241
Clinica Chimica Acta, 99 (1979) 241-245 @ ElsevierlNorth-Holland Biomedical Press
CCA 1179
QUANTITATION OF ORGANIC ACIDS IN AMNIOTIC FLUID BY GAS CHROMATOGRAPHY
S.P. WILKINSON,
S.E. NIVEN and R. HaHNEL
*
University of Western Australia, Department of Obstetrics and Gynaecology, Memorial Hospital for Women, Subiaco 6008 W.A. (Australia) (Received
King Edward
May 22nd, 1979)
Summary
The concentrations of 13 major organic acids in normal human amniotic fluid have been determined by gas chromatography. Each acid showed a large range and an irregular frequency distribution.
Introduction
The application of combined gas chromatography-mass spectrometry to the study of inborn errors of metabolism has enabled the discovery of several new metabolic diseases [l]. The availability of multicomponent analyses at the microgram level has led to the concept of metabolic profiles. We have previously described the qualitative organic acid composition of normal human amniotic fluid [2]. This paper presents the results of quantitative organic acid analyses of normal human amniotic fluid at different gestations using gas chromatography . Materials and methods
Extraction of amniotic fluid samples Amniotic fluid samples were obtained from amniocenteses performed in cases of therapeutic abortion, caesarian sections and inductions of labour by amniotomy. Specimens were centrifuged and the supematant stored at -20°C if not used immediately. Aliquots (10 ml) were saturated with NaCl and acidified (pH l-2) with HCl (5 mol/l). o-Hydroxyphenylacetic acid (50 pg) was added as internal standard and the mixture extracted with ethyl acetate (3 X 10 ml). The combined extracts were dried over sodium sulphate and evaporated * To whom correspondence should be addressed.
242
to dryness under nitrogen. The samples were stored in a desiccator prior to derivatisation. The residue was silylated with bis( trimethylsilyl)acetamide (50 ~1) for 1 h at 70°C. The solutions were stored overnight before ahquots (2 ~1) were analysed by gas chromatography. Gas chromatography-mass
spectrometry
This was carried out as described previously [ 21. Quantitative gas chromatography (GC) was carried out using a Hewlett-Packard 5840A microprocessor controlled gas chromatograph fitted with an autosampler. This instrument was programmed to produce a plot of the gas chromatogram and a listing of absolute areas, and areas relative to the internal standard, for each peak. Results Fig. 1 shows a typical metabolic profile of the organic acids extracted from a pooled amniotic fluid sample and separated as trimethylsilyl (TMS) derivatives by GC. Each component of the profile was characterised by its mass spectrum and GC retention time. About 36 acids were identified and a normal pattern established. Altogether 35 specimens of early gestation and 21 specimens of late gestation were examined. A complete description has been published earlier [ 21. The following 13 acids have been quantitated (peak numbers in brackets correspond to the numbers in Fig. 1): 2-hydroxybutyric (4), 3-hydroxybutyric and 3-hydroxyisobutyric (6), 3-hydroxyisovaleric (8), succinic (13), glyceric (14), 2-methyl-3-hydroxybutyric (15A), unidentified (16), malic (17), homovanillic (22), citric (24), 4-hydroxyphenyllactic (26), palmitic (32) and stearic (39). These acids were chosen because they were present in most samples. The chromatograms were electronically integrated and the concentrations of the acids calculated according to the method of internal standardisation with response factors and extraction efficiencies measured for authentic acid sam-
2
6
IS sTD
15A
:
4
’
22 I;
Fig. 1. Metabolic profile of acids extracted from pooled amniotic fluid and separated as TMS derivatives by GC. Column: 1.8 m X 2 mm I.D. glass, packed with 3% SE-30 on Chromosorb W, AW-DMCS. Temperature program: isothermal at 6O’C for 10 min. then heated at 4’C min-1 to 29O’C. Carrier gas: N2 at 20 ml . min-’ .
243
Fig. 2. Frequency
distribution
of concentration
of 2-hydroxybutyric
acid in early gestation
amniotic
fluid.
ples. Where the response factor was not experimentally determined, the value of a structurally similar acid was used. Concentrations were expressed as (i) pmol acid per litre of amniotic fluid, (ii) pmol acid per gram protein or pmol acid per total amniotic fluid volume (using volumes of amniotic fluid calculated by Fuchs [ 31). Histograms of concentration were plotted for each acid according to gestation (early or late), scale (linear or logarithmic) and units for expression of concentration as described above. No normal distributions were exhibited by any of these plots. Chalmers [4] has recently published data concerning the distribution of organic acids in adult urine. He was able to classify concentration distributions as unimodal, bimodal or irregular when plotted on a semi-logarithmic scale. This was not the case for organic acids in amniotic fluid. As an example, histograms for 2-hydroxybutyric acid (early and late gestation) are shown in Figs. 2 and 3. These distributions are typical for the amniotic fluid organic
P-Hydmxybtyric Fig. 3. Frequency
acid - late gestation
distribution
of concentration
of P-hydroxybutyric
acid in late gestation
amniotic
fluid.
