Distribution and metabolism of (5-hydroxymethyl) furfural in male F344 rats and B6C3F1 mice after oral administration

Article abstract of DOI:10.1080/009841099157764

(5-Hydroxymethyl)furfural (HMF), a heat-induced decomposition product of hexoses, is present in food and drink. Recent reports have shown HMF to be an in vitro mutagen after sulfate conjugation and to be a promoter as well as a weak initiator of colonic aberrant foci in rats. In order to investigate the metabolic activation further and to provide information for HMF toxicology studies, the disposition of [¹⁴C]-HMF had been investigated in male F344 rats and B6C3F1 mice following po administration of either 5, 10, 100 or 500 mg/kg. Tissue distribution results indicated that absorption of HMF was rapid in male rats and mice and that tissue concentrations in male mice at the earliest time point are not linearly proportional to dose. Excretion was primarily via the urine in both, with 60-80% of the administered dose excreted by this route in 48 h. Tissue/blood ratios of HMF-derived radioactivity were greater than 1 for liver and kidney. Three metabolites were identified and quantitated in urine. Formation of one of the metabolites, N-(5-hydroxymethyl-2-furoyl)glycine, was inversely proportional to dose in rats but not mice. None of the metabolites were sulfate conjugates nor likely to be formed from sulfate conjugates. There were relatively low levels of nonextractable radioactivity in liver, kidney, and intestines, indicating that some reactive intermediate(s) may be formed.

Full text of DOI:10.1080/009841099157764

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DISTRIBUTION AND
METABOLISM OF (5-
HYDROXYMETHYL)FURFURAL
IN MALE F344 RATS AND
B6C3F1 MICE AFTER ORAL
ADMINISTRATION
Veronica B. Godfrey, Ling-Jen Chen, Robert J. Griffin,
Edward H. Lebetkin, Leo T. Burka
Published online: 30 Nov 2010.
To cite this article: Veronica B. Godfrey, Ling-Jen Chen, Robert J. Griffin,
Edward H. Lebetkin, Leo T. Burka (1999) DISTRIBUTION AND METABOLISM OF (5-
HYDROXYMETHYL)FURFURAL IN MALE F344 RATS AND B6C3F1 MICE AFTER ORAL
ADMINISTRATION, Journal of Toxicology and Environmental Health, Part A: Current
Issues, 57:3, 199-210, DOI: 10.1080/009841099157764
To link to this article: http://dx.doi.org/10.1080/009841099157764
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DISTRIBUTION AND METABOLISM
OF (5-HYDROXYMETHYL)FURFURAL IN MALE F344 RATS
AND B6C3F1 MICE AFTER ORAL ADMINISTRATION
Veronica B. Godfrey, Ling-Jen Chen, Robert J. Griffin,
Edward H. Lebetkin, Leo T. Burka
Laboratory of Pharmacology and Chemistry, National Institute
of Environmental Health Sciences, Research Triangle Park,
North Carolina, USA
(
5-Hydroxymethyl)furfural (HMF), a heat-induced decomposition product of hexoses, is
present in food and drink. Recent reports have shown HMF to be an in vitro mutagen
after sulfate conjugation and to be a promoter as well as a weak initiator of colonic aber-
rant foci in rats. In order to investigate the metabolic activation further and to provide in-
1
4
formation for HMF toxicology studies, the disposition of [ C]-HMF has been investigated
in male F344 rats and B6C3F1 mice following po administration of either 5, 10, 100, or
500 mg/kg. Tissue distribution results indicated that absorption of HMF was rapid in male
rats and mice and that tissue concentrationsin male mice at the earliest time point are not
linearly proportional to dose. Excretion was primarily via the urine in both, with 60–80%
of the administered dose excreted by this route in 48 h. Tissue/blood ratios of HMF-
derived radioactivity were greater than 1 for liver and kidney. Three metabolites were
identified and quantitated in urine. Formation of one of the metabolites, N-(5-hydroxy-
methyl-2-furoyl)glycine, was inversely proportional to dose in rats but not mice. None
of the metabolites were sulfate conjugates nor likely to be formed from sulfate conju-
gates. There were relatively low levels of nonextractable radioactivity in liver, kidney,
and intestines, indicating that some reactive intermediate(s) may be formed.
(
5-Hydroxymethyl)-2-furfural (HMF) is a common decomposition
product of hexose sugars and is formed during cooking and heat steriliza-
tion of sugar-containing materials. HMF is one of the major intermediate
compounds in the Maillard reaction, a nonenzymatic browning reaction
of amino acids and sugars. HMF is found in a large variety of foods, in-
cluding milk (Morales et al., 1992), fruit juices (Meydav & Berk, 1978;
Blanco Gomis et al., 1991), cognac, and honey (Jeuring & Kuppers, 1980).
Another route of possible human exposure is from heat-sterilized parenteral
solutions (Ulbricht et al., 1984). It has been estimated that humans may
ingest up to 150 mg HMF/d (Ulbricht et al., 1984).
A review by Ulbricht et al. (1984) cites numerous in vivo toxicology
studies with HMF, but the toxicity is, for the most part, unremarkable.
Received 30 November 1998; sent for revision 14 December 1998; accepted 22 January 1999.
We acknowledge the assistance of Leesa Deterding in obtaining mass spectra and the assistance
of Joseph Vance in obtaining NMR spectra for the identification of metabolites. Statistical assistance
from Dr. Joseph Haseman is also acknowledged.
Address correspondence to Dr. Leo T. Burka, National Institute of Environmental Health Sciences,
PO Box 12233, Research Triangle Park, NC 27709, USA.
1
99
Journal of Toxicology and Environmental Health, Part A, 57:199–210, 1999
Copyright © 1999 Taylor & Francis
0
098-4108/99 $12.00 + .00

