Methods for diagnosing and detecting sulfhemoglobin in blood

Abstract

This disclosure relates to methods and devices for diagnosing and detecting sulfliemoglobin in blood.

Description

MBHB Ref. No.23-1256-WO METHODS FOR DIAGNOSING AND DETECTING SULFHEMOGLOBIN IN BLOOD STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under grant number P20GM103475. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE Field of Invention

[0002] This disclosure relates to methods and devices for diagnosing and detetecting sulfhemoglobin in blood. Background

[0003] Sulfhemoglobinemia is a rare condition in which there is excess sulfhemoglobin (SulfHb) in the blood. The pigment is a greenish derivative of hemoglobin which cannot be converted back to normal, functional hemoglobin. Normal levels of physiological sulfhemoglobin have been estimated to be below 0.037 g / dL (5.5 μM, ~ 0.28%), whereas 0.5 g / dL (74.4 μM, ~ 3.8%) in the blood is enough to present clinically detectable cyanosis symptoms. Toxic agents like oxidizing chemicals or drugs such as nitrites, nitrates, aniline dyes, aniline derivatives, sulfonamides, and lidocaine also convert oxy-Hb into met-aquo hemoglobin causing cyanosis. It is a challenge to determine precise concentrations of sulfheme in an oxyhemoglobin red cells environment. Gas oximeters have been the best accepted economic technique in the biomedical and toxicology community to identify sulfHb and met-aquo Hb in the presence of oxy-Hb. However, one concern for this heme quantification is the overlap between the characteristic electronic transitions of sulfHb (623 nm) and met-aquo Hb (635 nm).

[0004] Therefore, there is a practical need, like other human hemoglobin derivatives, to quantify, with high accuracy and precision, the sulfHb percentage present in the blood in the presence of met-aquo Hb and higher oxy-Hb concentrations to diagnose correctly sulfhemoglobinemia in blood samples of patients.MBHB Ref. No.23-1256-WO SUMMARY OF THE DISCLOSURE

[0005] This disclosure describes methods and devices for diagnosing and detetecting sulfhemoglobin in blood.

[0006] In a first aspect, this disclosure provides a method for measuring an amount of sulfhemoglobin in a biological sample comprising: providing a biological sample comprising hemoglobin; measuring UV-Vis absorbance of the biological sample at a first wavelength in the range of about 600 to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of 450 to 470 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample.

[0007] In one embodiment of the first aspect disclosed herein, the biological sample is a blood sample.

[0008] In one embodiment of the first aspect disclosed herein, the biological sample is human blood.

[0009] In one embodiment of the first aspect disclosed herein, the biological sample is animal blood.

[0010] In one embodiment of the first aspect disclosed herein, the method further comprises separating red blood cells from the blood sample and lysing the red blood cells

[0011] In one embodiment of the first aspect disclosed herein, the measuring a UV-Vis absorbance comprises determining a first intensity at the first wavelength in a UV-Vis absorption spectrum of the biological sample

[0012] In one embodiment of the first aspect disclosed herein, the first wavelength maximum is 623 nm in the 600 nm to 750 nm region.

[0013] In one embodiment of the first aspect disclosed herein, the measuring a fluorescence emission comprises determining a second intensity at the second wavelength in a fluorescence emission spectrum of the biological sample.

[0014] In one embodiment of the first aspect disclosed herein, the the fluorescence emission spectrum is measured using a fluorescence excitation at 420 nm.

[0015] In one embodiment of the first aspect disclosed herein, the second wavelength is 460 nm in the 450 nm to 470 nm region.MBHB Ref. No.23-1256-WO

[0016] In one embodiment of the first aspect disclosed herein, the correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve comprises: determining if the first intensity and the second intensity correspond to a point on the first standard curve; and if the point is on the first linear standard curve, correlating the fluorescence emission with a second standard curve to determine the amount of sulfhemoglobin present in the biological sample; or if the point is not on the first linear standard, determining that the amount of sulfhemoglobin present in the biological sample is less than a detection threshold.

[0017] In one embodiment of the first aspect disclosed herein, the first standard curve is a linear standard curve of the fluorescence emission versus UV-Vis absorbance of standard samples comprising known amounts of sulfhemoglobin.

[0018] In one embodiment of the first aspect disclosed herein, the second standard curve is a linear standard curve of the fluorescence emission versus sulfhemoglobin concentration of standard samples comprising known amounts of sulfhemoglobin.

[0019] In a second aspect, this disclosure provides a device for measuring an amount of sulfhemoglobin in a biological sample comprising: a biological sample holder; a UV-Vis light source configured to pass light through the biological sample holder; a fluorescence excitation light source configured to pass light through the biological sample holder; an absorbance detector; and an emission detector perpendicular to the fluorescence excitation light source; wherein the absorbance detector collects transmitted UV-Vis light passed from the UV-Vis light source and through the biological sample holder; and wherein the emission detector collects fluorescence emission.

[0020] In one embodiment of the second aspect disclosed herein, the biological sample holder is configured to allow transmittance of the UV-Vis light source.

[0021] In one embodiment of the second aspect disclosed herein, the biological sample holder is configured to allow for detection of fluorescence emission.

[0022] In one embodiment of the second aspect disclosed herein, the UV-Vis light source transmits light through the biological sample holder.MBHB Ref. No.23-1256-WO

[0023] In one embodiment of the second aspect disclosed herein, the UV-Vis light source excitation of 420 nm emits UV-Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

[0024] In one embodiment of the second aspect disclosed herein, the light emitted by the UV-Visible light source can be tuned to a desired UV-Visible wavelength.

[0025] In one embodiment of the second aspect disclosed herein, the UV-Vis light source emits light in the range of 600 to 750 nm.

[0026] In one embodiment of the second aspect disclosed herein, the UV-Vis light source emits light at 623 nm in the 600 nm to 750 nm region.

[0027] In one embodiment of the second aspect disclosed herein, the fluorescence excitation light source transmits light through the biological sample holder.

[0028] In one embodiment of the second aspect disclosed herein, the fluorescence excitation at 420 nm light source emits UV-Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

[0029] In one embodiment of the second aspect disclosed herein, the light emitted by the fluorescence excitation source can be tuned to a desired UV-Visible wavelength.

[0030] In one embodiment of the second aspect disclosed herein, the fluorescence excitation light source emits light in the range of about 440 to about 475 nm.

[0031] In one embodiment of the second aspect disclosed herein, the fluorescence excitation light source emits light at 460 nm upon excitation with 420 nm light.

[0032] In one embodiment of the second aspect disclosed herein, the UV-Vis light source and the fluorescence excitation light source are the same light source.

[0033] In one embodiment of the second aspect disclosed herein, the UV-Vis light source and the fluorescence excitation light source are different light sources.

[0034] In one embodiment of the second aspect disclosed herein, the emission detector is configured to collect fluorescence emission from the biological sample holder.

[0035] In one embodiment of the second aspect disclosed herein, the fluorescence excitation light source and the emission detector are separated by 90 degrees.

[0036] In a third aspect, this disclosure provides a method for measuring an amount of sulfhemoglobin in a biological sample using the device of any of claims 14-30 comprising:MBHB Ref. No.23-1256-WO providing a biological sample comprising hemoglobin; measuring a UV-Vis absorbance of the biological sample at a first wavelength in the range of 623 nm in the range of about 600 nm to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of about 440 nm to about 475 nm upon excitation at 420 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample.

[0037] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0039] Figure 1 provides a visualization of the sulfhemoglobin system containing partial sulfheme structures.

[0040] Figures 2A-2D depict the sulfheme complex as a function of hydrogen sulfideconcentration (165 M - 1155 M) and time. Fig. 2A shows the sulfmyoglobin productspectra. Figure 2B shows that sulfmyoglobin 618 nm absorbance changes as a function of hydrogen sulfide concentration through four hours. Fig.2C shows the sulfhemoglobin product spectra and Fig.2D shows the sulfhemoglobin 623 nm absorbance as a function of hydrogen sulfide concentration through four hours.

[0041] Figures 3A-3B show a sulfmyoglobin A618 / A582 (Fig.3A) and sulfhemoglobin A623 / A576 (Fig. 3B) ratios as a function of hydrogen sulfide concentration over seven hours’ reaction time.

[0042] Figure 4 shows sulfhemoglobin absorption spectra (open circles) and emission spectra (squares).MBHB Ref. No.23-1256-WO

[0043] Figures 5A-5B show UV-Vis Absorption (Fig.5A) and fluorescence (Fig.2B) spectra of oxy-hemoglobin (55.0 μM), met-aquo hemoglobin (55.0 μM), and sulfhemoglobin (5.63 μM).

[0044] Figures 6A-6C show UV-Vis (Fig.6A) and fluorescence (Fig.6B) spectra of sulfhemoglobin formation upon the reaction of oxy-hemoglobin (55 M) in the presence of met-aquo hemoglobin (5.5 M) and H2S solution (1,155 M) as a function of time. (Fig.6C) Same conditions but in the absence of met-aquo hemoglobin.

[0045] Figure 7 shows sulfhemoglobin fluorescence (460 nm) and visible absorbance (623 nm) intensity relationship.

[0046] Figures 8A-8B show fluorescence intensity at 460 nm and percentage of sulfhemoglobin in a sample. Fig.8A shows sulfhemoglobin from oxy-hemoglobin (55 M) in the presence of met-aquo hemoglobin (5.5 M) and H2S solution (1,155 M). Fig.8B shows the sulfhemoglobin upon oxy-hemoglobin reaction (55 M) with H2S solution (1,155 M). DETAILED DESCRIPTION

[0047] It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented, and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0048] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of” can be replaced with either of the other two terms, while retaining their ordinary meanings.MBHB Ref. No.23-1256-WO

[0049] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indictates otherwise.

[0050] In some embodiments, percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated disclosure.

[0051] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0052] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. For example, “about 5%” means “about 5%” and also “5%.” The term “about” can also refer to ± 10% of a given value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example.

[0053] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0054] As us used herein, the term “patient” refers to an animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein.

[0055] As used herein, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the present disclosure is a human.

[0056] As used herein, the terms “detect,” “detecting,” and all variations thereof refer to any method used to quantitatively determine the presence, absence, or amount of hemoglobin, met-aquo hemoglobin, oxyhemoglobin, sulfhemoglobin, myoglobin, and / or sulfmyoglobin in a biological sample.

[0057] As used herein, the terms “contacting,” “contact,” and all variations thereof, refer to any means that directly or indirectly cause placement together of moieties or components, such that the moieties or components come into physical contact with each other. ContactingMBHB Ref. No.23-1256-WO thus includes physical acts such as placing the moieties or components together in a container, combining the moieties or components, or mixing the moieties or components.

