281
Clinica Chimica Acta, 82 (1978) 281-284 @ Elsevier/North-Holland Biomedical Press
CCA 9112
LANTHIONINE
S.K. WADMAN
DETECTED
*, P.K. DE BREE and J.P. KAMERLING
University Children’s Hospital IJtrecht (The Netherlands)
(Received
August
IN HUMAN URINE
lOth,
‘Het
Wilhelmina
Kinderziekenhuis”,
Nieuwe
Gracht
137,
1977)
summary
Lanthionine has been detected incidentally in the urine of a patient with an abdominal pseudo-tumor, possibly caused by a Yersiniu infection. The L- and/ or D-form together with the meso-form were present. The identity of the amino acid was confirmed by GC-MS of the N-trifluoroacetyl methyl ester derivative. The abnormal amino acid was thought to be not of endogenous origin. The possibility that it was produced by gut bacteria is considered.
Introduction The natural occurrence of the sulphur-containing amino acid lanthionine is rather peculiar. It has been found in the free amino acid pool of the chick embryo (L-form) and was obtained as a constituent of heat-treated chick embryo protein (both the L- and meso-form) [l]. It is known to be present in locust muscle protein [ 21, in various insects (free; L-form) [ 31 and in plant pollen [ 41. It is a constituent of polypeptide antibiotics like subtilin from Bacillus subtilis (meso-form) [5] and nisin from Streptococcus Zactis [6]. A mixture of stereoisomers can be obtained from proteins rich in cystine, such as keratine, by alkaline treatment followed by acid hydrolysis. As far as we know, lanthionine has not so far been found in man. Here we report the incidental occurrence of this amino acid in the urine of a patient with an abdominal pseudo-tumor, possibly caused by a Yersinia infection. Methods Amino acid screening was performed by micro thin-layer after initial oxidation with H,Oz, as described before [ 71. * To whom correspondence
should be addressed.
chromatography,
282
~0~~~~ ~~~ro~rlatogru~~ly was carried out using a Technicon TSM 1 analyzer programmed for physiological fluids. Preparative paper chromatography was performed on Whatmann 3 MM-filter paper (22 X 22 cm), using the same solvents as described for thin-layer chromatography of amino acids. In this case H202 oxidation was omitted. The lanthionine was eluted from the paper with 2 M ammonia and the solution was evaporated in vacua. Deriuatization of lanthionine was performed as follows. The residue was esterified in 1 ml methanolic 3 M HCI for 3 h at 100°C in a closed ampoule. Subsequently the solution was evaporated to dryness. Traces of HCI were removed by three-fold co-evaporation with methanol The residue was dissolved in 1 ml trifluoroacetic anhydride and was kept overnight at room temperature. After evaporation of the anhydride under a gentle stream of nitrogen, the residue was taken up in ethyl acetate and was investigated by CC-MS. The same procedure was followed for commercial lanthionine (Calbiochem; mixture of L- and meso-forms). Gas chromatography combined with mass spectrometry (GC-MS) was carried out on a Jeol JGC-20KP/JMS-DlOO/WJMA combination; ionization energy 70 eV; ion source temperature 150°C; accelerating voltage 3 kV; ionizing current 300 PA; glass column packed with 3.8% SE-30 on Chromosorb W/AW-DMCS, HP, 80-100 mesh; oven temperature 165°C. Results and discussion On the two-dimensional thin-layer chromatogram of the urinary amino acids, oxidized lanthionine appeared in the same position as oxidized cystathionine. Therefore it can easily be confused with cystathionine. In the column chromatogram only a small cystathionine peak was present but two unusual peaks near the position of a-amino butyric acid were observed, the first being mesolanthionine and the second L- and/or D-lanthionine [8]. The concentrations were 0.2 mmol/l and 0.3 mmol/l, respectively, presuming that the colour yield with the ninhydrin reagent is the same for the isomers. In order to verify the presence of lanthionine the compound was isolated by preparative two-Dimensions paper chromatography and subsequently the N-trifluoroacetyl methyl ester derivative analyzed by GC-MS. The gas chromatogram showed only one peak of the derivatized lanthionine isomers. Also,
I 100
I
200
x
369
300
428
-7
400
-m/e Fig. urine.
