Proc. Natl. Acad. Sci.
USA
Vol. 90, pp. 9988-9992, November 1993
Medical Sciences
D-chiro-Inositol metabolism in diabetes mellitus
(insulin/mass spectrometry)
RICHARD E. OSTLUND, JR.*t, JANET B. MCGILL*, IAN HERSKOWITZ*, DAVID M. KIPNIS*,
JULIO V. SANTIAGO*, AND WILLIAM R. SHERMAN
*Departments of Medicine, *Pediatrics, and Psychiatry, Washington University, St. Louis, MO 63110
Contributed by David M. Kipnis, July 19, 1993
ABSTRACT D-chiro-Inositol is a rare inositol isomer pres-
ent in inositol phosphoglycans which are proposed mediators of
insulin action. To study D-chiro-inositol metabolism in diabetes
mellitus, a sensitive and specific assay was developed using
negative-ion chemical ionization gas chromatography/mass
spectrometry. Median urinary D-chiro-inositol excretion,
which was 2.1 ,umol/day in nondiabetics, was substantially
increased to 12 ,umol/day in non-insulin-dependent diabetes (P
< 0.0001) and to 74 j,mol/day in insulin-dependent diabetes (P
< 0.0001). Urinary D-chiro-inositol was strongly correlated
with fasting plasma glucose (r = 0.568, P < 0.0001), glycated
hemoglobin (r = 0.529, P < 0.0001), and urinary glucose (r =
0.368, P = 0.01). The renal clearance of D-chiro-inositol was
selectively elevated in both non-insulin-dependent and insulin-
dependent diabetes when compared with the clearances of
L-chiro-inositol or myo-inositol and exceeded the glomerular
filtration rate in 71% of the diabetics but in none of the
nondiabetics. In poorly controlled diabetic patients insulin
treatment reduced urinary D-chiro-inositol losses by 63% and
increased plasma levels by 8.8-fold. The metabolism of D-chiro-
inositol is abnormal in diabetes and appears to be influenced by
short- and long-term metabolic control.
One of the most important problems in endocrinology is the
rigorous description of the mechanism of insulin action.
Previous work suggests that some (but not all) of the actions
of insulin may be mediated through inositol phosphoglycan
molecules released from cell membranes (1-4). When such
putative mediators were hydrolyzed and analyzed chemically
it was found unexpectedly that in some preparations much or
all of the inositol present was not myo-inositol (which is, by
far, the most common mammalian inositol) but rather the rare
epimer D-chiro-inositol (5, 6). Kennington et al. (7) reported
abnormally low or unmeasurable levels of D-chiro-inositol in
urine and muscle from patients with non-insulin-dependent
diabetes mellitus (NIDDM) and suggested that D-chiro-
inositol deficiency might be related to the insulin resistance
observed in NIDDM.
However, further study of D-chiro-inositol metabolism has
been delayed by lack of a suitable.assay and complicated by
the low levels found in body fluids. Here we report an
analytical method for D-chiro-inositol which utilizes hexa-
deuterated chiro-inositol as an internal assay standard. The
use of negative-ion chemical ionization gas chromatography/
mass spectrometry (NICI GC/MS) enables us to measure
both chiro- and myo-inositol in plasma and urine of diabetic
and control subjects and to compare their levels with indices
of metabolic control. Our results, which differ from those of
Kennington et al. (7), show that both NIDDM and insulin-
dependent diabetes mellitus (IDDM) patients have large
urinary losses of D-chiro-inositol which are strongly related
to metabolic control.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. 1734 solely to indicate this fact.
9988
METHODS
Materials and Assays. D-chiro-Inositol was prepared by
Calbiochem and purchased -25 years ago. L-chiro-Inositol
was the gift of Laurens Anderson (University of Wisconsin,
Madison). DL-chiro-[1,2,3,4,5,6-2H6]Inositol (8) was the gift
of Ken Sasaki (Connaught Centre for Biotechnology Re-
search, Toronto). myo-[1,2,3,4,5,6-2H6]Inositol was pur-
chased from MSD Isotopes. Plasma insulin (9) and urinary
albumin (Diagnostic Products, Los Angeles) were deter-
mined by radioimmunoassay, and glycated hemoglobin was
determined by affinity chromatography (Pierce; normal range
4.4-6.3%).