200
V. B. GODFREY ET AL.
However, it was recently reported that HMF not only acts as a promoter
of colonic aberrant foci in rats initiated by azoxymethane, but may itself
act as a weak initiator (Zhang et al., 1993). Colonic aberrant foci are
putative precursors of colon cancer. Additional in vitro mutagenicity studies
have shown that HMF can be activated to an electrophilic species by sul-
fation of the hydroxymethyl group (Surh & Tannenbaum, 1994; Lee et al.,
1
995). These two observations, along with the fact that HMF is a poten-
tially reactive aldehyde found in many foods, led to the nomination of
HMF to the National Toxicology Program for toxicology/carcinogenicity
studies. In support of these studies, a metabolism/distribution study has
been conducted. While a metabolism/distribution study in rats has been
reported (Germond et al., 1987), there was interest in comparing the fate
of HMF in rats and mice. In addition, from the results of the previous
study, no evidence for (or against) formation of electrophilic species in
the in vivo metabolism of HMF was apparent. Studies were undertaken to
explore further the possibility of metabolism of HMF to reactive species.
MATERIALS AND METHODS
Chemicals
14
[
C]-HMF, uniformly labeled, specific activity 8 mCi/mmol, radiochemi-
cal purity 97.5%, was obtained from NEN Research Products (Boston).
Unlabeled HMF and 2,5-furan dicarboxylic acid (FDCA) were obtained
from Aldrich (Milwaukee, WI). 5-Hydroxmethyl-2-furoic acid (HMFA) and
N-(5-hydroxymethyl-2-furoyl)glycine (HMFG) were obtained from Mid-
west Research Institute (Kansas City, MO).
Apparatus
HPLC The high-performance liquid chromatography (HPLC) system
consisted of two Waters (Milford, MA) pumps, an automated system con-
troller, and a Waters model 481 ultraviolet (UV) detector at 260 nm.
Radiochemical detection was accomplished using an IN\US (Tampa, FL)
beta-Ram flow detector. A Rainin (Varian Associates, Walnut Creek, CA)
C18 5-µm column was used. System 1 utilized an isocratic solvent system
consisting of 0.1% trifluoroacetic acid (TFA) and 12% acetonitrile (AN) in
H O, 3 ml/min, on a 10 × 250 mm column. System 2 utilized an isocratic
2
solvent system (0.1% TFA, 12% AN, 1.6 ml/min) on a 4.6 × 250 mm col-
umn. System 3 utilized an isocratic solvent system (0.1% TFA, 8% AN,
1
.6 ml/min) on a 4.6 × 250 mm column. System 4 utilized an isocratic
solvent system (0.1% TFA, 8% AN, 1.0 ml/min) on tandem 4.6 × 250 mm
columns.
1
NMR Spectra
H-NMR spectra were acquired on either a Nicolet
NT-360 NB (Freemont, CA) or a Varian 500 (Palo Alto, CA) spectrometer.
Chemical shifts are reported in ppm relative to solvent peaks.

