WO2009153565A1 - Method for aiding the diagnosis of biliary atresia by determining the level of adma in a blood sample - Google Patents

Abstract

The invention relates to a method for aiding the diagnosis of bilial atresia in a subject, said method comprising (i) determining the level of ADMA in a blood sample from said subject, wherein said blood sample comprises a dried blood spot; (ii) comparing the level of ADMA determined in (i) to a reference ADMA value, wherein a higher level of ADMA in the sample from said subject compared to to said reference value identifies said subject as having an increased likelihood of bilial atresia.

Description

METHOD FOR AIDING THE DIAGNOSIS OF BILIARY ATRESIA BY DETERMINING THE LEVEL OF ADMA IN A BLOOD SAMPLE

Background to the Invention

Post-translational modification of arginine residues in proteins and subsequent proteolysis result in release of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Interest in the dimethylarginines has focussed on the inhibitory effect of ADMA on nitric oxide synthase and its metabolism by dimethylarginine dimethylaminohydrolase. However, increased plasma ADMA in patients with renal disease has been attributed to an important role of the kidney in excreting ADMA. SDMA is considered an end product of metabolism excreted by the kidney.

Biliary atresia is a congenially acquired condition in which the biliary tree become progressively sclerosed and occluded. The main intrahepatic ducts become obliterated and death occurs in 98% of individuals by two years old. Individuals undergoing surgery within about seven to eight weeks of birth are split into two groups. The first group, approximately one third of subjects receiving surgery, sees no success and will need a subsequent liver transplant. The remaining subjects (approximately two thirds) experience an initial success. Of this group, approximately 50% revert and will need a subsequent liver transplant. Thus, only about one third (50% of the group showing initial success following the operation) will display a ^"full recovery. Surgery conducted after approximately eight weeks of age is of almost no benefit since the disease is established at such an advanced stage to the extent that the-only available therapy is liver transplantation.

There has been interest in ADMA as a biomarker of certain conditions in the prior art. ADMA is widely regarded as a marker of cardiovascular disease and/or of endothelial function. This has been based upon the reasoning that ADMA is an inhibitor of nitric oxide synthase. Another view in the art is that ADMA is a marker of subsequent preeclampsia. Other publications have identified ADMA increase as being associated with renal failure. A further study has observed increased ADMA in children experiencing liver failure. Moreover, a study of liver transplant patients has shown thdt within one hour of the transplant being successfully completed, ADMA levels in the transplant recipient can return to normal. However, ADMA has not been widely perceived as a liver function marker, but it is an established cardiovascular indicator.

Current screening for newborn liver defects relies on the "yellow alert". The yellow alert is a rather crude observational measure designed to detect prolonged jaundice. Prolonged jaundice can be an indication of newborn liver disease. However, prolonged jaundice can be an indicator of an extremely wide range of underlying liver disorders. Furthermore, jaundice takes some days to present itself as a symptom. In addition, it is the prolonged nature of the jaundice which is key to using this yellow alert test as an indicator of disease, since newborn babies frequently display a short and temporary jaundice in the days after birth. Thus, by its very nature, the yellow alert test is only capable of identifying newborns with potential liver disorders at time points many weeks after their birth. Furthermore, no disease-specific information is obtained from a newborn infant presenting with prolonged jaundice. Moreover, if it is subsequently discovered that that subject has bilial atresia, their advancing age decreases the prospects of success due to the disease being progressive and requiring the earliest possible intervention coupled to the fact that the yellow alert test is necessarily only able to identify subjects possibly in need of intervention at a time point several weeks after their birth. This is a problem in the art.

In the prior art, the focus has always been adult liver disease. Typically, the emphasis is placed on cirrhosis of the liver, liver failure, or other serious adult presenting liver complaints. Child liver disease has received barely any attention. This is a problem in the art.

A study entitled "Plasma asymmetric dimethylarginine: a true liver function test?" described a retrospective cross-sectional study of plasma concentrations of ADMA in a range of liver diseases in children. Frozen liquid samples were used. The results confirmed that plasma ADMA is increased in a wide range of liver diseases including biliary atresia. These data for ADMA levels in children have not addressed bilial atresia in newborns. Furthermore, those data have been collected from children of several years of age, and not from newborns. Moreover, patients within the data collected from children include those who have had Kasai surgery. No distinctions have been drawn between treated and non-treated individuals in the prior art data. It is not possible to conclude from looking at the prior art data the associations which are disclosed herein.

The invention seeks to overcome problem (s) associated with the prior art.

