(Received July 10; accepted for publication July 19, 1971.)
This study was supported by grants from the Quebec Medical Genetics Network and the National
Genetics Foundation (New York).
ADDRESS FOR REPRINTS: (CR5.) Montreal Children’s Hospital, 2300 Tupper Street, Montreal 108,
Quebec, Canada.
80
PEDIATRICS, Vol. 49, No. 1, January 1972
ACID:CREATININE
RATIO
IN
URINE
Normal
Values
in the
Newborn
Roderick Mclnnes, M.D., Peter Lamm, Carol L. Clow, and Charles R. Scriver, M.D.
From time deBelle Laboratory for Biochemical Genetics, McGill Unieersity-Montreal Children’s hospital Research In.stitute, Montreal, Quebec, Canada
ABSTRACT. A simple method is described for de-termining the uric acid:creatinine ratio (UA/C
ra-tio) in urine collected on filter paper. Uric acid and creatinine can be eluted quantitatively from
Schleicher and Schuell 903 filter paper in alkaline
buffer; the eluate is analyzed for the two compounds
simultaneously by an automated method.
The mean UA/C ratio was defined in 820 new-born infants in the postnatal period of life. After
the third day, the average is 1.49 ± 0.60 (mean ± 1SD), falling from a high value of 2.25 ± 0.85 on the second day. There is no significant sex
differ-ence or diurnal variation at this age and neither
gestational age nor birth weight apparently affects the ratio.
The technique can be used to screen population, particularly the neonate after the 3rd day of life, for disorders of purine metabolism. An abnormal ratio (i.e., above 3.29 or >3SD above the mean)
would indicate such a disorder. Transient high UA /C ratios can be anticipated in at least 0.5% of
samples often because of acquired perinatal corn-plications.
Pediatrics, 49:80, 1972, NEWBORN INFANT,
SCREENING, URIC ACID, URIC ACID/CREATININE RATIO,
FILTER PAPER METHOD.
T
HE uric acid:creatinine ratio(
UA/C) in morning or 24-hour urine was de-scribed by Kaufman, Green, and Seegmiller’ as a useful index of some disorders of pu-rifle metabolism. Liquid urine was used in their study and older patients with completeor partial activity of the enzyme
phos-phoribosyl transferase (PRT) were revealed
through UA/C ratios above the normal
range. The collection and processing of liquid urine in any large screening study which might involve the newborn infant would become a problem in logistics. There-fore, we investigated the use of a filter paper method for collection and transport of urine and for determination of the UA/C ratio. This communication reports a reliable tech-nique which was used to determine the
nor-mal UA/C ratio in the first week of life.
UA/C: uric PRT: phosp
Abbreviations
acid: creatinine ratio horibosyl transferase
METHODS AND MATERIALS
Urine Collection
A coded square of Schleicher and Schuell 903 filter paper
(
Keene, N.H.)
is placed in the anterior fold of the diaper, without di-rect skin contact, to avoid fecal soiling. The latter should be avoided since it may affect the results. After the paper is wet with urine, it is removed, air-dried, and deliveredto the laboratory.
Analysis
A circle of urine-impregnated paper is cut with scissors, or five 3” discs are
punched out and placed in a sampling cup (2-ml capacity) of the type used for the
Autoanalyzer.#{176} An alkaline solution is used to elute the papers; we employed 0.2 M
phosphate buffer pH 7.6. The caps are capped and elution is performed at 37#{176}Cfor 4 hours. Other schedules of buffer choice and elution time may be more convenient in
32
ARTICLES 81
83
I
males
females
mean
±1SD
34
79
117
T
48
27
‘4
I
1
69
4#{244}
102.35
3
2 >10
0
I-4
U
4
DLU
z
z
I-4
LU
U
U
4
U
4
6
7
DAYS
AFTER
BIRTH
FIG. 1. The uric acid/creatinine ( UA/C) ratio in urine eluted from filter
paper discs. The filter paper (Schleicher and Schuell 903) is placed in the
anterior folds of the diaper and when moist with urine, it is air-dried and
sent to the laboratory for analysis with an Autoanalyzer using the N-30p
module for simultaneous analysis of uric acid and creatinine. Elution of
urine from five h” punched discs is performed in alkaline buffer (0.2 M
sodium phosphate pH 7.6) at 37#{176}Cfor 4 hours. The UA/C ratio (mean ±
1SD) is shown on various days after birth. Numbers indicate sample size
for male and female infants on each day. Gestational age, birth weight, and
time of day do not significantly change the age-dependent variation in the
UA/C ratio shown here. The normal ratio after the 3rd day of life is 1.49 ± 0.60.
different laboratories; for example, pH 9,
0.01 M ammonium hydroxide solution is said to elute uric acid from urine of the newborn more rapidly and with 90% recovery.2 Uric
acid and creatinine in the eluate are then determined with an N-30p module fitted with sampling tubing (size 0.051) on a
Technicon Autoanalyzer. Modified Archi-bald3 and Jaffe4 reactions are used for
colorimetric analysis. We performed 25 analyses/hour hut the rate of analysis can
be increased with appropriate adjustments.
