Chemistry: analytical and immunological testing – Heterocyclic carbon compound – Hetero-o
Reexamination Certificate
2000-07-28
2002-09-17
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Heterocyclic carbon compound
Hetero-o
C436S103000, C436S104000, C436S811000, C436S173000, C435S014000, C435S072000, C435S105000
Reexamination Certificate
active
06451611
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of detecting hexosemonophosphates (HMPs) in biological samples and uses therefor. In a preferred embodiment, the method of the invention can be used to screen newborns for galactosemia.
BACKGROUND OF THE INVENTION
Galactosemia is a life-threatening disorder with severe symptoms in the neonatal period. It is caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT) (EC. 2.7.7.12), (OMIM 230400).
In this disorder ingestion of milk causes accumulation of galactose in blood and urine and leads to a high intracellular concentration of galactose-1-phosphate (Gal-1-P). Gal-1-P is considered toxic for several tissues, especially the liver, brain, and renal tubules (12). The main symptoms appear shortly after ingestion of milk and include vomiting, failure to thrive, jaundice due to liver damage, and lethargy. The patients become comatose, and if treatment is not initiated early, death often occurs during the first weeks of life. Treatment with a galactose-free diet causes regression of symptoms and signs within a week or two. However, due to the low frequency of the disease (approximately 1:35000 in Denmark) and sub-optimal clinical awareness, diagnosis is often delayed or symptoms are misinterpreted as septicemia or isoimmunization, causing sequelae due to late intervention. While these considerations seem to advocate neonatal screening, this is still controversial as illustrated by two recent comprehensive reviews with opposing conclusions (11, 9). There are no controversies over the serious implications of untreated galactosemia:neonatal death or severe mental retardation. The major issues in the discussion are the low frequency of the disease affecting the cost benefit ratio, sub-optimal screening tests, and sequelae in the long-term outcome despite early diagnosis and treatment, such as intellectual impairment, speech disorders, cataracts, and hypergonadotrophic hypogonadism (13, 14, 5).
Several methods exist for neonatal screening for galactosemia. The simplest of all is examining the urine for reducing substances (i.e, using Fehling's and Benedict's solution). The commonly employed tests for neonatal screening use dried blood spot samples (DBSS). Paigen's (8) quantifies galactose and Gal-1-P by a microbiological assay. However, it is not suitable for automation, is sensitive to antibiotic otic treatment of the newborns or their mothers, and careful bacterial maintenance procedures are required. Measurement of GALT activity is the basis of the Beutler test (1, 2). GALT is sensitive to inactivation by heat or humidity, causing false-positive screening results. The Paigen and Beutler assays in combination are used by many screening laboratories (11) and can detect deficiencies in GALT, galactokinase, and galactose epimerase, but have relatively high false-positive rates (11). These disorders can also be detected by the alkaline phosphatase-galactose dehydrogenase assay (3, 4).
There is a need for an improved, more accurate, rapid, cost effective and easier method for screening for and diagnosing galactosemia.
SUMMARY OF THE INVENTION
The present inventors have developed a simple, fully automated, rapid and cost effective screening method to detect and quantify Hexosemonophosphates (HMPs). The method can be used to detect and quantify HMP in a biological sample. As such the method can be used as an indicator e.g. to diagnose screen for or identify patients at risk, for a disorder related to abnormal HMP levels (either relative or absolute). Such disorders include, but are not necessarily limited to: galactosemia, fructosuria, hereditary fructose intolerance, fructose 1,6, biphosphatase deficiency, glucose-6-phosphate dehydrogenase deficiency and diabetes meritus. In a preferred application, the method can be used as an indicator of glactosemia in newborns. In an embodiment of the invention, the method can be used in the diagnosis of galactosemia in newborns. In another embodiment, the method of the invention can be used in screening for HMP related disorders such as galactosemia. In another embodiment the screening can be followed by or used in conjunction with other diagnostic methods, such as gene analysis.
Quantify or quantitative analysis, as used herein, is meant to encompass any manner of determining the relative or absolute (for instance, concentration) amount of hexose monophosphate and/or a species of hexose monophosphate in a sample. HMP level(s) as used herein also refers to both the absolute and relative amounts of HMP, as the context permits.
In the preferred embodiment, the invention provides a new rapid technique for quantitatively analysing HMP levels from a blood sample, preferably from dried blood spots, preferably on filter paper or other suitable medium, using tandem mass spectrometry (MS/MS). This new method can be used in the diagnosis of galactosemia. The new method overcomes many of the limitations in the prior art methods used for screening for the disease.
In another preferred embodiment, the invention provides a method for detecting and quantifying HMP levels in a biological sample by obtaining an HMP extract of the biological sample in a form suitable for ionization. “HMP extract” as used herein refers to an extract of the biological sample with a solvent suitable for extracting HMPs. The HMP extract does not necessarily contain any HMPs. In a preferred embodiment, the sample is extracted with a solvent that is more hydrophilic than methanol. Suitable solvents could be acetonitrile in water, tetrahydrofuran in water, methanol in water, ethyl acetate in water or isopropanol in water. More preferably the solvent is or has properties similar to that of acetonitrile in water, most preferably the solvent is 30-70% acetonitrile (v/v) in water.
In another embodiment, the biological sample is dried prior to extraction. In yet another embodiment, the biological sample is blotted onto a suitable medium, such as filter paper, dried and then extracted as above.
In another embodiment, a number of biological samples can be used in the method of the invention where applicable, such as whole blood, fractionated blood components, tissue (such as liver biopsy specimens), urine, stool, bile, saliva, sweat or other glandular secretions. Preferably, the biological sample is whole blood that is blotted onto a suitable medium, dried and then extracted.
Subsequent to extraction, the HMP extract is then ionized, using techniques such as, electrospray, or pneumatically assisted electrospray (e.g. IonSpray), to form a stream of gas phase ions. Precursor molecular ions, molecular gas phase ions having a mass to charge ratio of HMP, i.e., 259.02 Da/e, are then selected and fragmented to obtain product ions of HMP. Product ions of interest, those most indicative of HMP levels or a particular HMP, such as Gal-1-P or fructose-1-P are selected by their mass to charge ratios then detected and their ion intensities obtained. The ion intensities are preferably recorded in negative-ion mode.
The ion intensities of the product ions can be used to determine by spectrum analysis the quantitative abundance of HMPs in the biological sample; and/or to determine the concentration of HMPs, or a particular species thereof, by comparing the ion intensities of the sample with that of a control comprising a known amount or concentration of HMP or species thereof; and/or to determine the ion abundance ratios (IAR) indicating the relative amount of HMP species present within the biological sample.
In a further embodiment the method of the invention can be used to screen for and/or diagnose galactosemia in a patient by comparing the level of HMP in a blood sample with that of a cutoff level for normal individuals. In another embodiment, the invention provides a method of creating a database and a database of HMP levels (concentrations and/or IAR) of normal individuals who do not have an HMP related disorder and abnormal individuals with an HMP related disorder. In yet another embodiment, the invention provide
Brandt Niels Jacob
Christensen Ernst
Jensen Ulrich Glümer
Simonsen Henrik
Gakh Yelena
Statens Serum Institute
Warden Jill
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