HMF DISPOSITION IN RATS AND MICE
201
Mass Spectra Electrospray (ESI) mass spectra were obtained on a
Micromass Platform II (Altrincham, UK) single-quadrapole mass spec-
trometer equipped with an electrospray ionization interface.
UV Spectra Spectra were obtained on a Beckman DU 640 spectro-
photometer (Fullerton, CA).
Synthesis
Preparation of 5-(Chloromethyl)furfural (CMF) CMF was prepared
according to the procedure of Surh and Tannenbaum (1994), except the
purification was achieved by column chromatography on silica gel (70–230
mesh, EM Science) with ethyl ether–hexane (1:1) as the eluting solvent
(
yield 57%). This compound has an R of 0.36 when analyzed by thin-layer
f
chromatography (silica gel, ethyl ether–hexane, 1:1).
Preparation of 5-(N-Acetyl-L-cystein-S-methyl)furfural To a stirred
mixture of CMF (80.7 mg, 0.56 mmol) and N-acetyl-L-cysteine (91.3 mg,
0
.56 mmol) in CH CN (15 ml) at room temperature was added a solution
3
of N,N-diisopropylethylamine (147.8 mg, 1.14 mmol) in CH CN (5 ml).
3
The mixture was stirred at room temperature overnight; the CH CN was
3
evaporated under reduced pressure and the residue was dissolved in H O
2
(10 ml). HPLC analysis of the aqueous solution using system 1 showed
formation of HMF (retention time 6.3 min) and a new product (retention
time 13 min) in roughly a 1:2 ratio. The peak at 13 min was collected
from a portion of the reaction mixture and the solvents removed by Speed-
Vac. Nuclear magnetic resonance (NMR) and mass spectroscopy (MS)
data of this compound are consistent with formation of 5-(N-acetyl-L-cys-
1
tein-S-methyl)furfural (estimated yield 90 mg): H-NMR (500 MHz, D O):
2
d9.44 (s, 1H, CHO), 7.53 (d, J = 3.6 Hz, 1H, C -H), 6.67 (d, J = 3.6 Hz, 1H,
3
C -H), 4.47 (dd, J = 8.4 and 4.8 Hz, 1H, Cys a-CH), 3.96 (s, 2H, C -CH ),
4
5
2
3
.14 (dd, J = 14.4 and 4.8 Hz, 1H, Cys b-CH ), 2.97 (dd, J = 14.4 and 8.4
a
Hz, 1H, Cys b-CH ), 2.04 (s, 3H, COCH ). Positive ion ESI-MS m/z 272
b
3
(
M + 1). UV (H O): l 292 nm. The pH of the remaining reaction mix-
2
max
ture was changed to 6 by adding 5% NaHCO and HMF was removed by
3
extraction with ethyl ether (5 × 20 ml). The aqueous solution containing
5
-(N-acetyl-L-cystein-S-methyl)furfural was then acidified with 2 N HCl to
pH 2 and used for the following reaction without further purification.
Preparation of 5-(N-Acetyl-L-cystein-S-methyl)furoic acid Dimethyl
sulfoxide (DMSO) (3 ml) was added to an aqueous solution of crude 5-
(
N-acetyl-L-cystein-S-methyl)furfural (5 ml, pH 2, ~45 mg, 0.17 mmol). A
solution of sodium chlorite (22.2 mg, 0.20 mmol) in H O (1 ml) was then
2
added dropwise at room temperature. The mixture was stirred at room tem-
perature for 2 h and then analyzed with HPLC system 1. Two new peaks
with retention times 5 and 12.4 min were observed along with 5-(N-acetyl-
L-cystein-S-methyl)furfural (retention time 13 min). Unreacted 5-(N-acetyl-L-
cystein-S-methyl)furfural was removed by addition of hydroxylamine
hydrochloride (6.4 mg, 0.09 mmol) to 1 ml of the reaction mixture. The

