ANALYTICAL
BIOCHEMISTRY
M),
671-683 (1978)
Assay and Specific Radioactivity Determination Metabolites of Propionic Acid by Gas Chromatography and Liquid Scintillation CLAUDE Luhoratoire
of
JEAN-BLAIN
de Biochimie Dynamique. Universit6 Clnude 43 Bd du II Novemhre 1918, 69621 Villeurbanne,
Bernurd Frunce
de Lyon.
Received May 24, 1978 Assay and specific radioactivity of products of the metabolism of propionic acid were determined by gas chromatography of the trimethylsilyl derivatives on a column coated with 3% SE 52 silicon, with a chromatograph incorporating a stream splitter and a gas fraction collector. With a temperature program rate of 2”Cimin between 90 and 200°C it is possible to separate lactic acid, hydroxypropionic acid, methylmalonic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, a-ketoglutaric acid, citric acid, and glucose on a single chromatogram. Glutaric acid is used as an internal standard. The determination of the radioactivity of each substrate which is recovered with the gas collector is made using labeled glutaric acid as a standard and not by using the volume of sample injected in the column. This determination has been used for metabolic studies with liver slices. It can be used also for studies in bacteria.
The in vitro study of propionate metabolism by various tissues with radioactive propionate necessitates the isolation and the measurement of the specific radioactivity of glucose and lactic acid in which the largest part of the radioactivity is recovered after incubation. It also requires the isolation of intermediates which are specific to propionic acid metabolism such as methylmalonic acid, or belong to the Krebs cycle. Gas chromatography is used to separate Krebs acids as methylesters (1,6,9,1 I) or trimethylsilyl derivatives (1,4,5,7,8) or quinoxalones (2). Trimethylsilyl derivatives are also usually employed for sugar separation (10). We have taken and modified the technique used by Horii et al. (3) and have adapted it for the separation of the different metabolites of propionic acid and for the measurement of their specific radioactivity. With this method, it is possible to separate very accurately in a single chromatogram glucose, lactic acid, and intermediates of propionic acid metabolism and to measure simultaneously their radioactivity after collection by liquid scintillation. 671
0003-2697/78/0902-0671$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
672
CLAUDE
JEAN-BLAIN
METHODS Materials
Gas chromatograph: Intersmat IGC 120 DFL, equipped with a stream splitter. Flame ionization detector. Recorder: Sefram Servotrace, 1 mV. Packard gas fraction collector for gas chromatography, Model 852. Glass cartridge for collection, Model 6001144. Scintillation spectrometer, Intertechnique SL 40. Reagents
for Column Preparation
Chromosorb W, acid-washed, dimethylchlorosilanized, 80 to 100 mesh, Intersmat. Silicon SE GE 52, Merck, methylphenylsilicon, phenyl 5%. Dimethylchlorosilan, Merck. Silyl 8 column conditioner, Pierce Chemical Company. Silylation
Reagents
Pyridine R. P. Prolabo. Trimethylchlorosilan, Merck. 1,1,1,3,3,3-hexamethyldisilazane, Merck. Control acids, glucose, hydroxylamine, Sigma. [1-14C]Glutaric acid, specific radioactivity 41 &i/mmol, 1’Energie Atomique, Saclay, France. Preparation
Commissariat
a
of the Column
Packing percentage of the stationary phase: 3% GE 52. Silicon SE 52 (0.75 g) is dissolved in 100 ml of toluene. Twenty-five grams of Chromosorb W is added to this solution. The mixture is gently stirred for 6 h. The toluene is evaporated under vacuum. Then the coated support is dried at 100°C. A glass column (length, 2.2 m; outside diameter, 6.35 mm) is first silanized by filling it with a 5% dimethylchlorosilanized solution in toluene overnight. The column is then emptied, rinsed out with methanol, and dried with a nitrogen stream. It is filled with the coated supporz and conditioned with a nitrogen flow for 3 days at 300°C. The last day, 100 ~1 of sily18 Pierce conditioner is injected to improve the silanization of the column walls and the glass wool. Experimental
Conditions
Carrier gas: nitrogen Air Liquide, U quality. Gas pressure at 200°C: 3 bars. Gas pressure at 90°C: 2.2 to 2.3 bars.
DETERMINATION
673
OF METABOLITES
D
d k
I: a
b
i
1
FIG. 1. Chromatography of trimethylsilyl derivatives of nonketoacids and glucose. Standard solution. Concentration ofacids 0.2 to 1 mgml. Concentration ofglucose is 2 mgiml. D is the solvent peak. a, lactic acid; b, P-hydroxypropionic acid; c, P-hydroxybutyric acid; d, methylmalonic acid; e, succinic acid; f, fumaric acid; g. glutaric acid; h, malic acid; i, citric acid: j, a-glucose; and k, P-glucose.
