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A SIMPLE BIOLOGICAL TEST FOR CATECHOL-AMINES IN URINE
1154 puncture or from air encephalography, it was found that the thickness of the pallium was not related to intelligence. As examples, a child with cortical thickness of 0-5 cm. estimated from a ventriculogram, had an i.Q. of 85, and another of 100. (3) No significant relationship was found between the intelligence quotient and the length of time that the hydrocephalus had been progressive. (4) A small correlation (significant at the 5% but not at the 1% level) existed between the intelligence quotient and age, the scores tending to rise slightly with age, presumably because a child overcomes some of his physical handicaps, and is therefore able to have more intelligence available for measurement by the tests employed. (5) A high correlation (0-77) was found between intelligence quotient and the degree of physical handicap; those with least handicap scoring higher. This relationship will be partly caused by the failure of tests to measure the remaining ability.
that should a hydrocephalic child survive to spontaneously, he has a 75% chance of being educable, and a 57% chance of being taught, apart from his physical handicap, in a school for normal children. In fact, of the 81 cases with arrested hydrocephalus which were traced, 25 are children of normal intelligence with negligible or no physical handicap. A more detailed analysis of the results, together with the prognosis of the various ætiological groups, will be published elsewhere. It
arrested spontaneously, 9 remain progressive, and 3 have been traced, but are probably alive. Of the traced survivors with spontaneous arrest, 75% are educable, and the intelligence quotient of 57% falls within the normal range (i.Q. 85 and above). The relationship between intelligence and other features (head size, cortical thickness, and physical condition and age) is briefly discussed. The prognosis and the results of surgery in this condition may have to be reconsidered. I am greatly indebted to Mr. Wylie McKissock for permission to use his cases, to Mr. Stephen Coates for his part in this investigation, not
and to Prof. A. A. Moncrieff and Dr. M. Bodian for advice and criticism. My thanks are also due to Miss K. M. Hare, Miss J. C. Marson, and Miss M. Crawford for help in finding the case-records; to Miss M-L. Lepine for invaluable secretarial assistance; to Mr. G. Lyth for the charts; and to the research committee of The Hospital for Sick Children, Great Ormond Street for encouragement and ’ financial suooort.
seems
arrest
REFERENCES
Ford, F. R. (1952) Diseases of the Nervous System in Infancy, Childhood and Adolescence; p. 255. Springfield, Ill. Forrest, D. M., Laurence, K. M., Macnab, G. H. (1957) Lancet, i, 1274. Holt, L. E., McIntosh, R. (1953) Pediatrics; p. 1055. New York. Ingraham, F. D., Matson, D. D. (1954) Neurosurgery of Infancy and Childhood; p. 117. Springfield, Ill. Jolly, H. (1955) Proc. R. Soc. Med. 48, 843. Macnab, G. H. (1955) ibid. p. 846.
A SIMPLE BIOLOGICAL TEST FOR CATECHOL-AMINES IN URINE
Discussion
generally believed that few children with hydrocephalus survive, and that those who do are doomed to permanent institutional care. Ford (1952) agrees that in some children the disease seems to become arrested, but adds that in such cases the child may go through life with more or less mental defect, optic atrophy, and spastic paralysis. Holt and McIntosh (1953) state that congenital hydrocephalus may become arrested at any
