- Email: [email protected]
The stability of tryptophan metabolites prior to urine analysis
247
Clinica Chimica Acta, 102 (1980) 247-251 0 Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 1299
THE STABILITY ANALYSIS
F. GEERAERTS
OF TRYPTOPHAN
*, L. SCHIMPFESSEL
METABOLITES
and R. CROKAERT
Laboratorium voor Biochemie, Vrije Uniuersiteit Brussel, Farmacie, Laarbeeklaan 103, B-l 090 Brussels (Belgium) (Received
July 30th,
PRIOR TO URINE
Fakulteit
Geneeskunde
en
1979)
Summary A method for the quantitative preservation of tryptophan metabolites is described. Analysis of the samples was done by reversed phase HPLC. Results both for a standard solution and biological samples (urine are presented.
Introduction Tryptophan metabolism has been extensively studied by measuring the urinary excretion of its metabolites before and after tryptophan loading tests and in several pathological conditions. Using reversed-phase liquid chromatography coupled to ultraviolet and fluorescence detectors, the tryptophan metabolites can be separated quickly. However, the routine application of this analysis in clinical chemistry is retarded by the problems caused by the instability of the metabolites, especially in a mixture as complex as urine. Until now, little attention was paid to the storage conditions of the sample and their effects on the compounds under investigation. As changes in urine samples occur [1,2], the stability of tryptophan and six of its metabolites has been checked under different conditions of pH and temperature, using a mixture of standards, human and rat urine. The metabolites chosen are representative for the two main metabolic pathways of tryptophan.
* To whom correspondence
should be addressed.
248
Materials and methods The standard solution contained tryptophan (TRP) (Difco Lab., Detroit, U.S.A.); serotonin (SER) and 5-hydroxyt~ptophan (5-HTRP) (Fluka, Buchs, Switzerland); 5-hydroxyindoleacetic acid (5-HIAA) (Merck, Darmstadt, F.R.G.); indolelactic acid (ILA), kynurenine (KYN) and kynurenic acid (KYNA) (Sigma Chem. Comp., St. Louis, U.S.A.). The samples were stored both at pH 5.6 in a 0.2 mol/l phosphate buffer and pH 2, at room temperature (ta), 4°C and -18°C for a maximuln of 168 h. HCl (12 mol/l) was used for acidification. The solutions were analysed every 24 h by high performance reversed-phase chromatography on two ii-Bondapak C-18 colmmns in series (Waters Ass., Milford, U.S.A.), with an eluent system described by Sentfleber et al. [3] and Veening (personal communication). The low concentration eluent was a 0.025 mol/l sodium acetate buffer, pH 4.4, and the high concentration eluent a 0.1 mol/l acetic acid solution in methanol. The eluents were degassed with a water-vacuum line before use. The flow rate was 40 ml/h (corresponding to a linear velocity of 0.117 cm/s) and the temperature was ambient. The compounds were detected at 260 nm with a variable wavelength l_J.V.-detector (Model LC 55, Perkin-Elmer, Oakbrook, U.S.A.) and by native fluorescence (Fluorimeter 2000, Perkin Elmer} (excitation wavelength, 280 nm; emission wavelength, 360 nm). The peaks were quantified by measuring the peak area on the chromatogram by the height times width at half-height method. Results (a) Standard
solution
The reversed-phase HPLC separation of the seven components of the mixture is shown in Fig. 1. In the conditions used, kynurenine does not show any natural fluorescence. The peaks were identified by retention times, co-chromatography, U.V.-, and if possible, fluorescence spectra. The results of the stability study of the standard are summarized in Table I. At pH 5.6 and at ta, the compounds were stable for 48 h, except for kynurenic acid. Under these conditions this metabolite can only be stored for a maximum of 36 h, but 5-HIAA and ILA are stable during the whole period. Lowering the temperature to 4°C assures good stability of the sample solutes for at least 168 h, except for serotonin for which a loss of 20% is found if stored longer than 48 h. Similar results are obtained at -18°C but with even a greater loss of SER. The acidified samples (pH 2) are seen to be more stable than the corresponding original ones, especially at the lower temperatures. At t,, the KYN concentration decreases by 50% if stored for 168 h, 72 h beinb the maximum time for which this compound can be stored under these conditions without loss. TRP can be stored for 120 h, after 168 h of storage at pH 2 and at ta, there is a loss of nearly 40%. At -18”C, there is a small loss of 5-HIAA (15%) if stored longer than 144 h, but under these conditions, all other compounds were stable for at least 168 h. However, the best results were obtained with acidification at pH 2 and storage at 4°C. With this method, all the tryptophan metabolites tested were preserved for at least eight days.
