JP2014503197A - High-throughput, sensitive detection of glucose-6-phosphate dehydrogenase - Google Patents

Abstract

Provided herein is a method for determining the amount of glucose-6-phosphate dehydrogenase (“G6PDH”) in a biological sample. Also provided herein are methods for detecting G6PDH deficiency and methods for diagnosing G6PDH-related disorders such as acute hemolytic anemia and pre-eclampsia. In some embodiments, Applicants' teachings provide a method of detecting G6PDH deficiency in a subject. The method includes, for example, determining the amount of G6PDH in a biological sample obtained from a subject by mass spectrometry and comparing the amount of G6PDH in the biological sample to an appropriate control, wherein G6PDH deficiency is detected when the amount of G6PDH in the sample is less than the appropriate control.

Description

RELATED APPLICATION This application is related to and claims priority to US Provisional Patent Application No. 61 / 416,957, filed Nov. 24, 2010. The entire contents of this US provisional patent application are expressly incorporated herein by reference.

Background Glucose-6-phosphate dehydrogenase ("G6PD" or "G6PDH") is a cytoplasmic enzyme in the pentose phosphate pathway (also called the phosphogluconate pathway). This metabolic pathway produces pentose and nicotinamide adenine dinucleotide phosphate (“NADPH”) in cells such as erythrocytes and thus supplies reducing energy. NADPH also maintains the level of glutathione in cells that helps protect red blood cells from oxidative damage. Glucose-6-phosphate dehydrogenase is stimulated to form 6-phosphoglucono-δ-lactone by its substrate, glucose-6-phosphate (“G6P”). Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the pentose phosphate pathway.

  Genetic deficiencies of G6PDH in humans predispose certain disorders, such as non-immune hemolytic anemia. The present methods for determining the presence of G6PDH and / or G6PDH activity include qualitative or quantitative fluorescent screening. However, such methods are often subjective and in general often do not detect partial deficiencies of G6PDH. For example, see Non-Patent Document 1 and Non-Patent Document 2.

Reclos GJ et al., “Glucose-6-Phosphate Dehydrogenase Defensive Neonatal Screening” J Med Screen, 7: 46-51 (2000) Kaplan M et al., “Comparison of Commercial Screening Tests for Glucose-6-Phosphate Dehydrogenase Dependency in the Neo-Period”, Vol. 37-43 (97-37).

SUMMARY Accordingly, in some embodiments, Applicants' teachings provide a high-throughput method for detecting the amount of G6PDH in a biological sample. This method includes, for example, reacting a biological sample with G6P and NADP in the presence of a surfactant and buffer under conditions suitable to form 6-phosphogluconic acid, quenching the biological sample, Forming a quenched sample and detecting by mass spectrometry the presence or amount of 6-phosphogluconic acid in the quenched sample, wherein 6-phosphoglucone in the quenched sample The amount of acid is related to the amount of G6PDH in the biological sample.

  In some embodiments, quenching is achieved using an enzyme denaturant.

  In some embodiments, the mass spectrometry is tandem mass spectrometry. In some embodiments, mass spectrometry is achieved using a mass spectrometer with a thermally assisted electrospray ionization probe, such as a TurboIonSpray® (TIS) probe. In some embodiments, mass spectrometry is achieved using a mass spectrometer with a reverse phase liquid chromatography column.

  In some embodiments, detection occurs in less than about 5 minutes. In some embodiments, detection occurs in less than about 3 minutes. In some embodiments, the method allows for detection of G6PDH of about 0.01 milliunit or less. In some embodiments, detection is not substantially affected by temperature variations.

  In some embodiments, Applicants' teachings provide a method of detecting G6PDH deficiency in a subject. The method includes, for example, determining the amount of G6PDH in a biological sample obtained from a subject by mass spectrometry and comparing the amount of G6PDH in the biological sample to an appropriate control, wherein G6PDH deficiency is detected when the amount of G6PDH in the sample is less than the appropriate control.

  In some embodiments, the subject is a newborn or infant. In some embodiments, the subject is a pregnant woman.

  In some embodiments, an amount of G6PDH in the sample that is 10% less than the control indicates G6PDH deficiency in the subject. In some embodiments, an amount of G6PDH in the sample that is 25% less than the control indicates G6PDH deficiency in the subject. In some embodiments, an amount of G6PDH in a sample that is 50% less than a control indicates G6PDH deficiency in the subject.

  In some embodiments, detection of G6PDH deficiency comprises reacting a biological sample with G6P and NADP in the presence of a detergent and buffer under conditions suitable to form 6-phosphogluconic acid. Quenching the biological sample to form a quenched sample, and detecting the presence or amount of 6-phosphogluconic acid in the quenched sample by mass spectrometry, wherein the quench The amount of 6-phosphogluconic acid in the prepared sample is related to the amount of G6PDH in the biological sample.

  In some embodiments, Applicants' teachings provide a method of diagnosing acute hemolytic anemia in a subject. The method includes, for example, detecting G6PDH deficiency in a subject according to the applicant's teaching, wherein G6PDH deficiency in a subject indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample that is 25% less than the control indicates acute hemolytic anemia in the subject.

  In some embodiments, Applicants' teachings provide a method of diagnosing pre-eclampsia in a subject. The method includes, for example, detecting G6PDH deficiency in a subject according to the applicant's teaching, wherein G6PDH deficiency in a subject indicates pre-eclampsia in a subject. In some embodiments, an amount of G6PDH in the sample that is 25% less than the control indicates pre-eclampsia in the subject.

