Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Supplemental Materials:
MATERIALS & METHODS
SOLVENT STUDIES
Dansyl Azide Trialed in Various Solvents
100mM dansyl azide (DnAz) standard solutions were prepared at room temperature. Solvents
tested included acetonitrile, ethanol (EtOH), ethylacetate, and dimethylformamide; attempts at
aqueous dilution of these solvents was met with DnAz precipitation. 100mM DnAz in EtOH
(DnAz-EtOH) or 50mM DnAz in EtOH (DnAz-EtOHdil) was prepared for all subsequent studies
and stored at room temperature under a UV protective covering.
Sodium Sulfide Nonahydrate Trialed in Various Solvents
A stock solution of sodium hydrosulfide in deionized water was prepared for initial internal
standard trials but questioning of reagent purity led to subsequent disuse of this compound. A
new preparation of sodium sulfide nonahydrate (Sigma-Aldrich: >98%, Na2S-9H2O, CAS 131384-4) was trialed and utilized for the duration of the study. 1mM sodium sulfide nonahydrate
stock solution was prepared fresh daily by mixing of the crystal with the chosen solvent in a
sealed plastic container at room temperature. Solvents tested included deionized water, 0.9%
normal saline (NS), 5% dextrose in water (D5), lactated ringers (LR), and 5% dextrose in 0.225%
normal saline (D5NS). 1mM sodium sulfide nonahydrate in NS was utilized for all further
studies (H2S-Stan).
1
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
INTERNAL STANDARD STUDIES
Internal Standards in Human Serum
Human whole blood was collected into either lithium microtainers (BD Microtainer®TM tube
with lithium heparin, #365971) (MT) or into heparinzed 3mL vacuum tubes (Greiner Bio-One
3mL Lithium Heparin Separation Vacuette, Ref#454247) (vial). Serum was separated after
centrifugation at 3000rpm for five minutes; each MT yielded approximately 250μL of serum,
each vial typically yielded 1.5mL of serum. The serum was separated into 200μL aliquots and
placed into individual eppendorf tubes for further treatment.
Prior to application of H2S-Stan to eppendorf aliquots DnAz-EtOH(dil) was added either to
whole blood prior to centrifuging (sample ID-pre), to samples after serum separation (sample
ID-fol), or both to whole blood and then subsequently again to serum (sample ID-pre&fol).
Unless prepared specifically as a background fluorescent intensity scan for changes with
application of H2S-Stan (no DnAz added) all blood collections were treated with DnAz-EtOH(dil)
to a final target concentration of 200μM or 400 μM. H2S-Stan was added to DnAz-EtOH(dil)
treated serum samples in concentrations of 0μM to 100μM. Containers were then sealed and
stored at room temperature under UV protective covering until read by fluoroscopy, typically
one to three hours.
HUMAN SERUM STUDIES
Protocol Development
2
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
The temporal order of application of the DnAz dilution to whole blood and serum samples was
investigated as described in the Internal Standards and Human Serum section (sample IDpre/post). Variations are described here. Whole blood collection syringes preloaded with pure
DnAz oil (sample ID-syringe) were trialed for gas trapping integrity. Pure DnAz oil was also
trialed as a smear along the walls of the MT (sample ID-pure) prior to addition of whole blood.
A single volunteer (Subject V) was utilized for serial blood collection and serum analysis during
the protocol development phase of the project. All samples from Subject V were drawn from
antecubital sites, including those taken for comparison under the variable oxygen conditions
discussed in the Human Volunteer Study section.
Solubility and Stability Assessment of DnAz-EtOH
Increase in baseline fluorescence during human subject trials led to interrogation of the DnAz
and laboratory tools being used. Solubility of the reagents, unintentional reduction of the
azide, and equipment failure were investigated.
i) Solubility: Increased difficulty in dissolving of the DnAz in EtOH when prepared as a
100mM stock was noted as was questionable precipitation upon injection of the
concentrate to both whole blood and buffer samples. Subsequent to deterioration of
internal standard reliability in Subjects C and E, following previously successful collections
in Subjects A and B, the solid DnAz being used was interrogated under nuclear magnetic
resonance (NMR) spectroscopy in deuterated dimethylsulfoxide (DMSO). Samples of
DnAz in DMSO (DnAz-DMSO)(100mM), DnAz in EtOH (100mM), and DnAz in EtOH(dil)
3
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
(50mM) were prepared. The reduced dansyl amide in DMSO (250μM)(DnNH2-DMSO) was
made available for fluorescent intensity comparisons. A sample of the prior batch of DnAz
oil used during protocol development was also prepared as DnAz in EtOH (100mM) but it
was noted that during the intermittent storage period the oil had converted to a solid and
no longer demonstrated its prior solubility profile in the EtOH. Selected samples prepared
in EtOH were subjected to microfiltration to remove insoluble particulates not visible to
the naked eye (DnAz-EtOH filter).