1. The
mass
spectrum
of
the N-trifluoroacetyl
methyl
ester
derivative
of lanthionine
isolated
from
the
283
the commercial product, being a mixture of the L- and meso-forms, gave rise to one gas chromatographic peak. The mass spectrum of the lanthionine derivative (see Fig. 1) compared well with that of the commercial product. For literature data see ref. 9. The presence of lanthionine in the patient’s urine is difficult to interpret. This amino acid, normally not occurring in man, was detected only in one 24-h sample; it was absent in samples collected 8 and 10 weeks later. This points to an incidental event rather than a regular metabolic abnormality. There seems to be no direct connection with the patient’s pseudo-tumor, which was still present at the time of collection of the second and third samples. During the following half year the tumor gradually disappeared. At the time of sample collection the patient received the following medications: doxycycline; salazapyrine; prednisone and lasix. The lanthionine found is probably not a drug metabolite, neither was it induced by the drugs mentioned, because lanthionine is not seen in other patients receiving this medication. Artificial formation from urinary cystine due to the analytical manipulations was excluded by running cystine through the procedures used. Moreover, no pretreatment was used in the column chromatography experiment, except acidification. In man two homologous thioethers are synthesized. (1) L-Cystathionine is formed endogenously from L-homocysteine and L-serine. This reaction is catalyzed by cystathionine synthetase. No allo-cystathionine is produced in this process. (2) L-Homolanthionine is produced continuously in patients with homocystinuria [lo]. The occurrence of meso-homolanthionine was not mentioned. The enzyme system has not yet been identified. In insects 35S-labeling studies [ 31 have shown that L-cystine is a precursor of L-lanthionine, indicating that an enzyme system for the synthesis of lanthionine exists. The same follows from the occurrence of free L-lanthionine in chick embryo, whilst no meso-lanthionine was detected. In the yolk of incubated hen’s eggs the presence of a “cysteine lyase” has been demonstrated, which catalyses the formation of lanthionine from cysteine [ 111 via a-aminoacrylic acid as transition state. In theory a mixture of L- and meso-lanthionine can be formed from L-cysteine, but no data about any specificity were reported. We do not know whether in bacteria a similar process of desulfuration and recombination can occur. Similar mechanisms were described for the chemical formation of a mixture of stereoisomers of lanthionine from cystine in alkali-treated wool [12]. A mixture of L- and meso-lanthionine can be synthesized from L-cysteine and cr-acetyl aminoacrylic acid [ 131. An acceptable hypothesis for the origin of the lanthionine in the patient’s urine seems to presume formation by the bacterial flora in the gut or in vitro formation by urinary bacteria. The latter is improbable because no signs of excessive bacterial activity were present. Streptococcus lactis can occur in the human intestine and it is known that this organism is able to synthesize a lanthionine containing antibiotic [ 61. Bacillus su btilis, a soil bacteria species, produces a meso-lanthionine containing antibiotic [ 51. Other gut micro-organisms may have the same ability. However, it is not probable that Yersiniu were the source of the lanthionine found, because at the time of collection of the first
284
urine sample no gastro-intestinal infection was present, and in the patient’s blood the antibodies against yersinia antigen were no longer elevated. Formed by the gut bacteria, lanthionine may be expected to pass through the patient’s extracellular fluid for a part and to be excreted in the urine without reabsorption in the kidney. Encouraged by the present observation, we should try to get more information about the occurrence of urinary lanthionine and to assess whether there exists a relationship with an intestinal micro-organism. References 1
Sloane,
2
Stein,
N.H. J.M.
3
Rae,
4
Rossetti,
D.R.,
and
Untch, Chem.
Ennor,
A.H.
V. (1966)
5
Alderton, Berridge,
NJ.,
Newton.
7
Wadman.
S.K.,
de Bree,
8
G. and
Clin.
Williams,
9
Harpp,
Chim.
M.J.
Monograph Perry,
11
Chapeville,
12
Cuthbertson,
13
SehGberl,
and
Thorpe,
Chim. H.L. P.K., 59.
B. (1967)
Rome and
5, 2658-2665
774
(1951)
G.G.F.
Acta
Biochemistry
London,
56,
Biochemistry
J. Am,
Chem.
Abraham,
van Sprang,
6.1208-1216
935 Sot.
E.P.
F.J.,
73,
(1952)
463464
Biochem.
Kamerling,
J. 52,
,J.P.. Haverkamp,
T.L.,
and
Woodhouse,
Gleason,
Hansen, F. and W.R. A.
J.G.
S. and
J.M.
(1967)
in 5th
Colloquium
and Wagner,
J. Org.
Chem.
36,
P. (1961)
Phillips, A.
(1971)
MaeDoug&,
Fromageot, and
529-535 J. and
Vliegenthart.
J.F.G.
313-320 in Amino
2.96-100
D.N.
10
Fevold,
(1966)
Ind. and
Ann.
6
(1975)
K.G.
(1955)
Science
Biochim.
Biophys.
H. (1945)
(1947)
73-80
L. (1966)
Chem.
Biochem. Ber.
