Screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios
11650212 · 2023-05-16
Assignee
Inventors
Cpc classification
G01N2560/00
PHYSICS
International classification
Abstract
The invention relates to a method of diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency in an animal comprising: determining in a biological sample the intensity signal by mass analysis of at least a bile alcohol glucuronide and a C24- or C27-bile acid or a conjugate thereof, comparing the intensity signals to a control sample or control value, and determining 27-hydroxylase (CYP27A1) deficiency based on said comparison.
Claims
1. A method of identifying 27-hydroxylase (CYP27A1) deficiency in an animal, the method comprising: receiving a dried blood spot comprising a blood sample from said animal; enriching the blood sample by liquid chromatography, protein precipitation, liquid-liquid extraction, solid-liquid extraction, or ultrafiltration; detecting, by mass analysis of the enriched blood sample, an intensity signal of at least a bile alcohol glucuronide and a C27- or C24-bile acid or a conjugate thereof, wherein the C27- or C24-bile acid or conjugate thereof is selected from the group consisting of: cholic acid (CA), tauro-cholic acid (t-CA); glyco-cholic acid (g-CA); chenodeoxycholic acid (CDCA); tauro-chenodeoxycholic acid (t-CDCA); glyco-chenodeoxycholic acid (g-CDCA); trihydroxycholestanoic acid (THCA); tauro-trihydroxycholestanoic acid (t-THCA); glyco-trihydroxycholestanoic acid (g-THCA); dihydroxycholestanoic acid (DHCA); tauro-dihydroxycholestanoic acid (t-DHCA); and glyco-dihydroxycholestanoic acid (g-DHCA); calculating a ratio between the intensity signals of said bile alcohol glucuronide and said C24- or C27-bile acid or conjugate thereof; comparing said ratio between the intensity signals of said bile alcohol glucuronide and said C24- or C27-bile acid or conjugate thereof with a ratio of a control sample or control value from a control animal of the same species as the animal but without CYP27A1 deficiency; and identifying the animal as having hydroxylase (CYP27A1) deficiency, based on said comparison of said ratios.
2. The method according to claim 1, wherein said bile alcohol glucuronide is cholestanetetrol glucuronide (tetrol).
3. The method according to claim 1, wherein the intensity signal is determined of a stable isotope labeled compound selected from the group consisting of: bile alcohol glucuronide, a C24- or C27-bile acid, or a conjugate thereof, and wherein said intensity signal of said stable isotope labeled compound is compared to the intensity of said at least a bile alcohol glucuronide and a C24- or C27-bile acid or conjugate thereof.
4. The method according to claim 1, wherein at least the intensity signal is determined of a C24-bile acid or conjugate thereof and of a C27-bile acid or conjugate thereof, wherein said C24-bile acid or conjugate thereof is selected from the group consisting of: cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), and glyco-chenodeoxycholic acid (g-CDCA), and said C27-bile acid or conjugate thereof is selected from the group consisting of: trihydroxycholestanoic acid (THCA), tauro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA).
5. The method according to claim 1, wherein said mass analysis is selected from the group consisting of: mass spectrometry (MS), High-resolution MS, liquid chromatography—mass spectrometry (LC-MS), matrix-assisted laser desorption/ionization—time of flight mass spectrometry (MALDI-TOF), quadrupole time of flight mass spectrometry (Q-TOF), Orbitrap-MS, and flow-injection MS.
6. The method according to claim 1, wherein said animal is a human.
7. The method according to claim 1, wherein said animal is a newborn.
8. The method according to claim 1, further comprising measuring the intensity signal of said bile alcohol glucuronide or C24- or C27-bile acid or a conjugate thereof, relative to the intensity signal of an isotopically labeled standard.
9. The method according to claim 8, wherein said isotopically labeled standard comprises a stable isotope labeled C24- or C27-bile acid or a conjugate thereof selected from the group consisting of: cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), glyco-chenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), tauro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA).
10. The method of claim 9, wherein said method comprises a step of ionizing the biological sample using a cone voltage of about 60 to about 90 V.
11. A kit comprising a stable isotope labeled C24- or C27-bile acid or a conjugate thereof selected from the group consisting of: cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), glyco-chenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), tau ro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA); and further comprising a positive control sample, wherein the positive control sample is a dried blood spot comprising a blood sample from a control animal of the same species as the animal but with cerebrotendinous xanthomatosis (CTX).
