trmnsic obstruction of the duodenum. Amer.
J.
Surg., 114:190, 1967.
8. Tandler, J.: Zur entwicklungsgeschichte des
menschlichen duodenums in fruhen
emby-onal stadium. Morph. Jahrb., 29:187, 1900.
9. Mishalany, H. G., and Najjar, F. B. : Familial
jejunal atresia: Three cases in one family. J.
Pediat., 73:753, 1968.
Reducing Substances in Neonatal
Stools Detected
by ClinitestKerry and Anderson1 have described a sim-ple test for fecal reducing substances which uses Clinitest tablets and which is useful as a
screening test to detect diarrhea that may be
due to sugar malabsorption. Results of this test are graded from 0 to 4 + and values greater than 1 + in children are considered abnormal and strongly suggestive of the presence of in-testinal malabsorption of sugar. Although sugar
malabsorption can also be a serious problem in
the neonatal period, there has been no ade-quate data to indicate whether Kerry and An-derson’s test is similarly applicable to this age group, and there have been conflicting reports as to the amounts of sugars normally found in the neonate’s stools.2,3 In order to determine whether Clinitest results over 1+ should be considered abnormal in neonates also, we have applied this test to 145 stool specimens from 114 normal babies. We have also done chromat-ographic and enzymatic assays of lactose, glu-cose, and galactose on 22 of these specimens
in an attempt to show whether detectable
amounts of the sugars are sometimes found in the stools of normal neonates.
SUBJECTS AND METHODS
Stools were collected from apparently
nor-mal full-term neonates in the newborn nurser-ies of the Vancouver General Hospital,
Van-couver, British Columbia, Canada, and the
Sorrento Maternity Hospital, Birmingham,
En-gland. There were 73 stools from 57 breast-fed
infants and 60 stools from 45 infants fed
for-mulas containing approximately 6.7 to 7% lac-tose. Twelve stools were tested from infants fed formula containing 4.8% lactose and 2%
su-crose. Six meconium stools were also obtained
from infants aged under 24 hours who had
been fed glucose-water only. The stools were
collected in polythene-lined diapers and were
tested as soon as possible after collection
(
never over 2 hours) . Contamination of thestools with urine occurred very rarely, and any
stools in which this had occurred were
dis-carded. The test was done as follows:
One part stool was mixed with two parts water and
then centrifuged. Fifteen drops of the supernatant
were placed in a clear test tube and a Clinitest
tablet was added. The resulting reaction was
al-lowed to proceed undisturbed. Fifteen seconds after reaction had subsided, the tube was gently
shaken and the color was compared to that on the
urine color chart provided with the Clinitest tablets.
Results were read as 0, trace, +, --+, --++, and
++++ for reducing substances (equivalent in
reducing power to 0, li, 3, %, 1, and 2% glucose respectively ) .
Heating the stool extract with HC1 before
add-ing the Clinitest tablet enables sucrose also to be detected, but since no sucrose was present in the babies’ diets, no attempt was made to detect it.
Paper chromatography was used to look for lactose, glucose, and galactose in 22 of the
spec-imens. The stool extracts were prepared as for
the Clinitest test and 5 or 10 jzl were spotted on Whatman paper along with standard solution of the sugars. Descending chromatograms were run in three different solvent systems, i.e.,
iso-propanol-water, 4: 1; ethyl
acetate-pyridine-water, 65: 25: 20; and butanol-acetic
acid-wa-ter, 120:30:50. Location of the spots was done with two different reagents, i.e., silver nitrate and aniline diphenylamine. Identification of the sugars was made on the basis of their mo-bility in these three solvent systems and their
reactions with these location reagents. A
semi-quantitative assay of the amounts of the
van-ous sugars present was also obtained by
corn-panison of the size and staining density of the
unknown spots with standards of known
con-centration.
Specific estimation of glucose and galactose was also done on these 22 fecal extracts using
tnis-glucose oxidase and galactose
dehydrogen-ase6 methods, respectively.
Stool pH was measured using pH paper.
RESULTS
The Clinitest results from all the breast-fed
babies and those on the formulas containing
over 6.7% lactose are shown in Table I.