Inositol Analysis. Twenty-four-hour urine specimens were
collected with cooling but without preservatives and aliquots
were stored frozen at -20C. D-chiro-Inositol levels changed
<3% after incubation of nonsterile urine from two poorly
controlled diabetics and two normal subjects for 24 hr at
25C, indicating that urinary D-chiro-inositol levels are stable
during collection. To 0.25 ml of urine were added 10 nmol of
deuterated DL-chiro-inositol and 20 nmol of deuterated myo-
inositol as internal standards. The samples were then purified
by a modification of a procedure (7) in which the sample was
first passed over a 0.6-ml column of water-washed Amberlite
IR-120(+) (Aldrich) and then over a 0.6-ml column of washed
Amberlite IRA-400 hydroxide 16-50 mesh (Sigma). The
highly basic IRA-400 hydroxide resin removed 99% of glu-
cose from diabetic urine, in agreement with a previous report
(10), resulting in a significant improvement of sample purity.
In a final step the deionized samples were passed over C18
Sep-Pak cartridges (Waters) that had been washed with 2 ml
of methanol followed by 5 ml of water. The columns were
washed with an additional 0.5 ml of water to remove remain-
ing sample, and the combined aqueous eluates were lyoph-
ilized.
Fasting EDTA-anticoagulated plasma samples were col-
lected on ice and stored frozen at -20C. To each sample (0.5
ml) was added 1.25 nmol of deuterated DL-chiro-inositol and
17.5 nmol of deuterated myo-inositol. The samples were then
deproteinized by ZnSO4 using Method I of Somogyi as
modified for plasma (11). The sample was diluted until its
conductivity was <2.5 pS and then passed over a 1-ml
column of prewashed AG501-X8(D) 20-50 mesh (Bio-Rad)
mixed-bed resin to remove ionic substances. The column was
washed twice with 0.6 ml of water and to the combined
eluates was added 1 g (wet weight) of prewashed Amberlite
IRA-400 hydroxide resin. The samples were shaken for 30
min, the supernatant was removed, the resin was washed
with 0.5 ml of water, and the samples were lyophilized.
Abbreviations: NICI GC/MS, negative-ion chemical ionization gas
chromatography/mass spectrometry; NIDDM, non-insulin-depen-
dent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus;
PFP, pentafluoropropionic; El GC/MS, electron ionization gas chro-
matography/mass spectrometry.
tTo whom reprint requests should be addressed.
 Medical Sciences: Ostlund et al.
The processed samples were converted to hexakis[pen-
tafluoropropionic (PFP)] esters by reaction overnight at 65C
with 150,ul of a 1:1 (vol/vol) solution of PFP anhydride and
acetonitrile. Most of the derivatizing reagent was removed
prior to chromatography in order to avoid damage to the GC
column. Plasma samples of 10-60,ul were reduced to a few
microliters under a stream of nitrogen and taken up in 7-15
Al of acetonitrile, whereas urine samples of 5-10,ul were
similarly reconstituted in 10-30,ll of acetonitrile. Analyses
were then performed as rapidly as possible to minimize
time-dependent losses. Because of the large difference in
concentrations of chiro-inositol and myo-inositol, adjust-
ments in the volume injected sometimes had to be made in
order to measure each inositol.
NICI GC/MS was performed on a Hewlett-Packard 5988A
with an ion-source temperature of 100C using a Vector-1
data system (Teknivent, St. Louis). Methane was used as
reagent gas. Sufficient sensitivity for the analysis of plasma
chiro-inositol was achieved by injecting chiro-inositol PFP
ester and adjusting the source pressure of methane to achieve
maximum response.