ii) Stability: DnAz-EtOH (100mM) stock as prepared fresh that morning (sample ID-fresh)
or from the prior day (sample ID-1d old) was added to a final concentration of 200μM to
vials of human whole blood either immediately after sampling (imm DnAz) or following a
fifteen minute delay after collection (15min DnAz). DnAz-EtOH (100mM) stock and DnAzEtOHdil (50mM) were compared in side-by-side runs on whole blood subjected to variable
oxygen conditions. DnAz-EtOHdil (50mM) was then compared in side-by-side runs as a
whole blood only addition (DnAz-EtOHdil pre) versus DnAz-EtOHdil (50mM) whole blood
and then serum addition (DnAz-EtOHdil pre/post) under variable oxygen conditions.
iii) Equipment and Supplies: Human whole blood samples collected in vials were treated
with DnAz-EtOH to final concentrations of 200μM or 400μM following exposure to the
variable oxygen conditions tested (see section involving human volunteer study). After
serum separation samples were treated with H2S-Stan to final concentrations of 0μM to
80μM and then read on either the 96-well plate utilized for prior experiments (old plate:
4
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
sample ID) or a new 96-well plate reader (new plate: sample ID). New pipette tips were
trialed to rule out reductive contamination by the stored internal standard solution
(sample ID old/new pip). Reagents were evaluated by new preparation of 20mM
phosphate/0.5% Tween 20 buffer compared to the prior batch (Buffer: old/new Tween),
and new EtOH reagent with >99% purity (sample ID-pure solv) compared to prior EtOH
supply.
RESULTS
SOLVENT STUDIES
DnAz Standard
DnAz in acetonitrile and ethylacetate caused frank distortion or melting of sample wells in the
plate reader and treated samples had to be discarded; dimethylformamide exhibited similar
instability with laboratory supplies though with a slower onset (Supplemental Figure 1). DMSO
was trialed while addressing solubility concerns during the human volunteer study but
considering general complaints surrounding handling of DMSO by laboratory staff, and a lack of
significant improvement in internal standard trendlines (Supplemental Table 1), EtOH was
chosen as the DnAz solvent for the duration of the study.
Internal Standard
Sodium sulfide nonahydrate (Na2S-9H2O) was chosen as the reagent for internal standard
testing due to concerns about the purity of our sodium hydrosulfide reagent; upon changing to
Na2S-9H2O improved correlation coefficients in buffer were obtained and the associated
5
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
pungent odor of hydrogen sulfide gas was more readily appreciated. Mixing of both sulfide
agents into deionized water (1mM) resulted in notable gas bubble formation on the sides of
glass vessels which instigated concerns of hydrogen sulfide gas loss or reagent consumption.
This concern was satisfactorily addressed by changing to low head-space plastic containers for
mixing and storage. ZnCl2 was trialed as a hydrogen sulfide gas-quenching agent but in human
plasma failed to show reliability and so was discontinued during the human volunteer study
(Supplemental Table 2).
As localized hemolysis was noted upon application of the Na2S-9H2O in deionized water to
whole blood alternative solvents common to the clinical environment were investigated (NS,
D5, D5NS, and LR). NS proved to be stable when applied to serum collected in MT (no vial
samples were treated in that test run with NS to compare in side-by-side analysis). The
dextrose solutions (D5 and D5NS) also yielded acceptable correlation coefficients when applied
to serum collected from vials. LR use resulted in clotting and loss of some samples, making the
resultant trendline appear artificially favorable. All samples that were treated in the MT after
DnAz addition but as whole blood prior to centrifugation and plasma collection showed clear
indications of hemolysis when compared to samples treated with the tested Na2S-9H2O solution
as a separated serum which remained a pale yellow (Supplemental Figure 2). As NS compared
favorably to deionized water in buffer and human serum (Supplemental Table 3) it was chosen
as the solvent for subsequent 1mM Na2S-9H2O stock preparations (H2S-Stan).
