US20050048499A1

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Publication number: US20050048499A1
Application number: US10/652,732
Authority: US
Inventors: Blas Cerda
Original assignee: PerkinElmer Life Sciences Inc
Current assignee: Revvity Health Sciences Inc
Priority date: 2003-08-29
Filing date: 2003-08-29
Publication date: 2005-03-03
Also published as: US8318417B2; EP1664325B1; US8399186B2; CA2537034C; EP1664325A1; JP2007503818A; CA2537034A1; EP1664325A4; AU2004269410B2; US20060234326A1; US20090111137A1; JP4838129B2; WO2005021779A1; US20120122131A1; AU2004269410A1; JP2011167208A; ES2527306T3

Abstract

A method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry is provided. The method comprises the steps of: (1) placing a blood spot from said newborn into a microtiter plate well; (2) adding a solution of polar solvent to said well so as to reconstitute enzymes in said blood spot and so as to extract polar analytes, thereby forming a sample; (3) incubating said sample for a predetermined period of time, so as to extract metabolites and allow enzyme activity to take place; (4) adding a second solution to said sample so as to stop enzymatic reactions and extract remaining analytes; (5) mixing said sample for a predetermined period of time; (6) measuring said sample by tandem mass spectrometry so as to identify analyte concentration and enzyme activity; and (7) using said analyte concentration and said enzyme activity to determine whether said newborn is affected by any inborn errors of metabolism. The solution of polar solvent may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, a substrate for at least one enzyme, and a buffer. The second solution may contain at least one of the following: an internal standard for a polar analyte, an internal standard for a non-polar analyte, an internal standard for an enzymatic product, an internal standard for at least one substrate, and a buffer.

Description

TECHNICAL FIELD OF THE INVENTION

The present invention is in the field of spectroscopy. Specifically, the present invention is related to methods for use with mass spectrometry.

BACKGROUND OF THE INVENTION

Inborn errors of metabolism (IEM) are genetic disorders in which key enzymes necessary for the completion of metabolic pathways are inactive or lacking. Thus affected individuals will either accumulate certain metabolites to toxic levels and/or lack enzymatic products required for the completion of essential metabolic pathways. The effects of inborn errors of metabolism range from mental retardation to premature death.^1-3.

As shown inscheme 1, the lack or inactivity of key enzymes due to IEM can result in the accumulation of precursor metabolites, the accumulation of secondary product metabolites, and/or the lack of primary product metabolites. Because the overriding result of genetic mutations in IEM is the lack or inactivity of essential enzymes, there are at least two possible ways of detecting these disorders. The first is to measure the amounts of marker metabolites, i.e. measure the accumulation of the precursor or alternate product metabolites and/or measure the decrease of the primary product metabolite. The second possibility is to measure the enzyme activity of the unknown patient sample.
The first approach is generally performed by tandem mass spectrometry (MS/MS) methods. With these MS/MS methods, a small disc from a patient sample in the form of a blood spot dried on filter paper is punched into a microtiter plate well. A solvent containing a set of internal standards is added to each well containing a blood spot punch to extract the marker metabolites from the sample. The solution containing the extracted metabolites and internal standards is then transferred to a new microtiter plate and the solution is dried under heated nitrogen. To the dried sample, a new solution containing acid and butanol is added to create butyl ester derivatives of the extracted metabolites. The derivatized sample is then dried under heated nitrogen again to remove the excess derivatizing agent and reconstituted with a third solvent type. Finally, the concentration of the desired metabolites is then measured by isotope dilution MS/MS. This approach is used for measuring the concentrations of acylcarnitines and amino acids (such as shown in U.S. Pat. Nos. 6,258,605 B1 and 6,455,321 B1 to Chace; hereby incorporated herein by reference). Below is a full protocol for such MS/MS method for the analysis of amino acids and acylcarnitines:

