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Urinary excretion of tyrosine metabolites in spina bifida
Clinica Chimica Acta, oo(Ig73) ooo-ooo 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed in The Netherlands
249
cc.4 5290
URINARY
EXCRETION
T. DUCKWORTH*,
OF TYROSINE
P. A. BOND
METABOLITES
** , B. McKIBBIN*
BIFIDA
AND E. C. MITCHELL*
*University Department of Orthopaedics, The Royal Infirmary, **the M.R.C. Unit, Middlewood Hospital, Shefield (U.K.) (Received
IN SPINA
Shefield and
July 7, 1972)
SUMMARY
In a carefully matched series of children suffering from myelomeningocele and normal controls, a significant increase was found in the levels of the tyrosine metabolites para-hydroxyphenylacetic acid, vanilmandelic acid and homovanilic acid,when these were expressed in terms of creatinine output. However, creatinine levels also showed a significant variation between patients and controls, being lower in the group of patients. It is suggested that the creatinine variations probably account for the whole of the variation in the other three constituents and that the common practice of expressing urine constituents on random specimens in terms of creatinine excretion be reconsidered, particularly in spina bifida. The reasons for the low levels of creatinine excretion are considered.
INTRODUCTION
Abnormalities in the excretion of para-hydroxyphenylacetic acid (pHPAA), an intermediary product of tyrosine metabolism, were first reported in spina bifida in an investigation into Vitamin C metabolismr. A further study of the metabolism of tyrosine in this condition showed the presence in the urine of abnormal quantities of the catecholamine metabolites, vanilmandelic acid (VMA) and homovanilic acid (HVA) in a very high proportion of the children investigated2. It was felt that these results might indicate an error of tyrosine metabolism associated either primarily or secondarily with spina bifida. The point was made, however, that the levels of the constituents measured were necessarily expressed as a ratio of the creatinine excretion because of the almost insurmountable difficulty of obtaining 24-h urine specimens in these children. It was noticed that some of the patients tested showed unusually low levels of creatinine in individual urine specimens, and also that significant variations could be found during the course of a day, such variations not having been found in a control group previously reported %*. Changes in creatinine excretion would of course produce apparent variations in the other constituents mentioned. It was thought, therefore, that in order to assess the presence or extent of any abnormality of tyrosine metabolism that these effects should be subjected to further investigation. The present study was designed to assess the influence on the urinary levels of
DucKwomH et al.
250
pHPAA, VMA and HVA of three factors: the age of the child, the time of day at which the specimen was collected, and the effect of creatinine variation. Carefully matched groups of controls and patients were set up in order to subject these factors to statistical analysis. MATERIAL
AND
Specimens
METHOD
of urine were obtained
from a group of children suffering from spina
bifida and a group of controls, whose ages ranged between two and twelve years. The spina bifida children were all cases of myelomeningocele and were unselected except that they were easily available for study, either being in hospital or attending for follow-up. Such a group will inevitably include a high proportion of the more severely affected children who tend to be seen more frequently. None of the children were examined in the immediate post-operative period, that is within a period of less than four weeks following any surgical procedure. The control group consisted mainly of normal children but included a small number of children who were in hospital for reasons other than spina bifida: most of these children being in the late stages of Perthes’ disease. Their results did not appear to differ significantly from those of the truly normal children. The urine specimens were divided into two groups, those collected in the morning and those collected in the afternoon. The morning specimens were true early morning specimens, being the first specimen passed on waking. The afternoon specimens were collected at 2.0 p. m. Analysis pHPAA, HVA and VMA were estimated by the method of Sprinkle et al.5 with slight modifications. One ml of urine was acidified with 6 N HCI saturated with sodium chloride and extracted with IO ml, then twice with 5 ml of ether. The combined extracts were dried over sodium sulphate, then taken to dryness with dry nitrogen, The residue was reacted with 0.2 ml of bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane at 70~ for 20 min. Gas chromatography was carried out on a Pye model 84 instrument with a flame ionisation detector. A g ft. x t” column of 4% OV I on “Celite” (acid-washed and silanised) was used at a temperature of 200~ with a carrier gas (nitrogen) flow rate of 60 ml per min. Two ,~l of the silylated mixture was injected directly on to the column. The amount of the three acids present in urine was calculated from the peak height of standards added to urine and carried through the procedure. Gas chromatography-mass spectrometry on a Perkin-Elmer model 240 instrument was used to examine the specificity of the method. The gas chromatographic peak due to pHPAA appeared free from other substances. Those peaks which corresponded with HVA and VMA, although they contained other compounds, were mainly accounted for by HVA and VMA. RESULTS
The results obtained are shown in Tables I to V. Each sub-group of patients and controls consisted of 5 individuals and the table shows the mean value and standard deviation for each constituent within each group of 5.