249
A
FI
‘S
;20
I/
1 ...-_-..~~_ , ~.
KYN
~_
L__-.==_h G
Fig.
10
20
1. Reversed-phase
0.21
mmol/l;
0.17
mmol/I;
ple volume,
HPLC
SER.
0.10
ILA.
0.23
100
30
40
separation
mmol/l;
of
5-HTRP.
mmol/l.
For
~1. Fluorescence
50
the
0.23
standard
mmol/l;
abbreviations
scale
60
solution.
TRP,
used
is in arbitrary
7o
and
0.19
MIN
”
Composition mmol/l;
conditions.
of
KYNA,
the
0.33
see Materials
mixture: mmol/l;
and
KYN, 5-HIAA,
methods.
Sam-
units.
(b) Urine samples Fig. 2 shows the chromatogram obtained from a human urine spiked with the tryptophan metabolites under investigation. Additional fluorescent peaks are indican (IND) and unknowns (U). The preservation method giving the best results for the standard solution has been applied to human and rat urine samples (Table II). For both biological samples, pH 2, combined with a temper-
TABLE
I
COMPARISON
OF
STANDARDS (Percentage which
the
Compound
THE
UNDER of
peak
compound
STABILITY
OF
DIFFERENT area
STORAGE
remaining
is stable
% of control
under
after
168
the given
(control
TRYPTOPHAN
IN
A
SOLUTION
hours
of
storage.
Between
brackets,
the
time
50(48
h)
90
SER
70(72
h)
80(48
5-HTRP
70(48
h)
95
TRP
80(48
h)
100
85(36
h)
100
-18’C
tR
100 h)
70(48 95 100 95
50(72 h)
h)
4Oc
-18’C
100
100
90
100
90
95
95
60(120
h)
95
100
100 100
100
100
5-HIAA
100
100
100
100
100
85(144
ILA
100
100
100
100
100
95
temperature,
h)
= 100%)
4Oc
KYN
room
(in
PH 2
tR
tR,
OF
condition.)
PH 5.6
KYNA
METABOLITES
CONDITIONS
h)
for
250
t .L lV.abs.
~
Fig.
2.
tions.
Chromatogram see Materials
of
spiked
and methods.
human
urine.
Sample
volume,
IND, 100
indican;
U, unknown:
~1. Fluorescence
other
scale
abbreviations
is in arbitrary
and
corldi-
units.
ature of 4°C preserved the tryptophan metabolites for at least eight days, confirming and extending the results obtained for the standard solution. Discussion Preliminary attempts to work out a preservation method for all urinary U.V.absorbing compounds having failed [2], attention was focused on tryptophan and some of its urinary metabolites. The preservation of whole urine rather than extracts was tried as the tryptophan metabolites belong to different classes of chemical compounds so that the quantitative extraction of all products of interest is difficult to achieve. The results obtained at pH 2, 4°C and pH 2, -18°C are quite similar, but the
TABLE
II
STABILITY
OF
TRYPTOPHAN
(Conditions.
pH
2; temperature,
Compound
METABOLITES 4’C;
% of control Human
urine
IN
percentage
(control
URINE
of peak
= 100%) Rat
KYN
100
SER
95
100 95
5-HTRP
95
95
TRP
100
100
KYNA
100
100
5-HIAA
100
100
ILA
100
95
urine
SAMPLES
area remaining
after
168
hours
of storage.)
251
loss of 15% of 5-HIAA in the latter makes the former the method of choice. The method described guarantees a quantitative preservation of the metabolites tested prior to rapid chromatographic separation. Acknowledgement This work was supported by Grant 3.0016.75 kundig en Wetenschappelijk Onderzoek”.
of the “Fonds
voor Genees-
References 1
Grushka,
2
Geeraerts,
3
Sentfleber,
E.,
Kikta,
F..
Jr., E.J.
Schimpfessel,
F.C..
HaIIine,
and
Naylor,
L. and A.G.,
E.W.
Crokaert,
Veening,
(1977) R.
H. and
J. Chromatogr.
(1979) Dayton,
Arch. D.A.
Int.
143, Physiol.
(1976)
Clin.
5146 Biochem. Chem.
87, 22.