INTERNAL STANDARD STUDIES
6
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Internal Standards in Buffer: Solubility and Correlation Coefficients
A correlation coefficient (R2≥0.95) was chosen for evaluation of DnAz-EtOH(dil) integrity as a
H2S gas trapping agent in buffer in the setting of H2S-Stan application as an internal standard.
At least one of two buffer runs in a side-by-side analysis with serum samples on the same plate
read needed to yield R2≥0.95 for serum samples to be included in final data analysis. Trends
toward acceptable internal standardization were notable during the protocol development
phase of the experiment with gradual improvement in correlation coefficient values as
collection systems, solvents, and dilutions were trialed. The return of poor trendline
correlation (R2<0.95) in Subject C and E heralded the change to solubility of the DnAz that was
noted at that time (Supplemental Table 4).
Internal Standards in Human Serum: Solubility and Correlation Coefficients
A correlation coefficient (R2≥0.90) was chosen for evaluation of DnAz-EtOH(dil) integrity as a
H2S gas trapping agent in serum in the setting of H2S-Stan application as an internal standard.
Changes to solubility and the resulting decline in reliability of the DnAz-EtOH during the human
volunteer study instigated alterations to both concentration and temporal application of the
fluorescent trapping agent as the source of the new errors were investigated. Subject A and B
had whole blood samples treated a single time with 200μM DnAz-EtOH: both subjects showed
acceptable response to internal standard addition to serum with Subject B in particular
demonstrating what was likely full consumption of the 200μM of fluorescent agent during the
hypoxic exposure trial (Supplemental Table 4). Subject C and E, however, failed to yield
adequate internal standard profiles in buffer (R2<0.95) and furthermore demonstrated greatly
7
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
increased baseline fluorescent intensities from that which was seen previously; solubility of the
reagents, unplanned or premature reduction of the DnAz, fluorescent impurities, and
equipment contamination were all investigated as sources for this change. The conclusions
from this work were that neither pipette tips nor plates were the source for the increased
background fluorescence, nor was there significant new impurity in the DnAz-EtOH that was
causing high background (as demonstrated by the minimal increase in intensity change despite
doubling of the DnAz-EtOH (Supplemental Table 5). NMR studies under various solvents were
also undertaken upon the prepared DnAz with no evidence of accumulation of any significant
impurities (data not shown).
Solubility of the solid DnAz was, however, noted to be considerably different than experienced
with earlier runs where it was a pale yellow oil. While background fluorescent changes did not
return to previous numbers with dilution of the DnAz to a 50mM stock it did maintain or
improve internal standard capturing in buffer, in addition to showing overall lower background
intensity. Subject F and G reflect the changes seen with dilution of the azide (DnAz-EtOHdil)
which allowed for initial gas capture in whole blood (suggested by a higher 15%0 2 intensity),
but then failed to show significant change upon addition of internal standards to the separated
serum component. Considering the changes to solubility there was concern for precipitation of
the azide during the immediate period following sample collection. To test this theory a second
spike of the DnAz-EtOHdil (~200μM) was applied to the serum; this yielded return of internal
standard capturing (R2≥0.90) for the majority of the subsequent volunteer subjects
(Supplemental Table 4).
8
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Figure 1: DnAz trialed in various solvents
Legend: Frank melting of the bottom of the plate wells was noted upon addition of many of the
tested solvents. In particular rapid deterioration was seen with acetonitrile, ethylacetate, and
dimethylformamide. (Red arrows point to perforations in the reading windows of the wellplate.)
Figure 2: Human Serum Following Alternative Dosing Interval of DnAz
Legend: Differing levels of hemolysis was noted with variation of order in addition of the DnAz to human
whole blood and serum samples. Image shows both vial and microtainer (MT) collections.
