Documente Academic
Documente Profesional
Documente Cultură
on Brain Physiology and Metabolism, National Institute on Aging, National Institutes of Health,
Bethesda, Maryland, U.S.A., and 2Department of Radiology, State University of New York at Stony Brook,
Stony Brook, New York, U.S.A.
Abstract: Cells utilize myo-inositol for osmoregulation and phosphatidylinositol signaling. Mass
spectrometric and in vivo magnetic resonance spectroscopic techniques have been
complementarily used in our laboratories to investigate brain myo-inositol metabolism. Mass
spectrometric quantitation methods are surveyed focusing primarily on derivatization reactions,
gas chromatographic separation and detection of ions. Monitoring of the m/z 373 fragment ion
generated from acetate derivative provides precise quantitation of myo-inositol in biological
matrices. The technique and its clinical applications are discussed. Measurement of myo-inositol
transport using a stable isotope technique is illustrated for cultured neurons. In addition, the
possible use of the technique in probing phosphatidylinositol turnover is discussed.
An in vivo 1H magnetic resonance spectroscopic technique is described for measuring the
absolute concentration of myo-inositol in human brain. Magnetic resonance spectroscopy with
short echo-time enables detection of the resonance peak of myo-inositol (3.56 ppm) when the
water resonance peak is suppressed by narrow band radio-frequency pulses. The review
focuses on an external reference method involving collection of data from the human subject
and the phantom containing aqueous myo-inositol standard solution in the same scanning
session. The method takes into account differences in longitudinal and transverse relaxation
time constants of myo-inositol between brain tissue and aqueous solution. Application of the
technique is illustrated by measuring brain myo-inositol in Down syndrome adults and Alzheimer
disease patients. Advantages and limitations of this noninvasive technique in monitoring
metabolic processes are discussed.
Introduction
myo-Inositol is an optically inactive stereoisomer of
hexahydroxycyclohexane where C-2 hydroxyl is axial
and the remaining five groups are equatorial (Fig. (1 )).
Other stereoisomers have been designated as cis-,
epi-, allo-, muco-, neo-, (+)chiro- and (-)chiro-inositol.
myo-Inositol, the major isomer found in mammalian
cells, is important for signal transduction and
osmoregulation. scyllo-Inositol and chiro-inositol occur
in low concentrations and their physiological roles have
not been elucidated. Cells take up and concentrate
myo-inositol to varying degrees by means of a
Na+ /myo-inositol cotransporter [1, 2]. Most of the myoinositol pool in the body is of dietary origin. However,
myo-inositol de novo synthesis via cyclization of
Fig.
(1). myo-Inositol structure and phosphatidylinositol signaling: PtdIns, phosphatidylinositol; PtdIns(4,5)P2,
phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; DAG, diacylglycerol; PKC, protein kinase C; Ins(1,4,5)P3,
inositol 1,4,5-trisphosphate; Ins(1)P, inositol 1-phosphate.
phosphatidylinositol
synthesis.
A
number
of
neurotransmitters, hormones, growth factors and other
agonists
have
been
shown
to
influence
phosphatidylinositol
turnover
during
membrane
signaling [7, 8].
The
myo-inositol
homeostasis
and
the
phosphatidylinositol signaling may be altered in certain
pathophysiological
conditions.
These
include
diabetes, where the myo-inositol pool utilized for
phosphatidylinositol synthesis is depleted in peripheral
neurons [9, 10]. An excessive accumulation of myoinositol in the brain is a phenotype of Down syndrome
(trisomy 21) and of an animal model of this disorder [1113]. Metabolite profiling by magnetic resonance
spectroscopy indicates elevated concentrations of
myo-inositol and myo-inositol metabolites in the
Alzheimer disease brain [14, 15]. Also, fibroblasts from
Alzheimer disease patients show augmented inositol
1,4,5-trisphosphate-mediated Ca2+ signaling [16] and
the Alzheimer presenilin-1 mutation is reported to
Gas
Chromatographic/mass
Spectrometric (GC/MS) Analysis of myoInositol
Derivatization
Silylation is a commonly used derivatization
procedure to render polyols and sugars volatile for GC
analysis [20]. A number of quantitation procedures for
myo-inositol involve analysis of trimethylsilyl derivative
by GC with thermal conductivity [21], flame ionization
[22] or mass spectrometric [23] detection. A mixture of
hexamethyldisilazane
and
trimethylchlorosilane
(2:1;v:v) in pyridine is used to prepare hexatrimethylsilyl
derivative of myo-inositol (Fig. (2a)). The reaction does
not occur without trimethylchlorosilane [20]. Silylating
reagent
such
as
N,O-bis(trimethylsilyl)trifluoroacetamide can be used in place of
hexamethyldisilazane [23]. The reaction is carried out in
an anhydrous condition and the trimethylsilyl derivative
is analyzed avoiding contact with moisture. The
trimethylsilyl derivative of myo-inositol is unstable and
the procedure is not suitable for analyzing a large batch
of samples.
