GB2394770A - Assay for s-adenosyl methionine (SAM) and s-adenosyl homocysteine (SAH) - Google Patents

Abstract

A method for assaying SAM and SAH in a sample, said method comprising the steps of contacting a first aliquot of the sample with a ligand capable of binding to both SAM and to SAH, removing or degrading either SAM or SAH in a second aliquot of said sample, contacting said second aliquot with said ligand, and assessing the concentrations of SAM and/or SAH in said first and second aliquots. A kit for use in the above method is also disclosed.

Description

- 1 - Assay The present invention relates to an assay for S 5 adenosyl

homocysteine and S-adenosyl methionine in biological samples, optionally together with homocysteine. S-adenosyl homocysteine (SAM) is a metabolic intermediate and arises from reactions transferring 10 methyl groups from S-adenosyl methionine (SAM). SAH is hydrolyzed to adenosine and homocysteine by the enzyme S-adenosyl homocysteine hydrolase. SAH has been suggested to be an intermediate of homocysteine toxicity, particularly as a mediator for hyperhomo 15 cysteinemia related vascular changes.

Homocysteine is a sulphur-containing amino acid that is closely related to methionine and cysteine.

Homocysteine is not a building block of naturally occurring proteins. Homocysteine is formed in the 20 metabolism of the essential amino acid methionine.

Hyperhomocysteinemia is generally defined as fasting total plasma homocysteine (tHcy) above 12-15 Remold. It has been shown to be a risk factor for cardiovascular disease and for complications in 25 pregnancy and congenital malformations. A link between impaired homocysteine metabolism and neuropsychiatric disorders and cognitive impairment in the elderly has also been shown.

Normalisation of homocysteine levels may be 30 achieved by life-style changes (e.g. quitting smoking, taking exercise, reducing coffee consumption, improving diet, etc.) or by vitamin supplementation.

S-adenosyl-methionine (SAM) is a derivative of the amino acid methionine. SAM is a methyl donor in more 35 than 35 different methylation reactions where it donates its methyl group to a large and varied group of acceptor molecules, including methylation of deoxyribose nucleic ............

- 2 - acid (DNA), and thereby forming its demethylated short-

lived metabolite SAH, which is metabolized to homocysteine. SAM has been used clinically in the treatment of liver disease (Friedel et al. Sadenosyl-L 5 Methionine: a review of its therapeutic potential in liver dysfunction and affective disorders in relation to its physiological role in cell metabolism. Drugs 1989; 38: 389-416), arthritis (Di Padova, Sadenosylmethionine in the treatment of osteoarthritis: review of the 10 clinical studies; Am. J.med.83 (suppl 5); 1987: 50-65), and depression (Kagan et al. Oral S-adenosylmethionine in depression: a randomized, double blind, placebo controlled trial. Am.J.Psychiatry 147; 1990: 591-5).

The clinical potential of SAM in other neurological 15 disorders has been reviewed by Bottiglieri et al. (Bottiglieri T. Hyland K, Reynolds, E.H. The Clinical Potential of Ademetionine (S-Adenosylmethionine) in Neurological Disorders, Drugs 48; 1994: 137-152). It has recently been introduced as a mood enhancer in the 20 American food supplementation market. However, the potential risk of increased heart failure theoretically associated with increased SAM levels has not been investigated. SAH, homocysteine and SAM are active participants 25 in the methionine cycle (Figure 1). Both SAH and SAM are intermediates in the synthesis of homocysteine and have regulatory effects on some of the enzymes in the methionine cycle. For example, SAM acts as an al losteric inhibitor of MTHFR (methyl tetrahydrofolate 30 reductase EC1.1.1.68) which is crucial for 5 methyltetrahydrofolate synthesis (Jencks DA, Matthews RG. Allosteric inhibition of methylenetetrahydrofolate reductase by adenosylmethionine. J.Biol.Chem.1987; 262: 2485-93.) and as an activator for cystathionine 0 35 synthase (EC 4.2.1.22) (Finkelstein JD, Kyle WE, Martin JJ, Pick AM. Activation of cystathionine synthase by adenosylmethionine. Biochem Biophys Res Commun 1975; 66:

- 3 81-7) whereas high concentrations of SAH will have a strong inhibitory effect on the enzyme S-adenosyl methionine synthase, catalysing the demethylation of SAM to SAH, and will therefore be a competitive inhibitor of 5 methylation reactions in general.

