Research

Antibodies and Immunoassays

Our team of scientists performs integrated research, development, and technical support to market the antibodies against S-adenosylmethionine, or the SAM molecule. We are dedicated to develop several formats of immunoassays to detect SAM and S-adenosylhomocysteine (SAH) from biosamples efficiently. Immunoassays have been approved to be easy to use without requiring costly equipment, accurate, sensitive, specific and fast.

With our antibodies one can determine the concentration of the SAM molecule in samples without costly equipment. Arthus Biosystems of Richmond, California, offers a complete series of research tools for research laboratories worldwide to measure SAM effectively using immunoassay technology.

An Important Research Tool

Antibodies are known to be very useful in the process of discovering many biological aspects of the specific antigens with which the antibodies bind to specifically, including quantification, qualification, localization, diagnostics, etc. Commonly used areas are Enzyme Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC) , Immunofluorescence (IF), Flow Cytometry(FCM), Immunoprecipitation (IP), Western Blot, Dot Blot, Enzyme Linked Immunospot (Elispot), Fluorescence Activated Cell Sorting (FACS). Scientists have not had any anti-SAM antibody available until now. With our unique series of products, more and more important scientific findings relating to SAM methylation, transsufuration, aminopropylation in important biological processes including DNA repair, carcinogenesis, neurodegeneration, tissue differentiation,inflammation,aging,general pathogenesis will be revealed.

Antibodies against SAH

We have successfully generated several mouse monoclonal antibodies against S-adenosylhomocysteine (SAH). Compared with other SAH monoclonal antibodies, our mouse monoclonal antibody against SAH has a better sensitivity, can detect free SAH molecule, and can be used for measuring SAH from bio-samples.

Our products make it possible to measure the levels of S-adenosalmethionine and and S-adenosylhomocysteine at the same time in biosamples quickly and easily by common technicians in a lab with inexpensive instruments. This opens a door for the research communities to effectively measure methylation index in a variety of their research areas.

Clinical observations

By using quick, easy and convenient immunoassays, clinical labs and research labs are now able to perform more clinical observations on how SAM, SAH and methylation index relate to diseases and health status. We have measured SAM, SAH levels from plasma, serum and saliva samples from normal subjects and patients. Some results are summarized in here. Elevated serum levels of S-adenosylhomocysteine, but not SAM and homocysteine, are associated with cardiovascular disease in stage 5 chronic kidney disease patients (Clin Chim Acta. 2008; 395:106). More systematic studies are underway, which will speed up the process of properly applying these biomarkers in clinical practice and help assist in usage of SAMe as a medicine.

Antibodies against SAM and SAH

It is difficult to generate antibodies against this biologically extremely active metabolite. Our researchers have developed and produced monoclonal and polyclonal antibodies against S-adenosylmethionine, or SAM. The cross-reactions to its closely related analogs, such as S-Adenosylhomocysteine, or SAH, as well as methionine (Met) and adenosine, ATP, ADP and methythioadenosine (MTA) have been tested to be very trivial. To further verify the specificity of our anti-SAM antibodies, we proved the SAM sythesized by methionine adenosyltransferase (MAT) from ATP and Met can competitively bind the antibodies in a dose-dependent manner (Figure to the right).

For anti-SAH antibodies, cross reaction to S-Adenosylmethionine is below 3%; to Homocysteine, L-Cysteine, Adenosine, Glutathione, L-Cystathionine, ADP and ATP are < 1%; to MTA is about 5%. The relatively higher cross-reaction with SAM and MTA should not be concerned as physiological levels of SAM and MTA are much lower than 1uM, at which level no cross reaction with the antibody is observed. Our products make it possible to measure the level of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in samples quickly, accurately and easily by common technicians in a lab with inexpensive instruments.

How to measure it?

Since SAM is an intrinsically unstable molecule, the determination of its concentration in various biological fluids and tissues has been challenging. The current ways to quantifying SAM and SAH are LC, MS, and combinations of MS. HPLC was used but was proven to have obvious advantages.