152.1750-1752 Aeta
J. 39.7-17
80.379-390
49.328-334
Acid
Analysis,
Technicon
Clinica Chimica Acta, 82 (1978) 281-284 @ Elsevier/North-Holland Biomedical Press
CCA 9112
LANTHIONINE
S.K. WADMAN
DETECTED
*, P.K. DE BREE and J.P. KAMERLING
University Children’s Hospital IJtrecht (The Netherlands)
(Received
August
IN HUMAN URINE
lOth,
‘Het
Wilhelmina
Kinderziekenhuis”,
Nieuwe
Gracht
137,
1977)
summary
Lanthionine has been detected incidentally in the urine of a patient with an abdominal pseudo-tumor, possibly caused by a Yersiniu infection. The L- and/ or D-form together with the meso-form were present. The identity of the amino acid was confirmed by GC-MS of the N-trifluoroacetyl methyl ester derivative. The abnormal amino acid was thought to be not of endogenous origin. The possibility that it was produced by gut bacteria is considered.
Introduction The natural occurrence of the sulphur-containing amino acid lanthionine is rather peculiar. It has been found in the free amino acid pool of the chick embryo (L-form) and was obtained as a constituent of heat-treated chick embryo protein (both the L- and meso-form) [l]. It is known to be present in locust muscle protein [ 21, in various insects (free; L-form) [ 31 and in plant pollen [ 41. It is a constituent of polypeptide antibiotics like subtilin from Bacillus subtilis (meso-form) [5] and nisin from Streptococcus Zactis [6]. A mixture of stereoisomers can be obtained from proteins rich in cystine, such as keratine, by alkaline treatment followed by acid hydrolysis. As far as we know, lanthionine has not so far been found in man. Here we report the incidental occurrence of this amino acid in the urine of a patient with an abdominal pseudo-tumor, possibly caused by a Yersinia infection. Methods Amino acid screening was performed by micro thin-layer after initial oxidation with H,Oz, as described before [ 71. * To whom correspondence
should be addressed.
chromatography,
282
~0~~~~ ~~~ro~rlatogru~~ly was carried out using a Technicon TSM 1 analyzer programmed for physiological fluids. Preparative paper chromatography was performed on Whatmann 3 MM-filter paper (22 X 22 cm), using the same solvents as described for thin-layer chromatography of amino acids. In this case H202 oxidation was omitted. The lanthionine was eluted from the paper with 2 M ammonia and the solution was evaporated in vacua. Deriuatization of lanthionine was performed as follows. The residue was esterified in 1 ml methanolic 3 M HCI for 3 h at 100°C in a closed ampoule. Subsequently the solution was evaporated to dryness. Traces of HCI were removed by three-fold co-evaporation with methanol The residue was dissolved in 1 ml trifluoroacetic anhydride and was kept overnight at room temperature. After evaporation of the anhydride under a gentle stream of nitrogen, the residue was taken up in ethyl acetate and was investigated by CC-MS. The same procedure was followed for commercial lanthionine (Calbiochem; mixture of L- and meso-forms). Gas chromatography combined with mass spectrometry (GC-MS) was carried out on a Jeol JGC-20KP/JMS-DlOO/WJMA combination; ionization energy 70 eV; ion source temperature 150°C; accelerating voltage 3 kV; ionizing current 300 PA; glass column packed with 3.8% SE-30 on Chromosorb W/AW-DMCS, HP, 80-100 mesh; oven temperature 165°C. Results and discussion On the two-dimensional thin-layer chromatogram of the urinary amino acids, oxidized lanthionine appeared in the same position as oxidized cystathionine. Therefore it can easily be confused with cystathionine. In the column chromatogram only a small cystathionine peak was present but two unusual peaks near the position of a-amino butyric acid were observed, the first being mesolanthionine and the second L- and/or D-lanthionine [8]. The concentrations were 0.2 mmol/l and 0.3 mmol/l, respectively, presuming that the colour yield with the ninhydrin reagent is the same for the isomers. In order to verify the presence of lanthionine the compound was isolated by preparative two-Dimensions paper chromatography and subsequently the N-trifluoroacetyl methyl ester derivative analyzed by GC-MS. The gas chromatogram showed only one peak of the derivatized lanthionine isomers. Also,
I 100
I
200
x
369
300
428
-7
400
-m/e Fig. urine.
