Método Disgnóstico

1. Inositol as a
Biomarker for Type II
Diabetes

2. - Discovered in the 1850’s by Scherer in a meat extract
made by Liebig
- There are nine inositol isomers, of which two are
enantiomers of each other, namely D- and L-chiro-Inositol
Why Inositol
Fisher, S. K. J Neurochem 2002; 82(4): 736-754

3. The Cori Discovery That Lead to
The 1947 Nobel Prize in Medicine
In their study of how glycogen is broken down in the body
Carl and Gerty Cori showed that the reaction of glycogen to
glucose 6-phosphate was probably reversible.
G1P → G6P

4. Glucose 6-phosphate is also a Precursor
in the myo-Inositol Biosynthesis
Pathway

5. Dr. Joseph Larner, a graduate student in the Cori
Laboratory discovered the glycogen “debranching”
enzyme, amylo-1,6-glucosidase, along with Gerty Cori
Cori, G.T. JBC 1951; 188(1): 17-29

6. The defect in Type II diabetics may be partially due to the
body’s inability to process glucose properly, known as
insulin resistance. Perhaps enzymatic defect(s) of one or
more reactions somewhere along the pathway from blood
glucose to the mediator’s final action inside the cell to lower
blood glucose (glucose disposal, both oxidative and non-
oxidative pathways).
The quest for that insulin mediator was initiated
I worked with Dr. Larner for over 30 years. It was during this
time that he showed a dual action of insulin (Larner, J. Adv.
Second Messenger Phosphoprotein Research 1990; 24: 290-
294) suggesting an intermediary, or mediator, which works
to lower blood glucose.

7. Partial structures for two of those mediators have been
determined through isolation from rat liver. One of the early
chromatography steps (AG1x8 resin) separates the two
putative mediators by elution with pH 2.0 HCl and then pH 1.3
HCl
1. chiro-Inositol + galactosamine + mannose(s) +
ethanolamine
2. myo-Inositol + glucosamine + mannose(s)) +
ethanolamine
The chiro mediator was shown to stimulate PDHP whereas the
myo mediator was shown to inhibit cyclic AMP kinase

8. The pH 2.0 eluate contains the putative chiro-Inositol mediator
The pH 1.3 eluate contains the putative myo-Inositol mediator
Larner, J. BBRC 1990; 151(3): 1416-1426
Figure 3

9. Larner, J. BBRC 1990; 151(3): 1416-
1426
Figure 6

10. pH 1.3 Putative myo-Inositol Mediator Acts
to Inhibit cAMP Kinase
Larner, J. BBRC 1990; 151(3): 1416-1426

11. Alison Kennington, a graduate student in Dr.
Larner’s laboratory, discovered the presence of
chiro-inositol, mainly the D-epimer, in human urine.
A study involving both University of Virginia and Pima
Indian subjects was subsequently undertaken

12. Pima Indian vs. University of Virginia Subjects
Type II Diabetics vs. Normals
Bogardus and Kennington Phoenix AZ and Charlottesville VA
M/C
57
M/C
1.7
M/C
27
M/C
2.8
Kennington, A. New England J Med 1990; 323(6): 373-378
Kennington, A. New England J Med 1990; 323(6): 373-378

13. In 1996, Larner and Craig in their paper, “Urinary myo-
lnositol-to-chiro-lnositol Ratios and Insulin
Resistance” first suggested the use of myo-inositol to
chiro-inositol ratio as opposed to examining the chiro-
inositol content
Larner, J. Diabetes Care 1996; 19(1): 76-78
Ratio (n)

14. Structure determined by 2D NMR and chemical synthesis as 4-O-(2-amino-2-
deoxy-β-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol (Larner, J. J Med
Chem 2003; 46(15): 3283-3291)
Free amino group on galactosamine and the rare inositol pinitol suggested two
potential precursors – GPI-Phospholipids and GPI-Proteins
Galactosamine
β-1,4 Linkage
Pinitol
Rubenstein - S. Africa
Alfalfa (Medicago sativa)
The Lancet 1962; 280(7270): 1348-1351
Narayanan - India
Bougainvillea spectabilis
Curr Sci (India) 1987; 56(3): 139-141
Compound synthesized
Named INS-2
a pseudo-disaccharide
a DCI glycan
Partial Structure For a chiro-Inositol
Containing Mediator Synthesized