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Definitions
(7) As used herein, the terms “diagnosis” “diagnose” and “diagnosing” refer to the process of attempting to determine or identify whether or not a subject is suffering from a given disease or condition. The term “diagnosis” does not refer to the ability to determine the presence or absence of a particular disease with 100% accuracy, or even that a given course or outcome is more likely to occur than not. Instead, the skilled artisan will understand that the term “diagnosis” refers to an increased probability that a certain disease is present in the subject. Diagnosis as used herein also includes methods for preliminary diagnosis which could be confirmed by diagnosis using other methods known in the art. Additionally, it is contemplated that the methods as described herein may be used for monitoring the progression of a condition and/or the efficacy of a therapy being used to treat a condition.
(8) The term “bile acid,” as used herein, includes steroid acids (and/or the carboxylate anion thereof), and salts thereof, found in the bile of an animal (e.g., a human), including, by way of non-limiting example, cholic acid, cholate, deoxycholic acid, deoxycholate, hyodeoxycholic acid, hyodeoxycholate, glycocholic acid, glycocholate, taurocholic acid, taurocholate, chenodeoxycholic acid, chenodeoxycholate, lithocholic acid, lithocholate, and the like.
(9) The term “primary” bile acid refers to any of cholic acid (3α,7α,12α-trihydroxy-5β-cholanoic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanoic acid).
(10) The term “secondary bile acid” as used herein refers to any of primary bile acid and conjugate thereof that has been modified by bacteria in the gut, including but not limited to lithocholic acid and deoxycholic acid.
(11) The term “C24 bile acid” as used herein refers to any primary and secondary bile acid and conjugates thereof having 24 carbon atoms.
(12) The term “C27 bile acid” as used herein refers to any bile acid intermediate and conjugate thereof having 27 carbon atoms, which can be formed in the bile acid synthesis before and during peroxisomal beta-oxidation.
(13) The term “conjugate of a bile acid” refers to any primary bile acid conjugated to the amino acids glycine and taurine.
(14) The term “bile acid intermediate” refers to any metabolite between cholesterol and the primary bile acids that are formed by normal or pathological bile acid metabolism/synthesis.
(15) As used herein the term “bile alcohol glucuronide” refers to a conjugate of a bile alcohol with glucuronic acid.
(16) The term “tetrol” as used herein refers to 5β-cholestane-3α,7α,12α,25-tetrol glucuronide and the isomers of cholestanetetrol glucuronide. Preferably, tetrol is 5β-cholestane-3α,7α,12α,25-tetrol glucuronide.
(17) The term “control sample” as used herein refers to a biological sample or samples of a patient suffering from CTX, or another positive or negative control sample for determining control intensity signals.
(18) The term “control value” as used herein refers to a predetermined intensity signal or ratio for said at least a bile alcohol glucuronide and a C27- or C24-bile acid bile acid or a conjugate thereof and/or a predetermined cut-off level.
(19) Preferably, a control value refers to control level of said bile alcohol glucuronide and a C27- or C24-bile acid bile acid or a conjugate thereof and/or a predetermined intensity signal level or ratio of the intensity signals between said at least a bile alcohol glucuronide and a C27- or C24-bile acid bile acid or a conjugate thereof and/or a predetermined cut-off level. The control value can for example be a reference intensity signal to which test sample intensity signals are compared, and/or a predetermined intensity signal or signals for example as a numerical value and/or range (e.g. control range) corresponding to the intensity signal levels in such sample or samples. For example, as demonstrated herein, control samples with a known outcome (such as suffering from CYP27A1 deficiency, Zellweger, cholestasis or from a healthy control) can be used to determine a cut-off above or below which subjects are predicted to suffer from CYP27A1 deficiency. Test samples are then compared to the predetermined value determined using control samples. The control value can be an average, median, or calculated cut-off intensity value (e.g. threshold) for said bile alcohol glucuronide and a C27- or C24-bile acid bile acid or a conjugate thereof and/or a composite thereof (e.g. ratio, sum) above or below which value a subject can be classified with an outcome class—e.g. CYP27A1 deficiency or not.
(20) In an embodiment, the control can be a ratio of the intensities of said bile alcohol glucuronide and a C27- or C24-bile acid bile acid or a conjugate thereof and the mass intensity of one or more internal standardization markers in a control sample. The control ratio is compared to a corresponding ratio determined for the sample.
(21) The term “ratio” as used herein refers to the mathematical relationship between two quantities. For instance, a ratio of quantities A and B is a quotient of value A divided by value B or of value B by A. As used herein, the ratios A/B and B/A can be used interchangeably. Therefore, if it is stated herein that the ratio between the mass intensities of C24- or C27-bile acid or a conjugate thereof is determined, said ratio comprises the mass intensity of said C24-bile acid divided by the mass intensity of said C27-bile acid and the mass intensity of said C27-bile acid divided by the mass intensity of said C24-bile acid.
