From the Departments of Foods and Nutrition

(GPS, CH) and Oral Biology (CD), University of M an-

itoba, W innipeg, M anitoba, Canada.
2Supported by M anitoba M edical Services Founda-
tion and M edical Research Council of Canada.
3Address reprint requests to: Dr Gustaaf P Seven-
huysen, Department of Foods and Nutrition, University
of M anitoba, W innipeg, M anitoba, Canada R3T 2N2.
Present address: Kellogg Salada Canada, 6700
Finch Street, Rexdale, Ontario, M gW 5P2.
Received July 15, 1983.
Accepted for publication November 15, 1983.
The American Journa/ of C/inica/ Nutrition 39: APRIL 1984, pp 584-588. Printed in USA
1984 American Society for Clinical Nutrition
584
Development of salivary a-amylase in infants from
birth to 5 months1-3
Gustaaf P Sevenhuysen, P/iD, Christine Holodinsky,4 M SC, and
Cohn Dawes, BSC, BDS, PhD
ABSTRACT The a-amylase activity in whole saliva of two groups of infants was investigated
from birth to 5 months at monthly intervals. Foods used in infant feeding as well as height and
weight were recorded at each monthly collection period. a-Amylase activity was found to increase
rapidly from low values at birth to approximately two-thirds of adult values by 3 months. Large
variation in a-amylase activity, either per ml of saliva or per mg of protein, was recorded. No
significant relationships of a-amylase activity with weight, weight for height, growth rate, or
presence of starch-containing foods in the diet were found. Introduction of starch-containing
food before 3 months of age would likely lead to inadequate hydrolization of starch in some
infants. Am J C/in Nuir 1984;39:584-588.
KEY W ORDS a-Amylase, infant, saliva, growth, infant diet
Introduction
Starch and other complex carbohydrates
are hydrolyzed by a-amylase, end-products
of this hydrolysis being maltose, glucose,
maltotriose, and dextrins. a-Amylase (1,4-
a-D-glucan glucanohydrolase, EC 3.2.1.1),
which is found in both saliva and pancreatic
secretion, ensures complete hydrolysis of
starch in the alimentary canal in most indi-
viduals. Incomplete hydrolysis will reduce
the energy available from ingested starch
and may result in bacterial fermentation of
undigested carbohydrate in the colon, caus-
ing decreased absorption of other nutrients
(1). Inadequate hydrolysis can occur when
activity levels of salivary and pancreatic am-
ylase are insufficient.
In the newborn infant a-amylase activity
levels in saliva are very low compared with
those of adults and a-amylase activity levels
in pancreatic secretion are negligible (2, 3,
3a). a-Amylase activity in both secretions
has been found to be significant by 6 months
of age (4). Infants have been found to main-
tain gastric pH and pepsin concentration at
levels insufficient for protein hydrolysis, for
longer periods of time than adults (5-7).
Gastric pH of 4.0 and above allows salivary
a-amylase to remain active in the small in-
testine (2, 7) and it has been postulated that
for infants salivary a-amylase has an impor-
tant role in starch hydrolysis in the duo-
denum (8). A similar role has been suggested
for mammary a-amylase (6).
The first solid food introduced into the
infants diet is most commonly cereal (9), in
which starch is the major constituent. Sali-
vary a-amylase activity level is an important
indicator of the ability to hydrolyze starch
in infancy. The rate of development of a-
amylase activity in saliva may be relevant to
recommendations made to parents regard-
ing the introduction of starch-containing
foods to infants, in view of the complications
recorded with inadequate carbohydrate
digestion (1, 9a).
Assays for a-amylase activity in human

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4 5
AGE I MONThS
SALIVARY a-AM YLASE IN INFANTS 585
FIG 1. Salivary a-amylase activity from birth to 5
months.
saliva have used a variety of methods, the
results ofwhich are not always directly corn-
parable. The method chosen for this study
allows data to be expressed in ways compa-
rable with a number of previous studies.
