Immunoassay of S-adenosylmethionine and S-adenosylhomocysteine: the methylation index as a biomarker for disease and health status
© The Author(s) 2016
Received: 12 February 2016
Accepted: 15 November 2016
Published: 28 November 2016
S-Adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are relevant to a variety of diseases. Previous reports that quantified SAM and SAH were based on HPLC or LC–MS/MS. No antibody against SAM has been generated, and the antibody against SAH cannot be used with blood samples. Immunoassays have not been used to measure SAM and SAH. In this study, ELISA was used to measure blood SAM and SAH levels.
Specific antibodies against SAM were produced for the first time using a stable analog as the antigen. The monoclonal antibodies against SAM and SAH were characterized. No cross-reactivity was detected for the analyzed analogs. For the anti-SAM antibodies, the ELISA sensitivity was ~2 nM, and the affinity was 7.29 × 1010 L/mol. For the anti-SAH antibodies, the sensitivity was ~15 nM, and the affinity was 2.79 × 108 L/mol. Using high-quality antibodies against SAM and SAH, immunoassays for the detection of SAM and SAH levels in blood and tissue samples were developed. Clinical investigations using immunoassays to measure SAM, SAH and the methylation index (MI) in normal and diseased samples indicated that (1) the SAM level is age and gender dependent; (2) the SAM level is associated with the severity of liver diseases, inflammatory reactions and other diseases; and (3) the methylation index (MI) is significantly reduced in many diseases and may serve as a screening biomarker to identify potentially unfavorable health conditions.
It is possible to generate antibodies against active small biomolecules with weak immunogenicity, such as SAM and SAH, using traditional hybridoma technology. The antigens and antibodies described here will contribute to the development of immunoassays to measure SAM, SAH and related molecules. These assays enable the MI to be measured specifically, accurately, easily and quickly without costly equipment. This preliminary study indicates that the MI could be an effective indicator of general health, except under conditions that may alter the value of the MI, such as special diets and medications.
S-Adenosylmethionine (SAM, SAMe, or AdoMet) is a naturally occurring compound found in all living cells. SAM is a critical metabolite derived from adenosine triphosphate (ATP) and methionine (Met) via methionine adenosyltransferase (MAT EC184.108.40.206). SAM is an essential, metabolically pleiotropic molecule that participates in multiple cellular reactions as the precursor for the synthesis of glutathione and the major methyl donor for the methylation of nucleic acids, phospholipids, histones, biogenic amines, and proteins. SAM is the key molecule in the Met cycle and plays important roles in transmethylation, transsulfuration and aminopropylation. Methylation processes are the central biochemical basis of the neuropsychiatry of folate and B12 metabolism. The de novo synthesis of Met requires vitamin B12, which is involved directly in the transfer of the methyl group to homocysteine (HCys). Deficiency in either vitamin B12 or folate is considered the cause of low methylation index (MI). Metabolic hypomethylation might be one mechanism related to adverse clinical outcomes in B12-deficient individuals . SAM provides the methyl group during the production of essential biomolecules such as carnitine (fat burner), acetyl-l-carnitine (neuro-nutrient, membrane-transporting agent), phosphocreatine (primary ATP reservoir), epinephrine/adrenalin (endogenous catecholamine, stress hormone and neurotransmitter), phosphatidylcholine (the most important membrane phospholipid), and melatonin (circadian rhythm modulator). Combined administration of N-acetyl cysteine (an antioxidant and glutathione precursor that protects against Abeta neurotoxicity), acetyl-l-carnitine (increases ATP levels, protects mitochondria, and buffers Abeta neurotoxicity), and S-adenosylmethionine (facilitates glutathione usage and maintains acetylcholine levels) enhances or maintains cognitive function and attenuates or prevents aggression, in mouse models of aging and neurodegeneration. Treatment with this nutraceutical combination can compensate for a lack of dietary folate and vitamin E . Transsulfuration begins with SAH, the residual structure of SAM after donating the methyl group (trans-methylation). Hydrolysis of SAH yields HCys, which is converted to cystathionine, cysteine, and then glutathione, the hepatocellular antioxidant and life-saving detoxification agent. Aminopropylation is another process that is initiated by SAM through decarboxylation. Decarboxylated SAM is coupled to putrescine to generate spermidine and spermine, which are critical for cell growth, differentiation and DNA and RNA stability. Methylthioadenosine (MTA), the by-product of polyamine synthesis, is a powerful analgesic and anti-inflammatory agent that may be responsible for the clinical benefits observed in the treatment of osteoarthritis, rheumatoid arthritis and fibromyalgia with SAM [3, 4].
