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Determination of α-acyl-α-hydroxy acids in biological fluids by head-space gas chromatography
ANALYTICAL
37, 368-377 (1970)
BIOCHEMISTRY
Determination Fluids
of by
cw-Acyl-a-hydroxy
Head-Space
Gas
Acids
in Biological
Chromatography
J. HEJGAARD Department
of Biochemistry
and Nutrition Polytechnic &300 Lyngby, Denmark
Institute
of Denmark,
Received March 25, 1970
The first enzyme in the combined pathway to the branched-chain amino acids, ,acetohydroxy acid synthetase, simultaneously synthesizes a-acetolactate and a-aceto-.a-hydroxybutyrate, whi’ch are precursors of valine (leucine) and isoleucine, respectively (1). The a-aceto-a-hydroxy acids may be determined by the method of Westerfeld (2) after decarboxylation by strong acid to the corresponding acyloins. When studying the synthetase in crude cell-free extracts the assay may be complicated by the presence of acetohydroxy acid decarboxylating enzymes and other aceboin forming enzymes. Such sidereactions have been considered in the differential assay procedure of Kuwana et al. (3). When both pyruvate and a-ketobutyrate ‘are present as substrates the sum of the two acetohydroxy acids formed is determined by these procedures. A microbiological assay of a-aceto-a-hydroxybutyrate has been developed by Leavitt and Umbarger (4). Recently Ronkainen et al. (5) published a gas chromatographic procedure for the separate ,determination of the two acetohydroxy acids in fermentation solutions. The two acids were separated from the fermented medium by column chromatography and analyzed after conversion to a-diketones. The method to be described involves no previous separation from the biological material. It has been used in studies on the metabolic regulation of acetohydroxy acid synthetase from Saccharomyces carlsbergensis (6), and permits a quick and direct ,determination of a-acyl-a-hydroxy acids with 5-7 carbon atoms (C&,-C,) formed by incubation of cell-free enzyme extracts with C&-C& a-keto acids. PRINCIPLE
OF THE
METHOD
The method is based upon the eonversion of a-aceto-cu-hydroxy acids, and other 8a-acyl-a-hydroxy acids, to the corresponding a-diketones (7) 368
GLC
OF
a-ACYL-a-HYDROXY
ACIDS
369
and subsequent determination of these compounds by gas chromatography (8). After the enzyme reaction the incubation mixture is quickly adjusted to pH 4.1 by addition of buffer and then heated to 60” in a closed bottle in the presence of air. By this procedure the enzyme is immediately inactivated and, after incubation for a stated time, the a-aceto-a-hydroxy acids are quantitatively converted to a-diketones by oxidative decarboxylation (7). The solution is cooled, and a measured amount of a higher cY-diketone is added as internal standard. The vapor pressure of the a-diketones in the bottle is increased by saturation of the solution with Nan inorganic salt. The bottle is then closed tightly and incubated at constant temperature for a fixed time. An exactly measured gas sample is removed from the gas space over the solution and injected into the gas chromatograph, where the diketones are separated and analyzed using an electron capture detector as ‘described by Harrison (8). A precolumn back-flush aystem prevents contamination of the detector with water, thus increasing sensitivity, reproducibility, and rapidity of analysis. MATERIALS
The ethyl esters of cu,O-diacetyllactic acid and a,O-diacetyl-a-hydroxybutyric acid were prepared by the method of Krampitz (9). The ,esters were hydrolyzed by standing overnight with 2 equiv NaOH at 5”. The resulting aqueous ‘solutions of a-acetolactate Nor a-aceto-a-hydroxybutyrate were used directly. Diacetyl (puriss., Fluka AG, Buchs, Switzerland), acetylpropionyl (pract., Fluka) , and acetyl-n-butyryl (Fritzsche Brothers Inc., New York) were redistilled before use. Dipropionyl was prepared by oxidation of propionoin (10) and isolated by distillation. Similarly, acyloins formed from an equimolar mixture of ethyl Iacetate and ethyl n-valerate (11) were oxidized to a-8diketones, and acetyl-nValery1 was isolated by fractional distillation. The purity of all a-diketone preparations was controlled by GLC using flame ionization and electron capture detection. a-Ketobutyric acid and cu-ketovaleric acid were prepared by the method of Vogel and Schinz (12). The yeast used was Saccharom~ces carlsbergensis (U-strain, Carlsberg Breweries, Denmark). Cell-free extracts were prepared from yeast cells grown on minimal medium and harvested in the exponential growth phase. The cells were suspended in 0.1 M phosphate-Tris buffer (pH 7.3, 0.5 M sucrose, 1mM EDTA) at a cell density of 0.3 gm (wet weight) per milliliter and disintegrated in an X-press (BIOX, Sweden) (13) at -20”. Cells and cell debris were removed by centrifugation at 150009 and 0” for 15 min, sand the clear supernatant was used immediately.
370
J.