M. A. FLOYER M.A., M.D. Cantab., M.R.C.P.
It is
stage, but maintain that such an outcome is rare. Clinicians who commonly deal with this problem are well aware that natural arrest is quite common. Ingraham and Matson (1954) briefly refer to it but make no mention of its incidence, and Jolly (1955) discusses only its importance. On the other hand Macnab (1955) reports 19 cases with spontaneous remission among his 160 patients. Forrest, Laurence, and Macnab (1957), when following up their patients seen between 1954 and 1955, found that 30 of their 70 cases had arrested spontaneously, but no reference was made to their physical or mental condition. It may be thought that the high rate of arrest is based on an unrepresentative series of less severe cases, the more progressive being selected for operation. This in fact is not so, since those patients receiving surgical treatment tended to have less advanced hydrocephalus. It should be re-emphasised, however, that this is a series based on the neurosurgical practice of a large children’s hospital and that it is not entirely representative of the worst cases of hydrocephalus, which are fatal in the first three months, nor of the mildest forms of hydrocephalus. This investigation suggests a more hopeful prognosis, for an appreciable number of these cases have not only survived but are now growing into useful citizens capable of earning their living. Further, in view of the 46% spontaneous-arrest rate, the reported results of surgery in hydrocephalus may have to be reassessed.
Summary 182 unselected and unoperated cases of hydrocephalus seen by one surgeon during the past twenty years have been followed up. Of these, 89 have died, 81 have
ASSISTANT DIRECTOR, MEDICAL UNIT, THE LONDON
HOSPITAL, E.1
With the technical assistance of A. R. REYNAUD
Euler and Hellner (1951) described a biological method for estimating catechol-amines in the urine, After adsorption on alumina the catechol-amines are eluted and assayed in a cat. Hamilton et al. (1953) described a similar method in which the assay was car. ried out in the rat. A more rapid approximate estimation of urinary catechol-amines can be made by measuring the rise in bloodpressure after direct intravenous injection of urine into an anæsthetised rat. This method is simple, requires little apparatus, and has proved suitable for screening large numbers of patients with hypertension to exclude chaeochromocvtoma. Since we began using this techvon
nique
over
a
year ago,
Masson et al. (1957) have described a similar method. Method A Wistar albino rat (about 250 g.) is anaesthetised by
intraperitoneal injection of 015 ml. of 50%solution of
urethane; simultaneously two
separate subcutaneous
injections of 0.2 ml. are given to maintain anxsthesia for several hours. A simple manometer is made by filling a’Polythene ’ tube (external diameter 1 mm,. internal diameter 05 mm. with heparinised saline 5L’ units per ml.) and drawing mercury
(about
20
cm.
into one end see The part of the tube containing the mercury
column) figure).
1155 is bent to form a U and mounted vertically on the scale; the other end is inserted into the rat’s carotid artery. Injections are made with a 1 ml. graduated tuberculin syringe through a 16-gauge needle connected to a short polythene cannula placed in the external jugular vein. After injection of 0-2 ml. of heparinised saline, 0.50% solution of pentolmium tartrate (’Ansolysen’) is infused slowly until there is no further fall in blood-pressure. Usually 0-2 to 0-3 ml. is required and reduces the blood-pressure to 50 or 60 mm. Hg. A standard solution containing 0-1 µg. of noradrenaline per ml. is prepared by diluting noradrenaline (’ Levophed’) 1 in 10,000 in normal saline. 01ml. of this solution gives a sharp but transient rise of blood-pressure varying from 5 to 20 mm. Hg in different rats; in the same animal the rise following repeated injections is fairly constant over a period of 1/2-1hour. acidified by adding 1 to 2 ml. concentrated HCl per 100 ml. urine, can be kept for several days in a refrigerator (2’C). Just before use solid sodium bicarbonate is added in amounts sufficient to bring the pH to between 6 and 7 (tested by ’Universal’ indicator paper). The urine is then infused cautiously into the external jugular vein of the test animal; if there is no sharp pressor response an injection of 0.1ml. is made. If this causes a significant rise in blood-pressure (more than half that following 0-1 ml. of noradrenaline solution) a rough assay is made by finding the amount of noradrenaline solution needed to give the same rise as a known amount of urine (usually 0-1 ml.). Urine which has a marked pressor effect is suitably diluted in normal saline before making the assay. Evidence that the pressor substance is noradrenaline or adrenaline is obtained by injecting 0-1 ml. of 0-05% phentolamine (’Rogitine ’ diluted 1 in 10 in saline) into the rat; this inhibits the pressor effect of subsequent injections of urine containing catechol-amines. Up to 15 samples can be tested in the same rat (before phentolamine is given) provided that responsiveness is maintained; this is tested by injection of noradrenaline solution after each urine sample. If the response to noradrenaline is small, larger injections of urine (up to 0-4 ml.) are required so that a smaller number of samples can be tested.