Defects in methylation lead to cell dysfunction and accompanying changes in SAM levels. Poor methylation or SAM deficiency has been implicated or related to the development of birth defects , cardiovascular disease , cancers , liver disease , and many other diseases [9–14, 18, 20, 21]. SAM levels have been identified as a diagnostic marker for Pneumocystis carinii pneumonia (PCP) in patients with immune-compromised conditions . SAM has been used clinically for the treatment of liver disease, arthritis , and depression . SAM produced cognitive improvement in patients with Alzheimer’s disease . Treatment with SAM has been confirmed to be as effective as prescription tricyclic antidepressants  and non-steroidal anti-inflammatory drugs (NSAIDS) , with efficacy for the treatment of some liver conditions, such as cholestasis in pregnancy and intrahepatic cholestasis associated with liver diseases [23, 24]. More significantly, SAM is well tolerated, and no serious side effects have been observed. SAM provides rapid relief and is superior to tricyclic antidepressant treatment .
A deficiency of the major dietary sources of methyl donors, Met and choline, leads to the formation of liver cancer in rodents . Extra-hepatic tumor formation increased in carcinogen-treated animals fed diets low in methyl donors including Met, choline, and folic acid . Several mechanisms explain the enhancing effects of dietary methyl deprivation on carcinogenesis , of which the most accepted is that dietary methyl deficiency results in abnormal DNA methylation . A critical metabolite in this hypothesis is SAM, the sole methyl donor in the body. Studies have linked deficiencies in folic acid, vitamin B12, and Met with an increased risk of cancer in various organs . The question thus arises whether such deficiencies exert their activities through diminished availability of SAM. Studies have also demonstrated the utility of determining erythrocyte SAM and SAH and of plasma HCys in assessing disease status .
Due to the importance of SAM and SAH, an easy and reliable method is needed to measure their concentrations in a biological sample. A classic method for measuring SAM and SAH in rat liver utilizes the tripolyphosphatase activity associated with SAM synthetase in rat liver . Tripolyphosphatase activity is stimulated by low concentrations of SAM . The reported sensitivity of this method is 0.1 nmol of SAM in an assay volume of 0.1 mL (i.e., 10−6 M). The samples are lyophilized, homogenized in acid, and centrifuged. The supernatant is then passed through Dowex 1 to remove endogenous inorganic phosphate and other potential interferons in the tissue. Great care is needed to avoid inorganic phosphate contamination from all reagents, including the enzyme preparation and glassware. The disadvantages of this assay are a clear lack of specificity, low sensitivity (1 µM), and difficulties related to controls and comparisons among assays in different laboratories. Another common method for measuring SAM in tissues or biological fluids is HPLC or electrophoresis after sample preparation, which normally comprises protein precipitation and/or extraction . Post-column detection may include derivatization followed by measurement by absorption, fluorescence, or electrochemical changes and, more recently, by LC–MS/MS . These methods are laborious, time-consuming and require expensive equipment. LC–MS/MS does not consider the biological relevance of the detected metabolites. Therefore, the use of LC–MS/MS to measure SAM and SAH may not be accurate or complete from a biological perspective. Chemical methods can only detect the free form of SAM or SAH at the time of sample collection and exclude any SAM or SAH associated with other biomolecules. The inability of LC–MS/MS to detect SAM molecules that fall within the specific molecular weight range defined by LC–MS/MS in a sample does not indicate that SAM has been completely degraded, lost or is not functional. Furthermore, the SAM standard used to train LC–MS/MS is not identical to SAM from living cells. However, for a technology such as LC–MS/MS, a molecule identical to the molecule of interest is required as the training standard. Recent studies have suggested that GC–MS and LC–MS analytical methods may not accurately measure metabolites due to changes caused by lengthy and high-temperature manipulation processes during sample extraction, preprocessing and measurement .