16. C3 hydroxyl oxidized to
keto group
C3 keto reduced to hydroxyl with
consequent bond inversion
3 3 3

17. Urinary chiro-Inositol Decreases Progressively from Normal
to Impaired Glucose Tolerance to Type II Diabetes in
Japanese subjects
Decreased urinary chiro-Inositol is directly related to insulin
sensitivity
Suzuki, S. Diabetes Care 1994; 17(12): 1465-1468

18. Dr. Manassés Fonteles found that infusion treatment
with INS-2 (putative chiro-inositol mediator) in
streptozotocin-diabetic rats significantly lowered blood
glucose
Fonteles, M. C. Diabetologia 1996; 39(6): 731-
734

19. Shashkin, P. N. Diabetologia 1997; 40(5): 557-563
The PDHP bioactivity of the partially purified pH 2.0
mediator in serum was tested by Katz’s group in
Sweden
n = 6 healthy males
n = 7 insulin resistant
obese an diabetic males

20. Ku, B. J. Korean J Med 2007; 72(1): 29-36
Twelve Week Treatment with Pinitol Showing
Changes in Various Glucose Control
Parameters

21. Connection Between Diabetes and Hypertension
Some of the common variables between hypertension, or
high blood pressure, and diabetes are insulin resistance,
inflammation, increased oxidative stress, dyslipidemia,
hypercoagulability and endothelial damage
Zhou, M.-S. Diabetol Metab Syndr 2014(1); 6: 12-19

22. Analytical Techniques used in the
Fonteles Laboratory, Fortaleza, Brazil
2015 to 2018

23. Sample Cleanup Protocol
1. If the sample contains a considerable amount of
protein, such as saliva, then it is first deproteinized - 1
mL aliquot of sample + 3 mL ice-cold abs. ethanol in
glass centrifuge tube. Vortex thoroughly and incubate
in refrigerator for a minimum of 2 hours, preferably
overnight. Centrifuge at high speed using a clinical
centrifuge. Decant supernatant and speedvac (vacuum
centrifuge) the decanted supernatant to dryness.
2. One mL sample is loaded onto 3 mL bed volume
mixed-bed ion exchange medium, (TMD-8), which has
been pre-equilibrated with HPLC grade H2O. Collect
pass-through (PT). Wash column with 3 mL H2O,
collect PT. Pool PT and speedvac to dryness.
3. Pre-equilibrate a C18 SepPak cartridge with 3 mL
methanol and then 3 mL H2O. Reconstitute sample in 1
mL H2O. Pass through C18. Collect PT. Wash column
with 3 mL H2O. Collect PT. Pool PT and speedvac to
dryness.

24. A method to analyze urinary and salivary
myo-inositol for use as a potential biomarker
for Type II Diabetes had to be found

25. First assay found was a UV method using benzoyl
chloride as the UV absorbing and derivitizing agent.
myo-Inositol is mixed with benzoyl chloride in an
alkaline organic medium. Inositols are not
chromophoric and hence are not detected by UV
methods. However, once derivitized with a
chromophoric agent the derivitized compound will
theoretically absorb in the UV light spectrum. The
system consisted of a Shimadzu CLC C18 HPLC
column, two gradient pumps, column heater, UV
monitor, Rheodyne manual injector and a Shimadzu
Chromatopac recorder.
Frieler, R. A. J Chromat B 2009; 877(29): 3667-3672

26. Method was attempted a number of times without success.
After consultation with Dr. Rosenberger, the PI for the
group that published the article, some suggestions were
made:
Oven: 55°C Allow for column equilibration time
Derivitization: No heating
After adding 4N NaOH be sure to allow time
for sample dissolution. Gently rotate tubes
so
as to provide as large a surface area and
agi-
tation at the organic - H2O interface
Resolvation: Add acetonitrile first. Make sure sample is
dissolved and then slowly add water.
Throughly mix the samples before injecting.
The protocol was followed as closely as possible but
continued to get a white insoluble ppt. That ppt was
insoluble in H2O, acetonitrile and n-hexane. The injected
sample still did not yield adequate chromatography. The
method was then scrapped and another method was