  In some embodiments, Applicants' teachings provide a prognostic method for increased mortality and / or morbidity due to pre-eclampsia or acute hemolytic anemia in a subject. The method includes, for example, detecting G6PDH deficiency in a subject according to applicant's teachings, wherein the amount of G6PDH in a biological sample that is 50% or less of a control is mortality and / or in the subject. Shows the prognosis of increased morbidity.

  In some embodiments, a suitable control is a control based on G6PDH levels in a normal population. In some embodiments, the biological sample is a dry blood sample.

  In some embodiments, Applicants' teachings provide a kit for detecting the amount of G6PDH in a biological sample. The kit may be, for example, glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate, optionally a buffer, optionally a surfactant, nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH. And instructions for preparing a reaction mixture that facilitates the reaction. In some embodiments, the kit also includes an enzyme denaturing agent. In some embodiments, the kit further comprises instructions for preparing a sample for analysis with a mass spectrometer. In a further embodiment, the kit also includes a calibration curve comprising a plot in millimeters of [6-phosphogluconic acid / glucose-6-phosphate area] versus G6PDH.

  These and other features of Applicants' teachings are set forth herein.

FIG. 1 represents a mass spectrometric reading on MRM (Multiple Reaction Monitoring) obtained from the exemplary method of applicants' teaching. FIG. 2 represents a comparative mass spectrometry reading in MRM obtained from the exemplary method of applicants' teachings. FIG. 3 represents an exemplary standard curve obtained using various methods of applicants' teachings. 4A and 4B represent mass spectrometric readings in MRM obtained from an exemplary method of applicants' teachings. FIG. 5 represents exemplary standard curves obtained using various methods of applicants' teachings. 6A and 6B represent mass spectrometric readings in MRM obtained from an exemplary method of applicants' teachings. FIGS. 7A and 7B are a correlation plot and a Brand and Altman plot, respectively, comparing the exemplary method of Applicants' teaching with a commercially available assay.

Description Applicants' teachings determine, at least in part, the amount of G6PDH in a biological sample using mass spectrometry, eg, MS / MS, LC / MS or LC / MS / MS, quickly and accurately. It is based on the knowledge that it can be done. While not wishing to be bound by any particular theory, the ability to determine the amount of G6PDH (eg, not simply its presence or absence) is useful, for example, in detecting partial G6PDH deficiency in a subject. It is believed that there is.

  G6PDH converts glucose-6-phosphate to 6-phosphogluconolactone and simultaneously reduces NADP to NADPH. 6-phosphogluconolactone is then converted to 6-phosphogluconic acid by gluconolactonase. Previous methods for determining G6PDH activity, including the Beutler method, utilize NADPH formation as an indicator of enzymatic reactions. However, because the C13 isotope isomer of NADP overlaps with the first C12 isotope isomer of NADPH, the amount of NADPH can be difficult to determine in assays such as by mass spectrometry. Accordingly, the amount of 6-phosphogluconic acid is monitored in the methods provided herein.

  Accordingly, in some embodiments, Applicants' teachings provide a high-throughput method for detecting the amount of G6PDH in a biological sample. Such a method involves reacting a biological sample with G6P and NADP (eg, in the presence of a surfactant and buffer) under conditions suitable to form 6-phosphogluconic acid and quenching the biological sample. Forming a quenched sample and detecting the presence or amount of 6-phosphogluconic acid in the quenched sample by mass spectrometry. In such a method, the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.

  As used herein, the term “high throughput” refers to the process of assaying a large number of samples in a relatively short period of time. High throughput assays can be accomplished using, for example, 96 well plates, 384 well plates and 1536 well plates. In some embodiments, the purpose of the high-throughput method is about 250 samples per week, about 300 samples per week, about 350 samples per week, about 400 samples per week, about 450 samples per week, about 500 samples per week, About 550 samples per week, about 600 samples per week, about 650 samples per week, about 700 samples per week, about 750 samples per week, about 800 samples per week, about 850 samples per week, about 900 samples per week, about 900 samples per week Screening about 950 samples, about 1000 samples per week, about 1100 samples per week, about 1200 samples per week, about 1300 samples per week, about 1400 samples per week, about 1500 samples per week . In some embodiments, the purpose of the high-throughput method is to screen samples at a rate greater than about 1000 samples per week.

  In some embodiments, the methods described herein allow detection of low levels of G6PDH. For example, by the methods described herein, G6PDH of about 1.00 millimeters or less, G6PDH of about 0.90 millimeters or less, G6PDH of about 0.80 millimeters or less, about 0.70 millimeters or less. G6PDH, G6PDH of about 0.60 millimeters or less, G6PDH of about 0.50 millimeters or less, G6PDH of about 0.45 millimeters or less, G6PDH of about 0.40 millimeters or less, G6PDH, about 0.35 millimeters or less G6PDH, G6PDH of about 0.30 mm or less, G6PDH of about 0.25 mm or less, G6PDH of about 0.20 mm or less, G6PDH of about 0.15 mm or less, G6PDH of about 0.10 mm or less G6PDH, about 0.05 mm or less G6PDH, about 0.04 mm or less G6PDH, about 0.03 mm or less G6PDH, about 0.02 mm It is possible to even the detection of the following G6PDH or about 0.01 millimeters or less of G6PDH units. In some embodiments, the methods described herein allow detection of G6PDH in about 0.10 milliunits or less. In some embodiments, the methods described herein allow detection of G6PDH of about 0.05 milliunit or less. In some embodiments, the methods described herein allow detection of G6PDH of about 0.01 milliunit or less. In some embodiments, the methods described herein allow detection of a level of G6PDH of at least about 0.01 milliunit of G6PDH. In some embodiments, the methods described herein allow detection of a level of G6PDH of at least 0.01 milliunit of G6PDH.