I
Retention
time
ACIDS
14
15A
16
2-Methyl-3-hydroxybutyric
Unidentified
8
6
4
No.
Peak
13
24
26
32
39
Citric
4-HydroxyphenyUactic
Pahnitic
Steatic
1.56
1.36
1.27
1.23
1.16
0.95
0.90
0.85
0.80
0.11
0.70
0.62
or mg
FLUID
g-1
0.35
0.47
0.82
88
0.7
45
0.086
47
3.8
19
20
51
30
protein.
O-2.0
O-2.96
O-4.3
C+460
0-
O-150 5
25
85
o--o.44
O-220
0-
0-
O-140
O-290
(t-150
0.105
0.15
0.27
28
0.25
14
0.027
14
12
6
8.5
19
10
Mean
Mean
Range
l~mol
gestation .
g-l
acid):
/.lmol .1-’
Early
(n = 35)
(2-hydroxyphenylacetic
Concentration
standard
AMNIOTIC
0.58
tR
are in mg . I-1
22
HomovaniIlic
* AU concentrations
17
MaIic
*
IN
to the internal
Glyceric
for this acid
relative
ORGANIC
Succinic
3-H~drox~isovaIeric
OF
of acid
and
3-hydroxyisobutyric
3-Hydroxybutyric
2-Hydroxybutyric
Acid
tR:
CONCENTRATION
TABLE
10
o--o.8
04.78
O-2.4
O-l
O-2.4
050
97
0.17
0.16
0.093
4
0.22
11
0.0039
10
13 -
8
85
O-l.7
o-2.1
04.88
o-
(t2.2
o-
50
82
(to.048
0-
O-110 -
O-120
O-134
O-130
25 24
Range
Mean
. 1-l
(n = 21)
samples.
gestation
fluid
/.mlol
Late
of amniotic
35
O-+.25
O-140
o-
0-
O-130
O-260
O-140
Range
protein
n: No.
2.2
0.00053
0.018
0.024
0.012
0.5
0.024
23
2.5
1.2
2.9
0.5
Mean
fimol.
g-l
7
o--o.16
O-O.38
04.13
o-5.7
w.29
O-20
O-0.0068
(t28
O-19
O-18
O-l
o-36
Range
protein
245
acids and are markedly skewed to the right. Only one or two samples in the population showed very high concentrations. This trend is exemplified in the means and ranges shown in Table I. The mean concentrations for all acids are lower in the late gestation samples. We have previously reported [5] the concentration of lactic acid (peak 2) in amniotic fluid to be 9.33 mmol - 1-r (range 5.22-11.0 mmol - 1-l). There has been much discussion recently concerning the method of choice for extracting organic acids from biological fluids [6]. The two main methods are (i) ion exchange chromatography using DEAE Sephadex and (ii) liquidliquid extraction using solvents such as diethyl ether or ethyl acetate. The ion exchange method has proven to be much more sensitive for polyhydroxy acids but suffers from the disadvantage of being time-consuming. As our interest in measuring organic acids in amniotic fluid was in establishing reference values for a rapid screening program, we chose the faster and simpler of the two techniques - solvent extraction. Hagenfeldt [7] has reported the concentrations of some organic acids in human amniotic fluid. Our values for 3-hydroxybutyric and lactic acids are in very good agreement while the value for succinic acid is about twice, and the value for citric acid about one third of the values obtained by Hagenfeldt. The results presented in this study form the basis of a normal metabolic acid profile which may be useful for the identification of organic acidurias. References 1 2 3 4
Jellurn. E. (1977) J. Chromatogr. 143.427462 Nicholls, T.M.. Hglmel, R. and Wilkinson, S.P. (1978) Clin. Chim. Acta 84.11-17 Fuchs, F. (1966) Clin. Obstet. Gynec. 9.449460 Chalmers, R.A.. Healy. M.J.R.. Lawson, A.M., Hart. J.T. and Watts, R.E. (1976) Clin. Chem. 22. 1292-1298 6 Nicholls, T., Hiihnel, R.. Wilkinson. S. and Wysocki. S. (1976) Clin. Chim. Acta 69.127-130 6 Thompson, J.A. and Markey. S.P. (1975) Anal. Chem. 47.1313-1321 7 Hagenfeldt, L. and Hagenfeldt. K. (1972) Clin. Chim. Acta 42. 219
Clinica Chimica Acta, 99 (1979) 241-245 @ ElsevierlNorth-Holland Biomedical Press
CCA 1179
QUANTITATION OF ORGANIC ACIDS IN AMNIOTIC FLUID BY GAS CHROMATOGRAPHY
S.P. WILKINSON,
S.E. NIVEN and R. HaHNEL
*
University of Western Australia, Department of Obstetrics and Gynaecology, Memorial Hospital for Women, Subiaco 6008 W.A. (Australia) (Received
King Edward
May 22nd, 1979)
Summary
The concentrations of 13 major organic acids in normal human amniotic fluid have been determined by gas chromatography. Each acid showed a large range and an irregular frequency distribution.