Summary of the Invention

The present inventors disclose that elevated ADMA levels can be indicative of bilial - atresia in newborns. Furthermore, the inventors disclose that this symptom can be detected very early following birth of the baby. The combination of. extremely early detection coupled to the connection of elevated ADMA with bilial atresia leads to an advantageously early diagnosis. This permits effective medical interventions to be deployed at a time when conventional modes of diagnosis could not yet have reported. The invention is based upon these surprising findings.

Moreover, since bilial atresia is a progressive disease, and since surgical intervention beyond approximately eight weeks of life has almost no value in terms of improving outcome, the earlier a subject is treated the greater prospect they of survival. It is a key benefit of the present invention that the earliest possible diagnosis is enabled, thereby directly improving the outcomes of patients suffering from biiial atresia.

Thus, in one aspect the invention provides a method for aiding the diagnosis of bilial atresia in a subject, said method comprising

(i) determining the level of ADMA in a blood sample from said subject, wherein said blood sample comprises a dried blood spot;

(ii) comparing the level of ADMA determined in (i) to a reference ADMA value,

wherein a higher level of ADMA in the sample from said subject compared to to said reference value identifies said subject as having an increased likelihood of bilial atresia.

In another aspect, the invention relates to a method as described above further comprising the step of carrying out a secondary analysis in order to confirm a diagnosis of bilial atresia. Suitably the invention relates to a method as described above further comprising the step of analysing an image of the liver, of the subject identified as having an increased likelihood of bilial atresia, wherein detection of transposition of vessels in said image verifies the diagnosis of bilial atresia.

Suitably said sample comprises blood which was collected from said subject 8 weeks or less from birth. Surgical intervention beyond 8 weeks has poor outcomes, therefore it is an advantage of the invention to detect bilial atresia before this threshold age.

Suitably said sample comprises blood which was collected from said subject 1 to 8 days from birth. By detecting bilial atresia within the first few weeks of life, surgical interventions become a realistic life saving treatment option. Furthermore, standard practice in developed countries is to sample newborn blood within this period. Thus, by using samples of this age there is advantageously avoided the need for a further sampling event to take place. In other words, labour and costs are saved by using samples already rountinely collected, as well as coupling this to early detection.

Suitably said sample comprises blood which was collected from said subject 5 to 7 days from birth. This has the extra benefit of avoiding possibly confounding effects of birth trauma on the ADMA levels.

Suitably the ADMA level is determined in said sample by mass spectrometric analysis.

Suitably said mass spectrometric analysis comprises a chromatography step followed by positive ion mass spectrometry.

Suitably ADMA is selectively analysed by fragmentation specificity. This has the advantage that ADMA and SDMA need not be separated in order to analyse ADMA, thereby saving time and cost in the analysis.

Secondary Testing

It must be emphasised that detection of elevated ADMA is not in isolation (ie, on its own) diagnostic for bilial atresia. In the newborn screening context, ADMA is an important biomarker for congenital biliary atresia, because it is the primary hepatic problem at that age. However, testing a blood sample from a newborn infant and detecting an elevated ADMA could in fact indicate one of a range of liver disorders. However, as explained herein, bilial atresia is the most important and the most significant of these disorders. Nevertheless, clearly before diagnosis is completed and/or before surgery is undertaken, a secondary test must be deployed in order to confirm a diagnosis of bilial atresia. For this reason, suitably a method of diagnosis disclosed herein comprises a further step of assessing an image of the liver of the subject for the condition of bilial atresia.

Suitably, subjects who are identified according to the methods of the invention as possibly having bilial atresia are subjected to a secondary test in order to complete the diagnosis. Suitably, this secondary test is to image the liver. Suitably, the secondary test is to inspect an image of the subject's liver and to determine whether or not said image shows signs indicative of bilial atresia. Such signs are well-known in the art, as the clinical condition of bilial atresia is well-defined. However, in case further guidance is needed, typically a secondary test will comprise analysis of an MRI image of the subject's liver. Suitably, the method of -the invention does not comprise the actual collection of the image, but rather comprises only its analysis in vitro. The image is then analysed to look for transposition of vessels within the liver, which is indicative of bilial atresia.

Any other secondary test capable of confirming the diagnosis of bilial atresia may be employed by the person skilled in the art.

Sample

The sample is suitably a blood sample. The sample is suitably a dried blood sample, such as a dried blood spot. Although the precise details of collection of dried blood spot blood samples can vary from country to country, in principle a small amount of blood is collected from the newborn infant, and this is spotted or patched onto an absorbent substrate such as a filter paper. This sample is then dried, typically by allowing it to air dry at room temperature (typically at 18⁰C to 25⁰C) immediately after collection.