[0058] As used herein, the term “biological sample” means any biological sample including, but are not limited to, a blood sample, biopsy specimen, tissue explant, organ culture, biological fluid or any other tissue or cell preparation, or fraction or derivative thereof or isolated therefrom. Biological samples can be obtained from any subject or biological source including, for example, human or non-human animals, including mammals and non-mammals, vertebrates and invertebrates, and can also be any multicellular organism or single-celled organism such as a eukaryotic (including plants and algae) or prokaryotic organism, archaeon, microorganisms (e.g., bacteria, archaea, fungi, protists, viruses), and aquatic plankton.

[0059] The terms “hemoglobin” or “Hb” as used herein refer generally to the protein within red blood cells that transports oxygen. Each molecule of Hb has 4 subunits, 2 -chain subunits and 2 -chain subunits, which are arranged in a tetrameric structure. Each subunit also contains one heme group, which is the iron-containing center that binds the ligands O2, NO and CO. Thus, each Hb molecule can bind up to 4 ligand molecules. Hemoglobin or Hb as used herein includes variant forms such as natural or artificial mutant forms differing by the addition, deletion and / or substitution of one or more contiguous or non-contiguous amino acid residues, or modified polypeptides in which one or more residues is modified, and mutants comprising one or more modified amino acid residues. Hb also includes chemically modified forms as well as genetically altered forms, such as fusion proteins, and truncated forms. It also includes Hbs of all animal species and variant forms thereof. The biological and / or chemical properties of these variant Hbs can be different from those of hemoglobins which are found natu rally occurring in animals. The term “hemoglobin” includes, but not limited to total hemoglobin, oxyhemoglobin, reduced hemoglobin, carboxyhemoglobin, methemoglobin, met-aquo hemoglobin, or sulfhemoglobin.

[0060] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need.MBHB Ref. No.23-1256-WO OVERVIEW

[0061] Disclosed herein are methods and devices for detecting and diagnosing sulfhemoglobin in the presence of met-aquo Hb and oxy-Hb in blood.

[0062] Hydrogen sulfide (H2S), a highly lipophilic gas endogenously produced in tissues and organs by enzymes, has been associated with several conditions and diseases (Whiteman, M., et al., Emerging Role of Hydrogen Sulfide in Health and Disease: Critical Appraisal of Biomarkers and Pharmacological Tools. Clin Sci 2011, 121 (11), 459–488; Li, Q., et al. Chemical Foundations of Hydrogen Sulfide Biology. Nitric Oxide 2013, 35, 21–34.; Rajendran, S., et al. Nitric Oxide and Hydrogen Sulfide Regulation of Ischemic Vascular Growth and Remodeling. Compr Physiol 2019, 9 (3), 1213–1247; Olson, K. R. Is Hydrogen Sulfide a Circulating “Gasotransmitter” in Vertebrate Blood? Biochim Biophys Acta Bioenerg 2009, 1787 (7), 856–863; Wang, R. Physiological Implications of Hydrogen Sulfide: A Whiff Exploration That Blossomed. Physiol Rev 2012, 92 (2), 791–896; Mathai, J. C. et al., No Facilitator Required for Membrane Transport of Hydrogen Sulfide. Proc Natl Acad Sci U S A 2009, 106 (39), 16633–16638; Murphy, B., et al., Hydrogen Sulfide Signaling in Mitochondria and Disease. FASEB Journal 2019, 33 (12), 13098–13125; Cirino, G., et al. (2022). Physiological Roles of Hydrogen Sulfide in Mammalian Cells, Tissues, and Organs. Physiol Rev 2022; Gopi K. Kolluru, et al., Hydrogen Sulfide Chemical Biology: Pathophysiological Roles and Detection. Nitric Oxide 2013, 35, 5–20; Guo, W. et al., Hydrogen Sulfide and Translational Medicine. Acta Pharmacol Sin 2013, 34 (10), 1284– 1291; Szabo, C. Novel Regulatory Roles of Hydrogen Sulfide in Health and Disease. Biomolecules 2022, 12 (10), 10–13). H2S plasma values in the 100 M range have been associated with the fibrosis phenomena (Zhang, S. et al., Hydrogen Sulfide as a Potential Therapeutic Target in Fibrosis. Oxid Med Cell Longev 2015, 2015). SARS-CoV-2 survivorsshow serum H2S levels higher ( 150 M) than those of non-survivors (G Renieris, et al.,Serum Hydrogen Sulfide and Outcome Association in Pneumonia by The Sars-Cov-2 Corona Virus. Shock 2020, 54 (5), 633–637; Yang, G. H2S as a Potential Defense against COVID- 19? Am J Physiol Cell Physiol 2020, 319 (2), C244–C249); production of H2S has been suggested to act as a defense mechanism against COVID-19 (Gorini, F., et al., H2S as a Bridge Linking Inflammation, Oxidative Stress and Endothelial Biology: A Possible DefenseMBHB Ref. No.23-1256-WO in the Fight against SARS-CoV-2 Infection? Biomedicines 2021, 9 (9); Iciek, M., et al.,Reactive Sulfur Compounds in the Fight against COVID 19. Antioxidants 2022, 11 (6)).Reduced H2S levels are also related to Parkinson's disease, Alzheimer's disease, andatherosclerosis (Peng, S.-Y., et al., Research Progress of Hydrogen Sul de in Alzheimer’sDisease from Laboratory to Hospital: A Narrative Review. Med Gas Res 2020, 10 (3), 125– 129; Nagpure, B. V. et al., Brain, Learning, and Memory: Role of H2S in Neurodegenerative Diseases. Chemistry, Biochemistry, and Pharmacology of Hydrogen Sulfide 2015, 230, 193– 215). Additionally, H2S has been correlated with certain chronic diseases (Yang, N., et al. Role of Hydrogen Sulfide in Chronic Diseases. DNA Cell Biol 2020, 39 (2), 187–196; Merz, T., et al. H2S in Critical Illness—A New Horizon for Sodium Thiosulfate? Biomolecules 2022, 12 (4), 1–15; Zhang, H., et al., Protective Effect of Hydrogen Sulfide on the Kidney (Review). Mol Med Rep 2021, 24 (4), 1–11), inflammation (Wang, Y., et al., Hydrogen Sulfide Alleviates Particulate Matter-Induced Emphysema and Airway Inflammation by Suppressing Ferroptosis. Free Radic Biol Med 2022, 186, 1–16; Sun, H. J., et al., Role of Endothelial Dysfunction in Cardiovascular Diseases: The Link Between Inflammation and Hydrogen Sulfide. Front Pharmacol 2020, 10, 1–15; Xu, M.; Liu, X., et al., H2S Protects Against Immobilization-Induced Muscle Atrophy via Reducing Oxidative Stress and Inflammation. Front Physiol 2022, 13, 1–13), immune system regulation (Dilek, N., et al., Hydrogen Sulfide: An Endogenous Regulator of the Immune System. Pharmacol Res 2020, 161; Pozzi, G. , et al., Buffering Adaptive Immunity by Hydrogen Sulfide. Cells 2022, 11 (3), 1–16), cancer (Khattak, S., et al., Hydrogen Sulfide Biology and Its Role in Cancer; 2022; Vol.27; M. R. Hellmich, et al., The Therapeutic Potential of Cystathionine -Synthetasehydrogen Sul de Inhibition in Cancer. Antioxidants Redox Signal 2015, 22 (5),424–448), oxidative stress (Li, L., et al., Hydrogen Sulfide Suppresses Skin Fibroblast Proliferation via Oxidative Stress Alleviation and Necroptosis Inhibition. Oxid Med Cell Longev 2022, 2022; Parsanathan, R, et al., Hydrogen Sulfide Regulates Irisin and Glucose Metabolism in Myotubes and Muscle of HFD-Fed Diabetic Mice. Antioxidants 2022, 11 (7); Sun, Y, et al., Implications of Hydrogen Sulfide in Development of Pulmonary Hypertension. Biomolecules 2022, 12 (6), 1–8; Ahmad, A. Physiological, Pathological and Pharmacological Interactions of Hydrogen Sulphide and Nitric Oxide in the Myocardium of Rats with LeftMBHB Ref. No.23-1256-WO Ventricular Hypertrophy. Current Issues in Molecular Biology.2022, pp 433–448; Zhu, S. et al., Hydrogen Sulfide Protects Retina from Blue Light-Induced Photodamage and Degeneration via Inhibiting ROS-Mediated ER Stress-CHOP Apoptosis Signal. Redox Report 2022, 27 (1), 100–110; Barrow, K., et al., H2S Protects from Oxidative Stress-Driven ACE2 Expression and Cardiac Aging. Mol Cell Biochem 2022, 477 (5), 1393–1403), oral diseases (Wu, D. D., et al., Role of Hydrogen Sulfide in Oral Disease. Oxid Med Cell Longev 2022, 2022), glaucoma (Feng, Y., et al., Current Perspective of Hydrogen Sulfide as a Novel Gaseous Modulator of Oxidative Stress in Glaucoma. Antioxidants 2021, 10 (5)), subarachnoid hemorrhage (Lu, D., et al., Role of Hydrogen Sulfide in Subarachnoid Hemorrhage. CNS Neurosci Ther 2022, 28 (6), 805–81), infertility in men (F. Akbarian, et al., Down-Regulated Expression of Cystathionine -Synthase and Cystathionine -Lyase in Varicocele, and Infertile Men a Case-Control Study. Cell J 2022, 24 (4), 176–181), arterial oxygen saturation (Huang, Y, et al., Endogenous Hydrogen Sulfide Is an Important Factor in Maintaining Arterial Oxygen Saturation. Front Pharmacol 2021, 12 May, 1–12), and skin diseases (Xiao, Q., et al., Hydrogen Sulfide in Skin Diseases: A Novel Mediator and Therapeutic Target. Oxid Med Cell Longev 2021, 2021). Furthermore, clinical studies have emphasized the potential of modulating H2S synthesis for therapeutic use (Wallace, J. L., et al., Hydrogen Sulfide-Based Therapeutics: Exploiting a Unique but Ubiquitous Gasotransmitter. Nat Rev Drug Discov 2015, 14 (5), 329–345; E. Zaorska, et al. HydrogenSul de in Pharmacotherapy, Beyond the Hydrogen Sul de-Donors. Biomolecules 2020, 10(2); Powell, C. R., et al., A Review of Hydrogen Sulfide (H2S) Donors: Chemistry and Potential Therapeutic Applications. Biochem Pharmacol 2018, 149, 110–123). However, the investigation results have been limited by the lack of reliable H2S measurements in body fluids and tissues and the absence of specific biomarkers (Whiteman, M., et al., Emerging Role of Hydrogen Sulfide in Health and Disease: Critical Appraisal of Biomarkers and Pharmacological Tools. Clin Sci 2011, 121 (11), 459–488).