It has been established that high concentrations of homocysteine represent a risk factor for cardiovascular disease. It is however not yet known whether the homocysteine itself is the causal factor or whether it 10 is a marker of high concentrations of another factor which causes cardiovascular problems. The level of homocysteine can be correlated to the risk of cardiovascular disease. A high homocysteine level is correlated with a dramatically elevated cardiovascular 15 risk, and a slightly elevated level is thus correlated with a moderately elevated risk. However this risk could be amplified in conjunction with other risk factors (Graham et al., Plasma homocysteine as a risk factor for vascular disease; the European Concerted 20 Action project, JAMA 277; 1997: 177581), and the homocysteine level is therefore useful in monitoring cardiovascular risk. Several studies have shown that a marked elevation in risk is associated with homocysteine differences as small as 1-2 mol/L, equivalent to an 25 elevation of 10 to 20% over normal levels.

SAH has been suggested to be the mediator for hyperhomocysteinemia related vascular changes, which changes are generally associated with endothelial damage and SAH has been shown to modulate endothelial cell 30 apoptosis. Increased levels of plasma SAH have been shown to be associated with DNA hypomethylation (James et al., Third International Conference on Homocysteine Metabolism, Sorrento, July 2001). Recent studies upon a population of patients that have undergone a 35 cardiovascular event have shown that elevation of plasma SAH levels may be amplified compared to the elevation of homocysteine in the plasma (Kerins et al., Am J Clin

- 4 - Nutr 2001; 74: 723-9). The average homocysteine concentration in the patient group was 15% higher than that for the control group, however when SAH was measured it was 48% higher in the patient group than in 5 the control group. The difference in the concentration of SAM was of the same magnitude as that for homocysteine. James et al. (supra) found that a decrease in the ratio of SAM concentration to SAH concentration was predictive of reduced methylation 10 capacity when associated with an increase in SAH. A decrease in this ratio due to SAM depletion alone was not sufficiently predictive.

In methionine loading experiments where the homocysteine concentration shows an approximately 4-fold 15 transient increase, the SAM concentration increases approximately 6-fold, whereas no significant increase has been observed for SAH (Loehrer FMT, Haefli WE, Angst CP, Browne G. Frick G, Fowler B; Effect of methionine loading on 5- methyltetrahydrofolate, S 20 adenosylmethionine and S- adenosylhomocysteine in plasma of healthy humans, Clin Sci 1996; 91: 79- 86).

The inventors consider that the ratio of SAH to SAM (or vice versa) is a better marker for cardiovascular risk than is the concentration of homocysteine alone.

25 Additionally the ratio of SAH to SAM in conjunction with the level of homocysteine can be used to assess risk of cardiovascular disease.

However, the current methods of measuring SAM and SAH concentrations rely upon cumbersome HPLC methods and 30 to enable use of these parameters as risk indicators, simpler methods suitable for routine screening are required. The current methods are time-consuming and the chromatographic separation requires highly specialized and sophisticated equipment. Since SAM and 35 SAH are very similar compounds, distinguished only by one methyl group, it is important to provide an assay that can distinguish the two entities.

_......

A need therefore exists for an improved assay for SAM and SAH which is simple, specific, quick to perform, readily adapted for use in clinical settings and, above all, which avoids the need for costly and timeconsuming 5 chromatographic separation. The present invention provides such an assay.