Thus, the sample is suitably a dried blood spot.

Suitably the sample is not liquid blood plasma.

Suitably the sample is not liquid blood.

Timing

Suitably the sample is collected at eight weeks of age or less, suitably the sample is collected at seven weeks of age or less, suitably the sample is collected at three weeks of age or less, suitably the sample is collected at two weeks of age or less. Most suitably, the sample is collected between one and eight days of age.

In the United States and mainland Europe, newborn blood samples are typically collected within about 24 to 48 hours of birth. Such samples are suitable for use in the present invention.

In the United Kingdom, blood samples from newborns are typically taken approximately five to eight days after birth. Suitably, the sample is a sample which was taken approximately five to eight days after birth. Most suitably, the sample is a sample which was taken at approximately five to seven days after birth. The advantage of samples taken at these particular time points is that certain birth traumas, such as birth asphyxia, may give rise to certain characteristics of the sample which might lead to false positives in certain situations. In particular, samples taken in the first day or two after birth can suffer from increased false positives due to effects of birth traumas. Thus, by using samples which were taken from the subjects at approximately five to seven days qfter birth, this problem may be advantageously avoided and a greater specificity is provided to the methods of the invention.

It should be noted that in the newborn period ADMA becomes more specific for biliary atresia, as this is the time it presents. The majority of other liver disorders that might present with increased ADMA will be outside the newborn period. Thus, the timing of the collection of the sample tested actually makes a specific further technical contribution in aiding diagnosis of biliary atresia, and has the advantage of bringing further specificity to the methods of the invention.

Moreover, where ADMA is raised in the newborn period, for reasons other than biliary atresia, the early clinical diagnosis will still be valuable. The confirmation of biliary atresia or differential diagnosis may be by routine investigation of liver disease; the blood spot ADMA analysis taught herein is a screening test, or a tool for aiding diagnosis.

It is an advantage of the invention that the diagnosis is reached earlier in the life of the subject being tested than with known techniques. This facilitates the earliest possible intervention. Since bilial atresia is a progressive disease, early intervention is better. As explained above, if intervention is not achieved within approximately eight weeks from birth, outcomes are so bleak that for all practical purposes liver transplantation is the only option.

Without wishing to be bound by theory, the present inventors advance the idea that ADMA can be viewed as a measure of cardiac output or blood flow to the liver. The reason is that ADMA is processed/removed by the liver. Therefore, if blood flow to the liver is increased, the blood concentration of ADMA is reduced.

Sample Format

As noted above, suitably the sample is a dried blood spot. Assessment of ADMA levels has been tested in liquid blood samples. However, it was observed that storage of the samples at -20⁰C overnight can lead to an elevation of ADMA levels, such as a three to four-fold elevation of ADMA levels. Initially, it appeared that the storage of liquid blood samples at -80ºC overnight had no effect. However, it was subsequently discovered that a thawing of blood samples from storage at -80⁰C also lead to confusingly high ADMA readings. The reason for these observations is that there are enzymes present in liquid blood which are capable of generating ADMA if stored in liquid form. Thus, to counteract this enzymatic activity, samples are prepared as blood spots (dried blood spots). Permitting the blood to dry in this manner prevents continued enzymatic activity taking place and confounding the determination of ADMA levels. Indeed, the present inventors have demonstrated that use of dried blood spots as the sample provides robust equivalence to the plasma concentration (whole blood concentration) of ADMA in the initial sample. This is a further benefit of the sample being a dried blood spot format.

These observations regarding fluctuating ADMA levels illustrate the difficulties in utilising ADMA as a marker. These may have contributed reason (s) why ADMA is not perceived as a good marker in the art. As disclosed herein, the inventors have addressed and solved this problem by use of blood spots as the sample. This is a key benefit of measuring ADMA in blood spots according to the present invention.

It should be noted that there is no known disclosure of ADMA measurement in blood spots before the present invention. In the prior art, ADMA has only ever been measured in liquid blood or plasma.

It is a further advantage of the invention that the methods described herein are conducted on dried blood spots, which dried blood samples are already routinely collected throughout the developed world as part of monitoring the health of newborn infants.

In addition, the findings disclosed herein which demonstrate that excess ADMA is not produced as the blood spot dries further demonstrate the benefits of this robust approach to aiding diagnosis.

It is a further advantage of the invention that the methods disclosed relate to blood spots which means that the amount of sample and hence amount of marker that is being studied is very small, so requiring only small amounts of blood. Determination of ADMA Levels

ADMA levels are suitably determined from a dried blood spot sample. Any suitable method known to those skilled in the art for measurement of ADMA in a dried blood spot sample may be used. Most suitably, mass spectroscopy analysis is used to assess ADMA levels in the dried blood spot sample as is disclosed in more detail in the examples section.