HEJGAARD
PROCEDURE
Conversion of a-Aceto-a-hydroxy
Acids to wDiketones
The incubation with enzyme extract is performed directly in the headspace bottle. The bottle (20 ml capacity, 18 mm id.) generally contains a total volume of 1.2 ml incubation mixture. The enzymic reaction is stopped quickly cooling the bottle to 0”. Then 1.0 ml 1 M citric acid/ sodium ,citrate buffer (pH 4.1) is added, and the stoppered bottle is incubated in a thermostated water bath at 60” for 40 min. During this period the bottle is frequently shaken. After conversion of the acetohydroxy acids to a-diketones the bottle may be stored at -20” until analyzed. Head-Space Analysis
Prior to the gas chromatographic analysis, 2.0 gm (NH,)zSO, and 0.5 ml internal standard solution (5 mg acetylbutyryl/liter water, 5% ethanol) is added to the cold mixture. The bottle is then closed by a silicone rubber membrane and incubated in a mechanical shaker at 30”. After (at least) 40 min equilibration time, a 1 ml head-space sample is removed from the bottle by means of a 1 ml gastight syringe (Hamilton) equipped with ‘a 40 X 0.5 mm needle with ‘a side hole. The needle is introduced through the membrane to the middle of the gas space. The syringe is slowly filled and emptied back into the bottle, 5 times during 30 set to achieve adsorption equilibrium. By the sixth filling, exactly 1 ml of gas is withdrawn and immediately injected into the gas chromatograph. Before being used again the syringe is cleaned by evacuation of cylinder, piston, and needle at 0.1 mm Hg and 80” for 5 min. The syringe parts are then transferred t,o close-fitting glass chambers and placed in a 30” water bath for exactly 5 min for quick temperature equilibration. Gas Chromatographic Conditions
The Beckman GC-4 gas chromatograph was equipped with an 8 port, 2 position switching valve (Carle) placed in a separate thermostated oven to permit back-flush of water from the preoolumn (Fig. 1). Columns. l/s”’ steel tubing. Precolumn: 1’ 20% diglycerol on 60-80 mesh GAS-CHROM P. Analytical column: 6’ 20% 1,2,3-tris (2-cyanoethoxy) propane (TCEP) on 100-120 mesh GAS-CHROM CLA or 3’4” TCEP followed by 3’4*’ 15% polypropylene glycol on 80-100 mesh Chromosorb W. Temperatures. Injection line: 80”. Columns: 70”. Back-flush valve: 130”. Detector line: 130”. Detector: 180”.
GLC
‘”
1
OF
2
a-ACYL-a-HYDROXY
371
ACIDS
‘zrx’
1
2
3 L 51
A
B
FIG. 1. Gas chromatographic system with precolumn back-flush (valve position B) : (1) Carrier and purge helium. (2) Discharge helium. (3) Split 1:l. (4) 8 port, 2 position valve. (5) Injector. (6) Precolumn. (7) Analytical column. (8) CO, (carrier diluent). (9) Electron capture detector. (10) Purge helium restrictor. (11) Compensator needle valve. (12) Vent.
Gus flow rates. Discharge helium: 100 ml/min. Carrier helium: 35 ml/min (in both valve positions), Carbon dioxide (carrier diluent) : optimal (3-4 ml/min) . Detector settings. Source current: 7 mA. Polarization voltage: optimal (about 70 V). Bias voltage: optimal (about 0 V) . Electrometer. Current sensitivity: 5 X lo-lo A full scale. Registration. Servo/riter II, 1 mV (Texas Instruments, Inc.). Valve activation. Time from injection to start of back-flush is determined ‘experimentally (about 2 min) . Analysis time. 5-20 min, depending on number of cu-diketones determined and #choice of internal standard. Stundurdixution
Before the head-space analysis, the standard solutions (1.2 ml) were treated exactly as the enzyme incubations. The standards were based on incubation mixture (with boiled ‘enzyme extract) to which cu-acetolactate and a-aceto-la-hydroxybutyrate or C,-C, a-diketones were added. In most determinations, acetylbutyryl was used as internal standard. In experiments in which this compound or dipropionyl was formed or suspected, acetylvaleryl was used. However, it is possible to make the analysis without internal standard by accurate sampling and careful
372
J. HE'JGAAFD
standardization, thereby reducing analysis time and calculations. Internal standardization based on other compounds than cr-diketones or alkylhalogenides gave unsatisfactory results. The air in the sample affects the stability and sensitivity of the detector. By the previous injection of 34 head-space samples taken above a 2% solution of ethanol in water the background current may be stabilized and kept constant for several hours of analysis with injection of a sample every 10 min. The method as described here is sensitive to about 0.2 nmole a-diketone per 1.2 ml incubation mixture. Calculations The ,electron capture response is a reduction in a (background) current and the linear range is narrow (14). Because of detector and electronic noise the nonlinear region of the response curve has to be used in most practical applications of the electron capture detector. Under these conditions, multicomponent analysis with internal standardization may give problems. The