EMBODIMENTS
(22) Based on the mass intensity signals of the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, we developed a new DBS screening assay for CTX that is suitable for neonatal screening. Using our assay, a good separation was achieved between DBS of CTX patients and DBS of controls, Zellweger patients and newborns with (potential) cholestasis. Initially, we set out to solely measure tetrol in untreated CTX patients. However, despite a fairly good separation between the mean response in controls and CTX patients, there was overlap between the responses for the patient and control groups (see
(23) We found that a combination of mass measurements of bile alcohol glucuronide and a C24- or C27-bile acid or a conjugate thereof, resulted in a good sensitivity and specificity.
(24) The invention therefore provides a method of diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency in an animal comprising: a) determining in a biological sample the intensity signal by mass analysis of at least a bile alcohol glucuronide and a C27- or C24-bile acid or a conjugate thereof, b) determining the ratio between said bile alcohol glucuronide and said C24- or C27-bile acid or a conjugate thereof, c) comparing said ratio with the ratio of a control sample or control value, d) determining 27-hydroxylase (CYP27A1) deficiency based on said comparison.
(25) The method is suitable for diagnosis or screening of animals, in particular of mammals, and preferably it is used for humans. Preferred embodiments as described herein include diagnosing or screening methods for newborn screens. However, the methods as described herein are equally useful for other populations and screening/diagnosis such as toddlers, and adults.
(26) The present teachings can provide methods for determining the relative concentration of the analytes selected from bile alcohol glucuronides and C24- or C27-bile acids or a conjugate thereof, in one or more samples and provide methods whereby the relative or absolute concentration, can be determined using mass spectrometry.
(27) Preferably, ratios between the mass intensities of bile alcohol glucuronides and C24- or C27-bile acids and conjugates thereof, more preferably between the mass instensities of bile acid t-CDCA and tetrol, and the bile acid intermediate t-THCA and tetrol are used. Alternatively a look up table of the results can be searched and used as input for a formula that includes other parameters, for example plasma cholestanol or a scoring system of clinical readouts.
(28) Any biological sample which comprises bile acid synthesis metabolites may be used in the method of the invention. For example, urine, and other bodily fluids are suitable. It is preferred that said biological sample is a blood, serum, or plasma. In particular, dried blood spots (DBS) are preferably used, as these samples are routinely used in newborn screening assays.
(29) A very suitable biological sample comprises a quarter-inch (6.4 mm) diameter DBS punch (or for most CTX DBS; two eighth-inch DBS, which equals half the material in one quarter-inch DBS) whereas most neonatal screening laboratories prefer a one eighth-inch (3.2 mm) diameter DBS punch. A modern triple quadrupole tandem mass spectrometers will readily detect the three metabolites used in our assay in eighth-inch DBS punched. Conversely, our invention also shows that laboratories that have older machines still can screen for CTX with our method when using a quarter-inch diameter DBS punch (or two eighth-inch DBS).
(30) In a preferred embodiment, said biological sample is a DBS not bigger than ¼ inch, ⅛ inch.
(31) The biological sample can be subjected to chromatographic separation, for example, by LC such as by HPLC, followed by mass analysis.
(32) The method can also comprise the step of extracting the analyte using either liquid-liquid extraction, solid-liquid-extraction or protein precipitation using hydrophobic solvents such as methanol prior to the mass analysis step.
(33) In some preferred embodiments the mass spectrometer can be used in tandem MS mode with multiple-reaction monitoring (MRM) to detect the transitions of the analytes preferably, but not restricted to the bile alcohol glucuronides and C24- or C27-bile acids or a conjugate thereof listed in table 2. In these embodiments and in other preferred embodiments, a stable isotope labeled internal standard corresponding to the unlabeled ion to be detected is preferably used to calculate the concentration or abundance of these metabolites.
(34) In some preferred embodiments, a mass spectrometer is used with high resolution capacities enabling the measurement of the exact mass in full scan mode to directly determine the abundance of the required ions with exact mass precision. During the analysis, the exact mass trace of ions listed in, but not restricted to those in, table 3 can be used for detection of bile acid metabolites. In these embodiments and in other preferred embodiments, a stable isotope labeled internal standard corresponding to the unlabeled ion to be detected is preferably used to calculate the concentration or abundance of these metabolites.
