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A sensitive and specific method for the measurement of monophosphoinositide at a microregional level in brain
ANALYTICAL
BIOCHEMISTRY
A Sensitive
and
54,
32-39
(1973)
Specific
Method
of Monophosphoinositide
Washington
University
Received
Measurement
in Brain1 AND
W. R. SHERMAN
School of Medicine, St. Louis. Missouri July
the
at a Microregional
Level T. J. CICERO
far
24, 1972;
accepted
Department 63110 February
of Psychiatry,
9, 1973
A method is described wherein monophosphoinositide is quantitatively extracted from as little as 1 mg of brain, deacylated to glycerophosphoryl inositol, trimethylsilylated and gas chromatographed. Amounts of glycerophosphoryl inositol as small as 5 ng (15 pmoles) have b>en chromatographed. The flame ionization detector response is linear from this level to 10 pg. This method gives values for monophosphoinositide in excellent agreement with those obtaind with much larger tissue samples using the previously available techniques. Other advantages of the gas chromatographic method are its high degree of specificity, due to the increased resolution of the separation technique, and the considerable increase in the speed with which multiple determinations can be carried out.
Recently, a good deal of research interest has focused on the role of the phosphoinositides, particularly monophosphoinositide (MPI), in brain function (l-3). Most studies conducted up to this time, however, have utilized whole brain or gross regional samples for analysis and have thus ignored the marked heterogeneity of brain with respect to biochemistry, ultrastructure and function. It seems clear that, our understanding of the role of MPI in neuronal function will be subst’antially improved if analyses are performed only in histologically well-defined regions or subcellular fractions of brain. The principal problem associated with the microassay of MPI in brain, however, is that relatively large amounts of tissue are required using the available techniques for the separation (thin-layer or ion-exchange chromatography) and detection (phosphorus determination) of the phospholipid. In the present paper, we wish to report the development of a highly sensitive and specific gas chromato‘This research was supported in part by USPHS grants MH20717 and NS05159. T. J. C. is recipient of Research Scientist Development Award I-KZ-MH-70180 and W. R. S. of Research Career Program Award GM-21,863. The mass spectrometer was purchased by Health Sciences Advancement Award 5S04PR06115. Copyright All rights
32 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.
MEASUREMENT
OF
MONOPHOSPHOINOSITIDE
33
graphic (GC) method for the assay of NIP1 in as little as 1 mg (wet weight) of brain. We (4) and others (5), have previously reported that the dcacylated product of MPI, glycerophosphoryl inositol (GPI), can be gas chromatographed as its completely trimethylsily lated derivative. We have now developed a micro GC technique to extract and quantitatively determine MPI levels in brain. METHODS
Purification of Glycerophosphoryl InositoZ. The GPI standard obtained from the supplier (Supelco, Inc., Bellefonte, Pa.1 was found to contain a limited amount of impurities (2-3s) on receipt, principally ?nyoinositol and vlyo-inositol l-phosphate. Consequently, the GPI standard was purified prior to use by a slight modification of the method of Wells and Dittmer (6,lO). A 0.4 X 80-cm column was packed with AGl-X2 anion-exchange resin in the formate form. The sample of GPI (l-2 mg) was applied to the column, which had been equilibrated with 0.2 M CHOONH, (pH = 8.5)) and was then eluted with a formnte gradient. A three-chamber gradlent mixer was used, the first and second chambers contaimng 20 ml of 0.2 >l CHOONH, (pH = 8.5) and the third chamber 20 ml of 0.625 M CHOONH., (pH = 8.5). The column fom rate was 0.5 ml per min and l-ml fractions were collected. Vncler these ccnditions, GPI was completely eluted in fractions 29-35. A gas clromatograph (see below for details) was used to detect the cluted GPI. The fractions containing the GPI were then pooled and the phosphorus content wa: determined by the Bartlett method ( 7 1. GC analysis of the purified GPI standard revealed a single peak corresponding to the GPI with only trace amounts of nbyo-inositol and l>tyo-inositol 1-phospl~ate (total of less than 17%) contaminants. This standard was usetl in all subsequent quantitative work. However, WC have focal it useful in routine analysis to employ commercially available GPI as a Ltanclard after fir.st determining its concentration with our “lmrificcl” standard. This elim’nate5 the timeconsuming step of repeatedly purifying GPI on ion-exchange columns prior to its use. Extraction
of MPI from Bmin.