  In some embodiments, provided herein are methods wherein detection of the amount of G6PDH is substantially unaffected by variations in temperature, pH and / or salt concentration. In some embodiments, provided herein are methods wherein detection of the amount of G6PDH is substantially unaffected by temperature variations. In some embodiments, provided herein are methods wherein detection of the amount of G6PDH is not substantially affected by temperature variations during detection. In some embodiments, provided herein are methods wherein detection of the amount of G6PDH is substantially unaffected by temperature fluctuations during sample storage.

  In some embodiments, an internal standard is used to provide a more accurate measurement of the amount of 6-phosphogluconic acid detected in the sample. In some embodiments, the internal standard is a compound / substance added to the sample that does not react with other substances in the sample and thus provides a constant value. In some embodiments, the internal standard is the level of one of the other compounds / substances that are naturally present in the sample. In some embodiments, glucose-6-phosphate can be used as an internal standard.

  As used herein, the term “biological sample” refers to, but is not limited to, a sample of the host body. Biological samples useful in the applicant's teachings can be fresh (eg, collected within a few hours prior to analysis) or refrigerated or frozen (eg, collected and stored in a refrigerator or freezer until analysis). It may have been dried (eg, collected and placed on Whatman paper or spotted directly on Whatman paper). Exemplary biological samples include blood, interstitial fluid, spinal fluid, saliva, urine, tears, sweat, and the like. Additional biological samples include, but are not limited to, cells or tissues or cultures (or subcultures), crude cell lysates or treated cell lysates (including whole cell lysates), body fluids, tissues Fluids derived from extracts or cell extracts, feces, cerebrospinal fluid, amniotic fluid, lymph fluid or glandular secretions. The sample may be processed by any method known in the art prior to contact with the substrate described herein. For example, the sample may be subjected to a precipitation step, a column chromatography step, a heating step, and the like. If the sample contains other enzymes that can interfere with the activity of G6PDH, inactivators (eg, irreversible inhibitors directed at the active site) are added to the sample to inactivate unwanted activity May be. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a dry blood sample.

  “Detect” and “detect” are intended to include detection, measurement and / or characterization of the G6PDH enzyme or its enzyme activity. For example, enzyme activity can be detected in the course of screening to detect or characterize a modulator of enzyme activity.

  In some embodiments, detection of the amount of G6PDH occurs in less than about 10 minutes. In some embodiments, detection of the amount of G6PDH is performed in less than about 7.5 minutes. In some embodiments, detection of the amount of G6PDH is performed in less than about 5 minutes. In some embodiments, detection of the amount of G6PDH occurs in less than about 4 minutes. In some embodiments, detection of the amount of G6PDH is performed in less than about 3 minutes. In some embodiments, detection of the amount of G6PDH is performed in less than about 2.5 minutes. In some embodiments, detection of the amount of G6PDH is performed in less than about 2 minutes.

  As used herein, the term “quenching” refers to inactivating a reagent or stopping a chemical reaction. It is understood that quenching refers to inactivating or stopping regardless of the mechanism by which inactivation or stopping is achieved. As a specific, non-limiting example, quenching can also react with G6PDH or G6P, even if it is due to a change in pH, so that G6PDH or G6P is no longer available for reaction. There is also. Thus, a quenched sample refers to a sample in which G6PDH or G6P is no longer reacting. As used herein, the term “quenching reagent” is any reagent that can interact with G6PDH or G6P, so that G6PDH or G6P is quenched and thus cannot react further. In some embodiments, the quenching agent is an enzyme denaturing agent. For example, in some embodiments, the quenching agent denatures G6PDH. Enzyme modifiers usually cause non-covalent changes in the structure of the enzyme (eg, secondary, tertiary or quaternary structure). Examples of quenching reagents include, but are not limited to, acetonitrile, methanol, and mixtures thereof.

  The term “buffer” usually refers to a solution consisting of a weak acid and its conjugate base or weak base and its conjugate acid, where the pH of the solution changes little upon addition of a small amount of acid or base. Suitable buffers include the “Biological Buffers” section of the Sigma catalog as well as sigmaaldrich. com. And those described online. Exemplary buffers include, but are not limited to, potassium phosphate buffer, MES, MOPS, HEPES, Tris (Trizma), bicine, TAPS, CAPS, and the like. The buffer is present in an amount sufficient to produce and maintain the desired pH. For example, the pH can be 2-12, 4-11 or 6-10.

The term “surfactant” refers to a molecule that has a polar head, usually energetically favoring solvation with water, and a hydrophobic tail, usually not fully solvated by water. Surfactants may be ionic (ie, anionic, cationic) or nonionic. Exemplary surfactants include, but are not limited to, polysorbates / tweens (eg, Tween 20 and Tween 80), pluronics (eg, F68 and F88), Tritons (eg, TRITON® X-114, X- 102, X-45, X-15), poloxamers (eg poloxamer 188), sorbitan esters or spans (eg span 20 and span 80), lipids (eg phospholipids, lecithin, phosphatidylcholine, phosphatidylethanolamine) ), Alcohol ethoxylates (eg BRIJ® 56, C ₁₆ H ₃₃ (OCH ₂ CH ₂ ) ₁₀ OH, BRIJ® 58, C ₁₆ H ₃₃ (OCH ₂ CH ₂ ) ₂₀ OH), fatty acids And fatty acid Tellurium, steroid (eg, cholesterol), chelating agent (eg, EDTA), amine ethoxylate, glucoside, glucamide, polyalkylene oxide, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glycoside , Lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamide Propyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, Midpropyl-betaine, isostearamidopropyl-betaine, lauroamidopropyl-betaine, myristamidopropyl-dimethylamine, palmidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methylcocoyl-taurate, disodium methyloleyl-taurate , MONAQUAT ™ surfactant, polyethylene glycol, polypropylene glycol and copolymers of ethylene and propylene glycol. Other surfactants are described, for example, in McCutcheon's Emulsifiers and Detergents, Manuf. Confectioners Pub. Co. Glen Rock, N .; J. et al. 2000, Kirk-Othmer, Encyclopedia of Chemical Technologies, 2nd edition, volume 19, pages 512-564.