Flow rate: 8.5 ml/min. Injector block temperature: 250°C. Detector block temperature: 250°C. Initial temperature of the oven: 90°C. Final temperature of the oven: 200°C. Program rate: 2Wmin. Preparation of Standard Solutions
It is necessary to use free acids for the preparation of standard solutions, as sodium salts of these acids have very poor solubility in pyridine and are incompletely dissolved. However, for lactic acid, it is possible to use
DETERMINATION
OF METABOLITES TABLE
SEPARATION
OF THE
Lactic acid Hydroxypropionic acid Hydroxybutyric acid Methylmalonic acid and malonic acid Succinic acid Fumaric acid Glutaric acid Malic acid Citric acid a-Glucose /3-Glucose Oxime derivatives Oxaloacetic acid a-Ketoglutaric acid
1
DIFFERENT
Retention
675
time
ACIDS
(min)
AND
GLUCOSE
Retention time relative to glutaric acid (96)
3.6 5.8 6.4
16 26 29
8.8 13.2 15.2 22.4 24 42.8 46.8 51.8
39 59 68 100 107 191 209 231
28.8 31.6
129 141
cadmium lactate, which is very soluble in pyridine. The concentration of each acid in the standard solution is 0.2 to 1 mg/ml. The concentration of glucose is 2 mg/ml. Interrtcrl
Standard
Glutaric acid was chosen because it is not an intermediary for animal tissues and also gives no interference with the other peaks. Its position in the middle of the chromatogram gives a very precise plot of the peaks. A solution of glutaric acid, 10 mgiml, with radioactive glutaric acid gives a final radioactivity of 1 &i/ml. Glucose and nonketoacid solutions can be kept for 3 months at 0°C. For ketoacids there is a progressive destruction. The standard solutions for these acids must be prepared just before use. Preparation
of
SampleJ
The incubations (1 g of liver for 5 ml of medium) are stopped by cooling in ice and by introducing 0.2 ml of 2 N hydrochloric acid. The tissues are crushed with a Vir Tis homogenizer. Perchloric acid is added. After FIG. 2. Chromatography of trimethylsilyloxime glutaric acids. Standard solution. Concentration, acid; and C. cu-ketoglutaric acid.
derivatives of oxalacetic I mgiml. A. Glutaric acid;
and oc-ketoB, oxalacetic
676
CLAUDE
JEAN-BLAIN
centrifugation the supematant is neutralized with KOH. The precipitate of potassium perchlorate is eliminated by centrifugation. If the amino acids are wanted for analysis the supernatant is poured on a Dowex 50-X8 column; the amino acids are retained and can be eluted with 10 ml of NH,OH M. The supernatant, or the supematant after elution from the column, is neutralized with potassium hydroxide and lyophilized. The
D k a dtm
j
Ji C
i :
1.
/
FIG. 3. Incubation of [Z-Wlpropionate (50 pmol, 80 &i/mmol) with rat liver slices (1 g of fresh liver) in presence of malonate 10 mM. Total radioactivity (dpm) and specific radioactivity, (dpminmol) are given, respectively. (a) Lactic acid-24,800 dpm, 5 dpm/nmol; (c) hydroxybutyric acid-0 dpm, 0 dpm/nmol; (d + m) malonic + methylmalonic acids60,200 dpm (a. methylmalonic); (e) succinic acid-353,000 dpm, 102 dpm/nmol; (g) glutaric acid-internal standard; (i) citric acid-51,000 dpm, 72 dpm/nmol; (j + k) glucose1.044,OOO dpm. 105 dpminmol. Note: Peaks are not all recorded with the same amplification.
DETERMINATION
OF
METABOLITES
677
lyophilisate is diluted in 2 ml of pyridine. When oxime derivatives are needed, 5 mg of hydroxylamine are put into the pyridine 1 h before introducing silylation reagents. Two-tenths milliliter of the internal standard of glutaric acid, 0.4 ml of hexamethyldisilazane, and 0.4 ml of trimethylchlorosilane are added. The silylation continues for 1 h at room temperature. Ten microliters of the mixture is injected in the column. It is necessary to use an excess of reagents to obtain a complete silylation. For each case, tests must be performed to determine the right quantity of silylating reagents. One must be sure that a new quantity of reagent does not increase the size of the present peaks or give new ones. RESULTS Separcrtion
of the Different
AND DISCUSSION
Species
The chromatograms shown in Figs. 1 and 2 were made with a standard solution of glucose and of the different acids which can be met in the study of propionate metabolism in animals or in bacteria. Hydroxybutyric acid which is not an intermediate of propionate metabolism can also be separated. Glucose gives two peaks corresponding to (II and p anomers. Table 1 indicates the retention times of glucose and the different acids. There is sometimes a slight displacement of the position of the acid peaks in relation to the solvent peak, if the initial temperature is not exactly 90°C. Therefore the retention time relative to the glutaric acid is a more accurate measure. Figures 3 and 4 show chromatograms obtained from liver extracts after incubation of [2-‘4C]propionate with malonate or succinate. When malonate is used methyl malonate does not appear as a distinct peak but it is possible to count the corresponding radioactivity. Amy
To test the proportionality
between the ratio
height of the peak of the acid to be determined height of the glutaric acid peak
(a)
and the acid concentration, various chromatograms with different concentrations of a mixture of pure acids and glucose were made. Three experiments were done for each concentration. Figure 5 indicates that there is a linear relationship between the acid concentration and the ratio (a). The regression coefficient was in the range of 0.98 to 0.99. A similar result was obtained for glucose when the ratio height of a-glucose peak + height of P-glucose peak height of glutaric acid peak
678
CLAUDE
JEAN-BLAIN
h
9 a
i
FIG. 4. Incubation of [2-‘Tlpropionate (SO @mol. 80 &i/mmol) with rabbit liver slices (1 g of fresh liver) in the presence of succinate 10 mM. (a) Lactic acid-191,600 dpm, 7.6 dpm/nmol; (c) butyric acid-O, 0; (f) fumaric acid-35,000 dpm. 23 dpminmol; (g) glutaric acid-internal standard; (h) malic acid-218,000 dpm. II dpminmol; (i) citric acid- 14,000 dpm, 20 dpm/nmol; Cj + k) glucose-85,000 dpm, 24 dpminmol (in the normal metabolism no methylmalonate accumulates). Note: Peaks are not all recorded with the same amplification.