The basis of LC-MS/MS and HPLC to measure SAM and SAH is not ideal for biologically active and unstable molecules. Therefore, these methods have limitations. These methods are laborious, time consuming and requires expensive equipment. LC-MS/MS is the prevailing method currently yet it has no consideration of biological activity-relevance of the metabolites it detects. Therefore, its usage in measuring SAM or SAH may not be accurate and complete from the biological perspectives. The chemical methods can only detect free form of SAM or SAH excluding any form of SAM or SAH associated with other biomolecules. Just because LC-MS/MS cannot detect from a sample the SAM molecules that falls within that narrow molecular weight specification may not always mean SAM has been totally degraded, disappeared and non-functioning. Furthermore, the SAM standard used in training LC-MS/MS are not the same as the SAM in living cells, yet in technology like LC-MS/MS, the exact same molecule as the one to be measured is required as the standard. Recently, there is a dispute on the GC-MS and LC-MS analytical methods that may not accurately measure metabolites due to unwanted changes caused by manipulation processes including sample extraction, preprocessing and during the measurement (http://cen.acs.org/articles/93/i42/Heated-Dispute-Over-Analytical-Method.html).

A simple, convenient method that does not require costly instrumentation, but take into considerations of boiological finctions such as capability of bingding relevant melecules definitely worths the efforts to indulge into. We are now able to provide all the basic reagents that are needed to quantitatively measure SAM and SAH with immunoassays and help solve technical issues.

Using our products to measure SAM levels in plasma and serum, many clinical studies are made possible to confirm the above statements. Many studies remain there for scientists of different fields worldwide to get into. We believe more and more important findings related to methylation index will come out in the near future.

Inconsistent data status quo

The normal range of SAM concentration in blood, serum, and plasma has not been determined accurately. Due to population, sample handling, pre-treatment, and detection methods, published results have been inconsistent. It has been recently reported that SAM level in human plasma is about 60nM-140 nM as measured by LC-MS/MS method (Klepacki J, Clin Chim Acta, 2013), whereas > 1.5 uM as measured by HPLC (Carolynk K, J. Chromatography, 1997). Even laboratories utilizing similar techniques have reported wide ranges of concentration for healthy patients. The data is, in general, most useful for comparison within the context of the particular studies. In view of the importance of SAM, it is desirable to have an easy and reliable method to measure its concentration in a biological sample. In immunoassays with high quality antibody against SAM, it is certain that the molecule which specifically and sensitively binds to the antibody is SAM. That is to say, specificity makes the detection method more reliable at the least.

More interesting areas of SAM application

Considerable research has been conducted in the past regarding the functions of SAM. For example SAM-e, incubated in vitro with human erythrocytes, penetrates the cell membrane and increases ATP within the cell thus restoring the cell shape. SAM-e is clinically useful in many apparently unrelated areas because of its important function in basic metabolic processes. One of its most striking clinical uses is in the treatment of alcoholic liver cirrhosis that, until now, remained medically untreatable. SAM-e has been administered to patients with peripheral occlusive arterial disease and was shown to reduce blood viscosity, chiefly via its effect on erythrocyte deformability.

Sam-e attenuates the damage caused by tumor necrosis factor alpha (TNFα) and can also decrease the amount of TNFα secreted by cells. SAM-e has been studied for its ability to reduce the toxicity associated with administration of cyclosporine A, a powerful immunosuppressor. It has also been studied in patients suffering from migraines and was found to be of benefit.

Pneumocystis pneumonia (PCP) occurs when the host is immunosuppressed. The PCP in humans is associated with advanced HIV disease, severe malnourishment in children, and treatments for cancers, advanced cancers, rheumatic disease, and the prevention of organ transplant rejection (Perez-Leal et al. Am J Respir Cell Mol Biol Vol 45, PP1142-1146, 2011). It is fatal if untreated. Therefore early diagnosis is very important. Studies have been done regarding S-adenosylmethionine (SAM) levels in the diagnosis of Pneumocystis Carinii Pneumonia (PCP) in patients with HIV Infection. Because S-adenosylmethionine is required by Pneumocystis carinii in vitro, Pneumocystis infection depletes plasma SAM of rats and humans, nicotine reduces SAM of guinea pig lungs, and smoking correlates with reduced episodes of Pneumocystis pneumonia (PCP) in AIDS patients. Chronic nicotine treatment increases lung polyamine catabolic/anabolic cycling and/or excretion leading to increased SAM-consuming polyamine biosynthesis and depletion of lung SAM (J. Biological Chemistry 2005; 280(15):15219-15228). Therefore, severely decreased plasma SAM level helps to predict occurrence of PCP in patients with immunocompromised conditions only. The best treatment regimens for PCP should include keep SAM level low since lowered SAM level helps to kill PCP pathogen, whereas, increasing SAM level is recommended for better outcomes of treating other diseases (not PCP) when SAM or methylation index is low.