SAM is an intrinsically unstable molecule, and its optical density maximum of 258–260 nm is not unique. Thus, the determination of the concentration of SAM in various biological fluids and tissues is a challenging task. A simple, convenient method that does not require costly instrumentation is clearly desirable for determining the biological concentration of SAM and monitoring changes in SAM levels in body fluids, tissues and organelles. The MI is defined as the ratio of the concentration of SAM to the concentration of SAH. Given the important roles of SAM and SAH in various pathological processes, it is desirable to conveniently measure the levels of SAM and SAH using methods that can be performed in typical research and clinical laboratories. The availability of specific antibodies against SAM and SAH has enabled the development of various forms of immunoassays to satisfy different needs. In this study, the generation and characterization of anti-SAM and anti-SAH antibodies as well as their applications are described. The usefulness of blood SAM and MI as markers for evaluating the roles of SAM in the onset and development of human diseases was also evaluated. This investigation included the determination of SAM, SAH, and MI in blood samples collected from normal and diseased subjects.
The hapten used to generate anti-SAM antibodies
Many SAM analogs were synthesized as shown in US patent No. 8,344,115 and evaluated to identify the best analog to generate the best anti-SAM antibodies. One of the most promising analogs, 5′-N-methyl, 5′-N-butyryl-5′-deoxyadenosine, or 5′-((3-carboxypropyl)methylamino)-5′-deoxy-adenosine, abbreviated as aza-deamino-SAM or shortened as aza-SAM, was employed to immunize Balb/c mice. All asymmetric carbons in aza-SAM maintained their chirality.
Preparation of KLH-aza-SAM
To a flask containing 15 mg of aza-SAM that was vacuum-dried overnight, appropriate amounts (proportional to aza-SAM) of DCC (N,N′-dicyclohexylcarbodiimide), NHS (N-hydroxysuccinimide) and KLH (keyhole limpet hemocyanin) were added. Approximately 1.5 mL of dry DMF (dimethylformamide) was then added to the flask. The solution was stirred at room temperature. Formation of the NHS ester was confirmed by thin layer chromatography. The final volume after dialysis was 29.5 mL with a concentration of 0.6 mg/mL.
Preparation of PLL-aza-SAM (poly-l-lysine-conjugated aza-SAM)
A 4.72-mg quantity of aza-SAM was dissolved in 1 mL of DMF with 6.5 mg EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Next, HCl and 4 mg of NHS were added, followed by stirring at room temperature in the dark overnight. Then, 1.5 mg of PLL was dissolved in 1 mL of 10 mM PBS pH 8.2. Aza-SAM was then added slowly to the PLL solution, and the mixture was incubated overnight in the dark. The reaction mixture was dialyzed for 48 h against 10 mM PBS, pH 7.3.
Preparation of BSA-SAH
A 3.8-mg quantity of SAH (Sigma) was dissolved in 1.5 mL of PBS. Then, 10 mg of EDC HCl (Sigma) and 4.5 mg of NHS were added, and the mixture was stirred at room temperature for 24 h. We then dissolved 12.9 mg of BSA in PBS. The SAH was added to the BSA solution, and the mixture was incubated at 4 °C in the dark overnight.