Separation of D- and L-chiro-inositol PFP esters was
achieved on a Chirasil-Val fused-silica capillary column [25 m
x 0.32 mm (i.d.) Alltech Associates] by modification of a
published procedure (12). Typical chromatographic condi-
tions were injection-port temperature, 180C, and pressure, 5
psi helium (1 psi = 6.89 kPa); injector split ratio, 12:1;
temperature program, 100C for 3 min, then 40C/min to
165C for 3 min, then 60C/min to 185C and hold for 3 min.
Under these conditions D- and L-chiro-inositol PFP esters
were eluted at 174 and 186 sec, respectively (Fig. 1), and
myo-inositol PFP ester was eluted at 514 sec (data not
shown). Quantitation was carried out by monitoring m/z 1036
and m/z 1041, the analyte inositols and the deuterium-labeled
internal standards, respectively (these ions are highly specific
for the PFP inositols). Chromatographic peak heights were
used for quantitation. Calculation of the unknown inositols
was done by reference to a standard curve produced by
plotting the ratio of analyte peak height to internal-standard
peak height versus the amount of inositol in each standard.
The standard curve was linear over the range 15-8000 nmol
of D-chiro-inositol per liter of plasma or urine (r2 0.999).
This procedure measures only free inositols. Analysis of
PLASMA chiro-INOSITOL
m/z 1036 D-chiro
r>,N
4000-1/
1 / \
2000- // \ L-chiro
z and Fig. 2 illustrates D-chiro-inositol levels observed. Uri-
170 180
INTERNAL STANDARD chiro-INOSITOL
cc L-chiro
D-chiro
I-.
4c
180
SECONDS
FIG. 1. NICI GC/MS of a diabetic patient plasma with simulta-
neous monitoring of the PFP derivatives of natural chiro-inositols at
m/z 1036 (Upper) and internal standard chiro-inositols at m/z 1041
(Lower). The slightly earlier elution time of the internal standard is
characteristic of deuterium-labeled chiro-inositol PFP esters.
Proc. Natl. Acad. Sci. USA 90 (1993) 9989
normal and diabetic samples before and after hydrolysis in 6
M HCl for 24 hr at 110C showed that D-chiro-inositol was
92% free in plasma and 93% free in urine; myo-inositol was
25% free in plasma and 92% free in urine.
Subjects. Table 1 gives characteristics of the subjects and
values for plasma and urinary inositols. Diabetic subjects
were recruited from the Washington University Diabetes
Registry, and controls from students and medical-center
personnel. Subjects with serum creatinine > 1.7 mg/dl were
excluded. The effect of insulin treatment was studied in two
groups of diabetics with poor control. The first consisted of
seven NIDDM subjects taking maximum doses of sulfonyl-
ureas who were judged by their physicians to require insulin
treatment and who had glycated hemoglobin of 15.4 2.8%
(mean SD). They received 0.45 unit of insulin per kg daily
by subcutaneous injection, and urinary inositols were mea-
sured before and after 1-20 days (mean, 11 days) of insulin
therapy. Fasting plasma glucose declined from 293 34 to
160 67 mg/dl. The second group (see Fig. 4) consisted of
six insulin-treated and one diet-treated diabetic subjects with
glycated hemoglobin of 11.8 2.5% (four with IDDM and
three with NIDDM). They were admitted to the Washington
University Clinical Research Center where fasting samples
were taken at 8:00 a.m. followed at 2:00 p.m. by an intrave-
nous insulin infusion beginning at 1 unit/hr with hourly
adjustments so as to normalize the plasma glucose in =6 hr.
Meals were given as usual. The-infusion was continued for 18
hr, and then overnight fasting plasma was again taken.
Statistical Methods. Plasma and urinary chiro-inositol lev-
els were not normally distributed. Measured values of
D-chiro-inositol were replaced with ranks for correlation and
multiple regression analyses by using the general linear model
of the statistical analysis system (SAS) (SAS Institute, Cary,
NC). Groups were compared with the Wilcoxon two-sample
test. Two-tailed probabilities were employed.
