EP2643474A1 - Détection sensible, à haut débit, de la glucose-6-phosphate déshydrogénase - Google Patents

Détection sensible, à haut débit, de la glucose-6-phosphate déshydrogénase

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Publication number
EP2643474A1
EP2643474A1 EP11843953.8A EP11843953A EP2643474A1 EP 2643474 A1 EP2643474 A1 EP 2643474A1 EP 11843953 A EP11843953 A EP 11843953A EP 2643474 A1 EP2643474 A1 EP 2643474A1
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EP
European Patent Office
Prior art keywords
g6pdh
subject
sample
amount
less
Prior art date
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EP11843953.8A
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German (de)
English (en)
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EP2643474A4 (fr
Inventor
Bruno Casetta
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DH Technologies Development Pte Ltd
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DH Technologies Development Pte Ltd
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Publication of EP2643474A1 publication Critical patent/EP2643474A1/fr
Publication of EP2643474A4 publication Critical patent/EP2643474A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • G6PD Glucose-6-phosphate dehydrogenase
  • G6PDH Glucose-6-phosphate dehydrogenase
  • Glucose-6-phosphate dehydrogenase is stimulated by its substrate, Glucose-6-phosphate ("G6P"), to form 6-phosphoglucono- ⁇ -lactone. It is the rate-limiting enzyme of the pentose phosphate pathway.
  • G6PDH Genetic deficiency of G6PDH in humans predisposes them to certain disorders, such as non-immune hemolytic anemia.
  • 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 are generally incapable of detecting a partial deficiency of G6PDH in many cases. See, e.g., Reclos GJ, et al.
  • applicant's teachings provide high throughput methods for detecting an amount of G6PDH in a biological sample.
  • the methods include, for example: reacting a biological sample with G6P and NADP in the presence of a surfactant and a buffer under suitable conditions to form 6- phosphogluconic acid; quenching the biological sample to form a quenched sample; and detecting by mass spectrometry a presence or amount of 6-phosphogluconic acid in the quenched sample, wherein the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.
  • the quenching is accomplished using an enzyme denaturant.
  • the mass spectrometry is tandem mass spectrometry.
  • the mass spectrometry is accomplished using a mass spectrometer equipped with a thermally assisted electrospray ionization probe, e.g. , a
  • the mass spectrometry is accomplished using a mass spectrometer equipped with a reversed phase liquid chromatography column.
  • the detecting occurs in less than about 5 minutes. In some embodiments, the detecting occurs in less than about 3 minutes. In some embodiments, the method allows for detection of about 0.01 milliUnits of G6PDH or less. In some embodiments, the detecting is not substantially affected by fluctuations in temperature.
  • applicant's teachings provide methods for detecting G6PDH deficiency in a subject.
  • the methods include, for example: determining by mass spectrometry an amount of G6PDH in a biological sample from a subject; 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.
  • the subject is a neonate or an infant. In some embodiments, the subject is a neonate or an infant. In some
  • the subject is a pregnant woman.
  • an amount of G6PDH in the sample which is 10% less than the control indicates a G6PDH deficiency in the subject.
  • an amount of G6PDH in the sample which is 25% less than the control indicates a G6PDH deficiency in the subject. In some embodiments, an amount of G6PDH in the sample which is 50% less than the control indicates a G6PDH deficiency in the subject.
  • detecting G6PDH deficiency further includes: reacting the biological sample with G6P and NADP in the presence of a surfactant and a buffer under suitable conditions to form 6-phosphogluconic acid; quenching the biological sample to form a quenched sample; and detecting by mass spectrometry a presence or amount of 6-phosphogluconic acid in the quenched sample, wherein the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.
  • applicant's teachings provide methods for diagnosing acute hemolytic anemia in a subject.
  • the methods include, for example: detecting G6PDH deficiency in a subject in accordance with applicant's teachings, wherein a G6PDH deficiency in the subject indicates acute hemolytic anemia in the subject.
  • an amount of G6PDH in the sample which is 25% less than the control indicates acute hemolytic anemia in the subject.
  • applicant's teachings provide methods for diagnosing pre-eclampsia in a subject.