O
C4 H9
OR
RO
RO
C4H9
O
B
OR
C4 H9
d
Si
CH 3
CH 3
CH 3
CH 3
O
c
O
OR
OR
CF 3
Gas Chromatography
Packed columns have been used in the past to
analyze trimethylsilyl derivative of myo-inositol [20-23,
31]. The stationary phases used in these columns
include
polyethylene
glycol
succinate,
dimethylpolysiloxane, phenylmethylpolysiloxane, nitrile
silicone and carbowax. Capillary columns coated with
these and other stationary phases have a higher
resolving capacity. Such columns have been used for
the analysis of myo-inositol extracted from biological
samples
[32,
33].
The
analysis
involved
chromatographic separation of the trimethylsilyl
derivative on a capillary column bonded with (50%phenyl)methylpolysiloxane or dimethylpolysiloxane
phase. Both packed and capillary columns are capable
of resolving inositol stereoisomers found in biological
systems.
Stationary
phases,
(50%phenyl)methylpolysiloxane
and
(50%
trifluoropropyl)methylpolysiloxane, have been used for
the analysis of ester derivatives (acetate and
trifluoroacetate) of myo-inositol [26, 27]. A 30-m long
capillary
long
bonded
with
(50%phenyl)methylpolysiloxane separates all polyols in
biological systems and is appropriate for the mass
spectrometric analysis. The column separates
stereoisomers such as chiro- and scyllo-inositol present
in low levels in tissues. Analysis of butaneboronic acid
ester of myo-inositol has been carried out using (50%phenyl)methylpolysiloxane phase in a packed column
[29, 30]. In a subsequent study,
involving
measurement of serum myo-inositol, the boronate
ester was chromatographed on a dimethylpolysiloxane
capillary column and detected by mass spectrometry
[34].
Mass Spectrometry
Because of its detection specificity, GC/MS is the
most appropriate technique to measure free myoinositol in the biological specimens. Mass spectrometric
detection can be performed with the trimethylsilyl,
acetate, trifluoroacetate or the n-butylboronate
derivative. An electron ionization or a chemical
ionization technique has been used to analyze these
myo-inositol derivatives. Ions of the trimethylsilyl
derivative suitable for monitoring and their abundances
are m/z 318 (52%), 305 (95%), 217 (100%), 191
(53%), 147 (65%) and 73 (122%) [35]. Quantitation is
usually performed by selected ion monitoring of the
m/z 305 ion. For instance, ions, m/z 305 for myoinositol and m/z 307 for the deuterium-labeled myoinositol (internal standard) were monitored in the
quantitation of myo-inositol in neurons [23]. The
method also has been used to measure inositol 1,4,5-
Fig. (3). Chemical ionization mass spectra of acetate derivatives of myo-inositol and [ 2H6]myo-inositol.
Extraction
Samples
of
myo-Inositol
in
Biological
Fig. (4). Ion chromatogram of plasma polyols and the added internal standard. Data from ref. [26].
* P < 0.005.
Data from ref. [11].
Internal
Control (n = 10)
Mean SD (ng/l)
Controls
in
Cerebrospinal
Fluid
Mannitol
0.872 0.248
0.947 0.622
Sorbitol
2.51 0.61
2.55 1.54
Galactitol
0.343 0.125
0.326 0.172
Ribitol
0.580 0.108
0.535 0.136
Arabitol
3.68 0.95
3.33 0.90
1,5-Anhydrosorbitol
15.40 3.75
18.13 5.25
myo-Inositol
24.35 4.20
36.32 8.02*
from
suggesting brain as the origin of the elevated myoinositol. GC/MS analysis of postmortem brain tissues
showed a similar increase in the concentration of myoinositol 1. Consistent with these findings, elevated myoinositol was also found in the trisomy 16 mouse, an
animal model of Down syndrome [13]. Thus, the mass
spectrometric
technique
enabled
unequivocal
identification of abnormal accumulation of myo-inositol
in Down syndrome brain. The Na+ /myo-inositol
cotransporter gene maps to the long arm of human
chromosome 21 [46] and thus an augmented transport
is likely the mechanism for myo-inositol elevation in
Down syndrome.
myo-Inositol
Study
Transport
and
Incorporation
Fig. (5). GC/MS detection of [2H6]myo-inositol/myo-inositol in cultured cortical neurons: [2H6]myo-inositol accumulation with
time (a and b); scyllo-inositol (internal standard) is shown for the 2 min uptake experiment only (a); negligible [2H6]myo-inositol
uptake at 0C (c); uptake in the presence of NaCl (d) and uptake when NaCl was replaced with choline chloride (e). Data from ref.