EXCRETIONS IN SPINA BIFIDA TABLE
I
URINARY EXCRETION
OF CHILDREN
AGED
2-4
YEARS
Controls Mean + I S.D. morning pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/roo ml TABLE URINARY
Patients Mean + I S.D. afternoon
morninn
22.5 +
8.8
39.4 k
6.7
31.74 +
8.3
68.2 & 27.4
17.5 &
6.2
30.3 +
2.9
27.4
I-t
4.4
46.1 f
23.4
6.7 i
3.6
13.5 +
6.7
16.8
f
5.2
21.4 f
9.0
36.8 i_ 15.0
38.8
i_ 17.4
71.2 & 18.2
EXCRETION
BY
THE
pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml
AGE
4-6
GROUP
YEARS
Patients Mean + I S.D.
Gzorning
afternoon
23.4 *
1z.o
22.6 +
9.9
29.8 *
9.2
93.2 + 46.3
18.5 *
4.5
18.2 &
7.2
27.6 + 14.4
44.4 k 19.3
10.2
5.7
20.4 + 17.6
Is.2
12.8
33.6 zt 27.2
26.0 +
59.8 & 16.6
&
83.6 i
67.5
afternoon
morning
9.7
f
7.6
21.2 &
III
URINARY EXCRETIONBY
THE
AGE
6-8
GROUP
YEARS
Patients Mean 5 I S.D.
Controls
Mean * I S.D. morning
pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml TABLE
37.0 + 13.2
II
Controls Mean 4 I S.D.
TABLE
afternoon
16.9 f
aftevnoon 5.3
17.9 * 10 8.4 & 117.6 f
8.6 55.0
morning
afternoon
13.6 f
2.0
38.4 i
29.6
89.7 h 97.8
18.5 f
4.6
27.0 + 20.8
32.8 I_t 25.8
17.4 + 11.0 29.6 f
14.9
8.0 &
6.7
41.0 + 27.2
21.2 +
IO.9
24.4 + 10.8
IV
URINARY EXCRETIONBY THE AGE GROUP 8-10 YEARS Controls Mean + I S.D. morning pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml
Patients Mean -J=I S.D. afternoon
morning
afternoon
25.8 &
7.1
35.8 +
9.6
28.9 & 12.6
50.8 & 17.1
25.3 5
I.2
21.2 i_
7.0
13.6 +
6.3
15.0 &
2.7
15.2 + 11.4
12.2 l
4.2
9.6 +
8.5
9.4 k
I.5
68.6 & 30.1
94.8 i
26.9
33.0 i
12.7
53.8 & 15.8
DUCKWORTH et al.
252 T,4BLE
V
URINARY EXCRETIOK
BY
THE
AGE
GROUP
IO-12
YEARS
Controls Mean
f
I
morning pHPAA Hc%/mg
creatinine
pg/mg creatinine V&IA pg/mg creatinine Creatinine mg/roo ml
Patients Mean + I S.D
S.D. afternoon
morning
afternoon
32.0 +
4.8
25.2 *
7.3
56.6 + 69.0
30.4 + 12.2
17.9
8.8
16.0 +
3.4
28.2 t
21.6
21.4 *
7.8
‘5.5 + 13.9
1r.4 +
3.6
23.6 + ‘3.9
14.1 f
7.0
39.8 t
45.6 + 31.4
f
106.6 + 66.2
84.8 zt 35.’
29.4
The statistical test employed was the Analysis of Variance using a three-factor model of the mixed (random and fixed) effects type. Patients/controls and age groupings were the random ings were taken as the fixed effect. TABLE
VI
SUMMARY
OF
STATISTICAL
FINDINGS
Subject/Control (Patient) effect
HVA
VMA
*
**
**
**
Controls
Controls
Controls >subjects N.S. (E) (increase with age) N.S. (E) higher in morning
Time effect
N.S. (E) higher in afternoon
Subject x age effect Subject x time effect Age x time effect
N.S. N.S. N.S.