Suitably, ADMA is identified by ion fragmentation specificity. The advantage of this approach is that there is no need to separate ADMA from SDMA when determining ADMA levels, since the information derived from the ions produced permits the specific determination of ADMA levels even when SDMA is present in the same sample.

Suitably, direct injection mass spectrometry analysis is avoided in order to reduce problems which can arise from ion suppression masking the low concentrations of ADMA which are typically present in the samples being analysed. Nevertheless, more sensitive instrumentation may mean that direct injection is an acceptable approach.

Analysis of eluant may be conducted using negative ion spray mass spectrometry.

Most suitably, a chromatographic step is used in order to facilitate positive ion spray mass spectrometry. This approach has the advantage of avoiding ion suppression in the analysis of ADMA levels.

Suitable chromatography columns for this application are commercially available. Details of particular columns may be found in the examples section. Suitably, unless otherwise indicated, such columns are used according to the manufacturer's recommendations.

Suitably, shorter chromatography columns are used. The advantage of shorter columns is a reduction in the time taken for analysis.

Further Applications

It is a key concept of the present invention to link the analysis of blood spot ADMA levels to the aiding of a diagnosis of congenital bilial atresia. It is surprising that this link has been made. ADMA has been established in the field as a cardiovascular indicator.

The invention is susceptible to application as part of a multiplex system i.e analysis of ADMA as one of several markers/analytes/activities determined simultaneously and/or from the same sample. In particular the invention may be applied as part of a cardiovascular multiplex screen which would include SDMA among the component parts. Most suitably such a multiplex screen would include at least two or more of:

The invention may be applied as part of a multiplex analytical system for the simultaneous measurement of analytes (e.g. octanoylcarnitine for MCADD), detection of proteins (sickle beta haemoglobin), and measurement of enzyme activities, on a single 3.2mm blood spot (the blood spot size may be bigger or smaller and liquid whole blood or plasma may be used but blood spots are preferred for reasons set out above), suitably using direct sample injection without chromatography and electrospray mass spectrometry-mass spectrometry (MSMS) detection. The system is described with reference to newborn screening but could be applied to a range of clinical diagnostic and monitoring situations, e.g. cancer, diabetes (type I and type 2), renal disease, and liver disease.

Further analytes which may be combined with asymmetric dimethylarginine (ADMA), include orotic acid, and 3-O-methyl-dihydroxyphenylalanine (3OMDOPA) with potential for screening for further inherited and congenital disorders. Disease screening with these metabolites has not previously been described. Although it -might be possible to subsequently incorporate these metabolites into the currently described multiplex, performance of the analyses using blood spot elution with 150μl methanol (containing the full range of stable isotopes for amino acid, acylcarnitine, creatinine, methylmalonic acid, orotic acid, ADMA, and symmetric dimethylarginine (SDMA) quantitation) for 30min followed by 2 injections, each of 5μl of the eluate, for rapid chromatography, electrospray in both positive and negative ion modes, and MRM analysis on a SCIEX API5000 (Applied Biosystems, Warrington, UK) is most suitable. SDMA has been proposed as a measure of glomerular filtration rate and the data demonstrate that SDMA can be measured with sufficient accuracy and precision in a blood spot, allowing accurate diagnosis of renal failure and monitoring of renal function in a wide range of clinical conditions, e,g diabetes and heart disease, associated with decline in renal function and consequent increase in cardiovascular risk.

Reference Value

Many methods of the present invention require comparison of ADMA level in the sample from the subject with ADMA reference value. This reference value may be a previously determined reference value. This reference value may be a mean or any other kind of average reference value for a normal or healthy subject.

Alternatively, the reference value may be determined in parallel with the determination of the ADMA level in the sample from the subject being analysed. In these embodiments, two samples would be analysed in parallel - one being the sample from the subject under investigation, the other being a reference blood value from a normal or healthy individual. Suitably, said sample and said reference sample would be age- matched, sex-matched, or matched for any other relevant criteria such as ethnic background or genetic make-up. In such embodiments, the two values obtained are simply compared.

ADAAA

ADMA, an endogenous nitric oxide synthase inhibitor, is gaining credibility as a significant risk factor for adverse cardiovascular events (1 ). The role of nitric oxide in the regulation of vascular tone and the potent effects of ADMA on nitric oxide synthase have resulted in significant developments in our understanding of vascular ADMA metabolism and a recognition that increased plasma ADMA may be a major cause of endothelial dysfunction (1). However, despite their importance, the factors controlling plasma ADMA concentrations are poorly understood.