use of internal standard by calculation of peak ‘area -or peak height-ratios is erroneous without previous linearization of the ,detector response. For the detector type used, a linear response function based on the measurement of peak heights has been derived. When all gas chromatographic parameters, including the background current, are carefully stabilized and kept constant during the analysis of a series of head-space samples, a nearly linear correlation between reciprocal peak height and reciprocal concentration of a-diketone in the 1.2 ml sample was found (Fig. 2). In kinetic studies, the reciprocal initial velocity as used in Lineweaver-Burk plots may be derived directly from such curves. By extrapolation to infinite concentration (Fig. 3) a quantity here termed the effective background current b, is determined. The response function h/be-h shows a perfect linear correlation with concentration (Fig. 3). For comparison the variation of peak height h with eoncentration is depicted. This response function is used in the head-space analysis calculations. After stabilization of the gas chromatographic system, ru-,diketone solutions, including the internal standard, with concentrations giving a 10-70s reduction of the background #current b at peak maximum are analyzed. The b, values are determined (Fig. 2), and the response functions are cal,culated for all peaks from the analysis of samples and standards. The response function values are divided by the corresponding internal standard value, linear standard curves are drawn, and the concentrations calculated. As previously mentioned, the head-space analy-
GLC OF a-ACYL-a-HYDROXY
ACIDS
373
0.5
Fro. 2. Reciprocal peak height vs. reciprocal concentration of diacetyl (DA) and acetylpropionyl (AP) in sample (head-space analysis). Determination of effective background current b. by extrapolation. FIG. 3. Response function h/(b, - h) and response h/b, (h as fraction of b.) vs. concentration of diacetyl (DA) and acetylpropionyl CAP). Data obtained from Figure 2.
sis may be performed without internal standard, and linear standard curves as shown in Figure 3 are then obtained. By the analysis of head-space samples giving ,a 2040% reduction of the background current at peak maxima, the standard deviation for diacetyl and acetylpropionyl was approximately 5-675 without and 3% with internal standardization. APPLICATION
OF THE
METHOD
The method has (been applied in studies of the conversion of lower cu-keto acids to a-acyl-a-hydroxy acids by cell-free extracts of Saccharomyces carlsbergensis. The standard incubation mixture (1.2 ml) contained 3 pmoles MgSO,, 20 ,pg thiamine pyrophosphate, O-100 pmoles sodium pyruvate, O-60 ,pmoles Icu-ketobutyric acid or cu-keto valeric acid (pH 7)) 20-100 ~1 enzyme extract (3 mg protein) ; and 0.6 ml 0.2 M potassium hydrogen phosphate adjusted to pH 7.3 with 1 M Tris buffer. The incubation time was 10-30 mm at 30”. RESULTS
AND
DISCUSSION
After enzyme incubation at pH 7.3, no cu-diketone peaks and no peaks with retention times near those of the cu-diketones were observed by
374
J.
HEJGAABD
electron capture gas chromatography. At pH 4.1 the oxidative decarboxylation of acetolactate to diacetyl and of acetohydroxybutyrate to acetylpropionyl was quantitative after ca. 100 ,min at 30” and in less than 30 min at 60”. The conversion as a function of time proceeds identically in incubation mixtures and standard solutions. Incubation at 60”, pH 4.1, immediately after addition of enzyme extract showed no formation ,of a-diketones, enzymic or thermal. The enzyme was immediately inactivated at 60” and activity never restored at lower temperatures. Lower aldehydes formed by enzymic decarboxylation ,of keto acids did not influence the cu-diketone concentrations in the head-space sample nor the gas chromatographic separation. This was demonstrated by GLC after addition of micromole amounts of C&C4 aldehydes to incubation mixtures. Peaks representing other compounds present, e.g., ethanol, ,did not interfere with the determinations. By analysis of 10 individually prepared, identical, 1.2 ,ml incubations the following standard deviations were found, using no internal standard: 17 nmoles + 5.5% acetolactic acid and 27 nmoles & 7% acetohydroxybutyric acid. The applicability of the method is illustrated ‘by a series of analytical results, The formation of ,a-acetolactic acid and a-aceto-cr-hydroxybutyric acid vs. a-ketobutyrate concentration with fixed pyruvate concentration is shown in Figure 4. At high a-ketobutyrate/pyruvate ratios, dipropionyl is found by GLC, corresponding to the presence of a-propionyl-crhydroxybutyric acid after enzyme incubation. From this series of experi-
cu-aceto-rr-hydroxybutyric FIG. 4. Formation of cr-acetolactic acid (01, and a-propionyl-cu-hydroxybutyric acid (m) from 100 pmoles pyruvate amounts of wketobutyrate (O-60 pmoles) in standard incubations.