  • the methods include, for example: detecting G6PDH deficiency in a subject in accordance with applicant's teachings, wherein a G6PDH deficiency in the subject indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is 25% less than the control indicates pre-eclampsia in the subject.
  • applicant's teachings provide prognostic methods for increased mortality and/or morbidity resulting from pre-eclampsia or acute hemolytic anemia in a subject.
  • the methods include, for example: detecting G6PDH deficiency in a subject in accordance with applicant's teachings, wherein an amount of the G6PDH in the biological sample which is 50% or less than the control indicates a prognosis of increased mortality and/or morbidity in the subject.
  • the appropriate control is a control based upon
  • the biological sample is a dried blood sample.
  • kits for detecting an amount of G6PDH in a biological sample can include, for example, glucose-6- phosphate, nicotinamide adenine dinucleotide phosphate, optionally a buffer, optionally a surfactant, and instructions for preparing a reaction mixture that facilitates a reaction of nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH.
  • the kit also includes an enzyme denaturant.
  • the kit further includes instructions for preparing a sample for analysis on a mass spectrometer.
  • the kit also includes a calibration curve which comprises a plot of [6-phosphogluconic acid/glucose-6-phosphate area] versus milliUnits of G6PDH.
  • Figure 1 depicts a mass spectrometric reading in MRM (Multiple
  • Figure 2 depicts comparative mass spectrometric readings in MRM obtained from exemplary methods of the applicant's teachings.
  • Figure 3 depicts an exemplary standard curve obtained using various methods of the applicant' s teachings.
  • Figures 4A and 4B depict mass spectrometric readings in MRM obtained from an exemplary method of the applicant' s teachings.
  • Figure 5 depicts an exemplary standard curve obtained using various methods of the applicant' s teachings.
  • Figures 6A and 6B depict mass spectrometric readings in MRM obtained from an exemplary method of the applicant' s teachings.
  • Figures 7A and 7B are a correlation plot and a Bland and Altman plot, respectively, comparing an exemplary method of the applicant' s teachings with a commercially available assay.
  • G6PDH converts glucose-6-phosphate to 6-phosphogluconolactone with the concurrent reduction of NADP to NADPH.
  • 6-phosphogluconolactone is then converted to 6-phosphogluconic acid by gluconolactonase.
  • Previous methods for determining G6PDH activity including the Beutler method, exploit the formation of NADPH as the indicator of enzymatic reaction.
  • amounts of NADPH may be difficult to determine in such an assay by mass spectroscopy because the C13- isotopomer of the NADP overlaps with the first C12 isotopomer of NADPH.
  • the applicant' s teachings provide a high throughput method for detecting an amount of G6PDH in a biological sample.
  • a method includes reacting a biological sample with G6P and NADP (e.g. , in the presence of a surfactant and a buffer) under suitable conditions to form 6- phosphogluconic acid; quenching the biological sample to form a quenched sample; and detecting by mass spectrometry a presence or amount of 6-phosphogluconic acid in the quenched sample.
  • the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.
  • high-throughput refers to the process of assaying a large number of samples in a relatively short period of time. High-throughput assays may be accomplished using, for example, 96-, 384-, and 1536-well plates.
  • the aim of high-throughput methods is to screen samples at a rate that exceeds 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 950 samples per week, 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,.
  • the aim of high-throughput methods is to screen samples at a rate that exceeds about 1000 samples per week.
  • the methods described herein allow for detection of low levels of G6PDH.
  • the methods described herein allow for detection of about 1.00 milliUnits of G6PDH or less, about 0.90 milliUnits of G6PDH or less, about 0.80 milliUnits of G6PDH or less, about 0.70 milliUnits of G6PDH or less, about 0.60 milliUnits of G6PDH or less, about 0.50 milliUnits of G6PDH or less, about 0.45 milliUnits of G6PDH or less, about 0.40 milliUnits of G6PDH or less, about 0.35 milliUnits of G6PDH or less, about 0.30 milliUnits of G6PDH or less, about 0.25 milliUnits of G6PDH or less, about 0.20 milliUnits of G6PDH or less, about 0.15 milliUnits of G6PDH or less, about 0.10 milliUnits of G6PDH or less, about 0.05 milliUnits
  • the methods described herein allow for detection of about 0.10 milliUnits of G6PDH or less. In some embodiments, the methods described herein allow for detection of about 0.05 milliUnits of G6PDH or less. In some embodiments, the methods described herein allow for detection of about 0.01 milliUnits of G6PDH or less. In some embodiments, the methods described herein allow for detection of levels of G6PDH of at least about 0.01 milliUnits of G6PDH. In some embodiments, the methods described herein allow for detection of levels of G6PDH of at least 0.01 milliUnits of G6PDH.