was plotted against the glucose concentration coefficient was 0.99. Percentage
(Fig. 6). The regression
Recovery of the Tested Species
There is always some glucose in the liver slices both before and after incubation. Therefore to test the recovery of glucose, 20 identical assays were performed with liver slices. At the end of the incubation 6 prnol of glucose was added to 10 of these assays. The determination of glucose was performed as usual. The difference between the two groups was calculated and compared with the known amount of glucose added.
DETERMINATION
679
OF METABOLITES
I.1 1
0.8. F
2 0.7. 5 = 0.6. a
= 0.5-
0.1
12
3
4
5
6
7
p moles/ml FIG. 5. Correlation between the height of the acid peak and acid concentration. HA. height of the acid peak; H glut, height of the internal standard peak (glutaric acid); L. lactic acid; MM, methylmalonic acid; S, succinic acid; F, fumaric acid; M. malic acid: and HP, hydroxypropionic acid. Each half-bar = 1 cr (SD of three measures).
The recovery of malonic and succinic acids was tested by adding them in known amounts to unincubated liver slices in the Krebs-Ringer medium. It was verified that neither acid was present in these slices. The following percentages of recovery were found: glucose, 89 2 5.3% (10 measurements); and acids, 84 2 2.8% (10 measurements). Determination of the Speci$c Radioactivit> Collection of the fractions corresponding to ecrchpeak. First, collection
was performed directly at the outlet of the stream splitter with the apparatus shown in Fig. 7. The Eppendorf tube is held firmly against the silicon rubber disk during the time of passage of a peak.
680
CLAUDE
5
JEAN-BLAIN
10
15
u moles /ml FIG. 6. Correlation between the ratio of glucose to glutaric acid peaks and the glucose concentration. H,, height of cy-glucose peak; HO, height of P-glucose peak; and H glut, height of the internal standard peak (glutaric acid). Each bar = 2 u.
Then the Packard gas collector was used. The heated inlet tube keeps the samples in the vapor state until they are collected. The sample is carried by this tube from the stream splitter outlet to the heated head equipped with a dispensing nozzle which protrudes through the head and is sealed with a rubber pad onto the collecting cartridge during collection. Collection is started as the pen of the recorder begins to rise for a peak and is stopped shortly after the pen has returned to the baseline. Rotation of the collector turntable is manual. Eppendorf vials or Packard cartridges are put in a liquid scintillation vial containing 20 ml of the scintillation mixture. Samples are then placed in the Intertechnique scintillation spectrometer for direct counting in disintegrations per minute. The plastic or glass tubes, filled up with glass wool, give no quenching, and the counting for all samples is done in a homogenous phase. The withdrawal of these vials does not alter the counting. The percentage of recovery for one column was measured as a function of the retention time or issue temperature of the derivative: recovery of [3H]glucose, 58.8 -+ 2.93; [14C]glutaric acid, 59.1 + 3.43; and [14C]malonic acid. 60.5 2 1.3.
DETERMINATION
OF METABOLITES
681
A variance analysis on these results shows no significant difference. The cooling of the collection cartridges with a mixture of dry ice and alcohol does not improve the recovery. Subsequently the collection was made at room temperature. The elimination from the calculation of the volume of the sample injected in the column is accomplished by using the glutaric acid as an internal standard to calculate the acid concentration. This volume was similarly eliminated in the determination of the specific radioactivity by means of the radioactive standard. Significant errors resulting from the variation in the injected volume due to leaks in the syringe or the septum are possible. Furthermore, it is more difficult to determine the quantity of derivative actually recovered. The percentage of recovery is in fact not exclusively a function of the geometric characteristics of the stream splitter. Therefore, the theoretical volume recovered by the collection cartridge is determined by an indirect method using the radioactive internal standard. If R is the recovered radioactivity of the peak of glutaric acid and p is the radioactivity of this acid introduced in the sample (dpm/pl), then the recovered theoretical volume is c’ = R/p. The entire radioactivity of a derivative x in the sample is then A(V/u) if V is the total volume of the sample, u is the recovered volume, and A is the recovered radioactivity of peak X. The specific radioactivity SR is then: SR (dpmi pmol) = A x V/u x M, with M = kmol of x in the sample. The smallest detectable radioactivity of the initial substrate recovered for each peak must be about 100 dpm. Then, the radioactivity of the derivative in the sample must be (with a 60% recuperation): R=lOOxfx~, V
if v = 10 ~1, V = 3000 ~1, and R = 5 x IO” dpm. If 1% of the radioactivity of the initial substrate is recovered in this derivative the minimum radioactivity of the initial substrate must be 5 x lo6 dpm. Therefore, it is necessary in each incubation to use a sufficient quantity of the radioactive substrate to recover a measurable radioactivity in each peak. The minimum size of the volume V is limited by the buffer salts which absorb pyridine and decrease the solubility of the other compounds. Figures 3 and 4 show examples of specific radioactivity determination in liver extracts. Calculation for the determination of the succinic acid specific rudiorrctivity in the exumple of Fig. 3. The following reagents were added to
the lyophilized extract of the liver slices: 2 ml of pyridine, silylation reagents, and 0.2 ml of [14C]glutaric acid, containing
0.8 ml of 2.2 x IO6
682
CLAUDE
JEAN-BLAIN
FIG. 7. Apparatus for direct collection: a. stream splitter outlet; b, silicon rubber disk; c. Eppendorf centrifugation tube, the bottom of which has been cut: and d. glass wool.