Urine,
Results
The results of the assay of 24-hour samples of urine from six patients suffering from phxochromocytoma are given in the table and are compared with the results obtained from the same samples of urine by a chemical method (Wright 1958). The diagnosis was confirmed at operation in all patients. We have examined 24-hour samples of urine in 59 other patients suffering from severe hypertension or with symptoms suggestive of phaEochromocytoma, in all but 2 the daily excretion was less than 100 µg. noradrenaline per day. One patient excreted 140 µg. and another 250 ug. but there was no clinical evidence of phxochromocytoma ; repeated examination showed no excess of noradrenaline in subsequent urine samples. We have also examined " casual " specimens of urine from over 100 outpatients suffering from different forms
hypertension. Concentrations of noradrenaline of 0-1 Lg. per ml. were found in 2 samples, but at subsequent examination urine from these patients was normal. No other sample gave a significant pressor response.
of
Discussion
It is not claimed that this is an accurate method of assay; it is, on the other hand, a quick method for screening using simple apparatus and needing little technical
skill. The assay results are expressed as noradrenaline. Added adrenaline does not increase the pressor effect of noradrenaline when the two are injected together into the that this method probably measures noradrenaline and only may give a low result for total catechol-amines in patients excreting a high proportion of adrenaline. In all 6 patients with proven phæochromocytoma the pressor effects of the urine on the rat left no doubt about the diagnosis, whereas in very few instances did urine from the hypertensive patients give a measurable pressor response. There may very occasionally be some doubt in differentiating the latter from cases of phæochromocytoma in whom the excretion is low, but in the great majority the separation is easy and reliable.
rat so
I am grateful to Professor Clifford Wilson preparation of this paper.
for his
help
in the
REFERENCES
W. S., Sowry, G. S. C. (1953) Brit. Heart J. 15, 241. Masson, G. M. C., Corcoran, A. C., Humphrey, D. C. (1957) J. Amer. med. Ass. 165, 1555. von Euler, U. S., Hellner, S. (1951) Acta physiol. scand. 22, 161. Wright, J. T. (1958) Lancet, ii, 1155.
Hamilton, M., Litchfield, J. W., Peart,
A RAPID QUANTITATIVE METHOD FOR CHEMICAL ESTIMATION OF URINARY CATECHOL-AMINES IN THE DIAGNOSIS OF PHÆOCHROMOCYTOMA
J. T. WRIGHT D.M.
Oxon., M.R.C.P.
ASSISTANT PHYSICIAN, LONDON HOSPITAL, LONDON, E.1
THE usual method for the extraction and preliminary purification of urinary catechols is to adsorb them with alumina (von Euler and Hellner 1951, Lund 1949, Pekharinen and Pitkanen 1955). The main disadvantage of this method is that alumina is difficult to standardise, and no two batches can be relied upon to give the same performance. Moreover, adsorption of catechol-amines on to alumina requires careful manipulation at pH levels which are unfavourable to their stability. The synthetic inorganic materialPermutit’ was first used by Whitehorn (1923) to extract adrenaline from solution, and more recently Bergstrom and Hansson (1951) and Hellberg (1955) have used the carboxylic cation-exchange resin, ’Amberlite ’ IRC 50, for the same ESTIMATED BY BIOASSAI 24-HOUR EXCRETION OF PRESSOR AMINES purpose. Weil-Malherbe and Bone (1957) and Burn and COMPARED WITH RESULTS OF CHEMICAL METHOD ON SAME SAMPLI Field (1956) use cation-exchange resins as a subsidiary means of purifying catechol-amines after preliminary extraction from urine with alumina columns. In the work recorded here I am indebted to Prof. C. J. 0. R. Morris for suggesting that catechol-amines could be extracted from solution as boric-acid complexes by means of anion-exchange resins. This has provided a method for extraction and estimation of these substances which can be carried out rapidly by a skilled ’This specimen had been kept for several months between chemical and bioassay. technician and which gives a degree of accuracy compar+1 am grateful to Dr. A. L. Jacobs, of the Whittington Hospital, for letting able to existing but more complicated methods. me have the urine from one of his patients.