Whole brain. Rats were decapitate:l, the brains were quickly removed from the cranial cavity and frozen on dry ice immediately to suppress the gradual increase iu MPI which occurs on standing at room tempcrature. They were then weighed while frozen and homogenized in glass hand homogenizers in 20 vol of CHCl, : MeOH (1: 1, v/v). The homogenate was then centrifuged at 2,000 rpm for 20 min. The supernatant was saved and the precipitate was resuspended in CHCI,:MeOH (2: 1) and
34
CICERO
AND
SHERMAN
spun again. This was repeated two times. The combined supernatant fluids were then taken to dryness in a stream of Nz. The lipids were then deacylated according to previously described procedures (6,lO) by suspending the dried lipid in 1 ml of CHCl,: MeOH (1:4, v/v) per 1.5 g of brain tissue and adding 200 ~1 of 1.2 N NaOH (MeOH: H,O [ 1: 1, v/v] ) . The mixture was then incubated for 10 min at 37°C. The reaction was quickly terminated first by cooling in ice and then by neutralizing with 1 N acetic acid. To this was added 1.0 ml of CHCl,:MeOH (9: 1, v/v), 1.0 ml of H,O and 0.5 ml of isobutyl alcohol. The mixture was then centrifuged at 2,500 rpm for 2 min. The aqueous layer was removed and the (lower) organic phase was then washed twice with MeOH :H,O (1: 1, v/v), The combined aqueous methanolic layers were then transferred to Dowex 50W (H+ form, 200400 mesh) columns to convert the deacylated lipids to the free acid form which is required for trimethylsilylation (4,5). The eluate was then placed under a stream of NZ for l-2 hr to remove excess MeOH so that the remaining solvent could be lyophilized. Small brain samples. MPI was extracted from tissue samples ranging from 1 to 100 mg by a micro-modification of the above procedure. The tissue samples were obtained in one of two ways: (a) Rats were decapitated, the brains were removed and dissected on ice. Samples, ranging from 1 to 20 mg, of caudate nucleus were obtained, frozen on dry ice and then weighed; (b) whole brains were obtained as described above and then homogenized in 4 vol of H,O. Aliquots of the HZ0 homogenate were taken corresponding to tissue weights from 1 to 100 mg. The purpose of this procedure was to provide a homogeneous source of tissue (for the linearity studies described below) which cannot be assured when fresh tissue samples of markedly different weights are obtained from histologically heterogeneous brain regions. Both sets of samples were treated identically. They were first homogenized in 50 vol of CHCl, : MeOH (1: 1, v/v) in micro glass homogenizers (Micro-Metric Instrument Co., Cleveland, Ohio). The homogenates were transferred to 3-ml test tubes and the homogenizers were washed four times with the same solvent. The homogenate and combined washings were then taken to dryness in a stream of N,. The dried tissue extract was taken up in 0.5 ml CHCl, : MeOH (1: 4, v/v) and 0.1 ml of 1.2 N NaOH (MeOH:HzO [l: 1, v/v]) was added. The mixture was incubated, as described above, for 10 min at 37°C. The reaction was terminated by cooling in ice and neutralizing with 1 N acetic acid. To this was added 0.5 ml CHCI,:MeOH (9: 1, v/v), 0.5 ml H20 and 0.25 ml isobutyl alcohol. The mixture was then centrifuged at 2500 rpm for 20 min, the aqueous layer collected and the bottom layer washed twice with 0.5 ml of MeOH: H,O (1: 1, v/v). The combined aqueous methanolic layers were then passed over ion exchange columns made from
MEASUREMENT
OF
MONOPHOSPHOINOSITIDE
35
5.75-in. disposable Pasteur pipettes which had been packed with 2 ml of Dowex 50W (H+). The columns were washed in succession with two 0.5ml aliquots of water. The eluate was placed under a stream of N, for one hour and the remainder then lyophilized. Gas Chromatography of GPI. The lyophilized, deacylated lipids and the GPI standard, purified as described above, were then converted to their trimethylsilyl (TMS) derivatives by reacting them in BSTFA (N,O-bis[trimethylsilyl] trifluoracetamide, Regis Chemical Co., Chicago, Ill.) : anhydrous pyridine: trimethylchlorosilane (1: 1: 0.1). For the whole brain samples 1 ml of silylating reagent was used ; whereas, for the smaller, regional samples 0.1-0.2 ml (depending on the tissue size) was employed. The samples were then mixed thoroughly on a mechanical shaker for at least 2 hr prior to GC analysis. Two gas chromatographs were employed: a Varian model 2100 and an F&M model 409. Although TMS GPI chromatographed when a variety of column sizes and packings were employed, excellent results were obtained using l-2-ft U-shaped glass columns (3.5 mm, i.d.) packed with 1% SE-30 on Supelcoport (Supelco, Inc.). Helium was used as carrier gas. The GC conditions were as follows: oven temperature, 190°C; injector and detector blocks, 220°C; carrier gas flow at 50 cc per min. As with many sugar phosphates (B), we generally found it necessary to “prime” our columns prior to use. This was accomplished by chromatographing TMS glucose-6-phosphate a sufficient number of times to give constant maximal peak heights prior to running the TMS GPI samples. RESULTS