In one aspect the present invention therefore provides a method for assaying SAM and SAH in a sample, said method comprising the steps of contacting a first 10 aliquot of the sample with a ligand capable of binding to both SAM and to SAH, removing or degrading either SAM or SAH in a second aliquot of said sample, contacting said second aliquot with said ligand, and assessing the concentrations of SAM and/or SAH in said first and 15 second aliquots.

The assay of the invention may be used to determine the amount of SAH and SAM in a sample, and thus the SAH: SAM ratio in the sample can be calculated.

By ''degrading" it is meant that the ability of the 20 SAM or SAH to bind to the ligand is removed, eg by chemical transformation or by complexation with a binding partner to produce a reaction product or complex to which the ligand does not bind.

In the method of the invention, the SAH and SAM 25 content of the sample will typically be determined indirectly by determining the amount of SAH:ligand and/or SAM: ligand complex. Any standard immunoassay format may be used as discussed further below.

The ligand (or ligands) used in the assay is (are) 30 preferably an antibody (or antibodies). Such antibodies may be polyclonal or more preferably monoclonal.

However the ligand may also be a single chain antibody, an antibody fragment (e.g. a Fab fragment), variants thereof, an oligopeptide, an oligonucleotide, or a small 35 organic molecule, etc. Where the desired ligand is not already commercially available it may be produced by standard techniques, eg oligopeptide phage display, ......

- 6 - chemical library panning, computer aided ligand design, and antibody raising techniques. Polyclonal antibodies can be raised in animals such as mice, goats, sheep and rabbits using a suitable antigen. Monoclonal antibodies 5 can be raised by fusing an antibody-producing cell with a myeloma cell line, known as the hybridoma method.

Methods for preparing monoclonal antibodies are described in Gaffre et al. ''Preparation of Monoclonal Antibodies: Strategies and Procedures", Methods 10 Enzymology, 31-46, 1987). Hybridoma cells must be sorted to select clones which bind to the correct hapten. The antibodies raised in the Example of WO 00/40973 (Axis-Shield ASA) are suitable for use in the assay of the invention. For the present assay, a ligand 15 is selected which can bind to both SAM and SAH.

In the production of an antibody ligand, it may be necessary to couple SAH or SAM to an immunogenic carrier protein to enable antibodies to be raised. The antigen is conveniently conjugated to a macromolecule such as 20 Bovine Serum Albumin (BSA) or hemocyanin. In the present invention, the antibody is preferably raised to SAH or SAM conjugated to a macromolecule. Suitable macromolecules are described in WO 93/15220 (Axis Research AS).

25 The assay of the invention preferably uses immunological techniques for analyte (i.e. SAM and SAH) assessment. As used herein the term "assessing" is intended to include both quantitative and qualitative determination in the sense of obtaining an absolute 30 value for the amount or concentration of the analyte present in the sample, and also obtaining an index, ratio, percentage, visual or other value indicative of the level or relative level of analyte in the sample.

Examples of suitable immunological techniques to 35 assess the concentration of the analyte in a sample include methods involving reaction of the analyte with specific ligands which are themselves assessable

- 7 - directly, which form ligand:analyte conjugates which are assessable directly, or which form ligand:analyte conjugates which can be reacted further to generate - detectable products. Two common immunoassays are 5 competitive immunoassays and sandwich assays. In a competitive immunoassay a known concentration of labelled SAH or SAM would be added to the sample, and the amount of bound or free label would be measured at the end, from which the amount of unlabelled SAM and SAH 10 in the sample can be calculated. Such a competitive assay forms a preferred aspect of the invention.

The detection methods most commonly used in immunological techniques are calorimetry, fluorescence and chemiluminescence. Colorimetric detection is 15 normally performed by use of enzyme labelled analyte for competitive methods, or by use of an enzyme-labelled secondary ligand for sandwich assays (e.g. enzyme-linked immunosorbent assays - ELISA). The most commonly used enzymes are alkaline phosphatase and horseradish 20 peroxidase, which liberate a coloured product when incubated with a suitable substrate. Alternatively, fluorescent or chemiluminescent labels can be used, either on the competing analyses or on the ligands.