dpm/ml, that is 2.2 x IO6 x 0.2 = 0.44 x lo6 dpm. This gives a total volume for the prepared sample of 3 ml. A sample with the standard solution of acids and glucose was prepared in the same manner. (a) Calculation of the amount of succinic acid in the sample. Glutaric acid was used as an internal standard. The height of the glutaric acid peak for the standard solution was 160 mm while that for succinic acid was 87 mm. The amount of succinic acid in the sample prepared with the standard solution was 8625 nmol. The height of the glutaric acid peak for the sample (Fig. 3) was 165 mm and that for succinic acid was 36 mm. Taking into account the different amplification of the recorder, this gives the amount of succinic acid in the sample as: 8625 x g
x $
= 3461 nmol.
(b) Calculation of the total radioactivity of the succinic acid in the sample. Radioactivity of the collected fraction corresponding to glutaric acid was 1413 dpm. Radioactivity of glutaric acid in 1 ~1 of the sample solution was: 0.44 x lOY3000 = 146.67 dpm/pl. The theoretical volume recovered was: 1413/146.67 = 9.63 ~1. Radioactivity of the collected fraction corresponding to succinic was 1133 dpm. This gives the radioactivity for the total succinic acid in the sample as: 3000 1133 x = 353,000 dpm. 9.63 (c) The specific radioactivity the previous two results:
of the succinic acid is calculated
from
DETERMINATION
353,000
~
346 1
683
OF METABOLITES
= 102 dpminmol.
The same calculations are made for each acid. For glucose, the (Y and /3 peaks are collected and counted together. The validity of the method was checked for glucose by the following technique. The perchloric extract of liver is first poured on a Dowex 50-X8 column, then on an Amberlite IR 4B column. Glucose is determined in the eluate with the glucose oxydase method. Then the eluate is chromatographed on Whatman No. 1 in a butanol-formic acid-water (75: 15: 10) mixture. Spots are revealed with anilin phtalate. cut up, and put in counting vials with PPO,’ POPOP, toluene for the determination of radioactivity. An internal standard of [*4C]glucose is used for the determination of quenching. Results obtained with this method did not differ by more than 8% from the gas chromatographic method, This method then permits a precise measurement, in only one analysis, of very different common intermediates. Its disadvantage is the length of time taken in chromatography to collect the fractions and also to modify the sensitivity of the recorder because the recovered substances are either in high (lactic acid) or in low concentrations (Krebs intermediates). ACKNOWLEDGMENTS The author is very grateful Financial support was obtained
to Professor from DGRST
D. C. Gautheron and INRA.
who
promoted
this
work.
REFERENCES I. Alcock, N. W., Methods in Enzymology (Lowenstein. Ed.), Vol. 13. pp. 397-415. Academic Press, New York, 1968. 2. Hoffman, N. E.. and Kilinger, T. A. (1969) Am/. Chern. 41, 162-3. 3. Horii, Z.. Makita, M.. and Namura, Y. (1965) Cham. and Id. 34, 1494. 4. Horning, Cl.. Boucher. E. A.. Moss, A. M., and Horning, E. C. (1968) AM/. Left. 1, 713. 5. Langenbeck. U.. and Seegmiller, J. E. (1973) J. Chromrrrogr. 78, 420-423. 6. Mensen De Silva. E. (1971) And. Chrm. 43, 1031-1035. 7. Von Nicolai. H., and Zilliken. F. (1974) J. Chromcrtogr. 92, 43 I-434. 8. Pinelli, A., and Colombo, A. ( 1976) J. Chvomntogr. 118, 236-239. 9. Simmonds. G.. Pettitt, B. C.. and Zlatkis, A. (1967) And. Chrm. 39. 163- 167. IO. Sweeley, C. C.. Bentley. R.. Makita. M., and Wells, W. W. (1963) J. Amrr. Chrm. Sot. 85, 2497-2507. Il. Warner. C. V.. and Vahouny. G. V. (1975) Anal. Bioc~hrm. 67, 122- 129.
’ Abbreviations benzene.
used:
PPO,
2.5.diphenyloxazole;
POPOP.
