Current Organic Chemistry, 2000, 4, 1-17

Measurement of myo-Inositol in Biological Systems by Mass

Spectrometric
and
In Vivo 1H
Magnetic
Resonance
Spectroscopic Techniques
H. Umesha Shetty*1 and Wei Huang2
1Section

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

*Address correspondence to this author at the Section on Brain

Physiology and Metabolism, National Institute on Aging, National
Institutes of Health, Building 10, Room 6C 103, 10 Center Drive Msc
1582, Bethesda, MD 20892-1582, U.S.A.; Telephone: 301-594-7752; Fax:
301-402-0074; E-mail: shetty@box-s.nih.gov
1385-2728/00 $19.00+.00

glucose-6-phosphate has been demonstrated in

kidney, testis, liver and other organs [3, 4]. The cyclitol
is catabolized in the kidney and excreted [5]. Thus,
myo-inositol homeostasis is maintained by a
combination of transport, synthetic and catabolic
processes. In brain and in kidney, myo-inositol is
osmoregulated by the Na+ /myo-inositol cotransporter
gene [2, 6].
In addition, myo-inositol is a structural component of
membrane phospholipids such as phosphatidylinositol
and phosphatidylinositol 4,5-bisphosphate (Fig. (1 )).
myo-Inositol is continuously incorporated into the
membrane to replenish the phosphatidylinositol pool
utilized for signal transduction. Receptor-mediated
activation of phospholipase C causes the hydrolysis of
phosphatidylinositol 4,5-bisphosphate and generates
inositol 1,4,5-trisphosphate and diacylglycerol. Inositol
1,4,5-trisphosphate, a potent second messenger, is
rapidly dephosphorylated in the cytosol and
regenerated myo-inositol is made available for
2000 Bentham Science Publishers B.V.

2 Current Organic Chemistry, 2000, Vol. 4, No. 1

Shetty and Huang

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

potentiate this signaling [17]. Certain therapeutic

agents may alter phosphatidylinositol turnover and
signaling. For instance, lithium inhibits inositol
monophosphatase at therapeutic levels used in the
treatment of bipolar disorder [18]. myo-Inositol
depletion leading to decreased phosphatidylinositol
turnover has been proposed as the mechanism of
action of this ion [19].
Powerful analytical techniques are required to
obtain a quantitative profile of tissue myo-inositol and
detect its alterations in disease. In addition,
investigation of the dynamics of myo-inositol
metabolism at cellular levels requires a sensitive
quantitation technique.
A
number
of
mass
spectrometric methods have been described to
measure low levels of myo-inositol in biological
samples. In this review, derivatization reactions, gas
chromatographic separations, mass spectrometric
detection techniques and other aspects of myo-inositol
quantitation are discussed. Biological application of the

Myo-Inositol in Biological Systems

Current Organic Chemistry, 2000, Vol. 4, No. 1

mass spectrometric technique is illustrated with the

data for a genetic disorder. Magnetic resonance
spectroscopy allows measurement of certain
metabolites in tissues noninvasively. The technique
involved in the quantitation of brain myo-inositol is
discussed and its clinical applications are cited. A
quantitative profile of the molecule is provided for
certain brain disorders.

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

myo-Inositol and other polyols can be readily

converted to acetate derivatives in the presence of
biological matrices. Acetylation is usually carried out
using acetic anhydride and a base such as pyridine, Nmethylimidazole or sodium acetate [24]. The reaction
with acetic anhydride and pyridine mixture is facilitated
by the addition of a catalytic amount of 4dimethylaminopyridine
[25,
26].
myo-Inositol
hexaacetate (Fig. (2 b )) is stable and withstands an
aqueous, post-derivatization cleanup procedure. It is
important to remove excess reagents and nonvolatile
residues when analyzing low levels of myo-inositol in
biological samples. The GC injector liner needs to be
replaced less frequently if the derivatized samples do
not leave residues. In addition, a large number of
samples may be analyzed without affecting the
performance of the capillary column.
After
derivatization, the sample may be dissolved in
chloroform and washed with ammonia solution [24] to
remove excess reagents. In the assay developed in our
laboratory, the acetate derivative of myo-inositol was
dissolved in hexane and ethyl acetate (80:20;v:v)
mixture and washed with 5% NaHCO3 solution [26].
The solvent mixture provided optimum recovery of the
analyte without trapping water.
The trifluoroacetate derivative (Fig. (2c)) allows
quantitation of myo-inositol by negative ion chemical
ionization mass spectrometry [27]. The derivative is
prepared by reacting the analyte with trifluoroacetic
anhydride and sodium trifluoroacetate in acetonitrile.
Alternatively,
N-methyl-bis(trifluoroacetamide)
in
pyridine can be used to derivatize the hydroxyl groups
of myo-inositol. The reaction mixture is injected into the
GC/MS
without
processing.
myo-Inositol
trifluoroacetate is labile and not amenable to postderivatization cleanup procedures.