  In some embodiments, G6PDH is combined with G6P for at least about 15 seconds, about 30 seconds, about 45 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes. Contact time for minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes or about 60 minutes. In some embodiments, G6PDH is contacted with G6P for a reaction time of at least about 15 minutes. In some embodiments, G6PDH is contacted with G6P for a reaction time of at least about 30 minutes. In some embodiments, G6PDH is contacted with G6P for a reaction time of at least 15 minutes. In some embodiments, G6PDH is contacted with G6P for a reaction time of at least 30 minutes.

  Applicants' teaching methods can be performed using tandem mass spectrometers and other mass spectrometers that have the ability to select and fragment molecular ions. Tandem mass spectrometers (and to some extent single stage mass spectrometers) have the ability to select and fragment molecular ions according to their mass-to-charge (m / z) ratio, and then the resulting fragments (Daughter) Record the ion spectrum. More particularly, daughter fragment ion spectra can be generated by subjecting selected ions to dissociation energy levels (eg, collision induced dissociation (CID)). For example, ions corresponding to a specific m / z ratio compound from a first mass analysis can be selected, fragmented, and reanalyzed in a second mass analysis. Representative instruments capable of performing such tandem mass spectrometry include, but are not limited to, a magnetic four sector type, a tandem time-of-flight type, a triple quadrupole type, an ion trap type, and a hybrid quadruple type. Polar time-of-flight (Q-TOF) mass spectrometer.

  These types of mass spectrometers can be used in conjunction with a variety of ionization sources including, but not limited to, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). An ionization source can be used to create a charged species for the first mass analysis, where the analyte does not already have a fixed charge. Additional mass spectrometric instruments and fragmentation methods include post-source decomposition in MALDI-MS instruments and high energy CID using MALDI-TOF-TOF MS. A review of tandem mass spectrometers can be found, for example, in R.A. Abersold and D.H. Goodlett, Mass Spectrometry in Proteomics. Chem. Rev. 101: 269 295 (2001).

  In some embodiments, the mass spectrometry is tandem mass spectrometry. In some embodiments, mass spectrometry is accomplished using a mass spectrometer with a thermally assisted electrospray ionization probe (eg, TurboIonSpray® (TIS), Turbo-V or the like). . In some embodiments, mass spectrometry utilizes multiple reaction monitoring (MRM). Exemplary methods for generating mass spectra can be found, for example, in US Pat. Nos. 6,930,305 and 7,145,133, both of which are incorporated herein by this reference. It is. In some embodiments, mass spectrometry utilizes an AB Sciex API 4000 ™ tandem mass spectrometer.

  In some embodiments, processing of the sample can include, for example, separation prior to mass spectrometry. For example, sample components can be separated and mass spectrometry can be performed on only a fraction of the sample mixture. In this way, the separated analytes can be analyzed individually, reducing the complexity of the analysis. Separation can be performed by chromatography. For example, liquid chromatography / mass spectrometry (LC / MS) or liquid chromatography / tandem mass spectrometry (LC / MS / MS) can be used to achieve such sample separation and mass analysis. In addition, any chromatographic separation method suitable for separating the analyte of interest may be used. For example, the chromatographic separation can be normal phase chromatography, reverse phase chromatography, ion exchange chromatography, size exclusion chromatography or affinity chromatography. Separation can also be performed electrophoretically. Non-limiting examples of electrophoretic separation techniques include, but are not limited to, 1D electrophoresis, 2D electrophoresis and / or capillary electrophoresis. In some embodiments, the mass spectrometry provided herein is accomplished using a mass spectrometer with a reverse phase liquid chromatography column.

  Also provided herein are methods for detecting G6PDH deficiency in a subject. Such a method includes determining the amount of G6PDH in a biological sample from a subject by mass spectrometry and comparing the amount of G6PDH in the biological sample to an appropriate control, wherein the sample G6PDH deficiency is detected when the amount of G6PDH in it is less than the appropriate control. G6PDH deficiency is generally an inherited genetic defect that is X-linked, usually due to a mutation in the G6PDH gene. This deficiency is common and is present in over 400 million people worldwide (usually African, Middle Eastern and South Asian).

  The G6PDH gene is found on the long arm of the X chromosome (band Xq28) and spans approximately 18.5 kilobases. In some embodiments, the G6PDH deficiency is due to a G6PDH gene mutation. The G6PDH gene mutation is described, for example, in Butler E. et al. "G6PD Deficiency" Blood, Vol. 84 (11): 3613-3636, (1994). In some embodiments, the G6PDH deficiency is due to one of the genetic mutations listed in Table 1.