Introduction
The application of combined gas chromatography-mass spectrometry to the study of inborn errors of metabolism has enabled the discovery of several new metabolic diseases [l]. The availability of multicomponent analyses at the microgram level has led to the concept of metabolic profiles. We have previously described the qualitative organic acid composition of normal human amniotic fluid [2]. This paper presents the results of quantitative organic acid analyses of normal human amniotic fluid at different gestations using gas chromatography . Materials and methods
Extraction of amniotic fluid samples Amniotic fluid samples were obtained from amniocenteses performed in cases of therapeutic abortion, caesarian sections and inductions of labour by amniotomy. Specimens were centrifuged and the supematant stored at -20°C if not used immediately. Aliquots (10 ml) were saturated with NaCl and acidified (pH l-2) with HCl (5 mol/l). o-Hydroxyphenylacetic acid (50 pg) was added as internal standard and the mixture extracted with ethyl acetate (3 X 10 ml). The combined extracts were dried over sodium sulphate and evaporated * To whom correspondence should be addressed.
242
to dryness under nitrogen. The samples were stored in a desiccator prior to derivatisation. The residue was silylated with bis( trimethylsilyl)acetamide (50 ~1) for 1 h at 70°C. The solutions were stored overnight before ahquots (2 ~1) were analysed by gas chromatography. Gas chromatography-mass
spectrometry
This was carried out as described previously [ 21. Quantitative gas chromatography (GC) was carried out using a Hewlett-Packard 5840A microprocessor controlled gas chromatograph fitted with an autosampler. This instrument was programmed to produce a plot of the gas chromatogram and a listing of absolute areas, and areas relative to the internal standard, for each peak. Results Fig. 1 shows a typical metabolic profile of the organic acids extracted from a pooled amniotic fluid sample and separated as trimethylsilyl (TMS) derivatives by GC. Each component of the profile was characterised by its mass spectrum and GC retention time. About 36 acids were identified and a normal pattern established. Altogether 35 specimens of early gestation and 21 specimens of late gestation were examined. A complete description has been published earlier [ 21. The following 13 acids have been quantitated (peak numbers in brackets correspond to the numbers in Fig. 1): 2-hydroxybutyric (4), 3-hydroxybutyric and 3-hydroxyisobutyric (6), 3-hydroxyisovaleric (8), succinic (13), glyceric (14), 2-methyl-3-hydroxybutyric (15A), unidentified (16), malic (17), homovanillic (22), citric (24), 4-hydroxyphenyllactic (26), palmitic (32) and stearic (39). These acids were chosen because they were present in most samples. The chromatograms were electronically integrated and the concentrations of the acids calculated according to the method of internal standardisation with response factors and extraction efficiencies measured for authentic acid sam-
2
6
IS sTD
15A
:
4
’
22 I;
Fig. 1. Metabolic profile of acids extracted from pooled amniotic fluid and separated as TMS derivatives by GC. Column: 1.8 m X 2 mm I.D. glass, packed with 3% SE-30 on Chromosorb W, AW-DMCS. Temperature program: isothermal at 6O’C for 10 min. then heated at 4’C min-1 to 29O’C. Carrier gas: N2 at 20 ml . min-’ .
243
Fig. 2. Frequency
distribution
of concentration
of 2-hydroxybutyric
acid in early gestation
amniotic
fluid.
ples. Where the response factor was not experimentally determined, the value of a structurally similar acid was used. Concentrations were expressed as (i) pmol acid per litre of amniotic fluid, (ii) pmol acid per gram protein or pmol acid per total amniotic fluid volume (using volumes of amniotic fluid calculated by Fuchs [ 31). Histograms of concentration were plotted for each acid according to gestation (early or late), scale (linear or logarithmic) and units for expression of concentration as described above. No normal distributions were exhibited by any of these plots. Chalmers [4] has recently published data concerning the distribution of organic acids in adult urine. He was able to classify concentration distributions as unimodal, bimodal or irregular when plotted on a semi-logarithmic scale. This was not the case for organic acids in amniotic fluid. As an example, histograms for 2-hydroxybutyric acid (early and late gestation) are shown in Figs. 2 and 3. These distributions are typical for the amniotic fluid organic
P-Hydmxybtyric Fig. 3. Frequency
acid - late gestation
distribution
of concentration
of P-hydroxybutyric
acid in late gestation
amniotic
fluid.