O
OR
OR

CF 3

Fig. (2). myo-Inositol derivatives used in the GC/MS

analysis.

n-Butylboronic acid reacts selectively with diols and

yields volatile diester derivatives [28]. The reagent has
been successfully used in the GC/MS analysis of
certain sugars and polyols [29]. Derivatization is carried
out by heating the analyte with excess n-butylboronic
acid in pyridine. The reaction tolerates low levels of
moisture and no special attention is needed to dry the
analyte or pyridine. Interestingly, the reagent reacts wih
myo-inositol and forms a trisbutaneboronate (Fig. (2 d ))
although there is only one set of cis OH groups. It has
been proposed that the initial attack of the reagent is 1,
2 (cis) and the cyclic diester intermediate facilitates
esterification of the remaining trans OH groups by
skewing the cyclohexane ring [30]. The derivative is
probably myo-inositol 1,2;3,5;4,6 trisdiester, as shown
in Fig. (2 d ). The sample is injected into the GC/MS and
excess reagent appears in the solvent front. The
boronate esters are not suitable for post-derivatization
processing.

4 Current Organic Chemistry, 2000, Vol. 4, No. 1

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-

Shetty and Huang

trisphosphate and other inositol phosphates in cardiac

myocytes [32]. The procedure involved treatment of
inositol phosphates with alkaline phosphatase and
analysis of the free myo-inositol.
Certain fragment ions (generated by electron
ionization) of myo-inositol acetate are appropriate for
monitoring. These ions and their abundances are m/z
210 (66%), 168 (100%), 157 (50%), 126 (58%), and
115 (65%) [35]. The m/z 210 ion has been monitored
in procedures developed for assaying myo-inositol in
clinical samples [36-38]. The internal standard was
[2H6]myo-inositol, which loses two deuterium atoms in
the corresponding m/z 214 ion. There are ions which
retain all deuteriums and these include the molecular
ion, m/z 432, and a fragment ion, m/z 373. However,
their abundances are very low 0.03% for the m/z 432
ion and 2.5% for the m/z 373 ion [35] and, thus, are
not suitable for monitoring. In contrast, the m/z 373
fragment ion from myo-inositol acetate was generated
in 100% abundance during a chemical ionization
process [26, 39]. The protonated molecule was not
detected and other fragment ions were in < 10%
abundance. Other stereoisomers of inositol also
yielded the m/z 373 ion in similar abundance. Under
the same ionization condition, [ 2H6]myo-inositol acetate
yielded analogous m/z 379 ion and thus retained all six
deuterium atoms. Fig. (3 ) shows chemical ionization
mass spectra for myo-inositol and [2H6]myo-inositol
acetates. These mass spectra were obtained in our
laboratory using acetonitrile vapor as a reagent gas in
an ion trap mass spectrometer.
Electron ionization of myo-inositol acetate yields the
m/z 373 fragment ion due to the loss of a CH3COO
group from the molecular ion [35]. However, during
chemical ionization, direct abstraction of the CH3COO
moiety or loss of stable, neutral CH3COOH from the
protonated molecule may be the mechanism involved
in the generation of m/z 373 ion in high abundance.
Stabilization of the m/z 379 ion via an oxonium ion (Fig.
(3 )) is feasible and hence the pathway leading to its
formation may be energetically favored under chemical
ionization conditions. Collision with acetonitrile,
isobutane or N2 ions as well as by self-chemical
ionization in an ion trap mass spectrometer generated
the m/z 373 ion [39].
In negative ion chemical ionization, myo-inositol
trifluoroacetate yields m/z 227 ion in 100% abundance
[27]. This ion retains only one deuterium from the
labeled internal standard and, therefore, is not suitable
for monitoring. However, another fragment ion, m/z
642 (abundance < 10%), retains five deuteriums of
[2H6]myo-inositol. Thus, in a reported mass
spectrometric procedure, m/z 642 ion for myo-inositol
and m/z 647 ion for the labeled internal standard were
monitored [27]. Low levels of inositol 1,4,5-

Myo-Inositol in Biological Systems

Current Organic Chemistry, 2000, Vol. 4, No. 1

Fig. (3). Chemical ionization mass spectra of acetate derivatives of myo-inositol and [ 2H6]myo-inositol.

trisphosphate and other phosphate derivatives were

isolated from pancreatic islets, dephosphorylated and,
subsequently, myo-inositol was detected by this
technique.
Electron ionization of myo-inositol n-butylboronate
yields molecular ion m/z 378 (approximately 8%
abundance) and a fragment ion m/z 321 (approximately
40% abundance) suitable for monitoring [30]. The
mass spectrometric characteristics of the nbutylboronate derivative were utilized in a GC/MS
quantitation procedure developed for the serum myoinositol [34]. Hexahydroxybenzene reacts with nbutylboronic acid and thus was used as an internal
standard in the assay. Ions m/z 378 and m/z 321 from
myo-inositol and analogous ions 372 and 316 from the
internal standard were acquired in the selected ion
monitoring assay of serum myo-inositol.