1,4-bis(5-phenyloxazol-2-yl)-
BIOCHEMISTRY
M),
671-683 (1978)
Assay and Specific Radioactivity Determination Metabolites of Propionic Acid by Gas Chromatography and Liquid Scintillation CLAUDE Luhoratoire
of
JEAN-BLAIN
de Biochimie Dynamique. Universit6 Clnude 43 Bd du II Novemhre 1918, 69621 Villeurbanne,
Bernurd Frunce
de Lyon.
Received May 24, 1978 Assay and specific radioactivity of products of the metabolism of propionic acid were determined by gas chromatography of the trimethylsilyl derivatives on a column coated with 3% SE 52 silicon, with a chromatograph incorporating a stream splitter and a gas fraction collector. With a temperature program rate of 2”Cimin between 90 and 200°C it is possible to separate lactic acid, hydroxypropionic acid, methylmalonic acid, succinic acid, fumaric acid, malic acid, oxaloacetic acid, a-ketoglutaric acid, citric acid, and glucose on a single chromatogram. Glutaric acid is used as an internal standard. The determination of the radioactivity of each substrate which is recovered with the gas collector is made using labeled glutaric acid as a standard and not by using the volume of sample injected in the column. This determination has been used for metabolic studies with liver slices. It can be used also for studies in bacteria.
The in vitro study of propionate metabolism by various tissues with radioactive propionate necessitates the isolation and the measurement of the specific radioactivity of glucose and lactic acid in which the largest part of the radioactivity is recovered after incubation. It also requires the isolation of intermediates which are specific to propionic acid metabolism such as methylmalonic acid, or belong to the Krebs cycle. Gas chromatography is used to separate Krebs acids as methylesters (1,6,9,1 I) or trimethylsilyl derivatives (1,4,5,7,8) or quinoxalones (2). Trimethylsilyl derivatives are also usually employed for sugar separation (10). We have taken and modified the technique used by Horii et al. (3) and have adapted it for the separation of the different metabolites of propionic acid and for the measurement of their specific radioactivity. With this method, it is possible to separate very accurately in a single chromatogram glucose, lactic acid, and intermediates of propionic acid metabolism and to measure simultaneously their radioactivity after collection by liquid scintillation. 671
0003-2697/78/0902-0671$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
672
CLAUDE
JEAN-BLAIN
METHODS Materials
Gas chromatograph: Intersmat IGC 120 DFL, equipped with a stream splitter. Flame ionization detector. Recorder: Sefram Servotrace, 1 mV. Packard gas fraction collector for gas chromatography, Model 852. Glass cartridge for collection, Model 6001144. Scintillation spectrometer, Intertechnique SL 40. Reagents
for Column Preparation
Chromosorb W, acid-washed, dimethylchlorosilanized, 80 to 100 mesh, Intersmat. Silicon SE GE 52, Merck, methylphenylsilicon, phenyl 5%. Dimethylchlorosilan, Merck. Silyl 8 column conditioner, Pierce Chemical Company. Silylation
Reagents
Pyridine R. P. Prolabo. Trimethylchlorosilan, Merck. 1,1,1,3,3,3-hexamethyldisilazane, Merck. Control acids, glucose, hydroxylamine, Sigma. [1-14C]Glutaric acid, specific radioactivity 41 &i/mmol, 1’Energie Atomique, Saclay, France. Preparation
Commissariat
a
of the Column
Packing percentage of the stationary phase: 3% GE 52. Silicon SE 52 (0.75 g) is dissolved in 100 ml of toluene. Twenty-five grams of Chromosorb W is added to this solution. The mixture is gently stirred for 6 h. The toluene is evaporated under vacuum. Then the coated support is dried at 100°C. A glass column (length, 2.2 m; outside diameter, 6.35 mm) is first silanized by filling it with a 5% dimethylchlorosilanized solution in toluene overnight. The column is then emptied, rinsed out with methanol, and dried with a nitrogen stream. It is filled with the coated supporz and conditioned with a nitrogen flow for 3 days at 300°C. The last day, 100 ~1 of sily18 Pierce conditioner is injected to improve the silanization of the column walls and the glass wool. Experimental
Conditions
Carrier gas: nitrogen Air Liquide, U quality. Gas pressure at 200°C: 3 bars. Gas pressure at 90°C: 2.2 to 2.3 bars.
DETERMINATION
673
OF METABOLITES
D
d k
I: a
b
i
1
FIG. 1. Chromatography of trimethylsilyl derivatives of nonketoacids and glucose. Standard solution. Concentration ofacids 0.2 to 1 mgml. Concentration ofglucose is 2 mgiml. D is the solvent peak. a, lactic acid; b, P-hydroxypropionic acid; c, P-hydroxybutyric acid; d, methylmalonic acid; e, succinic acid; f, fumaric acid; g. glutaric acid; h, malic acid; i, citric acid: j, a-glucose; and k, P-glucose.