The plasma ADMA concentration depends upon the balance between its production and excretion/metabolism. Production of ADMA from the breakdown of methylated nuclear associated proteins, although under intense investigation is unlikely to be significantly modulated except, perhaps, in acute and severe catabolic clinical conditions. The excretion/metabolism of ADMA offers significantly more opportunities for regulation. Initial measurements of plasma ADMA showed that concentrations were significantly increased in patients with renal failure (2), establishing a potential role for the kidney. Although the kidney clears some ADMA, two important observations suggest that the kidney is relatively unimportant in plasma ADMA regulation. Firstly, the lowest levels of glomerular function are often associated with normal ADMA concentrations. Secondly, following kidney transplantation plasma ADMA levels remain elevated (3), strongly suggesting that the original increase in ADMA may have been due to factors other than renal function.

The role of dimethylarginine dimethylaminohydrolase (DDAH), the enzyme converting ADMA to citrulline and dimethylamine, in the catabolism and regulation of ADMA is now fully agreed. However, research has tended to concentrate on impaired activity of vascular DDAH as the cause of increased plasma ADMA. The implications of the observation that the liver is the major site of DDAH activity and ADMA metabolism (4) have been less readily understood and embraced.

The first measurements of urinary ADMA and SDMA reported an increased ADMA/SDMA ratio in patients with liver disease (5). However, it is only recently that more substantive evidence of the effect of liver failure on plasma ADMA concentrations has been published (6,7,8). If ADMA is metabolised, principally in the liver by DDAH, then, for a given ADMA production rate the plasma level of ADMA depends on liver blood flow and DDAH activity, a situation analogous to sorbitol clearance used to measure effective liver blood flow. As such, plasma ADMA represents a true endogenous liver function test.

The importance of liver blood flow/function in determining plasma/whole blood ADMA concentrations is illustrated by the observation that ADMA is significantly increased in patients with end-stage liver failure, immediately pre liver transplantation and during the transplant operation. Crucially, plasma ADMA levels normalise within a few hours of successful liver transplantation (9).

Biliary Atresia

Biliary atresia is a congenially acquired condition in which the biliary tree become progressively sclerosed and occluded. The main intrahepatic ducts become obliterated and death occurs in 98% of individuals by two years old. The cause may be a congenital or perinatal infection, for example with reoviruses. It occurs once in 10 000 live births. 25% of patients have other congenital abnormalities. Correctable lesions may be treated by direct drainage. A Roux-en-Y jejunal loop is anastomosed to the patent portion of the extrahepatic biliary tree communicating with the intrahepatic ducts.

Where direct anastomosis of the biliary tree and gut cannot be achieved treatment is with hepatoportoenterostomy of Kasai.

Patients undergoing surgical correction before 8 weeks of age have a 70% chance of losing their jaundice.

50% of these successes are temporary and subsequent cholangiopathy will result in chronic liver failure.

The remainder will develop normally without liver disease.

Failure to respond to initial surgery is now a realistic indication for liver transplantation. Consequently, early diagnosis and appropriate surgery can dramatically improve patient outcome and prognosis.

Attempts have been made, using other biomarkers, e.g. bile salts, to screen for biliary atresia in newborn infants. They have not been successful. Consequently, in the UK, the Yellow Alert Campaign, promoted by the Childrens Liver Disease Foundation, has been used to increase awareness of the problem and, hopefully, earlier diagnosis.

We now demonstrate the ability to accurately quantify ADMA in blood spots using a chromatographic procedure requiring about 5min. Optimisation includes reducing column size and increasing flow rate to achieve appropriate chromatography within a 2min cycle. Consequently, when considering the data presented herein it is clear that blood spot ADMA can be used in newborn blood spot screening for congenital biliary atresia. The sensitivity and specificity of the test can be clearly defined. We now know that plasma ADMA is increased in the early newborn period and the clinical data presented herein indicate that ADMA will not be specific for biliary atresia and may pick up other, significant liver diseases and, potentially, anomalies associated with reduced cardiac output. These possibilities are easily eliminated using secondary testing, and ADMA is a valid biomarker for newborn screening.

Brief Description of the Drawings

Figure 1 shows chromatograms for a dried blood (no anticoagulant) spot. Figures 2 and 3 show graphs illustrating recovery. Figure 4 shows α graph.

The invention is now described by way of example. These examples are not intended to limit the scope of the appended claims, but are rather illustrative in nature.

