203
Clinica Chimica Acta, 59 (1975) 203-207 @ Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CCA 6870
THE DETERMINATION OF &AMINOISOBUTYRIC SERUM BY ION-EXCHANGE CHROMATOGRAPHY
E. SOLEMa,
D.P. AGARWALb
ACID IN HUMAN
and H.W. GOEDDE”
aInstitute bInstitute
of Clinical Biochemistry, University of Oslo, Oslo (Norway) and of Human Genetics, University of Hamburg, Hamburg (G.F.R.)
(Received
October
3, 1974)
Introduction Different investigators have studied the variability in urinary excretion of difference in fl-aminoisobutyric acid (BAIBA) in man. The inter-individual BAIBA excretion was found to be caused by genetic factors [l-3]. Several clinical conditions may also lead to a considerable increase in the urinary excretion of this amino acid [4,5]. Unfortunately most of the methods previously used for the determination of urinary BAIBA are time consuming and rather unspecific. Earlier investigations have provided evidence that the normal serum level of BAIBA is quite low [6]. Thus, the lack of a sensitive method made it difficult to examine the serum BAIBA level for clinical and genetical studies. In the present communication a rapid and sensitive method for the quantitative determination of BAIBA in serum is presented. Sera from 28 different healthy persons were analysed and the BAIBA concentration determined. In 20 of these subjects the estimation of the urinary BAIBA excretion was also made. The results show that the serum level of BAIBA seems to vary within a small range, in contrast to the great variability in the urinary BAIBA excretion in the same subjects. This discrepancy may be explained by the occurrence of both isomers of BAIBA in the serum samples, i.e. the different renal handling of the two BAIBA enantiomorphs. There was no significant correlation between the interindividual serum BAIBA concentration and urinary BAIBA excretion, found in the present material. Material and Methods Lithium citrate, lithium chloride, lithium hydroxide, sodium citrate, sodium acetate, sodium hydroxide, perchloric acid, ammonia, n-caprylic acid, hydrochloric acid and potassium hydroxide were obtained from E. Merck, Darmstadt. Detergent and thiodiglycol were made by F.A. Hedinger, Stuttgart. Dowex 50 resin was obtained from Serva, Heidelberg. Racemic BAIBA was a gift
204
from Hoffman La Roche, Basel. Ninhydrin and methyl cellosolve were supplied either from E. Merck, Darmstadt, or from Koch-Light Laboratories, Ltd, England. Tin (II) chloride was made by Ride1 de Haen, Hannover. The serum samples were collected in the morning from the antecubital vein. Urine samples were collected during 24 hours and the BAIBA excretion estimated during this period. A lo-ml serum sample was deproteinised with 1.0 ml cold perchloric acid (20%) and centrifuged at 29 000 X g for 10 minutes. The supernatant was neutralised with 5 N KOH and allowed to stand for 1 hour at 4°C before filtering out the precipitate of potassium perchlorate. The filtrate was desalted by passing through a column of Dowex 50 X 4 (1.5 cm X 4 cm, H’ form, 100-200 mesh). The column was washed with 200 ml of water and the adsorbed amino acids eluted with 50 ml of 2 M ammonia, and the eluate evaporated to dryness under reduced pressure. The urine samples, corresponding to 5 mg of creatinine were desalted directly, in the same manner. The desalted, dry serum and urine samples were redissolved in sample buffer (for ionexchange chromatography, pH 2.2, 0.3 M). The ion-exchange chromatography was carried out either on a Beckman, Unichrom (Beckman Instruments GmbH, Germany) or on a Jeol JLC-6AH (Jeol, Ltd, Tokyo, Japan) amino acid analyser, using a sodium citrate (0.38 M, pH 4.26) or lithium citrate buffer (0.3 M, pH 4.4) system, respectively. On the Beckman Unichrom analyser the recording unit was amplified to 0.1 mV ( /fulls&e). The analyses were carried out without any buffer change. After addition of different amounts of synthetic (racemic) BAIBA to identical serum samples, the recovery by ion-exchange chromatography was found to be quantitative, i.e. 95-105%. The identity of the BAIBA peak was verified by gas-liquid chromatography/mass spectrometry. Some of the urine samples were also examined by GLC. Details of this procedure have been published elsewhere [ 71. Results and Discussion The main advantage of ion-exchange chromatography with the one buffer system was a great shortening of the retention