RU2789156C2 - Method for screening cerebrotendon xanthomatosis using bile alcohol glucuronides and metabolite ratios - Google Patents

Abstract

FIELD: screening or diagnosing cerebrotendinous xanthomatosis (CTX).

SUBSTANCE: method for diagnosing or screening for 27-hydroxylase deficiency (CYP27A1) in an animal, comprising: a) determining the signal intensity in a biological sample, which is blood, serum or plasma, by mass spectrometric analysis of at least a bile alcohol glucuronide and a bile acid C27 or C24 or its conjugate, b) determining the ratio between the specified bile alcohol glucuronide and the specified bile acid C27 or C24 or its conjugate, c) comparison of said ratio with a reference value, which is the ratio of the mean, median or calculated threshold values of the intensities of the specified alcohol glucuronide and C27- or C24 bile acids or their conjugates, moreover, the control value is determined using control samples which are biological samples of a patient suffering from cerebrotendinous xanthomatosis (CTX), d) determination of 27-hydroxylase deficiency (CYP27A1) based on the specified comparison beyond which the subject would be considered to have a CYP27A1 deficiency. A kit for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency using the method described above, containing stable isotope labeled tetrol and stable isotope labeled C24 or C27 bile acid or its conjugate selected from the group consisting of cholic acid (CA), taurocholic acid (t-CA), glyco-cholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA), and glycodihydroxycholestanoic acid (g-DHCA).

EFFECT: above described method for diagnosing or screening for 27-hydroxylase deficiency (CYP27A1) in an animal is sensitive and specific for CTX detection at large scale screening, does not require a derivatization step.

13 cl, 6 dwg, 3 tab, 3 ex

Description

Scope of the invention

The concepts described herein relate to methods and kits related to the screening or diagnosis of cerebrotendon xanthomatosis in blood samples.

Prerequisites for the creation of the invention

Cerebrotendon xanthomatosis (CTX) is a rare autosomal recessive disorder caused by a deficiency of 27-sterol hydroxylase (encoded by the CYP27A1 gene), which plays a critical role in bile acid synthesis. CTX in adults is characterized by a progressive neurological phenotype. Symptoms during infancy and childhood include neonatal cholestasis, chronic diarrhea (may be a characteristic finding clinically soon after birth), bilateral cataracts, and developmental delay. After ten or twenty years, tendon xanthomas and neuropsychiatric symptoms appear, including pyramidal and cerebellar signs, peripheral neuropathy, and dementia (1, 2). A metabolic block in the bile acid synthesis pathway results in a deficiency of primary bile acids such as cholic acid (CA) and especially chenodeoxycholic acid (CDCA), in addition to accumulation of 7α-hydroxy-4-cholesten-3-one. 7α-hydroxy-4-cholesten-3-one is further converted to various metabolites, including cholestanol and characteristic bile alcohols. Cholestanol, together with cholesterol, accumulates in tissues. Although the pathophysiology of CTX is still poorly understood, cholestanol is believed to cause most of the observed pathological changes. Cholestanol has been shown to induce neuronal cell apoptosis and its accumulation is believed to be the cause of the observed neurodegeneration (3, 4). The development of symptoms and signs can be stopped or prevented by the addition of CDCA, which reduces bile acid synthesis through a feedback mechanism and, as a result, leads to inhibition of cholesterol 7α-hydroxylase, thus preventing the production and accumulation of cholestanol (5). The prognosis of CTX is good if therapy is started early, but less favorable if treatment is started later in life (6–9 years). Early initiation of therapy can completely prevent the development of a neurological phenotype, and patients are expected to be asymptomatic if treatment is started immediately after diagnosis in the neonatal period (7, 10). Using the allelic frequencies of pathogenic CTX variants in the ExAC database, it was calculated that the incidence of CTX in different ethnic groups ranges from 1 : 36072 to 1 : 263222 (11). This and other studies suggest that CTX may be underdiagnosed (11, 12). All of the above indicates that CTX is an excellent potential disease to screen for, especially in neonates. However, the detection of neonatal CTX patients is hampered by the lack of a suitable dry blood spot (DBS) neonatal screening method. DeBarber et al published a potential high-throughput method for screening CTX in newborns based on the quantitation of the ketosterol intermediate in DBS using the LC-ESI/MS/MS method (13, 14). A potential disadvantage is that a derivatization step is required prior to analysis, complicating the integration of neonatal screening into existing programs and increasing costs.

The aim of the present invention is to provide a screening diagnostic test that does not require a derivatization step and is sufficiently sensitive and specific to detect CTX at large screening scales.

Statement of the Invention

The present invention is based on the unexpected discovery that by determining the intensity of the mass spectral signals of bile alcohol glucuronide and C24 or C27 bile acid or its conjugate in a biological sample taken from a person, one can accurately determine whether that person suffers from 27-hydroxylase deficiency ( CYP27A1) using a method that does not require a derivatization step. Although bile alcohol glucuronides are known to be elevated in the plasma of CTX patients (15), concentrations of these metabolites vary between individuals. Moreover, the concentrations of these metabolites also deviate from normal levels in patients suffering from Zellweger's syndrome and in patients with cholestasis. Thus, it is not possible to predict whether mass spectral signal intensities will be informative regarding differentiation between CTX patients and normal controls or patients suffering from other diseases.

First, it was investigated whether the measurement of tetrol alone was sufficient to distinguish dry blood spots (DBS) of newborns (and adults) with CTX without treatment from DBS of control newborns. There is no stable isotopically labeled tetrol internal standard commercially available, and several commercially available compounds have been investigated as a potential internal standard, namely ² H ₄ -t-CDCA, ² H ₄ -g-CDCA, ² H ₄ -t-CA, ² H ₄ -g-XK and pregnandiol glucuronide (Fig. 2). The tetrol response in most patients was clearly elevated, but was not completely distinct from term/premature control neonates. As previously reported, patients with Zellweger's syndrome and patients with cholestatic liver disease also exhibited elevated concentrations of bile alcohols (16,17), and in this experiment, patients with Zellweger's syndrome could not be distinguished from those with CTX solely by measuring the tetrol response, regardless of used internal standard (Fig. 2).