[42].
1Abstract: Shetty, H.U.; Huang, W.; Schapiro, M.B. J. Neurochem., 1998, 70S, 66C.
Fig. (6). Time-course of [2H6]myo-inositol uptake by cortical neurons from the trisomy 16 mouse and diploid controls. Data
points are mean SEM of 4 or 5 determinations. Data from ref. [42].
11
(1)
(2),
(3)
Table 2.
Metabolite T 1 and T2 Values in Healthy Control and Down Syndrome Brains, and Standard
Solution at 1.5 T
Samples
Standard (n = 7)
Control brain (n = 3)
Metabolites
T1 (ms)
T2 (ms)
N-Acetylaspartate
1310 80
897 25
Creatine
1607 118
592 37
Choline
1954 72
1167 109
myo-Inositol
1036 55
343 24
N-Acetylaspartate
1314 170
313 52
Creatine
1383 197
177 11
Choline
1190 127
326 31
myo-Inositol
1014 141
151 18
N-Acetylaspartate
1210 171
353 64
Creatine
1472 215
178 19
Choline
1375 218
310 59
myo-Inositol
1150 194
145 23
syndrome adults had a significantly higher mean myoinositol concentration in the brain. There was a
significant group by age interaction for the myo-inositol
concentration in the occipital region. Follow-up simple
effects analysis using pairwise t-tests showed that the
older Down syndrome group had a significantly (P <
0.003) higher mean myo-inositol concentration
compared with the younger Down syndrome group and
that no such age-related alteration was observed in the
healthy controls.
We found myo-inositol concentrations of 6.5 1.0
mmol/liter wet brain tissue for the occipital region and
6.3 1.0 mmol for the parietal region in healthy
controls. These values, determined by in vivo 1H MRS
Down syndrome
Regions
Young (n = 8)
Old (n = 9)
Young (n = 8)
Old (n = 11)
Occipital
6.6 0.6
6.5 1.0
9.7 1.0*(+47%)
11.4 1.1*(+75%)
Parietal
5.6 0.9
6.3 1.0
8.4 1.2*(+50%)
10.1 1.0*(+60%)
13
Fig. (7). Representative 1H MR spectra (TR/TE = 3000/30 ms, 128 scan averages) from: a young Down syndrome subject, 36
years; a young control, 38 years; an old Down syndrome subject, 50 years; an old control, 53 years; the external standard
(metabolites in physiological saline). All brain spectra were collected from the occipital region. The identified resonance peaks
(in the spectrum from the young control) are: NAA, N-acetylaspartate at 2.02 ppm; Cr, creatine at 3.03 ppm; Cho, choline at
3.23 ppm; mI, myo-inositol at 3.56 ppm. The upward arrows indicate obvious increases of myo-inositol and choline peak
amplitudes in Down syndrome subjects.
Brain
myo-
Table 4.
Metabolite T 1 and T 2 Values in Healthy Control and Alzheimer Disease Brains at 1.5T
Brain
Control (n = 3)
Alzheimer disease (n = 3)
Metabolites
T1 (ms)
T2 (ms)
N-Acetylaspartate
1284 156
363 45
Creatine
1305 176
197 14
Choline
1117 137
340 25
myo-Inositol
984 123
161 15
N-Acetylaspartate
1189 121
383 54
Creatine
1352 184
190 20
Choline
1320 201
333 52
myo-Inositol
892 114
141 21
Table 5.
Brain (Regional)
Subjects
Subjects
myo-Inositol
Concentrations
Occipital
in
Alzheimer
Disease
and
Right parietal
Healthy
Control
Left parietal
Control
6.2 0.5
6.4 0.7
6.0 0.8
Alzheimer disease
8.2 1.8*(+32%)
8.8 2.0*(+38%)
9.2 2.3*(+53%)
Conclusions
Mass spectrometric techniques enable precise
measurement of myo-inositol and detection of diseaserelated alterations in its level in complex biological
systems. myo-Inositol transport, phosphatidylinositol
signaling and other dynamic processes in cells can be
probed using stable isotope tracer and mass
spectrometric detection. In addition, a liquid
chromatographic/mass spectrometric technique may
be appropriate to measure myo-inositol metabolites
and phosphoinositides involved in cellular processes.
Noninvasive measurement of myo-inositol in a living
system is possible by the recent advances in MRS.
15
References
[1]
[2]
Kwon, H.M.; Yamauchi, A.; Uchida, S.; Preston, A.S.; GarciaPerez, A.; Burg, M.B.; Handler, J.S. J. Biol. Chem., 1992, 267,
6297.