いくつかの実施形態では、被験体におけるＧ６ＰＤＨ欠乏症は、対照より約５％少ない、対照より約１０％少ない、対照より約１５％少ない、対照より約２０％少ない、対照より約２５％少ない、対照より約３０％少ない、対照より約３５％少ない、対照より約４０％少ない、対照より約４５％少ない、対照より約５０％少ない、対照より約５５％少ない、対照より約６０％少ない、対照より約６５％少ない、対照より約７０％少ない、対照より約７５％少ない、対照より約８０％少ない、対照より約８５％少ない、対照より約９０％少ない、対照より約９５％少ない、または対照より約１００％少ない、被験体由来の生体サンプル中のＧ６ＰＤＨの量によって示される。例えば、いくつかの実施形態では、被験体におけるＧ６ＰＤＨ欠乏症は、対照より少なくとも約１０％少ない、被験体由来の生体サンプル中のＧ６ＰＤＨの量によって示される。いくつかの実施形態では、被験体におけるＧ６ＰＤＨ欠乏症は、対照よりも少なくとも約２５％少ない、被験体由来の生体サンプル中のＧ６ＰＤＨの量によって示される。いくつかの実施形態では、被験体におけるＧ６ＰＤＨ欠乏症は、対照よりも少なくとも約５０％少ない、被験体由来の生体サンプル中のＧ６ＰＤＨの量によって示される。例えば、いくつかの実施形態では、被験体におけるＧ６ＰＤＨ欠乏症は、対照よりも少なくとも１０％、少なくとも２５％または少なくとも５０％少ない、被験体由来の生体サンプル中のＧ６ＰＤＨの量によって示される。

In some embodiments, G6PDH deficiency in the subject is about 5% less than the control, about 10% less than the control, about 15% less than the control, about 20% less than the control, about 25% less than the control, control About 30% less than control, about 35% less than control, about 40% less than control, about 45% less than control, about 50% less than control, about 55% less than control, about 60% less than control, than control About 65% less, about 70% less than the control, about 75% less than the control, about 80% less than the control, about 85% less than the control, about 90% less than the control, about 95% less than the control, or less than the control Indicated by the amount of G6PDH in a biological sample from a subject that is about 100% less. For example, in some embodiments, G6PDH deficiency in a subject is indicated by the amount of G6PDH in a biological sample from the subject that is at least about 10% less than the control. In some embodiments, G6PDH deficiency in a subject is indicated by the amount of G6PDH in a biological sample from the subject that is at least about 25% less than the control. In some embodiments, G6PDH deficiency in a subject is indicated by the amount of G6PDH in a biological sample from the subject that is at least about 50% less than the control. For example, in some embodiments, G6PDH deficiency in a subject is indicated by the amount of G6PDH in a biological sample from the subject that is at least 10%, at least 25%, or at least 50% less than the control.

  As used herein, the term “subject” includes, but is not limited to, a human, a primate, a cow, a sheep, a goat, a horse, a pig, a dog, a cat ( cat), rabbit, guinea pig, rat, mouse or other bovine, ovine, equine, canine, feline, rodent or murine species including rodent species Refers to animals such as animals. In some embodiments, the subject is a human. In some embodiments, the subject is a newborn or a child. In some embodiments, the subject is a pregnant woman.

  In some embodiments, Applicants' teachings provide a method of screening compounds for the ability to modulate G6PDH. In some embodiments, the effect of a modulator (eg, enhancer or inhibitor) of G6PDH activity can be examined using the methods described herein. In addition, the substrate specificity of G6PDH can also be examined using the methods described herein.

  In some embodiments, methods for diagnosing a G6PDH-related disorder are described herein. As used herein, the term “G6PDH-related disorder” refers to a disease and / or disorder resulting at least in part from G6PDH deficiency. In some embodiments, the G6PDH-related disorder is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80. %, At least about 90%, and about 100% of diseases and / or disorders resulting from G6PDH deficiency. In some embodiments, the G6PDH-related disorder comprises a disease and / or disorder resulting from at least 25% G6PDH deficiency. In some embodiments, the G6PDH-related disorder comprises a disease and / or disorder resulting from at least 50% G6PDH deficiency. In some embodiments, the G6PDH-related disorder comprises a disease and / or disorder resulting from at least 75% G6PDH deficiency.

  In some embodiments, the G6PDH-related disorder is selected from chronic hemolysis, acute hemolytic anemia, broad bean disease, neonatal hyperbilirubinemia and pre-eclampsia. Other G6PDH-related disorders include, for example, abnormal platelets, pedicle flap loss, dysfunction of motor ability, seizure disorders, increased frequency of cataracts, schizophrenia or depression, renin release failure, diabetes Abnormal insulin release, cardiovascular disease, cholelithiasis, myoglobinuria, increased susceptibility to infection, abnormal white blood cell function, increased jaundice in hepatitis, mental retardation or serum dehydroepiandrosterone sulfate An increase can be mentioned. For example, Butler, E .; , "G6PD Deficiency" Blood, Vol. 84 (11): 3613-3636, (1994).

  In some embodiments, the G6PDH-related disorder is a disorder caused by or induced by a substance capable of inducing G6PDH deficiency. Such substances include, but are not limited to, broad beans, antimalarials (eg, primaquine, pamaquin and chloroquine), sulfonamides (eg, sulfanilamide, sulfamethoxazole and maphenide), nitrofurantoin, acetylsalicylic acid , Acetophenetidine, thiazolesulfone, methylene blue, naphthalene, analgesics (eg, aspirin, phenazopyridine and acetanilide) and some non-sulfa antibiotics (eg, nalidixic acid, nitrofurantoin, isoniazid and furazolidone).

  In some embodiments, provided herein are methods for diagnosing acute hemolytic anemia in a subject. Such a method includes detecting G6PDH deficiency in a subject according to the method described above, wherein G6PDH deficiency in the subject indicates acute hemolytic anemia in the subject.

  In some embodiments, an amount of G6PDH in the sample that is at least about 90% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 80% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 70% less than a control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 60% less than a control is indicative of acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 50% less than a control is indicative of acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 40% less than a control is indicative of acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 30% less than a control is indicative of acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 20% less than a control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is at least about 10% less than a control indicates acute hemolytic anemia in the subject.