time, as demonstrated in Fig. 1. This approach enabled us to carry out 4-6 analyses in a day. Using sodium citrate buffer on the Beckman/Unichrom analyser, BAIBA elutes as the last ninhydrin-positive peak after 68 minutes. By the lithium citrate/Jeol analyser system the elution time of BAIBA was somewhat prolonged (not demonstrated). This method seems also to be suitable for rapid analyses of phenylalanine and tyrosine (total separation only with the lithium citrate system). Sera from 28 different persons were examined; 20 of these subjects were also examined for urinary BAIBA excretion (Table I). The concentration of serum BAIBA varied from 185 to 465 ng/ml (of serum). A comparison of the urinary BAIBA excretion in the same subjects showed that there was no significant correlation between serum level and urinary BAIBA excretion (r = 0.08, 0.7 < p < 0.8). One genetically “high excretor” of BAIBA was included in the study (subject No. 15). The genetically “high excretor” also showed the highest amount serum BAIBA of all examined persons. As recently was shown [8] , thymine is a precursor of the R-isomer of
205
B
2i
325
CO
i C 5
1
68
325
Retetion
time
in minutes
Fig. 1. (A) Ion-exchange chromatogram (with buffer change) from normal urine. Peak 1, threonine; peak 2, glycine; peak 3, tyrosine; peak 4, phenylanine: peak 5, BAIBA; instrumentation, Beckman/ Unichrom. (B) Ion-exchange chromatogram (one buffer system) of synthetic BAIBA (peak 5). Shortening of the retention time. Column, 68 cm X 0.9 cm. Resin, Beckman Custom Research Resin Type PA-28: flow 50 ml/hour; column temperature 30°C; buffer, sodium citrate, 0.38 M, pH 4.26; instrumentation as above. (C) Ion-exchange chromatogram (one buffer system) of a serum sample (subject No. 19, Table I). Conditions and instrumentation as above. Peak 5, BAIBA.
,-
I-
I-
IN =20 , =0.08 l0.7 < p < 0.8
I-
IO
20 URINARY
Fig. 2. Graph of urinary
30 EXCRETION
excretion
LO ( mg/2L
50
60
70
80
90
hours-1
and serum level of BAIBA.
0, normal
excretor;8.
high excretor.
206 TABLE
I
Subject NO.
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
F F M F
Serum BAIBA (m/m0
iv M M M M F M M M M F F M M M M M M F M M M M M
220 200 350 245 250 220 265 255 185 320 300 250 290 250 465 270 230 290 380 220 225 225 260 230 240 220 295 260
level
Urinary BAIBA (m&24 hours)
excretion
7.4 27.2 9.2 16.1 24.7 5.8 15.2 9.5 9.6 16.5 39.5 20.6 85.2 14.7 12.8 1.1 25.4 18.1 11.4
23.1
BAIBA, which is excreted in urine. On the other hand the S-form of BAIBA was recently detected in a serum pool from two persons [7]. Moreover, the ratio of the R- and S-enantiomorph in the serum pool was shown to be 1 : 4, respectively. The different renal handling of the two isomers makes it reasonable to assume that the low correlation between serum and urinary (total) BAIBA, found in this study, may be explained by the presence of both enantiomorphs in the serum samples. If relatively high amounts of S-BAIBA are present, this would lead to a “quantitative camouflage” of the R-form of BAIBA in serum. Only the latter enantiomorph has a metabolic relationship to the BAIBA excreted in urine; the metabolic precursor of S-BAIBA is unknown. Thus, only high amounts of the serum R-BAIBA should lead to a distinct elevation of the serum total BAIBA. It might be questionable however, if the urinary BAIBA excretion (mg/24 hours) is a useful parameter in this context, or connected with too many uncertainties. Further investigation is required to show if there is a significant correlation between the R-form of BAIBA in serum and the excreted R-form of BAIBA in urine.
207
Acknowledgements The authors are indebted to Dr. K. Altland for helpful discussions. This work has partly been supported from the University of Hamburg, the Deutsche Forschungsgemeinschaft and the Norwegian Cancer Society. References 1
K.
Fink,
2
Y.
Kakimoto.
R.B.
3
J. Yanai,
4
H. Brunschede,
5
M.
Y.
Abe,
(1973)
M.
Henderson K.
and
Taniguchi
Kakimoto,
R.M.
T. Tsujio
R. Hoffbauer Takahashi.
T.
Fink.
and I. Sam,
J. Biol. J. Biol.
and I. Sane,
and
H.W.
Nishidai.
Am.
Goedde,
S.
Chem., Chem.,
J. Hum.
Klin.
Suyama,
19’7 244
S.
(1952)
(1969) Genet..
Wochenschr..
Oshima
and
441 335 21
(1969)
2 (1965) T.
Teramatsu,
115 93 Int.
J. Radiat.
Chem.,
238
Biol.,
24
73
6
M.D.
7
E. Solem,
Armstrong,
E. Jellum
K.
8
E. Solem,
Clin.
Chim.
Yates. and
Y.
Kakimoto,
L. Eldjam,
Acta.
53 (1974)
Clin.
K. Chin
183
Taniguchi
and
Acta.