  • methods are provided herein where the detection of an amount of G6PDH is not substantially affected by fluctuations in temperature, pH and/or salt concentration. In some embodiments, methods are provided herein where the detection of an amount of G6PDH is not substantially affected by fluctuations in temperature. In some embodiments, methods are provided herein where the detection of an amount of G6PDH is not substantially affected by fluctuations in temperature during detection. In some embodiments, methods are provided herein where the detection of an amount of G6PDH is not substantially affected by fluctuations in temperature during storage of the sample.
  • an internal standard is used to produce a more accurate measurement of the amount of 6-phosphogluconic acid detected in the sample.
  • the internal standard is a compound/substance added to the sample which does not react with other substances in the sample, and thus provides a constant value.
  • the internal standard is a level of one of the other compounds/substances naturally occurring in the sample.
  • glucose-6-phosphate can be used as an internal standard.
  • biological sample refers, without limitation, to a sample of a host body. Biological samples useful in the applicant's teachings may be fresh (e.g.
  • Exemplary biological samples include, for example, blood, interstitial fluid, spinal fluid, saliva, urine, tears, sweat, or the like. Additional biological samples include, but are not limited to, cells or tissues, or cultures (or subcultures) thereof, crude or processed cell lysates (including whole cell lysates), body fluids, tissue extracts or cell extracts, feces, cerebral fluid, amniotic fluid, lymph fluid or a fluid from a glandular secretion.
  • the sample may be processed prior to contact with a substrate described herein by any method known in the art. For example, the sample may be subjected to a precipitation step, column
  • the biological sample is a blood sample.
  • the biological sample is a dried blood sample.
  • Detect and “detection” are intended to encompass detection, measurement and/or characterization of G6PDH enzyme or its enzyme activity.
  • enzyme activity may be detected in the course of screening for, detecting or characterizing modulators of the enzyme activity.
  • detecting the amount of G6PDH occurs in less than about 10 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 7.5 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 5 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 4 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 3 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 2.5 minutes. In some embodiments, detecting the amount of G6PDH occurs in less than about 2 minutes.
  • quench refers to inactivating a reagent or halting a chemical reaction. It is to be understood that quenching refers to the inactivation or halting regardless of the mechanism by which the inactivation or halting is achieved. As specific non-limiting examples, the quenching may be due to changes in pH or to the addition of a quenching agent, which may react with the G6PDH or G6P, such that it is no longer available for reaction. Thus, a quenched sample refers to a sample in which G6PDH or G6P are no longer undergoing a reaction.
  • the term “quench” refers to inactivating a reagent or halting a chemical reaction. It is to be understood that quenching refers to the inactivation or halting regardless of the mechanism by which the inactivation or halting is achieved. As specific non-limiting examples, the quenching may be due to changes in pH or to the addition of a quenching agent, which may react with the G6PDH or G6P
  • quenching reagent as used herein is any reagent which is able to interact with a G6PDH or G6P such that the G6PDH or G6P are quenched and therefore unable to further react.
  • the quenching agent is an enzyme denaturant.
  • the quenching agent denatures the G6PDH.
  • Enzyme denaturants typically trigger a non-covalent change in the structure (e.g., secondary, tertiary or quaternary structure) of the enzyme.
  • quenching reagents include, but are not limited to, acetonitrile, methanol, and mixtures thereof.
  • buffer refers to a solution, typically consisting of a weak acid and its conjugate base or a weak base and its conjugate acid, wherein the pH of the solution changes very little upon additions of small amounts of acid or base.
  • Suitable buffers include those described in the "Biological Buffers" section of the Sigma Catalog, as well as online at sigmaaldrich.com.
  • Exemplary buffers include, but are not limited to, potassium phosphate buffers, MES, MOPS, HEPES, Tris (Trizma), bicine, TAPS, CAPS, and the like.