RESULTS
Three experimental considerations were necessary to enable
the measurement of chiro-inositol in plasma. (i) An internal
standard deuterated chiro-inositol was included before sam-
ple purification to correct for recovery and to compensate for
=
variations in instrument performance. (ii) NICI GC/MS was
used rather than electron ionization (EI) GC/MS with posi-
tive-ion detection. In this way we achieved >300-fold in-
creased sensitivity compared with the positive-ion El
GC/MS methods previously reported (7). (iii) Glucose and
other reducing sugars were removed with IRA-400 hydroxide
resin. With these modifications it was possible to measure the
levels of D-chiro-inositol in all 90 plasma and urine samples
tested.
Table 1 presents the characteristics of the subjects studied
190 nary excretion of D-chiro-inositol was much higher in dia-
betics than in controls (Fig. 2A). Median urinary excretion
was 12.0 umol/day in NIDDM (P < 0.0001) and 74 ,umol/day
in IDDM (P < 0.0001), compared with only 2.09 ,umol/day in
nondiabetic controls. Fig. 2B shows that there was no
significant difference in median plasma D-chiro-inositol lev-
els between diabetics and normals. Fig. 2C demonstrates that
the calculated renal clearance of D-chiro-inositol was in-
creased in the diabetic subjects (median values, 12.3 ml/min
in nondiabetics and 160.4 ml/min in diabetics; P < 0.0001).
The magnitude and physiological meaning of increased
urinary D-chiro-inositol excretion in diabetes are seen more
clearly in Fig. 3, where D-chiro-inositol clearance (o) and
L-chiro-inositol clearance (o) are plotted as a fraction of the
creatinine clearance, a measure of the glomerular filtration
rate. In nondiabetics the fractional clearances of both chiro-
inositols were similar and small, the median values being
9990 Medical Sciences: Ostlund et al. Proc. Natl. Acad. Sci. USA 90 (1993)
Table 1. Characteristics of the subjects
Normal NIDDM IDDM
No. of subjects, women/men 19/23 22/13 6/7
Age, years 43.3 15.2 51.3 13.3 38.8 16.6
Body mass index, kg/M2 29.5 8.1 30.9 5.6 24.4 3.6
Fasting plasma glucose, mM 5.3 0.6 11.7 3.9 11.7 6.0
Glycated hemoglobin, % 5.1 0.9 11.7 3.2 10.2 3.5
Plasma insulin, pM 64.4 48.1 110.9 72.6* *
No. treated, insulin/sulfonylurea 0/0 6/22 13/0
Microalbuminuria/proteinuriat 7/2 1/4
Urine excretion
D-chiro-Inositol, ,umol/day 2.09 (0.14-160) 12.0 (0.28-341) 74.0 (1.5-320)
L-chiro-Inositol, ,mol/day 0.33 (0.0-8.96) 0.36 (0.0-4.92) 0.96 (0.16-3.42)
myo-Inositol, ,mol/day 88.0 (13-538) 789 (11-3606) 825 (159-1554)
Glucose, g/day 12.2 (0.1-208) 14.1 (1.1-101)
Plasma concentration
D-chiro-Inositol, nM 95.2 (19.3-5625) 97.6 (5.9-1761) 84.3 (28.0-1687)
L-chiro-Inositol, nM 26.6 (2.6-321) 34.4 (3.8-256) 10.1 (3.0-73.8)
myo-Inositol, AM 24.5 (17.5-40.7) 24.1 (13.2-40.9) 24.1 (14.9-36.2)
Renal clearance, ml/min
Creatinine 106 30 109 39 113 23
D-chiro-Inositol 12.3 (0.65-86.7) 145 (2.5-3301) 416 (14.1-5429)
L-chiro-Inositol 12.9 (0.0-208) 11.7 (0.0-200) 66.0 (7.8-482)
myo-Inositol 2.4 (0.44-13.6) 20.6 (0.43-96.7) 22.1 (4.4-50)
Median and range are presented for inositols and mean SD for other variables.