acid (A), and varied
GLC
OF a-ACYL-wHYDROXY
375
ACIDS
ments typical gas chromatographic separations are shown in Figure 5A. The chromatogram from an incubation with 3.75 pmoles cw-ketobutyrate demonstrates about equal amounts of diacetyl and acetylpropionyl formed from acetolactic acid and acetohydroxybutyric acid. Superimposed is part of the chromatogram from incubation with 60 ,pmoles ketobutyrate, showing in this case small amounts of diacetyl and a small dipropionyl peak. Figure 5B illustrates the formation of a-acetolactic acid and cu-aceto-cu-hydroxyvaleric acid by incubation with pyruvate and ‘cu-ketovalerate. Superimposed is a chromatogram from an identical incubation, but with valine, inhibitor of acetohydroxy acid synthetase, added. Threonine deaminase (L-threonine hydro-lyase (deaminating) , EC 4.2) 1.16 can also be studied in combined assays with acetohydroxy acid synthetase. The formation of a-acetohydroxybutyric acid vs. threonine concentration (Fig. 6) depicts the formation of a-ketobutyrate by threonine deaminase present in the extract. The inhibition of this enzyme by isoleucine is also demonstrated.
l- lr B
? I ‘ \ ‘,--.,\\ LL.L
I
I 0
I 5
I
T;RE
I
I 0
I 5
I
10
(MINUTES)
FIG. 5. Gas chromatographic separation of cY-diketones formed from o-acyl-cuhydroxy acids: (A) Standard incubation with 100 pmoles pyruvate and 3.75 pmoles cu-ketobutyrate (-); incubation with 100 pmoles pyruvate and 60 pmoles a-ketobutyrate (--). (B) Standard incubation with 60 pmoles pyruvate and 12 pmoles a-ketovalerate (--); same with Bamoles valine added (---). (1) Air. (2) Aldehydes. (3) Diacetyl. (4) Acetylpronionyl. (5) Acetyl butyryl. (6). Dipropionyl.
376
J.
HEJGAARD
FIQ. 6. Formation of a-aceto-cr-hydroxybutyric acid (A.) and u-acetolactic acid (0) from 100 amoles pyruvate and varied amounts of threonine (k-20 pmoles) in standard incubations at pH 8.0. Same with 50 amoles isoleucine added (A and 0).
Determination of acylhydroxy acids in enzyme incubation mixtures by direct head-space gas chromatography may thus be a valuable method in studies of the regulatory enzymes in valine-isoleucine biosynthesis. SUMMARY
A method has been developed for the determination of cu-acetolactic acid, a-aceto-a-hydroxybutyric, and other low molecular la-acyl-cu-hydroxy acids in biological fluids. The acylhydroxy acids are converted to iu-diketones by incubation at 60”, pH 4.1, for a specific time. A head-space gas sample is taken from above the incubation mixture and analyzed by gas chromatography with electron capture detection. Internal standardization is used, and calculations are facilitated by introduction of a linear response function based on peak height measurements. The method was applied to the ,determination of 0.2-60 nmoles of different a-aceto-a-hydroxy acids formed in 1.2 ml incubations of a-keto acids with cell-free enzyme extracts of Saccharomyces carlsbergensis. ACKNOWLEDGMENTS The author
is grateful
to Professor Robert
Djurtoft
for advice and encourage-
ment during this investigation. The
work
was partially
1. LEAVITT, R., 2. WESTERFELD,
supported
by a grant from the Tuborg
REFERENCES H. E., J. Bid. Chem. 236, W. W., J. Biol. Ckm. 161, 495 (1945).
AND
UMBARGER,
2486
Foundation.
(19611,
GLC
OF a-ACYL-a-HYDROXY
ACIDS
377
3. KUWANA, H., CAROLINE, D. F., -DINGI, R, W., AND WAGNER, R. P., Arch. B&hem. Biophys. 124 184 (1968). 4. LEAVITT, R., AND UMBAFNEX, H. E., J. Bacterial. 80, 18 (1960). 5. RONKAINEN, P., BRUMMER, S, AND SUOMALAINEN, H., Anal. B&hem. 34, 101 (1970) . 6. HEJQMRD, J., in preparation. 7. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., @ADA, K., AND K~ROIWA, Y., Rept. Res. Lab. Kirin Brewery Co. Ltd., No. 11, 9 (1968). 8. HARRISON, G. A. F., BYRNE, W. J., AND COLLINS, E., J. Inst. Brewing 71, t&6 11965). 9. KRAMPITZ, L. O., Arch. Biochem. 17, 81 (1948). 10. RIQBY, W., J. Chem. sot. 1951, 793. 11. Bwrr, A. H. (Ed.), “‘Organic Synthesis,” Vol. II. p. 114. Wiley, New York, 1966. 12. VOGEL, E., AND SCHINZ, H. Helv. Chim. Acta 33, 116 (1950). 13. EDEZB~, L., J. B&hem. Microbial. Techrwl. Eng. 2, 453 (1960). 14. WENTWORTH, W. E., CHEN, E., AND LOVELOCK, J. E., J. Phys. Chem. 70, 445 0966).