1. The
mass
spectrum
of
the N-trifluoroacetyl
methyl
ester
derivative
of lanthionine
isolated
from
the
283
the commercial product, being a mixture of the L- and meso-forms, gave rise to one gas chromatographic peak. The mass spectrum of the lanthionine derivative (see Fig. 1) compared well with that of the commercial product. For literature data see ref. 9. The presence of lanthionine in the patient’s urine is difficult to interpret. This amino acid, normally not occurring in man, was detected only in one 24-h sample; it was absent in samples collected 8 and 10 weeks later. This points to an incidental event rather than a regular metabolic abnormality. There seems to be no direct connection with the patient’s pseudo-tumor, which was still present at the time of collection of the second and third samples. During the following half year the tumor gradually disappeared. At the time of sample collection the patient received the following medications: doxycycline; salazapyrine; prednisone and lasix. The lanthionine found is probably not a drug metabolite, neither was it induced by the drugs mentioned, because lanthionine is not seen in other patients receiving this medication. Artificial formation from urinary cystine due to the analytical manipulations was excluded by running cystine through the procedures used. Moreover, no pretreatment was used in the column chromatography experiment, except acidification. In man two homologous thioethers are synthesized. (1) L-Cystathionine is formed endogenously from L-homocysteine and L-serine. This reaction is catalyzed by cystathionine synthetase. No allo-cystathionine is produced in this process. (2) L-Homolanthionine is produced continuously in patients with homocystinuria [lo]. The occurrence of meso-homolanthionine was not mentioned. The enzyme system has not yet been identified. In insects 35S-labeling studies [ 31 have shown that L-cystine is a precursor of L-lanthionine, indicating that an enzyme system for the synthesis of lanthionine exists. The same follows from the occurrence of free L-lanthionine in chick embryo, whilst no meso-lanthionine was detected. In the yolk of incubated hen’s eggs the presence of a “cysteine lyase” has been demonstrated, which catalyses the formation of lanthionine from cysteine [ 111 via a-aminoacrylic acid as transition state. In theory a mixture of L- and meso-lanthionine can be formed from L-cysteine, but no data about any specificity were reported. We do not know whether in bacteria a similar process of desulfuration and recombination can occur. Similar mechanisms were described for the chemical formation of a mixture of stereoisomers of lanthionine from cystine in alkali-treated wool [12]. A mixture of L- and meso-lanthionine can be synthesized from L-cysteine and cr-acetyl aminoacrylic acid [ 131. An acceptable hypothesis for the origin of the lanthionine in the patient’s urine seems to presume formation by the bacterial flora in the gut or in vitro formation by urinary bacteria. The latter is improbable because no signs of excessive bacterial activity were present. Streptococcus lactis can occur in the human intestine and it is known that this organism is able to synthesize a lanthionine containing antibiotic [ 61. Bacillus su btilis, a soil bacteria species, produces a meso-lanthionine containing antibiotic [ 51. Other gut micro-organisms may have the same ability. However, it is not probable that Yersiniu were the source of the lanthionine found, because at the time of collection of the first
284
urine sample no gastro-intestinal infection was present, and in the patient’s blood the antibodies against yersinia antigen were no longer elevated. Formed by the gut bacteria, lanthionine may be expected to pass through the patient’s extracellular fluid for a part and to be excreted in the urine without reabsorption in the kidney. Encouraged by the present observation, we should try to get more information about the occurrence of urinary lanthionine and to assess whether there exists a relationship with an intestinal micro-organism. References 1
Sloane,
2
Stein,
N.H. J.M.
3
Rae,
4
Rossetti,
D.R.,
and
Untch, Chem.
Ennor,
A.H.
V. (1966)
5
Alderton, Berridge,
NJ.,
Newton.
7
Wadman.
S.K.,
de Bree,
8
G. and
Clin.
Williams,
9
Harpp,
Chim.
M.J.
Monograph Perry,
11
Chapeville,
12
Cuthbertson,
13
SehGberl,
and
Thorpe,
Chim. H.L. P.K., 59.
B. (1967)
Rome and
5, 2658-2665
774
(1951)
G.G.F.
Acta
Biochemistry
London,
56,
Biochemistry
J. Am,
Chem.
Abraham,
van Sprang,
6.1208-1216
935 Sot.
E.P.
F.J.,
73,
(1952)
463464
Biochem.
Kamerling,
J. 52,
,J.P.. Haverkamp,
T.L.,
and
Woodhouse,
Gleason,
Hansen, F. and W.R. A.
J.G.
S. and
J.M.
(1967)
in 5th
Colloquium
and Wagner,
J. Org.
Chem.
36,
P. (1961)
Phillips, A.
(1971)
MaeDoug&,
Fromageot, and
529-535 J. and
Vliegenthart.
J.F.G.
313-320 in Amino
2.96-100
D.N.
10
Fevold,
(1966)
Ind. and
Ann.
6
(1975)
K.G.
(1955)
Science
Biochim.
Biophys.
H. (1945)
(1947)
73-80
L. (1966)
Chem.
Biochem. Ber.
152.1750-1752 Aeta
J. 39.7-17
80.379-390
49.328-334
Acid
Analysis,
Technicon