Fluorescence detection is normally performed by use 25 of a fluorescent antigen (tracer) which competes with the antigen (analyte) present in the sample for binding to the antibody. Such an assay is known as a fluorescence polarization immunoassay (FPIA), and the amount of polarized fluorescence emitted by the sample 30 is inversely proportional to the quantity of analyte in the sample.

Further to the use of calorimetric, fluorescent or chemiluminescent techniques, other photometric techniques may be used. Useful techniques also include 35 immunoprecipitation and particle agglutination techniques. Such techniques rely upon the use of ligands which on conjugation lead to precipitation or ......

- 8 - particle aggregation which can be detected by turbidimetric or nephelometric measurement. Where the ligand:competing analyte analog complex formation is inhibited by SAM or SAH, the content of SAM or SAH may 5 be assessed from the reduction in precipitation/aggregation. The assessment step in the process of the invention may alternatively use techniques which do not require labelling of a ligand or a competing analyte, e.g. 10 surface plasmon resonance (SPR). In one embodiment, SPR may be effected using a substrate-bound primary ligand (i.e. to bind the SAM and SAH in the first aliquot or the SAM or SAH in the second aliquot). In this embodiment a relatively low molecular weight ligand is 15 preferably used.

While analyte assessment may be by means of detection of a radioactive label on a ligand or a competing analyte, this is generally not preferred except in the case of 3H labelling and detection by 20 scintillation. The radiation emission from 3H labelled reagents is generally so low as to require no special handling, storing or packaging requirements.

In an especially preferred embodiment of the invention, the primary ligand is immobilized on the 25 membrane of a membrane-tipped pipette and a chromophore or fluorophore labelled secondary ligand capable of binding to conjugates of SAM or SAH and the primary ligand or to the primary ligand when unconjugated by SAM or SAH is used for analyte assessment. In this 30 embodiment, the aliquot is passed through the membrane, preferably repeatedly, to capture SAM and/or SAH "hereafter a liquid containing the secondary ligand is passed through the membrane, again preferably repeatedly, and the amount of secondary ligand captured 35 by the membrane is detected spectroscopically, preferably using a digital camera.

The assay of the invention utilizes a ligand that

_ 9 recognizes both SAM and SAH, and thus in the initial aliquot of the sample the concentration of both SAM and SAH will be measured. To enable the calculation of the concentration ratio of SAM to SAH it is necessary to 5 treat the second aliquot to remove or degrade one analyte, either SAH or SAM, and then to contact the aliquot with the same ligand. Thus, the concentration of the other ligand can be calculated. Using the concentration of both analyses from the first aliquot, 10 the concentration of the analyte removed from the second aliquot can easily be calculated. Either SAH or SAM can be removed or degraded to enable the assessment of the concentration of the remaining, unaffected analyte.

Preferably the method of removal or degradation is 15 enzymatic, e.g. using an SAH or SAM degrading enzyme.

Other chemical methods of removing one analyte can of course be used.

Preferably the second aliquot of the sample is subjected to a method that removes SAM and leaves SAH 20 unaltered in the sample. Enzymatic methods of removing SAM are preferred, and a suitable degrading enzyme is SAM decarboxylase. Any SAM degrading enzyme known in the art would be suitable, other than S-adenosyl-

methionine synthase, since this would result in 25 production of SAH. Alternatively, the SAM binding domain of methylene synthetase could be isolated from E. cold and added to the second aliquot to bind the SAM and prevent the subsequently added ligand from binding to SAM. Further, a chemical method could be used to 30 selectively remove SAM from the second aliquot of the sample, for example the pH of the aliquot could be raised by the addition of sodium hydroxide or another base. SAM is degraded in alkaline conditions. After alkali treatment, the pH may be reduced, e.g. returned 35 to normal (approximately pH 7), by any appropriate means, e.g. addition of acid. Alternatively, an aliquot of the sample can be heated to destroy SAM, since SAH is

- 10 more heat-stable than SAM. As another alternative the aliquot may be contacted with a substrate bound ligand specific for one of SAM and SAH. Such a substrate (e.g. antibody-loaded magnetic polymer beads) might then be 5 separated from the aliquot.

