Author: Nicole Collins, DO Title: Development Of A Clinically

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%, Na

2

S-9H

2

O, CAS 1313-

84-4) 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 (H

2

S-Stan).

1




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 H

2

S-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 H

2

S-Stan (no DnAz added) all blood collections were treated with DnAz-EtOH(dil) to a final target concentration of 200μM or 400 μM. H

2

S-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




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




(50mM) were prepared. The reduced dansyl amide in DMSO (250μM)(DnNH

2

-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 DnAz-

EtOHdil (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 H

2

S-Stan to final concentrations of 0μM to

80μM and then read on either the 96-well plate utilized for prior experiments (old plate:

4




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 (Na

2

S-9H

2

O) was chosen as the reagent for internal standard testing due to concerns about the purity of our sodium hydrosulfide reagent; upon changing to

Na

2

S-9H

2

O improved correlation coefficients in buffer were obtained and the associated

5



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. ZnCl

2

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 Na

2

S-9H

2

O 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 Na

2

S-9H

2

O 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 Na

2

S-9H

2

O stock preparations (H

2

S-Stan).

INTERNAL STANDARD STUDIES

6




Internal Standards in Buffer: Solubility and Correlation Coefficients

A correlation coefficient (R 2 ≥0.95) was chosen for evaluation of DnAz-EtOH(dil) integrity as a

H

2

S gas trapping agent in buffer in the setting of H

2

S-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 R 2 ≥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 (R 2 <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 (R 2 ≥0.90) was chosen for evaluation of DnAz-EtOH(dil) integrity as a

H

2

S gas trapping agent in serum in the setting of H

2

S-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 (R 2 <0.95) and furthermore demonstrated greatly

7



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 (R 2 ≥0.90) for the majority of the subsequent volunteer subjects

(Supplemental Table 4).

8




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 well- plate.)

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




Table 1: Assessment of Dansyl Azide

Sample ID R 2 Notes

Solvent Trials for DnAz

Buffer DnNH

2

-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

0.281

DnNH

0.977

DnAz-DMSO (100mM)

1.000

DnAz-EtOH (100mM)

1.000

DnAz-EtOH (100mM) dissolved & filtered; added to WB

0.089

EtOH added to WB, no DnAz

0.554

DnAz-DMSO (100mM) added to WB

0.039

DnAz-EtOH (100mM) added to WB

0.864

0.869

0.200

2

-DMSO (250μM) standard

DnAz-EtOH (100mM) to WB, purified EtOH

DnAz-EtOH (50mM standard) added to WB vial

DnAz-EtOH (100mM) dissolved & filtered; added to WB

Solubility/Dilution Trials of DnAz

Buffer

Buffer

Serum: PIVRA concentrated

0.962

DnAz-EtOH (100mM) to buffer

0.998

DnAz-EtOH (50mM) to buffer

0.025

DnAz-EtOH (100mM) to WB vial

Serum: PIVRA dilute

Serum: PIV15% concentrated

0.037


0.083


Serum: PIV15% dilute 0.315


Serum: PIV100% concentrated 0.906


Serum: PIV100% dilute 0.983


*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 ZnCl

2 as Quenching Agent

Sample ID

Buffer

Buffer

Buffer

Buffer

Serum-MT

R 2 (-)ZnCl R 2 (+)ZnCl Notes

DnAzpure-MT 0.226

Serum-MT

Serum-vial

0.908

0.893

0.990

0.983

0.981

0.967

0.967

Abbreviations: microtainer (MT)

0.902

ZnCl

2

>baseline ("zero-point")

0.914

0.992

0.980

ZnCl

2


0.917

0.182

ZnCl

2


0.968

ZnCl

2


0.575

10




Table 3: Assessment of Solvents for Na

2

S x 9H

2

O

Sample ID

Buffer

Serum-Water

Serum-NS

WB-water

WB-D5

WB-D5NS

WB-NS

WB-LR

D5-vial

D5NS-vial

LR-vial

R 2 Notes

0.983

DnAz (100mM)