  • the buffer is present in an amount sufficient to generate and maintain a desired pH.
  • the pH can be from 2 to 12, from 4 to 11, or from 6 to 10.
  • surfactant refers to a molecule having a polar head group, which typically energetically prefers solvation by water, and a hydrophobic tail which is typically not well solvated by water.
  • the surfactant may be ionic (i.e. , anionic, cationic) or nonionic.
  • exemplary surfactants include, but are not limited to, polysorbates/Tweens (e.g. , Tween 20 and Tween 80), pluronics (e.g., F68 and F88), Tritons (e.g. , TRITON ® X-114, X- 102, X-45, X-15), poloxamers (e.g.
  • sorbitan esters or Spans e.g. , Span 20 and Span 80
  • lipids e.g. , phospholipids, lecithin, phosphatidylcholines, phosphatidylethanolamines
  • alcohol ethoxylates e.g., BRIJ ® 56
  • EDTA amine ethoxylates, glucosides, glucamides, polyalkyleneoxides, sodium dodecyl sulfate (SDS), sodium laurel 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, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, is
  • the G6PDH is contacted with the G6P for a reaction time of 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, 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.
  • the G6PDH is contacted with the G6P for a reaction time at least about 15 minutes.
  • the G6PDH is contacted with the G6P for a reaction time at least about 30 minutes.
  • the G6PDH is contacted with the G6P for a reaction time at least 15 minutes.
  • the G6PDH is contacted with the G6P for a reaction time at least 30 minutes.
  • tandem mass spectrometers and other mass spectrometers that have the ability to select and fragment molecular ions.
  • Tandem mass spectrometers (and, to some degree, single- stage mass spectrometers) have the ability to select and fragment molecular ions according to their mass-to-charge (m/z) ratio, and then record the resulting fragment (daughter) ion spectra. More specifically, daughter fragment ion spectra can be generated by subjecting selected ions to dissociative energy levels (e.g. collision- induced dissociation (CID)).
  • CID collision- induced dissociation
  • ions corresponding to compounds of a particular m/z ratio can be selected from a first mass analysis, fragmented and reanalyzed in a second mass analysis.
  • Representative instruments that can perform such tandem mass analysis include, but are not limited to, magnetic four-sector, tandem time- of-flight, triple quadrupole, ion-trap, and hybrid quadrupole time-of-flight (Q-TOF) mass spectrometers.
  • Q-TOF hybrid quadrupole time-of-flight
  • mass spectrometers may 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).
  • ESI electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • Ionization sources can be used to generate charged species for the first mass analysis where the analytes do not already possess a fixed charge.
  • Additional mass spectrometry instruments and fragmentation methods include post-source decay 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. Aebersold and D. Goodlett, Mass Spectrometry in
  • the mass spectrometry is tandem mass
  • the mass spectrometry is accomplished using a mass spectrometer equipped with a thermally assisted electrospray ionization probe (e.g., TurboIonSpray® (TIS), Turbo-V, or similar).
  • a thermally assisted electrospray ionization probe e.g., TurboIonSpray® (TIS), Turbo-V, or similar.
  • the mass spectrometry utilizes multiple reaction monitoring (MRM). Exemplary methods for generating mass spectra may be found, for example, in U.S. Patent No. 6,930,305 and U.S. Patent No. 7,145,133, both of which are incorporated herein by this reference.
  • the mass spectrometry utilizes an AB Sciex API 4000TM tandem mass spectrometer.
  • the processing of a sample can involve separation, e.g., prior to mass analysis.
  • components of the sample can be separated and mass analysis performed on only a fraction of the sample mixture. In this way, the complexity of the analysis can be reduced because separated analytes can be
  • Separation can be performed by chromatography.
  • chromatography liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) can be used to effect such a sample separation and mass analysis.
  • any chromatographic separation process suitable to separate the analytes of interest can be used.
  • the chromatographic separation can be normal phase chromatography, reversed-phase chromatography, ion-exchange chromatography, size exclusion chromatography or affinity chromatography. Separation can also be performed electrophoretically.
  • electrophoretic separation techniques include, but are not limited to, ID electrophoresis, 2D
  • the mass spectrometry provided herein is accomplished using a mass spectrometer equipped with a reversed phase liquid chromatography column.