  In some embodiments, an amount of G6PDH in the sample that is 75% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in a sample that is 50% less than a control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample that is 25% less than the control indicates acute hemolytic anemia in the subject.

  In some embodiments, provided herein are methods for diagnosing pre-eclampsia in a subject. Such a method comprises detecting G6PDH deficiency in a subject according to the method described above, wherein G6PDH deficiency in the subject indicates pre-eclampsia in the subject.

  In some embodiments, an amount of G6PDH in the sample that is at least about 90% less than the control indicates preeclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 80% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 70% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 60% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 50% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 40% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 30% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 20% less than the control is indicative of pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is at least about 10% less than the control is indicative of pre-eclampsia in the subject.

  In some embodiments, an amount of G6PDH in the sample that is 75% less than the control indicates preeclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is 50% less than the control indicates preeclampsia in the subject. In some embodiments, an amount of G6PDH in the sample that is 25% less than the control indicates pre-eclampsia in the subject.

  In some embodiments, provided herein are prognostic methods for predicting increased mortality and / or morbidity due to pre-eclampsia or acute hemolytic anemia in a subject. Such methods include detecting G6PDH deficiency in a subject according to the methods described above, wherein the amount of G6PDH in a biological sample that is 50% or less of a control is mortality and / or morbidity in the subject. Shows the prognosis of the rate increase. In some embodiments, an amount of G6PDH in a biological sample that is 75% or less of a control indicates a prognosis of increased mortality and / or morbidity in the subject.

  As used herein, the term “appropriate control” is a control based on G6PDH levels in a normal population. That is, in some embodiments, a suitable control is a predetermined value. In some embodiments, a suitable control is the level detected in a single individual known to have normal G6PDH activity. In some embodiments, a suitable control is an average level obtained from a population of individuals with normal G6PDH activity. In some embodiments, a suitable control is the level detected in the patient prior to administration of a substance capable of inducing G6PDH deficiency.

  In some embodiments, the diagnostic or prognostic methods provided herein comprise any one of the following steps: under conditions suitable to form 6-phosphogluconic acid Reacting the biological sample with G6P and NADP in the presence of an activator and buffer; quenching the biological sample to form a quenched sample; and the presence of 6-phosphogluconic acid in the quenched sample Or detecting the amount by mass spectrometry. In such a method, the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.

Also provided are kits for performing the methods described herein. In some embodiments, the kit comprises glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate to prepare a reaction mixture that facilitates the reaction of nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH. And a surfactant and a buffer. The buffer and / or surfactant may be provided in a dry or liquid form in the container. Exemplary suitable buffers and surfactants are provided herein above. The buffer is usually present in the kit at least in an amount sufficient to produce a specific pH in the mixture. In some embodiments, the buffer is provided as a stock solution having a preselected pH and buffer concentration. In some embodiments, acids and / or bases are also provided in the kit to adjust the reaction mixture to the desired pH. The kit may further comprise a quenching solution, for example an enzyme denaturant such as an acetonitrile: methanol mixture. The kit can further comprise one or more diluents or solvents suitable for use in a mass spectrometry system, for example. Kits are beneficial for enzymatic activity of salts (eg KCl, NaCl or NaOAc), metal salts (eg Ca ²⁺ salts such as CaCl ₂ , MgCl ₂ , MnCl ₂ , ZnCl ₂ or Zn (OAc) etc.) And / or other components that may be useful for the G6PDH enzyme. These other components may be provided in dry or liquid form, separately from one another or mixed together.

  Glucose-6-phosphate and / or nicotinamide adenine dinucleotide phosphate can be provided in dry or liquid form with or separately from the buffer. Glucose-6-phosphate and / or nicotinamide adenine dinucleotide phosphate can be used in aqueous solutions, partially aqueous solutions, or non-aqueous storage solutions that are miscible with other components of the reaction mixture to facilitate dissolution in the reaction mixture. May be provided.

In some embodiments, the kit further comprises instructions for use. For example, the kit can include instructions for preparing a reaction mixture that facilitates the reaction of nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH. In some embodiments, the kit includes instructions for preparing a sample for analysis with a mass spectrometer. In some embodiments, the kit further comprises a pre-prepared calibration curve, such as the curve shown in FIG. 3, whereby the user can determine the 6-phosphogluconic acid determined using a mass spectrometer. It is possible to determine the amount of G6PDH in the sample based on the amount. For example, a pre-prepared calibration curve may be a plot of [6-phosphogluconic acid / glucose-6-phosphate area] versus G6PDH in millimeters. In some embodiments, the kit includes a standard sample with a predetermined amount of G6PDH so that the user can create his own calibration curve. In some embodiments, the kit further comprises instructions for how to prepare the calibration curve. Instructions for use are:
a. Mixing glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate in the presence of a buffer and / or surfactant for a set period of time (eg, 15 minutes, 20 minutes, 25 minutes, 30 minutes, etc.);
b. Quenching the sample using a quenching solution;
c. Analyzing the quenched sample using a mass spectrometer;
d. Preparing a calibration curve;
e. Any combination of comparing the analysis (eg, mass spectrum) of the quenched sample to a calibration curve may be included.

  The operation of the various compositions and methods may be further understood in light of the following non-limiting examples, which should in no way be construed as limiting the scope of applicants' teachings.