Preparation of HRP-conjugated antibodies
To develop a more efficient ELISA, HRP (horseradish peroxidase, Sigma) was directly conjugated to mouse anti-SAM and mouse anti-SAH antibodies. Five milligrams of HRP (dissolved in deionized water at 10 mg/mL) was added to 0.5 mL of freshly prepared 0.06 M sodium periodate and incubated at 4 °C for 30 min. Approximately 5 mg of antibody was added to the HRP solution and dialyzed overnight against 50 mM carbonate buffer, pH 9.6. Approximately 2 mg of sodium borohydride was added to the reaction mixture and incubated in the dark at 4 °C for 2 h with shaking once every 30 min. The sample was then dialyzed against 0.01 M PBS, pH 7.2, for at least 18 h.
Procedures for generating monoclonal antibodies against SAM and SAH
Mouse monoclonal antibodies were produced based on procedures developed in a previous work . Immunization was performed via subcutaneous injection of 0.1 mg of antigen at multiple sites into Balb/c mice. The initial injection consisted of a 1:1 mixture of complete Freund’s adjuvant and aza-SAM-KLH or SAH-BSA conjugate solutions in PBS upon emulsification. The subsequent 3 injections were similar except for the use of incomplete Freund’s adjuvant. Blood was collected from each immunized mouse at 1–2 weeks. The obtained antisera were then evaluated to determine the immune response and the antibody titer. Mice with satisfactory titers were primed via intravenous injection with immunogen three days prior to its sacrifice. The spleens of the mice were harvested and homogenized. The spleen cells were then fused with myeloma SP2/0 cells, and the fused cell suspension was plated out in 96-well microtiter plates. Hybridomas were grown in RPMI 1640 medium enriched with 18% fetal bovine serum, HAT and HT supplements and then screened. Clones that were positive for SAM or SAH were selected for further studies, including cross-reactivity tests. After the clones were obtained, the cells were injected into mice for ascite production. The antibody was then purified from the ascites using a protein A affinity column.
Methionine adenosyltransferase (MAT)-catalyzed SAM biosynthesis
Solutions of 1 mM ATP and L-Met (Sigma) were prepared in 100 mM Tris, 100 mM KCl, 20 mM MgSO4, and 1% ProClin, pH 7.42. Recombinant E. coli MAT was added to the above substrates at a concentration of 0.6 mg/mL or 1 mg/mL, respectively. The reaction mixture was analyzed in duplicate using the SAM cELISA described in this section. The synthesized SAM was measured every 10 min after incubation at 37 °C for 20–80 min.
Blood sample collection
Informed consent for sample collection and publication of clinical data was obtained from the participants. Sera were obtained from 81 normal volunteers, 310 plasma samples from the Changsha Blood Center and sera from 505 patients. For the plasma samples, peripheral venous blood was collected in tubes with EDTA and mixed well. The tubes were immediately cooled to 4 °C and centrifuged at 2000 g or higher for 15 min within 30 min after blood collection to obtain plasma. The plasma was either used for the measurements or frozen at −20 °C for future use. Serum samples were collected in serum separating tubes and placed in the refrigerator for approximately 2 h until blood coagulation occurred. The remaining steps were similar to those used for plasma collection. Plasma from the blood center was stored in the refrigerator for 1–7 h before centrifugation and freezing.
The Supplemental Data (Additional file 1) provides details regarding disease information. Patient samples were collected from clinical laboratories with no further information about the patient and disease conditions, status and treatments. Statistical analyses were performed using R by Dr. Huaitian Liu (National Cancer Institute, National Institute of Health, Rockville, MD) and Sydney Wong (University of California, Berkeley, CA).