M ethods
A total of 29 infants was investigated. Observations
on 10 of these were made at birth, 1, 2, 3, 4, and 5
months, 7 days (group I). Observations on the re-
maining 19 were made at 3, 4, and 5 months (group
II), 7 days. All infants were born at the Health
Sciences Centre in W innipeg, M anitoba, Canada, and
were considered healthy after routine medical exami-
nation. Subjects investigated were all infants for whom
consent from physicians and parents was obtained,
providing a nonrandom sample. To control for the
circadian rhythms in saliva flow rate and composition
(10) associated with the various sleep-wake patterns of
the infants, all collections of saliva were taken at the
adult peak flow time of between 1 and 6 PM . All
samples were collected 1#{189} to 2 h past feeding to ensure
that samples were collected at periods ofresting flowrate
and composition. Dawes (10) concluded that in adults
collection of saliva should take place 2 h after feeding.
The contribution of different salivary glands to the
volume of saliva changes with the level of oral stimu-
lation. Oral stimulation will also cause the concentra-
tions of protein and a-amylase in the secretions to
change by varying amounts between different salivary
glands (10). All sample collections were made from
unstimulated saliva to avoid these changes in concen-
trations. No chemical stimulants were used, nor was
the oral cavity intentionally mechanically stimulated.
W hole saliva was collected by suction catheter (Argle
De Lee Suction Catheter, size IOFR, trap size 10 ml,
Sherwood M edical Industries, St Louis, M O). The trap
attached to the catheter was not used. Saliva was drawn
up by suction from the floor of the mouth. Saliva
samples were stored in plastic microcentrifuge tubes
and refrigerated to 4#{176}C within 1 or 2 h. a-Amylase
activity was assayed within 24 h of refrigeration. On
completion of the assay samples were frozen and sub-
sequently analyzed for total protein.
Height and weight were recorded each month at the
time of saliva collection. Height was measured to the
nearest 0.5 cm using an Infantometer (Grafco Infanto-
meter, no 2867-1334, Graham-Field Surgical Co Inc,
New Hyde Park, NY). The mean of two measurements
was recorded. W eight was measured to the nearest g
with a portable beam balance. W eight of diapers was
accounted for. Procedures for height and weight meas-
urements were those described by Jelliffe (11).
An interview with the mother provided a list of foods,
including breast milk or formula, fed to the infant at
the time measurements were taken. All measurements,
interviews, and saliva collection took place in the home.
All procedures were approved by the University of
M anitoba Ethical Review Committee.
Assays of a-amylase activity were carried out using
the method of Bernfeld (12). A unit of activity was
expressed as 1 mg of maltose released per minute at
30#{176}C, pH 6.9 from starch solution. The starch solution
was modified as described by Strumeyer (13). The
phosphate buffer contained 0.06 M NaC1. Absorbance
was read at 540 nm on a SP6-300 Pye Unicam Spectro-
photometer.
Estimation of the protein content of saliva samples
was carried out according to the method of Lowry et al
(14). Absorbance was read at 600 nm and values were
expressed as mg of protein per 100 ml of saliva. Specific
activity of infant saliva was defined as the a-amylase
activity recorded per mg of total protein. Activity per
ml of saliva and activity per mg of protein were recorded
for each observation.
Results
Development of a-amylase activity per ml
of whole saliva varied considerably between
infants as shown in Figure 1 . At birth the
majority of infants had negligible a-amylase
activity in saliva as shown in Table 1. By 2
months the majority of infants had appre-
ciable levels of a-amylase and by 3 months
several individuals had reached 90 or more
of a-amylase activity U/mI. It would seem
that 90 U/ml is equivalent to adult levels,
which range from 70 to 300 U/ml (15, 16).
Figure 1 demonstrates the great variability
in a-amylase content. At 5 months the ma-
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TABLE 1
M ean values (SD) for protein content and cv-amylase
activity by age-group I
Age Amylase activity Protein Specific activity
no U/mi sal i va mg//CO mi U/mg proiein
Birth 1 1 9.9 9.2 7 294.0 70.9 7* 4 5 5.03
1 11 28.327.3 9 240.498.9 9 14.3 11.1
2 1 1 48.7 46.4 10 143.3 76.6 10 34.5 26.0
3 1 1 57.2 40.7 10 140.2 45.5 10 43.0 26.6
4 10 52.6 37.4 10 1 12.5 34.9 10 50. 1 40.8
5 10 58.128.3 10 138.558.0 10 51.129.8
a Numbers of subjects.
