311
total N-methyl-formamide (NMF) in urine using a method rec-
ommended by the Deutsche Forschungsgemeinschaft (DFG
1997a). The NMF levels were between 8.8 and 84.6 mg/l. More-
over, in ten control individuals (six male and four female, eight
non-smokers, two smokers) without any occupational exposure to
DMF the N-methylcarbamoylated Hb-adducts were determined
simultaneously.
Materials and methods
Synthesis of 3-methyl-5-isopropylhydantoin
MIH was synthesized by reaction of 936 mg
D/L-valine (8 mmol)
with 430 ll MIC (8 mmol) in 20 ml bidistilled water. The proce-
dure and the further clean-up is described in detail in another
publication (Ramachandran et al. 1988). MIH was identi®ed by
mass spectrometry (MS) and gas chromatography mass spec-
trometry (GC/MS) after electron impact ionization (EI). The major
fragment ion of the standard is m/z 114. It is formed by a
McLaerty rearrangement of the molecular radical ion m/z 156.
The purity of the standard determined by nuclear magnetic reso-
Results and discussion
In the globin samples of exposed workers as well as
control individuals, the fragment ion m/z 114 could be
detected. Only in the samples of the DMF exposed
persons was the radical molecular ion m/z 156 also de-
tectable. Moreover, the correct ratio between m/z 114
and m/z 156 (160:1) was observed. Although varying the
GC temperature program, the retention times of the
fragment ion m/z 114 for MIH remained identical in
both investigated groups. GC/MS/EI-chromatograms of
a control person and a worker exposed to DMF are
shown in Fig. 1. In comparison to the adduct levels of
the controls the N-methylcarbamoylated Hb concen-
trations in samples of the DMF exposed persons are up
to 100-fold higher. Neither in exposed or in control
persons were the N-methylcarbamoylated globin adduct
levels in¯uenced by sex and smoking habits. MIH could
not be analysed in blank samples, which were also in-
cluded in each analytical series. The concentrations of
MIH are given in Table 1.
These results show that DMF exposure leads to
identical Hb-adducts as those found after exposure to
MIC (Scheme 1). The formation of N-methyl-
carbamoylated Hb in humans, however, is still unknown
although there are two possible explanations. Firstly,
MIC may be formed during the metabolism of DMF
and react with the N-terminal valine in a direct manner.
As there is an excess of other nucleophilic substances,
e.g. sulfhydryl compounds (e.g. glutathione, etc.) in the
human body, N-methylcarbamoylation of these sub-
stances is much more likely. N-methylcarbamoylated
glutathione was actually determined (Slatter et al. 1991).
As this molecular species is known to function as a
`carrier' substance (Pearson et al. 1991; Baillie and
Slatter 1991), the second possibility for the formation
nance (NMR) spectroscopy at 400 MHz H-NMR and 13C-NMR
1
was found to be >95%. The resulting standard was used to check
the retention time and characteristic fragment ions of MIH during
GC/MS/EI analysis, and was, moreover, used for calibration pur-
poses.
Sample preparation and gas chromatography-mass
selective detection
Blood samples (10 ml) were taken by venipuncture using a dis-
posable syringe containing ethylenediaminetetraacetic acid
(EDTA) as an anticoagulant. The red blood cells were separated by
centrifugation; isolation and puri®cation of globin is described in
detail elsewhere (Bader et al. 1995). One hundred milligrams of
globin was dissolved in 3 ml of a mixture of concentrated acetic
acid and concentrated hydrochloric acid (1:1) and incubated for 1 h
at 100 °C. The samples were extracted with 5 ml ethyl acetate and
the organic phase was evaporated to dryness under a stream of
nitrogen. The samples were dried in a vacuum desiccator over so-
dium hydroxide overnight and again resolved in ethyl acetate
(1 ml), 1 ll of this solution was analysed by GC/MS/EI in the
multiple ion detection mode (MID). Recorded ion traces for MIH
were m/z 156 and 114 (ratio 1:160). GC/MS was performed on a
gas chromatograph HP 5890 Series II ®tted with a mass spec-
trometer HP 5989A. Samples analysed by GC/MS/EI were injected
on a capillary column with the stationary phase DB-WAX (J&W
Scienti®c, Folsom, USA). The column length was 60 m with an
inner diameter of 0.32 mm and a ®lm thickness of 0.25 lm. Helium
was used as the carrier gas. The initial temperature of 120 °C was
programmed to rise to 180 °C at 10 °C/min, held at that tem-
perature for 25 min and then revised to 240 °C at 20 °C/min. That
temperature was held for 25 min again.
Calibration process
Three millilitres of standard calibration solutions of MIH in a
mixture of concentrated acetic acid and concentrated hydrochloric
acid (10 mg/l±10 lg/l) were analysed similarly to the globin sam-
ples. These concentrations represent MIH levels between 1.92 lmol of the adducts found is the transfer of the reactive
and 1.92 nmol MIH/g globin, respectively. The area counts of the
peaks of MIH were plotted as a function of the concentration and
this calibration curve was used for the calculation of the MIH
content in each blood sample. The concentration of MIH is given
N-methylcarbamoyl group from glutathione to the
N-terminal valine of Hb.
The method used in our pilot study was not sensitive
enough to identify N-methylcarbamoylated N-terminal
valine in control persons de®nitively. Nevertheless, the
occurrence is possible of N-methylcarbamoylated globin
in individuals not exposed to DMF. However, the for-
mation of such an adduct would aord two subsequent
reactions: ®rst of all carbamoylation of Hb by car-
bamoylphosphate which occurs in the human body in
high amounts. Carbamoylphosphate is known to trans-
fer the carbamoyl group to proteins in humans (Crist
et al. 1973). A subsequent methylation at the nitrogen of
in nmol/g globin. The detection limit calculated from a signal to
noise ratio of 3:1 was 1.92 nmol MIH/g globin, which is a con-
centration of ꢀ 10 lg/l.
Investigated subjects
Blood samples were investigated of ten male persons (six smoker,
four non-smoker) who were exposed to DMF during the manu-
facture of ®bres. The concentration of DMF in the air of the
workplace was monitored during on 8-h shift by personal air
samplers and ranged between 2.2 ppm and 53.7 ppm, respectively.
Urine samples of these workers were also analysed to determine the the carbamoyl group would yield the adduct in question.
Angerer, Juergen