  • G6PDH deficiency includes determining by mass spectrometry an amount of G6PDH in a biological sample from a subject; 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.
  • G6PDH deficiency is generally an X-linked, hereditary genetic defect, typically due to a mutation in the G6PDH gene. This deficiency is common, being present in more than 400 million people (typically of African, Middle Eastern and South Asian ancestry) worldwide.
  • the G6PDH gene is found on the long arm of the X chromosome (band
  • the G6PDH deficiency is due to a G6PDH gene mutation.
  • G6PDH gene mutations can be found, for example, in Beutler E., "G6PD Deficiency" Blood, 84(11): 3613-3636, (1994).
  • the G6PDH deficiency is due to one of the gene mutations listed in Table 1.
  • G6PDH deficiency in a subject is indicated by an amount of G6PDH in a biological sample from the subject which 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, about 30% less than the control, about 35% less than the control, about 40% less than the control, about 45% less than the control, about 50% less than the control, about 55% less than the control, about 60% less than the control, about 65% less than the control, 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 about 100% less than the control.
  • G6PDH deficiency in a subject is indicated by an amount of G6PDH in a biological sample from the subject which is at least about 10% less than the control. In some embodiments, G6PDH deficiency in a subject is indicated by an amount of G6PDH in a biological sample from the subject which is at least about 25% less than the control. In some embodiments, G6PDH deficiency in a subject is indicated by an amount of G6PDH in a biological sample from the subject which is at least about 50% less than the control. For example, in some embodiments, G6PDH deficiency in a subject is indicated by an amount of G6PDH in a biological sample from the subject which is at least 10%, at least 25%, or at least 50% less than the control.
  • the term "subject” refers to animals such as mammals, including, but not limited to, humans, primates, cows, sheep, goats, horses, pigs, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species.
  • the subject is a human.
  • the subject is a neonate or an infant. In some embodiments, the subject is a pregnant woman.
  • the applicant's teachings provide methods for screening compounds for the ability to modulate G6PDH.
  • the effects of modulators of G6PDH activity e.g., enhancers or inhibitors
  • substrate specificities of G6PDH can also be studied using the methods described herein.
  • G6PDH-associated disorders refers to diseases and/or disorders which are at least partially attributable to a deficiency of G6PDH.
  • G6PDH-associated disorders include diseases and/or disorders which are attributable to a deficiency of G6PDH of 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%, about 100%.
  • G6PDH-associated disorders include diseases and/or disorders which are attributable to a deficiency of G6PDH of at least 25%. In some embodiments, G6PDH- associated disorders include diseases and/or disorders which are attributable to a deficiency of G6PDH of at least 50%. In some embodiments, G6PDH- associated disorders include diseases and/or disorders which are attributable to a deficiency of G6PDH of at least 75%.
  • the G6PDH-associated disorder is selected from chronic hemolysis, acute hemolytic anemia, favism, neonatal hyperbilirubinemia and pre-eclampsia.
  • Other G6PDH- associated disorders may include, for example, platelet abnormalities, skin pedicle flap loss, impairment of athletic performance, seizure disorders, increased incidence of cataracts, schizophrenia or depression, impaired renin release, diabetes, abnormal insulin release, cardiovascular disease, cholelithiasis, myoglobuinuria, increased susceptibility to infection, abnormal leukocyte function, increased jaundice in hepatitis, mental retardation or increased serum
  • the G6PDH-associated disorder is a disorder caused or induced by a substance capable of inducing G6PDH deficiency.
  • substances include, but are not limited to, fava beans, antimalarial drugs such as primaquine, pamaquine and chloroquine, sulfonamides such as sulfanilamide, sulfamethoxazole and mafenide, nitrofurantoin, acetylsalicylic acid, acetophenetidine, thiazolesulfone, methylene blue, naphthalene, analgesics such as aspirin,
  • methods for diagnosing acute hemolytic anemia in a subject include detecting G6PDH deficiency in a subject in accordance with the methods described above, wherein a G6PDH deficiency in the subject indicates acute hemolytic anemia in the subject.
  • an amount of G6PDH in the sample which 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 which is at least about 80% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 70% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 60% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 50% less than the control indicates acute hemolytic anemia in the subject.