*
*
N.S. (E) N.S. (E)
N.S. **
Age effect
p value (0.1% p p value
Creatinine
FHPAA
Contr 01s
*:I
effects, and time group
(decrease with age) N.S. (E) higher in afternoon
N.S. N.S. (E) N.S. (E)
Table VI summarises the statistical findings. The upper half of the table shows the results of comparing subjects with controls, the subjects in this case being the patients; the effect of increasing age, and the effects of comparing specimens taken in the morning and in the afternoon. It will be seen that for all three constituents there is a statistically significant difference between the subjects and the controls, the values in the subjects being higher than those in the controls, the significance being at the 5% level in the case of pHPAA and at the 1.0% level in the case of HVA and VMA. There was no significant variation with age, except in the case of VMA where the values tended to be lower with increasing age, at a significance level of 5%. The statistical tests showed in the case of HVA that given a larger series it would be likely
EXCRETIONS
253
IN SPINA BIFIDA
that here also there would be a lowering of the values with increasing significance
level. There was no significant
difference
age at a 5%
between morning and afternoon
values, although again the statistical test suggested that a larger series might reveal a tendency to higher values in the afternoon. The bottom half of the table shows the interactions between two variables and reveals how two effects which, when taken individually, produce barely significant changes in the values can, when allowed to interact, in certain cases produce variations which achieve significance. In order to eliminate these interactions, therefore, it is necessary for comparability between subjects and controls, that both groups should be of the same age and that all specimens should be collected at the same time of day. The statistical
analysis of the creatinine
values was also carried out in the same
way and a summary of the results is shown in the right-hand half of Table VI. It can be seen that there is a statistically significant difference at the 1.0% level between patients and controls, the patients having lower values than the controls, and that the age and time effects, although not significant at the 5.0% level, do suggest trends for the values of this constituent which are in the opposite direction from those of the other constituents. DISCUSSION
The possibility
of an abnormality
of tyrosine
metabolism
in spina bifida was
first raised in a paper2 describing the finding of increased amounts of catecholamine metabolites in random specimens of urine taken from children suffering from myelomeningocele. Raised values of pHPAA, an intermediary product of tyrosine metabolism, had already been described in a previous investigation1 into ascorbic acid metabolism in spina bifida. The urinary excretion of pHPAA was being investigated as a possible indicator of Vitamin C unsaturation, as described by Boscott and Cookea. However, since it had been shown that pHPAA excretion could be raised in conditions other than Vitamin C deficiency7, and because it was considered possible that the pHPAA abnormalities might reflect a more widespread disorder of tyrosine metabolism, attention was directed to the metabolic end products, HVA and VMA. Here again values were found to be considerably raised. It is the usual practice to record the level of urinary constituents such as these as a ratio of creatinine excretion. It is assumed that the urinary excretion of creatinine, which is produced at a virtually constant rate, by degradation of creatine in muscles, will be reasonably constant from day to day and show minimal diurnal variation, and will therefore serve as a reasonable measure of urine dilution in a random specimen. This method of recording the results was adopted in the previous publications although some doubts were felt and expressed concerning its general validity. It would obviously be preferable to estimate constituents such as these on complete 24-h specimens of urine, but in the case of young children, in particular children suffering from spina bifida, and incontinence of urine, the difficulties of collecting 24-h specimens are considerable, and could only be solved by using a technique of continuous catheterisation. Because of the overwhelming dangers of infection of the urinary tract in such children this was not felt to be justified. The present paper is an attempt to overcome this difficulty by making a statistical analysis of groups of
2.54
DUCKWORTH
et ad.