The determination of the concentration of four bile acids (t-CA, g-CA, t-CDCA, r-CDCA) and the taurine conjugate of the bile acid intermediate trihydroxycholestanoic acid (t-THCA) was added to the protocol to determine whether the combination of bile alcohol glucuronide would allow and bile acid to distinguish patients with CTX from patients with Zellweger syndrome and patients with other causes of cholestatic liver disease. In FIG. 3 shows that these five metabolites can be measured in DBS using the appropriate internal standards labeled with a stable isotope. Bile acid and t-THCA concentrations in patients with CTX were at the lower end of the control range, and t-THCA clearly accumulated in patients with Zellweger's syndrome. Other combinations with other primary bile acid conjugates are shown in FIG. 4. It was possible to differentiate CTX patients from controls based on the ratio between the concentrations of bile alcohol glucuronide and C24 or C27 bile acid conjugates.

Thus, the invention provides a method for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency, comprising: a) determining the signal intensity in a biological sample by mass spectrometric analysis of at least a bile alcohol glucuronide and bile acids C24 or C27 or their conjugate, b a) comparing the intensity ratio of said signals with a control sample or control value; and c) determining a 27-hydroxylase (CYP27A1) deficiency based on said comparison.

Tetrol has been found to provide the best differentiation capability when plasma, blood or serum is used. Thus, in a preferred embodiment, said bile alcohol glucuronide comprises cholesteranetetrol glucuronide (tetrol).

Preferably said C24 or C27 bile acid or its conjugate is selected from: cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid acid (g-HDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCHA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCHA) . The advantage is that these compounds provide very good differentiating power in the method of the present invention.

Preferably, at least the signal intensity of the first and second compounds is determined, said first compound being selected from:

cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA) and glycochenodeoxycholic acid (g-CDCA),

and said second connection is selected from:

trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA). Using the intensity of the mass spectral signals of the specified first and specified compounds, at least the first ratio between the intensities of the mass spectral signals of the specified bile alcohol glucuronide and the intensities of the mass spectral signals of the specified first compound is determined, and the second ratio is determined between the intensities of the mass spectral signals of the specified bile glucuronide alcohol and the intensities of the mass spectral signals of the specified first compound. The advantage of using at least two ratios is that it results in fewer false positives.

The most preferred bile acids are t-CDCA and t-THCA because they have the most differentiating power when measured in combination with tetrol.

Additionally, the t-THCA/tetrol ratio was found to have a high specificity for CTX detection with respect to Zellweger's syndrome and cholestatic liver disease (see FIG. 1a). Indeed, the t-THCA/tetrol ratio allowed us to separate all CTX cases from the three cases of Zellweger syndrome and controls in the pilot study (Table 2A). Thus, in a preferred embodiment, the method includes determining the ratio of t-THCA/tetrol.

In addition, it was additionally found that in CTX, tetrol concentrations are high and t-CDCA concentrations are low, resulting in a marked increase in the tetrol/t-CDCA ratio in CTX DBS, while this ratio is expected to be (almost) normal in patients with Zellweger's syndrome or decrease in patients with cholestatic liver disease. This ratio showed excellent ability to separate all DBS CTX from DBS of all control subjects (Fig. 1B), including patients with hypercholanemia and Zellweger's syndrome. The control sample with the highest tetrol/t-CDCA ratio was 13.1 times lower than the lowest tetrol/t-CDCA ratio at CTX. This indicates that this ratio is an excellent potential biomarker for detecting CTX patients in DBS as part of newborn screening. Thus, in another preferred embodiment, the method includes determining the ratio of tetrol/t-CDCA.

In a preferred embodiment, said first and said second ratios are tetrol/t-CDCA and t-THCA/tetrol. The ratios t-CDCA/tetrol and tetrol/t-THCA are considered equivalent and are also included.

An additional advantage of these ratios is that they are different in cholestatic liver disease, further increasing the specificity for CTX. In FIG. 1 shows the scientific rationale for the two ratios (Fig. 1A), as well as the results of evaluating the ratio of metabolites in term (n = 150) and preterm (n = 50) controls, patients with Zellweger's syndrome (n = 3), and patients with CTX ( n = 14) (Fig. 1B and Table 2(A)).

Using the tetrol/t-CDCA ratio, 100% differentiation was achieved between CTX patients (in dried blood spots (DBS) of neonatal and older patients) and controls (term/premature) subjects and patients with Zellweger's syndrome. The t-THCA/tetrol ratio allowed separating DBS CTX from DBS patients with Zellweger syndrome and DBS control subjects. In addition, hypercholanemic controls were correctly separated from CTX patient samples using both the tetrol/t-CDCA ratio (hypercholanemic range: 0.000–0.018) and the t-THCA/tetrol ratio (hypercholanemic range: 2.5–311) , thereby increasing specificity for CTX by excluding subjects with hypercholanemia.

The DBS of a CTX patient (P9) with a tetrol/t-CDCA ratio at the lower end of the spectrum was used to test the accuracy of the metabolite(s) ratio method of the present invention. For the DBS assay, the inter-assay accuracy of the tetrol/t-CDCA ratio was acceptable at 14% (0.64±0.09, n=10). The inter-analytical accuracy of the t-THCA/tetrol ratio was higher (63%) due to the low concentration of t-THCA in the CTX sample, resulting in low t-THCA/tetrol values (0.07 ± 0.04, n = 10), but this change was small compared to the difference between the control group DBS and DBS CTX and therefore should not affect the CTX specificity of the method. Thus, in a preferred embodiment, the method includes determining the ratio between the signal intensities of tetrol and t-THCA in dry blood spot samples using mass spectrometric analysis.

Preferably said mass spectrometric analysis includes MS, MS-ESI, flow-injection MS.