Extraction
Samples

of

myo-Inositol

in

Biological

Quantitative data for myo-inositol have been

obtained for brain and peripheral tissues, cultured cells,
and for biofluids such as plasma, cerebrospinal fluid
and urine. myo-Inositol levels and biological matrices
are different in each of these samples. The choice of
extraction procedure is determined by the quantity of
sample, the nature of biological matrices as well as the
derivatization procedure. Sherman et al. used a hot
water extraction procedure to release myo-inositol in
brain tissues [22]. Protein was removed by zinc sulfate
treatment and centrifugation. The supernate was
lyophilized and used for the preparation of the
trimethylsilyl derivative. In contrast, submicrogram
quantities of brain cells have been directly lyophilized

6 Current Organic Chemistry, 2000, Vol. 4, No. 1

and then treated with the derivatizing agent [23, 40]. In

another procedure, the tissue was homogenized with a
large excess of methanol [13] and centrifuged to
remove the protein precipitate. Using a Savant
evaporator, the methanolic extract was evaporated to
complete dryness and treated with acetylating
reagents.
Sample preparation for assaying plasma myo-inositol
involved deproteinization using perchloric acid [37],
trichloroacetic acid [34], or methanol [26]. Perchloric
acid and trichloric acid interfere in the derivatization
steps and ion-exchange resins have been used to
neutralize acidified samples. If necessary, the lipid
component in the deproteinized extract may be
removed by a chloroform wash [34]. The protein
content of cerebrospinal fluid is low, and cerebrospinal
fluid need not be deproteinized to prepare a myoinositol derivative. In addition, GC/MS analysis can be
performed using microliter quantities of cerebrospinal
fluid [38, 39]. Furthermore, it is important to recognize
that derivatization reaction such as acetylation is not
affected by the presence of light sample matrices.
Internal Standards for Quantitation
Methyl -D-mannopyranoside has been used as an
internal standard in the GC quantitation of myo-inositol
[41]. Such sugar analogs have fragmentation patterns
different from that of myo-inositol and thus are not
useful as internal standards for GC/MS assays. The nbutyl boronate derivative of hexahydroxybenzene
displays mass spectral characteristics similar to those of
the myo-inositol derivative [34]. Hexahydroxybenzene
has been used as an internal standard, although its
physicochemical properties are not comparable to
those of myo-inositol. chiro-Inositol [33] and scylloinositol [42] can be used used as internal standards, if
these stereoisomers are absent or present in trace
levels in the samples. Chemical ionization of scylloinositol acetate generates m/z 373 ion in abundance
comparable to that obtained for the myo-inositol
derivative. A stable isotope labeled analog such as
[2H6]myo-inositol is the most suitable internal standard
for the quantitation of myo-inositol in biological samples
[23, 26, 27, 37-39]. The commercially available
[2H6]myo-inositol is practically free of unlabeled myoinositol and thus its mass shifted ions are produced in
same abundances as those by the analyte.
Reproducibility of the Analysis
A validated GC/MS method is required for the
routine measurement of myo-inositol in biological
samples. Some of the procedures, reported in the
literature, have been tested for reproducibility.

Shetty and Huang

Quantitation of myo-inositol has been performed using

nanogram quantities of tissues from rat cerebellar
granular layer and relative standard deviation of the
measurement ranged from 13 to 27% [23]. Using
GC/MS, Andersen et al. analyzed myo-inositol in
biological samples such as plasma, urine and
erythrocytes [37]. Measurements were performed
using deuterium labeled internal standard and the
correlation coefficient for the standard curve ranged
from 0.994 to 0.999. Data were not provided to support
reproducibility of the quantitation. However, using a
similar technique, myo-inositol in amniotic fluid has
been measured with a relative standard deviation of
10% [36]. Reproducibility data are not available for the
GC/MS technique involving measurement of myoinositol as its n-butylboronate ester derivative [34].
A highly precise quantitation can be achieved by
using a stable isotope labeled internal standard. The
standard curve for myo-inositol was highly linear and
reproducible when [2H6]myo-inositol was used as an
internal standard [26, 39]. The relative standard
deviation in the measurement of myo-inositol was < 2%
for cerebrospinal fluid and plasma samples [26].
Reproducibility of the assay was judged by both withinday and between-day precision data. Data were
reproducible even when less than 100 l of sample was
used. In addition, myo-inositol recovery was complete
and there was virtually no effect of cerebrospinal fluid or
plasma matrices on the quantitation.
Clinical Applications
GC/MS methods have been used to examine
possible alterations in the level of myo-inositol in clinical
conditions. Serum myo-inositol has been shown to be
elevated in diabetic patients and a GC/MS technique
was used to obtain quantitative data [34]. Decreased
cellular uptake and utililization may account for this
abnormality, consistent with the decreased myoinositol concentrations found in peripheral neurons [9]
and cerebrospinal fluid [43]. The kidney plays an
important role in myo-inositol homeostasis as this organ
is the site for catabolism, synthesis
and
osmoregulation. Using a GC/MS technique, a severalfold increase in serum myo-inositol has been
demonstrated in kidney failure [44]. Also, mass
spectrometric techniques have been used to probe
myo-inositol in certain brain disorders. The
cerebrospinal fluid myo-inositol was found to be
unaltered in schizophrenia [38] and Alzheimer disease
[45]. Allison et al. examined the acute effect of lithium
on the regional myo-inositol levels of the rat brain [40].
GC/MS analysis showed decrease (21 to 32%) in the
myo-inositol level in several layers of the frontal cortex.
In contrast, lithium caused no change in cerebellar myoinositol.