controls and patients in terms of the excretion of pHPAA, HVA and VMA expressed in the usual way as a ratio of creatinine excretion, and also of creatinine excretion itself. Analysis of the results has shown that while the levels of pHPAA, HVA and VMA are significantly increased in patients compared with controls, creatinine values are decreased; and whereas the levels of the three acids show a tendency to decrease with age and to be higher in the afternoon than in the morning, creatinine behaves in the opposite way. It follows that abnormalities in the excretion of the three acids, when expressed in terms of creatinine excretion, could be accounted for solely by the variations in creatinine levels. It is possible of course that the three constituents themselves may show true variations from normal, and that this could be masked by the creatinine variations, but there is very little evidence for this and it seems likely that the only true abnormality detected is one of creatinine excretion. This is in accord with the fact which was noticed during the analysis that those children with the highest levels of the three constituents were those children in whom the creatinine levels appeared to be abnormally low. Although the three constituents are significantly raised in the present series, the levels are by no means as high as those recorded in the previous paperz. The authors believe that the present method for the determination of the tyrosine metabolites gives a superior and more specific estimate of the levels of the individual constituent; the peaks on the gas chromatogram being particularly clear and well defined. Several issues are raised by the present findings. In the first place it would appear that the widely accepted method of expressing the values of urinary constituents in random specimens as a ratio of creatinine levels is not only unreliable but can be grossly misleading. This is particularly the case in spina bifida, and we would suspect also in children in general. In this respect we support the views of previous variations in the 24-h excretion of workers8~9, who have described considerable creatinine from day to day in the same individual, and thoselOtll who have demonstrated variations in creatinine excretion through the 24-h period. We are now convinced that in spina bifida variations in creatinine levels from the usually accepted normals are common, and can be of considerable magnitude. It is interesting to speculate on the reason for this. It is well known of course that whenever the total quantity of intra-cellular creatine or phospho-creatine is diminished as by loss of muscle mass, the quantity of creatinine formed each day, and excreted in the urine, must also be reduced. Many children suffering from spina bifida with extensive paralysis do have significantly diminished muscle bulk, not only because the muscles themselves may be wasted, but also because the child as a whole may be much smaller than normal and have an abnormal distribution of the body tissues. It was not possible in the present series to assess the results of degrees of paralysis on creatinine levels, but this is obviously a topic for further investigation. Another factor which may interfere with creatinine excretion and particularly of course with creatine clearance is the presence of severe renal disease, and there is little doubt that many of the patients in the present study would have at least a moderate degree of impairment of renal function, Since the excretion of creatinine in the urine is a function of the kidney, largely at glomerular level, impaired glomerular filtration rate may be associated with diminished excretion of creatinine. Usually,
EXCRETIONS
IN SPINA BIFIDA
255
however, in severe renal disease, there is a rise in plasma level of creatinine and the 24-h output remains constant, so that it becomes difficult to predict what overall effect there might be on random samples. All these factors need to be assessed more carefully, but in the meantime it is suggested that a re-appraisal of the widely accepted method of expressing urinary constituents in terms of creatinine excretion is necessary and overdue. ACKNOWLEDGEMENTS
We acknowledge with gratitude the assistance of Dr. R. J. Pollitt, who carried out the mass spectrometry investigations, and Mr. D. Phipps who was responsible for the statistical analysis. We would also like to express our gratitude to Mr. W. J. W. Sharrard for allowing us access to his patients. REFERENCES I B. MCKIBBIN,
P. A. TOSELAND AND T. DUCKWORTH, Develop.
Med.
Child Neural.,
(1968) 55. 2 B. MCKIBBIN, L. O’GORMAN AND T. DUCKWORTH, J. Clin. Pathol., 22 (1969) 687. 3 L. P. O’GORMAN, C&z. Chim. Acta, 19 (1968) 485. 4 S. GITLOW, M. MENDLOWITZ, E. K. WILK, R. WOLF AND J. GLICK, J. Clin. Invest., 5 6 7 8 g IO II
Supp.
15
44 (1965)
1049. T. J. SPRINICLE, A. H. PORTER, M. GREER AND C. M. WILLIAMS, Clin. Chim. Acta, 25 (1969) 4og. R. J. BOSCOTT AND W. T. COOKE, Quart. J. Med.. 23 (1954) 307. E. ROBINSON AND F. G. WARBURTON, Nature, 212 (1966) 1605. 0. M. EDWARDS, R. I. S. BAYLISS AND S. MILLER, Lancet, ii (1969) 1165. B. R. BAILEY AND H. E. DE WARDENER. Lancet, i (1970) 145. P. A. ZORAR, ST. CLARK AND A. HARRISON, kzncet, ii (1969) 1254. M. MOHAN RAM AND R. VINODINI, Lancet, ii (1970) 674.