[0063] Similarly, interactions with H2S with metal-driven and heme proteins metabolism, protection against toxicity, and detection have also been previously disclosed (Mantle, D., et al., Hydrogen Sulfide and Metal Interaction: The Pathophysiological Implications. Mol Cell Biochem 2022, 477, 2235–2248; Pietri, R., et al., Factors Controlling the Reactivity ofMBHB Ref. No.23-1256-WO Hydrogen Sulfide with Hemeproteins. Biochemistry 2009, 48 (22), 4881–4894; Doman, A., et al., Interactions of Reactive Sulfur Species with Metalloproteins. Redox Biol 2023, 60). Hydrogen sulfide also interacts with oxy-myoglobin and oxy-hemoglobin to generate sulfmyoglobin (sulfMb) and sulfhemoglobin (sulfHb) complexes. These processes have led to sulfhemoglobinemia, a bluish skin color associated with lack of oxygen and cyanosis. Furthermore, common causes of sulfhemoglobinemia-induced cyanosis include a wide range of overdoses involving drugs including acetanilide metoclopramide, phenacetin, dapsone, sulfanilamide, cimetidine, paracetamol, ibuprofen, naproxen, and exposure to sulfur compound (A. Rangan, et al., Interpreting Sulfhemoglobin and Methemoglobin in Patientswith Cyanosis: An Overview of Patients with M hemoglobin Variants. Int J Lab Hematol2021, 00, 1–8). This phenomenon has also have observed in newborns (Tangerman, A., et al., The Origin of Hydrogen Sulfide in a Newborn with Sulfhaemoglobin Induced Cyanosis. J Clin Pathol 2002, 55 (8), 631–633), from gamma-ray irradiation (Attia, A. M. M., et al., Assessment of Absorbed Dose of Gamma Rays Using the Simultaneous Determination of Inactive Hemoglobin Derivatives as a Biological Dosimeter. Radiat Environ Biophys 2019, 59 (1), 131–144; Attia A., et al., Determination of Human Hemoglobin Derivatives. Hemoglobin 2015, 39 (5), 371–374), in cancer and neurodegenerative disease (Padovani, D., et al., Sulfheme Formation during Homocysteine S-Oxygenation by Catalase in Cancers and Neurodegenerative Diseases. Nat Commun 2016, 7, 1–13), after dimethyl sulfide dermal applications (Burgess JL, et al., Sulfhemoglobinemia after Dermal Application of DMSO. Vet Hum Toxicol.1998, 40 (2), 87–89), from sodium nitrate formulations (Greenway, F. L., et al., Single-Dose Pharmacokinetics of Different Oral Sodium Nitrite Formulations in Diabetes Patients. Diabetes Technol Ther 2012, 14 (7), 552–560), sulfur dioxide (Lovati, M. R., et al., Effects of Sub-Chronic Exposure to SO2 on Lipid and Carbohydrate Metabolism in Rats. Arch Toxicol 1996, 70 (3–4), 164–173; Docherty, S., et al., The Diagnosis of Sulfated Hemoglobin (SulFHB) Secondary to Sulfur Dioxide Poisoning Using Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS)—a Novel Approach to an Unusual Clinical Problem. Diagnostics 2020, 10 (2)), hydroxylamine sulfate (Gharahbaghian, L., et al., Methemoglobinemia and Sulfhemoglobinemia in Two Pediatric Patients after Ingestion of Hydroxylamine Sulfate. Western Journal of emergency medicine 2009, 10 (3),MBHB Ref. No.23-1256-WO 197–201), hydrogen sulfide (Saeedi A, et al., Effects of Long-Term Exposure to Hydrogen Sulfide on Human Red Blood Cells. Int J Occup Environ Med 2015, 6, 20–25), chronic constipation (George, A., et al., A Case of Sulfhemoglobinemia in a Child with Chronic Constipation. Respir Med Case Rep 2017, 21, 21–24), urinary tract infections (Campagna, G., et al., A Case of Sulfhemoglobinemia Secondary to a Urinary Tract Infection. J Pediatr Hematol Oncol 2019), thiocolchicoside (Miorel) (Dupouy, J., et al., Une Cause Rare de Cyanose: Sulfhémoglobinémie Imputable Au Thiocolchicoside (Miorel®). Rev Mal Respir 2010, 27 (1), 80–83), metoclopramide and N-acetylcysteine (Langford, J. S., et al., An Adolescent Case of Sulfhemoglobinemia Associated with High-Dose Metoclopramide and N- Acetylcysteine. Ann Emerg Med 1999, 34 (4), 538–541). The mechanism of drugs and chemical-induced sulfhemoglobin has not yet been elucidated but has been postulated to be derived from H2S produced by intestinal bacteria (Wu, C., et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO- Oximeter. Clin Chem 1997, 43 (1), 162–166). However, hydrogen sulfide produced by mammalian tissues and organs by enzymes suggests that the chemical-induced sulfhemoglobin and sulfmyoglobin mechanism is more complex than previously hypothesized.

[0064] The problem could be further visualized from red blood cell (RBC) analysis. For example, a hemoglobin level of 13.5 g / dL is equivalent to a 2.0 mM Hb concentration. Normal levels of physiological sulfhemoglobin have been estimated to be below 0.037 g / dL (5.5 μM, ~ 0.28%), whereas a concentration of 0.5 g / dL (74.4 μM, ~ 3.8%) in the blood is enough to present clinically detectable cyanosis symptoms (Whiteman, M., et al., Emerging Role of Hydrogen Sulfide in Health and Disease: Critical Appraisal of Biomarkers and Pharmacological Tools. Clin Sci 2011, 121 (11), 459–488; Saeedi A, et al., A. Effects of Long-Term Exposure to Hydrogen Sulfide on Human Red Blood Cells. Int J Occup Environ Med 2015, 6, 20–25; George, A.; Goetz, D. A Case of Sulfhemoglobinemia in a Child with Chronic Constipation. Respir Med Case Rep 2017, 21, 21–24; Campagna, G.; Espaillat, A.; Pfeiffer, T., et al., A Case of Sulfhemoglobinemia Secondary to a Urinary Tract Infection. J Pediatr Hematol Oncol 2019; Dupouy, J., et al., Une Cause Rare de Cyanose: Sulfhémoglobinémie Imputable Au Thiocolchicoside (Miorel®). Rev Mal Respir 2010, 27MBHB Ref. No.23-1256-WO (1), 80–83; Langford, J. S., et al., An Adolescent Case of Sulfhemoglobinemia Associated with High-Dose Metoclopramide and N-Acetylcysteine. Ann Emerg Med 1999, 34 (4), 538– 541), with severe sulfHb cyanosis seldom exceeding 10% (~ 200 μM). However, SulfHb measurements of 16% and 23% have also been reported (Wu, C., et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO- Oximeter. Clin Chem 1997, 43 (1), 162–166).

[0065] Analogously, methemoglobinemia is a blood disorder characterized by a higher- than-average level of met-aquo Hb. Toxic agents like oxidizing chemicals or drugs such as nitrites, nitrates, aniline dyes, aniline derivatives, sulfonamides, and lidocaine can also convert oxy-Hb into met-aquo Hb (Dupouy, J., et al., Une Cause Rare de Cyanose: Sulfhémoglobinémie Imputable Au Thiocolchicoside (Miorel®). Rev Mal Respir 2010, 27 (1), 80–83; Langford, J. S, et al., An Adolescent Case of Sulfhemoglobinemia Associated with High-Dose Metoclopramide and N-Acetylcysteine. Ann Emerg Med 1999, 34 (4), 538– 541; Wu, C. , et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO-Oximeter. Clin Chem 1997, 43 (1), 162–166; Aravindhan, N., et al., Sulfhemoglobinemia Presenting as Pulse Oximetry Desaturation. Anesthesiology 2000, 93 (3), 883–884). Met-aquo hemoglobin levels of 1.5g / dL (223.0 μM) in the blood also cause detectable cyanosis; these values represent approximately 10% met- aquo Hb in total blood hemoglobin (Aravindhan, N., et al., Sulfhemoglobinemia Presenting as Pulse Oximetry Desaturation. Anesthesiology 2000, 93 (3), 883–884). At the same, it has been estimated that 3.0% of oxy-heme (FeII-O2) groups autoxidize to met-aquo heme (FeIII- H2O) and superoxide (O2-), leading to reactive oxygen species (ROS) involved reactions and free radical chemistries (Nagababu, E., et al., Formation of Fluorescent Heme Degradation Products during the Oxidation of Hemoglobin by Hydrogen Peroxide. Biochem Biophys Res Commun 1998, 247 (3), 592–596). Overall, only 0.5 g / dL (74.4 μM) sulfhemoglobin is needed to cause clinically detectable cyanosis, compared with 1.5 g / dL (223.0 μM) met-aquo hemoglobin and 5.0 g / dL (744.0 μM) of deoxygenated hemoglobin in a matrix of ~2.0 mM oxy-Hb (Aravindhan, N., et al., Sulfhemoglobinemia Presenting as Pulse Oximetry Desaturation. Anesthesiology 2000, 93 (3), 883–884). Thus, it has been a challenge to determine precise concentrations of sulfheme in oxy-hemoglobin red cells environment.MBHB Ref. No.23-1256-WO