  • an amount of G6PDH in the sample which is at least about 40% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 30% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 20% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 10% less than the control indicates acute hemolytic anemia in the subject.
  • an amount of G6PDH in the sample which is 75% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is 50% less than the control indicates acute hemolytic anemia in the subject. In some embodiments, an amount of G6PDH in the sample which is 25% less than the control indicates acute hemolytic anemia in the subject.
  • methods for diagnosing pre-eclampsia in a subject include detecting G6PDH deficiency in a subject in accordance with the methods described above, wherein a G6PDH deficiency in the subject indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is at least about 90% less than the control indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is at least about 80% less than the control indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is at least about 70% less than the control indicates preeclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 60% less than the control indicates pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 50% less than the control indicates pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 40% less than the control indicates pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 30% less than the control indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is at least about 20% less than the control indicates pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is at least about 10% less than the control indicates pre-eclampsia in the subject.
  • an amount of G6PDH in the sample which is 75% less than the control indicates pre-eclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is 50% less than the control indicates preeclampsia in the subject. In some embodiments, an amount of G6PDH in the sample which is 25% less than the control indicates pre-eclampsia in the subject.
  • prognostic methods for predicting increased mortality and/or morbidity resulting from pre-eclampsia or acute hemolytic anemia in a subject are provided herein. Such methods include detecting G6PDH deficiency in a subject in accordance with the methods described above, wherein an amount of the G6PDH in the biological sample which is 50% or less than the control indicates a prognosis of increased mortality and/or morbidity in the subject. In some embodiments, an amount of the G6PDH in the biological sample which is 75% or less than the control indicates a prognosis of increased mortality and/or morbidity in the subject.
  • the appropriate control is a predetermined value.
  • the appropriate control is a level detected in a single individual known to have normal G6PDH activity.
  • the appropriate control is an average level from a population of individuals having normal G6PDH activity.
  • the appropriate control is a level detected in a patient prior to administration of a substance capable of inducing a G6PDH deficiency.
  • the diagnostic or prognostic methods provided herein include any one of the following steps: reacting the biological sample with G6P and NADP in the presence of a surfactant and a buffer under suitable conditions to form 6-phosphogluconic acid; quenching the biological sample to form a quenched sample; and detecting by mass spectrometry a presence or amount of 6-phosphogluconic acid in the quenched sample.
  • the amount of 6-phosphogluconic acid in the quenched sample is related to the amount of G6PDH in the biological sample.
  • kits for performing the methods described herein comprises glucose-6-phosphate, nicotinamide adenine dinucleotide phosphate, a surfactant and a buffer for preparing a reaction mixture that facilitates the reaction of nicotinamide adenine dinucleotide phosphate, glucose-6- phosphate and G6PDH.
  • the buffer and/or the surfactant may be provided in a container in dry form or liquid form. Exemplary suitable buffers and surfactants are provided hereinabove.
  • the buffer is typically present in the kit at least in an amount sufficient to produce a particular pH in the mixture.
  • the buffer is provided as a stock solution having a pre-selected pH and buffer concentration.
  • acids and/or bases are also provided in the kit in order to adjust the reaction mixture to a desired pH.
  • the kit may additionally include a quenching solution, e.g. , an enzyme denaturant, such as an acetonitrile: methanol mixture.
  • the kit may additionally include one or more one or more diluents, e.g. , solvents suitable for use in a mass spectroscopy system.
  • the kit may additionally include other components that are beneficial to enzyme activity, such as salts (e.g., KC1, NaCl, or NaOAc), metal salts (e.g.
  • Ca 2+ salts such as CaCl 2 , MgCl 2 , MnCl 2 , ZnCl 2 , or Zn(OAc), and/or other components that may be useful for the G6PDH enzyme.
  • These other components can be provided separately from each other or mixed together in dry or liquid form.
  • the glucose-6-phosphate and/or nicotinamide adenine dinucleotide phosphate can be provided in dry or liquid form, together with or separate from the buffer.
  • the glucose-6-phosphate and/or nicotinamide adenine dinucleotide phosphate can be provided in an aqueous solution, partially aqueous solution, or non-aqueous stock solution that is miscible with the other components of the reaction mixture.