Competitive ELISA (cELISA)
To quantify plasma SAM and SAH levels, direct cELISA was developed by immobilizing antigens to a solid plate first, followed by the addition of SAM or SAH and HRP-labeled anti-SAM or anti-SAH antibodies. Antigens from a sample or standards competed with the corresponding fixed amount of immobilized antigens for binding to specific HRP-labeled antibodies. The final HRP substrate absorption values at 450 nM were inversely proportional to the amount of detected antigen, i.e., the higher the OD450 value, the lower the level of SAM or SAH in a sample. The wells of a 96-well ELISA plate (Corning high-affinity strips for ELISA) were coated with 0.05 μg/mL PLL-aza-SAM or 0.5 μg/mL BSA-SAH at 4 °C overnight. A series of SAM or SAH standards (aza-SAM from 0 to 960 nM and SAH-Na from 0 to 100 nM) and samples were added, followed by a 1:30,000 dilution of HRP-anti-SAM to a final volume of 100 μl. The plate was sealed and incubated at 37 °C for 60 min. The plate was then washed 3 times with PBST (10 mM PB, 150 mM NaCl, pH 7.4, 0.1% Tween 20). TMB (3,3′,5,5′-tetramethylbenzidine) was added to the wells (InnoReagents), and the plate was sealed and incubated for 15 min at 37 °C. The reaction was then stopped, and the OD450 values were read using a Multiskan FC reader (Thermo Fisher). The standards were generated using heat-inactivated pooled normal human plasma to minimize the matrix effect.
All analogs used in the cross-reactivity tests were from Sigma.
Results and discussion
Antibodies and immunoassays
Characterization of the anti-SAM monoclonal antibodies 118-6 and 84-3
Cross-reactivity with SAM analogs was tested using up to 100-fold higher dosages of analogs than that of SAM antigen with the established cELISA method. Figure 2 shows a cELISA using the anti-SAM monoclonal antibody 118-6. In this assay, 0.1 µg/mL PLL-aza-SAM was used to coat a 96-well plate. Serial dilutions of Aza-SAM, SAH, adenosine, L-Met, ADP (adenosine diphosphate), ATP, MTA and 1:35,000 of 118-6 antibody were added. HRP-conjugated goat anti-mouse IgG antibody and TMB substrate were used to develop the results. The cross-reactivity of antibody 84-3 was similar to that of 118-6. At concentrations exceeding 10 μM Met, SAH, MTA, adenosine, ADP, and ATP, competition for the coated antigen (PLL-aza-SAM) with the HRP-antibody did not occur (data not shown). No competitive inhibition of the HRP signal was observed using these analogs, whereas inhibition was clearly evident when SAM was added at a much lower dosage than the analogs. These data indicate that the cross-reactivity of all analogs was <1%.
When the antibodies were adjusted to 1 mg/mL in an indirect ELISA assay, the titer of the anti-SAM monoclonal antibodies ranged from 1:10,000 to 1:100,000 depending on the criteria and conditions used.
To demonstrate the ease and affinity of antigen and antibody binding, ELISA assays were performed using different amounts of coated antigens. For the anti-SAM clone 118-6, when antigen was coated at a higher concentration of 0.2 μg/mL, half the maximum OD was obtained at a dilution of 1:54,800. The corresponding antibody concentration was (Ab) = (1 mg mL−1/160,000 g mol−1)/54,800 = 1.14 × 10−10 M, where 160,000 is the molecular weight of the antibody. When the coating antigen concentration was less than 0.1 μg/mL, half the maximum OD was observed at 1:44,300. The corresponding antibody concentration was (Ab)t = (1 mg mL−1/160,000 g mol−1)/44,300 = 1.41 × 10−10 M, where n = 0.2/0.1(μg/mL) = 2, Ka = (n − 1)/2*(n(Ab) − (Ab)t) = 5.75 × 109 L/mol = 1.74 × 10−10 M. For clone 84-3, when the coating antigen was 0.1 μg/mL, half the maximum OD was observed at approximately 1:13,0000. When the coating antigen was 0.05 μg/mL, half the maximum OD was detected when 84-3 was diluted at approximately 1:70,000. Using the same method as above, Ka = (n − 1)/2*(n[Ab] − [Ab]t) = 7.29 × 1010 L/mol = 1.37 × 10−11 M.