37, 368-377 (1970)
BIOCHEMISTRY
Determination Fluids
of by
cw-Acyl-a-hydroxy
Head-Space
Gas
Acids
in Biological
Chromatography
J. HEJGAARD Department
of Biochemistry
and Nutrition Polytechnic &300 Lyngby, Denmark
Institute
of Denmark,
Received March 25, 1970
The first enzyme in the combined pathway to the branched-chain amino acids, ,acetohydroxy acid synthetase, simultaneously synthesizes a-acetolactate and a-aceto-.a-hydroxybutyrate, whi’ch are precursors of valine (leucine) and isoleucine, respectively (1). The a-aceto-a-hydroxy acids may be determined by the method of Westerfeld (2) after decarboxylation by strong acid to the corresponding acyloins. When studying the synthetase in crude cell-free extracts the assay may be complicated by the presence of acetohydroxy acid decarboxylating enzymes and other aceboin forming enzymes. Such sidereactions have been considered in the differential assay procedure of Kuwana et al. (3). When both pyruvate and a-ketobutyrate ‘are present as substrates the sum of the two acetohydroxy acids formed is determined by these procedures. A microbiological assay of a-aceto-a-hydroxybutyrate has been developed by Leavitt and Umbarger (4). Recently Ronkainen et al. (5) published a gas chromatographic procedure for the separate ,determination of the two acetohydroxy acids in fermentation solutions. The two acids were separated from the fermented medium by column chromatography and analyzed after conversion to a-diketones. The method to be described involves no previous separation from the biological material. It has been used in studies on the metabolic regulation of acetohydroxy acid synthetase from Saccharomyces carlsbergensis (6), and permits a quick and direct ,determination of a-acyl-a-hydroxy acids with 5-7 carbon atoms (C&,-C,) formed by incubation of cell-free enzyme extracts with C&-C& a-keto acids. PRINCIPLE
OF THE
METHOD
The method is based upon the eonversion of a-aceto-cu-hydroxy acids, and other 8a-acyl-a-hydroxy acids, to the corresponding a-diketones (7) 368
GLC
OF
a-ACYL-a-HYDROXY
ACIDS
369
and subsequent determination of these compounds by gas chromatography (8). After the enzyme reaction the incubation mixture is quickly adjusted to pH 4.1 by addition of buffer and then heated to 60” in a closed bottle in the presence of air. By this procedure the enzyme is immediately inactivated and, after incubation for a stated time, the a-aceto-a-hydroxy acids are quantitatively converted to a-diketones by oxidative decarboxylation (7). The solution is cooled, and a measured amount of a higher cY-diketone is added as internal standard. The vapor pressure of the a-diketones in the bottle is increased by saturation of the solution with Nan inorganic salt. The bottle is then closed tightly and incubated at constant temperature for a fixed time. An exactly measured gas sample is removed from the gas space over the solution and injected into the gas chromatograph, where the diketones are separated and analyzed using an electron capture detector as ‘described by Harrison (8). A precolumn back-flush aystem prevents contamination of the detector with water, thus increasing sensitivity, reproducibility, and rapidity of analysis. MATERIALS
The ethyl esters of cu,O-diacetyllactic acid and a,O-diacetyl-a-hydroxybutyric acid were prepared by the method of Krampitz (9). The ,esters were hydrolyzed by standing overnight with 2 equiv NaOH at 5”. The resulting aqueous ‘solutions of a-acetolactate Nor a-aceto-a-hydroxybutyrate were used directly. Diacetyl (puriss., Fluka AG, Buchs, Switzerland), acetylpropionyl (pract., Fluka) , and acetyl-n-butyryl (Fritzsche Brothers Inc., New York) were redistilled before use. Dipropionyl was prepared by oxidation of propionoin (10) and isolated by distillation. Similarly, acyloins formed from an equimolar mixture of ethyl Iacetate and ethyl n-valerate (11) were oxidized to a-8diketones, and acetyl-nValery1 was isolated by fractional distillation. The purity of all a-diketone preparations was controlled by GLC using flame ionization and electron capture detection. a-Ketobutyric acid and cu-ketovaleric acid were prepared by the method of Vogel and Schinz (12). The yeast used was Saccharom~ces carlsbergensis (U-strain, Carlsberg Breweries, Denmark). Cell-free extracts were prepared from yeast cells grown on minimal medium and harvested in the exponential growth phase. The cells were suspended in 0.1 M phosphate-Tris buffer (pH 7.3, 0.5 M sucrose, 1mM EDTA) at a cell density of 0.3 gm (wet weight) per milliliter and disintegrated in an X-press (BIOX, Sweden) (13) at -20”. Cells and cell debris were removed by centrifugation at 150009 and 0” for 15 min, sand the clear supernatant was used immediately.
370
J.