Myo-Inositol in Biological Systems

Current Organic Chemistry, 2000, Vol. 4, No. 1

Fig. (4). Ion chromatogram of plasma polyols and the added internal standard. Data from ref. [26].

In Down syndrome, gene products of the extra

chromosome 21 may cause abnormal levels of certain
metabolites due to altered synthesis, transport,
catabolism or regulatory pathways. Screening of certain
metabolites by mass spectrometry might lead to
identification of molecules relevant to the Down
syndrome pathophysiology. We measured myo-inositol
and several other polyol species in cerebrospinal fluid
as well as plasma from control and Down syndrome
adults [11]. Microlitre quantities of these biofluids were
processed and acetate derivatives were analyzed by
GC/MS (ion trap mass spectrometer) [26]. Fig. (4 ) is a
representative ion chromatogram generated for the
plasma polyols. The chromatogram includes peak for
Table 1.

[2H6]myo-inositol, added as an internal standard. A

qualitatively similar chromatographic profile was
obtained for the cerebrospinal fluid polyols.
Table 1 shows concentrations of polyols in the
cerebrospinal fluid for control and Down syndrome
subjects. These GC/MS data demonstrated a selective
increase in the level of myo-inositol in Down syndrome.
Polyol internal controls were not altered and thus a
generalized defect in carbohydrate metabolism was not
the cause for 50% elevation in myo-inositol. However,
plasma myo-inositol was not elevated in Down
syndrome. The cerebrospinal fluid to plasma
concentration ratio was significantly increased,

Concentrations of myo-Inositol and Polyol

Down Syndrome and Control Subjects
Polyols

* P < 0.005.
Data from ref. [11].

Internal

Control (n = 10)
Mean SD (ng/l)

Controls

in

Cerebrospinal

Fluid

Down syndrome (n = 10)

Mean SD (ng/l)

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

8 Current Organic Chemistry, 2000, Vol. 4, No. 1

Shetty and Huang

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

In eukaryotes, myo-inositol is actively transported

and a high concentration (5 to 6 mM in brain) is
maintained for cellular processes such as osmotic
volume regulation and phosphatidylinositol signaling.
Na+ /myo-Inositol cotransporter mRNA is expressed in
neurons and other brain cells [6]. Cellular transport of
myo-inositol has been studied using a radioisotope
tracer [1]. We developed a stable isotope technique to

investigate myo-inositol transport [42] and metabolism

in cultured cells. The procedure involved incubation of
cultured cortical neurons (from mouse fetuses) with a
physiological concentration of [2H6]myo-inositol,
replacing unlabeled myo-inositol. At the end of the
incubation period, washed cells were lysed and scylloinositol (internal standard) was added to the intracellular
material which contains labeled myo-inositol taken up
by the cells as well as the endogenous, unlabeled myoinositol. The GC/MS technique allowed quantitation of
both labeled and unlabeled myo-inositol in the cytosol
(Fig. (5 )). Cellular accumulation of [2H6]myo-inositol
increased with time as evident from the increase in the
peak height of the m/z 379 ion. Also, there was no
entry of myo-inositol into the cell at 0C. Depletion of
Na+ in the medium resulted in ten-fold decrease in the
uptake of [2H6]myo-inositol as indicated by its peak
height. Thus myo-inositol uptake was found to be
active and Na+ -dependent in cultured cortical neurons.
[2H6]myo-Inositol uptake rate was examined in
cortical neurons from the trisomy 16 mouse and
compared with that of control neurons. The uptake was
linear between 2 and 20 min for both types of neurons
(Fig. (6 )). myo-Inositol uptake rate was found to be
significantly increased (40%) in cortical neurons from

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.

Myo-Inositol in Biological Systems

Current Organic Chemistry, 2000, Vol. 4, No. 1

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].

the trisomy 16 mouse compared to control neurons.