Optionally, the SAH can be removed enzymatically, and a suitable enzyme is S-adenosyl hemocysteine hydrolase which catalyses the reaction: 10 SAH ---------> Homocysteine + adenosine In order to drive the reaction in the shown direction, adenosine deaminase is preferably added to remove adenosine from the reaction mix.

15 Once SAM or SAH has been removed or degraded, the aliquot is contacted with the ligand and the concentration of the remaining analyte is assessed.

Preferably, the analyte removed is SAM.

To calculate the amount of SAM and/or SAH in the 20 aliquots of the sample, the results obtained from the assay can be compared to a calibration curve obtained using the assay with known concentrations of SAM and/or SAH. Standard or Calibration curves will thus preferably be constructed for SAH and SAM individually.

25 If SAM is removed in the second aliquot, the concentration of SAM is calculated by determining the concentration of SAH from the second aliquot signal, subtracting from the first aliquot signal the fraction of that signal caused by SAH, and comparing the 30 remaining portion of the first aliquot signal with a calibration curve for SAM.

The normal concentration of SAH in various human tissues varies between 0. 5-30 nmol/g, whereas SAM concentration varies between 2.5-300 nmol/g. Values in 35 plasma have been reported at different levels possibly affected by non-standardised methods of analyses. The levels published by most scientists for healthy people

- 11 are around 25 nmol/L for SAH and 100 nmol/L for SAM.

Thus the normal ratio between SAM and SAH in plasma is about 4 in healthy individuals. In patients with chronic renal disease this ratio is decreased to below 5 0.4. The concentration of SAH in red blood cells increases about seven times whereas the concentration of SAM remains the same. A plasma SAH: SAM molar ratio of < 2 can thus be used as a disease indicator while a ratio of < 1 can be used as an indicator of severe disease.

10 It will thus be appreciated that in the method of the invention it is not necessary to calculate absolute SAM or SAH concentrations or even absolute values of the SAH: SAM molar ratio; it may instead be sufficient to determine whether or not the SAH: SAM ratio is below or 15 above one or more such values indicative of disease or disease severity. This is particularly relevant where the assay method is used as a general screening tool patients may be identified as at risk, diseased or seriously diseased and referred for further diagnosis, 20 treatment or advice.

In a further embodiment of the invention homocysteine is also assessed in the sample. The advantage of additionally measuring homocysteine in the sample is to assess whether the individual is vitamin B 25 deficient, whereas the SAM/SAM ratio gives an estimation of the risk of cardiovascular disease. Conventional homocysteine assays, e.g. as described in WO 01/77670 (Axis-Shield), may be used in this regard.

The "sample" assessed using the assay of the 30 invention may be any biological or biologically derived sample. The sample may be of any biological fluid or tissue and may be pretreated prior to assay. Preferred samples are blood, plasma, serum, urine and tissue extracts, especially plasma.

35 As used herein the term "aliquot" relates to a sub sample of the biological sample or derivative thereof, or a portion or fraction thereof. The aliquots are

- 12 preferably of uniform volume, or size.

If homocysteine is to be assessed in the method of the invention, significant proportions of the homocysteine, particularly in plasma or urine, may be 5 bound by disulphide linkage to circulating proteins, such as albumin, and homocysteine may also be present in the form of other disulphide derivatives (generally homocysteine - cysteine conjugates). Thus, to obtain an estimation of total homocysteine it may be desirable to 10 use a reducing agent (e.g. dithiothreitol) to liberate covalently bound homocysteine prior to assessment.