[0066] Structurally, the sulfheme product chromophore is a covalent heme modified by incorporating one sulfur within carbon atoms to generate a sulfur-carbons ring moiety acrossthe - double bond of heme pyrrole B. The sulfmyoglobin structure is supported by X-ray(Evans, S. V, et al., Three-Dimensional Structure of Cyanomet-Sulfmyoglobin C. Proc. Natl.Acad. Sci 1994, May 24, 91, 4723–4726), NMR (M.J. Chat eld, et al., Proton NMRCharacterization of Isomeric Sulfmyoglobins: Preparation, Interconversion, Reactivity Patterns, and Structural Features. Biochemistry 1987, 26 (22), 6939–6950; Chatfield, M. J., et al., Identification of the Altered Pyrrole in the Isomeric Sulfmyoglobins: Hyperfine Shift Patterns as Indicators of Rine Saturation in Ferric Chlorins. Biochemistry 1988, 27 (5), 1500– 1507; Chatfield, M. J., et al., 1H Nuclear Magnetic Resonance Study of the Prosthetic Group in Sulfhemoglobin. Arch Biochem Biophys 1992, 295 (2), 289–296; Timkoviclr, R., et al., Proton NMR Spectroscopy of Sulfmyoglobin. Biochemistry 1985, 24 (19), 5189–5196), and resonance Raman (Andersson LA, et al., Sulfmyoglobin. Resonance Raman Spectroscopic Evidence for an Iron-Chlorin Prosthetic Group. J Biol Chem 1984, 259 (24), 15340–15349; Román-Morales E, et. al. Structural Determinants for the Formation of Sulfhemeprotein Complexes. Biochem Biophys Res Commun 2010, 400 (4), 489–492) spectroscopy. Figure 1 shows a visualization model of the sulfhemoglobin system containing a partial sulfheme structure in the different subunits. The sulfhemoglobin and sulfmyoglobin Soret chromophore transitions in the Soret region occur from 402 nm to 423 nm, coupled with a unique charge transfer band in the 565 nm to 623 nm region. These transition energies depend on the nature of the hemeprotein, iron oxidation, ligation, spin states, and pH (J.A. Berzofsky, et al., Sulfheme Proteins. The Reversible Oxygenation of Ferrous SulfMyoglonin. J Biol Chem 1971, 246 (23), 7366–7372; Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30– 40; J.A. Berzofsky, et al., Sulfheme Proteins. Optical and Magnetic Properties of Sulfmyoglobin and Its Derivates. J Biol Chem 1971, 246 (10), 3367–3377.; Berzofsky, J. A., et al., Sulfheme Proteins. The Stoichiometry of Sulfur Incorporation and the Isolation of Sulfhemin, the Prosthetic Group of Sulfmyoglobin. Journal of Biological Chemistry 1972, 247 (12), 3783–3791; J.A. Berzofsky, et al., Sulfheme Proteins III. Carboxysulfmyoglobin: The Relation between Electron Withdrawal from Iron and Ligand Binding. J Biol ChemMBHB Ref. No.23-1256-WO 1972, 247 (12), 3774–3782; Michel, H. A Study of Sulfhemoglobin. J. Biol. Chem 1938, 126, 323–348; Nicholls, P. The Formation and Properties of Sulphmyoglobin and Sulphcatalase. Biochem. J.1961, 81, 374–383; Romero, F. J., et al., The Reactivity of Thiols and Disulfides with Different Redox States of Myoglobin. Redox and Addition Reactions and Formation of Thiyl Radical Intermediates. Journal of Biological Chemistry.1992, pp 1680–1688; Brittain, T. Studies on Ligand Binding to Sulphaemoglobin. Biochim Biophys Acta 1981, 673, 253– 258; Bondoc, L. L., et al., Structure of a Stable Form of Sulfheme. Biochemistry 1986, 25 (26), 8458–8466). The most prominent transitions for oxy-sulfhemoglobin are the Soret bandat 412 nm, characteristic of a to * transition, and the 623 nm band characterized by to d(dyz, dxz) charge transfer transition associated with the sulfur ring attached to pyrrole B and the heme iron (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; Carrico, R. J., et al., The Reversible Binding of Oxygen to Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (20), 7212–7215; H. Arbelo-López, et al. Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955.

[0067] Roman-Morales et al. demonstrated that distal histidine in the E7 position (HisE7) is essential in sulfheme formation (Román-Morales E, et. al., Structural Determinants for the Formation of Sulfhemeprotein Complexes. Biochem Biophys Res Commun 2010, 400 (4), 489–492), it being the only amino acid in the heme distal position that catalyzes the reaction. The hydrogen bond between heme Fe(III)-H2O2 or heme Fe(II)-O2, heme distal His64, and H2S regulates this process. Mechanistically, the sulfheme formation for met-aquo Mb (Fe(III))-H2O2adduct and oxy-Mb(Fe(II)) in the presence of H2S shows different heme intermediates and energy barriers towards the reaction intermediate heme Cpd 0 (Heme Fe(III)-OOH) and the generation of a thiyl radical (HS•) (H. Arbelo-López, et al. Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955; Arbelo-Lopez, H. D., et al., Homolytic Cleavage of Both Heme-Bound Hydrogen Peroxide and Hydrogen Sulfide Leads to the Formation of Sulfheme. Journal of Physical Chemistry B 2016, 120 (30), 7319–7331; Rodriguez- Mackenzie, A. D., et al., A Reaction Pathway to Compound 0 Intermediates in Oxy- Myoglobin through Interactions with Hydrogen Sulfide and His64. J Mol Graph Model 2020,MBHB Ref. No.23-1256-WO 94; A.D. Rodriguez-Mackenzie, et al., Mechanistic Inside into PH Dependent SulfMyoglobin Formation: Spin Control and His64 Proton Relay. J. Chemistry). This reactive sulfur species, suggested previously (Romero, F. J., et al., The Reactivity of Thiols and Disulfides with Different Redox States of Myoglobin. Redox and Addition Reactions and Formation of Thiyl Radical Intermediates. Journal of Biological Chemistry.1992, pp 1680–1688; Nichol, A. W., et al., Mechanism of Formation of Sulphhaemoglobin. Biochim Biophys Acta 1967, 156, 97–108), explicitly attacks the heme pyrrole B at the - double bond to generate the heme-sulfurfive-member ring formation. Yet, favorable energy pathways of -135.3 kcal / mol and - 69.1Arbelo -López, et al . Charge Transfer and to * Transitions in the Visible Spectra ofSulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955; Arbelo - Lopez, H. D., et al .,Homolytic Cleavage of Both Heme - Bound Hydrogen Peroxide and Hydrogen Sulfide Leads to the Formation of Sulfheme. Journal of Physical Chemistry B 2016, 120(30), 7319–7331; Rodriguez- Mackenzie, A. D., et al ., A Reaction Pathway to Compound 0Intermediates in Oxy - Myoglobin through Interactions with Hydrogen Sulfide and His64. JMol Graph Model 2020, 94;A.D. Rodriguez -Mackenzie, et al ., Mechanistic Inside into PHDependent SulfMyoglobin Formation: Spin Control and His64 Proton Relay. J. Chemistry ) . These mechanisms support the antioxidant role of hydrogen sulfide by avoiding the strainedformation of the highly reactive heme compound I(Fe(IV)=O+•, Cpd I ) .

[0068] Upon forming sulfhemoglobin (sulfHb) and sulfmyoglobin (sulfMb), there is an oxygen reduction affinity of hemoglobin and myoglobin by 135 and 2,500 times, respectively (J.A. Berzofsky, et al., Sulfheme Proteins. The Reversible Oxygenation of Ferrous SulfMyoglonin. J Biol Chem 1971, 246 (23), 7366–7372; Carrico, R. J., et al., The Reversible Binding of Oxygen to Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (20), 7212–7215). Experimental results suggest that the difference in charge transfer intensity between sulfMb and sulfHb in the 620 nm region could be attributed to the lack of sulfheme formation in oxy-Hb heme centers upon reaction with H2S (C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579– 1584; C.M. Park, R.L. Nagel, W. E. B. et al. Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810; Tomoda, A., et al., OxidativeMBHB Ref. No.23-1256-WO and Reductive Reactions of Sulphhaemoglobin with Various Reagents Correlated with Changes in Quaternary Structure of the Protein. Biochem J 1984, 221 (3), 587–591). As time progresses, the autoxidation process leads to the formation of met-aquo-, hydroxyl, deoxy-, and oxy-sulfheme species, resulting in energy shifts in the Soret transitions, intensity decrease, and a broad 623 nm band (J.A. Berzofsky, et al., Sulfheme Proteins. The Reversible Oxygenation of Ferrous SulfMyoglonin. J Biol Chem 1971, 246 (23), 7366–7372; Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; J.A. Berzofsky, et al., Sulfheme Proteins. Optical and Magnetic Properties of Sulfmyoglobin and Its Derivates. J Biol Chem 1971, 246 (10), 3367–3377; Berzofsky, J. A., et al., Sulfheme Proteins. The Stoichiometry of Sulfur Incorporation and the Isolation of Sulfhemin, the Prosthetic Group of Sulfmyoglobin. Journal of Biological Chemistry 1972, 247 (12), 3783–3791; J.A. Berzofsky, et al., Sulfheme Proteins III. "Carboxysulfmyoglobin: The Relation between Electron Withdrawal from Iron and Ligand Binding. J Biol Chem 1972, 247 (12), 3774–3782; Michel, H. A Study of Sulfhemoglobin. J. Biol. Chem 1938, 126, 323–348; Nicholls, P. The Formation and Properties of Sulphmyoglobin and Sulphcatalase. Biochem. J.1961, 81, 374–383; Romero, F. J., et al., The Reactivity of Thiols and Disulfides with Different Redox States of Myoglobin. Redox and Addition Reactions and Formation of Thiyl Radical Intermediates. Journal of Biological Chemistry.1992, pp 1680–1688; Brittain, T. Studies on Ligand Binding to Sulphaemoglobin. Biochim Biophys Acta 1981, 673, 253–258; Bondoc, L. L., et al., Structure of a Stable Form of Sulfheme. Biochemistry 1986, 25 (26), 8458–8466). Similarly, a sulfHb change in the transition state from R to the T and ligand gates can play a role in such an outcome (Tomoda, A., et al., Oxidative and Reductive Reactions of Sulphhaemoglobin with Various Reagents Correlated with Changes in Quaternary Structure of the Protein. Biochem J 1984, 221 (3), 587–591). These results have been beneficial in explaining the 10% to 15% sulfHb yield from a patient-derived sample (C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579–1584; C.M. Park, et al. Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810), but the mechanistic pathway controlling such behavior also remains unknown. Therefore, there is a practical need, similar to other human hemoglobin derivatives (Hopp, M.MBHB Ref. No.23-1256-WO T., et al., Heme Determination and Quantification Methods and Their Suitability for Practical Applications and Everyday Use. Anal Chem 2020, 92 (14), 9429–9440; Yoshida, T., et al., Red Blood Cell Storage Lesion: Causes and Potential Clinical Consequences. Blood Transfusion 2019, 17 (1), 27–52; D’Alessandro, A., et al., Proteomic Analysis of Red Blood Cells and the Potential for the Clinic: What Have We Learned so Far? Expert Rev Proteomics 2017, 14 (3), 243–252; D’Alessandro, A., et al., The Red Blood Cell Proteome and Interactome: An Update. J Proteome Res 2010, 9 (1), 144–163; Rashighi, M., et al., Handling Heme: The Mechanisms Underlying the Movement of Heme within and between Cells. Free Radic Biol Med.2019, 133, 88–100), to quantify, with high accuracy and precision, the sulfHb percentage present in the blood in the presence of met-aquo Hb and higher oxy-Hb concentrations (Wu, C., et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO-Oximeter. Clin Chem 1997, 43 (1), 162–166; Aravindhan, N., et al., Sulfhemoglobinemia Presenting as Pulse Oximetry Desaturation. Anesthesiology 2000, 93 (3), 883–884; Nagababu, E., et al., Formation of Fluorescent Heme Degradation Products during the Oxidation of Hemoglobin by Hydrogen Peroxide. Biochem Biophys Res Commun 1998, 247 (3), 592–596; P. Demedts, et al. Pitfalls in Discriminating Sulfhemoglobin from Methemoglobin. Clin Chem 1997, 43 (6), 1098–1099; Van Leeuwen, et al., Three-Wavelength Method for the Optical Differentiation of Methemoglobin and Sulfhemoglobin in Oxygenated Blood. Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS 2017, 4570–4573; P. Dijkhuizen, et al. Sulfhaemoglobin. Absorption Spectrum, Millimolar Extinction Coefficient at = 620 Nm, and Interference with the Determination of Haemiglobin and Haemiglobincyanide. Clinica Chimica Acta 1977, 78 (3), 479–487; Noor, M., et al., Acquired Sulfhemoglobinemia. Alert, Notices, and Case Reports 1998, 169 (6), 386–389; Siggaard- Andersen O, et al., Hemoglobin Pigments. Spectrophotometric Determination of Oxy-, Carboxy-, Met-, and Sulfhemoglobin in Capillary Blood. Clinica Chimica Acta 1972, 42, 85– 100; Norgaard-Pedersen B, et al., Mixing Technique for Preparation of Known Fractions of Hemoglobin Pigments. Clinica Chimica Acta 1972, 42, 109–113).