202
V. B. GODFREY ET AL.
peak at 12.4 min was collected, solvent removed under vacuum, and fur-
ther purified by HPLC system 2 (retention time 8.1 min). NMR and MS
data of this compound are consistent with the formation of 5-(N-acetyl-L-
1
cystein-S-methyl)furoic acid: H-NMR (500 MHz, D O): d7.24 (d, J = 3.5
2
Hz, 1H, C -H), 6.53 (d, J = 3.5 Hz, 1H, C -H), 4.44 (dd, J = 8.2 and 4.7 Hz,
3
4
1
H, Cys a-CH), 3.91 (s, 2H, C -CH ), 3.10 (dd, J = 14.2 and 4.7 Hz, 1H,
5
2
Cys b-CH ), 2.96 (dd, J = 14.2 and 8.3 Hz, 1H, Cys b-CH ), 2.04 (s, 3H,
a
b
COCH ). Positive ion ESI-MS m/z 288 (M + 1) . UV (H O): l
259 nm.
3
2
max
The NMR spectrum of the peak at 5.0 min is consistent with sulfoxide iso-
1
mers of 5-(N-acetyl-L-cystein-S-methyl)furoic acid: H-NMR (360 MHz,
D O): d7.31 (d, J = 3.5 Hz, C -H), 6.69 (m, C -H), 4.51 (dd, J = 14.4 and
2
3
4
7
.3 Hz, Cys b-CH ), 4.32 (dd, J = 14.6 and 5.6 Hz, Cys b-CH ), 3.54 (dd,
a
b
J = 13.8 and 5.6 Hz, Cys b-CH ) , 3.23 (dd, J = 13.8 and 7.3 Hz, Cys b-
a
CH ), 2.03 (s, COCH ), 2.02 (s, COCH ), Cys a-CH and C -CH overlap
b
3
3
5
2
with the HOD peak.
Animals and Treatments
All animal procedures were approved by the Institutional Animal Care
and Use Committee. Male F344 rats and B6C3F1 mice (Taconic) were
used. The mean age and weight were 11 wk and 225 g for rats and 5 wk
and 25 g for mice when obtained. Animals were acclimated for 1 wk
prior to use.
Doses used included 5, 10, 100, or 500 mg/kg administered by gav-
1
4
age. [ C]-HMF, approximately 10 µCi/animal, was diluted with sufficient
unlabeled HMF in distilled water to give the desired dose. The dose vol-
ume was 4 ml/kg and 10 ml/kg for rats and mice, respectively. All HMF
doses were in solution.
Sample Collection and Analysis
The rates and routes of elimination of HMF-derived radioactivity over
time were determined by collection and analysis of urine and feces at
time points ranging from 8 to 48 h following administration of a single dose
of HMF. Additionally, expired volatiles and CO were measured in a pre-
2
liminary study that consisted of one rat or mouse per dose using methods
previously described (Sanders et al., 1998). All animals were individually
housed in metabolism cages with food (NIH number 31) and distilled water
provided ad libitum.
Following collection, urine was immediately stored at –20°C until
analyzed by HPLC. Feces were air-dried, weighed, and ground to a fine
powder using a mortar and pestle. Animals were euthanized with CO2.
Blood was drawn by cardiac puncture and tissues were collected.
Representative rat and mouse urine samples were thawed, centrifuged
at 10,000 × g, and aliquots of the supernatant were individually injected
directly on the HPLC system. Tissues and feces (triplicate 50- to 100-mg
samples) were weighed, oxidized in a model 306 biological tissue oxidizer

HMF DISPOSITION IN RATS AND MICE
203
(
Packard Instruments Co., Meriden, CT), and counted in a Beckman (Fuller-
ton, CA) model LS6500 or LS9800 scintillation counter for determination
1
4
of total C content. All tissue weights were determined gravimetrically,
except blood, adipose tissue, skin, and muscle, which were estimated to
be 8, 11, 16, and 50% of total body weight, respectively (Matthews &
Anderson, 1975; Birnbaum et al., 1980).
Covalent Binding to Protein
Tissue samples were homogenized 1:3 in 50 mM Tris buffer, pH 7.4.
Protein pellets were washed in a series of solvents (5 ml/wash) and cen-
trifuged at low g in a desktop centrifuge. The order of washes was 80%
methanol × 3, 0.4 M TCA (trichloroacetic acid) × 1, 3:1 ethanol:ether × 1,
and 80% methanol × 1. The sequence was repeated until the total activity
in the supernatant was less than 3 times background. The pellets were
solubilized in 0.5 N NaOH, the protein content was determined (Lowry
et al., 1951), and aliquots were counted in the scintillation counter.
Metabolite Identification
Urine samples from mice or rats treated with HMF were analyzed by
HPLC system 3. Three major metabolites with retention times 3.6 min
(
HMFG), 4.6 min (HMFA), and 6.2 min (FDCA) were observed. The metab-
olites were identified by comparison of HPLC retention times and by com-
parison of NMR spectra of isolated metabolites with standards. No peak
corresponded to the 5-(N-acetyl-L-cystein-S-methyl)furoic acid standard,
which eluted at 19.5 min in this system. HPLC system 4 was used to quan-
titate the metabolites. The retention times of these metabolites are as fol-
lows: 11.5 min (HMFG), 14.2 min (HMFA), and 19.3 min (FDCA). Radio-
activity in each metabolite peak was integrated to give the amount of each
metabolite formed.
Statistics
Analysis-of-variance (ANOVA) procedures were used to assess the sig-
nificance of differences among groups. The variance-stabilizing logarithmic
transformation was used where appropriate. If significant overall differences
were found, then pairwise comparisons were made by Fisher’s least sig-
nificant difference (LSD) test. Values were considered to be statistically
significant at p £ .01.
RESULTS
Disposition
The total radioactivity recovered in excreta in the 48 h following
administration of a 5- to 500-mg/kg dose was 82 to 91% in male rats and
8
0 to 92% in male mice. The majority of radioactivity in mice and rats