The stable isotope/mass spectrometric technique
allowed detection of altered uptake due to Na+ /myoinositol cotransporter overexpression. Thus an
increased transport may be the physiological basis for
increased accumulation of myo-inositol in Down
syndrome brain.
The dynamics of myo-inositol turnover in
phosphatidylinositol may be altered in certain
pathophysiological conditions. A method to quantitate
myo-inositol turnover (in phosphatidylinositol) will be
useful in investigating pathophysiological conditions or
agents that modulate this dynamic process. Preliminary
studies indicate that a mass spectrometric technique
can be used to probe myo-inositol turnover in cultured
cortical neurons2. The method involved incubation of
cells in a culture medium where myo-inositol was
replaced with [2H6]myo-inositol. A mass spectrometric
technique was used to measure the changes in
specific activities of the tracer in cytosol and in
phosphatidylinositol.
Phosphatidylinositol
was
digested with phospholipase C and the product
containing inositol phosphate was hydrolyzed with
2Abstract: Chikhale, E.G.; Rapoport, S.I.; Shetty, H.U. American
Association for Pharmaceutical Scientists, San Francisco, 1998.

alkaline phosphatase. scyllo-Inositol (internal standard)

was added and the [2H6]myo-inositol and myo-inositol,
released from phosphatidylinositol, were quantitated
by mass spectrometry. Incorporation of the tracer into
the membrane lipid was linear up to 10 h. The rate of
incorporation of myo-inositol into phosphatidylinositol
was calculated using specific activity data for cytosolic
myo-inositol at steady state and for membrane
phosphatidylinositol. Protein content of the cell extract
was measured and the labeled and unlabeled
phosphatidylinositol concentrations were normalized.
Using these data and the rate of incorporation, the
basal
turnover
rate
for
myo-inositol
in
phosphatidylinositol was computed.
1H
In
vivo
Magnetic
Resonance
Spectroscopic Measurement of Brain
Metabolite Concentrations

In recent years, in vivo 1H magnetic resonance

spectroscopy (MRS) is being increasingly used in
clinical research because of its unique ability to monitor
metabolic processes noninvasively in living systems on
a regional basis. Also, these
spectroscopic
measurements can be performed in most clinical sites
where MR scanners are available. There is a growing

10 Current Organic Chemistry, 2000, Vol. 4, No. 1

body of evidence that suggests that MRS techniques

can contribute in the clinical evaluation and biological
understanding of a number of pathologies in the brain.
MRS has been used to examine brain metabolites in
pathologies such as ischemia [47-50], cancer [51-53],
multiple sclerosis [54-56], Alzheimer disease [14, 57,
58], Down syndrome [12] and epilepsy [59-62].
In vivo 1H MRS quantitation of metabolites can be
performed using low field strength ( 2 Tesla) clinical
MR scanners. The most commonly quantitated brain
metabolites and their chemical shifts (for the prominent
resonance peaks) are: lactate, 1.33 ppm; Nacetylaspartate, 2.02 ppm; creatine (phosphocreatine
and
creatine),
3.03
ppm;
choline
(mostly
phosphocholine and glycerophosphocholine), 3.23
ppm; and myo-inositol, 3.56 ppm. Glucose, glutamine
and glutamate, and other metabolites detectable with
short echo time (TE) on a clinical scanner, are rarely
reported in a quantitative manner. The resonance
peaks for these compounds appear as broad, lowamplitude multiplets and greatly overlap the very broad
resonances of macromolecules. Thus it is difficult to
quantify such brain metabolites accurately. The
concentration of lactate is low in healthy, adult human
brain and thus rarely visible in 1H MR spectra. However,
lactate is readily detectable in pathologies such as
ischemia and cancer.
Techniques of in vivo 1H MRS Quantitation
Most investigators use creatine as an internal
reference in the 1H MRS quantitation of brain
metabolites and thus report the metabolite to creatine
ratio. However, creatine concentration may not always
remain constant in different brain regions or between
comparison groups in a study. A change in the
denominator without a change in the numerator may
result in artifactual group differences. Cultured brain
cells have been probed by 1H MRS and creatine
concentration was found to be higher in astrocytes and
oligodendrocytes than in neurons [63]. Thus, any brain
disorder that affects the composition of brain cell types
may cause changes in the brain creatine concentration,
leading to a false conclusion from a ratio comparison of
metabolite to creatine between the healthy control
group and patient group.
Using absolute MRS quantitation method, we
showed that brain creatine concentration is elevated in
old Down syndrome adults (> 40 years) compared to
age-matched controls [12], and that the brain creatine
concentration is significantly higher in Alzheimer
disease patients compared to healthy controls [64].
Others have reported that the brain creatine level is
increased in gray matter heterotopia disorder [65].
Therefore, in vivo 1H MRS measurement of absolute