(35) In a preferred embodiment, the abundance of each transition (or in case of a mass spectrometer with high-resolution capacity, the exact mass trace) during the elution/infusion of the sample can be averaged followed by correction for background signal by subtracting the average intensity prior or after the sample elution/infusion. This yields the intensity/abundance of the chosen analytes.
(36) The intensity/abundance of the analytes can then be used to calculate the metabolite ratios. In these embodiments and in other preferred embodiments, if dedicated (stable-isotope labeled) internal standards are used, the internal standard can be used to correct for matrix effects, after which the resulting response can be used to perform metabolite calculations.
(37) In preferred embodiments, the collision fragment energy is selected to be in the range of 35-60 eV.
(38) In a preferred embodiment, flow injection mass analysis (HA) is used. An advantage thereof is that HA equipment belongs to standard newborn screening equipment.
(39) Quantitation can be enabled by relative or absolute measurement of the signal derived from one or more analytes and standards. The negative charge can be transferred to the analyte which functions as the fragment ion to be detected by mass spectrometry.
(40) In a preferred embodiment, an internal standard solution is added to the sample comprising the analytes. Preferably, said solution comprises one or more of pregnanediol glucuronide, stable isotope-(.sup.13C, .sup.2H, etc.)-labeled tetrol-glucuronide, .sup.2H.sub.4-t-CDCA, .sup.2H.sub.4-g-CA, .sup.2H.sub.4-g-CDCA, preferably dissolved in methanol.
(41) In some preferred embodiments, the “Parent-daughter ion transition monitoring” or “PDITM” is used as the method of analysis and workflow status. PDITM refers to a technique whereby the transmitted mass-to-charge (m/z) range of a first mass separator (often referred to as “MS” or the first dimension of mass spectrometry) is specifically selected to transmit a molecular ion (often referred to as “the parent ion” or “the precursor ion”) to an ion fragmentor (e.g. a collision cell, photodissociation region, etc.) to produce fragment ions (often referred to as “daughter ions”) and the transmitted m/z range of a second mass separator (often referred to as “MS/MS” or the second dimension of mass spectrometry) is selected to transmit one or more daughter ions to a detector which measures the daughter ion signal. This technique offers unique advantages when the detection of daughter ions in the spectrum is focused by “parking” the detector on the expected daughter ion mass. The combination of parent ion and daughter ion masses monitored can be referred to as the “parent-daughter ion transition” monitored. The daughter ion signal at the detector for a given parent ion-daughter ion combination monitored can be referred to as the “parent-daughter ion transition signal”. For the parent ion-daughter combinations used in the method described herein, see Table 2.
(42) In one embodiment of parent-daughter ion transition monitoring is multiple reaction monitoring (MRM) (also referred to as selective reaction monitoring). In various embodiments of MRM, the monitoring of a given parent-daughter ion transition comprises using the first mass separator (e.g., a first quadrupole parked on the parent ion m z of interest) to transmit the parent ion of interest and using the second mass separator (e.g., a second quadrupole parked on the daughter ion m/z of interest) to transmit one or more daughter ions of interest. In various embodiments, a PDITM can be performed by using the first mass separator (e.g., a quadrupole parked on a parent ion m/z of interest) to transmit parent ions and scanning the second mass separator over a m/z range including the m/z value of the one or more daughter ions of interest.
(43) In some preferred embodiments, a tandem mass spectrometer (MS/MS) instrument or, more generally, a multidimensional mass spectrometer instrument, can be used to perform PDITM, e.g., MRM. Examples of suitable mass analyzer systems comprise, but are not limited to, those that comprise one or more of a triple quadrupole, a quadrupole-linear ion trap, a quadrupole TOF, and a TOF-TOF, ion trap, orbitrap, quadrupole ion trap, quadrupole orbitrap.
(44) In an embodiment a step of subjecting at least a portion of the combined sample to PDITM is applied, which comprises introducing the combined sample directly into a mass analyzer system, e.g., by introduction of the combined sample in a suitable solution using an electrospray ionization (ESI) ion source or atmospheric pressure ionization (APCI) source.
(45) According to various preferred embodiments, samples containing one or more compounds may be enriched by various methods prior to analysis. The enrichment method can depend upon the type of sample, such as blood (fresh or dried), plasma, serum, etc. Exemplary enrichment methods can comprise, without limitation, protein precipitation, liquid-liquid extraction, solid-liquid extraction, and ultrafiltration. Other enrichment methods, or a combination of two or more enrichment methods may be used.