EXPERIENCE AND REASON-BRIEFLY RECORDED
TABLE I
RESULTS OF CLINITEST TEST ON STOOLS Faoi INFANTS FED EITHER BREAST MILK
OR Cow’s MILK BASED FORMULAE CONTAINING 6.7% OR MORE LACTOSE
Age of
Baby
Type of Feed
Number of Stools u’ith (‘linitest Result
633
Total
0 ‘I’I’ace + ++ +++ ++++
Day 3 Day 4 - 6 2 - 7 5 i --- I ‘3 1 1 ‘2 4 ‘2 3 -- I 3 --- I 9 6 17 16
0 6 0 0 0
Day 0
Glucose-water
Day 1 Breast
Formula
Day 2 Breast
Formula Breast Formula Breast Formula
Day 5 Breast
Formula
I)ay 6 Breast
Formula
I)ay 7 Breast
Formula
I)ay 8-14 Breast
Formula
Day 15-30 Breast
Formula Total Breast Formula Glucose-water ---
___-
--- - - 4
- - 1 - 8
1 - 1 9
I - - -- 5
I - S ‘3 10
3 1 4 - 13
3 3 4 3 16
3 1 - 1 8
- 3 5 4 1’2
2 I 3 -- 1
- 2 4 5 11
3 1 -
--
71 - 6
- - - 1 4
- - ‘2 - ‘3
- - I - I
- 1 - I
- - -
-8 11 20 19 73
I2 4 9 2 60
tested, the results are not appreciably altered if only one specimen from each baby was consid-ered. Of 133 stools in Table I, 65 gave a Clini-test result above the 1 + which has been
con-sidered normal in older children and 50 of
these registered more than 2+ . Twenty-one
specimens gave the maximum reading of 4+
and, of these, 19 were from breast-fed infants. Only 3 of 32 specimens collected before 3 days of age gave results over 1 + , but five of eight
specimens collected between 8 and 30 days of
age had results over 1 + , with one 4+ result found at 30 days. None of the 12 stools from the infants fed on the lower lactose formula had Clinitest results as high as 1+.
Chromatography of 22 of the fecal extracts
demonstrated varying amounts of lactose, ga-lactose, and glucose as well as several
unidenti-fled reducing substances which had low Rg
val-ues (i.e., low mobility with the solvent systems
used) . The unidentified substances were
pres-ent in almost all specimens, often appearing in
quite high concentrations. No attempt was
made to positively identify these substances,
but their chromatognaphic characteristics
sug-gested that they were probably
oligosaccha-rides.
The results of the quantitative chromato-graphic assay of lactose are given in Table II.
This system was not capable of detecting
lac-tose unless its concentration exceeded 50 mg/
100 ml but it found lactose in 4 of the 22
spec-imens in concentrations from 200 to 300+
mg/100 ml.
The chromatographic assays of glucose and
galactose agreed with the results of the specific
assays for these sugars, and the results of the
specific assays are given in Table II. Of the 22
specimens tested, galactose was found in 20
(Ii,i-NUIIIhCI t(st I 0 ‘2 0 3 0 4 ‘Ii’ .5 ‘I’i#{149} (1 + 7 + S + 9 + 10 ++ 11 ++ H ++ 13 ++ 14 +++ 15 16 ‘+++ 17 18 !+++ 19 ‘20 ‘21 ‘22
++-f-‘Fr = trace.
+=1+.
++++ =4+.
‘I’AIILI 11
(‘oIl’AItIsoN OF CLINITEST RESULTS AND CONTENT OF
LACTOSE, GALACTOE 011 (iLucosF IN ST00L’ Fitoi
INFANTS FED BREAST MILK OR FORMULAE (‘oNT&INING 6.79 on MORE LAUTOE
Lactose , (alactosc (JIU(OSe
log lOt) 1111 IIlg/ 100 [II
) Hg 10() JIll
(Iopel ((OlliletosI
((iluose
( I1IOIIE1- I )eliyuro-()xidase)
togiaphy) gellase)
0 0 0
- 0 17
(I (1
-
<10 <1010 37
68 153
<10 58
0 157
.
‘27 (14<10 I3
54 75
‘200 275 768
-
14 18138 I,’248
‘2’27 459
--
95 1753()0’ 2’20 681
-
‘24 411-
16 34--
‘238 4’203(1(1 54 ‘251
20() 10 94
250 mg/100 ml in 7 specimens, with a highest
value of 1,248 mg/100 ml. Glucose
concentra-tions were lower than galactose, but it was
de-tected in all but four stools and up to 275 mg/
100 ml was found.
Although the highest concentrations of these three reducing sugars were found in stool
spec-imens with high Clinitest results, and the
low-est concentrations were found in stools with
Clinitest results of 0 or trace, some specimens
with high Clinitest results appeared to contain
little glucose, galactose, or lactose. Two of
these specimens tested 3+ to Clinitest but had
no lactose and less than 100 mg/100 ml of
glucose or galactose. In these, the unidentified
“oligosacchanide” components were prominent
in the chromatograms.
The pH of the stools ranged from 5.5 to 7.5.
No consistent relation between reducing
sub-stance concentration and pH was noted. The
appearance of the stools ranged from yellow
soft paste to yellow soft curds with moderate
amounts of mucousy liquid. There (lid not
seem to be any close correlation between the
description of the stool and the reducing
sub-stance concentration, although the highest
Clin-itest results seemed more often found in the
stools with most liquid.