In another preferred embodiment, the intensity of a compound labeled with a stable isotope selected from tetrol, a bile alcohol glucuronide, a C24 or C27 bile acid, or a conjugate thereof is determined, and said intensity of said stable isotope is compared with a specified signal intensity of said bile alcohol glucuronide and said bile acid C24 or C27 or its conjugate. The advantage is that the stable isotope can be used to accurately quantify the concentrations of said bile alcohol glucuronide and C24 or C27 bile acid or conjugate thereof.

The invention further provides a kit for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency, containing stable isotope labeled tetrol and stable isotope labeled C24 or C27 bile acid or its conjugate selected from the group consisting of cholic acid (CA), taurocholic acid ( t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA).

Preferably said kit further comprises a positive control sample, preferably a blood sample from a CTX patient.

Brief description of graphic materials

In FIG. 1 shows that the ratios increase the selectivity and specificity of the CTX screening assay. (A) Scientific rationale for the two selected ratios. Tetrol/t-HDCA ratio. With CTX, tetrol accumulates and CDCA decreases, leading to a strong increase in the ratio. In Zellweger's syndrome, tetrol is elevated, but CDCA remains at normal levels, resulting in a normal or slightly elevated ratio, while during cholestasis, CDCA (and CA) is elevated, keeping the ratio low. The ratio of t-TGCA/tetrol. In CTX, this ratio is significantly reduced, while in Zellweger's syndrome and cholestasis it is normal or increased. (B) Tetrol/t-CDCA and t-THCA/tetrol ratios in term/preterm controls (triangle), Zellweger syndrome patients (diamond), neonates with CTX, and untreated CTX patients (open/closed circle). The ratio of tetrol/t-CDCA has a high differentiating power, also for Zellweger's syndrome and CTX. The t-THCA/tetrol ratio also differentiates between Zellweger syndrome and CTX.

In FIG. 2 shows the response of tetrol in term/premature controls (triangle), Zellweger syndrome patients (diamond), newborns with CTX, and untreated CTX patients (open/closed circle) calculated using different internal standards: (A) pregnandiol glucuronide, (B) ² H ₄ -t-CA, (C) ² H ₄ -g-CA, (D) ² H ₄ -t-CDCA and (E) ² H ₄ -g-CDCA. Despite a clear rise in tetrol response for all internal standards used, the separation of controls and CTX patients was unsatisfactory. Patients with Zellweger's syndrome showed a significantly elevated tetrol response that overlapped with the CTX range.

In FIG. 3 shows the concentrations of bile acid and bile acid intermediate t-THCA in term/premature controls (triangles), Zellweger syndrome patients (diamond), CTX neonates, and untreated CTX patients (open/filled circle) calculated from using different internal standards: (A) t-QC, (B) g-QC, (C) t-TGCA, (D) t-CDCA, and (E) g-CDCA. Bile acid and t-THCA concentrations in CTX patients were at the lower end of the control range. As expected, t-THCA clearly accumulated in patients with Zellweger syndrome.

In FIG. 4 shows other studied metabolite ratios in term/premature controls (triangle), Zellweger syndrome patients (diamond), newborns with CTX, and untreated CTX patients (open/filled circle): (A) tetrol/t-CA, (B) tetrol/g-CA and (C) tetrol/g-CDCA. The ratio tetrol/t-CDCA has the best differentiating ability.

In FIG. 5 shows total bile acid concentrations in term/premature controls (triangle), Zellweger syndrome patients (diamond), newborns with CTX, and untreated CTX patients (open/filled circle).

In FIG. 6 shows 10,000 blood drops screened by HPLC-MS/MS analysis. Of note is the logarithmic scale for the tetrol/t-CDCA ratio, low results for screened blood spots (•), and a positive control from a patient with confirmed CTX (X).

Detailed description

Definitions

As used herein, the terms "diagnosing", "diagnosing", and "diagnosing" refer to attempts to determine or detect whether a subject is suffering from a given disease or condition or not. The term "diagnosis" does not refer to the ability to determine the presence or absence of a particular disease with 100% accuracy, or even to determine whether a given course or outcome is likely to occur. Instead, one of skill in the art will appreciate that the term "diagnosis" refers to an increased likelihood of a subject having a particular disease. As used herein, diagnostics also includes pre-diagnosis methods that can be confirmed by diagnostics using other methods known in the art. In addition, it is contemplated that the methods described herein can be used to monitor the progression of a condition and/or the effectiveness of the therapy used to treat the condition.

As used herein, the term "bile acid" includes steroid acids (and/or their carboxylate anion) and their salts present in animal (e.g., human) bile, 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.

The term "primary" bile acid refers to any of cholic acid (3α,7α,12α-trihydroxy-5β-cholanic acid) and chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholanic acid).

As used herein, the term "secondary bile acid" refers to any of the primary bile acids and their conjugates that have been modified by bacteria in the gut, including, without limitation, lithocholic acid and deoxycholic acid.

As used herein, the term "C24 bile acid" refers to any primary and secondary bile acid and its conjugates having 24 carbon atoms.

As used herein, the term "C27 bile acid" refers to any bile acid intermediate and its conjugate having 27 carbon atoms that can be formed during bile acid synthesis prior to and during peroxisomal beta-oxidation.

The term "bile acid conjugate" refers to any primary bile acid conjugated to the amino acids glycine and taurine.

The term "bile acid intermediate" refers to any metabolite between cholesterol and primary bile acids formed through normal or abnormal bile acid metabolism/synthesis.

As used herein, the term "bile alcohol glucuronide" refers to a bile alcohol glucuronic acid conjugate.

As used herein, the term "tetrol" refers to 5β-cholestan-3α,7α,12α,25-tetrol glucuronide and cholestantetrol glucuronide isomers. Preferably, tetrol is 5β-cholestan-3α,7α,12α,25-tetrol glucuronide.

As used herein, the term "control sample" refers to a biological sample or samples of a patient suffering from CTX, or other positive or negative control sample to determine the intensity of the control signals.

