bitches to go on heat. The pathological basis for this is presumably the infection of the hippocampus by the virus; Negri bodies were indeed seen on section of this patient’s
was
the
patients.
extremely low IgA values in borderline-psychotic This is at present under investigation.
Psychochemistry Institute, Rigshospitalet, Copenhagen, and Protein Laboratory, University of Copenhagen, Denmark.
ELISABETH BOCK B. WEEKE O. J. RAFAELSEN.
LITHIUM DISTRIBUTION IN THE BRAINS OF TWO MANIC PATIENTS SiR,-Lithium levels in serum, liver, and brain have been measured in animals treated with lithium salts, but there is little published information about the distribution of lithium in the human brain. We have measured lithium concentrations in various parts of the brains of two patients treated with lithium for mania. Both patients had received lithium carbonate, 900 mg. daily in three divided doses. Both died the same morning -the first after 4 days’ treatment, the second after 3 days’ treatment, the causes of death being respectively asthma and cardiac disease (probably beriberi, because of a grossly dilated heart and very fatty liver). Necropsies were performed 30 and 29 hours after death. Slices were taken from BRAIN LITHIUM LEVELS
hippocampus. Newton Abbot Hospital, Devon.
A. M. N. GARDNER.
IMMUNOGLOBULINS IN SCHIZOPHRENIC PATIENTS SIR,-Dr. Strahilevitz and Dr. Davis (Aug. 15, p. 370) detected significantly raised IgA levels in schizophrenic patients, but unlike Solomon et all were unable to find increased IgM. We have measured 20 serum proteins in 32 psychiatric patients and in 32 healthy medical-staff members matched for age and sex.2 The immunoglobulins were determined by the method of Laurell.3 All blood-samples were drawn during the first two weeks after admission, and all patients had been off drugs for at least one month. As shown in the accompanying table, we found normal IgA and low IgM values in the schizophrenic patients. We agree that it is important that the patients tested should be newly admitted, to avoid the possible influence on serum proteins of infection and malnutrition, but we believe it is of similar importance to eliminate the influence of psychotropic drugs. Perhaps our most interesting finding 1.
2. 3.
Solomon, G. F., Allansmith, M., McClellan, B., Amkraut, A. Archs gen. Psychiat. 1969, 20, 272. Bock, E., Weeke, B., Rafaelsen, O. J. Unpublished. Laurell, C. B. Anal. Biochem. 1966, 15, 45.
the frontal lobe, the lateral aspect of the cerebellum, and through the pons (transverse section), and were frozen pending testing. For testing they were thawed enough to allow specimens of grey and white matter to be cut out. (Specimens from cerebellum and pons contained both grey and white matter.) Lithium content was determined by the method of Schou,l using a UnicamSP90’ atomicabsorption spectrophotometer and lithium sulphate as standard solution. Later, the sodium and potassium levels were measured in the same specimens; but the results of these estimations are less reliable because no special precautions were taken to prevent contamination. The results are shown in the accompanying table. Of particular interest are the lithium levels in the pons, which Since the release of lithium were higher than elsewhere. 1.
Schou,
M. Acta
IMMUNOGLOBULIN LEVELS IN PATIENTS AND CONTROLS
pharmac.
tox.
1958, 15, 115.