50 (1974)
T. Kappe, 393
J. Biol.
(1963)
1447
Clinica Chimica Acta, 59 (1975) 203-207 @ Elsevier Scientific Publishing Company,
Amsterdam
-Printed
in The Netherlands
CCA 6870
THE DETERMINATION OF &AMINOISOBUTYRIC SERUM BY ION-EXCHANGE CHROMATOGRAPHY
E. SOLEMa,
D.P. AGARWALb
ACID IN HUMAN
and H.W. GOEDDE”
aInstitute bInstitute
of Clinical Biochemistry, University of Oslo, Oslo (Norway) and of Human Genetics, University of Hamburg, Hamburg (G.F.R.)
(Received
October
3, 1974)
Introduction Different investigators have studied the variability in urinary excretion of difference in fl-aminoisobutyric acid (BAIBA) in man. The inter-individual BAIBA excretion was found to be caused by genetic factors [l-3]. Several clinical conditions may also lead to a considerable increase in the urinary excretion of this amino acid [4,5]. Unfortunately most of the methods previously used for the determination of urinary BAIBA are time consuming and rather unspecific. Earlier investigations have provided evidence that the normal serum level of BAIBA is quite low [6]. Thus, the lack of a sensitive method made it difficult to examine the serum BAIBA level for clinical and genetical studies. In the present communication a rapid and sensitive method for the quantitative determination of BAIBA in serum is presented. Sera from 28 different healthy persons were analysed and the BAIBA concentration determined. In 20 of these subjects the estimation of the urinary BAIBA excretion was also made. The results show that the serum level of BAIBA seems to vary within a small range, in contrast to the great variability in the urinary BAIBA excretion in the same subjects. This discrepancy may be explained by the occurrence of both isomers of BAIBA in the serum samples, i.e. the different renal handling of the two BAIBA enantiomorphs. There was no significant correlation between the interindividual serum BAIBA concentration and urinary BAIBA excretion, found in the present material. Material and Methods Lithium citrate, lithium chloride, lithium hydroxide, sodium citrate, sodium acetate, sodium hydroxide, perchloric acid, ammonia, n-caprylic acid, hydrochloric acid and potassium hydroxide were obtained from E. Merck, Darmstadt. Detergent and thiodiglycol were made by F.A. Hedinger, Stuttgart. Dowex 50 resin was obtained from Serva, Heidelberg. Racemic BAIBA was a gift
204
from Hoffman La Roche, Basel. Ninhydrin and methyl cellosolve were supplied either from E. Merck, Darmstadt, or from Koch-Light Laboratories, Ltd, England. Tin (II) chloride was made by Ride1 de Haen, Hannover. The serum samples were collected in the morning from the antecubital vein. Urine samples were collected during 24 hours and the BAIBA excretion estimated during this period. A lo-ml serum sample was deproteinised with 1.0 ml cold perchloric acid (20%) and centrifuged at 29 000 X g for 10 minutes. The supernatant was neutralised with 5 N KOH and allowed to stand for 1 hour at 4°C before filtering out the precipitate of potassium perchlorate. The filtrate was desalted by passing through a column of Dowex 50 X 4 (1.5 cm X 4 cm, H’ form, 100-200 mesh). The column was washed with 200 ml of water and the adsorbed amino acids eluted with 50 ml of 2 M ammonia, and the eluate evaporated to dryness under reduced pressure. The urine samples, corresponding to 5 mg of creatinine were desalted directly, in the same manner. The desalted, dry serum and urine samples were redissolved in sample buffer (for ionexchange chromatography, pH 2.2, 0.3 M). The ion-exchange chromatography was carried out either on a Beckman, Unichrom (Beckman Instruments GmbH, Germany) or on a Jeol JLC-6AH (Jeol, Ltd, Tokyo, Japan) amino acid analyser, using a sodium citrate (0.38 M, pH 4.26) or lithium citrate buffer (0.3 M, pH 4.4) system, respectively. On the Beckman Unichrom analyser the recording unit was amplified to 0.1 mV ( /fulls&e). The analyses were carried out without any buffer change. After addition of different amounts of synthetic (racemic) BAIBA to identical serum samples, the recovery by ion-exchange chromatography was found to be quantitative, i.e. 95-105%. The identity of the BAIBA peak was verified by gas-liquid chromatography/mass spectrometry. Some of the urine samples were also examined by GLC. Details of this procedure have been published elsewhere [ 71. Results and Discussion The main advantage of ion-exchange chromatography with the one buffer system was a great shortening of the