Gas Chromatography of TMS GPI. Figure 1 presents two gas chromatograms showing the separation of a TMS GPI standard (left tracing), purified as described in the Methods section, and TMS GPI extracted from 50 mg of whole brain (right tracing; see text for details). As shown in this figure, TMS GPI chromatographed as a single, symmetrical peak in both the standard solution and that prepared from brain and was free of overlapping peaks. Mass spectral analysis revealed that the spectrum of TMS GPI derived from brain was identical to that obtained with “standard” TMS GPI. Multiple scans through the GC peak revealed no mass spectrometric peaks other than those of TMS GPI and SE-30 column bleed. Further, we have previously shown (4,ll) that TMS GPI is gas chromatographically resolved from deacylated TMS di- and triphosphoinositide and from deacylated TMS cardiolipin. We have also found (12) that TMS GPI elutes from SE-30 columns later than all of the major deacylated glycerophospholipid TMS derivatives found in brain tissue.
36
CICERO
AND
FIG. 1. Two gas chromatograms showing (left tracing) and TMS GPI derived from are given in Methods.
SHERMAN
the separation of a TMS brain (right tracing). The
GPI standard GC conditions
To determine that the GC detector response was linear over a range of TIMS GPI levels, varying amounts of TIMS GPI were gas chromatographed. The GC conditions were identical to those described in the Methods section. The results are shown in Fig. 2. The detector response was linear over a range of GPI values from 0.005 to 10 pg. Extrapolation of the linear portion of this plot intersected both axes at zero. Below 0.005 pg, the detector response decreased sharply and was nonlinear. Consequently, a calibration curve would be necessary when GPI levels fall below this point.
FIG. 2. Relationship of detector response to the amount of GPI graphed as its TMS derivative. The ordinate is the chromatographic calculated for an unattenuated electrometer.
gas chromatopeak height
MEASUREMENT
OF
37
MONOPHOSPHOINOSITIDE
Extraction and Estimation of MPI in Brain. Preliminary work indicated that MPI was quantitatively extracted when either fresh tissue or a water homogenate of whole brain was used. Consequently, H,O homogenates of brain, which provide a homogeneous source of tissue, were employed in most of the experiments requiring small tissue samples. A departure from the established procedure for the extraction and deacylation of MPI from brain was employed in the present studies. Specifically, we found it unnecessary to centrifuge the CHC1,:MeOH (1: 1) homogenate of fresh brain tissue when tissue samples less than 1uO mg were employed. Rather, we simply deacylated the entire t,issue homogenate. A comparison of the two methods revealed no differences in the yield of MPI (2.45 pmoles/g wet weight when the homogenate was not centrifuged as compared to 2.36 pmole/g wet weight with centrifugation) although the results tended to be more variable when centrifugation was employed. Consequently, the extraction and deacylation of MPI in 100 mg samples of brain or less were carried out as described in the Methods section, i.e., without centrifugation of the brain homogenate. To determine that the extraction of MPI from brain was linear over a range of tissue weights, aliquots of a water homogenate of brain were taken which corresponded to from 1 to 100 mg of brain tissue. The MPI was extracted, converted to GPI and trimethylsilylated as described in the Methods. Figure 3 presents the results of these studies. Two things are apparent in this figure: first, the extraction of MPI was, in fact, linear over the range of tissue samples examined; secondly, the sensitivity of the GC technique was more than ample to measure MPI in 1 mg of brain tissue. Table 1 presents MPI levels determined in whole brain and in caudate nucleus by the micro-GC method described in this paper. For the whole
Nonomoles
FIQ. 3. Plot showing the yield of MPI from 1 to 100 mg wet weight and measured See Methods for experimental details.
MP!
extracted from tissue by gas chromatography
samples weighing as TMS GBI,
38
CICERO
AND
SHERMAN
TABLE 1 MPI Levels (pmoles/g wet weight) in Whole Brain and Caudate Nucleus of Bat Braina Whole brain W.B. W.B. W.B. W.B.