The second possibility, to measure enzyme activity, is generally performed by punching a small disc of a blood spot sample into a microtiter well. Subsequently, a solution containing buffers is added to reconstitute an enzyme of interest. Once the enzyme is reconstituted, a second solution containing a fluorescent analog to the natural substrate is added and the sample is incubated for a predetermined amount of time to allow the reconstituted enzyme to act on the artificial substrate. Once sufficient time has elapsed, a third type of solvent is added to stop the reaction (precipitate.the enzyme). The plate containing the sample is then placed in a fluorometer and the fluorescence of the artificial product is measured. The fluorescence can then be correlated to a particular level of enzyme activity.^4-5
The advantages the MS/MS approach are that it allows for the simultaneous analysis of several metabolite markers (acylcarnitines and amino acids) from a single sample and it alleviates environmental factors that affect enzyme activity such as exposure to light, temperature ranges, and humidity. The disadvantages are that the sample preparation is lengthy and complicated as well as that it employs harmful solvents such as butanol in HCI that can be very deleterious to personnel and equipment. Additionally and more importantly, is the fact that certain IEM cannot be screened for by MS/MS because the marker metabolites have many natural isomers also present in blood that prevent the measurement of their concentration. A case in point is galactose metabolism in which the measurement of the marker galactose-1-phosphate is impeded because several other phosphorylated sugars present in blood such as glucose-1-phospahate are isomeric with galactose-1-phosphate. Further, in many IEM, the levels of potential metabolite markers are too low in blood to be measured effectively by MS/MS at a stage in which the affected individual still does not show any symptoms. In these cases MS/MS would only be able to detect the marker metabolites at a clinical stage that may be too late to administer effective preventive treatment.
Measuring enzyme activity by fluorometry alleviates some of the disadvantages suffered by MS/MS. In case of isomeric interferences, there is no better discriminator of isomers than the natural enzymes. Enzymes have great specificity for their natural substrates such that isomeric interferences are generally not an issue. Additionally, in cases in which the levels of markers are too low to be measured directly by MS/MS, one can reconstitute the enzyme of interest and add enough artificial substrate to drive the reaction and be able to discriminate patients with normal versus decreased enzyme activity. The disadvantages of fluorometric methods are that one has to employ artificial substrates that are tagged with a fluorescent group. Because of this, the substrates are different enough from their natural counter parts that enzyme activity using such substrates is inherently reduced and thus the sensitivity of the assays. Additionally, because one estimates enzyme activity by measuring fluorescence it is not practical to fully take advantage of multiplex analysis. In some cases, tags that would fluoresce at different wavelengths can be used, but instruments capable of reading multiple fluorescent labels can only handle a few of such markers. Because of this, most enzyme activity assays employing some type of light emission for measurement are single analyte assays.
There is need for improvements in the current methodologies used in clinical newborn screening laboratories. With the current technologies, these laboratories are forced to master several different technologies to provide comprehensive testing. Additionally, as more and more analytes are added, the mainly single analyte-single test format available requires that the patients donate more and more blood for testing (a traumatic experience for the patient). Further, laboratories are forced to spend more and more time and resources when new analytes are added because the technologies available do not effectively allow for multiplexing and expansion. Therefore, it is desirable to have access to technology and methodology that can consolidate the two clinical methods described above.

SUMMARY OF THE INVENTION

The present invention provides methods for simultaneous measurement of enzyme activity and relatively polar and/or relatively non-polar analyte concentration, thereby overcoming the deficiencies of prior art methodologies. More specifically, the present invention does not require sample derivatization or substrate tagging with fluorescent groups. The present invention measurement of enzyme activity and metabolite elution may be performed in the same mixture, followed by tandem mass spectrometry assay.

In general terms, the present invention is a method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry, the method comprising the steps of: (a) obtaining a dried blood sample containing at least one enzyme, at least one relatively polar metabolic analyte, and at least one relatively non-polar metabolic analyte; (b) adding a solution of a relatively polar solvent to the dried blood sample so as to reconstitute the at least one enzyme in the dried blood sample and so as to extract the at least one relatively polar analyte therefrom, thereby forming a first solution; (c) incubating the first solution with a substrate for the at least one enzyme so as to create an enzymatic product; (d) adding a solution of a relatively non-polar solvent (this solution may contain acid at the required concentration to precipitate the enzyme) to the first solution so as to stop the reaction of the at least one enzyme and so as to extract the at least one relatively non-polar analyte, so as to form a second solution; (e) measuring the second solution by tandem mass spectrometry so as to assay the presence or concentration of the at least one relatively polar analyte, the enzymatic product, and the at least one relatively non-polar analyte.

The solution of a relatively polar solvent additionally may also comprise at least one component selected from the group consisting of: an internal standard for the at least one relatively polar analyte, an internal standard(s) for the enzymatic product(s) to be measured, an internal standard for the at least one relatively non-polar analyte, one or more substrates for the enzyme(s), and a buffer. These internal standard components may be labeled.

The solution of a relatively non-polar solvent may additionally comprise at least one component selected from the group consisting of: an internal standard for at least one relatively polar analyte, an internal standard(s) for the enzymatic product(s) to be measured, an internal standard for at least one relatively non-polar analyte, and a buffer. These internal standard components may be labeled.