The UA/C ratio is computed from the values
for uric acid and creatinine expressed as milligrams per 100 ml urine.
Subjects
in-eluded in the study. Eight hundred and
twenty subjects were examined during a period of 2 months. Urine was collected on or after the 2nd day of life, and at different
times of the day. Sex, gestational age, birth weight, and postnatal age of the subject
and time of collection were marked in pen-cil, by the nurse, on the filter papers placed
in the diaper. This information and the UA
I
C ratio were recorded on data processing cards and the relevant correlations and sta-tistical work obtained by computer analysis.RESULTS
Volume of Urine on Filter Paper Disc
Dry discs were weighed, soaked in urine, blotted, and reweighed. The specific
gray-ity of the urine samples was also deter-mined. The 3” disc of S and S 903 filter pa-per contains about 16.4 .l of urine. Since newborn urine is dilute, five discs were re-quired for analysis of the UA/C ratio by the present method; fewer discs are re-quired in older subjects.
Recovery of Uric Acid and Creatinine
Both substances are recovered equiva-lently from urine-inipregnated filter paper discs. When urine from infants is used, re-covery is 88 ± 3% for discs incubated in
pH 7.6 phosphate buffer at 37#{176}Cfor 4 hours. Elution for shorter periods impairs recovery and prolonged elution alters the
ratio. Recovery is 100% from the more
concentrated adult urine.
Reproducibility
UA/C ratios were obtained and com-pared from liquid urine (16 samples) and
from discs soaked in the same urine sam-ples. The mean ratio obtained from the 16 discs was only 3.5% higher than from the liquid samples. The mean error for each pair of samples was 5%.
UA/C Ratio in Urine
EFFECT OF MAILING: The UA/C ratio is the same for aliquots of urine-impregnated filter paper analyzed freshly in the
labora-tory, and for the same samples sent to our-selves through the post, or kept at room temperature and analyzed daily up to 14 days. The UA/C ratio can change mark-edly either upwards or downwards in liquid urine samples handled in a similar fashion.
EFFECT OF POSTNATAL AGE: The UA/C ratio is higher on the 2nd and 3rd day of life than on subsequent days
(
Fig. 1). The mean UA/C ratio is 1.49 ± 0.60 after the 3rd day of life. There is no difference be-tween male and female subjects at this age.OTHER VARIABLES: The age-dependent
UA/C ratio shown in Figure 1 is not sig-nificantly influenced by gestational age; 71 infants born at less than 37 weeks’ gestation were examined. Birth weight did not in-fluence the ratio either. Diurnal variation
in the UA/C ratio was not evident in the
postnatal period, as it is in adults.
EFFECT OF Iwss: The UA/C ratio after
the 3rd day of life was greater than 3.29 (i.e., >3SD above the mean) in four in-fants in the survey or the equivalent of about 0.5% of samples. These subjects were
in the regular nurseries, all were apparently healthy and none had hyperuricaciduria on
follow-up investigation. We also found an increased prevalence of high UA/C ratios among the newborn infants in the intensive care nursery. Further investigation showed
that the abnormal ratio reflected acquired metabolic imbalance, the former returning to normal with correction of the latter.
We were able to obtain and test urine samples which had been collected during the first week of life from known patients with the Lesch-Nyhan Syndrome.
DISCUSSION
Children with hyperuricemia due to al-most total absence of hypoxanthine
gua-nine-phosphoribosyl transferase activity
ARTICLES 83
then that the urine UA/C ratio can be used in the newborn for early detection of
inher-ited disorders of purine metabolism, since it is known that the serum uric acid level can
be abnormally elevated from the first day of
56 We have now defined the normal
uri-nary UA/C ratio for Caucasian infants in
the first week of life.