(46) The sample workup of our method is straightforward as no derivatization is required and since a standard methanol extraction is used, the assay can suitably be combined with other neonatal screening assays. Therefore, in a preferred embodiment, said method is combined with other neonatal screening assays. The measurement uses preferably flow injection, as it only takes a few minutes per run.
(47) Preferably, the method comprises the step of measuring specific transitions for cholestanetetrol glucuronide, tauro-chenodeoxycholic acid (t-CDCA) and tauro-trihydroxycholestanoic acid (t-THCA).
(48) The invention further provides a kit for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency comprising a stable isotope labeled tetrol and a stable isotope labeled C24- or C27-bile acid or a conjugate thereof selected from the group consisting of cholic acid (CA), tauro-cholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), tauro-chenodeoxycholic acid (t-CDCA), glyco-chenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), tauro-trihydroxycholestanoic acid (t-THCA), glyco-trihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), tauro-dihydroxycholestanoic acid (t-DHCA), and glyco-dihydroxycholestanoic acid (g-DHCA). A skilled person in the art knows how to synthesize stable isotope labelled bile acid. Methods to synthesize tetrol are known in the art (19).
(49) Preferably, the kit further comprises a positive control sample, preferably a blood sample of a CTX patient.
(50) In a further aspect, persons diagnosed with 27-hydroxylase (CYP27A1) deficiency, preferably in the newborn period, using the method of the invention, can be effectively treated with chenodeoxycholic acid (CDCA) supplementation administered orally, thereby preventing the development of any symptoms associated with cerebrotendinous xanthomatosis (CTX; 27-hydroxylase (CYP27A1) deficiency). In a preferred embodiment, CDCA supplementation comprises 5 mg/kg/day divided in three equal dosages. In another preferred embodiment, CDCA supplementation comprises 15 mg/kg/day in three equal dosages. This regime is preferred for children. In another preferred embodiment, CDCA supplementation comprises up to 250 mg three times daily. This regime is preferred for adult patients.
(51) While the above description provides examples and specific details of various embodiments, it will be appreciated that some features and/or functions of the described embodiments admit to modification without departing from the scope of the described embodiments. The above description is intended to be illustrative of the teachings herein, the scope of which is limited only by the language of the claims appended hereto.
Example
(52) Patient and Control Samples
(53) Anonymized DBS of 150 term (mean gestational age (range): 39.9 (37.0-42.0) weeks and 50 preterm newborns (mean gestational age (range): 34.5 (26.7-36.9) weeks) were obtained from the biobank of the Dutch newborn screening program (Dutch National Institute for Public Health and the Environment, Bilthoven, The Netherlands). All parents gave informed consent for the anonymized use of DBS from the Guthrie card of their child at the time of collection (standard procedure in the Dutch newborn screening program; all Guthrie newborn screening cards in the Netherlands are stored for five years after which they are destroyed). Fifteen DBS, including 2 original neonatal DBS, were collected from 14 individual untreated CTX patients. These included DBS from participants enrolled in Institutional Review Board approved studies at Oregon Health & Science University with written informed consent provided by the participants for use of the DBS. De-identified diagnostic samples submitted to the Sterol Laboratory for biochemical confirmation of CTX were also used with Institutional Review Board approval. Table 1 lists all relevant details concerning the CTX patients and their DBS. All patients or their parents/legal representatives gave written informed consent for the use of their DBS/the DBS of their child. Three anonymized Zellweger DBS were obtained from the biobank at the Academic Medical Center (Amsterdam).
(54) Materials
(55) Solvents: methanol and acetonitrile were purchased from Biosolve. .sup.2H.sub.4-g-CDCA, .sup.2H.sub.4-g-CA, .sup.2H.sub.4-t-CDCA and .sup.2H.sub.4-t-CA were purchased from CDN Isotopes. H.sub.2O was MilliQ® purified water. 5β-Pregnane-3α,20α-diol glucuronide (Pregnanediol glucuronide) was from Sigma-Aldrich and filter paper Whatman 903 was purchased from Drukkerij PAL.