*Plasma insulin was not measured in the 13 IDDM and 7 NIDDM subjects receiving insulin treatment.
tNo. of subjects with microalbuminuria (24-hr albumin excretion, 30-300 mg/day) or gross proteinuria (24-hr albumin
excretion, >300 mg/day).
0.126 and 0.154 for the D and L isomers, respectively. In gender, and whether or not sulfonylureas were given. There
NIDDM the median fractional clearance of L-chiro-inositol was no relation between plasma insulin and urinary D-chiro-
was essentially unchanged at 0.147. However, the median inositol.
fractional clearance of D-chiro-inositol in NIDDM was in- Urinary D-chiro-inositol excretion was particularly ele-
creased to 1.39 (P < 0.0001). In IDDM patients the median vated in sulfonylurea-treated NIDDM patients in poor con-
fractional clearance of L-chiro-inositol was also increased trol in whom insulin treatment was about to be instituted.
above normal to 0.51 (P = 0.0004), but that for D-chiro- Seven such subjects were studied before and after the sub-
inositol was much higher at 4.12 (P < 0.0001). The clearance cutaneous administration of 0.45 unit of NPH insulin per kg
of D-chiro-inositol exceeded the clearance of creatinine in per day. Prior to insulin treatment, median urinary D-chiro-
71% of the diabetics and none of the normals. While myo- inositol excretion was 174 ,umol/day (range, 97-250), 80-fold
inositol clearance was also increased in diabetics (Table 1), higher than the median value of 2.1 gmol/day in 42 normal
the median fractional myo-inositol clearance was only 0.21 in subjects (P < 0.0001). After insulin treatment for 1-20 days,
diabetics and did not exceed creatinine clearance in any the median urinary level fell 63% to 64.9 umol/day (range,
diabetic patient. Taken together, these data suggest that 2.9-162.5; P = 0.002 for the change).
urinary loss of D-chiro-inositol in diabetes is far greater than Intensive insulin treatment appeared to raise plasma
the loss of either L-chiro-inositol or myo-inositol. D-chiro-inositol levels selectively. In a second group of seven
The increased excretion of urinary D-chiro-inositol was poorly controlled diabetic subjects, six of whom were already
related to diabetic control. The correlation coefficient (r) in the taking insulin, an intravenous insulin infusion was given and
entire group of subjects between urinary D-chiro-inositol ex- plasma glucose was normalized by hourly measurement of
cretion and glycated hemoglobin was 0.529 (P < 0.0001), and plasma glucose levels and adjustment of the infusion rate.
that between D-chiro-inositol excretion and fasting plasma Plasma inositols were measured in the fasting state before
glucose was 0.568 (P < 0.0001). In an analysis of covariance and 18 hr after the insulin infusion was started (Fig. 4).
both diabetes classification (nondiabetic, NIDDM, IDDM) Median plasma D-chiro-inositol was initially low, at 10 nM
and glycated hemoglobin level were independent predictors of (range, 6-74), compared with the nondiabetic median of 95
urinary D-chiro-inositol excretion (P = 0.0033 and P = 0.0034, nM (range, 19-5625; P = 0.0003). After prolonged insulin
respectively). In diabetics, urinary D-chiro-inositol excretion infusion, plasma D-chiro-inositol levels increased 8.8-fold to
was significantly correlated with urinary glucose excretion (r a median of 180 nM (P = 0.02), whereas there was no change
= 0.368, P = 0.01), but proteinuria (microalbuminuria plus in the L-chiro-inositol levels (5 nM before and 6 nM after).
gross proteinuria; Table 1) was not related to either urinary or Plasma myo-inositol decreased 12% after insulin treatment (P
plasma D-chiro-inositol (P > 0.58). D-chiro-Inositol excretion = 0.02), consistent with a previous report (13). The one
and plasma levels were not associated with body mass index, patient who did not respond to intravenous insulin was a
age, or gender. substantially overweight (body mass index = 41) 42-year-old
Fasting plasma insulin was measured in the 70 subjects woman with NIDDM who had severe insulin resistance
who were not receiving insulin treatment (Table 1). There manifested by an intravenous insulin requirement of 3
was no relation of plasma D-chiro-inositol to plasma insulin units/hr to maintain normoglycemia.