1 2 3 4
4w 10 50
mg mg 100 mg
Mean
(+SEM)
Caudate nucleus 2.40 2.47
3.42 3.52
2.21
3.61
2.40 2.43 2.34 2.35
3.18 3.29
2.49 2.39 (+ .03)
3.40
(k.08)
a The whole brain values were determined either in whole brain samples (fresh tissue, designated W.B.) or in aliquots of a II20 homogenate of brain corresponding to various tissue weights (designated by the tissue weight). See Methods section for experimental details.
brain determinations, two types of samples were employed: aliquots of whole brain H,O homogenates corresponding to 4-100 mg of tissue or alternatively fresh tissue (whole brain). The samples of caudate nucleus were obtained as described in Methods and ranged in weight from 6.7 to 24.8 mg. As shown in Table 1, there was little variability among the individual measurements. The values of MPI determined in whole brain are in excellent agreement with those reported by other workers, e.g., Wagner et al. (2.6 pmoles/g wet) (9) and Dittmer and Douglas (2.2 pmoles/g wet) (10). There have been no previous repor& on the levels of MPI in rat caudate nucleus. DISCUSSION
The method presented in this paper provides a highly sensitive and specific assay for the measurement of MPI in brain. The present GC method has numerous advantages over existing techniques, perhaps the most commonly used being ion-exchange chromatography of the deacylated phospholipid (6,lO). The most obvious advantage of the present technique is the subst,antial amount of time saved in the extraction, deacylation and estimation of MPI. In our hands, ion-exchange chromatography of deacylated phospholipids requires three full days from the initial extraction to the detection and measurement of MPI. The amount of time and effort involved is greatly increased by the need to use a separate column for each sample to he analyzed. With the present technique, we found that up to 20 samples can be handled efficiently and with
MEASUREMENT
OF
39
MONOPHOSPHOINOSITIDE
an expenditure of time less than half that required to analyze one sample with the former procedure. An additional, and perhaps more important, advantage of the present technique is the high degree of specificity inherent in GC analysis which exceeds, in resolution, bot,h ion-exchange and thin-layer chromatography by a large margin. A further significant advantage of the present technique is ins high degree of sensitivity. The lower limit of sensitivity with the GC method described in this paper is less than 15 pmoles of MPI. This contrasts sharply with a lower limit, of 10 nmoles when the Bartlett procedure for phosphorus determination is employed. Consequently, the present GC method is over 500 times more sensitive than the best quantitative technique previously available. Since the amount of MPI contained in 1 mg of brain tissue was found to be 2.56 nmoles, which is 100 times greater than the lower limit of sensitivity of the method, it is probable that MPI levels could be measured in as little as 10 pg of brain tissue. With the use of mass spectrometry, particularly if the mass spectrometric multiple ion detection method is employed, we hope that MPI in single cells can be measured. In conclusion, the GC method described in this paper offers a rapid, highly sensitive and specific quantitative method for the estimation of MPI levels in micro-regional or subcellular samples of brain tissue. REFERENCES 1. HOKIN, L. E. (1969) N. Y. Acad. Sci. 165, 695. 2. DURELL, J. AND GARLAND, J. T. (1969) Ann. N. Y. Acad. Sci. 3. LEVEY, G. S. (1971) J. Biol. Chem. 246, 7405. 4. CICBXO, T. J. AND SHERMAN, W. R. (1971) Biochem. Biophys.
165, 743. Res.
Comm.
42,
428. 5. DUNCAN, J., LENNAFLZ, W. AND FENSELAU, C. (1971) Biochemistry 10, 6. WELLS, M. A. AND DITTMER, J. C. (1966) Biochemistry 5, 3405. 7. BARTLETT, G. (1959) J. Biol. Chem. 234,466. 8. SHERMAN, W. R., GOODWIN, S. L. AND ZINBO, M. (1971) J. Chromatogr.
927. Sci. 9,
363. 9. WAGNER,
H., HOLZL,
J., LISSAU,
A. AND HORHAMMER,
I.
(1963) Bioehem.
Z. 339,
34. 10. DITTMER, 11. CICERO,
J. C. AND DOUGLAS, T. J. AND SHERMAN,
451. 12. SEHGAL,
R. K. AND SHERMAN,
M. G. (1969) Ann. N. Y. Acad. W. R. (1971) Biochem. Biophys. W. R., unpublished
observations.