The present invention may be applied to a method for genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry. The method comprises placing a blood spot from the newborn into a microtiter plate well. A solution of a polar solvent is then added to the well so as to reconstitute enzymes in the blood spot and to extract polar analytes, thereby forming a sample. The sample is then incubated for a predetermined period of time to extract metabolites and to allow substrate to product conversion by the reconstituted enzyme(s). A second solution is then added to the sample to stop enzymatic reactions. At this point the sample is mixed for a predetermined period of time (optional step) to allow additional metabolite extraction. The sample is then measured by tandem mass spectrometry so as to identify analyte concentration and enzyme activity. The analyte concentration and enzyme activity are then used to determine whether the newborn is affected by any inborn errors of metabolism.

As used herein, the term “metabolite ” shall mean (1) a substance produced by metabolism; (2) a substance necessary for or taking part in a particular metabolic process; (3) a product of metabolism; (4) a substance produced by metabolic action, as urea; or (5) any substance involved in metabolism (either as a product of metabolism or as necessary for metabolism).

As used herein the term “labeled ” shall include isotopic labeling, fluorescent labeling, and other forms of chemical labeling as are known to those of ordinary skill in the art.

It is preferred that the solution of polar solvent comprises at least one component selected from the group consisting of: an internal standard for an analyte, an internal standard for an enzymatic product to be measured, a substrate, and a buffer. It is most preferred that at least one of the at least one components are labeled.

The internal standard(s) may be one or more internal standards for each class of analyte, or even one internal standard per analyte. The analyte(s) may then be compared to the internal standard(s).

It is preferred that the second solution is less-polar than the solution of polar solvent. It is preferred that the second solution comprises at least one component selected from the group consisting of: an internal standard for an analyte, an internal standard for an enzymatic product to be measured, and a buffer. It is most preferred that at least one of the at least one component is labeled.

The method may additionally comprise the step of adding at least one detergent to the sample prior to mixing.

A second method for the genetic screening of inborn errors of metabolism in a newborn by tandem mass spectrometry of the present invention comprises placing a blood spot sample from the newborn into a microtiter plate well so as to form a sample. The microtiter plate well is treated with at least one pre-treater selected from the group consisting of: an internal standard for the at least one relatively polar analyte, an internal standard(s) for the enzymatic product(s) to be measured, an internal standard for the at least one relatively non-polar analyte, one or more substrates for the enzyme(s), and a buffer. These internal standard components may be labeled. These pre-treaters are dry-coated onto the microtiter well.

The method may additionally comprise the step of adding a polar solvent to the well after placing the blood spot therein. Additionally, the method may comprise adding a solution after incubation so as to stop enzymatic reactions. Optionally, the method may comprise mixing the sample for a predetermined period of time.

The sample (in the well) is incubated for a predetermined period of time before being measured by tandem mass spectrometry. Tandem mass spectrometry is used to determine analyte concentration and enzyme activity. The analyte concentration and enzyme activity data are used to determine whether the newborn is affected by any inborn errors of metabolism.

Accordingly, the present invention includes both a combined metabolite and enzyme activity sample preparation method, with a single tandem mass spectrometry analysis in which the internal standards and substrates are incorporated within the solvents or are already present on the microtiter well as dry-coated materials. The present invention consolidates the measurements of enzyme activity and metabolite concentration measurement into a single assay that does not require sample derivatization. The present invention thus represents an improvement over current newborn screening methodologies that are performed independently from each other.

Some of the many applications for the methods of the present invention include but are not limited to assays for biotinidase deficiency, assays pertaining to disorders in carbohydrate metabolism (such as galactosemia), assays for lyposomal storage disorders (such as mucopolysaccharidoses, sphingolipodoses, oligosaccharidoses and mucolipidoses), fatty acid β-oxidation and organic acid metabolic disorders, amino acid metabolism disorders, and congenital adrenal hyperplasia. Other substances that may be determined from the methods of the present invention include but are not limited to carbohydrates, bile acids, very long chain fatty acids and steroids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of a newborn screening method for metabolite concentration measurement by MS/MS in accordance with the prior art.

FIG. 2 is a schematic of a newborn screening method for enzyme activity measurement by fluorometry in accordance to prior art.

FIG. 3 is a schematic of a newborn screening method for combined enzyme activity and metabolite concentration measurement by MS/MS in accordance with one embodiment of the present invention.