in the nondiabetic subjects (P = 0.41). However, plasma
insulin was significantly inversely correlated with plasma DISCUSSION
D-chiro-inositol in the 29 NIDDM subjects not receiving
exogenous insulin (r = -0.423, P = 0.02). This relation was The data presented here show that D-chiro-inositol metabo-
independent of plasma glucose, glycated hemoglobin, age, lism is substantially altered in diabetes mellitus. Both
 Medical Sciences: Ostlund et al. Proc. Natl. Acad. Sci. USA 90 (1993) 9991
I~~~~~~~~~~~~~~~~~~~~~~~~~
A B 0
0
0 0
0

0
8 0O
100 oO 8 8

1
0
0

8 o
8
0~~~~~~~~0

0 80

la 10 I-1
*o
0 I- 0.1 F
I., 0
~0
go
0 'U
0 * 0
r._ 0
0
o 0
0~~~~~~~~
0
O
1 0.01 F 0
0
0

8
0.1 0.001
NORMAI, NIDDM IDDM NORMAL NIDDM IDDM

C I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0
0

iooo1 0

c)
0
'U
s
3-
8
C)
cU0a 100i
8
co
-3
C1
0

.2
S~~~~~~~~
o 0
10 [ 8 i
a
8 FIG. 2. Box plots of urinary D-chiro-inositol excretion
0 (A), plasma D-chiro-inositol level (B), and D-chiro-inositol
0 clearance (C). The box encloses the 75th to 25th percen-
tiles, the single horizontal line depicts the median, and the
1 error bars enclose the 10th and 90th percentiles. Note that
NORMAL NIDDM IDDM ordinate values are plotted on a logarithmic scale.
NIDDM and IDDM patients had large increases in urinary diabetes is unusually large. Clearances exceeding the glo-
D-chiro-inositol excretion compared with normal subjects, merular filtration rate can occur if D-chiro-inositol is secreted
and the most potent predictor of increased urinary levels was by the renal tubules or produced de novo by the kidney or if
diabetic control as measured either by glycated hemoglobin plasma levels of D-chiro-inositol change markedly during the
or by plasma glucose. However, a careful examination of the day. The current data do not allow a distinction between
data shows that the urinary loss of D-chiro-inositol was rather these possibilities.
specific and out of proportion to that expected in diabetic The effects of diabetes and short-term insulin treatment
glycosuria. Fig. 3 demonstrates that D-chiro-inositol clear- can be seen most clearly in two smaller groups of diabetics
ance exceeded the creatinine clearance in 71% of the diabet- who were selected for inadequate control of the plasma
ics but none of the nondiabetics. In contrast, L-chiro-inositol glucose and who were studied more intensively. The first
clearance exceeded the creatinine clearance in only 2% of the group consisted of NIDDM patients in whom sulfonylurea
nondiabetics and 15% of the diabetics, and myo-inositol treatment had failed. Baseline urinary D-chiro-inositol was
clearance did not exceed the creatinine clearance in any of found to be increased 80-fold over nondiabetic controls. After
the subjects. Thus, the urinary loss of D-chiro-inositol in subcutaneous insulin treatment urinary levels were reduced
9992 Medical Sciences: Ostlund et al.
1
S
*1 0~~~~~~~~~~~~
'5 0.10
0~~~~~~~~~~
* 0
- 0 S~~~~~~~~~~
go
C.., 0~~~~~
S of urine comparing our standard method with positive-ion El
0.01 0
NORMAL NIDDM IDDM
FIG. 3. chiro-Inositol clearance expressed as a fraction of cre-
atinine clearance. *, L-chiro-Inositol; 0, D-chiro-inositol. Boxes are
described in the legend to Fig. 2.
