Biology Reference
In-Depth Information
comparison of the amount of MC-LR content from single fi laments isolated from natural samples
and those of laboratory cultures revealed that the content of MC-LR was below MDL in majority
of the cases. Of the two species,
P
.
agardhii
from Bassenthwaite Lake (England) exhibited lowest
MC concentration (0.7 fg µm
-3
) whereas
P
.
rubescens
from Iznik Lake showed highest MC content
(2.9 fg µm
-3
).
P
.
agardhii
showed a better positive correlation between fi lament biovolume and MC
content than
P
.
rubescens
from environmental samples. On the other hand,
P
.
rubescens
showed a
good relationship between fi lament biovolume and MC content under cultural conditions (Akcaalan
et al
., 2006).
iv)
Inhibition of acetylcholinesterase activity
:
Anatoxin-a(s) can be detected by estimating the
activity of acetylcholinesterase. Since acetycholinesterase degrades acetylcholine into acetyl group and
choline, the acetyl group can be assayed colorimetrically by complexing with dithiobisnitrobenzoic
acid. A reduction in the absorbance at 410 nm over a period of 5 minutes would indicate the
presence of anatoxin-a(s). However, it is not a specifi c inhibition assay for anatoxin-a(s) because
organophosphorus pesticides also inhibit acetycholinesterase activity. If this assay is employed, it
has to be substantiated by other confi rmatory tests for toxins of cyanobacterial origin.
2) Instrumental methods of Analysis:
(i) High Precision Liquid Chromatography (HPLC)
:
Routine
analysis of peptide toxins has been traditionally carried out by HPLC (Harada
et al
., 1996a,b; Meriluto,
1997; Meriluto
et al
., 1996). Sample separation is followed by sample detection. Sample separation
is achieved by a reversed phase C18 packed column (Krishnamurthy
et al
., 1986; Harada
et al
.,
1988a,b; Flett and Nicholson, 1991; Rositano and Nicholson, 1998; Lawton
et al
., 1994; Wirsing
et
al
., 1999), amide C16 column (Spoof
et al
., 2001), internal surface reversed phase column (Meriluto
and Eriksson, 1988) or an ion exchange column (Gathercole and Thiel, 1987) and an aqueous mobile
phase containing methanol or acetonitrile. The use of an internal surface-reversed-phase column
minimized the time compared to others (Meriluto and Eriksson, 1988; Meriluto
et al
., 1990; Watanabe
et al
., 1988; Kondo
et al
., 1992b). Good resolution of the toxins from one another and other co-extracted
compounds depends on the mobile phase used. As for example, MC-LR and MC-YR can be better
resolved from one another with methanol-based mobile phase rather than with acetonitrile/
ammonium acetate mobile phase (Harada
et al
., 1988a).
Detection of MCs has been traditionally carried out with UV-absorbance (Lawton
et al
., 1994,
1995; ISO, 2005). Most of the MCs and nodularins have UV-absorption maxima at 238 nm. However,
those MCs with aromatic amino acids such as tryptophan (MC-LW) have absorption maxima at
lower wavelengths, i.e. 222 nm (Lawton
et al
., 1994, 1995). The problems arising out of the co-eluted
compounds and the use of plasticware have to be kept in view. As MC-LR is the standard available,
the concentration of the rest of the MCs could only be expressed in terms of MC-LR equivalents.
However, the total MC content can be determined with high degree of accuracy by UV-detection.
Alternatively, the use of a photo-diode array (PDA) detection not only helps in the detection of
a MC but also provides its absorption spectrum thus indicating the presence of MC in a given
sample with confi rmation. PDA detection also allows the determination of concentrations of MCs
lower than 1µg L
-1
. Semi-quantitative analyses of benthic cyanobacterial communities from Fresh
Pond, McMurdo Ice Shelf of Antarctica by using HPLC-DAD and PP-inhibition assay revealed the
presence of MC-LR and [D-Asp
3
] MC-LR equivalent to 11.4 ng MC-LR mg
-1
dry weight (Jungblut
et
al
., 2006). A survey of 40 ponds, lakes and hydroterrestrial environments in Bratina Island and four
dry valleys of Antarctica by PCR amplifi cation of 16S rRNA gene and
mcyE
gene brought to light
that a
Nostoc
sp. is responsible for the production of novel MCs. Detected by ELISA, LC-MS and PP