FIG. 4 is a schematic of a newborn screening method for combined enzyme activity and metabolite concentration measurement by MS/MS in accordance with another embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT(S)

There is a need for improvement in the methodologies used in the clinical newborn screening laboratories. The present state of the art requires laboratories to master several different technologies to provide comprehensive testing. Additionally, as more and more analytes are added, the mainly single analyte-single test format available may require that the patients donate more and more blood for testing (a traumatic experience for the patient). Further, laboratories are forced to spend more and more time and resources when new analytes are added because the technologies available do not effectively allow for multiplexing and expansion. Therefore, it is desirable to have access to technology and methodology that can consolidate the two clinical methods described above.⁶

With the current state of these technologies, it is not possible to combine the two approaches. The main problem is that MS/MS methods require sample derivatization. The conditions required to carry out this chemical modification of the sample may destroy both the enzymatic products as well as the enzymes; thus it may be impossible to measure enzyme activity with the current MS/MS methods. Additionally, many of the natural substrates and/or products involved in the metabolic pathways currently probed by enzyme activity assays belong to complex isomeric classes and/or are present in very low concentration or in conjugated forms. Therefore, it is not practical with the current MS/MS newborn screening methodology to accurately measure their concentration. Transforming these enzyme activity assays into MS/MS metabolite concentration assays is thus currently impractical.

Previously, there has not existed a comprehensive methodology that could perform both enzyme activity and analyte concentration measurements simultaneously. However, the present invention provides a method that does not require sample derivatization or substrate tagging with fluorescent groups and that consolidates the two types of assays into a single sample preparation. The invention is a very simple and easy to use comprehensive clinical method.

First, a blood spot from a newborn is placed into microtiter plate wells (any plate format). To each well is added a solution of a polar solvent containing internal standards for a multitude of analytes (whichever are desired) and a multitude of substrates (whichever needed). In addition, this solution can contain internal standards for the enzymatic products to be measured. This step accomplishes two tasks: a) enzyme reconstitution and b) extraction of all polar analytes present in the sample. Buffers can also be added if necessary.

The internal standards are analogs of the analytes (preferably, but not required, isotopically labeled analogs). A multitude of standards can be added (whichever are desired). The internal standards can also include (isotopically or other wise labeled) analogs of the enzymatic products.

The substrates (whichever needed) do not need to be tagged with fluorescent groups; they can be isotopically or other wise labeled analogs to the natural substrate so that they have more physiologic affinity to their corresponding enzyme. In this case, the labels are different than in the enzymatic product internal standard to avoid confusion.

The solution in this step is polar (for instance, any solvent varying from pure water to any mixture of polar solvents and buffers). With this solution we can reconstitute any enzyme and we can extract polar compounds such as (not comprehensive list) amino acids, sugars, nucleobases, nucleotides, nucleosides, etc.

Next, the sample is incubated for a predetermined amount of time. During this time, the reconstituted enzymes are acting on the specific added substrates and the metabolites are being extracted simultaneously.

After the required incubation/extraction time is fulfilled, add a non-polar (or less polar than the “polar solution ” above) solvent to precipitate the enzymes to stop the enzymatic reactions. At the same time, this second solvent allows the extraction of non-polar to less polar analytes present in the sample. This solvent can also contain a multitude of non-polar (less polar) internal standards (including internal standards for the products of the enzymatic reactions). For example, if we add methanol at this step, enzymes will denature while at the same time we can extract less polar analytes such as acylcarnitines, bile acids, fatty acids, less polar amino acids, organic acids, etc. The solution in this step is less polar than the polar solution previously added. At some point (depending on polarity differences), the two solutions may not be miscible with each other. In these instances, detergents can be added to homogenize the solutions.

It is preferred to mix the sample for a predetermined amount of time to homogenize and extract the remaining analytes.

Next, the sample is measured by tandem mass spectrometry using isotope dilution MS/MS. Analyte concentration is measured by comparing intensities of the analytes against the intensities of the corresponding internal standards of known concentration. Enzyme activity is measured by determining the concentration of the labeled product. The concentration of the labeled product is measured by comparing the intensity of the product against the intensity of the corresponding internal standard of known concentration. The concentration of the enzymatic product is then related to the incubation time to determine enzyme activity.

By this method one can measure both analyte concentrations and enzyme activities for a multitude of analytes and enzymes out of a single sample punch and sample preparation. With this method, one can potentially screen from 25 to 50 IEM simultaneously.

It should be noted that all of the internal standards and substrates mentioned could already be coated on the well of the microtiter plates so that the solutions (i.e. the polar solution and the less-polar/non-polar solution) do not need to include them.

Further, the proportions of the polar and less-polar/non-polar solution can be adjusted to ensure proper extraction of each class of analytes and enzyme reconstitution. This will allow new analytes to be added to the test menu with only minor changes to the method.