by 63% but still remained elevated, as was true for most of the
general diabetic population shown in Fig. 2A. Fasting plasma
glucose, while significantly improved, also remained ele-
vated at 160 + 67 mg/dl. The second group consisted
principally of diabetics with poor control despite prior treat-
ment with insulin (Fig. 4). Here the plasma D-chiro-inositol
levels were measured and found to be reduced by 89%
compared with the levels in nondiabetics. However, after
intensive intravenous insulin treatment for 18 hr, plasma
D-chiro-inositol levels rose to the normal range. Taken to-
gether, these data suggest that the effect of insulin is to
decrease renal losses of D-chiro-inositol, with secondary
increases occurring in the plasma. It is possible that insulin
treatment may also cause shifts in D-chiro-inositol between
plasma and tissue compartments. The independent inverse
correlation between plasma insulin and plasma (but not
urinary) D-chiro-inositol levels in NIDDM patients is evi-
dence for a link between insulin (or insulin resistance) and
D-chiro-inositol metabolism which is independent of the
amount of D-chiro-inositol excreted in the urine. If the
hypothesis that D-chiro-inositol and insulin metabolism are
interrelated is correct, then the large renal losses of D-chiro-
inositol may be an adverse metabolic effect of the diabetic
state.
Our results for D-chiro-inositol urine analyses are different
from those reported in two previous studies (7, 14). Those
investigators found that urinary D-chiro-inositol was reduced
rather than increased in NIDDM. The reason for this dis-
crepancy is not known, but it is possible that analytical
differences might be responsible in part. The previous
method relied on quantitative recovery through several pu-
rification steps and did not employ an internal recovery
standard (7). We experienced losses of chiro-inositol both in
the purification procedure and when the samples stood after
being transferred from the derivatizing reagent to acetonitrile
in preparation for injection into the mass spectrometer.
Nonquantitative recovery was also observed when deriva-
Proc. Natl. Acad. Sci. USA 90 (1993)
soo 500 30
0
^400 _ 400
-a00 .0 20
0
: 300 a 300 10
._ Q
.-
g 200 "I 200 cc
CU
._
EI? 100 8 100
cU 0 oo 0
0
Pre Post Pre Post Pre Post
FIG. 4. Inositol levels in plasma before and after treatment of
seven poorly controlled diabetic subjects with intravenous insulin for
18 hr.
tized samples were extracted with cold hexane according to
the procedure described (7). Disagreement in our results does
not appear to be related to the derivatives made or to the mass
spectrometers used, since we have performed parallel assays
GC/MS using trimethylsilyl and heptafluorobutyryl deriva-
tives and found that the levels measured were not substan-
tially different with the various techniques (data not shown).
Our nondiabetic group was composed of individuals with a
wide range of body mass index and age. However, both
stratification by those variables and use of them in multiple
regression analyses indicated that they were not closely
related to D-chiro-inositol levels in plasma or urine and are
not likely to account for our different results. However, the
large urinary losses we report here are entirely consistent
with the deficient D-chiro-inositol content noted previously in
inositol phosphoglycan fractions purified from human dia-
betic muscle (7).
The current work shows that both insulin and diabetes
have specific effects on D-chiro-inositol metabolism and
supports the notion that D-chiro-inositol-containing com-
pounds or the inositol itself may have biological relevance as
markers or even effectors of insulin action. Whether and how
disordered metabolism of D-chiro-inositol relates to insulin
action and putative insulin modulators containing D-chiro-
inositol remains to be elucidated.
We thank Carolyn Fritschle, Nels Holmberg, and Eldon Koch for
important technical contributions. This work was supported by
National Institutes of Health Grant HL29229 and an American
Diabetes Association Clinical Research Grant to R.E.O. and by the
Washington University Diabetes Research and Training Center
(AM20579), General Clinical Research Center (RR00036), and Mass
Spectrometry Resource (RR00954).
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