Example (Example 1)
DBS Sample A filter paper with a dried blood spot (DBS) obtained from a neonatal screening program was utilized in this example. A 3 mm diameter disc was removed from the filter paper and the blood sample on this disc was added to a 1.5 mL Eppendorf tube using 50 μL “Reagent 1”. “Reagent 1” consists of 3 volumes of double distilled water, 3 volumes of 250 mM K ₂ HPO ₄ , 2 volumes of 1% saponin for Molecular Biology, 1 volume of 7.5 mM nicotinamide adenine dinucleotide phosphate and 1 volume of 10 mM. Formulated by mixing glucose-6-phosphate. After closing the Eppendorf tube and gently mixing, the tube was placed in a 37 ° C. incubation oven for 30 minutes. The reaction was quenched by adding 100 μL of 1: 1 acetonitrile: methanol (LC-MS grade) mixture. After centrifugation, 10 μL of the clear supernatant was diluted with 240 μL of 20 mM butyl-dimethyl-ammonium bicarbonate (BDMAC) aqueous solution.

2 μL of this solution was injected into the LC / MS / MS system, which included an Agilent 1200 LC system with a Phenomenex C18 column and an AB Sciex API 4000 ™ tandem mass spectrometer with a TIS probe. . Liquid chromatography was performed by injecting the sample into a column maintained at 50 ° C. and flowing 300 μL / min LC-eluent (20 mM BDMAC + 3% MetOH) for 2.5 minutes in isocratic mode. The mass spectrometer was operated with MRM, Negative Ion Mode, using a nominal gun temperature of 350 ° C. using a TIS of −4500 volts. The transitions used are as follows:
Glucose-6-phosphate 259.2> 79.0 using DP: -60 V and CE: -70 eV
65.2-> 79.0 of 6-phospho-gluconic acid using DP: -60V and CE: -70eV
NADP 742.1> 79.0 using DP: -80V and CE: -130eV.

  In "Quant" or "MultiQuant" mode, traces corresponding to transitions 275.2> 79.0 and 259.2> 79.0 are the analyte (6-phosphogluconic acid) and internal standard (G6P), respectively. Processed. The resulting ratio was compared to a calibration curve obtained using commercially available G6PDH as described in Example 2.

  Since enzyme deficiency is accompanied by a high signal of NADP, the transition 742.1> 79.0, where NADP was monitored, was used as an internal check for false positive results. Thus, if both the 6-phosphogluconic acid and NADP traces are low or absent, the result is considered false.

  FIG. 1 shows exemplary mass spectrometric readings with MRM obtained from dried blood samples and shows levels of G6P, 6-phosphogluconic acid and NADP. FIG. 1 is representative of normal levels of G6PDH. FIG. 2 compares a normal sample with a sample without G6PDH activity.

(Example 2)
Enzyme calibration A commercially available enzyme standard (eg, Sigma G5885) was dissolved using a solution of 50% double-distilled water, 30% 250 mM K ₂ HPO ₄ and 20% 1% saponin for Molecular Biology. Milliunits / mL were obtained. 0, 1, 2, 4, 10, 20 and 40 μL of this solution (corresponding to 0, 0.1, 0.2, 0.4, 1.0, 2.0 and 4.0 milliunits of enzyme) Was added to 50 μL of “Reagent 1” (described in Example 1) in a 1.5 mL Eppendorf tube. After closing the Eppendorf tube and gently mixing, the tube was placed in a 37 ° C. incubation oven for 30 minutes. The reaction was quenched by adding 100 μL of 1: 1 acetonitrile: methanol (LC-MS grade) mixture. After centrifugation, 10 μL of the clear supernatant was diluted with 240 μL of 20 mM butyl-dimethyl-ammonium bicarbonate (BDMAC) aqueous solution. As in Example 1, 2 μL of this solution was injected into the LC / MS / MS system. In "Quant" or "MultiQuant" mode, traces corresponding to transitions 275.2> 79.0 and 259.2> 79.0 were processed as "analyte" and "internal standard", respectively. Quantitative evaluation of the sample is done by absolute readings in millimeters and is referenced to the volume of blood on the punched disc (3.1 μL). As a reference, an enzyme activity of approximately 3 milliunits / DBS was retained as normal. The results are shown in Table 2 below.

サンプリングされた実際の血液容量に基づいて、これらの方法の検出性は、正常サンプルの平均酵素活性の約１／３００であると思われる。約３〜４ミリ単位／スポットまでの線形性が観察されたが（図３を参照のこと）、必要に応じて、インキュベーション時間を減少することによって拡張することができる。

Based on the actual blood volume sampled, the detectability of these methods appears to be about 1/300 of the average enzyme activity of normal samples. Linearity up to about 3-4 millimeters / spot was observed (see FIG. 3), but can be extended by reducing the incubation time if necessary.

(Example 3)
Analysis of Dry Blood Samples Correlation studies were constructed using anonymous surplus samples obtained from routine analysis in hospitals, collected and used so far, according to local ethical committee guidelines. The blood sample was spotted on a gas ree card, dried and sent for measurement by LC / MS / MS. Analysis by LC / MS / MS was performed as described above for Example 1. As a reference, the samples were used with the Bio Vision kit (Bio Vision Research Products, 980 Linda Vista Avenue, Mountain View, CA 94043 USA, catalog number K757-100) as performed in routine hospital laboratories. Measured first.

  FIGS. 4A and 4B show two exemplary mass spectrometric readings with MRM obtained from two dry blood samples corresponding to high and low G6PDH activity as measured by the BioVision kit (BV). As can be seen by these figures, LC-MS / MS can measure both high and low G6PDH activity. Since there is no uniform calibration available for both assays, the above sample corresponding to the extreme activity values was used to generate a calibration curve for the LC-MS / MS assay. The calibration used as the LC-MS / MS standard is shown in FIG.

