M. Ferrali et al. / Bioorg. Med. Chem. 9 (2001) 3041–3047
3047
measurements were repeated after further additions of
, 10 and 20 mmol of MCOH, respectively.
After the treatment, rats were placed in metabolic cages
and re-fed after the second hour. At the times reported
in Table 2 rats were anaesthetised and their livers with-
drawn and homogenised (1g) in two volumes of ethyl
acetate. Urine samples were collected and (1mL) simi-
larly extracted with ethyl acetate. After centrifugation
the organic solvent was collected, evaporated and resi-
due dissolved in about 50 mL of ethyl acetate. Using
glass capillary tubes, a few mL were applied on silica gel
plates (250 mm) with fluorescein. After running with
ethyl acetate: petroleum ether (30:70) as element, a pale
5
Mass spectrometry of the MCOH–Fe3+ complex. One
drop of a solution obtained by dissolving few micro-
grams of MCOH and ferric chloride or sulphate in ace-
tone was put on the stainless steel tip of the LSI-MS
probe, mixed with glycerol as a matrix, and exposed to
+
the Cs beam for the desorption. In other experiments,
electron ionisation was also used. Metastable decom-
positions occurring in the first field-free region were
studied by computer-controlled B/E linked scans.
blue fluorescent spot (R ’0.6), was detected under irra-
f
diation at 360 nm in urine and liver of treated animals.
Lipophylicity of MCOH
Octanol–water repartition. 3 mL of octanol were added
to a 100 mm solution of MCOH in water (3 mL, tripli-
cate) and vigorously shaken. The two phases allowed to
separate by gentle centrifugation. The concentration of
MCOH was determined spectrophotometrically using
appropriate standards in octanol and water. The mea-
sures were repeated with saline-phosphate buffer (pH
References and Notes
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1
2
3
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7.4) instead of water. The respective repartition coeffi-
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Arthritis Rheum. 1986, 29, 1087.
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5
stock solution of MCOH–Fe
3
+
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5
3
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3
+
00 nmol of MCOH and 150 nmol of MCOH
com-
plex withdrawn from these solutions, were pipetted into
the flasks, the solvent evaporated and 3 mL of 50% (v/
v) rat erythrocyte suspension in Tris-buffered saline
solution added. Alternatively, MCOH and its iron
complex, at the same concentrations, were dissolved in
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ꢃ
flasks were shaken for 1h at 37 C. Then the samples
were centrifuged (2500 RPM for 10 min) and the super-
natants separated from erythrocytes. Aliquots of both,
supernatant and red cells were extracted by two volumes
of ethyl acetate and MCOH and its complex measured
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3
+
spectrophotometrically. One mL of MCOH–Fe com-
ꢃ
plex loaded cells were re-incubated for 1h at 37 C after
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Enzymol.; Packer, L., Glazer, A.N., Eds; Academic: New
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York, 1990, 186, 343.
2
2. Hertog, M. G. L.; Feskens, E. J. M.; Hollman, P. C. H.;
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In vivo test
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Male Sprague–Dawley rats, between 220 and 250 g body
weight and maintained by a common chow, were fasted
for 16 h before the treatment. MCOH was dissolved in
dimethylsulfoxide at the concentration of 50 mg/mL.
Rats were treated intraperitoneally at the dose of 50 mg
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2
1
2
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