0.983

DnAz (100mM) added to MT after spin

0.990

DnAz (100mM) added to MT after spin

0.271

DnAz (100mM) added to WB in MT

0.344


0.439


0.521


0.420


0.949

DnAz added to serum in eppendorf tubes

0.836

DnAz added to serum in eppendorf tubes

N/A 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 R 2 Zero-Point Intensity Internal Standard Intensity Means Internal Standard R 2 *

RA 15%O

2

100%O

2

RA 15%O

2

100%O

2

(RA/15%/100%)

DnAz-EtOH: whole blood only †

0.98 21324 V - PIV

V - PIV 21207

V - PIV 25859

V - PIV 0.96

A - PIV 0.99

22433

5496

27174

28529

36768

30377

5416 13470

23729

23955

12218

20425

3661

A - IJ

B - PIV 0.97 5077 15625 3875

B - IJ

C - PIV/IJ

5727 16168 4556

0.91 Did not meet inclusion criteria

21718 27792 26548

20956 28590 25528

26750

22077

36671

30938

14398

21129

7543

6376 14438

6845 15877

7661 16632

6049

5354

6121

0.37/0.03/0.75

0.78/0.68/0.85

0.91/0.89/1.00

0.02/0.08/0.91

0.98/ - /0.96

0.98/0.99/ -

1.00/0.84/1.00

1.00/0.08/0.99

11




E - PIV/IJ 0.94 Did not meet inclusion criteria

DnAz-EtOHdil: whole blood only ††

18892 V - PIV

F - PIV

0.96

0.97 15595

13307 F - IJ

G - PIV

G - IJ

1.00 20258

18088

17288

15445

13840

20599

17602

6147

15628

13589

12630

11432

18978 17048

15667

13383

20380

17959

15637

13619

20711

16816

7559

15316

13406

13401

13401

0.04/0.31/0.98

0.04/0.49/0.25

0.08/0.10/0.93

0.21/0.02/0.71

0.27/0.33/0.92

DnAz-EtOHdil: whole blood and serum †††

G - PIV 1.00 19045 16984

0.99 H - PIV

H - IJ

15433

17930

17981

17743

0.99 I - PIV

I - IJ

18180

17087

17810

16649

J - PIV

J - IJ

1.00 17287

17649

17100

16267

0.99 K - PIV

K - IJ

15767

17107

17071

17262

L - PIV 0.99 27084 22540

12209

18704

15983

17947

18447

17323

16137

18115

15007

18241

17434

19332

19190

17938

19136

18729

17268

18520

18314

19773

19194

18903

17231

18509

17719

18596

18423

13447

20950

17628

18468

19375

19251

17561

19438

16507

0.99/0.99/0.91

0.91/0.87/0.85

0.94/0.86/0.97

0.82/0.91/0.04

0.09/0.64/0.65

0.95/0.97/0.74

0.90/0.99/0.98

0.94/0.98/0.99

0.95/0.97/0.95

21423 27094 24057 22823 0.80/0.94/0.95

L - IJ 22583 21556

23013

21499 23760 22824 22701 0.98/0.91/0.82

D - PIV

D - IJ

1.00 21543

19418

20424

21600

22550

20790

24014

24596

21704

22866

0.99/0.98/1.00

0.85/0.98/0.95 23446

† 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




Table 5: Assessment of Plates, Pipette Tips, and Reagents

Sample ID R 2 Notes

Buffer: old Tween 0.997

old buffer solution

Buffer: new Tween 0.999

new buffer solution

New Plate:PIV RA 200μM 0.366

DnAz (200μM) added to WB only

New Plate:PIV 15% 200μM 0.030


New Plate:PIV 100% 200μM 0.754


Old Plate:PIV RA 200μM 0.775


Old Plate:PIV 15% 200μM 0.678




Old Plate:PIV RA 400μM 0.912






Serum old pip-200μM

Serum new pip-200μM

Serum old pip-400μM

Serum new pip-400μM

0.985

0.988

0.975

0.976

DnAz (200μM); old pipette tips and eppendorfs




*All DnAz-EtOH from 100mM stock solution

13

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