Transcript Measurement of exposure to indoor fungal growth using

PURPOSE OF RESEARCH

Project as a whole:
By using several methods of measuring fungal biomass
and speciating fungi, we wish to compare existing and
modified tests to determine which one or combination of tests,
best correlate with health measures

Personal Project:
Quantitatively measure 1,3--D-glucan by developing
chemical procedures to adapt the Limulus amebocyte lysate
assay which is commonly used for LPS endotoxin
INTRODUCTION
Research to Date

Since 1982, ~30 questionnaire studies on the
association of dampness, mold and respiratory health in
Europe and North America

Dose-Effect has shown that more visible mold
yielded more symptoms in children

Unfortunately, acceptance of association between
indoor fungal growth and health as causal is prevented
by uncertainty in measuring the exposure
Types of Measurement
Airborne
Settled
Ergosterol - information on total mold
biomass in air
Dust Collection - information on taxa
Measurement
Spore
of Fungi and Fungal Products in Dust
-1,3--D-glucan and ergosterol
Collection - examined by microscopy with a
number of stains used to visualize
a) sticky surface
b) impaction on agar
Indoor Air
Indicator Biochemicals - for either
a) bacteria  endotoxin and peptidyl glycan
b) fungi  1,3--D-glucan and ergosterol
1,3--D-glucan
A polyglucose
compound found in the cell wall of fungi,
certain bacteria, and plants
Can
form triple helices, single helices and single chains
Some
forms show anti-tumor activity, while others show
a relationship between glucan concentration and the
extent of airways inflammation
Treatment
of 1,3--D-glucan with urea, NaOH, or by
heating causes a change in conformation
EXPERIMENTAL
Use
a modified form of the classical limulus amebocyte
lysate test for determination of 1,3--D-glucan
Endotoxin
Factor C
1,3--D-glucan
Activated Factor C
Factor B
Activated Factor B
Activated Factor G
Proclotting Enzyme
Clotting Enzyme
Boc-Leu-Gly-Arg-pNA
Boc-Leu-Gly-Arg
PNA (p-nitroaniline)
Factor G
Perform
various control tests using LAL on glucan and
glucan + endotoxin standards to obtain optimal modified
test specific to 1,3- -D-glucan
Extract
1,3--D-glucan from field samples using the
above optimal conditions (exposure expressed as ng/m3)
1,3- -D-glucan content of common fungal
species encountered in indoor air using modified LAL
Determine
Remove
and extract ergosterol from field sample filters
using microwave-digestion technique (Young, 1995)
Analyze
extract by means of HPLC or GC-MS to
determine amounts of ergosterol (exposure expressed as
ng/m3)
Young, JC., “Microwave-assisted extraction of the fungal metabolites ergosterol and total fatty acids” in J.Agric. Food
Chem., 43:2904-2910 (1995)
RESULTS
LAL chromogenic test is approximately 1000 times more
sensitive to endotoxin (LPS) than to 1,3--D-glucan
Curdlan (unbranched linear 1,3--D-glucan) retains
activity after NaIO4 oxidation
 indicates std is pure within LOD of test
Upon addition of Tween, no significant change in
glucan activity over a range of 0.1-0.005% added
Determined that endotoxins’ reactivity (10 ng) with LAL
could be inactivated up to 100% using various NaOH
concentrations (i.e. 0.15N - 0.50N)
 0.15N (100%), 0.3N (78%), 0.5N (52%)
From above, determined time scale of endotoxin
destruction for 0.15 N NaOH
Endotoxin in 0.15 N NaOH at various times
2.5
y = 0.3531x + 0.4206
R2 = 0.9984
Absorbance Units (405 nm)
2
1.5
1
0.5
0
0
1
2
3
4
5
6
Concentration (ng/ml)
t=0 control
t=0.5 hrs
t=1 hr
t=1.5 hrs
t=2 hrs
t=2.5 hrs
Linear (t=0 control)
Curdlan in 0.15N NaOH shows increase in LAL activity
of up to 5x (for 2.5hrs) compared to neutralized curdlan
 therefore, greater test sensitivity
LAL activity of curdlan showed no increase after 2.5 hrs,
up to 25 hours in length
After neutralization, a decrease in LAL activity was
observed at 10 and 23 hours, at which time activity was
equal to that of controls (0.3125-2.5 ng/ml)
LAL test provides r2 values of greater than .990
consistently for 1,3--D-glucan standard curves
Glucan in 0.15 N NaOH
vs. Time
2.5
y = 0.0609x + 0.0667
R2 = 0.9966
Absorbance
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Concentration (ng/ml)
control
t=12 hrs
t=25 hrs
Linear (control)
Conversion of Glucan Back to Triple Helix
Conformation vs. Time
2.5
Absorbance UnIts
t=2.5 hrs
y = 0.0609x + 0.0667
R2 = 0.9966
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Concentration (ng/ml)
t=0 control
t=2.5 hrs
t=2.5 after 10 hrs neutralization
t=2.5 after 25 hrs neutralization
1,3--D-glucan structure in linear form
CH2OH