As used herein, the term “reference value” refers to a predetermined signal intensity or ratio of at least a bile alcohol glucuronide and a C27 or C24 bile acid or conjugate thereof and/or a predetermined threshold level.

Preferably, the control value refers to a control concentration of said bile alcohol glucuronide and C27 or C24 bile acid or conjugate thereof and/or a predetermined signal intensity level or signal intensity ratio between at least said bile alcohol glucuronide and C27 or C24 bile acid or conjugate thereof and /or a predefined threshold level. The reference value may, for example, be a reference signal intensity against which the signal intensities of the test sample are compared and/or a predetermined signal or signal intensity, such as a numerical value and/or a range (e.g., a reference range) corresponding to signal intensity levels. in such a sample or samples. For example, as shown herein, control samples with a known diagnosis (e.g., from patients suffering from CYP27A1 deficiency, Zellweger syndrome, cholestasis, or from healthy controls) can be used to determine a threshold level beyond which a subject will be considered to have CYP27A1 deficiency. The test samples are then compared to a predetermined value determined using control samples. The reference value may be the mean, median, or calculated intensity threshold (e.g., limit) for the specified bile alcohol glucuronide and C27 or C24 bile acid or conjugate and/or combination thereof (e.g., ratio, sum), out of range which will be regarded as the presence or absence of a certain condition in the subject, for example, CYP27A1 deficiency.

In one embodiment, the control value may be the ratio of the intensities of said bile alcohol glucuronide and C27 or C24 bile acid or conjugate thereof and the intensity of the mass spectral signal of one or more internal standardization markers in the control sample. The control ratio is compared with the corresponding ratio determined for the sample.

As used herein, the term "ratio" refers to the mathematical relationship between two quantities. For example, the ratio of A to B is the quotient of A value divided by B value or B value divided by A value. A/B and B/A ratios may be used interchangeably herein. Thus, where it is indicated herein that a ratio between the mass spectral signal intensities of a C24 bile acid and a C27 bile acid or its conjugate, said ratio contains the mass spectral signal intensity of said C24 bile acid divided by the mass spectral signal intensity of said bile acid C27, and the mass spectral signal intensity of said C27 bile acid divided by the mass spectral signal intensity of said C24 bile acid.

Embodiments

Based on the mass spectral signal intensities of accumulative cholestantetrol glucuronide (tetrol) and certain bile acids/bile acid intermediates, a new screening assay for CTX in DBS has been developed that is suitable for neonatal screening. With the present analysis, a good separation was achieved between the DBS of CTX patients and the DBS of control subjects, patients with Zellweger syndrome, and newborns with (potential) cholestasis. Initially, only tetrol was measured in untreated CTX patients. However, despite a fairly good separation between mean responses in controls and CTX patients, overlap was observed between patient and control responses (see FIG. 2). In addition, other disorders such as Zellweger's syndrome (16) and cholestatic liver disease (17) also lead to accumulation of tetrol or similar isobaric steroid alcohol glucuronides, which also occurred in the three DBS with Zellweger's syndrome in which elevated tetrol concentrations were found. . Thus, since this method lacks sensitivity and is not specific enough to detect only CTX patients, tetrol alone quantification is not suitable as a neonatal screening assay for CTX.

It was found that the combination of mass spectrum measurements of bile alcohol glucuronide and bile acid C24 or C27 or its conjugate provided good sensitivity and specificity.

Thus, the present invention provides a method for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency in an animal, comprising:

a) determining the signal intensity in the biological sample by mass spectrometric 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 its conjugate,

c) comparing the specified ratio with the control sample ratio or control value,

d) determination of 27-hydroxylase (CYP27A1) deficiency based on said comparison.

This method is suitable for diagnosing or screening animals, in particular mammals, and is preferably used in humans. Preferred embodiments, as described herein, include methods for diagnosing or screening newborns. However, the methods described herein are equally applicable to other populations and types of screening/diagnosis, such as children aged 1-3 years and adults.

The present concepts provide methods for determining the relative concentration of analytes selected from bile alcohol glucuronides and C24 or C27 bile acids or a conjugate thereof in one or more samples and provide methods by which relative or absolute concentration can be determined using mass spectrometry.

Preferably, ratios are used between the intensities of the mass spectral signals of bile alcohol glucuronides and bile acids C24 or C27 and their conjugates, more preferably between the intensities of the mass spectral signals of bile acid and t-CDCA and tetrol and the intermediate t-THCA and tetrol. In an alternative embodiment, a lookup table of results can be searched and used as input to a formula that includes other parameters, such as plasma cholestanol, or a clinical data scoring system.

Any biological sample that contains bile acid synthesis metabolites can be used in the method of the present invention. For example, urine and other body fluids are suitable. Preferably, said biological sample is blood, serum or plasma. In particular, it is preferable to use dried blood spots (DBS), as these samples are often used in newborn screening tests.

The most appropriate biological sample contains a quarter-inch (6.4 mm) DBS circle (or for most DBS with CTX, two eighth-inch DBS circles, each containing half of the material found in the quarter-inch DBS), while most neonatal screening labs prefer the one-eighth inch (3.2 mm) DBS beaker. Modern triple quadrupole tandem mass spectrometers will readily detect the three metabolites used in this assay in an eighth inch diameter DBS mug prepared with a perforator. In addition, the present invention also shows that labs with older machines can still screen CTX with the present method using a quarter inch DBS circle (or two eighth inch DBS).

In a preferred embodiment, said biological sample is a DBS no larger than 1/4 inch, 1/8 inch.

The biological sample can be subjected to a chromatographic separation, for example by means of an LC such as HPLC followed by a mass spectrometric analysis.

The method may also include the step of extracting the analyte by liquid-liquid extraction, solid-liquid extraction, or protein precipitation using hydrophobic solvents such as methanol prior to the mass spectrometric analysis step.

In some preferred embodiments, the mass spectrometer can be used in tandem MS mode with multiple reaction monitoring (MRM) to detect analyte transitions such as, preferably, but not limited to, 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 is preferably used to calculate the concentration or relative abundance of these metabolites.