TABLE 2
M ean values (SD) for a-amylase activity and
protein content by age-group II
Age Amyiase activity Protein Specific activity
U/mi sal i va mg/HX.I ml U/mg prolein
3 19* 31.322.0 11 75.428.2 11 35.022.2
4 18 41.028.8 11 90.825.9 18 44.426.8
5 19 59.2 39.7 19 106.2 62.0 19 59.9 35.9
a Numbers of subjects.
TABLE 3
M ean wt (SD) and growth rate at
each collection period
wt
Age
Groupi G r ou p li
Growth#{176} NCHS standard
G r ou p i G r ou p li F M
no g g g/da, g/da; mean g
Birth 3627 567 3230 3270
1 5 2 2 1 8 3 2 3 3 1 3 3 9 8 0 4 2 9 0
2 5 7 5 4 8 1 8 3 0 1 5 4 7 0 0 5 3 0 0
3 6 2 9 9 8 8 2 6 1 0 8 1 0 3 4 2 5 8 2 1 1 1 5 4 0 0 5 9 8 0
4 7 1 0 2 1 0 2 1 6 9 7 4 1 0 4 7 2 3 8 2 2 2 0 6 0 3 0 6 8 0 0
. 5 7 6 4 4 9 7 7 7 4 0 1 1 2 5 7 2 0 7 9 2 0 6 6 3 0 7 3 0 0
TABLE 4
Linear regression analysis for group I
S Growth rates: the wt at the previous month subtracted from the wt at the current month divided by the
number ofdays between the two measurements.
TABLE 5
Linear regression analysis for group II
586 SEVENHUYSEN ET AL
Independent
De pe nde nt variables
Amylase activity Specific activity
variable (U/mi s a liva ) (U/mg protein)
r p r p
Age (days) 0. 16 0.0008 0.24 0.0001
W t (g) 0.26 0.0001 0.27 0.001
Growth (g/day) 0.00 0.8 1 5 0.003 0.70
jority of infants had achieved 85% or more
of adult levels (Table 2).
None of the variability in salivary a-am-
ylase observed between infants could be ex-
plained by variation in growth indicators or
the presence in the diet of foods containing
starch. Neither growth rates nor absolute
increases in weight, shown in Table 3, cor-
Independent
Dependen t variables
Amylase activity Specific activity
variable (U/mi saliva) (U /m g pro te in)
r p r p
Age (days) 0.42 0.001 0.05 0.10
W t(g) 0.27 0.001 0.002 0.71
Growth (g/day) 0.04 0.05 0.02 0.28
related with either a-amylase activity per ml
of saliva or per mg of total protein (specific
activity) in either of the groups studied. In-
creases in weight divided by increases in
height between monthly observations did
not correlate with absolute changes in a-
amylase activity. Regression of a-amylase
activity values over all 5 months simultane-

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SALIVARY a-AM YLASE IN INFANTS 587
ously with age, weight, and growth rate
showed significant relationships with both
age and weight as shown in Tables 4 and 5.
In addition, significant relationships were
found between age and weight and amylase
specific activities for the 0- to 5-month group
(Table 4). The presence of starch in infant
diets was not associated with higher values
of salivary a-amylase activity than were
found in infants without starch in the diet.
Starch-containing foods were fed as early
as 1 month of age. In both groups I and II
36% ofinfants consumed cereal at 3 months
(four and seven infants, respectively). At 5
months approximately 72% consumed cer-
cal in the two groups (eight and 14 infants,
respectively). Only three of 29 infants con-
sumed food items other than cereal as their
first solid food. The proportion of group I
infants breast-fed at 3 and 5 months was
80% . For group II infants proportions were
63 and 52% at 3 and 5 months, respectively.
Activities were not consistently higher dur-
ing the months after the first introduction o f
starch-containing foods.