Flow rate: 8.5 ml/min. Injector block temperature: 250°C. Detector block temperature: 250°C. Initial temperature of the oven: 90°C. Final temperature of the oven: 200°C. Program rate: 2Wmin. Preparation of Standard Solutions
It is necessary to use free acids for the preparation of standard solutions, as sodium salts of these acids have very poor solubility in pyridine and are incompletely dissolved. However, for lactic acid, it is possible to use
DETERMINATION
OF METABOLITES TABLE
SEPARATION
OF THE
Lactic acid Hydroxypropionic acid Hydroxybutyric acid Methylmalonic acid and malonic acid Succinic acid Fumaric acid Glutaric acid Malic acid Citric acid a-Glucose /3-Glucose Oxime derivatives Oxaloacetic acid a-Ketoglutaric acid
1
DIFFERENT
Retention
675
time
ACIDS
(min)
AND
GLUCOSE
Retention time relative to glutaric acid (96)
3.6 5.8 6.4
16 26 29
8.8 13.2 15.2 22.4 24 42.8 46.8 51.8
39 59 68 100 107 191 209 231
28.8 31.6
129 141
cadmium lactate, which is very soluble in pyridine. The concentration of each acid in the standard solution is 0.2 to 1 mg/ml. The concentration of glucose is 2 mg/ml. Interrtcrl
Standard
Glutaric acid was chosen because it is not an intermediary for animal tissues and also gives no interference with the other peaks. Its position in the middle of the chromatogram gives a very precise plot of the peaks. A solution of glutaric acid, 10 mgiml, with radioactive glutaric acid gives a final radioactivity of 1 &i/ml. Glucose and nonketoacid solutions can be kept for 3 months at 0°C. For ketoacids there is a progressive destruction. The standard solutions for these acids must be prepared just before use. Preparation
of
SampleJ
The incubations (1 g of liver for 5 ml of medium) are stopped by cooling in ice and by introducing 0.2 ml of 2 N hydrochloric acid. The tissues are crushed with a Vir Tis homogenizer. Perchloric acid is added. After FIG. 2. Chromatography of trimethylsilyloxime glutaric acids. Standard solution. Concentration, acid; and C. cu-ketoglutaric acid.
derivatives of oxalacetic I mgiml. A. Glutaric acid;
and oc-ketoB, oxalacetic
676
CLAUDE
JEAN-BLAIN
centrifugation the supematant is neutralized with KOH. The precipitate of potassium perchlorate is eliminated by centrifugation. If the amino acids are wanted for analysis the supernatant is poured on a Dowex 50-X8 column; the amino acids are retained and can be eluted with 10 ml of NH,OH M. The supernatant, or the supematant after elution from the column, is neutralized with potassium hydroxide and lyophilized. The
D k a dtm
j
Ji C
i :
1.
/
FIG. 3. Incubation of [Z-Wlpropionate (50 pmol, 80 &i/mmol) with rat liver slices (1 g of fresh liver) in presence of malonate 10 mM. Total radioactivity (dpm) and specific radioactivity, (dpminmol) are given, respectively. (a) Lactic acid-24,800 dpm, 5 dpm/nmol; (c) hydroxybutyric acid-0 dpm, 0 dpm/nmol; (d + m) malonic + methylmalonic acids60,200 dpm (a. methylmalonic); (e) succinic acid-353,000 dpm, 102 dpm/nmol; (g) glutaric acid-internal standard; (i) citric acid-51,000 dpm, 72 dpm/nmol; (j + k) glucose1.044,OOO dpm. 105 dpminmol. Note: Peaks are not all recorded with the same amplification.
DETERMINATION
OF
METABOLITES
677
lyophilisate is diluted in 2 ml of pyridine. When oxime derivatives are needed, 5 mg of hydroxylamine are put into the pyridine 1 h before introducing silylation reagents. Two-tenths milliliter of the internal standard of glutaric acid, 0.4 ml of hexamethyldisilazane, and 0.4 ml of trimethylchlorosilane are added. The silylation continues for 1 h at room temperature. Ten microliters of the mixture is injected in the column. It is necessary to use an excess of reagents to obtain a complete silylation. For each case, tests must be performed to determine the right quantity of silylating reagents. One must be sure that a new quantity of reagent does not increase the size of the present peaks or give new ones. RESULTS Separcrtion
of the Different
AND DISCUSSION
Species
The chromatograms shown in Figs. 1 and 2 were made with a standard solution of glucose and of the different acids which can be met in the study of propionate metabolism in animals or in bacteria. Hydroxybutyric acid which is not an intermediate of propionate metabolism can also be separated. Glucose gives two peaks corresponding to (II and p anomers. Table 1 indicates the retention times of glucose and the different acids. There is sometimes a slight displacement of the position of the acid peaks in relation to the solvent peak, if the initial temperature is not exactly 90°C. Therefore the retention time relative to the glutaric acid is a more accurate measure. Figures 3 and 4 show chromatograms obtained from liver extracts after incubation of [2-‘4C]propionate with malonate or succinate. When malonate is used methyl malonate does not appear as a distinct peak but it is possible to count the corresponding radioactivity. Amy
To test the proportionality
between the ratio
height of the peak of the acid to be determined height of the glutaric acid peak
(a)
and the acid concentration, various chromatograms with different concentrations of a mixture of pure acids and glucose were made. Three experiments were done for each concentration. Figure 5 indicates that there is a linear relationship between the acid concentration and the ratio (a). The regression coefficient was in the range of 0.98 to 0.99. A similar result was obtained for glucose when the ratio height of a-glucose peak + height of P-glucose peak height of glutaric acid peak
678
CLAUDE
JEAN-BLAIN
h
9 a
i
FIG. 4. Incubation of [2-‘Tlpropionate (SO @mol. 80 &i/mmol) with rabbit liver slices (1 g of fresh liver) in the presence of succinate 10 mM. (a) Lactic acid-191,600 dpm, 7.6 dpm/nmol; (c) butyric acid-O, 0; (f) fumaric acid-35,000 dpm. 23 dpminmol; (g) glutaric acid-internal standard; (h) malic acid-218,000 dpm. II dpminmol; (i) citric acid- 14,000 dpm, 20 dpm/nmol; Cj + k) glucose-85,000 dpm, 24 dpminmol (in the normal metabolism no methylmalonate accumulates). Note: Peaks are not all recorded with the same amplification.