27. The next method under investigation used refractive index
as the detector. System consists of a Shimadzu RID-6A
Refractive Index Detector, single isocratic pump,
Rheodyne manual injector and a Asahi Shodex NH2P (an
amino group attached to polyvinyl alcohol gel) HPLC
column. This is following a dissertation by Karla Alves
(Fortaleza 2012),
“Estudo dos Níveis Salivares de Mioinositol e
Quiroinositol em Crianças Saudavais e Portadoras de
Diabetes Infanto-Juvenil”.
The solvent used was 25% acetonitrile / 75% H2O with a
flow rate of 0.4 mL/min. A glucose standard was prepared.
According to the dissertation glucose had a retention time
of 5 min 20 sec.

28. After several attempts no peaks were observed. What were
her instrument operating parameters? Could not find so
had to make an educated guess. Followed the conditions
used for the Shodex test chromatogram, which was
included with the column
Some technical issues required thorough cleaning of
column and RI
Why 25% acetonitrile? It is customary to use 75%
acetonitrile

29. Once the instrument was fixed the following standard
curve for myo-Inositol was obtained:
Despite the nice standard curve for myo-inositol no
resolution for glucose, chiro-inositol and myo-inositol,
three compounds of interest, was observed. Therefore it
was decided to try another HPLC column: Tosoh Haas
Amide-80 4.6 mm × 10 cm. The solvent ratio was changed
to 80% acetonitrile / 20% H2O
0 200 400 600 800 1000
0
100000
200000
300000
400000
500000
RIU
Conc (µg/mL)
Myo Inositol RI
lowest data points removed
5-5-16
y = 376 x + 52915
r2
= 0.9598

30. The following chromatogram was obtained after adjusting
the parameters: 0.8 mL/min; 80% acetonitrile / 20% H2O;
column at room temperature; Speed 3; Attenuation 5 and
Range 2:
This method was checked several times and sure enough
very similar chromatograms were obtained. The following
validation tests were run: Recovery, Dose Response, and
CV. LLOQ and LLOD were calculated
.

31. For the Recovery test a pooled saliva sample was
prepared. myo-Inositol was spiked into the saliva

32. A Standard Curve was Prepared
Myo (µg/mL)
Area
(RI)
0 200 400 600
0
100000
200000
300000
400000
y = 721 x - 10581
r2 = 0.986
Myo Inositol
Standard Curve
July 2016
Amide-80
Serial 19533
4.6 mm x 100 mm
Acetonitrile / H2O 80% / 20%
0.8 mL/min

33. LLOQ 100 μg/mL LLOD 30 μg/mL

34. Finally, samples are analyzed

35. Rat Renal Cortex
Three Euglycemic and two Diabetic rat renal cortex samples
received for analysis. Two samples of pinitol (10 mg and 1
mg) and one sample of myo-inositol (10 mg) were also
submitted for analysis
A trend toward lower myo-inositol in the diabetic rat is
seen, despite low sample number and large variation.
There was good agreement with the myo-inositol sample
but the amount analyzed for pinitol was not in agreement
with what was measured out.

36. Rat Urine myo-Inositol
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0
1000
2000
3000
Myo
(mg/mL)
myo-Inositol
Rat Urine
7-12-16

37. Another seven rat renal cortex
samples submitted for analysis
No change in glucose. Both diabetic chiro and myo
levels higher than euglycemic

38. A 3 month × 20 mg/kg b.i.d. treatment in diabetic rats
with pinitol showed a slight lowering of tissue glucose
(ns)
Treatment with Pinitol Shows a Small Decrease in Tissue Glucose

39. Ten samples from CIDH (Centro Integrado de Diabetes e Hipertens
Samples prepared for analysis by RI HPLC

40. Seventy two more samples were received
from CIDH
All samples were cleaned up prior to starting
the analysis

43. FIM
Muito obrigado para todo