[0069] Gas oximeters have been the most widely-accepted and cost-effective devices in the biomedical and toxicology community for addressing the challenge of sulfHb and met-MBHB Ref. No.23-1256-WO aquo Hb quantification in the presence of oxy-Hb. However, one concern for this heme quantification method is overlap between the characteristic electronic transitions of sulfHb (623 nm) and met-aquo Hb (635 nm) (Wu, C., et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO-Oximeter. Clin Chem 1997, 43 (1), 162–166; Chan, E. D., et al., Pulse Oximetry: Understanding Its Basic Principles Facilitates Appreciation of Its Limitations. Respir Med 2013, 107 (6), 789–799; Haymond, S., et al., Laboratory Assessment of Oxygenation in Methemoglobinemia. Clin Chem 2005, 51 (2), 434–444; Gehring, H., et al., Hemoximetry as the “Gold Standard”? Error Assessment Based on Differences among Identical Blood Gas Analyzer Devices of Five Manufacturers. International Anesthesia Research Society 2007, 105 (6), S24–S30). Other techniques have also been explored (Docherty, S., et al., The Diagnosis of Sulfated Hemoglobin (SulFHB) Secondary to Sulfur Dioxide Poisoning Using Matrix-Assisted Laser Desorption Time-of- Flight Mass Spectrometry (MALDI-TOF MS)—a Novel Approach to an Unusual Clinical Problem. Diagnostics 2020, 10 (2); Hopp, M. T., et al., Heme Determination and Quantification Methods and Their Suitability for Practical Applications and Everyday Use. Anal Chem 2020, 92 (14), 9429–9440; Zwart, A., et al., Multicomponent Analysis of Hemoglobin Derivatives with Reversed-Optics Spectrophotometer. Clin Chem 1984, 30 (3), 373–379; N. Fogh-Andersen, et al. Diode-Array Spectrophotometry for Simultaneous Measurement of Hemoglobin Pigments. Clinica Chimica Acta 1987, 166, 283–289; Stepanenko T, et al. Sulfhemoglobin under the Spotlight – Detection and Characterization of SHb and HbFeIII–SH. Biochim Biophys Acta Mol Cell Res 2023, 1870 (1), 119378; Plevniak K, et al.3D Printed Auto-Mixing Chip Enables Rapid Smartphone Diagnosis of Anemia. Conf Proc IEEE Eng Med Biol Soc 2016, 267–270), for example, high-pressure liquid chromatography normal phase (NP-HPLC) and reverse phase (RP-HPCL) coupled with fluorescence detection are techniques for rapid heme quantification (Hopp, M. T., et al., Heme Determination and Quantification Methods and Their Suitability for Practical Applications and Everyday Use. Anal Chem 2020, 92 (14), 9429–9440). The heme Soret visible excitation ranges from 390 nm to 410 nm, while the emission is monitored in the 600 nm to 630 nm range. Other emission signals at 662 nm have also been observed (Marcero, J. R., et al., Rapid and Sensitive Quantitation of Heme in Hemoglobinized Cells. BiotechniquesMBHB Ref. No.23-1256-WO 2016, 61 (2), 83–91). Similarly, fluorescence spectroscopy with excitation in the 460 nm region and emission in the 475 nm to 580 nm has been used to investigate the reaction between the heme system and hydrogen peroxide-producing oxygen reactive species (Nagababu, E., et al., Formation of Fluorescent Heme Degradation Products during the Oxidation of Hemoglobin by Hydrogen Peroxide. Biochem Biophys Res Commun 1998, 247 (3), 592–596; Nagababu E, R. J. Heme Degradation by Reactive Oxygen Species. Antioxid Redox Signal 2004, 6 (6), 967–978; Nagababu E, et. al. Role of the Membrane in the Formation of Heme Degradation Products in Red Blood Cells. Life Sci 2010, 86 (3–4), 133– 138; Goodchild, C. G, et al., Fluorescent Heme Degradation Products Are Biomarkers of Oxidative Stress and Linked to Impaired Membrane Integrity in Avian Red Blood Cells. Physiol Biochem Zool 2020, 93 (2), 129–139; Jia, Y., et al., Stopped-Flow Fluorescence Method for the Detection of Heme Degradation Products in Solutions of Chemically Modified Hemoglobins and Peroxide. Anal Biochem 2002, 308 (1), 186–188; Sinclair, P. R., et al., Measurement of Heme Concentration. Curr Protoc Toxicol 1999, 00 (1), 1–7; Meng F, A. AI. Determination of Extinction Coefficients of Human Hemoglobin in Various Redox States. Anal Biochem.2017, 521, 11–19). However, due to fluorescence signal quenching by the heme group, UV-Vis absorption spectroscopy is also used to monitor the reactions (Nagababu, E., et al., Formation of Fluorescent Heme Degradation Products during the Oxidation of Hemoglobin by Hydrogen Peroxide. Biochem Biophys Res Commun 1998, 247 (3), 592–596; Chan, E. D., et al., Pulse Oximetry: Understanding Its Basic Principles Facilitates Appreciation of Its Limitations. Respir Med 2013, 107 (6), 789–799; Haymond, S., et al., Laboratory Assessment of Oxygenation in Methemoglobinemia. Clin Chem 2005, 51 (2), 434–444; Gehring, H., et al., Hemoximetry as the “Gold Standard”? Error Assessment Based on Differences among Identical Blood Gas Analyzer Devices of Five Manufacturers. International Anesthesia Research Society 2007, 105 (6), S24–S30).

[0070] The present disclosure discloses a unique combination of UV-Vis and Fluorescence spectroscopy to measure sulfhemoglobin formation in the presence of met-aquo Hb and oxy-Hb. The results set forth herein demonstrate a linear correction between the sulfHb electronic charge transfer transition at 623 nm and emission wavelength 460 nm upon Soret excitation at 420 nm. The data set forth below indicate no oxy-Hb or met-aquo HbMBHB Ref. No.23-1256-WO interference in this linear relationship. Furthermore, this approach enabled the measurement of sulfhemoglobin from 0.02 % to 13.5% in mixtures of met-aquo hemoglobin with excess oxy-hemoglobin. These results suggested that simultaneous monitoring sulfHb electronic transition at 623 nm and emission wavelength 460 nm upon Soret excitation at 420 nm is a technique for determining the percentage of sulfhemoglobin in blood. The data and techniques presented, indicate that the fluorescence spectroscopy coupling with UV-Vis spectroscopy provides a fast and accurate method for detecting sulfhemoglobin in the blood, facilitating the correct diagnosis of sulfhemoglobinemia in patients.

[0071] In some embodiments, the present disclosure contemplates a method for measuring an amount of sulfhemoglobin in a biological sample comprising: providing a biological sample comprising hemoglobin; measuring UV-Vis absorbance of the biological sample at a first wavelength in the range of about 600 to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of 450 to 470 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample.

[0072] In some embodiments, the biological sample is a blood sample.

[0073] In some embodiments, the biological sample is human blood.

[0074] In some embodiments, the biological sample is animal blood.

[0075] In some embodiments, the method further comprises separating red blood cells from the blood sample and lysing the red blood cells

[0076] In some embodiments, the measuring a UV-Vis absorbance comprises determining a first intensity at the first wavelength in a UV-Vis absorption spectrum of the biological sample

[0077] In o some embodiments, the first wavelength maximum is 623 nm in the 600 nm to 750 nm region.

[0078] In some embodiments, the measuring a fluorescence emission comprises determining a second intensity at the second wavelength in a fluorescence emission spectrum of the biological sample.MBHB Ref. No.23-1256-WO

[0079] In some embodiments, the the fluorescence emission spectrum is measured using a fluorescence excitation at 420 nm.

[0080] In some embodiments, the second wavelength is 460 nm in the 450 nm to 470 nm region.

[0081] In some embodiments, the correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve comprises: determining if the first intensity and the second intensity correspond to a point on the first standard curve; and if the point is on the first linear standard curve, correlating the fluorescence emission with a second standard curve to determine the amount of sulfhemoglobin present in the biological sample; or if the point is not on the first linear standard, determining that the amount of sulfhemoglobin present in the biological sample is less than a detection threshold.

[0082] In some embodiments, the first standard curve is a linear standard curve of the fluorescence emission versus UV-Vis absorbance of standard samples comprising known amounts of sulfhemoglobin.

[0083] In some embodiments, the second standard curve is a linear standard curve of the fluorescence emission versus sulfhemoglobin concentration of standard samples comprising known amounts of sulfhemoglobin.

[0084] In another embodiment, this disclosure provides a device for measuring an amount of sulfhemoglobin in a biological sample comprising: a biological sample holder; a UV-Vis light source configured to pass light through the biological sample holder; a fluorescence excitation light source configured to pass light through the biological sample holder; an absorbance detector; and an emission detector perpendicular to the fluorescence excitation light source; wherein the absorbance detector collects transmitted UV-Vis light passed from the UV-Vis light source and through the biological sample holder; and wherein the emission detector collects fluorescence emission.

[0085] In some embodiments, the biological sample holder is configured to allow transmittance of the UV-Vis light source.

[0086] In some embodiments, the biological sample holder is configured to allow for detection of fluorescence emission.MBHB Ref. No.23-1256-WO

[0087] In some embodiments, the UV-Vis light source transmits light through the biological sample holder.

[0088] In some embodiments, the UV-Vis light source excitation of 420 nm emits UV- Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

[0089] In some embodiments, the light emitted by the UV-Visible light source can be tuned to a desired UV-Visible wavelength.

[0090] In some embodiments, the UV-Vis light source emits light in the range of 600 to 750 nm.

[0091] In some embodiments, the UV-Vis light source emits light at 623 nm in the 600 nm to 750 nm region.

[0092] In some embodiments, the fluorescence excitation light source transmits light through the biological sample holder.

[0093] In some embodiments, the fluorescence excitation at 420 nm light source emits UV-Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

[0094] In some embodiments, the light emitted by the fluorescence excitation source can be tuned to a desired UV-Visible wavelength.

[0095] In some embodiments, the fluorescence excitation light source emits light in the range of about 440 to about 475 nm.