9
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Table 1: Assessment of Dansyl Azide
R2
Sample ID
Notes
Solvent Trials for DnAz
Buffer DnNH2-DMSO
Buffer DnAz-DMSO
Buffer DnAz-EtOH
Buffer DnAz-EtOH filter
Serum EtOH (no DnAz)
Serum DnAz-DMSO
Serum DnAz-EtOH
Serum DnAz-EtOH (pure solv)
Serum DnAz-EtOH dilute
Serum DnAz-EtOH filter
Solubility/Dilution Trials of DnAz
0.281
0.977
1.000
1.000
0.089
0.554
0.039
0.864
0.869
0.200
DnNH2-DMSO (250μM) standard
Buffer
Buffer
Serum: PIVRA concentrated
Serum: PIVRA dilute
Serum: PIV15% concentrated
Serum: PIV15% dilute
Serum: PIV100% concentrated
Serum: PIV100% dilute
0.962
0.998
0.025
0.037
0.083
0.315
0.906
0.983
DnAz-EtOH (100mM) to buffer
DnAz-DMSO (100mM)
DnAz-EtOH (100mM)
DnAz-EtOH (100mM) dissolved & filtered; added to WB
EtOH added to WB, no DnAz
DnAz-DMSO (100mM) added to WB
DnAz-EtOH (100mM) added to WB
DnAz-EtOH (100mM) to WB, purified EtOH
DnAz-EtOH (50mM standard) added to WB vial
DnAz-EtOH (100mM) dissolved & filtered; added to WB
DnAz-EtOH (50mM) to buffer
DnAz-EtOH (100mM) to WB vial
DnAz-EtOH (50mM) to WB vial
DnAz-EtOH (100mM) to WB vial
DnAz-EtOH (50mM) to WB vial
DnAz-EtOH (100mM) to WB vial
DnAz-EtOH (50mM) to WB vial
*DnAz-EtOH (xmM) listed as stock solution; added as 200μM to WB
Abbreviations: ethanol (EtOH), dimethylsulfoxide (DMSO), dansyl azide (DnAz), dansyl amide (DnNH 2), peripheral
intravenous catheter (PIV)
Table 2: Assessment of ZnCl2 as Quenching Agent
Sample ID
Buffer
Buffer
Buffer
Buffer
Serum-MT
DnAzpure-MT
Serum-MT
Serum-vial
R2 (-)ZnCl
0.908
0.893
0.990
0.983
0.981
0.226
0.967
0.967
R2 (+)ZnCl
0.902
0.914
0.992
0.980
0.917
0.182
0.968
0.575
Notes
ZnCl2>baseline ("zero-point")
ZnCl2>baseline ("zero-point")
ZnCl2>baseline ("zero-point")
ZnCl2>baseline ("zero-point")
Abbreviations: microtainer (MT)
10
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Table 3: Assessment of Solvents for Na2S x 9H2O
R2
Sample ID
Buffer
Serum-Water
Serum-NS
WB-water
WB-D5
WB-D5NS
WB-NS
WB-LR
D5-vial
D5NS-vial
LR-vial
Notes
0.983
0.983
0.990
0.271
0.344
0.439
0.521
0.420
0.949
0.836
N/A
DnAz (100mM)
DnAz (100mM) added to MT after spin
DnAz (100mM) added to MT after spin
DnAz (100mM) added to WB in MT
DnAz (100mM) added to WB in MT
DnAz (100mM) added to WB in MT
DnAz (100mM) added to WB in MT
DnAz (100mM) added to WB in MT
DnAz added to serum in eppendorf tubes
DnAz added to serum in eppendorf tubes
clotting noted upon addition to serum
Abbreviations: 0.9% normal saline (NS), 5% dextrose (D5), 5%dextrose/ 0.9%NS
(D5NS), lactated ringers (LR)
Table 4: Human Volunteer Plasma Trials
Subject ID
Buffer R2
Zero-Point Intensity
RA
15%O2
Internal Standard Intensity Means
100%O2
RA
15%O2
100%O2
Internal Standard R2*
(RA/15%/100%)
DnAz-EtOH: whole blood only†
21324
27174
23729
21718
27792
26548
0.37/0.03/0.75
V - PIV
21207
28529
23955
20956
28590
25528
0.78/0.68/0.85
V - PIV
25859
36768
12218
26750
36671
14398
0.91/0.89/1.00
30377
20425
22077
30938
21129
0.02/0.08/0.91
3661
7543
6049
0.98/ - /0.96
V - PIV
0.98
V - PIV
0.96
22433
A - PIV
0.99
5496
A - IJ
B - PIV
0.97
B - IJ
C - PIV/IJ
0.91
5416
13470
5077
15625
5727
16168
6376
14438
3875
6845
15877
5354
1.00/0.84/1.00
4556
7661
16632
6121
1.00/0.08/0.99
Did not meet inclusion criteria