was plotted against the glucose concentration coefficient was 0.99. Percentage
(Fig. 6). The regression
Recovery of the Tested Species
There is always some glucose in the liver slices both before and after incubation. Therefore to test the recovery of glucose, 20 identical assays were performed with liver slices. At the end of the incubation 6 prnol of glucose was added to 10 of these assays. The determination of glucose was performed as usual. The difference between the two groups was calculated and compared with the known amount of glucose added.
DETERMINATION
679
OF METABOLITES
I.1 1
0.8. F
2 0.7. 5 = 0.6. a
= 0.5-
0.1
12
3
4
5
6
7
p moles/ml FIG. 5. Correlation between the height of the acid peak and acid concentration. HA. height of the acid peak; H glut, height of the internal standard peak (glutaric acid); L. lactic acid; MM, methylmalonic acid; S, succinic acid; F, fumaric acid; M. malic acid: and HP, hydroxypropionic acid. Each half-bar = 1 cr (SD of three measures).
The recovery of malonic and succinic acids was tested by adding them in known amounts to unincubated liver slices in the Krebs-Ringer medium. It was verified that neither acid was present in these slices. The following percentages of recovery were found: glucose, 89 2 5.3% (10 measurements); and acids, 84 2 2.8% (10 measurements). Determination of the Speci$c Radioactivit> Collection of the fractions corresponding to ecrchpeak. First, collection
was performed directly at the outlet of the stream splitter with the apparatus shown in Fig. 7. The Eppendorf tube is held firmly against the silicon rubber disk during the time of passage of a peak.
680
CLAUDE
5
JEAN-BLAIN
10
15
u moles /ml FIG. 6. Correlation between the ratio of glucose to glutaric acid peaks and the glucose concentration. H,, height of cy-glucose peak; HO, height of P-glucose peak; and H glut, height of the internal standard peak (glutaric acid). Each bar = 2 u.
Then the Packard gas collector was used. The heated inlet tube keeps the samples in the vapor state until they are collected. The sample is carried by this tube from the stream splitter outlet to the heated head equipped with a dispensing nozzle which protrudes through the head and is sealed with a rubber pad onto the collecting cartridge during collection. Collection is started as the pen of the recorder begins to rise for a peak and is stopped shortly after the pen has returned to the baseline. Rotation of the collector turntable is manual. Eppendorf vials or Packard cartridges are put in a liquid scintillation vial containing 20 ml of the scintillation mixture. Samples are then placed in the Intertechnique scintillation spectrometer for direct counting in disintegrations per minute. The plastic or glass tubes, filled up with glass wool, give no quenching, and the counting for all samples is done in a homogenous phase. The withdrawal of these vials does not alter the counting. The percentage of recovery for one column was measured as a function of the retention time or issue temperature of the derivative: recovery of [3H]glucose, 58.8 -+ 2.93; [14C]glutaric acid, 59.1 + 3.43; and [14C]malonic acid. 60.5 2 1.3.
DETERMINATION
OF METABOLITES
681
A variance analysis on these results shows no significant difference. The cooling of the collection cartridges with a mixture of dry ice and alcohol does not improve the recovery. Subsequently the collection was made at room temperature. The elimination from the calculation of the volume of the sample injected in the column is accomplished by using the glutaric acid as an internal standard to calculate the acid concentration. This volume was similarly eliminated in the determination of the specific radioactivity by means of the radioactive standard. Significant errors resulting from the variation in the injected volume due to leaks in the syringe or the septum are possible. Furthermore, it is more difficult to determine the quantity of derivative actually recovered. The percentage of recovery is in fact not exclusively a function of the geometric characteristics of the stream splitter. Therefore, the theoretical volume recovered by the collection cartridge is determined by an indirect method using the radioactive internal standard. If R is the recovered radioactivity of the peak of glutaric acid and p is the radioactivity of this acid introduced in the sample (dpm/pl), then the recovered theoretical volume is c’ = R/p. The entire radioactivity of a derivative x in the sample is then A(V/u) if V is the total volume of the sample, u is the recovered volume, and A is the recovered radioactivity of peak X. The specific radioactivity SR is then: SR (dpmi pmol) = A x V/u x M, with M = kmol of x in the sample. The smallest detectable radioactivity of the initial substrate recovered for each peak must be about 100 dpm. Then, the radioactivity of the derivative in the sample must be (with a 60% recuperation): R=lOOxfx~, V
if v = 10 ~1, V = 3000 ~1, and R = 5 x IO” dpm. If 1% of the radioactivity of the initial substrate is recovered in this derivative the minimum radioactivity of the initial substrate must be 5 x lo6 dpm. Therefore, it is necessary in each incubation to use a sufficient quantity of the radioactive substrate to recover a measurable radioactivity in each peak. The minimum size of the volume V is limited by the buffer salts which absorb pyridine and decrease the solubility of the other compounds. Figures 3 and 4 show examples of specific radioactivity determination in liver extracts. Calculation for the determination of the succinic acid specific rudiorrctivity in the exumple of Fig. 3. The following reagents were added to
the lyophilized extract of the liver slices: 2 ml of pyridine, silylation reagents, and 0.2 ml of [14C]glutaric acid, containing
0.8 ml of 2.2 x IO6
682
CLAUDE
JEAN-BLAIN
FIG. 7. Apparatus for direct collection: a. stream splitter outlet; b, silicon rubber disk; c. Eppendorf centrifugation tube, the bottom of which has been cut: and d. glass wool.