I n t h e g r o u p of children investigated, no
significant effect on growth rate could be
observed from the introduction of starch-
containing foods into the diets of chil-
dren with low salivary a-amylase. Neither
changes in growth rate nor estimated varia-
tion with expected growth rates were found
to be different between children with high
or low levels of a-amylase. No effects were
observed wh e t h e r t h e s a mp l e wa s grouped
a c c o r d i n g t o h i g h o r l o w l e v e l s o f s a l i -
v a r y a - a my l a s e a b o v e a n d b e l o w 5 0 , 3 0 , o r
20 U/ml. Fishers exact test and M ann W hit-
ney U test were used wi t h e a c h o f t h e t h r e e
g r o u p s s e p a r a t e l y .
Discussion
Th e f i n d i n g s are characterized by large
variations in the a-amylase activity of the
saliva in infants. The activity levels show
that some individuals have developed adult
levels by 3 months. The rate of increase in
a-amylase activity appeared t o s l o w down
f r o m t h e 3 r d mo n t h o n . Ro s s i t e r e t a l ( 1 7 )
found a similar relationship between the
activity levels of stimulated saliva at the 3rd
a n d 6 t h mo n t h . Al t h o u g h Rossiter e t a l ( 1 7 )
used a different method for enzyme assay, it
appears that after conversion, the mean ac-
tivities reported are of comparable magni-
tude to those found in this study for those 2
months and for values at birth.
A wide variation of salivary a-amylase
levels between individuals has been found
for adults as well as for infants (15, 17, 18).
Understanding this variation could be useful
in formulating advice on the introduction of
starch-containing foods into the infant diet.
Only two relationships explained variations
in a-amylase activity in infants. Activities
were higher in older children and activities
were higher in children with higher body
weights. However, age and weight would be
highly correlated in any healthy group of
infants and the effect of either is difficult to
separate. In the group studied, age accounted
for 42% of variation observed, whereas
weight accounted for 27% . Furthermore no
significant relationship was found between
a-amylase activity and either absolute
weight of the infant or monthly increase in
weight. Age therefore appears to be the only
factor closely related to the development of
a-amylase. However, the relationship is not
strong enough to use age as a reliable mdi-
cator of salivary a-amylase levels.
Influence of dietary carbohydrate on the
a-amylase content of saliva in adults is un-
c l e a r . Hi g h carbohydrate diets were associ-
ated with increased amylolytic activity in
some studies (19, 20) but not in others (2!,
21a). The infants investigated in this study
did not show any significant positive rela-
tionship between salivary a-amylase activity
a n d starch-containing food in the diet. This
s t u d y wo u l d s u p p o r t t h e c o n c l u s i o n o f
Dawes ( 2 2 ) t h a t t h e r e is little evidence to
suggest that salivary a-amylase levels change
in response to dietary components.
Inadequate digestion o f dietary carbohy-
d r a t e h a s been f o u n d t o b e a c a u s e o f failure
to thrive and severe diarrhea in infants (1).
Su c h response is in all cases a f u n c t i o n o f
the total amount of carbohydrate fed. Dev-
izia et al (23) reported that 10 to 23 g of
starch was absorbed by 1- and 3-month-old
infants when served as cooked flours. Starch
in the amounts of 40 g/day or 25 tablespoons
has been reported to cause fermentative
diarrhea (23). The amount of starch fed daily

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588 SEVENHUYSEN ET AL
to the infant with severe growth failure re-
ported by Lillibridge and Townes (1) was 9,
l7,and70gfor0tol, lto2,and2to4
months, respectively. The highest level of
starch fed in the present study was approxi-
mately 32 g/day to a 5-month-old infant.
Since the introduction of starch-containing
foods had no measurable effect on growth,
it seems that the feeding practices of mothers
in this study supplied amounts ofstarch that
can be tolerated by most infants. It has been
suggested that mammary amylase may be
important in carbohydrate digestion of
breast-fed infants (6) and may have contrib-
uted to the tolerance shown by the infants
investigated.
Since some infants do not tolerate
amounts of starch recorded herein it is pru-
dent to delay the introduction of starch-
containing foods in the diet. Current rec-
ommendations, stating that infants should
not receive weaning foods before 4 months
old, would appear to be appropriate for most
infants. It seems likely that infants of 3
months or less do not produce sufficient
amounts of salivary a-amylase to digest even
small amounts of starch. a
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