11
0.98/0.99/ -
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
E - PIV/IJ
0.94
Did not meet inclusion criteria
DnAz-EtOHdil: whole blood only††
V - PIV
0.96
18892
17288
6147
18978
17048
7559
0.04/0.31/0.98
F - PIV
0.97
15595
15445
15628
15667
15637
15316
0.04/0.49/0.25
13307
13840
13589
13383
13619
13406
0.08/0.10/0.93
20258
20599
12630
20380
20711
13401
0.21/0.02/0.71
18088
17602
11432
17959
16816
13401
0.27/0.33/0.92
F - IJ
G - PIV
1.00
G - IJ
DnAz-EtOHdil: whole blood and serum†††
G - PIV
1.00
19045
16984
12209
18241
18314
13447
0.99/0.99/0.91
H - PIV
0.99
15433
17981
18704
17434
19773
20950
0.91/0.87/0.85
17930
17743
15983
19332
19194
17628
0.94/0.86/0.97
18180
17810
17947
19190
18903
18468
0.82/0.91/0.04
17087
16649
18447
17938
17231
19375
0.09/0.64/0.65
17287
17100
17323
19136
18509
19251
0.95/0.97/0.74
17649
16267
16137
18729
17719
17561
0.90/0.99/0.98
15767
17071
18115
17268
18596
19438
0.94/0.98/0.99
17107
17262
15007
18520
18423
16507
0.95/0.97/0.95
27084
22540
21423
27094
24057
22823
0.80/0.94/0.95
22583
21556
21499
23760
22824
22701
0.98/0.91/0.82
21543
23013
20424
22550
24014
21704
0.99/0.98/1.00
19418
23446
21600
20790
24596
22866
0.85/0.98/0.95
H - IJ
I - PIV
0.99
I - IJ
J - PIV
1.00
J - IJ
K - PIV
0.99
K - IJ
L - PIV
0.99
L - IJ
D - PIV
D - IJ
1.00
† 100mM DnAz-EtOH: 200μM to whole blood only. (DnAz when an oil was notably more soluble in runs for Subject A & B. Background endogenous
and internal standard capture with single DnAz-EtOH addition).
†† 50mM DnAz-EtOHdil: 200μM to whole blood only. (Background endogenous capture only; no internal standard capture).
††† 50mM DnAz-EtOHdil: 200μM to whole blood and then 200μM to serum. (Background endogenous and internal standards captured).
*Internal standard correlation coefficients ≥0.9 are marked with blue. Level of intensity implies increased number of qualifying oxidative exposure
runs from a specific collection site with none (white) to all three RA/15%/100% (dark blue).
12
Author: Nicole Collins, DO
Title: Development Of A Clinically Applicable Protocol For Assessment Of Hypoxic Response Through Measurement Of The
Endogenous Gasotransmitter Hydrogen Sulfide In Human Plasma
Table 5: Assessment of Plates, Pipette Tips, and Reagents
Sample ID
Buffer: old Tween
Buffer: new Tween
New Plate:PIV RA 200μM
New Plate:PIV 15% 200μM
New Plate:PIV 100% 200μM
Old Plate:PIV RA 200μM
Old Plate:PIV 15% 200μM
Old Plate:PIV 100% 200μM
Old Plate:PIV RA 400μM
Old Plate:PIV 15% 400μM
Old Plate:PIV 100% 400μM
Serum old pip-200μM
Serum new pip-200μM
Serum old pip-400μM
Serum new pip-400μM
R2
0.997
0.999
0.366
0.030
0.754
0.775
0.678
0.851
0.912
0.894
0.995
0.985
0.988
0.975
0.976
Notes
old buffer solution
new buffer solution
DnAz (200μM) added to WB only
DnAz (200μM) added to WB only
DnAz (200μM) added to WB only
DnAz (200μM) added to WB only
DnAz (200μM) added to WB only
DnAz (200μM) added to WB only
DnAz (400μM) added to WB only
DnAz (400μM) added to WB only
DnAz (400μM) added to WB only
DnAz (200μM); old pipette tips and eppendorfs
DnAz (200μM); old pipette tips and eppendorfs
DnAz (400μM); old pipette tips and eppendorfs
DnAz (400μM); old pipette tips and eppendorfs
*All DnAz-EtOH from 100mM stock solution
13