Example 1

In order to demonstrate the invention we addressed the following questions:

-Can we detect normal values of ADMA and SDMA on a dried blood spot?

-Can we detect alterations in dried blood spot ADMA, within the expected physiological range?

-Is dried blood spot ADMA stable over a time period expected for postage to a central laboratory for analysis?

Experiment:

2 adult volunteers provided 5ml blood samples. At the time of blood sampling a few drops of each blood sample, without coagulant, were allowed to drop onto standard

Schleicher & Schuell filter paper and allowed to dry at room temperature.

The remaining blood samples were transferred to heparinised blood collection tubes, mixed, and an aliquot, c.l ml, of each blood sample was centrifuged and the plasma removed and stored at -80oC.

ADMA standard material was the added to the whole blood samples from each volunteer as below:

ND/CT 90μl whole blood + lOμl deionised water

(final concentration, basal)

ND/CT 90μl whole blood + lOμl of 2.5μmol/l ADMA

(final concentration, basal + 0.25μmol/l)

ND/CT 90μl whole blood + lOμl of 5.0μmol/l ADMA

(final concentration, basal + 0.50μmol/l)

ND/CT 90μl whole blood + lOμl of 7.5μmol/l ADMA

(final concentration, basal + 0.75μmol/l)

ND/CT 90μl whole blood + lOμl of 1.Oμmol/I ADMA

(final concentration, basal + 1.Oμmol/I)

ND/CT 90μl whole blood + lOμl of 2.0μmol/l ADMA

(final concentration, basal + 2.0μmol/l)

ND/CT 90μl whole blood + lOμl of 5.0μmol/l ADMA

(final concentration, basal + 5.0μmol/l)

50μl of each sample was pipetted onto standard Schleicher & Schuell filter paper and allowed to dry at room temperature

The remaining heparinised blood was kept at room temperature on a mixing roller.

Assays were then performed 24h and 7days after initial spotting. 2.5μl αliquots of aqueous standards and plasma controls (already measured by our standard technique using a 50μl sample volume) and 3.2mm blood spots (approximately equivalent to 2.5μl of sample) were prepared. A methanolic precipitation/elution solvent was prepared (containing the full range of stable isotopes for amino acid, acylcarnitine, creatinine, methylmalonic acid, orotic acid, ADMA, and SDMA quantitation). 150μl of the precipitation/elution solvent was added to each sample, the samples mixed, the aqueous/plasma/whole blood samples centrifuged, and the blood spots mixed for 30min at 37oC before centrifugation. Supernatants were transferred to a polypropylene 96 deep well plate and a cover mat applied. ADMA, SDMA, valine, and phenylalanine were then measured following chromatography and stable isotope dilution positive ion electrospray mass spectrometry-mass spectrometry (MSMS) in multiple reaction monitoring (MRM) mode on a SCIEX API5000 (Applied Biosystems, Warrington, UK).

5 μL of supernatant was automatically injected using an HTS PAL autosampler (CTC Analytics AG, Switzerland) into a 250 μL/min mobile phase stream of acetonitrile:water (50:50 v/v) with 0.025% (v/v) formic acid. Chromatography was performed on a Chirobiotic T 100 x 2.1 mm column with a 2cm x 4.0mm guard column (Advanced Separation Technologies, Congleton, UK) and precursor/product ion pairs (m/z 203.1/46.2, 209.1 /52.2 for ADMA, m/z 203.1 /172.2, 209.1 /175.1 for SDMA, 1 18.1 /72.0, 126.1/80.0 for valine, and m/z 165.9/120.1 , 170.9/125.1 for phenylalanine) were acquired in positive ion MRM mode. Assay standardisation was based on aqueous standards at 0.25, 1.0, and 5.0 μmol/l ADMA/SDMA and 50 and 250 μmol/l valine/phenylalanine, stored at -80oC. Results were calculated using Analyst version 1 .4.3. Pooled and spiked plasma samples, previously analysed using 50μl sample were used to confirm the validity of the reduced volume assay.

Results:

Reference is made to Figure 1 ; the top panel is ADMA, then SDMA, valine, and phenylalanine.

Note the 2 internal standard peaks for ADMA; these are L- and D- forms of the internal standard,_,only the L-form was used for quantitation.

Inspection of the chromatogram demonstrates that ADMA and SDMA are indeed detectable in a blood spot using the method described above. Note the sensitivity, c.1800 cps, for normal concentrations of both ADMA and SDMA. Note the chromatography takes 6min at present. However, with a 50mm column and 400μl/min flow rate this will reduce to about 2min.