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
Zhu, X.; Eichberg, J. Proc. Natl. Acad. Sci. USA, 1990, 87, 9818.
[11]
[12]
[13]
[14]
[15]
[16]
Ito, E.; Oka, K.; Etcheberrigaray, R.; Nelson, T.J.; McPhie, D.L.;
Tofel-Grehl, B.; Gibson, G.E.; Alkon, D.L. Proc. Natl. Acad. Sci.
USA, 1994, 91, 534.
[17]
[18]
[19]
Berridge, M.J.; Downes, C.P.; Hanley, M.R. Cell, 1989, 59, 411.
[20]
[21]
[22]
[24]
[50]
Houkin, K.; Kamada, K.; Kamiyama, H.; Iwasaki, Y.; Abe, H.;
Kashiwaba, T. Stroke, 1993, 24, 1316.
[51]
[52]
[53]
[54]
[55]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
DaTorre, S.D.; Corr, P.B.; Creer, M.H. J. Lipid Res., 1990, 31,
1925.
[56]
Bruhn, H.; Frahm, J.; Merboldt, K.D.; Hanicke, W.; Hanefeld, F.;
Christen, H.J.; Kruse, B.; Bauer, H.J. Ann. Neurol., 1992, 32, 140.
[33]
Heathers, G.P.; Juehne, T.; Rubin, L.J.; Corr, P.B.; Evers, A.S.
Anal. Biochem., 1989, 176, 109.
[57]
Moats, R.A.; Ernst, T.; Shonk, T.K.; Ross, B.D. Magn. Reson.
Med., 1994, 32, 110.
[34]
Hasegawa, M.; Doi, K.; Baba, S. Clin. Chim. Acta, 1988, 176,
207.
[58]
[35]
[59]
[36]
[60]
[37]
[61]
[38]
[39]
[62]
[40]
[63]
[41]
[64]
Huang, W.; Alexander, G.E.; Chang, L.; Levine, B.; Ibanez, V.;
Rapoport ,S.I.; Schapiro, M.B. Proc. Intl. Soc. Magn. Reson.
Med., 1998, 1, 425.
[42]
[65]
[43]
[66]
[44]
Inouye, M.; Mio, T.; Sumino, K. Jpn. J. Med., 1988, 27, 29.
[45]
[67]
[68]
[46]
Berry, G.T.; Mallee, J.J.; Kwon, H.M.; Rim, J.S.; Mulla, W.R.;
Muenke, M.; Spinner, N.B. Genomics, 1995, 25, 507.
[69]
[47]
Soher, B.J.; Hurd, R.E.; Sailasuta, N.; Barker, P.B. Magn. Reson.
Med., 1996, 36, 335.
[70]
[48]
Penrice, J.; Cady, E.B.; Lorek, A.; Wylezinska, M.; Amess, P.N.;
Aldridge, R.F.; Stewart, A.; Wyatt, J.S.; Reynolds, E.O. Pediatr.
Res., 1996, 40, 6.
[71]
Alger, J.R.; Symko, S.C.; Bizzi, A.; Posse, S.; DesPres, D.J.;
Armstrong, M.R. J. Comput. Assist. Tomogr., 1993, 17, 191.
17
[72]
Hennig, J.; Pfister, H.; Ernst, T.; Ott, D. NMR Biomed., 1992, 5,
193.
[85]
Frahm, J.; Bruhn, H.; Gyngell, M.L.; Merboldt, K.D.; Hanicke, W.;
Sauter, R. Magn. Reson. Med., 1989, 11, 47.
[73]
[86]
[74]
[87]
[75]
[88]
[76]
Kreis, R.; Ernst, T.; Ross, B.D. J. Magn. Reson. B, 1993, 102, 9.
[89]
[77]
Ernst, T.; Kreis, R.; Ross, B.D. J. Magn. Reson. B, 1993, 102, 1.
[90]
[78]
Soher, B.J.; van Zijl, P.C.M.; Duyn, J.H.; Barker, P.B. Magn.
Reson. Med., 1996, 35, 356.
[91]
[79]
[92]
Young, L.T.; Kish, S.J.; Li, P.P.; Warsh, J.J. Neurosci. Lett., 1988,
94, 198.
Austin, S.J.; Connelly, A.; Gadian, D.G.; Benton, J.S.; Brett, E.M.
Magn. Reson. Med., 1991, 19, 439.
[93]
[94]
Albert, M.S.; Huang, W.; Lee, J.-H.; Balschi, J.A.; Springer, Jr.,
C.S. NMR Biomed., 1993, 6, 7.
[95]
[80]
[81]
[82]
[83]
[84]