Additionally, this approach is flexible enough so that if one wants just to measure metabolite markers, one can consolidate the first tow of steps into a single one by making a solution of polar/less polar solvents and in a single step extract both polar and non-polar analytes. We have found a mixture of 75/25 methanol/water (v/v) can extract both polar amino acids and less-polar acylcarnitines in one step and that they can be measured by MS/MS with no prior derivatization.

Alternatively, one can only measure enzyme activity if so desired by just adding the corresponding substrates and internals standards (internal standards for the rest of the metabolites are not added).

In summary, the present invention includes a two-step protocol that does not require sample derivatization and that allows for the simultaneous measurement of metabolite concentrations and enzyme activities and that allows for the simultaneous screening of a multitude of IEMs.

The method of the represent invention may also be practiced using the sample test containers and related methods described in co-pending U.S. patent application Ser. No. 09/474,604 by Ostrup, hereby incorporated herein by reference.

FIG. 1 is a schematic of a newborn screening method for metabolite concentration measurement by MS/MS in accordance with the prior art. This method is used to extract and quantitate amino acids and acylcarnitines from newborn blood dried on filter paper. The amino acids and acylcarnitines concentration profiles are used to screen fatty acid β-oxidation, organic acid, and amino acid metabolic disorders in newborns (such as shown in U.S. Pat. Nos. 6,258,605 B1 and 6,455,321 B1 to Chace; hereby incorporated herein by reference).

FIG. 2 is a schematic of a newborn screening method for enzyme activity measurement by fluorometry in accordance with the prior art. This method, developed by Akie Fujimoto, et. al⁷is designed to screen for galactosemia by reconstituting the enzyme galactose-1-phospate uridyltransferase (GALT) from newborn dried blood spots, adding a fluorescent artificial substrate and monitor the rate of substrate to product conversion by fluorometry. The enzyme activity information is used to determine whether a newborn may be afflicted with galactosemia.

FIG. 3 shows a schematic of an assay procedure in accordance with one embodiment of the invention.FIG. 3 shows that the approaches exemplified inFIGS. 1 and 2 are consolidated in the present invention using the following procedure: Dried blood spots (DBS) may be punched into one or more microtiter well(s). In the next step, the enzyme of interest and polar analytes (e.g. amino acids) are eluted with a relative polar solution (e.g. 100% water). This solution may contain all of the necessary internal standards and substrates. The mixture is then incubated to allow enzyme activity and analyte extraction. In the next step, a relatively non-polar solvent (e.g., 100% MeOH) is added which halts the enzyme reaction and simultaneously extracts non-polar analytes (e.g. acylcarnitines). This second solution may contain all of the necessary internal standards. Enzyme precipitation and analyte extraction are allowed to occur. The enzymatic product, polar analytes and non-polar analytes may then be analyzed/quantified by tandem mass spectrometry. The analyte concentration and enzyme activity information is then used to determine whether the newborn is afflicted with any inborn error of metabolism.

FIG. 4 shows a schematic of an alternative assay procedure in accordance with another embodiment of the invention.FIG. 4 shows that dried blood spots (DBS) may be punched into one or more microtiter well(s). The wells are already coated will all necessary internal standards and substrates. In the next step, the enzyme and the polar analytes (e.g. amino acids) are eluted with a relative polar solution (e.g. 100% water). The mixture is then incubated to allow enzyme activity. In the next step, a relatively non-polar solvent (e.g., 100% MeOH) is added which halts the enzyme reaction and simultaneously extracts non-polar analytes (e.g. acylcarnitines). Enzyme precipitation and analyte extraction are allowed to occur. The enzymatic product, polar analytes and non-polar analytes may then be analyzed/quantified by tandem mass spectrometry. The analyte concentration and enzyme activity information is then used to determine whether the newborn is afflicted with any inborn error of metabolism.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not to be limited to the disclosed embodiment(s), but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims, which are incorporated herein by reference.

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2. Rinaldo, P., D. Matern, and M. J. Bennett. 2002. Fatty Acid Oxidation Disorders. Annu. Rev. Physiol. 64:477-502.
3. McCaman, M W, and E. Robins. 1962. Fluorometric method for determination of phenylalanine in serum. J. Lab. Clin. Med 59: 885.
4. Chamoles, N. A., M. B. Blanco, D. Gaggioli, and C. Casentini. 2001. Hurler-like Phenotype: Enzymatic Diagnosis in Dried Blood Spots on Filter Paper. Clinical Chemistry 47(12):2098-2102.
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7. Fujimoto, A., Y. Okano, T. Miyagi, G. Isshiki, and T. Oura. 2000. Quantitative Beutler test for newborn mass screening of galactosemia using a fluorometric microplate reader. Clinical Chemistry 46(6):806-810.

The foregoing references are hereby incorporated herein by reference.