HEJGAARD
PROCEDURE
Conversion of a-Aceto-a-hydroxy
Acids to wDiketones
The incubation with enzyme extract is performed directly in the headspace bottle. The bottle (20 ml capacity, 18 mm id.) generally contains a total volume of 1.2 ml incubation mixture. The enzymic reaction is stopped quickly cooling the bottle to 0”. Then 1.0 ml 1 M citric acid/ sodium ,citrate buffer (pH 4.1) is added, and the stoppered bottle is incubated in a thermostated water bath at 60” for 40 min. During this period the bottle is frequently shaken. After conversion of the acetohydroxy acids to a-diketones the bottle may be stored at -20” until analyzed. Head-Space Analysis
Prior to the gas chromatographic analysis, 2.0 gm (NH,)zSO, and 0.5 ml internal standard solution (5 mg acetylbutyryl/liter water, 5% ethanol) is added to the cold mixture. The bottle is then closed by a silicone rubber membrane and incubated in a mechanical shaker at 30”. After (at least) 40 min equilibration time, a 1 ml head-space sample is removed from the bottle by means of a 1 ml gastight syringe (Hamilton) equipped with ‘a 40 X 0.5 mm needle with ‘a side hole. The needle is introduced through the membrane to the middle of the gas space. The syringe is slowly filled and emptied back into the bottle, 5 times during 30 set to achieve adsorption equilibrium. By the sixth filling, exactly 1 ml of gas is withdrawn and immediately injected into the gas chromatograph. Before being used again the syringe is cleaned by evacuation of cylinder, piston, and needle at 0.1 mm Hg and 80” for 5 min. The syringe parts are then transferred t,o close-fitting glass chambers and placed in a 30” water bath for exactly 5 min for quick temperature equilibration. Gas Chromatographic Conditions
The Beckman GC-4 gas chromatograph was equipped with an 8 port, 2 position switching valve (Carle) placed in a separate thermostated oven to permit back-flush of water from the preoolumn (Fig. 1). Columns. l/s”’ steel tubing. Precolumn: 1’ 20% diglycerol on 60-80 mesh GAS-CHROM P. Analytical column: 6’ 20% 1,2,3-tris (2-cyanoethoxy) propane (TCEP) on 100-120 mesh GAS-CHROM CLA or 3’4” TCEP followed by 3’4*’ 15% polypropylene glycol on 80-100 mesh Chromosorb W. Temperatures. Injection line: 80”. Columns: 70”. Back-flush valve: 130”. Detector line: 130”. Detector: 180”.
GLC
‘”
1
OF
2
a-ACYL-a-HYDROXY
371
ACIDS
‘zrx’
1
2
3 L 51
A
B
FIG. 1. Gas chromatographic system with precolumn back-flush (valve position B) : (1) Carrier and purge helium. (2) Discharge helium. (3) Split 1:l. (4) 8 port, 2 position valve. (5) Injector. (6) Precolumn. (7) Analytical column. (8) CO, (carrier diluent). (9) Electron capture detector. (10) Purge helium restrictor. (11) Compensator needle valve. (12) Vent.
Gus flow rates. Discharge helium: 100 ml/min. Carrier helium: 35 ml/min (in both valve positions), Carbon dioxide (carrier diluent) : optimal (3-4 ml/min) . Detector settings. Source current: 7 mA. Polarization voltage: optimal (about 70 V). Bias voltage: optimal (about 0 V) . Electrometer. Current sensitivity: 5 X lo-lo A full scale. Registration. Servo/riter II, 1 mV (Texas Instruments, Inc.). Valve activation. Time from injection to start of back-flush is determined ‘experimentally (about 2 min) . Analysis time. 5-20 min, depending on number of cu-diketones determined and #choice of internal standard. Stundurdixution
Before the head-space analysis, the standard solutions (1.2 ml) were treated exactly as the enzyme incubations. The standards were based on incubation mixture (with boiled ‘enzyme extract) to which cu-acetolactate and a-aceto-la-hydroxybutyrate or C,-C, a-diketones were added. In most determinations, acetylbutyryl was used as internal standard. In experiments in which this compound or dipropionyl was formed or suspected, acetylvaleryl was used. However, it is possible to make the analysis without internal standard by accurate sampling and careful
372
J. HE'JGAAFD
standardization, thereby reducing analysis time and calculations. Internal standardization based on other compounds than cr-diketones or alkylhalogenides gave unsatisfactory results. The air in the sample affects the stability and sensitivity of the detector. By the previous injection of 34 head-space samples taken above a 2% solution of ethanol in water the background current may be stabilized and kept constant for several hours of analysis with injection of a sample every 10 min. The method as described here is sensitive to about 0.2 nmole a-diketone per 1.2 ml incubation mixture. Calculations The ,electron capture response is a reduction in a (background) current and the linear range is narrow (14). Because of detector and electronic noise the nonlinear region of the response curve has to be used in most practical applications of the electron capture detector. Under these conditions, multicomponent analysis with internal standardization may give problems. The use of internal standard by