Amsterdam
- Printed in The Netherlands
249
cc.4 5290
URINARY
EXCRETION
T. DUCKWORTH*,
OF TYROSINE
P. A. BOND
METABOLITES
** , B. McKIBBIN*
BIFIDA
AND E. C. MITCHELL*
*University Department of Orthopaedics, The Royal Infirmary, **the M.R.C. Unit, Middlewood Hospital, Shefield (U.K.) (Received
IN SPINA
Shefield and
July 7, 1972)
SUMMARY
In a carefully matched series of children suffering from myelomeningocele and normal controls, a significant increase was found in the levels of the tyrosine metabolites para-hydroxyphenylacetic acid, vanilmandelic acid and homovanilic acid,when these were expressed in terms of creatinine output. However, creatinine levels also showed a significant variation between patients and controls, being lower in the group of patients. It is suggested that the creatinine variations probably account for the whole of the variation in the other three constituents and that the common practice of expressing urine constituents on random specimens in terms of creatinine excretion be reconsidered, particularly in spina bifida. The reasons for the low levels of creatinine excretion are considered.
INTRODUCTION
Abnormalities in the excretion of para-hydroxyphenylacetic acid (pHPAA), an intermediary product of tyrosine metabolism, were first reported in spina bifida in an investigation into Vitamin C metabolismr. A further study of the metabolism of tyrosine in this condition showed the presence in the urine of abnormal quantities of the catecholamine metabolites, vanilmandelic acid (VMA) and homovanilic acid (HVA) in a very high proportion of the children investigated2. It was felt that these results might indicate an error of tyrosine metabolism associated either primarily or secondarily with spina bifida. The point was made, however, that the levels of the constituents measured were necessarily expressed as a ratio of the creatinine excretion because of the almost insurmountable difficulty of obtaining 24-h urine specimens in these children. It was noticed that some of the patients tested showed unusually low levels of creatinine in individual urine specimens, and also that significant variations could be found during the course of a day, such variations not having been found in a control group previously reported %*. Changes in creatinine excretion would of course produce apparent variations in the other constituents mentioned. It was thought, therefore, that in order to assess the presence or extent of any abnormality of tyrosine metabolism that these effects should be subjected to further investigation. The present study was designed to assess the influence on the urinary levels of
DucKwomH et al.
250
pHPAA, VMA and HVA of three factors: the age of the child, the time of day at which the specimen was collected, and the effect of creatinine variation. Carefully matched groups of controls and patients were set up in order to subject these factors to statistical analysis. MATERIAL
AND
Specimens
METHOD
of urine were obtained
from a group of children suffering from spina
bifida and a group of controls, whose ages ranged between two and twelve years. The spina bifida children were all cases of myelomeningocele and were unselected except that they were easily available for study, either being in hospital or attending for follow-up. Such a group will inevitably include a high proportion of the more severely affected children who tend to be seen more frequently. None of the children were examined in the immediate post-operative period, that is within a period of less than four weeks following any surgical procedure. The control group consisted mainly of normal children but included a small number of children who were in hospital for reasons other than spina bifida: most of these children being in the late stages of Perthes’ disease. Their results did not appear to differ significantly from those of the truly normal children. The urine specimens were divided into two groups, those collected in the morning and those collected in the afternoon. The morning specimens were true early morning specimens, being the first specimen passed on waking. The afternoon specimens were collected at 2.0 p. m. Analysis pHPAA, HVA and VMA were estimated by the method of Sprinkle et al.5 with slight modifications. One ml of urine was acidified with 6 N HCI saturated with sodium chloride and extracted with IO ml, then twice with 5 ml of ether. The combined extracts were dried over sodium sulphate, then taken to dryness with dry nitrogen, The residue was reacted with 0.2 ml of bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane at 70~ for 20 min. Gas chromatography was carried out on a Pye model 84 instrument with a flame ionisation detector. A g ft. x t” column of 4% OV I on “Celite” (acid-washed and silanised) was used at a temperature of 200~ with a carrier gas (nitrogen) flow rate of 60 ml per min. Two ,~l of the silylated mixture was injected directly on to the column. The amount of the three acids present in urine was calculated from the peak height of standards added to urine and carried through the procedure. Gas chromatography-mass spectrometry on a Perkin-Elmer model 240 instrument was used to examine the specificity of the method. The gas chromatographic peak due to pHPAA appeared free from other substances. Those peaks which corresponded with HVA and VMA, although they contained other compounds, were mainly accounted for by HVA and VMA. RESULTS
The results obtained are shown in Tables I to V. Each sub-group of patients and controls consisted of 5 individuals and the table shows the mean value and standard deviation for each constituent within each group of 5.