(56) Sample Preparation
(57) A punch (quarter-inch (6.4 mm) diameter) of a DBS was used for all except for CTX DBS number 1 to 8, 12 and 13, where two 3.2 mm DBS were used (for these, the result was corrected by multiplying the result by a factor two). The DBS were transferred to a 1.5 ml tube, to which 500 μL methanol was added and this was incubated for 30 min at room temperature in a sonicator bath (Branson 3510). The filter paper was removed, and the extraction fluid was transferred to a 4-mL glass tube followed by the addition of 20 μL internal standard mixture [1 μM Pregnanediol glucuronide, 0.25 μM .sup.2H.sub.4-t-CA, 0.25 μM .sup.2H.sub.4-t-CDCA, 0.25 μM .sup.2H.sub.4-g-CA, 0.25 μM .sup.2H.sub.4-g-CDCA dissolved in methanol/H.sub.2O (3:1 vol/vol)]. The internal standard solution was intentionally not added during the sonication step as this solution contains water which extracts unwanted salts/interfering compounds that interfere with the MS measurement. After vortex-mixing, the sample was taken to dryness under nitrogen flow at 40° C. The residue was reconstituted in 120 μL of methanol/H.sub.2O (3:1 vol/vol), transferred to a sample vial, and capped.
(58) Flow Injection Ms/Ms
(59) We injected 10 μL using Acetonitrile/H.sub.2O (9:1, vol/vol) as mobile phase into a Waters Premier-XE tandem mass spectrometer (MS/MS) (Waters Cooperation) operated in the negative ion electrospray ionization mode. Nitrogen was used as both the nebulizing and the desolvation gas. The collision gas was argon, and the cell pressure was 2.7*10.sup.−3 mbar. The source temperature was set at 120° C., and the capillary voltage was maintained at 2.5 kV. The detector was used in tandem MS mode with multiple-reaction monitoring to detect the transition of a specific precursor ion to a fragment for each analyte. The transitions, cone voltages, and collision energies established for each compound are listed in Table 2. The analytical run time, from injection to injection, was 2 minutes. Quantification was performed using Masslynx 4.2 employing the NeoLynx™ program. NeoLynx combined and averaged the individual MRM transition intensities across the flow injection profile and subsequently corrected for background signal by subtracting the average intensity prior to sample infusion. The resulting peak heights of the measured compounds were exported for further calculations in MS Excel (Microsoft).
REFERENCES
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(61) TABLE-US-00001 TABLE 1 DBS and CTX patient information Patient DBS age at Plasma Plasma age Storage sampling cholestanol cholestanol DBS Patient.sup.a (y) conditions (y) (mg/dl) (μM) Diagnosis.sup.b Phenotype at diagnosis 1 P1 0.5 −20° C. 9 1.62 42 G/B unknown 2 P2 0.5 −20° C. 12 2.57 66 G/B unknown 3 P3 4 −20° C. 17 1.84 47 B neonatal jaundice, intractable diarrhea, xanthomas 4 P4 9 −20° C. 16 3.23 83 G/B juvenile cataracts, developmental delay, autistiform behavior, cognitive impairment 5 P5 4 −20° C. 31 1.65 42 G/B xanthomas, spastic gait, paraparesis 6 P6 0.17 −20° C. 45 3.44 88 B cognitive decline, psychiatric disorder, dementia 7 P7 0.17 −20° C. 46 4.79 123 B depression, paraparesis, cerebellar ataxia, dystonia 8 P8 7.5 −20° C. 36 4.52 116 G/B cataract, xanthomas, seizures, intractable diarrhea 9 P9 0.5 4° C. 6.2 1.52 39 G speech delay, altered stool pattern 10 P10 0.1 4° C. 9 2.06 53 B cataracts 11 P11 0.5 4° C. 11 4.52 116 G cataracts, developmental delay, autistiform behaviour, altered stool pattern 12 P12 6 −20° C. 56 0.84 22 G/B cataracts, xanthomas 13 NB1 16 Unknown, 0.01 — — G/B juvenile cataracts, xanthomas, intractable diarrhea last 8 years stored at −20° C. 14 NB2 4 4° C. 0.01 0.86 22 G none, see (10) .sup.aNB: original neonatal screening DBS .sup.bG: genetic, B: biochemical
