127

Clinica Chimica Acta, 69 (1976) 127-130 @ Elsevier Scientific Publishing Company,

Amsterdam

- Printed

in The Netherlands

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IDENTIFICATION OF 2-HYDROXYBUTYRIC IN HUMAN AMNIOTIC FLUID

T. NICHOLLS,

R. HAHNEL,

S. WILKINSON

ACID

and S. WYSOCKI

Department of Obstetrics and Gynaecology, University of Western Australia, King Edward Memorial Hospital for Women, Subiaco, Western Australia, 6008 (Australia) (Received

December

15, 1975)

Summary 2-Hydroxybutyric acid has been identified in human amniotic fluid by combined gas chromatography and mass spectrometry. It was present in all 22 specimens examined of gestation 15 to 20 weeks and 30 to 40 weeks. The concentration of 2-hydroxybutyric acid in amniotic fluid was about 100 times less than that of the predominant acid, lactic acid.

Metabolic profiles of amniotic fluid are studied in our laboratory to define the normal composition and describe the correlation between abnormal patterns and disease. In the course of the investigations an acid was found, which has not been previously described as a normal constituent of amniotic fluid. This acid was shown to be 2-hydroxybutyric acid. Amniotic fluid was centrifuged at 3000 r.p.m. for 10 min at 4°C to remove cells and debris. To the clear supernatant (12 ml) were added aliquots of solutions containing the internal standards a-hydroxyisocaproic acid (1.12 mg) or malonic acid (0.40 mg). The solution was saturated with sodium chloride (5 g), adjusted to pH 11 with a few drops of 1 M sodium hydroxide and extracted with freshly distilled ethyl acetate (2 X 60 ml) to remove neutral and basic compounds. The aqueous phase was brought to pH 2 with 1 M hydrochloric acid and extracted with ethyl acetate (3 X 60 ml). The pooled extracts were dried over anhydrous sodium sulphate (5 g), concentrated in vacua and evaporated to dryness in the silylation tube with a stream of nitrogen at 35°C. The dry residue was silylated with N,O-bis(trimethylsilyl)acetamide (60 1-11)at 70°C for 30 min. Aliquots of the silylated extract were chromatographed on 1.8 m X 2 mm i.d. glass columns packed with 3% SE-30 on Chromosorb W, AW-DMCS (So-100 mesh) using helium as a carrier gas. The column was kept at 60°C for

Fig. 1. Part of an organic acid profile of amniotic fluid, showing lactic acid (peak 2), 2-hydroxybutyric acid (peak 4) and 3-hydroxybutyric acid (peak 6) as well as internal standards (malonic acid. peak 8 and cu-hydroxyisocaproic acid, peak 10).

10 min and then heated to 240°C at 4°C per min. The gas chromatograph was a Varian 2740 and was interfaced to a Varian MAT 311 mass spectrometer using a Biemann-Watson helium separator at 250°C. The mass spectrometer was operated at resolution 1000, accelerating voltage 3 kV, ionisation energy 70 eV

TABLE

I

RETENTION ACID IN ADDED(*)

TIME RELATION

OF TO

THE BIS(TRIMETHYLSILYL)-DERIVATIVE OTHER ORGANIC ACIDS OF AMNIOTIC ~~~

Peak No.

Organic

2 4 6 8 10

Lactic acid 2-Hydroxvbutyric 3-Hydroxybutyric Malonic acid (*) 2-Hydroxyisocaproic

__.-..

-.

OF 2-HYDROXYBUTYRIC FLIJID AND STANDARDS

~_~ ..-.

__

_~_ .._... - --

Temperature Relative retention time (ol-hydroxyC’C) isocaproic acid = 1.00) _.___.~~~__ .__ ~~_._.. __ _. ._. ._-.-- -

Retention time (min)

acid

_ acid acid acid (*)

9.5 13.0 14.5 17.0 18.5

0.51 0.70 0.78 0.92 1.00

60 72.5 79 89 96

_ ---.~~~

129

73 131

I I

I. , , Il..

/

II

147

II

190 ,

205 I

A_

233

Fig. 2. Mass spectrum of peak 4 of the gas chromatogram.

and ion source temperature 170°C. Part of a typical chromatogram of the silylated extract of amniotic fluid is shown in Fig. 1; retention times and temperatures are given in Table I. The major component was lactic acid (peak 2) together with smaller amounts of 3-hydroxybutyric acid (peak 6). These acids had already been identified in amniotic fluid [l]. A previously unidentified component (peak 4) was present in all samples. This component was shown to be 2-hydroxybutyric acid by comparison of its mass spectrum with that obtained from the authentic compound. The mass spectrum (Fig. 2) shows fragmentations consistent with an cu-hydroxy acid derivative. Loss of a silicon-bound methyl group from the molecular ion gives the ion at m/e 233. This ion rearranges with loss of CO to give the ion at m/e 205 which then loses CH3CH2CH0 to form the abundant ion at m/e 147 (Table II). The molecular ion readily undergoes e-cleavage with loss of the ester moiety to give the large ion at m/e 131. The average concentration of 2-hydroxybutyric acid (0.05 mmol/l, range 0.03 to 0.10) was about half that of 3-hydroxybutyric acid (0.12 mmol/l, range 0.06 to 0.24) and about two orders of magnitude lower than that of lactic acid (8.40 mmol/l, range 5.22 to 11.20). There was no indication that the concentration of these three acids in amniotic fluid changed with gestation. Our levels

TABLE II RELATIVE ABUNDANCE OF THE MAJOR IONS OF THE MASS METHYLSILYL)-DERIVATIVE OF 2-HYDROXYBUTYRIC ACID -.-.-~-m/e 73 75 81 115

Relative abundance 100 14.5 5 2.5

117

1.8

131

71.5

147

63

190

4.5

205

8.5

219

1.0

233

4.5

SPECTRUM

OF THE BIS(TRI-

130

for lactic acid are almost identical to those reported by Hagenfeldt and Hagenfeldt [l], while the mean 3-hydroxybutyric acid concentration in our series was about twice as high as their value. The presence of 2-hydroxybutyric acid in urine of children with acidosis has been linked with elevated levels of lactic acid [ 21. An increased ratio of NADH, / NAD in cells appears to be the reason for accumulation of these metabolites [ 31. References 1

Hagenfeldt,

2

Pettersen,

3

Landaas,

L. and J.E.,

Hagenfeldt.

Landaas.

S. (1975)

Clin.

S. and Chim.

K.

(1972)

Eldjam, Acta

58,

Clin.

Chin

L. (1973) 23

Acta

Clin.

42.

Chim.

219 Acta

48.

213