204
V. B. GODFREY ET AL.
1
4
TABLE 1. Cumulative Excretion of Radioactivity 0–48 h After Administration of [ C]-HMF to
Male F344 Rats (% Dose)
Dose
_
_______________________________________
Excretion
Urine
5 mg/kg
100 mg/kg
500 mg/kg
0
0
0
0
–8 h
61.7 ± 2.6
73.0 ± 2.7
75.0 ± 3.0
76.8 ± 3.0
55.9 ± 23.2
66.3 ± 17.6
68.0 ± 17.9
70.2 ± 18.4
61.8 ± 13.6
80.0 ± 2.7
80.8 ± 2.5
81.9 ± 2.6
–24 h
–32 h
–48 h
Feces
0
0
–24 h
–48 h
6.7 ± 1.4
8.5 ± 1.0
9.3 ± 3.4
12.2 ± 5.8
6.2 ± 0.8
9.2 ± 2.4
Total (0–48 h)
85.3 ± 2.3
82.4 ± 13.2
91.1 ± 5.0
Note. Values are mean ± standard deviation (n = 4).
was excreted in urine at all dose levels, mostly in the first 8 h (Tables 1
and 2). Relatively more radioactivity was excreted in feces by mice, espe-
cially at the high dose. No radioactivity was eliminated in expired air in
previous investigations (Germond et al., 1987); preliminary studies in the
present work confirmed this observation.
The concentration of HMF-derived radioactivity in rat and mouse tis-
sues is presented in Tables 3 and 4. HMF-derived radioactivity is rapidly
cleared from the tissues with no evidence for prolonged accumulation in
any tissue. Tissue concentrations at comparable doses tend to be higher in
rats than in mice, especially at earlier times. In general, there appears to be
1
4
TABLE 2. Cumulative Excretion of Radioactivity 0–48 h After Administration of [ C]-HMF to
Male B6C3F
1
Mice (% Dose)
Dose
_
_______________________________________
Excretion
Urine
10 mg/kg
100 mg/kg
500 mg/kg
0
0
0
0
–8 h
38.4 ± 6.0
55.7 ± 10.4
58.3 ± 10.2
65.6 ± 0.6
52.7 ± 9.2
72.9 ± 3.3
74.4 ± 2.8
76.5 ± 2.4
22.2 ± 13.6
56.7 ± 8.6
57.9 ± 8.6
60.8 ± 9.8
–24 h
–32 h
–48 h
Feces
0
0
–24 h
–48 h
12.6 ± 7.6
14.8 ± 10.0
14.5 ± 1.9
15.0 ± 2.0
24.8 ± 6.0
25.5 ± 6.2
Total (0–48 h)
80.4 ± 12.0
91.5 ± 3.9
86.3 ± 5.4
Note. Values are mean ± standard deviation (n = 4).