DIscusSIoN
These results have shown that reducing
sub-stances can be frequently found in the stools of
normal infants. High Clinitest results were
common between 3 and 7 days of age and
were also seen in a specimen from the oldest infant tested, aged 30 days. Further
investiga-tion of these reducing substances showed that
glucose, galactose, and lactose were frequently
present although in variable amounts. Other
re-ducing substances were also present and in
some instances were in sufficient quantity to
cause high Clinitest results even though little glucose, galactose, or lactose were detected in the sample.
These results show that in the group of
nor-mal infants we tested, neither high Clinitest
re-sults nor the presence of glucose, galactose, or
lactose in the stools are unusual. In addition, in
this group, a high Clinitest result cannot be
taken to prove the presence of glucose,
galac-tose, or lactose. Therefore, in contrast to the
situation in older children, neither a high
Clini-test result nor the presence of these sugars in
the stools can here be considered in itself a
pathological sign of sugar malabsorption.
Al-though the Clinitest test remains extremely
use-ful, high (“positive”) results must be
inter-preted with due regard for the entire clinical
and laboratory picture, including the age of the
child and the type of reducing substance pres-ent. A low Clinitest result (i.e., below 1 + ) re-tains its usefulness as evidence against the
presence of sugar malabsorption.
It is also worth noting that the typically
pastey, or curdy, liquid stools described in our
series were different from the liquid, acid,
EXPERIENCE AND REASON-BRIEFLY RECORDED 635
children with “pathological” sugar
malabsorp-tion and that the description of the stools should
therefore also be considered when evaluating
the results of the Clinitest test.
Our results contrast with those of Gryboski,
et d2 who found glucose or galactose in only
6% of stools from infants aged 1 to 5 days, and
concluded that these sugars rarely occurred.
However, the negative results in their series
came from infants on a formula containing only
about 4% lactose; and, if lactose is a source of
the glucose and galactose found, it is possible
that their low results were due to this lower
lactose intake. This possibility is also suggested
by the lower Clinitest results found in our
in-fants fed lower lactose feeds. It is also not clear
whether Gryboski and coworkers2 took
precau-tions against the absorption into the baby’s dia-pens of any liquid in the stools. If absorption occurred, water soluble sugars would also be
lost and might account for the low results. Ford
and Haworth3 also used a lower lactose content
feed than we did, but their results are more in
agreement with ours although they did not
quantitatively measure galactose or lactose and
found only 1 of 19 specimens with oven 750
mg/100 ml reducing substance.
It seems probable that the sugars and other
reducing substances found in the stools we
have examined were of dietary origin. This
probably accounts for the low values seen in
the first 2 days of life. It is possible that the
glucose and galactose seen were produced by
the hydrolysis of dietary lactose in the gut.
However, it is also possible that they were at
least in part derived from oligosacchanides
since these are present in human milk in
quan-tities as high as 0.6% and under certain
condi-tions glucose and galactose can be also
pro-duced from them by hydrolysis.8
A. C. F. DAvIDsON, B.Sc., M.D.
Institute of Child Health
University of Birmingham
Francis Road
Birmingham 16, England
M. MULLINGER, M.D. Health Centre for Children University of British Columbia
715 W. 12th Avenue
Vancouver, British Columbia, Canada
Address reprint requests to Dr. Mullinger.
A.G.F.D. has received a fellowship from the Queen Elizabeth II Canadian Research Fund.
The authors would like to thank the nurses from
the newborn nurseries of the Vancouver General
hospital and the Sorrento Maternity Hospital for
their intelligent co-operation. The authors are very grateful to Professor C. M. Anderson for her com-ments regarding this paper, and to Dr. K. R. Kenny and Mr. G. A. Brown for their helpful suggestions.
REFERENCES
1. Kenny, K. R., and Anderson, C. M. : A ward test
for sugar in faeces. Lancet, 1 :981, 1964. 2. Cryboski, NI. D., Zillis. J., and Ma, 0. II. : A
study of faecal sugars by high voltage electro-phoresis. Castroenterology, 47:26, 1964. :3. Ford, J. D., and Haworth, J. C. : The fecal
ex-cretion of sugars in children. J. Pediat., 63:
988, 1963.
4. Smith, lyon, ed. : Chromatographic and
Electro-phoretic Techniques, Vol. I. London: William
Heinemann Medical Books Ltd., 1960.
5. Dahlqvist, A. : Method for assay of intestinal di-saccharidases. Anal. Biochem., 7:18, 1964. 6. Wallenfele, K., and Kurz, C. : On the specificity
of galactose dehydrogenase from
Pseudomo-nas saccharophilor and its use as an analytical
aid. Biochem. J., 335:559, 1962.
7. Townley, R. R. W. : Disacchanidase deficiency in
infancy and childhood. PEDIAmIc5, 38:127,
1966.
8. Stacey, M. : The oligosacchanidea of milk. In
Stacey, M., and Barber, S. A.: Carbohydrates of Living Tissues. London: D. van Nostrand