[0096] In some embodiments, the fluorescence excitation light source emits light at 460 nm upon excitation with 420 nm light.

[0097] In some embodiments, the UV-Vis light source and the fluorescence excitation light source are the same light source.

[0098] In some embodiments, the UV-Vis light source and the fluorescence excitation light source are different light sources.

[0099] In some embodiments, the emission detector is configured to collect fluorescence emission from the biological sample holder. [000100] In some embodiments, the fluorescence excitation light source and the emission detector are separated by 90 degrees.MBHB Ref. No.23-1256-WO [000101] In another embodiment, this disclosure provides a method for measuring an amount of sulfhemoglobin in a biological sample using the device of any of claims 14-30 comprising: providing a biological sample comprising hemoglobin; measuring a UV-Vis absorbance of the biological sample at a first wavelength in the range of 623 nm in the range of about 600 nm to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of about 440 nm to about 475 nm upon excitation at 420 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample. EXAMPLES [000102] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way. Materials and Methods and Results HEPES buffer 0.05M, pH 7.4 Preparation [000103] This buffer was prepared by adding 2.38 g of 99% HEPES salt (C8H18N2O4S) to a volumetric flask containing 150 mL of ultra-pure deionized water. The solution was stirred for about a minute until it completely dissolved. The pH of the solution was monitored, and NaOH was added until the solution had a pH of 7.4. Then, deionized water was added to a volume of 200 mL. The solution was stored in a crystal bottle in the refrigerator for up to 4 months. Oxy-Myoglobin, Oxy-hemoglobin, Met-aquo hemoglobin, and Hydrogen Sulfide Stock Solutions [000104] Oxy-Mb stock solution was prepared by dissolving lyophilized powder of equine muscle myoglobin (Sigma Aldrich) in 0.05 mM HEPES buffer (pH 7.4) in a sealed vial. The solution was degassed by purging with nitrogen. A minimum excess (~2 fold) of sodium dithionite (Na2O4S2) solution was added to the vial to obtain the deoxy Mb (MbFe(II)) form. Once the deoxy hemeprotein was obtained, the solution was purged for 15 min with 99%MBHB Ref. No.23-1256-WO oxygen, and a purple-to-red color change was observed, corresponding to oxy-Mb. Excess sodium dithionite was removed by passing the solution through Amicon® ultrafiltration devices from Millipore. The solution spectra were recorded on a UV-Vis Shimadzu 2700 Spectrophotometer using a 1cm path-length quartz cuvette (Starna Scientific) to confirm the presence of the oxy-myoglobin. Protein concentration was calculated utilizing the Beer- Lambert law, , where c, A, b, and represent concentration (mM), absorbance, path length (1cm), and extinction coefficient, respectively. The extinction coefficient used for oxy- myoglobin is 136 cm-1mM-1at 418nm (Millar, S. J., et al., Some Observations on the Absorption Spectra of Various Myoglobin Derivatives Found in Meat. Meat Sci 1996, 42 (3), 277–288). Oxy-hemoglobin stock solution was prepared using the same methodology as the oxy-myoglobin stock solution (Román-Morales E, et al. Structural Determinants for the Formation of Sulfhemeprotein Complexes. Biochem Biophys Res Commun 2010, 400 (4), 489–492). Lyophilized human hemoglobin was obtained from Sigma Aldrich, and the extinction coefficient used to calculate oxy-hemoglobin concentrations was 1251at 415nm (Antonini, E. and Brunori, M. Chapter 3: The Derivates of Ferric Hemoglobin and Myoglobin. In Hemoglobin and Myoglobin in the Reactions with Ligands; 1971; p 41). Met- aquo hemoglobin stock solution was prepared by dissolving the protein powder in HEPES buffer 0.05 mM (pH 7.4). A 10% molar excess of K3(Fe(CN)6), ACS reagent, 99.0%, was used to fully oxidize the hemeprotein to met-aquo Hb. A UV-vis spectrophotometer was used to confirm the presence of met-aquo Hb. Potassium ferricyanide excess in a solution was removed using Amicon® ultrafiltration devices. The met-aquo Hb concentration was determined using the molar extinction coefficient 179 cm-1mM-1at 405 nm (Antonini, E. and Brunori, M. Chapter 2: The Derivates of Ferrous Hemoglobin and Myoglobin. In Hemoglobin and Myoglobin in the Reactions with Ligands.; 1971; p 19). Hydrogen sulfide stock solution (83 mM) was prepared anaerobically in an amber vial by dissolving 0.020 g ofsodium sulfide nonahydrate salt (Na2S 9H2O, 99.99%, Alfa Aesar) in 1.0 mL of HEPES0.05M buffer at pH 7.4. The solution was purged with nitrogen. SulfMb and SulfHb Formation Reactions and Uv-Vis Spectrophotometric MeasurementsMBHB Ref. No.23-1256-WO [000105] Samples were prepared using 0.05M HEPES buffer at pH 7.4 and 25 °C. Oxy- myoglobin and oxy-hemoglobin stock solutions were used to prepare eight individual solutions, each with a concentration of 55 M in a 1 cm quartz cuvette with a cap from Starna Scientific. To generate sulfMb and sulfHb, aliquots of the hydrogen sulfide stock solution (83 mM) were added to the oxy-myoglobin or oxy-hemoglobin solutions in a quartz cuvette, resulting in individual hydrogen sulfide concentrations ranging from 55 M to 1,155 M. Formation of sulfMb and sulfHb was monitored by spectral scanning using a Shimadzu UV 2700 spectrophotometer every minute over four hours. The presence of sulfMb was evaluated by observing characteristic Q bands around 618nm, while sulfHb was identified by Q-bands around 623 nm as described in previous studies (J.A. Berzofsky, et al., Sulfheme Proteins. Optical and Magnetic Properties of Sulfmyoglobin and Its Derivates. J Biol Chem 1971, 246 (10), 3367–3377; Nicholls, P. The Formation and Properties of Sulphmyoglobin and Sulphcatalase. Biochem. J.1961, 81, 374–383; Carrico, R. J., et al., The Preparation and Some Physical Properties of Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (7), 2386–2391). [000106] Formation reactions of sulfHb and sulfMb using 3-21 fold H2S molar excess follow a pseudofirst-order reaction (Pietri, R., et al., Factors Controlling the Reactivity of Hydrogen Sulfide with Hemeproteins. Biochemistry 2009, 48 (22), 4881–4894). Using The OriginLab® Origin 9, the pseudofirst-order constants (kobs) values were determined byplotting Ln (A-A ) versus time (seconds) for each H2S concentration. The second order rateconstant was obtained from the slope of kobsversus H2S molar concentration plot. This process was performed in triplicate for each sample. The corresponding extinction coefficient used for analysis was 20.8 (P. Dijkhuizen, et al., Sulfhaemoglobin. Absorption Spectrum, Millimolar Extinction Coefficient at = 620 Nm, and Interference with the Determination of Haemiglobin and Haemiglobincyanide. Clinica Chimica Acta 1977, 78 (3), 479–487). Sulfheme Formation Reaction: Spectrofluorometric Measurements [000107] Fluorescence spectroscopy measurements were conducted using a Jasco FP-8500 spectrofluorometer with the following settings: excitation and emission slit: 10nm / 10nm,MBHB Ref. No.23-1256-WO excitation wavelength: 420 nm, and an emission wavelength of 460 nm. The experiments were performed at a temperature of 25 °C. [000108] Initially, an oxy-hemoglobin solution with a concentration of 55 M was mixed with the H2S solution in a concentration ratio 1:21. The reaction spectra for sulfhemoglobin formation reaction were recorded using both UV-vis and fluorescence spectroscopy over 65 minutes. Next, the spectra of the hemoglobin species were obtained. Three 1 cm quartzcuvettes with caps filled with met-aquo Hb (55.0 M), OxyHb (55.0 M), and SulfHb (5.6M), respectively, were analyzed using a spectrophotometer and spectrofluorometer at 25 ° C. Following that, the sulfhemoglobin formation reaction was initiated by mixing oxy- hemoglobin (55.0 M), met-aquo hemoglobin (5.5 M), and H2S solution (1,155 M) in a 1 cm quartz cuvette with a cap. The cuvette was filled with HEPES 0.05M buffer at pH 7.4 and maintained at 25 ° C. Progress of the reaction was monitored using UV-vis and fluorescence spectroscopy for the first 65 minutes. Example 1: Optical absorption of SulfMb and SulfHb formation at prolonged time [000109] UV-vis spectroscopy was utilized to understand the conversion from oxy-Mb and oxy-Hb to SulfMb and SulfHb in the presence of H2S and the progression of their heme products over longer reaction times. The aim was to evaluate the formation and stability of sulfMb and sulfHb over a four-hour reaction period at various concentrations. In Figure 2A, the visible transition of sulfmyoglobin is depicted, with peaks observed at 545 nm and 582 nm, which are assigned to to * excitations. A distinctive charge transfer bandis also seen at 618 nm, representing to d (dyz, dxz) charge transfer transitions (Johnson, E.A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; Carrico, R. J., et al., The Reversible Binding of Oxygen to Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (20), 7212–7215; H. Arbelo-López, et al., Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955). Changes in intensity of these three electronic transitions have been associated with formation of met- aquo sulfMb and a mixture of its heme ligand states, such as sulfMbSH2, deoxy-sulfMb, oxy- sulfMb, and MbSH2derivatives (J.A. Berzofsky, et al., Sulfheme Proteins. The ReversibleMBHB Ref. No.23-1256-WO Oxygenation of Ferrous SulfMyoglonin. J Biol Chem 1971, 246 (23), 7366–7372; Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; J.A. Berzofsky, et al., Sulfheme Proteins. Optical and Magnetic Properties of Sulfmyoglobin and Its Derivates. J Biol Chem 1971, 246 (10), 3367–3377.; Berzofsky, J. A., et al., Sulfheme Proteins. The Stoichiometry of Sulfur Incorporation and the Isolation of Sulfhemin, the Prosthetic Group of Sulfmyoglobin. Journal of Biological Chemistry 1972, 247 (12), 3783–3791; J.A. Berzofsky, et al., Sulfheme Proteins III. Carboxysulfmyoglobin: The Relation between Electron Withdrawal from Iron and Ligand Binding. J Biol Chem 1972, 247 (12), 3774–3782; Michel, H. A Study of Sulfhemoglobin. J. Biol. Chem 1938, 126, 323–348; Nicholls, P. The Formation and Properties of Sulphmyoglobin and Sulphcatalase. Biochem. J.1961, 81, 374–383; Romero, F. J., et al., The Reactivity of Thiols and Disulfides with Different Redox States of Myoglobin. Redox and Addition Reactions and Formation of Thiyl Radical Intermediates. Journal of Biological Chemistry.1992, pp 1680–1688; Brittain, T. Studies on Ligand Binding to Sulphaemoglobin. Biochim Biophys Acta 1981, 673, 253–258; Bondoc, L. L., et al., Structure of a Stable Form of Sulfheme. Biochemistry 1986, 25 (26), 8458–8466). Theoretical calculations also supported these observations (H. Arbelo-López, et al., Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955). Figure 2B displays the 618nm transition intensity variation as a function of H2S concentration and time. The analysis allowed the determination of the average reaction constant, which was found to be 0.61 M-1s-1for the process. This value provide insight into the rate at which sulfmyoglobin is formed in the presence of different H2S concentrations. [000110] Similarly, sulfHb formation was observed through the appearance of the 540 nmand 576 nm electronic transition, along with the characteristic to d charge transfer band at623 nm (Figure 2C) (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; Carrico, R. J., et al., The Reversible Binding of Oxygen to Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (20), 7212–7215; H. Arbelo-López, et al., Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–MBHB Ref. No.23-1256-WO 4955). As the reaction progressed, autoxidation occurred, forming different sulfheme derivatives, resulting in a decrease in intensity and a broadening of the 623 nm band. Furthermore, it is noted that not all monomeric subunits of Hb generate sulfheme, suggesting a mixture of heme and sulfheme within the tetrameric structure of Hb, which limits the intensity at 623 nm. Figure 2D illustrates changes in the strength of the 623 nm transition as a function of H2S concentration and time. The data obtained permitted a reaction constant of 1.22 M-1s-1to be calculated. It should be noted that these constants for sulfMb (0.61 M-1s-1) and sulfHb (1.22s-1) did not fully characterize the kinetics of sulfheme formation. Instead, they represent the long-term presence of unknown derivatives. A separate kinetic constant ~1.0x103M-1s-1, determined for the reaction between met-aquo Mb and hydrogen peroxide (H2O2) in the presence of H2S, supports the previous assignments (Nicholls, P. The Formation and Properties of Sulphmyoglobin and Sulphcatalase. Biochem. J.1961, 81, 374– 383). [000111] The energetic barrier to the heme intermediate compound 0 (Fe(III)-OOH, Cpd-0) is 1.9 kcal / mol for the peroxide reaction between met-aquo hemoglobin and H2O2, whereas for the oxy-Mb in the presence of H2S, the heme Cpd-0 has a higher energy of 23.3 kcal / mol (H. Arbelo-López, et al. Charge Transfer and to * Transitions in the Visible Spectra of Sulfheme Met Isomeric Structures. J Phys Chem 2018, 122, 4947–4955; Arbelo-Lopez, H. D., et al., Homolytic Cleavage of Both Heme-Bound Hydrogen Peroxide and Hydrogen Sulfide Leads to the Formation of Sulfheme. Journal of Physical Chemistry B 2016, 120 (30), 7319–7331; Rodriguez-Mackenzie, A. D., et al., A Reaction Pathway to Compound 0 Intermediates in Oxy-Myoglobin through Interactions with Hydrogen Sulfide and His64. J Mol Graph Model 2020, 94; A.D. Rodriguez-Mackenzie, et al., Mechanistic Inside into PH Dependent SulfMyoglobin Formation: Spin Control and His64 Proton Relay. J. Chemistry). However, the relatively small 21.4 kcal / mol difference in energy barrier cannot account for the kinetic factor difference of 103. This observation further supported the idea that low values in the reaction constant represented a mixture of sulfheme species in sulfMb and sulfHb. Nonetheless, it remained an open question to determine specific derivatives that could be present and explain the kinetic behavior of sulfheme in patients displaying symptoms of sulfhemoglobinemia.MBHB Ref. No.23-1256-WO [000112] After a reaction time of seven hours between oxy-Mb, oxy-Hb, and H2S, the spectral changes of sulfMb (Figure 3A) and sulfHb (Figure 3B) were analyzed by converting them into absorbance ratios, namely (A618 / A582) and (A623 / A576), as a function of hydrogen sulfide concentration. The ratios have been commonly used to estimate the yields and purity criteria for sulfheme in samples (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30– 40.; J.A. Berzofsky, et al., Sulfheme Proteins. Optical and Magnetic Properties of Sulfmyoglobin and Its Derivates. J Biol Chem 1971, 246 (10), 3367–3377; Nichol, A. W.; Hendry, I.; Morell, D. B. Mechanism of Formation of Sulphhaemoglobin. Biochim Biophys Acta 1967, 156, 97–108; C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579–1584; C.M. Park, et al. Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810; Carrico, R. J., et al., The Preparation and Some Physical Properties of Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (7), 2386–2391). [000113] Figure 3 shows that sulfHb exhibits smaller (A623 / A576) ratios than sulfMb (A618 / A582). Also, a nonlinear behavior, specifically a quasi-sigmoidal curve, was observed for the sulfhemoglobin formation reaction (Figure 3B), whereas a linear behavior was observed for sulfMb (Figure 3A). These observations support the understanding in the art, which suggests that these ratios do not follow a linear relationship with sulfheme concentration. Higher values (3.28 to 1.0) than those presented in Figure 3 have been reported for sulfheme formation (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30– 40). These values have been associated with different percentages of sulfMb purity (i.e., 90%) and sulfHb purity (40%, 60%, and 75%) (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; Nichol, A. W., et al., Mechanism of Formation of Sulphhaemoglobin. Biochim Biophys Acta 1967, 156, 97–108; C.M. Park, et al. Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810). It has been suggested that as the values of the ratios decrease, indicating a decrease in the absorbance at higher wavelengths, the mixture of the sulfheme derivatives increases (Nichol, A. W., et al.,MBHB Ref. No.23-1256-WO Mechanism of Formation of Sulphhaemoglobin. Biochim Biophys Acta 1967, 156, 97–108). These findings suggest that the absorbance ratios (A618 / A582) and (A623 / A576) indicate sulfheme formation but do not directly correlate with sulfheme concentration. The purity and mixture of sulfheme derivatives in the samples influence the ratios. [000114] The approach of analyzing the (A623 / A576) and (A618 / A582) absorbance ratios has also been applied to explain the yield of sulfHb in patient-derived samples, ranging from 10% to 15% (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579– 1584; C.M. Park, et al., Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810). Therefore, the smaller ratios observed for sulfHb (A623 / A576) and sulfMb (A618 / A582) in Figure 3, after seven hours of reaction times, suggested that this sulfheme preparation method could provide further insight into the patient-derived sulfHb samples. The data supports sulfHb in the presence of sulfHb with inhomogeneity heme groups, some in their native form and others with the sulfur atom incorporated. The factors determining the extent to which heme groups have become sulfurated or remain in their native state remain challenging to predict (C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579–1584; C.M. Park, et al., Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810). The presence of hybrid groups, consisting of sulfurated and native heme groups, could explain the observed behavior during the formation of sulfhemoglobin at different H2S concentrations. Various factors contribute to this phenomenon, including (i) the techniques used for sulfheme synthesis, (ii) the nature of the oxidizing agent (oxygen or hydrogen peroxide), (iii) reaction time, (iv) sulfheme ligand species, (v) autoxidation processes, and (vi) pH. Therefore, the (A618 / A582) and (A623 / A576) absorbance ratios suggest valuable insights into the presence of these sulfheme derivates in the red blood cells, but caution should be exercised in interpreting the results. In summary, analyzing the absorbance ratios can contribute to understanding the landscape of sulfheme derivates in red blood cells, considering the complexities and factors influencing their formation and presence.MBHB Ref. No.23-1256-WO Example 2: Sulfhemoglobin UV-Vis Absorption and Fluorescence Response Spectra [000115] In an in vitro reaction between oxy-hemoglobin in solution (55 M) and H2S at a concentration ratio of 1:21, sulfhemoglobin formation and additional sulfheme products were observed using UV-vis and fluorescence spectroscopy (Figure 4). The Soret spectrum, which corresponds to the absorption maximum at 416 nm, suggests the presence of a mixture of sulfheme derivatives. Pure oxy-, deoxy-, and met- aquo sulfhemoglobin has maximum absorption at 412 nm, 423 nm, and 403 nm, respectively (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; Carrico, R. J., et al., The Reversible Binding of Oxygen to Sulfhemoglobin. Journal of Biological Chemistry 1978, 253 (20), 7212–7215). Production of the observed mixture of sulfHb species was expected due to the long reaction times. Additionally, the excess hydrogen sulfide in the reaction played a significant role in product formation, as it can lead to heme reduction under these experimental conditions (Pietri, R., et al., Factors Controlling the Reactivity of Hydrogen Sulfide with Hemeproteins. Biochemistry 2009, 48 (22), 4881–4894). [000116] For sulfhemoglobin fluorescence detection, the parameters used were excitation and emission slits10 / 10, an excitation wavelength of 420 nm, and an emission wavelength of 460 nm. Figure 4 shows a fluorescence profile signal observed upon sample excitation at 420 nm, with a maximum of 460 nm. The normalized spectra show a characteristic shift stokes of 40 nm (UV-Vis absorption maximum minus fluorescence maximum), which indicated these processes. It is worth noting that the heme group typically quenches the fluorescence signal of hemoglobin. However, in the case of sulfhemoglobin, the sulfur ring attached to pyrrole B permits the fluorescence signal, to be observed potentially overcoming the quenching effect (albeit perhaps by other mechanisms). [000117] To investigate the potential of fluorescence spectroscopy for detecting sulfHb in solution, individual UV-Vis and fluorescence spectra were obtained for oxy-hemoglobin (55μM), met-aquo hemoglobin (55μM), and sulfhemoglobin (5.63 μM), as shown in Figure 5. In the 600 nm region, the absorption spectra of met-aquo hemoglobin and sulfhemoglobin showed similar electronic transitions between the two species. However, there was a concentration difference of approximately ten fold for these two species (between 55μM andMBHB Ref. No.23-1256-WO 5.53μM). This contrast was not observed in the case of oxy-Hb (Figure 5A), where the absorption spectrum differed significantly from those of met-aquo hemoglobin and sulfhemoglobin. [000118] The smaller intensity of the (A623 / A576) absorbance ratio suggested the presence of a mixture of sulfHb species. This ratio indicated the relative concentrations of the absorbance at 623nm and 576nm, respectively, corresponding to characteristic transitions for sulfHb. The different ratios observed for met-aquo hemoglobin further supported the presence of a mixture of sulfHb species in the solution. [000119] These findings suggested that fluorescence spectroscopy could be a valuable tool for detecting sulfHb in solution, as the fluorescence properties of sulfhemoglobin can differ from those of the other hemoglobin species. [000120] Upon excitation at 420 nm, which corresponds to the Soret to * transitions (415 nm oxy-Hb, 405 nm met-aquo Hb, and 421 nm sulfHb), a fluorescence response 460 nm was observed (Figure 5B). The fluorescence response of sulfhemoglobin was higher than those for met-aquo hemoglobin and oxy-hemoglobin, despite the sulfheme species having a concentration ten times lower than the other hemoglobin species. This finding further supported using fluorescence spectroscopy to detect sulfhemoglobin. To evaluate sulfhemoglobin formation in a mixture of oxy-hemoglobin (55.0 M) and met-aquo hemoglobin (5.5 M), hydrogen sulfide (1,155 M) was added, and the reaction was monitored for 65 minutes. The presence of H2S excess ensured formation of a mixture of sulfHb derivatives. Under these conditions, Figures 6A and 6B show an increase in intensity in the 623 nm and the 460 nm fluorescence spectra, respectively. The same experiment was performed without met-aquo hemoglobin, yielding similar results. These results strongly supported that excitation at the sulfheme Soret transition at 420 nm leads to an increase in fluorescence intensity of 460 nm, indicative of sulfhemoglobin formation. Example 3: Sulfhemoglobin Correlations of UV-Vis and Fluorescence Data [000121] Figure 7 illustrates the relationship between the charge transfer intensity of the transition at 623 nm (Figure 6A) and the fluorescence intensity at 460 nm (Figure 6B) for sulfhemoglobin. The data demonstrated a linear behavior between these spectroscopicMBHB Ref. No.23-1256-WO properties. A similar trend was observed for the relationship between the UV-Vis spectra of sulfhemoglobin formation over time, as oxy-hemoglobin (55 M) reacted with H2S solution (1,155 M), and the fluorescence intensity at 460 nm (not shown). The linear relation between these measurements further supported the significance of sulfhemoglobin fluorescence and its correlation with the classical 623 nm sulfheme transition characterizedby to d (dyz, dxz) charge transfer.[000122] Figure 8 demonstrates the relationship between fluorescence intensity at 460 nm and the percentage of sulfhemoglobin in the samples, as calculated from the information displayed in Figures 6A and 6B. Physiological levels of sulfhemoglobin have been estimated to be below 0.037 g / dL (5.5 μM, ~ 0.28%), while clinically detectable cyanosis symptoms can occur at levels around 0.5 g / dL (74.4 μM, ~ 3.8%) in the blood (Whiteman, M., et al., Emerging Role of Hydrogen Sulfide in Health and Disease: Critical Appraisal of Biomarkers and Pharmacological Tools. Clin Sci 2011, 121 (11), 459–488; Saeedi A, et al., A. Effects of Long-Term Exposure to Hydrogen Sulfide on Human Red Blood Cells. Int J Occup Environ Med 2015, 6, 20–25; George, A.; Goetz, D. A Case of Sulfhemoglobinemia in a Child with Chronic Constipation. Respir Med Case Rep 2017, 21, 21–24; Campagna, G.; Espaillat, A.; Pfeiffer, T., et al., A Case of Sulfhemoglobinemia Secondary to a Urinary Tract Infection. J Pediatr Hematol Oncol 2019; Dupouy, J., et al., Une Cause Rare de Cyanose: Sulfhémoglobinémie Imputable Au Thiocolchicoside (Miorel®). Rev Mal Respir 2010, 27 (1), 80–83; Langford, J. S., et al., An Adolescent Case of Sulfhemoglobinemia Associated with High-Dose Metoclopramide and N-Acetylcysteine. Ann Emerg Med 1999, 34 (4), 538– 541). Severe sulfHb cyanosis rarely exceeds 10% (200 μM) (Wu, C., et al., A Case of Sulfhemoglobinemia and Emergency Measurement of Sulfhemoglobin with an OSM3 CO- Oximeter. Clin Chem 1997, 43 (1), 162–166). Smaller (A623 / A576) absorbance ratios have been associated with a 10% to 15% sulfHb yield in patient-derived sample (Johnson, E. A. The Reversion to Haemoglobin of Sulphhaemoglobin and Its Coordination Derivatives. Biochim Biophys Acta 1969, 207, 30–40; C.M. Park, et al., Sulfhemoglobinemia Clinical and Molecular Aspects. N Engl J Med 1984, 310 (24), 1579–1584; C.M. Park, et al., Sulfhemoglobin. Properties of Partially Sulfurated Tetramers. J Biol Chem 1986, 261 (19), 8805–8810). Figure 8 also indicates a direct relationship between fluorescence data at 460 nmMBHB Ref. No.23-1256-WO and the percentage of sulfheme in the sample. These values supported the reported sulfhemoglobin range observed in patients. Therefore, the relationship between sulfHb Soret excitation at 420 nm, the induced fluorescence at 460 nm, and its correlation with the charge transfer absorption at 623 nm indicated that these relationships and their spectroscopic detection can be used effectively for detecting sulfhemoglobinemia in patients. Conclusion [000123] The data set forth herein indicate that a combination of fluorescence spectroscopy and UV-Vis spectroscopy can be a powerful and effective method for detecting sulfhemoglobin in the blood. The fluorescence response at 460nm, coupled with the absorbance ratios at specific wavelengths, provides valuable information about the presence and concentration of sulfhemoglobin. This approach beneficially offers a fast and accurate detection method for sulfhemoglobinemia in patients that can allow healthcare professionals to better understand and determine sulfhemoglobin levels, resulting in appropriate medical interventions and treatments as needed. [000124] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments claimed. Thus, it should be understood that although the present description has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed can be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. Although some aspects of the present disclosure can be identified herein as particularly advantageous, it is contemplated that the present disclosure is not limited to these particular aspects of the disclosure.MBHB Ref. No.23-1256-WO [000125] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [000126] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. [000127] It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. References (1) Whiteman, M.; Le Trionnaire, S. L.; Chopra, M.; Fox, B.; Whatmore, J. Emerging Role of Hydrogen Sulfide in Health and Disease: Critical Appraisal of Biomarkers and Pharmacological Tools. 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Claims