HMF DISPOSITION IN RATS AND MICE
205
1
4
TABLE 3. Disposition of [ C]-HMF-Derived Radioactivity in Male F344 Rats Following Oral
Administration (nmol HMF eq/g Tissue)
Hours postadministration
_
___________________________________________________
Tissue
2
8
24
48
5
mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
6.92 ± 0.88
20.9 ± 1.1
46.3 ± 1.9
9.92 ± 3.71
1.19 ± 0.48
1.71 ± 0.52
1.53 ± 0.51
0.56 ± 0.25
0.81 ± 0.32
0.56 ± 0.03
2.24 ± 0.82
2.43 ± 1.13
0.25 ± 0.04
0.47 ± 0.12
0.29 ± 0.06
0.25 ± 0.02
0.32 ± 0.03
0.44 ± 0.21
1.25 ± 0.15
1.17 ± 0.10
0.22 ± 0.05
0.38 ± 0.05
0.23 ± 0.06
0.14 ± 0.02
0.21 ± 0.03
20.6 ± 3.0
52.8 ± 6.0
2.69 ± 0.28
3.39 ± 0.14
0.86 ± 0.08
1.07 ± 0.36
2.85 ± 0.61
1
00 mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
142 ± 30
280 ± 87
889 ± 329
73.2 ± 27.6
111 ± 50
33.9 ± 8.2
46.9 ± 16.5
67.4 ± 17.7
25.4 ± 9.9
73.3 ± 33.5
172 ± 99
25.1 ± 20.2
14.9 ± 0.6
23.0 ± 17.1
6.10 ± 2.40
19.5 ± 4.5
10.4 ± 4.9
32.3 ± 4.1
33.4 ± 2.9
3.57 ± 0.71
8.01 ± 1.43
3.09 ± 1.09
3.01 ± 0.30
5.54 ± 0.18
7.69 ± 2.57
17.4 ± 4.4
21.5 ± 5.6
4.95 ± 1.42
7.73 ± 1.17
3.51 ± 0.66
2.76 ± 0.43
4.35 ± 0.41
5
00 mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
1070 ± 640
1390 ± 220
5350 ± 1100
639 ± 123
861 ± 131
124 ± 26
93.8 ± 23.8
213 ± 83
58.4 ± 0.6
166 ± 13
187 ± 29
16.8 ± 3.2
58.1 ± 16.5
44.4 ± 52.2
17.5 ± 0.5
28.0 ± 2.7
33.3 ± 14.8
93.6 ± 29.8
81.2 ± 21.3
19.4 ± 6.5
34.1 ± 6.1
19.4 ± 5.0
15.7 ± 5.9
16.2 ± 3.5
599 ± 248
86.1 ± 46.8
80.2 ± 17.5
75.2 ± 39.2
18.6 ± 2.9
92.9 ± 30.0
253 ± 86
574 ± 144
Note. Values are mean ± standard deviation (n = 4).
good correlation between administered dose and tissue concentration, with
the possible exception of the 2-h time point in mice. The tissue concentra-
tions after the 100-mg/kg dose at this time are uniformly more than 10
times the corresponding concentration in the tissues following the 10-
mg/kg dose, and tissue concentrations after the 500-mg/kg dose are uni-
formly more than 5 times the concentration following the 100-mg/kg dose.
At 8 h and subsequent times there is return to dose proportionality.
In mice the tissue/blood ratio was greater than 1 for most tissues mea-
sured at 2 and 8 h postdosing. At later times this ratio was more than 1 only
for liver, kidney, and skin. In contrast, the tissue/blood ratio was greater
than 1 in rats only for liver and kidney at most time points.