EXCRETIONS IN SPINA BIFIDA TABLE
I
URINARY EXCRETION
OF CHILDREN
AGED
2-4
YEARS
Controls Mean + I S.D. morning pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/roo ml TABLE URINARY
Patients Mean + I S.D. afternoon
morninn
22.5 +
8.8
39.4 k
6.7
31.74 +
8.3
68.2 & 27.4
17.5 &
6.2
30.3 +
2.9
27.4
I-t
4.4
46.1 f
23.4
6.7 i
3.6
13.5 +
6.7
16.8
f
5.2
21.4 f
9.0
36.8 i_ 15.0
38.8
i_ 17.4
71.2 & 18.2
EXCRETION
BY
THE
pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml
AGE
4-6
GROUP
YEARS
Patients Mean + I S.D.
Gzorning
afternoon
23.4 *
1z.o
22.6 +
9.9
29.8 *
9.2
93.2 + 46.3
18.5 *
4.5
18.2 &
7.2
27.6 + 14.4
44.4 k 19.3
10.2
5.7
20.4 + 17.6
Is.2
12.8
33.6 zt 27.2
26.0 +
59.8 & 16.6
&
83.6 i
67.5
afternoon
morning
9.7
f
7.6
21.2 &
III
URINARY EXCRETIONBY
THE
AGE
6-8
GROUP
YEARS
Patients Mean 5 I S.D.
Controls
Mean * I S.D. morning
pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml TABLE
37.0 + 13.2
II
Controls Mean 4 I S.D.
TABLE
afternoon
16.9 f
aftevnoon 5.3
17.9 * 10 8.4 & 117.6 f
8.6 55.0
morning
afternoon
13.6 f
2.0
38.4 i
29.6
89.7 h 97.8
18.5 f
4.6
27.0 + 20.8
32.8 I_t 25.8
17.4 + 11.0 29.6 f
14.9
8.0 &
6.7
41.0 + 27.2
21.2 +
IO.9
24.4 + 10.8
IV
URINARY EXCRETIONBY THE AGE GROUP 8-10 YEARS Controls Mean + I S.D. morning pHPAA pg/mg creatinine HVA pg/mg creatinine VMA pg/mg creatinine Creatinine mg/Ioo ml
Patients Mean -J=I S.D. afternoon
morning
afternoon
25.8 &
7.1
35.8 +
9.6
28.9 & 12.6
50.8 & 17.1
25.3 5
I.2
21.2 i_
7.0
13.6 +
6.3
15.0 &
2.7
15.2 + 11.4
12.2 l
4.2
9.6 +
8.5
9.4 k
I.5
68.6 & 30.1
94.8 i
26.9
33.0 i
12.7
53.8 & 15.8
DUCKWORTH et al.
252 T,4BLE
V
URINARY EXCRETIOK
BY
THE
AGE
GROUP
IO-12
YEARS
Controls Mean
f
I
morning pHPAA Hc%/mg
creatinine
pg/mg creatinine V&IA pg/mg creatinine Creatinine mg/roo ml
Patients Mean + I S.D
S.D. afternoon
morning
afternoon
32.0 +
4.8
25.2 *
7.3
56.6 + 69.0
30.4 + 12.2
17.9
8.8
16.0 +
3.4
28.2 t
21.6
21.4 *
7.8
‘5.5 + 13.9
1r.4 +
3.6
23.6 + ‘3.9
14.1 f
7.0
39.8 t
45.6 + 31.4
f
106.6 + 66.2
84.8 zt 35.’
29.4
The statistical test employed was the Analysis of Variance using a three-factor model of the mixed (random and fixed) effects type. Patients/controls and age groupings were the random ings were taken as the fixed effect. TABLE
VI
SUMMARY
OF
STATISTICAL
FINDINGS
Subject/Control (Patient) effect
HVA
VMA
*
**
**
**
Controls
Controls
Controls >subjects N.S. (E) (increase with age) N.S. (E) higher in morning
Time effect
N.S. (E) higher in afternoon
Subject x age effect Subject x time effect Age x time effect
N.S. N.S. N.S.