Addition of ADMA standard to whole blood within the physiological range (0.25- 5μmol/l) is easily accurately and precisely measured. This is demonstrated by the recovery graphs for each volunteer shown in figures 2 and 3. The R2 value is a crude measure of the analytical precision over the analytical range; 0.9929 and 0.9997. These are excellent values considering that we are using dried blood spots. The slope is not 1 in either case suggesting that we may not have 100% recovery of added ADMA. In Pl the major effect is almost certainly a low result for the +5.0μmol/l spot. In P2 the discrepancy is only 8% and can be explained by our use of the assumption that a 3.2mm blood spot is equivalent to 2.5μl of sample (note that the assay is standardised using the aqueous standards).

The precision of the ADMA assay can also be deduced from the precision of the SDMA assay, where no additional material was added; for Pl , 8.6% and 5.6% and P2, 5.4% and 6.1%. Note that the precision is not significantly better for valine or phenylalanine (used here as reference analytes to assess expected precision), where the signals are orders of magnitude higher, indicating that the major determinant of the precision relates to blood spot sampling or is inherent to the technique.

30/05/200805/06/200830/05/2008 05/06/2008 30/05/2008 05/06/2008 30/05/2008 05/06/2008

Sample Sample ADMA ADMA SDMA SDMA Valine Valine Phenylalanine Phenylalanine

Sample Name ID Type (μmol/l) (μmol/l) (μmol/l) (μmol/l) (μmol/l) (μmol/l) (μmol/l) (μmol/l)

CT plasma Unknown 0.435 0.430 0.449 0.483 334.0 364.0 72.0 81.5

ND plasma Unknown 0.540 0.435 0.653 0.601 214.0 218.0 71.7 74.7

CT WB 24h/7 days Unknown 0.409 0.601 0.370 0.439 284.0 338.0 67.1 116.0

ND WB 24h/7 days Unknown 0.415 0.550 0.441 0.497 189.0 222.0 68.6 108.0

CT BS NAC Unknown 0.484 0.431 0.456 0.507 359.0 379.0 81.2 90.2

ND BS NAC Unknown 0.511 0.415 0.551 0.572 210.0 219.0 74.0 77.4

CT BS Unknown 0.408 0.323 0.405 0.350 297.0 274.0 68.1 64.5

CT BS 0.25 0.25Unknown 0.586 0.615 0.373 0.351 263.0 301.0 60.7 72.2

CT BS 0.5 0.5 Unknown 0.836 0.837 0.382 0.380 269.0 318.0 62.0 76.9

CT BS 0.75 0.75 Unknown 1.030 1.010 0.351 0.333 252.0 279.0 59.5 65.0

CT BS 1 1 Unknown 1.220 1.150 0.316 0.367 254.0 286.0 58.5 66.5

CT BS 2' 2 Unknown 2.150 1.960 0.349 0.368 258.0 283.0 58.9 66.2

CT BS 5 5 Unknown 4.030 4.220 0.329 0.325 236.0 271.0 53.1 62.4

ND BS Unknown 0.399 0.338 0.424 0.423 164.0 170.0 56.0 61.1

ND BS 0.25 Unknown 0.639 0.606 0.454 0.466 178.0 180.0 60.8 65.2

ND BS 0.5 Unknown 0.841 0.757 0.468 0.387 167.0 161.0 54.8 57.0

ND BS 0.75 Unknown 1.030 1.010 0.414 0.409 158.0 167.0 53.7 58.7

ND BS 1 Unknown 1.340 1.120 0.484 0.401 165.0 163.0 56.7 58.2

ND BS 2 Unknown 2.230 2.040 0.445 0.429 165.0 183.0 54.5 69.3

ND BS 5 Unknown 5.010 4.480 0.446 0.435 173.0 178.0 58.0 62.4

Mean plasma 0.488 0.433 0.551 0.542 274.0 291.0 71.9 78.1 Mean WB 24h 0.412 0.576 0.406 0.468 236.5 280.0 67.9 112.0 Mean BS NAC 0.498 0.423 0.504 0.540 284.5 299.0 77.6 83.8 Mean BS (corrected) 0.448 0.367 0.461 0.429 256.1 246.7 68.9 69.8 Mean CT BS 0.358 0.353 261.3 287.4 60.1 67.7 Mean ND BS 0.448 0.421 167.1 171.7 56.4 61.7

Note that the plasma concentrations of ADMA and SDMA are the very similar to those measured on dried blood spots collected without anticoagulant, liquid whole blood left for 24h, and dried blood spots collected with anticoagulant. The small differences are not diagnostically clinically significant.