I
Retention
time
ACIDS
14
15A
16
2-Methyl-3-hydroxybutyric
Unidentified
8
6
4
No.
Peak
13
24
26
32
39
Citric
4-HydroxyphenyUactic
Pahnitic
Steatic
1.56
1.36
1.27
1.23
1.16
0.95
0.90
0.85
0.80
0.11
0.70
0.62
or mg
FLUID
g-1
0.35
0.47
0.82
88
0.7
45
0.086
47
3.8
19
20
51
30
protein.
O-2.0
O-2.96
O-4.3
C+460
0-
O-150 5
25
85
o--o.44
O-220
0-
0-
O-140
O-290
(t-150
0.105
0.15
0.27
28
0.25
14
0.027
14
12
6
8.5
19
10
Mean
Mean
Range
l~mol
gestation .
g-l
acid):
/.lmol .1-’
Early
(n = 35)
(2-hydroxyphenylacetic
Concentration
standard
AMNIOTIC
0.58
tR
are in mg . I-1
22
HomovaniIlic
* AU concentrations
17
MaIic
*
IN
to the internal
Glyceric
for this acid
relative
ORGANIC
Succinic
3-H~drox~isovaIeric
OF
of acid
and
3-hydroxyisobutyric
3-Hydroxybutyric
2-Hydroxybutyric
Acid
tR:
CONCENTRATION
TABLE
10
o--o.8
04.78
O-2.4
O-l
O-2.4
050
97
0.17
0.16
0.093
4
0.22
11
0.0039
10
13 -
8
85
O-l.7
o-2.1
04.88
o-
(t2.2
o-
50
82
(to.048
0-
O-110 -
O-120
O-134
O-130
25 24
Range
Mean
. 1-l
(n = 21)
samples.
gestation
fluid
/.mlol
Late
of amniotic
35
O-+.25
O-140
o-
0-
O-130
O-260
O-140
Range
protein
n: No.
2.2
0.00053
0.018
0.024
0.012
0.5
0.024
23
2.5
1.2
2.9
0.5
Mean
fimol.
g-l
7
o--o.16
O-O.38
04.13
o-5.7
w.29
O-20
O-0.0068
(t28
O-19
O-18
O-l
o-36
Range
protein
245
acids and are markedly skewed to the right. Only one or two samples in the population showed very high concentrations. This trend is exemplified in the means and ranges shown in Table I. The mean concentrations for all acids are lower in the late gestation samples. We have previously reported [5] the concentration of lactic acid (peak 2) in amniotic fluid to be 9.33 mmol - 1-r (range 5.22-11.0 mmol - 1-l). There has been much discussion recently concerning the method of choice for extracting organic acids from biological fluids [6]. The two main methods are (i) ion exchange chromatography using DEAE Sephadex and (ii) liquidliquid extraction using solvents such as diethyl ether or ethyl acetate. The ion exchange method has proven to be much more sensitive for polyhydroxy acids but suffers from the disadvantage of being time-consuming. As our interest in measuring organic acids in amniotic fluid was in establishing reference values for a rapid screening program, we chose the faster and simpler of the two techniques - solvent extraction. Hagenfeldt [7] has reported the concentrations of some organic acids in human amniotic fluid. Our values for 3-hydroxybutyric and lactic acids are in very good agreement while the value for succinic acid is about twice, and the value for citric acid about one third of the values obtained by Hagenfeldt. The results presented in this study form the basis of a normal metabolic acid profile which may be useful for the identification of organic acidurias. References 1 2 3 4
Jellurn. E. (1977) J. Chromatogr. 143.427462 Nicholls, T.M.. Hglmel, R. and Wilkinson, S.P. (1978) Clin. Chim. Acta 84.11-17 Fuchs, F. (1966) Clin. Obstet. Gynec. 9.449460 Chalmers, R.A.. Healy. M.J.R.. Lawson, A.M., Hart. J.T. and Watts, R.E. (1976) Clin. Chem. 22. 1292-1298 6 Nicholls, T., Hiihnel, R.. Wilkinson. S. and Wysocki. S. (1976) Clin. Chim. Acta 69.127-130 6 Thompson, J.A. and Markey. S.P. (1975) Anal. Chem. 47.1313-1321 7 Hagenfeldt, L. and Hagenfeldt. K. (1972) Clin. Chim. Acta 42. 219