Sci. 165, 515. Res. Comm. 43,
BIOCHEMISTRY
A Sensitive
and
54,
32-39
(1973)
Specific
Method
of Monophosphoinositide
Washington
University
Received
Measurement
in Brain1 AND
W. R. SHERMAN
School of Medicine, St. Louis. Missouri July
the
at a Microregional
Level T. J. CICERO
far
24, 1972;
accepted
Department 63110 February
of Psychiatry,
9, 1973
A method is described wherein monophosphoinositide is quantitatively extracted from as little as 1 mg of brain, deacylated to glycerophosphoryl inositol, trimethylsilylated and gas chromatographed. Amounts of glycerophosphoryl inositol as small as 5 ng (15 pmoles) have b>en chromatographed. The flame ionization detector response is linear from this level to 10 pg. This method gives values for monophosphoinositide in excellent agreement with those obtaind with much larger tissue samples using the previously available techniques. Other advantages of the gas chromatographic method are its high degree of specificity, due to the increased resolution of the separation technique, and the considerable increase in the speed with which multiple determinations can be carried out.
Recently, a good deal of research interest has focused on the role of the phosphoinositides, particularly monophosphoinositide (MPI), in brain function (l-3). Most studies conducted up to this time, however, have utilized whole brain or gross regional samples for analysis and have thus ignored the marked heterogeneity of brain with respect to biochemistry, ultrastructure and function. It seems clear that, our understanding of the role of MPI in neuronal function will be subst’antially improved if analyses are performed only in histologically well-defined regions or subcellular fractions of brain. The principal problem associated with the microassay of MPI in brain, however, is that relatively large amounts of tissue are required using the available techniques for the separation (thin-layer or ion-exchange chromatography) and detection (phosphorus determination) of the phospholipid. In the present paper, we wish to report the development of a highly sensitive and specific gas chromato‘This research was supported in part by USPHS grants MH20717 and NS05159. T. J. C. is recipient of Research Scientist Development Award I-KZ-MH-70180 and W. R. S. of Research Career Program Award GM-21,863. The mass spectrometer was purchased by Health Sciences Advancement Award 5S04PR06115. Copyright All rights
32 @ 1973 by Academic Press, Inc. of reproduction in any form reserved.
MEASUREMENT
OF
MONOPHOSPHOINOSITIDE
33
graphic (GC) method for the assay of NIP1 in as little as 1 mg (wet weight) of brain. We (4) and others (5), have previously reported that the dcacylated product of MPI, glycerophosphoryl inositol (GPI), can be gas chromatographed as its completely trimethylsily lated derivative. We have now developed a micro GC technique to extract and quantitatively determine MPI levels in brain. METHODS
Purification of Glycerophosphoryl InositoZ. The GPI standard obtained from the supplier (Supelco, Inc., Bellefonte, Pa.1 was found to contain a limited amount of impurities (2-3s) on receipt, principally ?nyoinositol and vlyo-inositol l-phosphate. Consequently, the GPI standard was purified prior to use by a slight modification of the method of Wells and Dittmer (6,lO). A 0.4 X 80-cm column was packed with AGl-X2 anion-exchange resin in the formate form. The sample of GPI (l-2 mg) was applied to the column, which had been equilibrated with 0.2 M CHOONH, (pH = 8.5)) and was then eluted with a formnte gradient. A three-chamber gradlent mixer was used, the first and second chambers contaimng 20 ml of 0.2 >l CHOONH, (pH = 8.5) and the third chamber 20 ml of 0.625 M CHOONH., (pH = 8.5). The column fom rate was 0.5 ml per min and l-ml fractions were collected. Vncler these ccnditions, GPI was completely eluted in fractions 29-35. A gas clromatograph (see below for details) was used to detect the cluted GPI. The fractions containing the GPI were then pooled and the phosphorus content wa: determined by the Bartlett method ( 7 1. GC analysis of the purified GPI standard revealed a single peak corresponding to the GPI with only trace amounts of nbyo-inositol and l>tyo-inositol 1-phospl~ate (total of less than 17%) contaminants. This standard was usetl in all subsequent quantitative work. However, WC have focal it useful in routine analysis to employ commercially available GPI as a Ltanclard after fir.st determining its concentration with our “lmrificcl” standard. This elim’nate5 the timeconsuming step of repeatedly purifying GPI on ion-exchange columns prior to its use. Extraction
of MPI from Bmin.