dpm/ml, that is 2.2 x IO6 x 0.2 = 0.44 x lo6 dpm. This gives a total volume for the prepared sample of 3 ml. A sample with the standard solution of acids and glucose was prepared in the same manner. (a) Calculation of the amount of succinic acid in the sample. Glutaric acid was used as an internal standard. The height of the glutaric acid peak for the standard solution was 160 mm while that for succinic acid was 87 mm. The amount of succinic acid in the sample prepared with the standard solution was 8625 nmol. The height of the glutaric acid peak for the sample (Fig. 3) was 165 mm and that for succinic acid was 36 mm. Taking into account the different amplification of the recorder, this gives the amount of succinic acid in the sample as: 8625 x g
x $
= 3461 nmol.
(b) Calculation of the total radioactivity of the succinic acid in the sample. Radioactivity of the collected fraction corresponding to glutaric acid was 1413 dpm. Radioactivity of glutaric acid in 1 ~1 of the sample solution was: 0.44 x lOY3000 = 146.67 dpm/pl. The theoretical volume recovered was: 1413/146.67 = 9.63 ~1. Radioactivity of the collected fraction corresponding to succinic was 1133 dpm. This gives the radioactivity for the total succinic acid in the sample as: 3000 1133 x = 353,000 dpm. 9.63 (c) The specific radioactivity the previous two results:
of the succinic acid is calculated
from
DETERMINATION
353,000
~
346 1
683
OF METABOLITES
= 102 dpminmol.
The same calculations are made for each acid. For glucose, the (Y and /3 peaks are collected and counted together. The validity of the method was checked for glucose by the following technique. The perchloric extract of liver is first poured on a Dowex 50-X8 column, then on an Amberlite IR 4B column. Glucose is determined in the eluate with the glucose oxydase method. Then the eluate is chromatographed on Whatman No. 1 in a butanol-formic acid-water (75: 15: 10) mixture. Spots are revealed with anilin phtalate. cut up, and put in counting vials with PPO,’ POPOP, toluene for the determination of radioactivity. An internal standard of [*4C]glucose is used for the determination of quenching. Results obtained with this method did not differ by more than 8% from the gas chromatographic method, This method then permits a precise measurement, in only one analysis, of very different common intermediates. Its disadvantage is the length of time taken in chromatography to collect the fractions and also to modify the sensitivity of the recorder because the recovered substances are either in high (lactic acid) or in low concentrations (Krebs intermediates). ACKNOWLEDGMENTS The author is very grateful Financial support was obtained
to Professor from DGRST
D. C. Gautheron and INRA.
who
promoted
this
work.
REFERENCES I. Alcock, N. W., Methods in Enzymology (Lowenstein. Ed.), Vol. 13. pp. 397-415. Academic Press, New York, 1968. 2. Hoffman, N. E.. and Kilinger, T. A. (1969) Am/. Chern. 41, 162-3. 3. Horii, Z.. Makita, M.. and Namura, Y. (1965) Cham. and Id. 34, 1494. 4. Horning, Cl.. Boucher. E. A.. Moss, A. M., and Horning, E. C. (1968) AM/. Left. 1, 713. 5. Langenbeck. U.. and Seegmiller, J. E. (1973) J. Chromrrrogr. 78, 420-423. 6. Mensen De Silva. E. (1971) And. Chrm. 43, 1031-1035. 7. Von Nicolai. H., and Zilliken. F. (1974) J. Chromcrtogr. 92, 43 I-434. 8. Pinelli, A., and Colombo, A. ( 1976) J. Chvomntogr. 118, 236-239. 9. Simmonds. G.. Pettitt, B. C.. and Zlatkis, A. (1967) And. Chrm. 39. 163- 167. IO. Sweeley, C. C.. Bentley. R.. Makita. M., and Wells, W. W. (1963) J. Amrr. Chrm. Sot. 85, 2497-2507. Il. Warner. C. V.. and Vahouny. G. V. (1975) Anal. Bioc~hrm. 67, 122- 129.
’ Abbreviations benzene.
used:
PPO,
2.5.diphenyloxazole;
POPOP.
1,4-bis(5-phenyloxazol-2-yl)-