In some preferred embodiments, a high resolution mass spectrometer is used that allows accurate mass measurement in full scan mode to directly determine the relative abundance of the desired ions with an accurate mass. The exact masses of ions listed in Table 3 can be used in the assay to detect bile acid metabolites, but should not be limited to them. In these embodiments, and in other preferred embodiments, a stable isotope labeled internal standard corresponding to the unlabeled ion is preferably used to calculate the concentration or relative abundance of these metabolites.

In a preferred embodiment, the relative content of each transition (or, in the case of a high resolution mass spectrometer, the accurate mass spectrum) during sample elution/infusion can be averaged and then corrected for background signal by subtracting the average intensity before or after elution/ sample infusion. This allows you to get the intensity / relative content of the selected analytes.

The intensity/relative abundance of analytes can then be used to calculate metabolite ratios. In these embodiments, and in other preferred embodiments, when special (labeled with a stable isotope) internal standards are used, these internal standards can be used to correct for matrix effects, after which the resulting response can be used for metabolite calculations.

In preferred embodiments, the fragment collision energy is chosen to be in the range of 35-60 eV.

In a preferred embodiment, flow injection mass spectrometry (FIA) is used. The advantage of this method is that the FIA equipment is standard equipment for newborn screening.

Quantification can be performed by relative or absolute measurement of the signal obtained from one or more analytes and standards. A negative charge can be imparted to an analyte that functions as a fragment ion detectable by mass spectrometry.

In a preferred embodiment, an internal standard solution is added to the sample containing the analyte. Preferably, said solution contains one or more of pregnanediol glucuronide labeled with a stable isotope ( ¹³ C, ² H, etc.) of tetrol glucuronide, ² H ₄ -t-CDCA, ² H ₄ -g-CA, ² H ₄ -g-HDCA, preferably dissolved in methanol.

In some preferred embodiments, Parent-to-Daughter Transition Monitoring, or PDITM, is used as a method of analyzing and evaluating the status of a workflow. PDITM refers to a technique whereby the mass-to-charge ratio (m/z) transfer range of the first mass separator (often referred to as "MS" or the first level of mass spectrometry) is specifically selected to transfer a molecular ion (often referred to as the "parent ion" or “precursor ion”) to an ion fragmenter (e.g., collision cell, photodissociation region, etc.) to produce fragment ions (often referred to as “daughter ions”), and the transferred m/z range of the second mass separator (often referred to as "MS/MS" or second level mass spectrometry) is selected to transmit one or more progeny ions to a detector that measures the progeny ion signal. This technique has unique advantages when the detection of progeny ions in a spectrum is focused by "pinning" the detector to the estimated mass of the progeny ion. The combination of monitored masses of the parent ion and the child ion is referred to as "parent-to-daughter transition" monitoring. The daughter ion signal at the detector for a given combination of parent ion and child ion is referred to as the "parent-to-daughter transition signal". Parent ion and child ion combinations used in the method described herein are shown in Table 2.

In one embodiment, parent-to-daughter ion transition monitoring is multiple response monitoring (MRM) (also referred to as selective response monitoring). In various MRM embodiments, monitoring a given parent-to-daughter ion transition includes using a first mass separator (e.g., a first quadrupole pinned to the parent ion m/z of interest) to transfer the parent ion of interest and using a second mass separator (e.g., a second quadrupole pinned to the parent ion m/z of interest) to transfer one or more daughter ions of interest. In various embodiments, the PDITM may be performed using a first mass separator (e.g., a quadrupole pinned to the m/z ratio of the parent ion) to transfer the parent ions and scan the second mass separator over an m/z range including the m/z value of one or more interesting daughter ions.

In some preferred embodiments, a tandem mass spectrometer (MS/MS) or, more broadly, a multidimensional mass spectrometer is used to perform PDITM, such as MRM. Examples of suitable mass analyzer systems include, without limitation, systems that comprise one or more of a triple quadrupole, a quadrupole linear ion trap, a quadrupole TOF detector and a TOF-TOF detector, an ion trap, an orbittrap, a quadrupole ion trap, a quadrupole orbittrap .

In one embodiment, a step is used in which at least a portion of the composite sample is subjected to PDITM, and this step includes introducing the composite sample directly into the mass analyzer system, for example, by introducing the composite sample into a suitable solution using an electrospray ionization source (ESI) or atmospheric pressure ionization source (APCI).

According to various preferred embodiments, samples containing one or more compounds may be enriched in various ways prior to analysis. The enrichment method may depend on the type of sample, eg blood (fresh or dried), plasma, serum, etc. Examples of enrichment methods may include, 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.

Sample handling in the present method is straightforward as no derivatization is required, and since standard methanol extraction is used, the assay can be appropriately combined with other neonatal screening assays. Thus, in a preferred embodiment, this method is combined with other newborn screening assays. Flow injection is preferably used for the measurement, since only a few minutes are required for one analysis.

Preferably, the method includes the step of measuring specific conversions for cholestantetrol glucuronide, taurochenodeoxycholic acid (t-CDCA) and taurotrihydroxycholestanic acid (t-THCA).

The invention further provides a kit for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency, containing stable isotope labeled tetrol and stable isotope labeled C24 or C27 bile acid or its conjugate selected from the group consisting of cholic acid (CA), taurocholic acid ( t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA). One skilled in the art knows how to synthesize stable isotope labeled bile acid. Methods for the synthesis of tetrol are known in the art (19).

Preferably, the kit further contains a positive control sample, preferably a blood sample from a CTX patient.

In a further aspect, individuals diagnosed with 27-hydroxylase (CYP27A1) deficiency, preferably in the neonatal period, can be effectively treated using the method of the present invention by oral administration of a chenodeoxycholic acid (CDCA) dietary supplement, thereby preventing the development of any symptoms associated with cerebrotendon xanthomatosis. (CTX, 27-hydroxylase deficiency (CYP27A1)). In a preferred embodiment, the CDCA dietary supplement is administered at 5 mg/kg/day divided into three equal doses. In another preferred embodiment, the CDCA dietary supplement is administered at 15 mg/kg/day divided into three equal doses. This mode is preferred for children. In another preferred embodiment, the CDCA dietary supplement is administered in an amount of 250 mg three times a day. This regimen is preferred for adult patients.