In alcoholic liver, SAM is reduced whereas SAH and Hcy levels are increased. Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT), which catalyzes the biosynthesis of S-adenosylmethionine (SAMe), the principal methyl donor and, in the liver, a precursor of glutathione. MAT1A is expressed mostly in the liver, whereas MAT2A is widely distributed. MAT2A is induced in the liver during periods of rapid growth and dedifferentiation. In human hepatocellular carcinoma (HCC) MAT1A is replaced by MAT2A. This is important pathogenetically because MAT2A expression is associated with lower SAMe levels and faster growth, whereas exogenous SAMe treatment inhibits growth (Lu, SC et al. Alcoho 35(3):227-34, 2005).

Implications of measuring SAM and methylation index

Biomarker and therapeutics for neurodegenerative diseases such as dementia, Parkinson's disease, amyotrophic lateral sclerosis (ALS), etc. [ 1, 2, 3, 4 ]
Serum SAM level for diagnosis of Peumoncystis Carinii Pneumonia (PCP) arising from immune compromised conditions [ 5, 6, 7, 8, 9
Liver and bile duct diseases [ 10, 11, 19, 23, 25 ]
Cancers [12]
Nutritional, metabolic disorders and inflammation [ 13, 14, 17 ]
Congenital diseases such as Down syndrome and congenital heart diseases [ 15, 16 ]
Monitored treatment for depression, osteoarthritis and liver disorders with SAM-e [ 18, 20, 21, 24 ]
Methylation index and disease [22]

Methylation Index

S-adenosylmethionine (SAM) is an important molecule in methionine cycle pathway and one carbon metabolism pathway that are implicated in many metabolic abnomalities found in humans. Methylation index is defined as the ratio of SAM and SAH, which is a better way to evaluate methylation status of humans and other organisms. The level of S-adenosylmethionine (SAM) fluctuates depending on age, gender, race, body weight, diet, medicines taken, health and disease conditions. The internal unstable nature of SAM molecule and the dynamics of methylation process from an in vivo environment make measuring its de-methylation product S-adenosylhomocysteine (SAH) critical in the process of evaluating the status and extent of biochemical methylation process in a living body.

Below is a simple diagram (Melnyk S, Clin. Chem. 2000) that illustrates methionine cycle (a part of one carbon metabolism pathway) that details factors involved in the methylation process. Genetic or nutritional disturbances (e.g. deficiency of folate, Vitamin B12, B6, methionine or choline) that prevent efficient product removal of homocysteine (Hcy) or adenosine will lead to S-adenosylhomocysteine (SAH) accumulcation. Excess SAH and Hcy is thought to readily cross the cell membrance into the plasma. Elevated SAH or Hcy has been associated with increased risk of cardiovascular diseases, colon cancers, birth defects, recurrent pregnancy loss, central nervous system demyelinization and neuropsychiatric disorders. Increased SAH leads to methyltransferase inhibition, which reduces methylation of essential molecules such as DNA, RNA, protein, neurotransmitters, etc. This means methylation index is much reduced in many pathological processes.

Measuring Methylation Index

We are proud to be able to provide an effective method to easily and quickly measure methylation index. The research tools we provide will improve our further unerstandings of methionine cycle and one carbon metabolism pathways.

Monitoring S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) changes in the body fluids, tissues, and organelles are important as it has been anticipated that SAM and methylation index (SAM/SAH) have clinical implications in a broad range of disease conditions. It is also critically necessary to understand the impacts and consequences when SAM is used daily as a nutritional supplement and over-the-counter medicine in order to avoid unwanted side effects (insomnia, high blood pressure, gas, nausea, vomitting, diarrhea) and ensure that the proper treatment dosages are applied or taken.

Implication of Measuring Methylation Index

As discussed in other Research section, most likely methylation index is expected to be at least the same as measuring SAM alone. Carefully designed and thorough studies in clinical settings remain to be performed to expand our current knowledge on actual methylation index levels in healthy and diseased individuals, and how it is related to various disease conditions and stages. More accurate measurements of SAM and methylation index, as obtained by using our products, can be valuable in helping to diagnose existing or hidden pathologies. Many interesting discoveries are under way given the new research tools we are offering.