Shetty and Huang

concentrations of metabolites is required for a

meaningful
interpretation
of
the
biochemical
abnormalities associated with brain disease.
The most commonly used quantitative in vivo 1H
MRS methods that provide absolute concentrations of
metabolites are: internal water reference method [6670]; external reference method [12, 71-77]; and
phantom replacement method [78-81]. Quantitation
using the internal water signal is a simple and
convenient way of determining brain metabolite
concentrations. The acquisition time to collect the
water signal is negligible compared to the time required
to acquire signals from brain metabolites. The brain
concentration of water is approximately 50 M, and
concentrations of metabolites are in the range of 1-10
mM. The internal water reference method is insensitive
to variations in B0 and B1 fields, and no preparation of
standard solution is needed. The differences in
longitudinal (T1) and transverse (T2) relaxation time
constants and number of protons between water and
metabolite molecules have to be corrected for accurate
quantitation. The main limitation of this method is the
difficulty in determining the exact concentration of the
water reference in different brain regions. Most
quantitations are based on the assumption of brain
water concentration 39.2 mmol/g wet weight for white
matter and 46.8 mmol/g wet weight for gray matter [68].
However, water content of the brain may be altered in
cancer and other pathological conditions.
In the external reference method, the external
standard can be water, one metabolite, or multiple
metabolites of known concentrations. The external
standard is usually placed within a container adjacent to
the subject undergoing MR scanning. Differences in T 1
and T2 relaxation time constants and number of protons
between compounds in the external standard and
metabolites in the brain also need to be corrected. The
chemical composition and concentrations of the
external standard can be carefully controlled. However,
this method is subject to systematic errors caused by
inhomogeneities of both the B0 and B1 fields. For
single-voxel localized 1H MRS, extra acquisition time is
needed to collect signal from the external reference.
No additional acquisition time is needed for multi-voxel
1H MRS, as long as the external reference is positioned
within the field of view (FOV) of the multi-voxel
acquisition.
In the phantom replacement method, MRS signals
are acquired from the brain and from a phantom
containing standard solution in different scanning
sessions. Brain metabolite concentrations are
calculated by comparing the signal intensity from the
brain with that from the standard solution. Besides
correcting for the differences in T1 and T2 time
constants and for the number of protons between

Myo-Inositol in Biological Systems

compounds in the standard and in the brain

metabolites, coil loading factor also needs to be
corrected to compensate for the difference between
the RF power required to achieve a 90 pulse in the
brain and in the phantom [82]. Furthermore, the
difference in receiver gains for the phantom and the
brain needs to be corrected. This method allows for the
control of the chemical composition and concentrations
of the standard solution in the phantom, and the
human subject does not have to endure additional
scanning time inside the magnet as in the case of the
external reference method. However, many correction
procedures have to be performed in this method,
creating potential sources of error.
In vivo 1H MRS Quantitation of myo-Inositol in
Down
Syndrome
Brain
Using
External
Reference Method
We measured brain myo-inositol concentrations in
nineteen nondemented trisomy 21 Down syndrome
(DS) adults (eight young, < 40 years; eleven old, 40
years) and seventeen healthy control subjects (eight
young, < 40 years; nine old, 40 years) [12]. MR
experiments were performed using a 1.5 T GE Signa
whole-body scanner and a standard bird-cage
quadrature head coil. Before 1H MRS data acquisition,
T1-weighted scout brain images were collected. The
STEAM (stimulated echo acquisition mode) pulse
sequence [83] was employed to acquire single-voxel in
vivo 1H MRS data with TE = 30 ms, mixing time (TM) =
13.7 ms, repetition time (TR) = 3000 ms, and 128 scan
averages. In each data frame, 2048 data points were
collected over a 2500 Hz (39.1 ppm) spectral width.
The proton spectral data were acquired sequentially
from three square voxels (2.0 2.0 2.0 cm3 = 8.0
cm3) placed in the occipital region, parietal region, and
external standard solution. The external standard was a
250-mL plastic bottle filled with physiological saline
solution containing 10 mM N-acetylaspartate, 8 mM
creatine, 2 mM choline chloride, and 5 mM myo-inositol.
The bottle was positioned adjacent to the subject's
head during MR scanning and was within the image
FOV. Water suppression was achieved during
spectroscopic data acquisition by three narrow band
(50 Hz) RF CHESS (chemical shift-selective) pulses
centered at the water resonance frequency before the
first 90 pulse, followed by three crusher gradients.
After MRS data acquisition, T1-weighted volumetric
imaging was performed to cover the spatial range of the
MRS voxels. A total of seven image slices (3 mm thick
per slice with no gap) were collected for each subject.
An algorithm developed by Ibanez et al. [84] was used
to segment the volumetric images to determine the
cerebrospinal fluid volume fraction (fCSF) in the
spectroscopic voxels in the brain.