Although the description above provides examples and specific information about various embodiments, it should be understood that some of the features and/or functions of the described embodiments can be modified without deviating from the scope of the described embodiments. The foregoing description is intended to illustrate the concepts set forth herein, the scope of which is limited only by the text of the claims appended hereto.

Example

Patient samples and control samples

Anonymized DBS from 150 term (mean gestational age (range): 39.9 (37.0–42.0) weeks) and 50 preterm infants (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 provided informed consent for the anonymous use of DBS from their child's Guthrie card at the time of collection (standard procedure in the Dutch newborn screening program; all Guthrie newborn screening cards in the Netherlands are kept for five years before being destroyed). Fifteen DBS, including 2 fresh neonatal DBS, were collected from 14 individual CTX patients without treatment. These included DBS from participants enrolled in Institutional Review Board-approved studies at Oregon Medical University. Participants provided written informed consent for the use of DBS. De-identified diagnostic samples submitted to the sterol laboratory for biochemical confirmation of CTX were also used with permission from the Institutional Review Board. Table 1 lists all relevant information regarding CTX patients and their DBS. All patients or their parents/legal representatives provided written informed consent for the use of their DBS/their children's DBS. Three anonymized DBS from patients with Zellweger syndrome were obtained from the biobank at the Academic Medical Center (Amsterdam).

materials

Solvents: methanol and acetonitrile were purchased from Biosolve. ² H ₄ -g-CDCA, ² H ₄ -g-CA, ² H ₄ -t-CDCA and ² H ₄ -t-CA were purchased from CDN Isotopes. H ₂ O was MilliQ® purified water. 5β-pregnan-3α,20α-diol glucuronide (pregnandiol glucuronide) was purchased from Sigma-Aldrich and Whatman 903 filter paper from Drukkerij PAL.

Getting a Sample

A perforated circle (quarter-inch (6.4 mm) diameter) DBS was used for all samples except DBS with CTX numbers 1-8, 12, and 13, for which two 3.2-mm DBS were used (in these cases, the result corrected by multiplying the result by two). DBS was transferred to a 1.5 ml tube, to which 500 μl of methanol was added and incubated for 30 min at room temperature in an ultrasonic bath (Branson 3510). The filter paper was removed, and the extractant was transferred to a 4 ml glass tube, followed by the addition of 20 μl of the internal standard mixture [1 μM pregnanediol glucuronide, 0.25 μM ² H ₄ -t-QA, 0.25 μM ² H ₄ -t-CDCA , 0.25 μM ² H ₄ -g-QC, 0.25 μM ² H ₄ -g-CDCA, dissolved in methanol/H ₂ O (3:1 v/v)]. The internal standard solution was intentionally not added during the sonication step because this solution contains water which extracts unwanted salts/interfering compounds that affect the MS measurement. After mixing in a vortex mixer, the sample was brought to dryness in a stream of nitrogen at 40°C. The residue was diluted in 120 μl of methanol/H ₂ O (3 : 1 v/v), transferred to a sample vial and capped.

Flow Injection MS/MS

A Waters Premier-XE tandem mass spectrometer (MS/MS) (Waters Cooperation) operating in electrospray ionization mode was injected with 10 μl using acetonitrile/H ₂ O (9 : 1, v/v) as the mobile phase. Nitrogen was used as the spraying and desolvating gas. The collision gas was argon and the cell pressure was 2.7 * 10 ^-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 from a specific precursor ion to a fragment ion for each analyte. The transitions, cone stresses, and collision energies found for each compound are shown in Table 2. The analytical study time (from injection to injection) was 2 minutes. Quantification was performed using Masslynx 4.2 and NeoLynx™ software. NeoLynx pooled and averaged the individual MRM transition intensities within the flow-injection profile and then corrected for background signal by subtracting the average intensity before sample infusion. The obtained values of the peak height of the measured compounds were exported for further calculations in MS Excel (Microsoft).

Table 1 Information on DBS and CTX Patients

DBS Patient ^a Age of DBS (years) Storage conditions Patient age at sampling (years) Plasma cholesta-nol (mg/dl) Cholesta-nol in plasma (µM) Diagnosis ^b Phenotype at the time of diagnosis 1 P1 0.5 9 1.62 42 G/B unknown 2 P2 0.5 12 2.57 66 G/B unknown 3 P3 4 17 1.84 47 B Neonatal jaundice, chronic diarrhea, xanthomas 4 P4 9 16 3.23 83 G/B Juvenile cataract, developmental delay, autistic behavior, cognitive impairment 5 P5 4 31 1.65 42 G/B Xanthomas, spastic gait, paraparesis 6 P6 0.17 45 3.44 88 B Cognitive decline, mental disorder, dementia 7 P7 0.17 46 4.79 123 B Depression, paraparesis, cerebellar ataxia, dystonia 8 P8 7.5 36 4.52 116 G/B Cataract, xanthoma, seizures, chronic diarrhea 9 P9 0.5 6.2 1.52 39 G Delayed speech development, altered bowel habits 10 P10 0.1 9 2.06 53 B Cataracts eleven P11 0.5 eleven 4.52 116 G Cataracts, developmental delay, autistic behavior, altered bowel habits 12 P12 6 56 0.84 22 G/B cataracts, xanthomas 13 NB1 16 Unknown, last 8 years kept under 0.01 - - G/B Juvenile cataracts, xanthomas, chronic diarrhea 14 NB2 4 0.01 0.86 22 G None, see (10)