  • the kit further includes instructions for use.
  • the kit can include instructions for preparing a reaction mixture that facilitates a reaction of nicotinamide adenine dinucleotide phosphate, glucose-6-phosphate and G6PDH.
  • the kit includes instructions for preparing a sample for analysis on a mass spectrometer.
  • the kit further comprises a pre- prepared calibration curve, such as the curve shown in Figure 3, which would allow a user to determine an amount of G6PDH in the sample based upon an amount of 6- phosphogluconic acid determined using a mass spectrometer.
  • the pre- prepared calibration curve may be a plot of [6-phosphogluconic acid/glucose-6- phosphate area] versus milliUnits of G6PDH.
  • the kit includes standard samples, with pre-determined amounts of G6PDH, such that a user may produce their own calibration curve.
  • the kit further includes instructions on how to prepare a calibration curve. The instructions may include any combination of:
  • a Mixing the glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate in the presence of a buffer and/or a surfactant for a set time period (e.g., 15 minutes, 20 minutes, 25 minutes, 30 minutes, etc.); b. Quenching the sample using a quenching solution;
  • Figure 1 shows an exemplary mass spectrometric reading in MRM from a dried blood sample, showing levels of G6P, 6-phosphogluconic acid and NADP.
  • Figure 1 is representative of a normal level of G6PDH.
  • Figure 2 compares a normal sample with a sample having no G6PDH activity.
  • a commercially-available enzyme standard (e.g., Sigma #G5885) was dissolved to obtain 100 milliUnits/mL with a solution of 50% bi-distilled water, 30% 250 mM K 2 HP0 4 and 20% of 1% Saponine for Molecular Biology. 0, 1, 2, 4, 10, 20 and 40 ⁇ ⁇ of this solution (corresponding to 0, 0.1, 0.2, 0.4, 1.0, 2.0 and 4.0 milliUnits of enzyme) were added to 50 ⁇ ⁇ of "Reagent 1" (described in Example 1) in a 1.5-mL Eppendorf tube. The Eppendorf tube was closed, and after gentle mixing, the tube was placed in an incubation oven at 37°C for 30 minutes.
  • "Reagent 1" described in Example 1
  • Figures 4A and 4B show two exemplary mass spectrometric readings in
  • LC-MS/MS is capable of measuring both high and low G6PDH activity. Because there is no homogeneous calibration available for both the assays, the calibration curve for the LC- MS/MS assay was generated using the above samples, which represented extreme activity values. The calibration used as standard for the LC-MS/MS is shown in Figure 5.
  • Figures 6A and 6B depict two exemplary mass spectrometric readings in
  • FIGS. 7A and 7B are the correlation plot and the Bland and Altman plot (measures the agreement between two methods of clinical measurement) generated using the values measured in the present study. As can be seen by the above data and plots, there is good agreement between the values obtained using LC/MS/MS and those obtained using the BV kit, particularly with regard to the low activity samples.

Abstract

La présente invention concerne des procédés de détermination d'une quantité de glucose-6-phosphate déshydrogénase ("G6PDH") dans un échantillon biologique. De plus, l'invention concerne des procédés pour la détection d'une déficience en G6PDH, ainsi que le diagnostic de troubles associés à la G6PDH, tels que l'anémie hémolytique aiguë et la pré-éclampsie.
EP11843953.8A 2010-11-24 2011-11-17 Détection sensible, à haut débit, de la glucose-6-phosphate déshydrogénase Withdrawn EP2643474A4 (fr)

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KR101808391B1 (ko) * 2015-12-31 2017-12-12 강원대학교산학협력단 G6pd의 활성도 측정용 조성물, 이를 포함하는 키트 및 g6pd의 활성도 측정방법
CN105588937B (zh) * 2016-01-26 2017-10-10 上海馥地检测技术有限公司 酶标板包被液、封闭液及酶标板制作方法
IT201600103909A1 (it) * 2016-10-17 2018-04-17 Marco Flavio Michele Vismara Sistema e metodo di acquisizione, trasmissione ed elaborazione di dati ematochimici
CN109142551A (zh) * 2017-06-15 2019-01-04 北京挑战生物技术有限公司 一种淬灭酶产业化生产中酶活力的快速精确检测方法
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