  The results obtained from the analysis of the samples are provided in Table 3, which also provides a list of values obtained using the BV kit. G6PDH activity is expressed in milliunits / mL (mU / mL) for both LC / MS / MS and BV kits, and there is no normalization to LC / MS / MS hemoglobin.

^＊サンプルｎ８、ｎ１０およびｎ１７は、低活性サンプルと考えられる。

^* Samples n8, n10 and n17 are considered low activity samples.

  FIGS. 6A and 6B represent two exemplary mass spectrometric readings with MRM obtained from two dry blood samples read as unknown. These readings correspond to low and high G6PDH activity, the LC / MS / MS assay gives measurements of 40 and 1202 milliunits / mL, respectively, and the BV assay measures 20 and 1230 mU / mL, respectively. Give value. In addition, FIGS. 7A and 7B are correlation plots and brand and Altman plots made using the values measured in this study (measuring the agreement between the two methods of clinical measurement). As can be seen by the data and plots above, there is a good agreement, especially for low activity samples, between the values obtained using LC / MS / MS and those obtained using the BV kit.

Equivalents Those skilled in the art will recognize, and be able to ascertain using no more than routine experimentation, equivalents to the specific embodiments of the teachings described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (27)

A high-throughput method for detecting the amount of G6PDH in a biological sample,
Reacting the biological sample with G6P and NADP in the presence of a surfactant and buffer under conditions suitable to form 6-phosphogluconic acid;
Quenching the biological sample to form a quenched sample;
Detecting the presence or amount of 6-phosphogluconic acid in the quenched sample by mass spectrometry,
Wherein the amount of the 6-phosphogluconic acid in the quenched sample is related to the amount of the G6PDH in the biological sample.   The method of claim 1, wherein the quenching step is accomplished using an enzyme denaturant.   The method according to claim 1 or 2, wherein the mass spectrometry is tandem mass spectrometry.   The method of any one of the preceding claims, wherein the mass spectrometry is accomplished using a mass spectrometer with a thermally assisted electrospray ionization probe.   The method according to any one of the preceding claims, wherein the mass spectrometry is achieved using a mass spectrometer equipped with a reverse phase liquid chromatography column.   The method of any one of the preceding claims, wherein the detecting step is performed in less than about 5 minutes.   The method of any one of the preceding claims, wherein the detecting step is performed in less than about 3 minutes.   The method according to any one of the preceding claims, which allows the detection of G6PDH of about 0.01 milliunit or less.   The method according to any one of the preceding claims, wherein the detecting step is substantially unaffected by temperature variations. A method for detecting G6PDH deficiency in a subject comprising:
Determining the amount of G6PDH in a biological sample obtained from a subject by mass spectrometry;
Comparing the amount of the G6PDH in the biological sample with an appropriate control;
Wherein the G6PDH deficiency is detected when the amount of G6PDH in the sample is less than the appropriate control.   The method of claim 10, wherein the subject is a newborn or an infant.   The method according to claim 10 or 11, wherein the subject is a pregnant woman.   13. The method of any one of claims 10 to 12, wherein an amount of G6PDH in the sample that is 10% less than the control indicates G6PDH deficiency in the subject.   14. The method of any one of claims 10 to 13, wherein an amount of G6PDH in the sample that is 25% less than the control indicates G6PDH deficiency in the subject.   15. The method of any one of claims 10 to 14, wherein an amount of G6PDH in the sample that is 50% less than the control indicates G6PDH deficiency in the subject. A method of diagnosing acute hemolytic anemia in a subject comprising:
Detecting G6PDH deficiency in a subject according to the method of claim 10;
Here, the method wherein G6PDH deficiency in the subject indicates acute hemolytic anemia in the subject.   17. The method of claim 16, wherein an amount of G6PDH in the sample that is 25% less than the control indicates acute hemolytic anemia in the subject. A method of diagnosing pre-eclampsia in a subject comprising:
Detecting G6PDH deficiency in a subject according to the method of claim 10;
Wherein the G6PDH deficiency in the subject is indicative of pre-eclampsia in the subject.   19. The method of claim 18, wherein an amount of G6PDH in the sample that is 25% less than the control is indicative of pre-eclampsia in the subject. A prognostic method for increased mortality and / or morbidity due to pre-eclampsia or acute hemolytic anemia in a subject comprising:
Detecting G6PDH deficiency in a subject according to the method of claim 10;
Wherein the amount of G6PDH in the biological sample that is 50% or less of the control indicates a prognosis of increased mortality and / or morbidity in the subject.   21. The method of any one of claims 10 to 20, wherein the suitable control is a control based on G6PDH levels in a normal population. Reacting said biological sample with G6P and NADP in the presence of a surfactant and a buffer under conditions suitable to form 6-phosphogluconic acid;
Quenching the biological sample to form a quenched sample;
Detecting the presence or amount of 6-phosphogluconic acid in the quenched sample by mass spectrometry;
22. The method according to any one of claims 10 to 21, wherein the amount of the 6-phosphogluconic acid in the quenched sample is related to the amount of the G6PDH in the biological sample.   The method according to any one of the preceding claims, wherein the biological sample is a dried blood sample. A kit for detecting the amount of G6PDH in a biological sample,
Glucose-6-phosphate,
Nicotinamide adenine dinucleotide phosphate;
Optionally, a buffer,
Optionally, a surfactant,
Instructions for preparing a reaction mixture that facilitates the reaction of nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH.   The kit according to claim 24, further comprising an enzyme denaturant.   26. The kit of claim 24 or claim 25, further comprising instructions for preparing a sample for analysis with a mass spectrometer.   27. The kit according to any one of claims 24 to 26, further comprising a calibration curve comprising a plot of [6-phosphogluconic acid / glucose-6-phosphate area] versus G6PDH in millimeters.

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