calculation of peak ‘area -or peak height-ratios is erroneous without previous linearization of the ,detector response. For the detector type used, a linear response function based on the measurement of peak heights has been derived. When all gas chromatographic parameters, including the background current, are carefully stabilized and kept constant during the analysis of a series of head-space samples, a nearly linear correlation between reciprocal peak height and reciprocal concentration of a-diketone in the 1.2 ml sample was found (Fig. 2). In kinetic studies, the reciprocal initial velocity as used in Lineweaver-Burk plots may be derived directly from such curves. By extrapolation to infinite concentration (Fig. 3) a quantity here termed the effective background current b, is determined. The response function h/be-h shows a perfect linear correlation with concentration (Fig. 3). For comparison the variation of peak height h with eoncentration is depicted. This response function is used in the head-space analysis calculations. After stabilization of the gas chromatographic system, ru-,diketone solutions, including the internal standard, with concentrations giving a 10-70s reduction of the background #current b at peak maximum are analyzed. The b, values are determined (Fig. 2), and the response functions are cal,culated for all peaks from the analysis of samples and standards. The response function values are divided by the corresponding internal standard value, linear standard curves are drawn, and the concentrations calculated. As previously mentioned, the head-space analy-
GLC OF a-ACYL-a-HYDROXY
ACIDS
373
0.5
Fro. 2. Reciprocal peak height vs. reciprocal concentration of diacetyl (DA) and acetylpropionyl (AP) in sample (head-space analysis). Determination of effective background current b. by extrapolation. FIG. 3. Response function h/(b, - h) and response h/b, (h as fraction of b.) vs. concentration of diacetyl (DA) and acetylpropionyl CAP). Data obtained from Figure 2.
sis may be performed without internal standard, and linear standard curves as shown in Figure 3 are then obtained. By the analysis of head-space samples giving ,a 2040% reduction of the background current at peak maxima, the standard deviation for diacetyl and acetylpropionyl was approximately 5-675 without and 3% with internal standardization. APPLICATION
OF THE
METHOD
The method has (been applied in studies of the conversion of lower cu-keto acids to a-acyl-a-hydroxy acids by cell-free extracts of Saccharomyces carlsbergensis. The standard incubation mixture (1.2 ml) contained 3 pmoles MgSO,, 20 ,pg thiamine pyrophosphate, O-100 pmoles sodium pyruvate, O-60 ,pmoles Icu-ketobutyric acid or cu-keto valeric acid (pH 7)) 20-100 ~1 enzyme extract (3 mg protein) ; and 0.6 ml 0.2 M potassium hydrogen phosphate adjusted to pH 7.3 with 1 M Tris buffer. The incubation time was 10-30 mm at 30”. RESULTS
AND
DISCUSSION
After enzyme incubation at pH 7.3, no cu-diketone peaks and no peaks with retention times near those of the cu-diketones were observed by
374
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HEJGAABD
electron capture gas chromatography. At pH 4.1 the oxidative decarboxylation of acetolactate to diacetyl and of acetohydroxybutyrate to acetylpropionyl was quantitative after ca. 100 ,min at 30” and in less than 30 min at 60”. The conversion as a function of time proceeds identically in incubation mixtures and standard solutions. Incubation at 60”, pH 4.1, immediately after addition of enzyme extract showed no formation ,of a-diketones, enzymic or thermal. The enzyme was immediately inactivated at 60” and activity never restored at lower temperatures. Lower aldehydes formed by enzymic decarboxylation ,of keto acids did not influence the cu-diketone concentrations in the head-space sample nor the gas chromatographic separation. This was demonstrated by GLC after addition of micromole amounts of C&C4 aldehydes to incubation mixtures. Peaks representing other compounds present, e.g., ethanol, ,did not interfere with the determinations. By analysis of 10 individually prepared, identical, 1.2 ,ml incubations the following standard deviations were found, using no internal standard: 17 nmoles + 5.5% acetolactic acid and 27 nmoles & 7% acetohydroxybutyric acid. The applicability of the method is illustrated ‘by a series of analytical results, The formation of ,a-acetolactic acid and a-aceto-cr-hydroxybutyric acid vs. a-ketobutyrate concentration with fixed pyruvate concentration is shown in Figure 4. At high a-ketobutyrate/pyruvate ratios, dipropionyl is found by GLC, corresponding to the presence of a-propionyl-crhydroxybutyric acid after enzyme incubation. From this series of experi-
cu-aceto-rr-hydroxybutyric FIG. 4. Formation of cr-acetolactic acid (01, and a-propionyl-cu-hydroxybutyric acid (m) from 100 pmoles pyruvate amounts of wketobutyrate (O-60 pmoles) in standard incubations.