that should a hydrocephalic child survive to spontaneously, he has a 75% chance of being educable, and a 57% chance of being taught, apart from his physical handicap, in a school for normal children. In fact, of the 81 cases with arrested hydrocephalus which were traced, 25 are children of normal intelligence with negligible or no physical handicap. A more detailed analysis of the results, together with the prognosis of the various ætiological groups, will be published elsewhere. It
arrested spontaneously, 9 remain progressive, and 3 have been traced, but are probably alive. Of the traced survivors with spontaneous arrest, 75% are educable, and the intelligence quotient of 57% falls within the normal range (i.Q. 85 and above). The relationship between intelligence and other features (head size, cortical thickness, and physical condition and age) is briefly discussed. The prognosis and the results of surgery in this condition may have to be reconsidered. I am greatly indebted to Mr. Wylie McKissock for permission to use his cases, to Mr. Stephen Coates for his part in this investigation, not
and to Prof. A. A. Moncrieff and Dr. M. Bodian for advice and criticism. My thanks are also due to Miss K. M. Hare, Miss J. C. Marson, and Miss M. Crawford for help in finding the case-records; to Miss M-L. Lepine for invaluable secretarial assistance; to Mr. G. Lyth for the charts; and to the research committee of The Hospital for Sick Children, Great Ormond Street for encouragement and ’ financial suooort.
seems
arrest
REFERENCES
Ford, F. R. (1952) Diseases of the Nervous System in Infancy, Childhood and Adolescence; p. 255. Springfield, Ill. Forrest, D. M., Laurence, K. M., Macnab, G. H. (1957) Lancet, i, 1274. Holt, L. E., McIntosh, R. (1953) Pediatrics; p. 1055. New York. Ingraham, F. D., Matson, D. D. (1954) Neurosurgery of Infancy and Childhood; p. 117. Springfield, Ill. Jolly, H. (1955) Proc. R. Soc. Med. 48, 843. Macnab, G. H. (1955) ibid. p. 846.
A SIMPLE BIOLOGICAL TEST FOR CATECHOL-AMINES IN URINE
Discussion
generally believed that few children with hydrocephalus survive, and that those who do are doomed to permanent institutional care. Ford (1952) agrees that in some children the disease seems to become arrested, but adds that in such cases the child may go through life with more or less mental defect, optic atrophy, and spastic paralysis. Holt and McIntosh (1953) state that congenital hydrocephalus may become arrested at any
M. A. FLOYER M.A., M.D. Cantab., M.R.C.P.
It is
stage, but maintain that such an outcome is rare. Clinicians who commonly deal with this problem are well aware that natural arrest is quite common. Ingraham and Matson (1954) briefly refer to it but make no mention of its incidence, and Jolly (1955) discusses only its importance. On the other hand Macnab (1955) reports 19 cases with spontaneous remission among his 160 patients. Forrest, Laurence, and Macnab (1957), when following up their patients seen between 1954 and 1955, found that 30 of their 70 cases had arrested spontaneously, but no reference was made to their physical or mental condition. It may be thought that the high rate of arrest is based on an unrepresentative series of less severe cases, the more progressive being selected for operation. This in fact is not so, since those patients receiving surgical treatment tended to have less advanced hydrocephalus. It should be re-emphasised, however, that this is a series based on the neurosurgical practice of a large children’s hospital and that it is not entirely representative of the worst cases of hydrocephalus, which are fatal in the first three months, nor of the mildest forms of hydrocephalus. This investigation suggests a more hopeful prognosis, for an appreciable number of these cases have not only survived but are now growing into useful citizens capable of earning their living. Further, in view of the 46% spontaneous-arrest rate, the reported results of surgery in hydrocephalus may have to be reassessed.