Other suitable reducing agents are known in the art (see for example WO 01/77670) and include dithiothreitol (DTT), dithioerythrol (DTE), methyl iodide, thioredoxin, 15 lipoic acid or a borohydride. It may be desirable to subsequently treat the sample with an agent which neutralizes the reducing agent, e.g. one which binds to it, oxidizes it or otherwise depotentiates it. Suitable neutralizing agents are discussed in WO 01/77670 the 20 contents whereof are hereby incorporated by reference.

The assay method of the present invention may be used for the diagnosis and monitoring of pathological or potentially pathological conditions which are related to or manifested in the SAH, SAM and optionally 25 homocysteine content of bodily fluids or tissues. These include cardiovascular diseases and conditions such as atherosclerosis, blood diseases, vitamin deficiencies and/or inborn errors or metabolism.

In another aspect the invention provides an 30 analytical product, preferably a kit, for use in the assay of SAH and SAM in a sample, said product comprising: a SAM and SAH binding primary ligand; optionally a secondary ligand; and means for removing SAM or SAH from a sample or for converting SAM or SAH to 35 a species not bound by said primary ligand.

The invention will now be described by way of non-

limiting examples with reference to the drawings in

which: Figure 1 shows schematically the methionine cycle, the interaction between various metabolites, and the enzymes that catalyse these reactions; 5 Figure 2 shows a calibration curve (OD450 versus concentration of SAM/SAM). The experimental method is outlined in Example 1, and samples with known SAM/SAM concentrations were analysed and the results plotted on the calibration curve.

Example 1

1. 25pL of sample were diluted with 27pL PBS buffer, pH8.5 15 2. 25L sample + AL SAHase (40U/mL) + 25L adenosine deaminase (0.lU/mL) were incubated for 30 minutes at 37 C 3. 25pL of diluted samples (from steps 1 and 2) and 251 of SAH and SAM calibrators were pipetted into 20 the wells of a microtitre plate precoated with SAH conjugated to BSA.

4. 200L of an anti-SAH UCUS 99.05 (also recognising SAM) added to each well, and the plates were incubated for 30 min at 37 C.

25 5. The liquid was removed and the wells were washed with 3 times 400pL washing buffer (PBS) 6. 200pL of an HRP conjugated rabbit-anti-mouse antibody were added to each well and the plate were incubated at room temperature for 20 min. 30 7. The liquid was removed and the wells were washed as above 8. 100L of TMB+ substrate was added to each well and the plate was incubated for 10 min at room temperature. 35 9. 100L of a stop solution (0.8M sulphuric acid) was added to each well 10. The plate was shaken and read in a plate reader

- 14 (spectrophotometer) at 450nm.

The concentration of SAM in the samples were calculated for samples after pre-treatment (2) from the SAM 5 calibration curve. Apparent concentrations from all samples were calculated from the SAH calibration curve and the values obtained after pre-treatment (2) are subtracted from the corresponding not pre treated sample (1). This difference will represent the concentration 10 of SAH in the sample.

Thus, in this example, SAH was degraded in the second aliquot using SAH hydrolase, allowing the SAM/SAM ratio to be calculated.

Claims (2)

- 15 Claims

1. A method for assaying SAM and SAH in a sample, said method comprising the steps of contacting a first 5 aliquot of the sample with a ligand capable of binding to both SAM and to SAH, removing or degrading either SAM or SAH in a second aliquot of said sample, contacting said second aliquot with said ligand, and assessing the concentrations of SAM and/or SAH in said first and 10 second aliquots.

2. A kit for use in the assay of SAH and SAM in a sample, said kit comprising: a SAM and SAH binding primary ligand; optionally a secondary ligand; and means for removing SAM or SAH from a sample or for converting SAM or SAH to a species not bound by said primary ligand.