206
V. B. GODFREY ET AL.
14
1
TABLE 4. Disposition of [ C)-HMF-Derived Radioactivity in Male B6C3F Mice Following Oral
Administration (nmol HMF eq/g Tissue)
Hours postadministration
_
_____________________________________________________
Tissue
2
8
24
48
10 mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
0.476 ± 0.048
2.40 ± 0.59
3.70 ± 1.53
0.753 ± 0.435
2.37 ± 2.76
0.769 ± 0.864
0.436 ± 0.190
0.492 ± 0.095
0.500 ± 0.143
2.18 ± 0.27
2.03 ± 0.47
1.91 ± 1.18
1.16 ± 0.47
2.45 ± 1.56
0.190 ± 0.056
1.78 ± 1.40
0.579 ± 0.381
1.66 ± 0.45
0.174 ± 0.016
0.642 ± 0.040
0.642 ± 0.048
0.151 ± 0.032
1.29 ± 0.88
0.167 ± 0.024
0.103 ± 0.016
0.151 ± 0.087
0.928 ± 0.198
0.230 ± 0.048
0.428 ± 0.278
0.182 ± 0.056
0.159 ± 0.079
0.151 ± 0.024
1
00 mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
19.4 ± 7.5
54.0 ± 17.1
149 ± 60
34.1 ± 10.0
38.0 ± 17.9
36.3 ± 40.9
8.92 ± 3.83
39.5 ± 23.8
6.23 ± 1.66
25.9 ± 4.9
48.8 ± 26.6
39.8 ± 21.8
21.8 ± 9.9
106 ± 63
4.77 ± 1.31
24.6 ± 3.3
24.0 ± 6.0
5.66 ± 0.79
8.41 ± 0.55
3.65 ± 1.23
3.04 ± 0.36
3.95 ± 0.31
3.13 ± 0.55
11.1 ± 2.1
11.4 ± 2.2
2.93 ± 0.27
5.48 ± 0.60
2.08 ± 0.36
1.71 ± 0.36
1.09 ± 0.36
4.27 ± 2.82
40.2 ± 24.0
5
00 mg/kg
Blood
Liver
Kidney
Muscle
Skin
Adipose
Brain
Testes
453 ± 174
650 ± 200
3220 ± 760
373 ± 94
481 ± 108
332 ± 84
34.7 ± 13.7
127 ± 35
177 ± 28
164 ± 115
116 ± 66
143 ± 117
12.3 ± 5.0
96.3 ± 87.7
30.7 ± 11.8
97.3 ± 14.1
224 ± 121
27.5 ± 8.2
42.8 ± 8.6
14.4 ± 3.7
11.6 ± 2.5
14.5 ± 0.5
18.2 ± 3.8
56.2 ± 8.0
62.0 ± 17.1
17.5 ± 4.8
31.2 ± 6.6
12.4 ± 2.5
9.93 ± 2.04
7.94 ± 3.02
205 ± 46
419 ± 53
Note. Values are mean ± standard deviation (n = 4).
Urinary Metabolites
An HPLC profile for HMF urinary metabolites from mice is shown in
Figure 1. HMFA, HMFG, and FDCA were identified by comparison of
HPLC retention times and NMR spectra to those of synthetic standards.
No HMF was detected in urine. 5-(N-acetyl-L-cystein-S-methyl)furoic acid,
a likely mercapturic acid metabolite if the sulfate conjugate of HMF were
formed and reacted with glutathione, was synthesized. No radioactive
peak corresponding to the HPLC retention time of this mercapturic acid
was present in urine.
The major urinary metabolite in both mice and rats was HMFA (Table
), constituting about 80% of the eluted radioactivity. Excretion of the
5

HMF DISPOSITION IN RATS AND MICE
207
FIGURE 1. HPLC radiochromatogram of mouse urine collected in the first 24 h following an oral dose
14
of [ C)-HMF. The identity and retention times of the metabolites are: N-(5-hydroxymethyl-2- fu royl)-
glycine (HMFG), 11.5 min; 5-hydroxmethyl-2-furoic (HMFA), 14.2 min; and 2,5-furan dicarboxylic
acid (FDCA), 19.3 min.
glycine conjugate appears to be inversely related to dose in rats, possibly
due to glycine depletion at the higher doses.
Covalent Protein Binding
Covalent binding to protein was in the picomoles to nanomoles per
milligram protein range in the tissues selected (Table 6). At 24 h the amount
of nonextractable radioactivity was similar in tissues from both species,
with the possible exception being large intestine where the radioactivity
in rat is decidedly less than in mouse. There are large decreases in the
concentrations of nonextractable radioactivity in rat intestinal tissue be-
tween 8 and 24 h. This could be due to residual intestinal contents being
1
4
TABLE 5. Urinary Metabolites 0–24 h Following Oral Administration of [ C]-HMF
Dose
HMFG
HMFA
FDCA
Mouse
Rat
1
0 mg/kg
5.2 ± 1.1
7.9 ± 1.4
7.8 ± 2.1
80.3 ± 5.9
80.9 ± 0.8
77.5 ± 2.0
2.0 ± 0.9
2.3 ± 0.5
3.5 ± 0.6
1
5
00 mg/kg
00 mg/kg
5
mg/kg
5.7 ± 0.6a
2.7 ± 0.1
1.3 ± 0.2
81.5 ± 1.2
82.6 ± 2.2
84.9 ± 2.0
4.0 ± 0.6
4.9 ± 0.7
5.9 ± 1.9
1
5
00 mg/kg
00 mg/kg
b
Note. Values are mean ± SD of percent of eluted radioactivity.
a
Statistically different from 5- and 500-mg/kg treatment.
Statistically different from 5- and 100-mg/kg treatment.
b