Summary 182 unselected and unoperated cases of hydrocephalus seen by one surgeon during the past twenty years have been followed up. Of these, 89 have died, 81 have
ASSISTANT DIRECTOR, MEDICAL UNIT, THE LONDON
HOSPITAL, E.1
With the technical assistance of A. R. REYNAUD
Euler and Hellner (1951) described a biological method for estimating catechol-amines in the urine, After adsorption on alumina the catechol-amines are eluted and assayed in a cat. Hamilton et al. (1953) described a similar method in which the assay was car. ried out in the rat. A more rapid approximate estimation of urinary catechol-amines can be made by measuring the rise in bloodpressure after direct intravenous injection of urine into an anæsthetised rat. This method is simple, requires little apparatus, and has proved suitable for screening large numbers of patients with hypertension to exclude chaeochromocvtoma. Since we began using this techvon
nique
over
a
year ago,
Masson et al. (1957) have described a similar method. Method A Wistar albino rat (about 250 g.) is anaesthetised by
intraperitoneal injection of 015 ml. of 50%solution of
urethane; simultaneously two
separate subcutaneous
injections of 0.2 ml. are given to maintain anxsthesia for several hours. A simple manometer is made by filling a’Polythene ’ tube (external diameter 1 mm,. internal diameter 05 mm. with heparinised saline 5L’ units per ml.) and drawing mercury
(about
20
cm.
into one end see The part of the tube containing the mercury
column) figure).
1155 is bent to form a U and mounted vertically on the scale; the other end is inserted into the rat’s carotid artery. Injections are made with a 1 ml. graduated tuberculin syringe through a 16-gauge needle connected to a short polythene cannula placed in the external jugular vein. After injection of 0-2 ml. of heparinised saline, 0.50% solution of pentolmium tartrate (’Ansolysen’) is infused slowly until there is no further fall in blood-pressure. Usually 0-2 to 0-3 ml. is required and reduces the blood-pressure to 50 or 60 mm. Hg. A standard solution containing 0-1 µg. of noradrenaline per ml. is prepared by diluting noradrenaline (’ Levophed’) 1 in 10,000 in normal saline. 01ml. of this solution gives a sharp but transient rise of blood-pressure varying from 5 to 20 mm. Hg in different rats; in the same animal the rise following repeated injections is fairly constant over a period of 1/2-1hour. acidified by adding 1 to 2 ml. concentrated HCl per 100 ml. urine, can be kept for several days in a refrigerator (2’C). Just before use solid sodium bicarbonate is added in amounts sufficient to bring the pH to between 6 and 7 (tested by ’Universal’ indicator paper). The urine is then infused cautiously into the external jugular vein of the test animal; if there is no sharp pressor response an injection of 0.1ml. is made. If this causes a significant rise in blood-pressure (more than half that following 0-1 ml. of noradrenaline solution) a rough assay is made by finding the amount of noradrenaline solution needed to give the same rise as a known amount of urine (usually 0-1 ml.). Urine which has a marked pressor effect is suitably diluted in normal saline before making the assay. Evidence that the pressor substance is noradrenaline or adrenaline is obtained by injecting 0-1 ml. of 0-05% phentolamine (’Rogitine ’ diluted 1 in 10 in saline) into the rat; this inhibits the pressor effect of subsequent injections of urine containing catechol-amines. Up to 15 samples can be tested in the same rat (before phentolamine is given) provided that responsiveness is maintained; this is tested by injection of noradrenaline solution after each urine sample. If the response to noradrenaline is small, larger injections of urine (up to 0-4 ml.) are required so that a smaller number of samples can be tested.