177-178
1522-1527
Clinica Chimica Acta, 102 (1980) 247-251 0 Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 1299
THE STABILITY ANALYSIS
F. GEERAERTS
OF TRYPTOPHAN
*, L. SCHIMPFESSEL
METABOLITES
and R. CROKAERT
Laboratorium voor Biochemie, Vrije Uniuersiteit Brussel, Farmacie, Laarbeeklaan 103, B-l 090 Brussels (Belgium) (Received
July 30th,
PRIOR TO URINE
Fakulteit
Geneeskunde
en
1979)
Summary A method for the quantitative preservation of tryptophan metabolites is described. Analysis of the samples was done by reversed phase HPLC. Results both for a standard solution and biological samples (urine are presented.
Introduction Tryptophan metabolism has been extensively studied by measuring the urinary excretion of its metabolites before and after tryptophan loading tests and in several pathological conditions. Using reversed-phase liquid chromatography coupled to ultraviolet and fluorescence detectors, the tryptophan metabolites can be separated quickly. However, the routine application of this analysis in clinical chemistry is retarded by the problems caused by the instability of the metabolites, especially in a mixture as complex as urine. Until now, little attention was paid to the storage conditions of the sample and their effects on the compounds under investigation. As changes in urine samples occur [1,2], the stability of tryptophan and six of its metabolites has been checked under different conditions of pH and temperature, using a mixture of standards, human and rat urine. The metabolites chosen are representative for the two main metabolic pathways of tryptophan.
* To whom correspondence
should be addressed.
248
Materials and methods The standard solution contained tryptophan (TRP) (Difco Lab., Detroit, U.S.A.); serotonin (SER) and 5-hydroxyt~ptophan (5-HTRP) (Fluka, Buchs, Switzerland); 5-hydroxyindoleacetic acid (5-HIAA) (Merck, Darmstadt, F.R.G.); indolelactic acid (ILA), kynurenine (KYN) and kynurenic acid (KYNA) (Sigma Chem. Comp., St. Louis, U.S.A.). The samples were stored both at pH 5.6 in a 0.2 mol/l phosphate buffer and pH 2, at room temperature (ta), 4°C and -18°C for a maximuln of 168 h. HCl (12 mol/l) was used for acidification. The solutions were analysed every 24 h by high performance reversed-phase chromatography on two ii-Bondapak C-18 colmmns in series (Waters Ass., Milford, U.S.A.), with an eluent system described by Sentfleber et al. [3] and Veening (personal communication). The low concentration eluent was a 0.025 mol/l sodium acetate buffer, pH 4.4, and the high concentration eluent a 0.1 mol/l acetic acid solution in methanol. The eluents were degassed with a water-vacuum line before use. The flow rate was 40 ml/h (corresponding to a linear velocity of 0.117 cm/s) and the temperature was ambient. The compounds were detected at 260 nm with a variable wavelength l_J.V.-detector (Model LC 55, Perkin-Elmer, Oakbrook, U.S.A.) and by native fluorescence (Fluorimeter 2000, Perkin Elmer} (excitation wavelength, 280 nm; emission wavelength, 360 nm). The peaks were quantified by measuring the peak area on the chromatogram by the height times width at half-height method. Results (a) Standard
solution
The reversed-phase HPLC separation of the seven components of the mixture is shown in Fig. 1. In the conditions used, kynurenine does not show any natural fluorescence. The peaks were identified by retention times, co-chromatography, U.V.-, and if possible, fluorescence spectra. The results of the stability study of the standard are summarized in Table I. At pH 5.6 and at ta, the compounds were stable for 48 h, except for kynurenic acid. Under these conditions this metabolite can only be stored for a maximum of 36 h, but 5-HIAA and ILA are stable during the whole period. Lowering the temperature to 4°C assures good stability of the sample solutes for at least 168 h, except for serotonin for which a loss of 20% is found if stored longer than 48 h. Similar results are obtained at -18°C but with even a greater loss of SER. The acidified samples (pH 2) are seen to be more stable than the corresponding original ones, especially at the lower temperatures. At t,, the KYN concentration decreases by 50% if stored for 168 h, 72 h beinb the maximum time for which this compound can be stored under these conditions without loss. TRP can be stored for 120 h, after 168 h of storage at pH 2 and at ta, there is a loss of nearly 40%. At -18”C, there is a small loss of 5-HIAA (15%) if stored longer than 144 h, but under these conditions, all other compounds were stable for at least 168 h. However, the best results were obtained with acidification at pH 2 and storage at 4°C. With this method, all the tryptophan metabolites tested were preserved for at least eight days.