Current Organic Chemistry, 2000, Vol. 4, No. 1

11

To measure T 1 and T 2 values of the metabolites in

Down syndrome and control brains and in standard
solution, MRS data were collected with different TE
(30, 50, 70, 100, 160, 250 ms) at constant TR, and with
different TR (1.5, 2.0, 3.0, 4.0, 6.0 s) at constant TE,
from three Down syndrome subjects (28, 39, and 46
years old) and three control subjects (22, 34, and 53
years old) during separate scanning sessions, and also
from a one liter standard solution alone (seven times).
The raw spectral data were processed with
exponential line broadening (3 Hz), zerofilling, Fourier
transformation and with phase and baseline
corrections. Marquardt functions were used to fit the
resonance peaks of N-acetylaspartate, creatine,
choline and myo-inositol, and peak areas were
measured. To determine metabolite concentrations,
the following equation of MR signal intensity, S
(represented by resonance peak area), for a STEAM
pulse sequence [75] was employed:
S = N exp{-(TE/T2)-(TM/T1)}{1-exp(-TR/ T 1)}

(1)

where N represents the number of metabolite

molecules per unit voxel. From Eq. (1), we can derive
the following equation to calculate any metabolite
molecule numbers per unit voxel in the brain:
Sb Ns exp{-(TE/T2,s)-(TM/T1,s)}{1-exp(-TR/T 1,s)}
Nb = _________________________________________________
Ss exp{-(TE/T2,b)-(TM/T1,b)}{1-exp(-TR/ T 1,b)}

(2),

where the subscripts s and b represent standard and

brain, respectively. Finally, the brain metabolite
concentration in each voxel, [Met], in units of mmol/liter
wet brain tissue, can be calculated:
[Met] = Nb /[(1-fCSF)Vvoxel]

(3)

where Vvoxel is the volume of spectroscopic voxel and

is 8.0 cm 3 in this case. Because of low concentrations
of metabolites in the cerebrospinal fluid, the
contribution to the MR signal from the cerebrospinal
fluid compartment of the spectroscopic voxel is
negligible. For example, cerebrospinal fluid myoinositol concentration is approximately 0.13 mM [11]
which is about fifty times lower than in the brain.
By fitting S and TR (or TE) values to Eq. (1) at
constant TE (or TR), metabolite T1 (or T2) values were
obtained. Because no significant difference was
observed in T1 and T2 values between the occipital
region and parietal region within the same subject
group, the mean T1 and T2 values for control and Down
syndrome groups (Table 2) represent averaged values
across the measurements from both brain regions.
These mean values were used to calculate metabolite
concentrations (Eq. (2)) for control and Down
syndrome subjects. The T 1 and T 2 values measured in

12 Current Organic Chemistry, 2000, Vol. 4, No. 1

Table 2.

Shetty and Huang

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)

Down syndrome 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

Values are in mean SD.

n: number of subjects or number of repetitions.
T1: longitudinal relaxation time constant.
T2: transverse relaxation time constant.

this study for control brain and standard aqueous

solution agree well with literature values [57, 85, 86]. T1
and T2 values for the metabolites in Down syndrome
brain have not been reported previously.
Fig. (7 ) shows representative 1H MR spectra
acquired from the occipital region of Down syndrome
and control brains and from the external reference. The
spectra clearly illustrate an increase in the amplitude of
myo-inositol and choline peaks in both Down syndrome
age groups compared with age-matched controls. myoInositol concentrations were calculated according to
Eqs. (2) and (3) and Table 3 lists mean SD values for
the occipital and parietal regions of Down syndrome
and control brains. Compared with controls, Down
Table 3.

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

Brain myo-Inositol Concentrations in Down Syndrome Adults and Healthy Controls

Control

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%)

Concentrations (mean SD) are in mmol/liter wet brain tissue.

*Mean SD in Down syndrome group differs significantly from that of age-matched control group (ANOVA, P < 0.0001).
Number in parenthesis represents mean percent change from age-matched controls.
Data from ref. [12].

Myo-Inositol in Biological Systems

Current Organic Chemistry, 2000, Vol. 4, No. 1

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.

measurements, are in close agreement with

concentrations obtained for the postmortem brain by a
GC technique [87]. 1H MRS data showed an
approximately
50%
increase
in
myo-inositol
concentration in Down syndrome brain, consistent with
the gene dosage effect of the Na+ /myo-inositol
cotransporter [46]. However, the older Down syndrome
subjects in the preclinical stages of Alzheimer disease
showed brain myo-inositol concentrations significantly
higher than the concentrations found in younger Down
syndrome subjects. Although an age-effect can not be
excluded, it is thought most likely that the difference
between young and older Down syndrome subjects
represents an Alzheimer disease effect, based on
neuropathological studies showing all Down syndrome
brains over 40 years have Alzheimer disease
neuropathology [88]. The neurobiological significance
of the further increase of myo-inositol in older Down
syndrome subjects remains to be determined. It has
been reported that the myo-inositol concentration in
glia is higher than in neurons [89, 90]. Loss of neurons
with resultant gliosis could hypothetically account for
the myo-inositol elevation in older Down syndrome

adults. According to a report, the concentration of brain

phosphatidylinositol is significantly decreased in
Alzheimer disease [91]. In addition, defects in
postsynaptic intracellular signal transduction have been
reported in Alzheimer disease [92]. It is possible that
defects in signaling coexist with abnormalities in
intracellular systems for myo-inositol homeostasis.
In vivo 1H MRS Measurement of
Inositol in Alzheimer Disease

Brain

myo-

We performed 1H MRS on twenty-one Alzheimer

disease patients (age, 71 11 years) and seventeen
healthy age-matched control subjects (age, 72 7
years). The in vivo MR scanning procedures, the
parameters for 1H MRS, and the metabolite quantitation
method were the same as those employed in the Down
syndrome study. 1H MR spectra were collected from
both the left parietal and the right parietal regions, in
addition to the occipital region and the external
standard. The metabolite T1 and T2 values were
measured from three Alzheimer patients and three
control subjects during separate scanning sessions.