CH2OH
O
CH2OH
O
O
OH
OH
OH
OH
O
O
OH
OH
n
A proposed mechanism of
conformational change
involving partially opened
triple-helix for 1,3--Dglucan.
Young, S.H., Dong, W.J., Jacobs, R.R.,
Journal of Biological Chemistry, v.275, n.16,
pg. 11874-11879 (2000)
Pure endotoxin + curdlan showed upto a 350% increase in expected
glucan activity, resulting in a “leveling effect” of the reagent
2:1 reagent:sample mix eliminated this effect
Longer hydrolysis times (upto 25 hrs) did not affect the
destruction of endotoxin in the presence of curdlan
Preliminary tests of extracting 1,3--glucan from real fungal
spores (Cladosporium cladosporioides) indicated a glucan
content of 3.09 ±0.6 pg/spore
Using above result, a known number of C. cladosporioides
spores were added to filters followed by endotoxin
 0.15N NaOH not sufficient to destroy endotoxin
activity, therefore increased [NaOH]
16
Amount of Glucan (by LAL)
14
12
10
8
6
4
2
0
0
0.625
1.25
2.5
5
10
Amount of LPS Added (ng/ml)
Enhancement of Glucan (0.15N)
Glucan Standard (1.25 ng/ml)
Enhancement of Glucan in 0.3 N NaOH
Enhancement of Glucan (0.5N)
No significant change (p<0.05) below 1.25ng and 5ng
endotoxin added for 0.3N and 0.5N NaOH, respectively
Curdlan in 0.5N NaOH showed a further increase in LAL
activity (and hence in sensitivity) compared to 0.15N
Using result for C. cladosporioides, LAL method was
applied to outdoor air samples to determine mean number
of spores/m3 (=2400 spores/m3)
comparable with results obtained by microscopy
Other experiments demonstrating LAL applicability for
accurate measurement of 1,3--D-glucan were performed on
pollen, Ambrosia atemisiifolia, and yeast
Pollen - 83ng/mg is comparabe to results by
Rylander et al. using 1,3- -D-glucan specific
test called the Fungitec G-Test
Yeast - 0.321ng/spore is comparable to fungal spores
Rylander, R., et al. “1,3--D-glucan may contribute to pollen sensitivity” Clin Exp Immunol 1999; 115:383-384
Correlation between ergosterol and 1,3--D-glucan from
field samples was 0.654 (n=108, Pearson Correlation)
Glucan content of 12 different common indoor fungal
species was obtained using LAL method, followed by spore
dimensioning using digital images
2
Aspergillus
sydowii
Stachybotrys
chartarum
pg glucan/spore/m
(ellipsoid)
-4
0.00268 ± 1.85x10
n=5
-4
0.00401 ± 3.65x10
n=6
-4
0.00501 ± 3.90x10
n=5
-4
0.00662 ± 6.54x10
n=5
-3
0.00830 ± 2.68x10
n=5
-4
0.00920 ± 9.66x10
n=7
-4
0.0102 ± 6.89x10
n=4
-3
0.0103 ± 2.28x10
n=6
-3
0.0116 ± 2.19x10
n=8
Aspergillus
versicolor
Cladosporium
cladosporiodies
0.0220 ± 4.61x10
n=6
-3
0.0598 ± 7.46x10
n=6
Penicillium
viridicatum
Eurotium
herbariorum
Penicillium
citrinum
Paecilomyces
variotii
Penicillium
aurantiogriseum
Penicillium
commune
Eurotium repens
Digital image of P.variotii used for spore
dimensioning and glucan content of different
indoor fungal species
-4
pg glucan/spore/ m
(x1000)
2.68 a, b, c, d, e
2
4.01 a, b, c, d, e, f, g, h
5.01 a, b, c, d, e, f, h
6.62 a, b, c, d, e, f, g, h, i
8.30 a, b, c, d, e, f, g, h, i
9.20 b, d, f, g, h, i
10.2 b, c, d, e, f, g, h, i
10.3 b, c, d, e, f, g, h, i
11.6 d, e, f, g, h, i
2
Spore Surface Area (m )
(9.95) 16.0 ± 3.14 (32.2)
n=132
(16.6) 27.4 ± 6.83 (49.1)
n=97
(5.98) 10.6 ± 2.79 (21.0)
n=149
(11.9) 26.4 ± 8.79 (67.5)
n=143
(7.31) 19.9 ± 4.41 (37.7)
n=112
(10.3) 16.6 ± 3.56 (35.0)
n=150
(12.5) 20.5 ± 5.10 (41.7)
n=121
(10.2) 20.4 ± 5.70 (39.3)
n=103
(37.9) 75.0 ± 17.7 (120)
n=116
22.0 j
(7.12) 13.5 ± 3.31 (27.6)
n=134
59.8 k
(7.98) 56.88  26.6 (125)
n=59
Table 1: (1-3)--D-Glucan concentrations for 11 species of mold normalized for
Surface Area
NOTE: Lower case letters indicate the values which are
statistically similar (ANOVA Tukey Post-hoc, p>0.05)
CONCLUSIONS
0.5
N NaOH showed best results as an extracting solvent for
samples containing both endotoxin and 1,3--D-glucan, with no
significant difference in LAL activity below 5ng of endotoxin
 satisfactory for indoor and outdoor air samples as endotoxin
rarely exceeds the 5ng range, based on previous results
Modified
LAL method demonstrated good applicability with pollen
results that are comparable with 1,3--D-glucan specific test
Physical
methods are currently being explored (ie. MALDI-MS,
SEC) with curdlan standards coupled with the LAL method to
confirm concentration of the insoluble 1,3--D-glucans
Statistical
analysis of normalized spore dimensions and glucan content
show a correltion of 0.604 (0.939 without C. cladosporioides)
ACKNOWLEDGMENTS: Funding provided by NSERC, CHRP, TSIR, Health
Canada. We thank John Bissett (AAFC) for his assistance with the digital imaging.