Whole brain. Rats were decapitate:l, the brains were quickly removed from the cranial cavity and frozen on dry ice immediately to suppress the gradual increase iu MPI which occurs on standing at room tempcrature. They were then weighed while frozen and homogenized in glass hand homogenizers in 20 vol of CHCl, : MeOH (1: 1, v/v). The homogenate was then centrifuged at 2,000 rpm for 20 min. The supernatant was saved and the precipitate was resuspended in CHCI,:MeOH (2: 1) and
34
CICERO
AND
SHERMAN
spun again. This was repeated two times. The combined supernatant fluids were then taken to dryness in a stream of Nz. The lipids were then deacylated according to previously described procedures (6,lO) by suspending the dried lipid in 1 ml of CHCl,: MeOH (1:4, v/v) per 1.5 g of brain tissue and adding 200 ~1 of 1.2 N NaOH (MeOH: H,O [ 1: 1, v/v] ) . The mixture was then incubated for 10 min at 37°C. The reaction was quickly terminated first by cooling in ice and then by neutralizing with 1 N acetic acid. To this was added 1.0 ml of CHCl,:MeOH (9: 1, v/v), 1.0 ml of H,O and 0.5 ml of isobutyl alcohol. The mixture was then centrifuged at 2,500 rpm for 2 min. The aqueous layer was removed and the (lower) organic phase was then washed twice with MeOH :H,O (1: 1, v/v), The combined aqueous methanolic layers were then transferred to Dowex 50W (H+ form, 200400 mesh) columns to convert the deacylated lipids to the free acid form which is required for trimethylsilylation (4,5). The eluate was then placed under a stream of NZ for l-2 hr to remove excess MeOH so that the remaining solvent could be lyophilized. Small brain samples. MPI was extracted from tissue samples ranging from 1 to 100 mg by a micro-modification of the above procedure. The tissue samples were obtained in one of two ways: (a) Rats were decapitated, the brains were removed and dissected on ice. Samples, ranging from 1 to 20 mg, of caudate nucleus were obtained, frozen on dry ice and then weighed; (b) whole brains were obtained as described above and then homogenized in 4 vol of H,O. Aliquots of the HZ0 homogenate were taken corresponding to tissue weights from 1 to 100 mg. The purpose of this procedure was to provide a homogeneous source of tissue (for the linearity studies described below) which cannot be assured when fresh tissue samples of markedly different weights are obtained from histologically heterogeneous brain regions. Both sets of samples were treated identically. They were first homogenized in 50 vol of CHCl, : MeOH (1: 1, v/v) in micro glass homogenizers (Micro-Metric Instrument Co., Cleveland, Ohio). The homogenates were transferred to 3-ml test tubes and the homogenizers were washed four times with the same solvent. The homogenate and combined washings were then taken to dryness in a stream of N,. The dried tissue extract was taken up in 0.5 ml CHCl, : MeOH (1: 4, v/v) and 0.1 ml of 1.2 N NaOH (MeOH:HzO [l: 1, v/v]) was added. The mixture was incubated, as described above, for 10 min at 37°C. The reaction was terminated by cooling in ice and neutralizing with 1 N acetic acid. To this was added 0.5 ml CHCI,:MeOH (9: 1, v/v), 0.5 ml H20 and 0.25 ml isobutyl alcohol. The mixture was then centrifuged at 2500 rpm for 20 min, the aqueous layer collected and the bottom layer washed twice with 0.5 ml of MeOH: H,O (1: 1, v/v). The combined aqueous methanolic layers were then passed over ion exchange columns made from
MEASUREMENT
OF
MONOPHOSPHOINOSITIDE
35
5.75-in. disposable Pasteur pipettes which had been packed with 2 ml of Dowex 50W (H+). The columns were washed in succession with two 0.5ml aliquots of water. The eluate was placed under a stream of N, for one hour and the remainder then lyophilized. Gas Chromatography of GPI. The lyophilized, deacylated lipids and the GPI standard, purified as described above, were then converted to their trimethylsilyl (TMS) derivatives by reacting them in BSTFA (N,O-bis[trimethylsilyl] trifluoracetamide, Regis Chemical Co., Chicago, Ill.) : anhydrous pyridine: trimethylchlorosilane (1: 1: 0.1). For the whole brain samples 1 ml of silylating reagent was used ; whereas, for the smaller, regional samples 0.1-0.2 ml (depending on the tissue size) was employed. The samples were then mixed thoroughly on a mechanical shaker for at least 2 hr prior to GC analysis. Two gas chromatographs were employed: a Varian model 2100 and an F&M model 409. Although TMS GPI chromatographed when a variety of column sizes and packings were employed, excellent results were obtained using l-2-ft U-shaped glass columns (3.5 mm, i.d.) packed with 1% SE-30 on Supelcoport (Supelco, Inc.). Helium was used as carrier gas. The GC conditions were as follows: oven temperature, 190°C; injector and detector blocks, 220°C; carrier gas flow at 50 cc per min. As with many sugar phosphates (B), we generally found it necessary to “prime” our columns prior to use. This was accomplished by chromatographing TMS glucose-6-phosphate a sufficient number of times to give constant maximal peak heights prior to running the TMS GPI samples. RESULTS