After the 3rd day following birth, the ra-tio is 1.49 ± 0.60
(
mean ± SD); this value compares well with that reported byKauf-man, et al.’ who examined liquid urine gathered from an unspecified number of
in-fants “in the first week after birth.” We ob-served a higher ratio in the first 3 days after birth. This correlates with the findings of Monkus, et al.6 who observed that serum uric acid rises significantly in the first day after birth, falling thereafter until a steady-state is reached on the 3rd day. Schloss and
Crawford7 observed that the uric acid in urine falls in the first 3 days of life, as cx-pected if serum urate declines also. The lat-ter reports, and our data, suggest that test-ing in the newborn infant for disorders of purine metabolism should be performed af-ter the 3rd day of life. We observed that the sex of the patient, gestational age, birth
weight, and circadian periodicity have little
influence on the UA/C ratio in the new-born; thus these factors need not be
consid-ered when screening at this age.
Acquired postnatal complications and
metabolic imbalance can influence the UA/ C ratio and about 0.5% of tests will yield abnormally high results. This finding cor-roborates the observation of Monkus and colleagues6 who found high serum uric acid levels in infants with perinatal
complica-tions.
Inherited disorders of purine metabolism are probably rare in frequency, in Cauca-sians at least. This conclusion is based, of
course, only on the present estimates de-rived from case finding of probands with fully expressed syndromes. Nonetheless, at this point in time, there is a need for case finding at an early age, in order to study the age-dependent criteria for diagnosis and the potential value of treatment procedures
begun before the clinical phenotype is cx-pressed.5’8 The present method can serve this purpose. It will be necessary to refer the urinary UA/C ratio back to the serum uric acid level; although normal variation in the latter has been determined in the new-born6’ and the older child,10 serum screen-ing for uric acid is not feasible on a massive
scale.b0 A method using filter paper for urine collection makes it possible to screen
the newborn easily and to transport the sample safely over time and distance to a
central laboratory. The same collection pa-per can also be used for other purposes such as screening for the hyperaminoacidu-rias. Levy and colleagueshl have clearly
shown the potential of amino acid screen-ing applied to urine-impregnated filter pa-per. Therefore, these potential and practi-cal reasons lead us to believe that screening for disorders of purine metabolism, if mdi-cated, could be established with the filter paper technique.
REFERENCES
1. Kaufman, J. M., Greene, M. L., and
Seegmil-ler, J. E. : Urine uric acid to creatinine ratio
-a screening test for inherited disorders of purine metabolism. J. Pediat., 73:583, 1968.
purine metabolism. J. Pediat., 73:583, 1968. 2. Shapcott, D., and Lemieux, B.: Personal
corn-munication.
3. Nishi, H. H.: Determination of uric acid: An adaption of the Archibald method on the
Autoanalyzer. Clin. Chem., 13:12, 1967. 4. Chassen, A. L., Grady, H. J., and Stanley,
M. A.: Determination of creatinine by means of automatic chemical analysis. Amer. J. Gun. Path., 35:83, 1961.
5. Marks, J. F.. Baum, J., Keele, D. K., Kay, J. L., and MacFarlen, A.: Lesch-Nyhan syndrome treated from the early neonatal period.
PEDIATRICS, 42:357, 1968.
6. Monkus, E. J., Nyhan, W. L., Fogel, B. J., and
Yankow, S.: Concentrations of uric acid in
the serum of neonatal infants and their
mothers. Amer. J. Obstet. Cynec., 108:91, 1970.
7. Schloss, 0. M., and Crawford, J. L.: The
me-tabolism of nitrogen phosphorous and the
punne substances in the newborn; with spe-cial references to the causation of the uric
8. Schulman, j. 0., Greene, M. L., Fujimoto,
W. Y., and Seegmiller, J. E.: Therapy for Lesch-Nvhan Syndrome. Pediat. Res., 5:277
1971.
9. Marks, J. F., Kay, J., Baum, J., and Curry, L.:
Uric acid levels in full term and
low-birth-weight infants. J. Pediat., 73:609, 1968.
10. Harkness, R. A., and Nicol, A. D.: Plasma uric acid levels in children. Arch. Dis. Child., 44:
773, 1969.
11. Levy, H. L., Shih, V. E., Madigan, P. M.,
Ka-rolkewicz, V., and MacCready, R. A.:
Re-suits of a screening method for free amino
acids. II. Urine. Clin. Biochem., 1:208,
1968.
Acknowledgment
We are grateful to the following nurses for their
interest and assistance in the field study: Miss
Bourdage, Miss Henderson, Miss Estioko, Miss
Stairs, and Miss Tessier. Dr. Michael Klein offered valuable assistance in the early stages of this work.
Dr. Jack L. Neal kindly performed the computer