acid (A), and varied
GLC
OF a-ACYL-wHYDROXY
375
ACIDS
ments typical gas chromatographic separations are shown in Figure 5A. The chromatogram from an incubation with 3.75 pmoles cw-ketobutyrate demonstrates about equal amounts of diacetyl and acetylpropionyl formed from acetolactic acid and acetohydroxybutyric acid. Superimposed is part of the chromatogram from incubation with 60 ,pmoles ketobutyrate, showing in this case small amounts of diacetyl and a small dipropionyl peak. Figure 5B illustrates the formation of a-acetolactic acid and cu-aceto-cu-hydroxyvaleric acid by incubation with pyruvate and ‘cu-ketovalerate. Superimposed is a chromatogram from an identical incubation, but with valine, inhibitor of acetohydroxy acid synthetase, added. Threonine deaminase (L-threonine hydro-lyase (deaminating) , EC 4.2) 1.16 can also be studied in combined assays with acetohydroxy acid synthetase. The formation of a-acetohydroxybutyric acid vs. threonine concentration (Fig. 6) depicts the formation of a-ketobutyrate by threonine deaminase present in the extract. The inhibition of this enzyme by isoleucine is also demonstrated.
l- lr B
? I ‘ \ ‘,--.,\\ LL.L
I
I 0
I 5
I
T;RE
I
I 0
I 5
I
10
(MINUTES)
FIG. 5. Gas chromatographic separation of cY-diketones formed from o-acyl-cuhydroxy acids: (A) Standard incubation with 100 pmoles pyruvate and 3.75 pmoles cu-ketobutyrate (-); incubation with 100 pmoles pyruvate and 60 pmoles a-ketobutyrate (--). (B) Standard incubation with 60 pmoles pyruvate and 12 pmoles a-ketovalerate (--); same with Bamoles valine added (---). (1) Air. (2) Aldehydes. (3) Diacetyl. (4) Acetylpronionyl. (5) Acetyl butyryl. (6). Dipropionyl.
376
J.
HEJGAARD
FIQ. 6. Formation of a-aceto-cr-hydroxybutyric acid (A.) and u-acetolactic acid (0) from 100 amoles pyruvate and varied amounts of threonine (k-20 pmoles) in standard incubations at pH 8.0. Same with 50 amoles isoleucine added (A and 0).
Determination of acylhydroxy acids in enzyme incubation mixtures by direct head-space gas chromatography may thus be a valuable method in studies of the regulatory enzymes in valine-isoleucine biosynthesis. SUMMARY
A method has been developed for the determination of cu-acetolactic acid, a-aceto-a-hydroxybutyric, and other low molecular la-acyl-cu-hydroxy acids in biological fluids. The acylhydroxy acids are converted to iu-diketones by incubation at 60”, pH 4.1, for a specific time. A head-space gas sample is taken from above the incubation mixture and analyzed by gas chromatography with electron capture detection. Internal standardization is used, and calculations are facilitated by introduction of a linear response function based on peak height measurements. The method was applied to the ,determination of 0.2-60 nmoles of different a-aceto-a-hydroxy acids formed in 1.2 ml incubations of a-keto acids with cell-free enzyme extracts of Saccharomyces carlsbergensis. ACKNOWLEDGMENTS The author
is grateful
to Professor Robert
Djurtoft
for advice and encourage-
ment during this investigation. The
work
was partially
1. LEAVITT, R., 2. WESTERFELD,
supported
by a grant from the Tuborg
REFERENCES H. E., J. Bid. Chem. 236, W. W., J. Biol. Ckm. 161, 495 (1945).
AND
UMBARGER,
2486
Foundation.
(19611,
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OF a-ACYL-a-HYDROXY
ACIDS
377
3. KUWANA, H., CAROLINE, D. F., -DINGI, R, W., AND WAGNER, R. P., Arch. B&hem. Biophys. 124 184 (1968). 4. LEAVITT, R., AND UMBAFNEX, H. E., J. Bacterial. 80, 18 (1960). 5. RONKAINEN, P., BRUMMER, S, AND SUOMALAINEN, H., Anal. B&hem. 34, 101 (1970) . 6. HEJQMRD, J., in preparation. 7. INOUE, T., MASUYAMA, K., YAMAMOTO, Y., @ADA, K., AND K~ROIWA, Y., Rept. Res. Lab. Kirin Brewery Co. Ltd., No. 11, 9 (1968). 8. HARRISON, G. A. F., BYRNE, W. J., AND COLLINS, E., J. Inst. Brewing 71, t&6 11965). 9. KRAMPITZ, L. O., Arch. Biochem. 17, 81 (1948). 10. RIQBY, W., J. Chem. sot. 1951, 793. 11. Bwrr, A. H. (Ed.), “‘Organic Synthesis,” Vol. II. p. 114. Wiley, New York, 1966. 12. VOGEL, E., AND SCHINZ, H. Helv. Chim. Acta 33, 116 (1950). 13. EDEZB~, L., J. B&hem. Microbial. Techrwl. Eng. 2, 453 (1960). 14. WENTWORTH, W. E., CHEN, E., AND LOVELOCK, J. E., J. Phys. Chem. 70, 445 0966).