14 Current Organic Chemistry, 2000, Vol. 4, No. 1

Table 4.

Shetty and Huang

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

Values are in mean SD.

n: number of subjects.
T1: longitudinal relaxation time constant.
T2: transverse relaxation time constant.

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%)

Concentrations (mean SD) are in mmol/liter wet brain tissue.

*Mean SD in Alzheimer disease group differs significantly from that of age-matched control group (unpaired t-test, P < 0.0001).
Number in parenthesis represents mean percent change from age-matched controls.

No significant difference was observed in T1 and T 2

values among the occipital, left parietal, and right
parietal regions within the same subject group. The
mean T1 and T2 values (Table 4) for control and
Alzheimer groups represent averaged values across
the measurements from three brain regions. These
values agree well with those reported by Moats et al.
[57]. The amplitude of the myo-inositol resonance peak
was increased in all three brain regions of the Alzheimer
disease patients compared with the age-matched
controls (Figure not shown). In addition, the peak
intensity of N-acetylaspartate was decreased in the
Alzheimer disease brain. Compared with controls,
Alzheimer patients had a significantly higher myoinositol concentration in the brain (Table 5).
Our 1H MRS data showing increased myo-inositol
and decreased N-acetylaspartate concentrations in the
Alzheimer brain are consistent with those reported by
others [14, 57, 58]. Abnormal concentrations of myoinositol were found even in the earliest stages of
Alzheimer disease and there were no additional
changes with dementia severity, as determined by
neuropsychological tests. On the other hand, an

abnormality in N-acetylaspartate was not found in mild

Alzheimer disease. However, the N-acetylaspartate
level was found to progressively decrease with
dementia severity. These results suggest that
abnormalities in myo-inositol precede abnormalities in
N-acetylaspartate and can be detected with 1H MRS.
Although the clinical diagnosis of Alzheimer disease
may be imprecise in the earliest stages of disease, by
using in vivo 1H MRS, we were able to show that
subjects with mild Alzheimer disease had abnormal
brain myo-inositol concentrations. These data are
consistent with abnormal myo-inositol concentration
found in older nondemented Down syndrome adults in
the predementia phases of Alzheimer disease. Thus 1H
MRS detection of abnormal myo-inositol profile may be
used for the early diagnosis of Alzheimer disease.
Comments
The T2 value for brain myo-inositol is relatively short
(about 150 ms) and 1H MRS data acquisition was
performed using a short TE time to achieve its
detection with a satisfactory signal-to-noise ratio. We

Myo-Inositol in Biological Systems

used the external reference method to quantitate

metabolite concentrations in Down syndrome and
Alzheimer disease brains. The aspect (length to radius)
ratio of our external standard was close to 10 and thus
the local magnetic field inside the standard was quite
homogeneous [93, 94]. Field homogeneity inside the
spectroscopic voxel placed in the standard was easily
achieved by shimming (the full width at half magnitude
of the water peak was usually reduced to 2 Hz). As a
result, excellent water suppression was obtained. B1
field homogeneity is also essential for accurate
quantitation. In a separate experiment with the same
pulse sequence and acquisition parameters, the
spectroscopic voxel was placed across the volume of a
one-gallon plastic bottle filled with the same standard
solution. Analysis of the spectra collected from
different locations of this large phantom showed less
than 5% variation in the peak intensity. This result is
consistent with another study [78] with the same
hardware configuration. Thus, it was practical for us not
to correct for the difference in B 1 field between the
brain and the external standard in metabolite
quantitation.
Since the contribution to the MR signal from the
metabolites in cerebrospinal fluid is negligible
compared with that from brain metabolites, partial
volume effects due to the loss of brain tissue that
occurs with Alzheimer disease, as well as aging, must
be taken into account in functional studies of the brain.
For example, an atrophy correction of 19-49% in
Alzheimer disease and 16-38% in controls has been
reported in a cerebral metabolic study using positron
emission tomography [95]. In our Down syndrome
study, the atrophy corrections were 9% for the occipital
region and 5% for the parietal region of the control
brain. These corrections were 10% for the occipital
region and 7% for the parietal region in the Down
syndrome brain. Quantitation without correcting for the
cerebrospinal fluid volume fraction in the spectroscopic
voxel will lead to underestimation of brain metabolite
concentrations.

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.

Current Organic Chemistry, 2000, Vol. 4, No. 1

15

Quantitative changes in myo-inositol and other

metabolites can be assessed during the progression of
a disease. Additionally, in vivo MRS techniques may be
useful in studying the effect of therapeutic agents on
metabolites of physiological significance.

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