*
*
N.S. (E) N.S. (E)
N.S. **
Age effect
p value (0.1% p p value
Creatinine
FHPAA
Contr 01s
*:I
effects, and time group
(decrease with age) N.S. (E) higher in afternoon
N.S. N.S. (E) N.S. (E)
Table VI summarises the statistical findings. The upper half of the table shows the results of comparing subjects with controls, the subjects in this case being the patients; the effect of increasing age, and the effects of comparing specimens taken in the morning and in the afternoon. It will be seen that for all three constituents there is a statistically significant difference between the subjects and the controls, the values in the subjects being higher than those in the controls, the significance being at the 5% level in the case of pHPAA and at the 1.0% level in the case of HVA and VMA. There was no significant variation with age, except in the case of VMA where the values tended to be lower with increasing age, at a significance level of 5%. The statistical tests showed in the case of HVA that given a larger series it would be likely
EXCRETIONS
253
IN SPINA BIFIDA
that here also there would be a lowering of the values with increasing significance
level. There was no significant
difference
age at a 5%
between morning and afternoon
values, although again the statistical test suggested that a larger series might reveal a tendency to higher values in the afternoon. The bottom half of the table shows the interactions between two variables and reveals how two effects which, when taken individually, produce barely significant changes in the values can, when allowed to interact, in certain cases produce variations which achieve significance. In order to eliminate these interactions, therefore, it is necessary for comparability between subjects and controls, that both groups should be of the same age and that all specimens should be collected at the same time of day. The statistical
analysis of the creatinine
values was also carried out in the same
way and a summary of the results is shown in the right-hand half of Table VI. It can be seen that there is a statistically significant difference at the 1.0% level between patients and controls, the patients having lower values than the controls, and that the age and time effects, although not significant at the 5.0% level, do suggest trends for the values of this constituent which are in the opposite direction from those of the other constituents. DISCUSSION
The possibility
of an abnormality
of tyrosine
metabolism
in spina bifida was
first raised in a paper2 describing the finding of increased amounts of catecholamine metabolites in random specimens of urine taken from children suffering from myelomeningocele. Raised values of pHPAA, an intermediary product of tyrosine metabolism, had already been described in a previous investigation1 into ascorbic acid metabolism in spina bifida. The urinary excretion of pHPAA was being investigated as a possible indicator of Vitamin C unsaturation, as described by Boscott and Cookea. However, since it had been shown that pHPAA excretion could be raised in conditions other than Vitamin C deficiency7, and because it was considered possible that the pHPAA abnormalities might reflect a more widespread disorder of tyrosine metabolism, attention was directed to the metabolic end products, HVA and VMA. Here again values were found to be considerably raised. It is the usual practice to record the level of urinary constituents such as these as a ratio of creatinine excretion. It is assumed that the urinary excretion of creatinine, which is produced at a virtually constant rate, by degradation of creatine in muscles, will be reasonably constant from day to day and show minimal diurnal variation, and will therefore serve as a reasonable measure of urine dilution in a random specimen. This method of recording the results was adopted in the previous publications although some doubts were felt and expressed concerning its general validity. It would obviously be preferable to estimate constituents such as these on complete 24-h specimens of urine, but in the case of young children, in particular children suffering from spina bifida, and incontinence of urine, the difficulties of collecting 24-h specimens are considerable, and could only be solved by using a technique of continuous catheterisation. Because of the overwhelming dangers of infection of the urinary tract in such children this was not felt to be justified. The present paper is an attempt to overcome this difficulty by making a statistical analysis of groups of
2.54
DUCKWORTH
et ad.