(62) TABLE-US-00002 TABLE 2 Transitions, cone voltages and collision energies for measured compounds Cone Collision MRM voltage energy Compound (m/z) (V) (eV) t-CDCA 498.fwdarw.80 90 60 .sup.2H.sub.4-t-CDCA 502.fwdarw.80 90 60 t-CA 514.fwdarw.80 90 60 .sup.2H.sub.4-t-CA 518.fwdarw.80 90 60 t-THCA 556.fwdarw.80 90 60 g-CDCA 448.fwdarw.74 60 40 .sup.2H.sub.4-g-CDCA 452.fwdarw.74 60 40 g-CA 464.fwdarw.74 60 40 .sup.2H.sub.4-g-CA 468.fwdarw.74 60 40 Pregnanediol glucuronide 495.fwdarw.75 45 35 Cholestane tetrol glucuronide 611.fwdarw.75 80 48
(63) TABLE-US-00003 TABLE 2(A) Table of individual tetrol/t-CDCA and t-THCA/tetrol ratios CTX patients and ranges for controls (term/preterm) and Zellweger patients. DBS Patient Tetrol/t-CDCA t-THCA/tetrol 1 P1 5 0.028 2 P2 12 0.035 3 P3 233 0.004 4 P4 702 0.003 5 P5 328 0.001 6 P6 866 0.005 7 P7 101 0.001 8 P8 94 0.002 9 P9 0.8 0.048 10 P10 18 0.018 11 P11 21 0.026 12 P12 25 0.023 13 NB1 22 0.047 14 NB2 138 0.004 Term (range) 0.000-0.057 0.19-250 Preterm (range) 0.000-0.061 0.09-377 Zellweger (range) 0.023-0.101 10.5-38.0
(64) TABLE-US-00004 TABLE 3 Overview of selected (exact) masses of bile acids, bile alcohols and bile acid intermediates Nominal Exact mass mass Compound name 375 375.2905 3β-hydroxy-5-cholanoic acid (litocholic acid) 389 389.2697 dihydroxy-cholenoic acid 391 391.2885 dihydroxy-cholanoic acids (e.g. chenodeoxycholic acid) 405 405.2646 trihydroxy-cholenoic acids, 3β,7α,12α-trihydroxy- 5-cholenoic acid 407 407.2803 trihydroxy-cholanoic acids (e.g. cholic acid) 431 431.3169 3β,7α-dihydroxy-cholestenoic acid 432 432.3119 3β-hydroxy-5-cholanoic acid (litocholic acid) glycine 433 433.3323 dihydroxy-cholestanoic acid (e.g. DHCA) 444 444.2755 7α-hydroxy-3-oxo-4-cholenoic acid glycine 446 446.2912 3β,7α-dihydroxy-5-cholenoic acid glycine 447 447.3116 3β,7α,12α-trihydroxy-cholestenoic acid 448 448.3069 dihydroxy-cholanoic acids (e.g. chenodeoxycholic acid, deoxycholic acid) glycine 449 449.3273 trihydroxy-cholestanoic acid (e.g. THCA) 453 453.2316 3β-hydroxy-5-cholenoic acid sulfate 460 460.2705 7α,12α-dihydroxy-3-oxo-4-cholenoic acid glycine 462 462.2861 3β,7α,12α-trihydroxy-5-cholenoic acid glycine 464 464.3018 trihydroxy-cholanoic acids glycine (e.g. cholic acid) 465 465.3222 tetrahydroxy-cholestanoic acid (e.g. OH-THCA, varanic acid) 465 465.3044 cholesterol sulfate 469 469.2265 3β,7α-dihydroxy-5-cholenoic acid sulfate 471 471.2422 dihydroxy-cholanoic acid sulfate 472 472.3432 3β-hydroxy-5-cholestanoic acid glycine 480 480.2789 3β-hydroxy-5-cholanoic acid taurine 480 480.2967 tetrahydroxy-cholanoic acids glycine 482 482.2946 3β-hydroxy-5-cholanoic acid (litocholic acid) taurine 485 485.2215 3β,7α,12α-trihydroxy-5-cholenoic acid sulfate 487 487.2371 trihydroxy-cholanoic acid sulfate 488 488.3381 3β,7α-dihydroxy-cholestenoic acid glycine 490 490.3538 dihydroxy-cholestanoic acid (e.g. DHCA) glycine 494 494.2582 7α-hydroxy-3-oxo-4-cholenoic acid taurine 498 498.2895 dihydroxy-cholanoic acids (chenodeoxycholic acid) taurine 506 506.3487 dihydroxy-cholestanoic acid (e.g. THCA) glycine 507 507.3327 C.sub.29-dicarboxylic acid 510 510.2531 7α,12α-dihydroxy-3-oxo-4-cholenoic acid taurine 510 510.2531 3β-hydroxy-5-cholenoic acid glycine and sulfate 514 514.2844 trihydroxy-cholanoic acids (cholic acid) taurine 522 522.3259 3β-hydroxy-5-cholestanoic acid taurine 526 526.2480 3β,7α-dihydroxy-5-cholenoic acid glycine and sulfate 528 528.2637 dihydroxy-cholanoic acids (e.g. chenodeoxycholic acid) glycine and sulfate 530 530.2793 tetrahydroxy-cholanoic acids taurine 540 540.3364 