(Note that in the liquid whole blood sample there are slight increases for SDMA and, particularly, ADMA over the week. However, this is a potentially artificial situation not expected in routine clinical analysis.)

The mean data on the dried blood spot analyses suggest that ADMA might fall slightly over the 7 days, 0.448 to 0.367 μmol/l. However, across the full range of blood spots the comparison of 24h v 7 days demonstrates no deterioration, slope = 0.95 and, and excellent agreement, R2O.9877 (see figure 4).

No change in dried blood spot SDMA, valine, or arginine.

Summary of examples section:

We demonstrate detection of normal values of ADMA and SDMA on a dried blood spot.

We demonstrate detection of alterations in dried blood spot ADMA, within the expected physiological range.

We demonstrate that dried blood spot ADMA is stable over a time period expected for postage to a central laboratory for analysis.

We demonstrate that dried blood spot SDMA is stable over a time period expected for postage to a central laboratory for analysis.

We have also provided compelling data suggesting that measuring ADMA in the newborn period, blood spots are taken at day 5-8 in the UK, allows early diagnosis of children with congenital biliary atresia.

The fragmentation specific ADMA/SDMA MSMS. assay is an especially advantageous embodiment.

Blood spot analyses of this type are disclosed herein for the first time and the inventors are not aware of any suggestion of plasma ADMA or dried blood spot ADMA as a screening test for congenital biliary atresia in any prior art. We have demonstrated that the assay works and is useful and that ADMA and SDMA are stable on the blood spot for at least the necessary time.

Dried blood spot ADMA/SDMA also finds application in a more general population cardiovascular risk screen.

References:

1. J.P. Cooke. Circulation 109 (2004) 1813

2. P. Vallance, A. Leone, A. Calver, J. Collier, S. Moncada. Lancet 339 (1992) 572

3. C. Fleck, A. Janz, F. Schweitzer, E. Karge, M. Schwertfeger, G. Stein. Kidney International - Supplement 78 (2001 ) Sl 4

4. RJ. Nijveldt, T. Teerlink, M.P.C. Siroen, A.A. van Lambalgen, J.A. Rauwerda, P.A.M. van Leeuwen. Clinical Nutrition 22 (2003) 17

5. P.R. Carnegie, F.C. Fellows, G.R. Symington. Metabolism 26 (1977) 531

6. RJ. Nijveldt, T. Teerlink, B. van der Hoven, M.P.C. Siroen, DJ. Kuik, J.A. Rauwerda, P.A.M. van Leeuwen. Clinical Nutrition 22 (2003) 23 -

7. P. Martin-Sanz, L. Olmedilla, E. Dulin, M. Casado, N.A. Callejas, J. Perez-Pena, I. Garutti, J. Sanz, J. Calleja, S. Barrigon, L.Bosca. Liver Transplantation 9 (2003) 40

8. D. Tsikas, I. Rode, T. Becker, B. Nashan, J. Klempnauer, J.C. Frohlich. Hepatology 38 (2003) 1063

9. R.P. Mookerjee, R.N. Dalton, N.A. Davies, SJ. Hodges, C. Turner, R. Williams, R. Jalan. Liver Transplantation 13 (2007) 400

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are apparent to those skilled in the art are intended to be within the scope of the following claims.

Claims

Claims

1. A method for aiding the diagnosis of bilial atresia in a subject, said method comprising

(i) determining the level of ADMA in a blood sample from said subject, wherein said blood sample comprises a dried blood spot;

(ii) comparing the level of ADMA determined in (i) to a reference ADMA value,

wherein a higher level of ADMA in the sample from said subject compared to to said reference value identifies said subject as having an increased likelihood of bilial atresia.

2. A method according to claim 1 further comprising the step of analysing an image of the liver of the subject identified as having an increased likelihood of bilial atresia, wherein detection of transposition of vessels in said image verifies the diagnosis of bilial atresia.

3. A method according to any preceding claim wherein said sample comprises blood which was collected from said subject 8 weeks or less from birth.

4. A method according to claim 3 wherein said sample comprises blood which was collected from said subject 1 to 8 days from birth.

5. A method according to claim 4 wherein said sample comprises blood which was collected from said subject 5 to 7 days from birth.

6. A method according to any of claims 1 to 5 wherein the ADMA level is determined in said sample by mass spectrometric analysis.

7. A method according to claim 6 wherein said mass spectrometric analysis comprises a chromatography step followed by positive ion mass spectrometry.

8. A method according to claim 7 wherein ADMA is selectively analysed by fragmentation specificity.