208
V. B. GODFREY ET AL.
1
4
TABLE 6. Covalent Binding in Selected Tissues Following a 500-mg/kg Dose of [ C]-HMF
pmol/mg Protein)
(
Tissue
Mouse—24 h
Rat—8 h
Rat—24 h
Liver
Kidney
Small intestine
Cecum
Large intestine
328 ± 29
540 ± 112
704 ± 156
100 ± 50
505 ± 160
607 ± 142
857 ± 104
356 ± 116
194 ± 43
413 ± 233
2020 ± 800
9180 ± 1870
3210 ± 410
637 ± 125
Note. Values are mean ± standard deviation (n = 4).
included in the homogenate, even though care was taken to clean the tis-
sue thoroughly, or it could be a result of rapid turnover of intestinal cells.
The amount of covalent binding in rat liver appears to be increasing with
time.
DISCUSSION
14
The results of this study indicate that [ C]-HMF is rapidly absorbed,
metabolized, and excreted by male rats and mice, with most of the radio-
activity being excreted in urine in the first 24 h after administration. The
rate of excretion in rats seems somewhat slower than previously reported,
greater than 85% in the first 8 h (Germond et al., 1987). The slower excre-
tion may be due to differences in strain, F344 versus Sprague-Dawley, but
is more likely due to the fact that the animals in the Germond et al. (1987)
study were fasted before administration of the HMF. Mice appear to excrete
14
[
C]-HMF-derived radioactivity more slowly than rats.
14
There is little evidence of bioaccumulation of [ C]-HMF-derived radio-
activity. As might be expected, the highest concentrations of HMF equiva-
lents appear in liver and kidney, major organs for metabolism and excretion.
There is some evidence that mice absorb less of the administered dose than
rats; that is, tissue concentrations are generally lower in mice. There is also
relatively more HMF-derived radioactivity detected in feces from mice com-
pared to rats, consistent with lesser absorption in mice. Another distribution
difference between mice and rats is that the tissue concentration data from
rats shows good dose proportionality, while, at least at the earliest time
point, mice appear to absorb proportionally more as the dose is increased.
A metabolic scheme for HMF is shown in Figure 2. The observed me-
tabolites result from initial oxidation of the aldehyde followed by either con-
jugation of the resulting carboxylic acid or oxidation of the alcohol group.
In this study as well as the Germond et al. (1987) study, there is evidence
that the high doses saturate glycine conjugation in rats. This pathway
does not appear to be similarly affected in mice, as the percent of radio-
activity excreted as the glycine conjugate changes little with dose. These
oxidative pathways seem to be effective in eliminating HMF. There is no

HMF DISPOSITION IN RATS AND MICE
209
evidence that conjugation of the alcohol group, either as the glucuronide
or sulfate, occurs. The most likely mercapturic acid derived from activation
of the hydroxymethyl group via sulfation and subsequent reaction with
glutathione could not be detected in urine of either rat or mouse. Thus, no
in vivo evidence of the activation pathway postulated by Suhr and Tannen-
baum (1994) was obtained from the metabolite profile.
There was evidence that some covalent binding to protein, as mea-
sured by unextractable radioactivity, occurs. The most compelling obser-
vation for formation of a reactive intermediate is the fact that more
nonextractable radioactivity was found in rat liver after 24 h than after 8
h, even though the total amount of radioactivity in that tissue decreased
dramatically over that time. Covalent binding to liver protein has been
determined for the HMF parent structure, furan (Burka et al., 1991). At 24
h following an 8-mg/kg dose of furan, 2.2 nmol/mg protein of nonex-
tractable radioactivity was found: 4- to 5-fold more radioactivity from a
dose about 40-fold less on a molar basis.
In conclusion, it appears that metabolism of HMF in male mice and
rats depends primarily on oxidation, and there is little in vivo evidence
for metabolic activation of the hydroxymethyl group by conjugation path-
FIGURE 2. Metabolic scheme for HMF in F344 rats and B6C3F1 mice.

210
V. B. GODFREY ET AL.
ways. Some reactive species may be present that reacts with protein, pos-
sibly the aldehyde group already present in HMF or formed by oxidation
of the hydroxymethyl group.
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