It has been shown that reduced methylation index is associated with vitamin deficiency in pregnant women and newborns, Down's Syndrome and Congenital Heart Disease (CHD).

Increasing evidence suggested that epigenetic mechanisms such as DNA methylation and histone tail modifications are dynamically regulated in neurons and play a fundamental role in learning and memory processes. Studies in animal models of neurodegenerative diseases (Alzheimer's disease, Parkinson’s disease, Huntington's disease, Amyotrophic Lateral Sclerosis) have highlighted the potential role of epigenetic drugs, including inhibitors of histone deacetylases and methyl donor compounds, in ameliorating the cognitive symptoms and preventing or delaying the motor symptoms of the disease, thereby opening the way for a potential application in human pathology (Coppede, F Frontiers in Genetics July 14, 2014).

Markers of neurodegeneration (APP, α-synuclein) are related to markers of methylation (SAM, SAH) in patients with Parkinson's disease. Better cognitive function was related to higher methylation potential, i.e. methylation index. Therefore, methylation index is a biomarker for Parkinson's disease (Obeid R, et al. Clinical Chemistry 55:10 1852–1860, 2009)

Highly conserved Methionine Adenosyltransferase (MAT) isoenzymes

It has been shown that changes of intracellular levels of S-adenosylmethionine (SAM) are related to the well beings of cells and organisms. The fluctuation of SAM level is cause by imbalance between SAM synthesis and metabolic pathways (methylation, transsulfuration and aminiopropylation) or degradation. S-adenosylmethionine biosynthesis is strictly and solely dependent on the activity of Methionine Adenosyltransferase (MAT, E.C.2.5.1.6), also known as S-adenosylmethionine synthetase. The methionine adenosyltransferase is encoded by two genes, MAT1A and MAT2A, encode for two homologous MAT catalytic subunits, α1 and α2. MAT1A is expressed in normal liver, and it encodes the α1 subunit found in two native MAT isozymes, which are either a dimer (MAT III) or tetramer (MAT I) of this single subunit. MAT2A encodes for a catalytic subunit (α2) found in a native MAT isozyme (MAT II), which is associated with a regulatory subunit (β) in encoded by MATA2B gene. MAT1A is expressed mostly in the adult normal hepatocytes, whereas MAT2A is widely distributed, of which mainly investigated are among highly proliferating liver cells, such as fetal liver and liver cancer cells. Except for parasites that rely on host for living, cells from all organisms have methionine adenyltransferase. MAT genes have been found to be exceptionally conserved throughout evolution. Enzymatic activity of MAT is critical in regulating the level of SAM and plays critical roles in methionine cycle and epigenetic study. In some situations expression of MAT1A or MAT2A have not changed yet the capability of synthesizing SAM are different. Therefore, measuring MAT activity is essential in evaluation the functions of MAT/SAM axis.

MAT-II is important for cell proliferation

Regulation of liver cell proliferation is a key event to control organ size during development and liver regeneration. MAT-II is an important enzyme in cellular metabolism and catalyzes the formation of S-adenosylmethionine (SAMe) from L-methionine and ATP. MAT2A is expressed in extrahepatic tissues. In adult liver, increased expression of MAT2A is associated with rapid growth or de-differentiation of the liver. The influence of MAT expression on liver growth and injury was further demonstrated using a MAT1A knockout mouse model (Martínez-Chantar, M.L, 2002. FASEB J. 16:1292). In this model, absence of hepatic MAT1A is compensated by induction of MAT2A. These animals exhibit chronic hepatic SAMe deficiency, are prone to liver injury and develop hepatocellular carcinoma (HCC).

In human HCC, both promoter hypomethylation and increased expression of c-Myb and Sp1 with subsequent trans-activation of the MAT2A promoter contribute to transcriptional up-regulation of MAT2A in HCC. Nuclear binding of NF-κB and AP-1 to the promoter of human glutamate-cysteine ligase catalytic subunit was increased in HCC. In HepG2 cells, a human hepatocellular carcinoma, both NF-kB and AP-1 are required for basal MAT2A expression and mediate the increase in MAT2A expression observed in response to TNF-α treatment (Yang H, Induction of Human Methionine Adenosyltransferase 2A Expression by Tumor Necrosis Factor α. J Biochem. 2003. 278:50887). Up-regulation of MAT2A provides growth improvement and S-adenosylmethionine and methylthioadenosine thus can block mitogenic signaling in colon cancer cells.