controls and patients in terms of the excretion of pHPAA, HVA and VMA expressed in the usual way as a ratio of creatinine excretion, and also of creatinine excretion itself. Analysis of the results has shown that while the levels of pHPAA, HVA and VMA are significantly increased in patients compared with controls, creatinine values are decreased; and whereas the levels of the three acids show a tendency to decrease with age and to be higher in the afternoon than in the morning, creatinine behaves in the opposite way. It follows that abnormalities in the excretion of the three acids, when expressed in terms of creatinine excretion, could be accounted for solely by the variations in creatinine levels. It is possible of course that the three constituents themselves may show true variations from normal, and that this could be masked by the creatinine variations, but there is very little evidence for this and it seems likely that the only true abnormality detected is one of creatinine excretion. This is in accord with the fact which was noticed during the analysis that those children with the highest levels of the three constituents were those children in whom the creatinine levels appeared to be abnormally low. Although the three constituents are significantly raised in the present series, the levels are by no means as high as those recorded in the previous paperz. The authors believe that the present method for the determination of the tyrosine metabolites gives a superior and more specific estimate of the levels of the individual constituent; the peaks on the gas chromatogram being particularly clear and well defined. Several issues are raised by the present findings. In the first place it would appear that the widely accepted method of expressing the values of urinary constituents in random specimens as a ratio of creatinine levels is not only unreliable but can be grossly misleading. This is particularly the case in spina bifida, and we would suspect also in children in general. In this respect we support the views of previous variations in the 24-h excretion of workers8~9, who have described considerable creatinine from day to day in the same individual, and thoselOtll who have demonstrated variations in creatinine excretion through the 24-h period. We are now convinced that in spina bifida variations in creatinine levels from the usually accepted normals are common, and can be of considerable magnitude. It is interesting to speculate on the reason for this. It is well known of course that whenever the total quantity of intra-cellular creatine or phospho-creatine is diminished as by loss of muscle mass, the quantity of creatinine formed each day, and excreted in the urine, must also be reduced. Many children suffering from spina bifida with extensive paralysis do have significantly diminished muscle bulk, not only because the muscles themselves may be wasted, but also because the child as a whole may be much smaller than normal and have an abnormal distribution of the body tissues. It was not possible in the present series to assess the results of degrees of paralysis on creatinine levels, but this is obviously a topic for further investigation. Another factor which may interfere with creatinine excretion and particularly of course with creatine clearance is the presence of severe renal disease, and there is little doubt that many of the patients in the present study would have at least a moderate degree of impairment of renal function, Since the excretion of creatinine in the urine is a function of the kidney, largely at glomerular level, impaired glomerular filtration rate may be associated with diminished excretion of creatinine. Usually,
EXCRETIONS
IN SPINA BIFIDA
255
however, in severe renal disease, there is a rise in plasma level of creatinine and the 24-h output remains constant, so that it becomes difficult to predict what overall effect there might be on random samples. All these factors need to be assessed more carefully, but in the meantime it is suggested that a re-appraisal of the widely accepted method of expressing urinary constituents in terms of creatinine excretion is necessary and overdue. ACKNOWLEDGEMENTS
We acknowledge with gratitude the assistance of Dr. R. J. Pollitt, who carried out the mass spectrometry investigations, and Mr. D. Phipps who was responsible for the statistical analysis. We would also like to express our gratitude to Mr. W. J. W. Sharrard for allowing us access to his patients. REFERENCES I B. MCKIBBIN,
P. A. TOSELAND AND T. DUCKWORTH, Develop.
Med.
Child Neural.,
(1968) 55. 2 B. MCKIBBIN, L. O’GORMAN AND T. DUCKWORTH, J. Clin. Pathol., 22 (1969) 687. 3 L. P. O’GORMAN, C&z. Chim. Acta, 19 (1968) 485. 4 S. GITLOW, M. MENDLOWITZ, E. K. WILK, R. WOLF AND J. GLICK, J. Clin. Invest., 5 6 7 8 g IO II
Supp.
15
44 (1965)
1049. T. J. SPRINICLE, A. H. PORTER, M. GREER AND C. M. WILLIAMS, Clin. Chim. Acta, 25 (1969) 4og. R. J. BOSCOTT AND W. T. COOKE, Quart. J. Med.. 23 (1954) 307. E. ROBINSON AND F. G. WARBURTON, Nature, 212 (1966) 1605. 0. M. EDWARDS, R. I. S. BAYLISS AND S. MILLER, Lancet, ii (1969) 1165. B. R. BAILEY AND H. E. DE WARDENER. Lancet, i (1970) 145. P. A. ZORAR, ST. CLARK AND A. HARRISON, kzncet, ii (1969) 1254. M. MOHAN RAM AND R. VINODINI, Lancet, ii (1970) 674.