^a NB: fresh DBS from newborn screening

^b G: genetic, B: biochemical

Table 2. Transitions, Cone Stresses and Collision Energies for Measured Joints

Compound MRM
(m/z) Cone voltage (V) collision energy
(eV) t-HDHC 498→80 90 60 ² H ₄ -t-HDHC 502→80 90 60 t-hk 514→80 90 60 ² H ₄ -t-hk 518→80 90 60 t-TGCC 556→80 90 60 Mr. HDHC 448→74 60 40 ² H ₄ -g-HDHC 452→74 60→ 40 Mr. HK 464→74 60 40 ² H ₄ -g-hk 468→74 60 40 Pregnandiol glucuronide 495→75 45 35 cholestantetrol glucuronide 611→75 80 48

Table 2(A). Table of individual tetrol/t-CDCA and t-THCA/tetrol ratios for CTX patients and ranges for controls (term/preterm) and patients with Zellweger syndrome

DBS Patient Tetrol/t-HDHC t-TGHC/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 eleven P11 21 0.026 12 P12 25 0.023 13 NB1 22 0.047 14 NB2 138 0.004 Full-term (range) 0.000–0.057 0.19–250 premature (range) 0.000–0.061 0.09–377 Zellweger syndrome (range) 0.023–0.101 10.5–38.0

Table 3. Overview of Selected (Exact) Masses of Bile Acids, Bile Alcohols, and Bile Acid Intermediates

Rated weight Accurate weight Connection name 375 375.2905 3β-hydroxy-5-cholanic acid (lithocholic acid) 389 389.2697 dihydroxycholenic acid 391 391.2885 dihydroxycholanic acids (eg chenodeoxycholic acid) 405 405.2646 trihydroxycholenic acids, 3β,7α,12α-trihydroxy-5-cholenic acid 407 407.2803 trihydroxycholanic acids (eg cholic acid) 431 431.3169 3β,7α-dihydroxycholesthenic acid 432 432.3119 3β-hydroxy-5-cholanic acid (lithocholic acid) glycine 433 433.3323 dihydroxycholestanic acid (e.g. DHCA) 444 444.2755 7α-hydroxy-3-oxo-4-cholenic acid glycine 446 446.2912 3β,7α-dihydroxy-5-cholenic acid glycine 447 447.3116 3β,7α,12α-trihydroxycholesthenic acid 448 448.3069 dihydroxycholanic acids (eg chenodeoxycholic acid, deoxycholic acid) glycine 449 449.3273 trihydroxycholestanic acid (eg, THCA) 453 453.2316 3β-hydroxy-5-cholenic acid sulfate 460 460.2705 7α,12α-dihydroxy-3-oxo-4-cholenic acid glycine 462 462.2861 3β,7α,12α-trihydroxy-5-cholenic acid glycine 464 464.3018 trihydroxycholanic acids glycine (eg cholic acid) 465 465.3222 tetrahydroxycholestanic acid (e.g. OH-THCA, varanoic acid) 465 465.3044 cholesterol sulfate 469 469.2265 3β,7α-dihydroxy-5-cholenic acid sulfate 471 471.2422 dihydroxycholanic acid sulfate 472 472.3432 3β-hydroxy-5-cholestanoic acid glycine 480 480.2789 3β-hydroxy-5-cholenic acid taurine 480 480.2967 tetrahydroxycholanic acids glycine 482 482.2946 3β-hydroxy-5-cholanic acid (lithocholic acid) taurine 485 485.2215 3β,7α,12α-trihydroxy-5-cholenic acid sulfate 487 487.2371 trihydroxycholanic acid sulfate 488 488.3381 3β,7α-dihydroxycholesthenic acid glycine 490 490.3538 dihydroxycholestanic acid (e.g. DHCA) glycine 494 494.2582 7α-hydroxy-3-oxo-4-cholenic acid taurine 498 498.2895 dihydroxycholanic acids (chenodeoxycholic acid) taurine 506 506.3487 trihydroxycholestanic acid (e.g. THCA) glycine 507 507.3327 C ₂₉ - dicarboxylic acid 510 510.2531 7α,12α-dihydroxy-3-oxo-4-cholenic acid taurine 510 510.2531 3β-hydroxy-5-cholenic acid glycine and sulfate 514 514.2844 trihydroxycholanic acids (cholic acid) taurine 522 522.3259 3β-hydroxy-5-cholestanoic acid taurine 526 526.2480 3β,7α-dihydroxy-5-cholenic acid glycine and sulfate 528 528.2637 dihydroxycholanic acids (eg chenodeoxycholic acid) glycine and sulfate 530 530.2793 tetrahydroxycholanic acids taurine 540 540.3364 dihydroxycholestanic acid (e.g. DHCA) taurine 542 542.2429 3β,7α,12α-trihydroxy-5-cholenic acid glycine and sulfate 552 552.3000 7α,12α-dihydroxy-3-oxo-4-cholestanoic acid taurine 554 554.3157 trihydroxycholesthenic acid (e.g. THCA:1) taurine 556 556.3313 trihydroxycholestanic acid (e.g. THCA) taurine 567 567.3175 dihydroxycholanic acids glucuronide 570 570.3106 tetrahydroxycholestenic acids (e.g. OH-THCA:1) taurine 572 572.3263 tetrahydroxycholestanic acids (e.g. OH-THCA) taurine 583 583.3124 trihydroxycholanic acid glucuronide 586 586.3055 pentahydroxycholestenic acids (e.g. diOH-THCA:1) taurine 588 588.3212 pentahydroxycholestanic acid taurine 609 609.3644 5β-cholestantriol-?-one glucuronide, 5β-cholestentetrol glucuronide 611 611.3801 5β-cholestantetrol glucuronide 611 611.3437 tetrahydroxy-27-nor-5β-cholestan-24-one glucuronide 613 613.3593 27-nor-5β-cholestanpentol glucuronide 625 625.3593 tetrahydroxy-5β-cholestan-?-one glucuronide, 5β-cholestenpentol glucuronide 627 627.3750 5β-cholestanpentol glucuronide 627 627.3386 pentahydroxy-27-nor-5β-cholestan-24-one glucuronide 629 629.3543 27-nor-5β-cholestanehexol glucuronide 641 641.3543 5β-cholestenpentol-?-one glucuronide, 5β-cholestenhexol glucuronide 643 643.3699 5β-cholestanehexol glucuronide 643 643.3335 hexahydroxy-27-nor-5β-cholestan-24-one glucuronide 645 645.3492 27-nor-5β-cholestangeptol glucuronide 651 651.3862 dihydroxycholanic acid glycine and N-acetylglucosamine 659 659.3648 5β-cholestangeptol glucuronide 701 701.3689 dihydroxycholanic acid taurine and N-acetylglucosamine

Example 2

Using UHPLC-MS/MS, the ratios of tetrol/t-CDCA and t-THCA/tetrol were determined in approximately 10,000 drops of newborn blood. There were no positive and therefore false positive reactions. Cut-off level was based on 5 patients with confirmed CTX. The results are shown in FIG. 6.