In H35 hepatoma cells, growth factors such as hepatocyte growth factor (HGF) and insulin up-regulated MAT2A expression. Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-phosphate kinase (PI 3-K) pathways were required for both HGF-induced cell proliferation and MAT2A up-regulation. The inhibition of these pathways was associated with the switch from the expression of fetal liver MAT2A to the adult liver MAT1A isoform. Treatment of H35 hepatoma cells with MAT2A antisense oligonucleotides decreased cell proliferation induced by HGF. Growth inhibitors such as transforming growth factor (TGFβ) blocked HGF-induced MAT2A up-regulation while increasing MAT1A mRNA levels in H35 cells. MAT2A expression is required for the process of liver cell proliferation (Paneda C, Liver cell proliferation requires methionine adenosyltransferase 2A mRNA up-regulation. Hepatology. 2002 35:1381).

The consequence of the choline-deficient and ethionine-supplemented (CDE) diet is depletion of hepatic S-adenosylmethionine (SAM). After 48 h of the CDE diet, SAM levels decreased about half and MAT1/III disappeared via post-translational mechanisms, whereas MAT-II increased via pretranslational mechanisms. CDE-fed young mice exhibited extensive necrosis, edema, and acute pancreatic inflammatory infiltration and treatment by SAM can help prevent and recover the injury. Old female mice consuming the CDE diet that do not develop pancreatitis showed a similar fall in pancreatic SAM level. Although the pancreatic SAM level fell by more than 80% in the MAT1A knockout mice, no pancreatitis developed. MAT1A is highly expressed in the normal pancreas as well as pancreatic acini, which is in contrary to what we commonly believe that MAT-I/III is liver-specific. The CDE diet causes dramatic changes in the expression of MAT isozymes by different mechanisms. In contrast to the situation in the liver, where absence of MAT1A and decreased hepatic SAM level can lead to spontaneous tissue injury, in the pancreas the roles of SAM and MAT1A appear more complex and remain to be defined (Lu SC, Role of S-adenosylmethionine in two experimental models of pancreatitis. FASEB J 2003 17(1): 56). It is interesting to see age-dependent pathological changes in tissues. It might be related to the fact that normal human SAM level is also age dependent. As SAM is the sole methyl donor for the proper methylation of DNAs, RNAs, hormones, lipid proteins and neurotransmitters, reduced or depleted SAM level will definitely affect the normal methylation of critical bio-molecules through epigenetic mechanisms.

Epigenetic regulation of methionine adenosyltransferase and HCC therapy

MicroRNAs (miRNAs) and MAT1A are dysregulated in HCC, and reduced MAT1A expression correlates with worse HCC prognosis. Expression of miR-664, miR-485-3p, and miR-495, potential regulatory miRNAs of MAT1A, is increased in HCC. Knockdown of these miRNAs individually in Hep3B and HepG2 cells induced MAT1A expression, reduced growth, and increased apoptosis, while combined knockdown exerted additional effects. Subcutaneous and intraparenchymal injection of Hep3B cells stably overexpressing each of this trio of miRNAs promoted tumorigenesis and metastasis in mice. Treatment with miRNA-664 (miR-664), miR-485-3p, and miR-495 siRNAs reduced tumor growth, invasion, and metastasis in an orthotopic liver cancer model. Blocking MAT1A induction significantly reduced the antitumorigenic effect of miR-495 siRNA, whereas maintaining MAT1A expression prevented miRNA-mediated enhancement of growth and metastasis. Knockdown of these miRNAs increased total and nuclear level of MAT1A protein, global CpG methylation, lin-28 homolog B (Caenorhabditis elegans) (LIN28B) promoter methylation, and reduced LIN28B expression. Upregulation of miR-664, miR-485-3p, and miR-495 contributes to lower MAT1A expression in HCC, and enhanced tumorigenesis may provide potential targets for HCC therapy (Yang H, HEPATOLOGY 2013. 57(5): 2081).

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