Urine,
Results
The results of the assay of 24-hour samples of urine from six patients suffering from phxochromocytoma are given in the table and are compared with the results obtained from the same samples of urine by a chemical method (Wright 1958). The diagnosis was confirmed at operation in all patients. We have examined 24-hour samples of urine in 59 other patients suffering from severe hypertension or with symptoms suggestive of phaEochromocytoma, in all but 2 the daily excretion was less than 100 µg. noradrenaline per day. One patient excreted 140 µg. and another 250 ug. but there was no clinical evidence of phxochromocytoma ; repeated examination showed no excess of noradrenaline in subsequent urine samples. We have also examined " casual " specimens of urine from over 100 outpatients suffering from different forms
hypertension. Concentrations of noradrenaline of 0-1 Lg. per ml. were found in 2 samples, but at subsequent examination urine from these patients was normal. No other sample gave a significant pressor response.
of
Discussion
It is not claimed that this is an accurate method of assay; it is, on the other hand, a quick method for screening using simple apparatus and needing little technical
skill. The assay results are expressed as noradrenaline. Added adrenaline does not increase the pressor effect of noradrenaline when the two are injected together into the that this method probably measures noradrenaline and only may give a low result for total catechol-amines in patients excreting a high proportion of adrenaline. In all 6 patients with proven phæochromocytoma the pressor effects of the urine on the rat left no doubt about the diagnosis, whereas in very few instances did urine from the hypertensive patients give a measurable pressor response. There may very occasionally be some doubt in differentiating the latter from cases of phæochromocytoma in whom the excretion is low, but in the great majority the separation is easy and reliable.
rat so
I am grateful to Professor Clifford Wilson preparation of this paper.
for his
help
in the
REFERENCES
W. S., Sowry, G. S. C. (1953) Brit. Heart J. 15, 241. Masson, G. M. C., Corcoran, A. C., Humphrey, D. C. (1957) J. Amer. med. Ass. 165, 1555. von Euler, U. S., Hellner, S. (1951) Acta physiol. scand. 22, 161. Wright, J. T. (1958) Lancet, ii, 1155.
Hamilton, M., Litchfield, J. W., Peart,
A RAPID QUANTITATIVE METHOD FOR CHEMICAL ESTIMATION OF URINARY CATECHOL-AMINES IN THE DIAGNOSIS OF PHÆOCHROMOCYTOMA
J. T. WRIGHT D.M.
Oxon., M.R.C.P.
ASSISTANT PHYSICIAN, LONDON HOSPITAL, LONDON, E.1
THE usual method for the extraction and preliminary purification of urinary catechols is to adsorb them with alumina (von Euler and Hellner 1951, Lund 1949, Pekharinen and Pitkanen 1955). The main disadvantage of this method is that alumina is difficult to standardise, and no two batches can be relied upon to give the same performance. Moreover, adsorption of catechol-amines on to alumina requires careful manipulation at pH levels which are unfavourable to their stability. The synthetic inorganic materialPermutit’ was first used by Whitehorn (1923) to extract adrenaline from solution, and more recently Bergstrom and Hansson (1951) and Hellberg (1955) have used the carboxylic cation-exchange resin, ’Amberlite ’ IRC 50, for the same ESTIMATED BY BIOASSAI 24-HOUR EXCRETION OF PRESSOR AMINES purpose. Weil-Malherbe and Bone (1957) and Burn and COMPARED WITH RESULTS OF CHEMICAL METHOD ON SAME SAMPLI Field (1956) use cation-exchange resins as a subsidiary means of purifying catechol-amines after preliminary extraction from urine with alumina columns. In the work recorded here I am indebted to Prof. C. J. 0. R. Morris for suggesting that catechol-amines could be extracted from solution as boric-acid complexes by means of anion-exchange resins. This has provided a method for extraction and estimation of these substances which can be carried out rapidly by a skilled ’This specimen had been kept for several months between chemical and bioassay. technician and which gives a degree of accuracy compar+1 am grateful to Dr. A. L. Jacobs, of the Whittington Hospital, for letting able to existing but more complicated methods. me have the urine from one of his patients.