Example 3

A pilot study was conducted using flow-injection MS analysis, which is also used for standard newborn screening. The Official Newborn Screening Laboratory at the Academic Medical Center (Academisch Medisch Centrum) performed routine extraction of 1/8 inch circles using the currently used Neobase II kit from the blood drops of 10 CTX patients (including 2 fresh samples from newborn screening), 3 patients with Zellweger syndrome and 200 controls (150 term and 50 preterm infants) (see table below). Analysis of the recovered samples was performed in the Genetic Metabolic Diseases Laboratory on an 11 year old Premier-XE mass spectrometer (Waters), which was technically less advanced than the mass spectrometers currently used in modern neonatal screening laboratories. This means that this method can certainly be used with the current neonatal screening system. For analysis, standard flow-injection analysis and Neolynx software were used to calculate the relative content of t-THCA (m/z 556.2), tetralglucuronide (m/z 611.3) and t-CDCA (m/z 498.2) with subsequent calculation of the ratios. The results are very clear, with tetrol/t-CDCA and t-THCA/tetrol ratios clearly identifying CTX patients with no overlap with controls (term and preterm). The combination of these two ratios makes it possible to unambiguously identify CTX samples and avoid possible false positive results (tetrolglucuronide may be elevated in patients with Zellweger syndrome).

Try t-TGHC/tetrol Tetrol/t-HDHC New CTX neonatal screening card 0.001 1919.00 New 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 Syndrome 1 4.5 0.33 Zellweger Syndrome 2 15.3 0.09 Zellweger Syndrome 3 17.2 0.08 Term (N = 150) 0.059–134 0–1.0 Premature (N = 50) 0.080–124 0–0.21

Links

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Claims (22)

1. A method for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency in an animal, comprising: a) determining the signal intensity in a biological sample, which is blood, serum or plasma, by mass spectrometric analysis of at least a bile alcohol glucuronide and a C27 or C24 bile acid or its conjugate, b) determining the ratio between said bile alcohol glucuronide and said C24 or C27 bile acid or its conjugate, c) comparing said ratio with a control value which is the ratio of the mean, median or calculated threshold intensities of said alcohol glucuronide and C27 or C24 bile acids or their conjugates, the control value being determined using control samples which are biological samples of the patient suffering from cerebrotendon xanthomatosis (CTX), d) determining a 27-hydroxylase (CYP27A1) deficiency based on said comparison, beyond which the subject is considered to be CYP27A1 deficient. 2. The method of claim 1 wherein said bile alcohol glucuronide is cholestantetrol glucuronide (tetrol). 3. The method according to claim 1 or 2, wherein said C27 or C24 bile acid or its conjugate is selected from: cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA ), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (r-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t -DHCA) and glycodihydroxycholestanoic acid (g-DHCA). 4. The method according to any one of paragraphs. 1-3, which further comprises determining the intensity of a compound labeled with a stable isotope selected from: tetrol, C24 or C27 bile acid, or a conjugate thereof, and said intensity of said compound labeled with a stable isotope is compared with the specified intensity of said bile alcohol glucuronide and said bile acid C24 or C27 or its conjugate. 5. The method according to any one of paragraphs. 1-4, in which at least the signal intensity of the first and second connections is determined, and the specified first connection is selected from: cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA) and glycochenodeoxycholic acid (g-CDCA), and said second connection is selected from: trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA). 6. The method according to any one of paragraphs. 1-5, wherein said mass spectrometric analysis includes MS, high resolution MS, LC-MS, MALDI-TOF, Q-TOF, Orbitrap-MS, flow-injection MS. 7. The method according to any one of paragraphs. 1-6, in which said animal is a human. 8. The method according to any one of paragraphs. 1-7, in which said animal is a newborn. 9. The method according to any one of paragraphs. 1-8, which further comprises measuring the intensity of said bile alcohol glucuronide or C24 or C27 bile acid or conjugate thereof relative to the intensity of the isotopically labeled standard. 10. The method of claim 9 wherein said isotopically labeled standard comprises stable isotope labeled tetrol or stable isotope labeled C24 or C27 bile acid or a conjugate thereof selected from the group consisting of: cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA). 11. The method of claim. 10, including the stage of ionization of the analyte using a voltage on the cone in the range from 60 to 90 V. 12. A kit for diagnosing or screening for 27-hydroxylase (CYP27A1) deficiency using the method of claim 1, comprising stable isotope labeled tetrol and stable isotope labeled C24 or C27 bile acid or a conjugate thereof selected from the group consisting of: cholic acid (CA), taurocholic acid (t-CA), glycocholic acid (g-CA), chenodeoxycholic acid (CDCA), taurochenodeoxycholic acid (t-CDCA), glycochenodeoxycholic acid (g-CDCA), trihydroxycholestanoic acid (THCA), taurotrihydroxycholestanoic acid (t-THCA), glycotrihydroxycholestanoic acid (g-THCA), dihydroxycholestanoic acid (DHCA), taurodihydroxycholestanoic acid (t-DHCA) and glycodihydroxycholestanoic acid (g-DHCA). 13. The kit of claim 12, further comprising a positive control sample, preferably a blood sample from a patient with cerebrotendon xanthomatosis (CTX).

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