Gas Chromatography of TMS GPI. Figure 1 presents two gas chromatograms showing the separation of a TMS GPI standard (left tracing), purified as described in the Methods section, and TMS GPI extracted from 50 mg of whole brain (right tracing; see text for details). As shown in this figure, TMS GPI chromatographed as a single, symmetrical peak in both the standard solution and that prepared from brain and was free of overlapping peaks. Mass spectral analysis revealed that the spectrum of TMS GPI derived from brain was identical to that obtained with “standard” TMS GPI. Multiple scans through the GC peak revealed no mass spectrometric peaks other than those of TMS GPI and SE-30 column bleed. Further, we have previously shown (4,ll) that TMS GPI is gas chromatographically resolved from deacylated TMS di- and triphosphoinositide and from deacylated TMS cardiolipin. We have also found (12) that TMS GPI elutes from SE-30 columns later than all of the major deacylated glycerophospholipid TMS derivatives found in brain tissue.
36
CICERO
AND
FIG. 1. Two gas chromatograms showing (left tracing) and TMS GPI derived from are given in Methods.
SHERMAN
the separation of a TMS brain (right tracing). The
GPI standard GC conditions
To determine that the GC detector response was linear over a range of TIMS GPI levels, varying amounts of TIMS GPI were gas chromatographed. The GC conditions were identical to those described in the Methods section. The results are shown in Fig. 2. The detector response was linear over a range of GPI values from 0.005 to 10 pg. Extrapolation of the linear portion of this plot intersected both axes at zero. Below 0.005 pg, the detector response decreased sharply and was nonlinear. Consequently, a calibration curve would be necessary when GPI levels fall below this point.
FIG. 2. Relationship of detector response to the amount of GPI graphed as its TMS derivative. The ordinate is the chromatographic calculated for an unattenuated electrometer.
gas chromatopeak height
MEASUREMENT
OF
37
MONOPHOSPHOINOSITIDE
Extraction and Estimation of MPI in Brain. Preliminary work indicated that MPI was quantitatively extracted when either fresh tissue or a water homogenate of whole brain was used. Consequently, H,O homogenates of brain, which provide a homogeneous source of tissue, were employed in most of the experiments requiring small tissue samples. A departure from the established procedure for the extraction and deacylation of MPI from brain was employed in the present studies. Specifically, we found it unnecessary to centrifuge the CHC1,:MeOH (1: 1) homogenate of fresh brain tissue when tissue samples less than 1uO mg were employed. Rather, we simply deacylated the entire t,issue homogenate. A comparison of the two methods revealed no differences in the yield of MPI (2.45 pmoles/g wet weight when the homogenate was not centrifuged as compared to 2.36 pmole/g wet weight with centrifugation) although the results tended to be more variable when centrifugation was employed. Consequently, the extraction and deacylation of MPI in 100 mg samples of brain or less were carried out as described in the Methods section, i.e., without centrifugation of the brain homogenate. To determine that the extraction of MPI from brain was linear over a range of tissue weights, aliquots of a water homogenate of brain were taken which corresponded to from 1 to 100 mg of brain tissue. The MPI was extracted, converted to GPI and trimethylsilylated as described in the Methods. Figure 3 presents the results of these studies. Two things are apparent in this figure: first, the extraction of MPI was, in fact, linear over the range of tissue samples examined; secondly, the sensitivity of the GC technique was more than ample to measure MPI in 1 mg of brain tissue. Table 1 presents MPI levels determined in whole brain and in caudate nucleus by the micro-GC method described in this paper. For the whole
Nonomoles
FIQ. 3. Plot showing the yield of MPI from 1 to 100 mg wet weight and measured See Methods for experimental details.
MP!
extracted from tissue by gas chromatography
samples weighing as TMS GBI,
38
CICERO
AND
SHERMAN
TABLE 1 MPI Levels (pmoles/g wet weight) in Whole Brain and Caudate Nucleus of Bat Braina Whole brain W.B. W.B. W.B. W.B.