MBHB Ref. No.23-1256-WO What is claimed is:

1. A method for measuring an amount of sulfhemoglobin in a biological sample comprising: providing a biological sample comprising hemoglobin; measuring UV-Vis absorbance of the biological sample at a first wavelength in the range of about 600 to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of 450 to 470 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample.

2. The method of claim 1, wherein the biological sample is a blood sample.

3. The method of claim 1, wherein the biological sample is human blood.

4. The method of claim 1, wherein the biological sample is animal blood.

5. The method of any of claims 2-4, further comprising separating red blood cells from the blood sample and lysing the red blood cells.

6. The method of any of claims 1-5, wherein measuring a UV-Vis absorbance comprising determining a first intensity at the first wavelength in a UV-Vis absorption spectrum of the biological sample.

7. The method of any of claims 1-6, wherein the first wavelength maximum is 623 nm in the 600 nm to 750 nm region.MBHB Ref. No.23-1256-WO 8. The method of any of claims 1-7, wherein measuring a fluorescence emission comprising determining a second intensity at the second wavelength in a fluorescence emission spectrum of the biological sample.

9. The method of any of claims 1-8, wherein the fluorescence emission spectrum is measured using a fluorescence excitation at 420 nm.

10. The method of any of claims 1- 9 wherein the second wavelength is 460 nm in the 450 nm to 470 nm region.

11. The method of any of claims 1-10, wherein correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve comprises: determining if the first intensity and the second intensity correspond to a point on the first standard curve; and if the point is on the first linear standard curve, correlating the fluorescence emission with a second standard curve to determine the amount of sulfhemoglobin present in the biological sample; or if the point is not on the first linear standard, determining that the amount of sulfhemoglobin present in the biological sample is less than a detection threshold.

12. The method of any of claims 1-11, wherein the first standard curve is a linear standard curve of the fluorescence emission versus UV-Vis absorbance of standard samples comprising known amounts of sulfhemoglobin.

13. The method of claims 11 or 12, wherein the second standard curve is a linear standard curve of the fluorescence emission versus sulfhemoglobin concentration of standard samples comprising known amounts of sulfhemoglobin.

14. A device for measuring an amount of sulfhemoglobin in a biological sample comprising:MBHB Ref. No.23-1256-WO a biological sample holder; a UV-Vis light source configured to pass light through the biological sample holder; a fluorescence excitation light source configured to pass light through the biological sample holder; an absorbance detector; and an emission detector perpendicular to the fluorescence excitation light source; wherein the absorbance detector collects transmitted UV-Vis light passed from the UV-Vis light source and through the biological sample holder; and wherein the emission detector collects fluorescence emission.

15. The device of claim 14, wherein the biological sample holder is configured to allow transmittance of the UV-Vis light source.

16. The device of claim 14 or 15, wherein the biological sample holder is configured to allow for detection of fluorescence emission.

17. The device of any of claims 14-16, wherein the UV-Vis light source transmits light through the biological sample holder.

18. The device of any of claims 14-17, wherein the UV-Vis light source excitation of 420 nm emits UV-Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

19. The device of any of claims 14-18, wherein light emitted by the UV-Visible light source can be tuned to a desired UV-Visible wavelength.

20. The device of any of claims 14-19, wherein the UV-Vis light source emits light in the range of 600 to 750 nm.MBHB Ref. No.23-1256-WO 21. The device of any of claims 14-20, wherein the UV-Vis light source emits light at 623 nm in the 600 nm to 750 nm region.

22. The device of any of claims 14 -21, wherein the fluorescence excitation light source transmits light through the biological sample holder.

23. The device of any of claims 14-22, wherein the fluorescence excitation at 420 nm light source emits UV-Visible light; and wherein the UV-Vis light source emits light in the range of about 440 nm to about 475 nm.

24. The device of any of clams 14-23, wherein light emitted by the fluorescence excitation source can be tuned to a desired UV-Visible wavelength.

25. The device of any of claims 14-24, wherein the fluorescence excitation light source emits light in the range of about 440 to about 475 nm.

26. The device of any of claims 14-25, wherein the fluorescence excitation light source emits light at 460 nm upon excitation with 420 nm light.

27. The device of any of claims 14-26, wherein the UV-Vis light source and the fluorescence excitation light source are the same light source.

28. The device of any of claims 14-26, wherein the UV-Vis light source and the fluorescence excitation light source are different light sources.

29. The device of any of claims 14-28, wherein the emission detector is configured to collect fluorescence emission from the biological sample holder.MBHB Ref. No.23-1256-WO 30. The device of any of claims 14-29, wherein the fluorescence excitation light source and the emission detector are separated by 90 degrees.

31. A method for measuring an amount of sulfhemoglobin in a biological sample using the device of any of claims 14-30 comprising: providing a biological sample comprising hemoglobin; measuring a UV-Vis absorbance of the biological sample at a first wavelength in the range of 623 nm in the range of about 600 nm to about 750 nm; measuring a fluorescence emission of the biological sample at a second wavelength in the range of about 440 nm to about 475 nm upon excitation at 420 nm; and correlating the UV-Vis absorbance and the fluorescence emission with a first linear standard curve to determine the amount of sulfhemoglobin present in the biological sample.

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