CARDIOVASCULAR JOURNAL OF AFRICA • Vol 23, No 9, October 2012
502
AFRICA
numerous clinical conditions has increased over the last
decade.
14,15
Recently, a high-sensitivity commercially available
enzyme-linked immunoassay (ELISA) adapted to measure CRP
in human saliva was validated against serum CRP.
7
The findings provided initial support for the use of salivary
CRP as an alternative marker of inflammation, with a moderate-
to-strong association (
r
=
0.72,
p
<
0.001)
between CRP measured
in saliva and serum.
7
In addition, salivary and plasma CRP were
shown to be moderately correlated (
r
=
0.61)
in females, and
salivary CRP was able to discriminate between high and low
levels of plasma CRP, using a clinically relevant cut-off point of
3
mg/l.
16
These results were consistent with strong correlations
between saliva and serum CRP levels in earlier animal studies,
specifically in pigs (
r
=
0.73),
17
and in healthy and diseased dogs
(
r
=
0.87
and
r
=
0.84,
respectively).
18
In addition to being non-invasive, the assessment of CRP in
the saliva allows data collection to take place in the participant’s
natural environments (home and school). Furthermore, CRP in
saliva is stable at room temperature for up to eight hours after
collection, making the sampling of saliva outside of research
facilities a viable option.
7
Previous studies have also shown that
serum CRP does not have a circadian rhythm
19,20
and therefore
saliva sampling aimed to measure salivary CRP does not require
one to follow a standardised time-collection schedule to avoid
diurnal variations.
7
Recently, salivary CRP was shown to be a good measure of
discrimination for the clinically relevant serum CRP cut-off point
in adults.
7
Participants with high salivary CRP levels were more
likely to have higher IL-6 levels and body mass index (BMI)
and to smoke, compared to participants with low salivary CRP
levels.
7
The results suggested that salivary CRP may represent
an alternative marker of cardiovascular risk in adults,
7
however
the association with a major risk factor for the development of
chronic diseases, low CRF,
21
was not included in the analysis.
There is limited research on the relationship between body
composition, CRF and salivary CRP in adults and specifically
in paediatric populations. The aim of this study was to examine
the relationship between salivary CRP, body composition and
cardio-respiratory variables in grade 3 to 7 children.
Methods
One hundred and seventy black South African children (100
females, 70 males) in grades 3 to 7 (age 9.41
±
1.55
years) partici-
pated in the study. Participants were recruited from an urban,
combined junior and senior primary school in Pietermaritzburg,
KwaZulu-Natal. Gate-keeper permission to perform the study was
obtained from the KwaZulu-Natal Department of Education, the
school’s headmaster and governing body. The study was approved
by the institution’s Biomedical Ethics Research Committee.
Once permission to continue was obtained, a meeting was
held with parents/guardians and children to discuss the research
details and expectations of the participants. Written informed
consent was obtained at the meeting.
The guardians/parents completed a medical history form
that included sections on infectious, immune and salivary gland
disorders. The parents/guardians were trained in the salivary
collection procedure and were provided with instructions
regarding brushing teeth and the intake of food and drink on the
morning of the saliva collection. These standardised instructions
are outlined below.
Saliva collection and C-reactive protein analysis
Each grade was tested on a separate morning over a week,
starting with grade 3 on Monday and ending with grade 7 on
Friday. Saliva samples were collected between 07:30 and 08:30,
approximately 90 minutes after waking.
The parents/guardians and children were requested to adhere
as closely as possible to the following standardised saliva
collection instructions (Salimetrics 2010): The children should
(1)
not eat a major meal (breakfast) within 60 minutes of sample
collection, (2) not brush their teeth prior to sample collection
(
this may cause the gums to bleed causing blood contamination
of the saliva), (3) avoid dairy products for 20 minutes before
sample collection, (4) avoid foods with high sugar, acidity, or
high caffeine content immediately before sample collection
(
these have all been shown to impact on the saliva pH, altering
assay results), (5) rinse their mouths with water to remove
food residue before sample collection, and swallow to increase
hydration, and (6) wait at least 10 minutes after rinsing before
collecting saliva to avoid sample dilution.
Upon arriving in the school hall at 07:00 the children sat for
20
minutes. Based on completion of a short health questionnaire
and interview with the researchers upon arrival, no participant
reported symptoms suggesting that he/she was sick (e.g. fever,
flu, diarrhoea) and there were no reports of ‘bleeding gums’ or
‘
tooth ache’ on the day of data collection.
Saliva samples were collected via unstimulated passive drool
over a time period of five minutes. While seated, the children
were asked to lean slightly forward, tilt their heads down and
accumulate saliva in the floor of the mouth for a minute, which
was subsequently swallowed. Following this, there was a four-
minute collection where the children dribbled saliva through a
5-
cm plastic straw into a pre-weighed polypropylene cryovial (5
ml capacity). Care was taken to allow saliva to dribble into the
collecting tubes with minimal orofacial movement.
After collection, the cryovial was weighed in order to
determine the saliva flow rate. The concentration of salivary CRP
was expressed as the secretion rate of salivary CRP (pg/min)
or the total amount of salivary CRP appearing on the mucosal
surface per unit time. Salivary CRP secretion rate was calculated
by multiplying absolute salivary CRP concentration (pg/ml) by
saliva flow rate (ml/min). This latter value was calculated by
dividing the total volume of saliva obtained in each sample (ml)
by the time taken to produce each sample (4 min).
Samples were placed on dry ice immediately and kept frozen
until reaching the laboratory, upon which they were stored at
–70
o
C until analysis. Salivary CRP concentration (pg/ml) was
determined in duplicate by the salivary C-reactive protein ELISA
kit (Salimetrics, State College, PA, USA). The coefficients of
variation (CV) of all duplicate samples were less than 20%.
Body composition, cardiovascular and cardio-
respiratory fitness
These measures were determined after saliva collection. Body
composition was assessed by measuring height and weight to
calculate BMI, skinfold thickness to predict body fat percentage,
and waist and hip circumferences to calculate waist-to-hip ratios.
Height was measured to the nearest millimetre using a
portable stadiometer (Nagata bw-1122h) and body mass was
measured to the nearest 0.1 kg using a calibrated electronic