1 2 3 4
4w 10 50
mg mg 100 mg
Mean
(+SEM)
Caudate nucleus 2.40 2.47
3.42 3.52
2.21
3.61
2.40 2.43 2.34 2.35
3.18 3.29
2.49 2.39 (+ .03)
3.40
(k.08)
a The whole brain values were determined either in whole brain samples (fresh tissue, designated W.B.) or in aliquots of a II20 homogenate of brain corresponding to various tissue weights (designated by the tissue weight). See Methods section for experimental details.
brain determinations, two types of samples were employed: aliquots of whole brain H,O homogenates corresponding to 4-100 mg of tissue or alternatively fresh tissue (whole brain). The samples of caudate nucleus were obtained as described in Methods and ranged in weight from 6.7 to 24.8 mg. As shown in Table 1, there was little variability among the individual measurements. The values of MPI determined in whole brain are in excellent agreement with those reported by other workers, e.g., Wagner et al. (2.6 pmoles/g wet) (9) and Dittmer and Douglas (2.2 pmoles/g wet) (10). There have been no previous repor& on the levels of MPI in rat caudate nucleus. DISCUSSION
The method presented in this paper provides a highly sensitive and specific assay for the measurement of MPI in brain. The present GC method has numerous advantages over existing techniques, perhaps the most commonly used being ion-exchange chromatography of the deacylated phospholipid (6,lO). The most obvious advantage of the present technique is the subst,antial amount of time saved in the extraction, deacylation and estimation of MPI. In our hands, ion-exchange chromatography of deacylated phospholipids requires three full days from the initial extraction to the detection and measurement of MPI. The amount of time and effort involved is greatly increased by the need to use a separate column for each sample to he analyzed. With the present technique, we found that up to 20 samples can be handled efficiently and with
MEASUREMENT
OF
39
MONOPHOSPHOINOSITIDE
an expenditure of time less than half that required to analyze one sample with the former procedure. An additional, and perhaps more important, advantage of the present technique is the high degree of specificity inherent in GC analysis which exceeds, in resolution, bot,h ion-exchange and thin-layer chromatography by a large margin. A further significant advantage of the present technique is ins high degree of sensitivity. The lower limit of sensitivity with the GC method described in this paper is less than 15 pmoles of MPI. This contrasts sharply with a lower limit, of 10 nmoles when the Bartlett procedure for phosphorus determination is employed. Consequently, the present GC method is over 500 times more sensitive than the best quantitative technique previously available. Since the amount of MPI contained in 1 mg of brain tissue was found to be 2.56 nmoles, which is 100 times greater than the lower limit of sensitivity of the method, it is probable that MPI levels could be measured in as little as 10 pg of brain tissue. With the use of mass spectrometry, particularly if the mass spectrometric multiple ion detection method is employed, we hope that MPI in single cells can be measured. In conclusion, the GC method described in this paper offers a rapid, highly sensitive and specific quantitative method for the estimation of MPI levels in micro-regional or subcellular samples of brain tissue. REFERENCES 1. HOKIN, L. E. (1969) N. Y. Acad. Sci. 165, 695. 2. DURELL, J. AND GARLAND, J. T. (1969) Ann. N. Y. Acad. Sci. 3. LEVEY, G. S. (1971) J. Biol. Chem. 246, 7405. 4. CICBXO, T. J. AND SHERMAN, W. R. (1971) Biochem. Biophys.
165, 743. Res.
Comm.
42,
428. 5. DUNCAN, J., LENNAFLZ, W. AND FENSELAU, C. (1971) Biochemistry 10, 6. WELLS, M. A. AND DITTMER, J. C. (1966) Biochemistry 5, 3405. 7. BARTLETT, G. (1959) J. Biol. Chem. 234,466. 8. SHERMAN, W. R., GOODWIN, S. L. AND ZINBO, M. (1971) J. Chromatogr.
927. Sci. 9,
363. 9. WAGNER,
H., HOLZL,
J., LISSAU,
A. AND HORHAMMER,
I.
(1963) Bioehem.
Z. 339,
34. 10. DITTMER, 11. CICERO,
J. C. AND DOUGLAS, T. J. AND SHERMAN,
451. 12. SEHGAL,
R. K. AND SHERMAN,
M. G. (1969) Ann. N. Y. Acad. W. R. (1971) Biochem. Biophys. W. R., unpublished
observations.
Sci. 165, 515. Res. Comm. 43,