249
A
FI
‘S
;20
I/
1 ...-_-..~~_ , ~.
KYN
~_
L__-.==_h G
Fig.
10
20
1. Reversed-phase
0.21
mmol/l;
0.17
mmol/I;
ple volume,
HPLC
SER.
0.10
ILA.
0.23
100
30
40
separation
mmol/l;
of
5-HTRP.
mmol/l.
For
~1. Fluorescence
50
the
0.23
standard
mmol/l;
abbreviations
scale
60
solution.
TRP,
used
is in arbitrary
7o
and
0.19
MIN
”
Composition mmol/l;
conditions.
of
KYNA,
the
0.33
see Materials
mixture: mmol/l;
and
KYN, 5-HIAA,
methods.
Sam-
units.
(b) Urine samples Fig. 2 shows the chromatogram obtained from a human urine spiked with the tryptophan metabolites under investigation. Additional fluorescent peaks are indican (IND) and unknowns (U). The preservation method giving the best results for the standard solution has been applied to human and rat urine samples (Table II). For both biological samples, pH 2, combined with a temper-
TABLE
I
COMPARISON
OF
STANDARDS (Percentage which
the
Compound
THE
UNDER of
peak
compound
STABILITY
OF
DIFFERENT area
STORAGE
remaining
is stable
% of control
under
after
168
the given
(control
TRYPTOPHAN
IN
A
SOLUTION
hours
of
storage.
Between
brackets,
the
time
50(48
h)
90
SER
70(72
h)
80(48
5-HTRP
70(48
h)
95
TRP
80(48
h)
100
85(36
h)
100
-18’C
tR
100 h)
70(48 95 100 95
50(72 h)
h)
4Oc
-18’C
100
100
90
100
90
95
95
60(120
h)
95
100
100 100
100
100
5-HIAA
100
100
100
100
100
85(144
ILA
100
100
100
100
100
95
temperature,
h)
= 100%)
4Oc
KYN
room
(in
PH 2
tR
tR,
OF
condition.)
PH 5.6
KYNA
METABOLITES
CONDITIONS
h)
for
250
t .L lV.abs.
~
Fig.
2.
tions.
Chromatogram see Materials
of
spiked
and methods.
human
urine.
Sample
volume,
IND, 100
indican;
U, unknown:
~1. Fluorescence
other
scale
abbreviations
is in arbitrary
and
corldi-
units.
ature of 4°C preserved the tryptophan metabolites for at least eight days, confirming and extending the results obtained for the standard solution. Discussion Preliminary attempts to work out a preservation method for all urinary U.V.absorbing compounds having failed [2], attention was focused on tryptophan and some of its urinary metabolites. The preservation of whole urine rather than extracts was tried as the tryptophan metabolites belong to different classes of chemical compounds so that the quantitative extraction of all products of interest is difficult to achieve. The results obtained at pH 2, 4°C and pH 2, -18°C are quite similar, but the
TABLE
II
STABILITY
OF
TRYPTOPHAN
(Conditions.
pH
2; temperature,
Compound
METABOLITES 4’C;
% of control Human
urine
IN
percentage
(control
URINE
of peak
= 100%) Rat
KYN
100
SER
95
100 95
5-HTRP
95
95
TRP
100
100
KYNA
100
100
5-HIAA
100
100
ILA
100
95
urine
SAMPLES
area remaining
after
168
hours
of storage.)
251
loss of 15% of 5-HIAA in the latter makes the former the method of choice. The method described guarantees a quantitative preservation of the metabolites tested prior to rapid chromatographic separation. Acknowledgement This work was supported by Grant 3.0016.75 kundig en Wetenschappelijk Onderzoek”.
of the “Fonds
voor Genees-
References 1
Grushka,
2
Geeraerts,
3
Sentfleber,
E.,
Kikta,
F..
Jr., E.J.
Schimpfessel,
F.C..
HaIIine,
and
Naylor,
L. and A.G.,
E.W.
Crokaert,
Veening,
(1977) R.
H. and
J. Chromatogr.
(1979) Dayton,
Arch. D.A.
Int.
143, Physiol.
(1976)
Clin.
5146 Biochem. Chem.
87, 22.
177-178
1522-1527