retention time, as demonstrated in Fig. 1. This approach enabled us to carry out 4-6 analyses in a day. Using sodium citrate buffer on the Beckman/Unichrom analyser, BAIBA elutes as the last ninhydrin-positive peak after 68 minutes. By the lithium citrate/Jeol analyser system the elution time of BAIBA was somewhat prolonged (not demonstrated). This method seems also to be suitable for rapid analyses of phenylalanine and tyrosine (total separation only with the lithium citrate system). Sera from 28 different persons were examined; 20 of these subjects were also examined for urinary BAIBA excretion (Table I). The concentration of serum BAIBA varied from 185 to 465 ng/ml (of serum). A comparison of the urinary BAIBA excretion in the same subjects showed that there was no significant correlation between serum level and urinary BAIBA excretion (r = 0.08, 0.7 < p < 0.8). One genetically “high excretor” of BAIBA was included in the study (subject No. 15). The genetically “high excretor” also showed the highest amount serum BAIBA of all examined persons. As recently was shown [8] , thymine is a precursor of the R-isomer of
205
B
2i
325
CO
i C 5
1
68
325
Retetion
time
in minutes
Fig. 1. (A) Ion-exchange chromatogram (with buffer change) from normal urine. Peak 1, threonine; peak 2, glycine; peak 3, tyrosine; peak 4, phenylanine: peak 5, BAIBA; instrumentation, Beckman/ Unichrom. (B) Ion-exchange chromatogram (one buffer system) of synthetic BAIBA (peak 5). Shortening of the retention time. Column, 68 cm X 0.9 cm. Resin, Beckman Custom Research Resin Type PA-28: flow 50 ml/hour; column temperature 30°C; buffer, sodium citrate, 0.38 M, pH 4.26; instrumentation as above. (C) Ion-exchange chromatogram (one buffer system) of a serum sample (subject No. 19, Table I). Conditions and instrumentation as above. Peak 5, BAIBA.
,-
I-
I-
IN =20 , =0.08 l0.7 < p < 0.8
I-
IO
20 URINARY
Fig. 2. Graph of urinary
30 EXCRETION
excretion
LO ( mg/2L
50
60
70
80
90
hours-1
and serum level of BAIBA.
0, normal
excretor;8.
high excretor.
206 TABLE
I
Subject NO.
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
F F M F
Serum BAIBA (m/m0
iv M M M M F M M M M F F M M M M M M F M M M M M
220 200 350 245 250 220 265 255 185 320 300 250 290 250 465 270 230 290 380 220 225 225 260 230 240 220 295 260
level
Urinary BAIBA (m&24 hours)
excretion
7.4 27.2 9.2 16.1 24.7 5.8 15.2 9.5 9.6 16.5 39.5 20.6 85.2 14.7 12.8 1.1 25.4 18.1 11.4
23.1
BAIBA, which is excreted in urine. On the other hand the S-form of BAIBA was recently detected in a serum pool from two persons [7]. Moreover, the ratio of the R- and S-enantiomorph in the serum pool was shown to be 1 : 4, respectively. The different renal handling of the two isomers makes it reasonable to assume that the low correlation between serum and urinary (total) BAIBA, found in this study, may be explained by the presence of both enantiomorphs in the serum samples. If relatively high amounts of S-BAIBA are present, this would lead to a “quantitative camouflage” of the R-form of BAIBA in serum. Only the latter enantiomorph has a metabolic relationship to the BAIBA excreted in urine; the metabolic precursor of S-BAIBA is unknown. Thus, only high amounts of the serum R-BAIBA should lead to a distinct elevation of the serum total BAIBA. It might be questionable however, if the urinary BAIBA excretion (mg/24 hours) is a useful parameter in this context, or connected with too many uncertainties. Further investigation is required to show if there is a significant correlation between the R-form of BAIBA in serum and the excreted R-form of BAIBA in urine.
207
Acknowledgements The authors are indebted to Dr. K. Altland for helpful discussions. This work has partly been supported from the University of Hamburg, the Deutsche Forschungsgemeinschaft and the Norwegian Cancer Society. References 1
K.
Fink,
2
Y.
Kakimoto.
R.B.
3
J. Yanai,
4
H. Brunschede,
5
M.
Y.
Abe,
(1973)
M.
Henderson K.
and
Taniguchi
Kakimoto,
R.M.
T. Tsujio
R. Hoffbauer Takahashi.
T.
Fink.
and I. Sam,
J. Biol. J. Biol.
and I. Sane,
and
H.W.
Nishidai.
Am.
Goedde,
S.
Chem., Chem.,
J. Hum.
Klin.
Suyama,
19’7 244
S.
(1952)
(1969) Genet..
Wochenschr..
Oshima
and
441 335 21
(1969)
2 (1965) T.
Teramatsu,
115 93 Int.
J. Radiat.
Chem.,
238
Biol.,
24
73
6
M.D.
7
E. Solem,
Armstrong,
E. Jellum
K.
8
E. Solem,
Clin.
Chim.
Yates. and
Y.
Kakimoto,
L. Eldjam,
Acta.
53 (1974)
Clin.
K. Chin
183
Taniguchi
and
Acta.
50 (1974)
T. Kappe, 393
J. Biol.
(1963)
1447