dihydroxy-cholestanoic acid (e.g. DHCA) taurine 542 542.2429 3β,7α,12α-trihydroxy-5-cholcnoic acid glycine and sulfate 552 552.3000 7α,12α-dihydroxy-3-oxo-4-cholestenoic acid taurine 554 554.3157 trihydroxy-cholestenoic acid (e.g. THCA:1) taurine 556 556.3313 trihydroxy-cholestanoic acid (e.g. THCA) taurine 567 567.3175 dihydroxy-cholanoic acids glucuronide 570 570.3106 tetrahydroxy-cholestenoic acids (e.g. OH-THCA:1) taurine 572 572.3263 tetrahydroxy-cholestanoic acids (e.g. OH-THCA) taurine 583 583.3124 trihydroxy-cholanoic acids glucuronide 586 586.3055 pentahydroxy-cholestenoic acids (e.g. diOH-THCA:l) taurine 588 588.3212 pentahydroxycholestanoic acid taurine 609 609.3644 5β-cholestanetriol-?-one glucuronide, 5β- cholestenetetrol glucuronide 611 611.3801 5β-cholestenetetrol glucuronide 611 611.3437 tetrahydroxy-27-nor-5β-cholestane-24-one glucuronide 613 613.3593 27-nor-5β-cholestanepentol glucuronide 625 625.3593 tetrahydroxy-5β-cholestane-?-one glucuronide, 5β-cholestenepentol glucuronide 627 627.3750 5β-cholestanepentol glucuronide 627 627.3386 pentahydroxy-27-nor-5β-cholestane-24-one glucuronide 629 629.3543 27-nor-5β-cholestanehexol glucuronide 641 641.3543 5β-cholestanepentol-?-one glucuronide, 5β-cholestcnchexol glucuronide 643 643.3699 5β-cholestanehexol glucuronide 643 643.3335 hexahydroxy-27-nor-5β-cholestane-24-one glucuronide 645 645.3492 27-nor-5β-cholestaneheptol glucuronide 651 651.3862 dihydroxy-cholanoic acid glycine and N-acetylglucosamine 659 659.3648 5β-cholestancheptol glucuronide 701 701.3689 dihydroxy-cholanoic acid taurine and N-acetylglucosamine
Example 2
(65) Using UPLC MS/MS, tetrol/T-CDCA and T-THCA/tetrol ratios were determined in approximately 10.000 blood spots of neonates. No positives and thus no false positives were observed. Cut off level was based on 5 confirmed CTX patients. Results are shown in
Example 3
(66) We performed a pilot with the flow-injection MS analysis which is also used for routine newborn screening. The official newborn screening laboratory in the Academisch Medisch Centrum performed the routine extraction of ⅛ inch punches using the currently used Neobase II kit on blood spots of 10 CTX patients (including 2 original newborn screening samples), 3 Zellweger patients and 200 controls (150 term and 50 preterm babies) (see Table below). Analysis of the extract was done in the Laboratory of Genetic Metabolic Diseases on a 11-year old Premier-XE mass spectrometer (Waters) that is technically less advanced compared the currently used mass spectrometers in modern newborn screening laboratories. This means that the current setup for neonatal screening will certainly be able to use this method. For our analysis we used routine flow-injection analysis and used Neolynx software to calculate the abundances of T-THCA (m/z 556.2), tetrol glucuronide (m/z 611.3) and T-CDCA (m/z 498.2) followed by the calculation of the ratio's. The results are very clear, the tetrol/T-CDCA and the T-THCA/tetrol ratio's clearly identify the CTX patients without overlap with the controls (term and preterm). Combining the two ratio's makes the identification of the CTX samples unambiguous and possible false positives (Zellweger patients can have elevated tetrol glucuronide) are avoided.
(67) TABLE-US-00005 Sample T-THCA/tetrol Tetrol/T-CDCA Original CTX neonatal screening card 0.001 1919.00 Original CTX neonatal screening card 0.001 121.45 CTX 0.003 351.00 CTX 0.003 332.00 CTX 0.003 63.00 CTX 0.003 62.40 CTX 0.004 264.00 CTX 0.005 2.92 CTX 0.007 2.31 CTX 0.032 3.00 Zellweger 1 4.5 0.33 Zellweger 2 15.3 0.09 Zellweger 3 17.2 0.08 Term (N = 150) 0.059-134 0-1.0 Preterm (N = 50) 0.080-124 0-0.21