OD450 measured using different amounts of coating antigens with antibody 84-3
SAM standard (nM)
PLL-aza-SAM 0.15 µg/mL
Sensitivity of mouse anti-SAM antibody 118-6
SAM standard (nM)
Characterization of the anti-SAH monoclonal antibody 301-1
Using the same method described above for affinity, when the coating antigen was 1 μg/mL, half the maximum OD was observed at a dilution of 1:1900. When the coating antigen was 0.5 μg/mL, half of the maximum OD was observed at approximately 1:1100. Thus, Ka = (n − 1)/2*(n[Ab] − [Ab]t) = 2.79 × 108 L/mol = 3.6 × 10−9 M. The titer of 301-1 was in the range of 1:4000–8000 when the antibodies were adjusted to a concentration of 1 mg/mL in an indirect ELISA assay.
SAH standard curve and sensitivity of antibody 301-1
SAH is normally low in humans, and thus the ability to measure low levels of SAH is essential. The sensitivities of the anti-SAM and anti-SAH antibodies reported herein are the best to date. Using direct cELISA, the minimum detection limit was approximately 2 nM for SAM and 15 nM for SAH. Other more sensitive immunological technologies can be developed to further reduce the minimum detection limit, increase the range of detection, or improve the assays via faster, easier and higher throughput and better quality control and precision. Sensitivity is essential for most IVD (in vitro diagnosis) applications.
SAH is metabolically linked to SAM, and the structure of SAH contains one less carbon (a methyl group) than SAM. The co-existence and structural similarity of SAM and SAH present a great challenge in the development of a method for the specific determination of the concentration of either molecule in a biological sample. The unstable nature of SAM further complicates its determination. As the immediate precursor of HCys produced in the body, SAH has recently been suggested to be a more sensitive indicator of the risk of vascular diseases than plasma HCys . The total plasma concentration of SAH is normally much lower than that of HCys. As with SAM, SAH has no distinct absorption characteristics, hindering its detection in serum or plasma. Because SAH is the product of all methylation reactions involving SAM as a methyl donor, an increased concentration of SAH in tissues is frequently accompanied by a decreased concentration of SAM. Therefore, the ratio of SAM to SAH is considered a more sensitive indicator than the concentration of either SAM or SAH alone, particularly when their changes are subtle during the early stages of dysfunction or under abnormal conditions. In this work, we established convenient immunoassays to measure the MI quickly without costly instrumentation.
The level of SAM varies with age and gender
Normal distribution of SAM and SAH levels in normal serum or plasma samples
Distribution of normal plasma SAM levels in different genders and age groups
Distribution of normal human plasma SAM levels by gender
Male no. (%)
Female no. (%)
Distribution of normal human plasma SAM levels in different age groups
Age 18–30 No. (%)
Age 31–40 No. (%)
Age 41–50 No. (%)
Age 51–60 No. (%)
Figure 7 shows the average SAM levels in different age and gender groups. In both genders, the SAM level continuously decreased with increasing age, indicating that SAM might be directly or indirectly involved in the aging process. Table 5 shows that a higher percentage of people older than 51 years had SAM levels of less than 60 nM, which indicates that older age was associated with decreased SAM levels. ANOVA analysis was performed with SAM, SAH and the MI as the response variables and age and gender groups as the explanatory variables. The p value was 0.0343 for the age group in terms of SAM (significant at level α = 0.05) and 0.0511 for the gender group in terms of SAM (significant at level α = 0.1). These results indicate that the decreasing SAM trends for age and gender, as shown in Fig. 7, are statistically significant but at different levels. The levels of SAM in females were significantly higher than those in males. In a normal population, the younger a person, the higher the level of SAM. The SAM level was significantly higher in the 18- to 30-year-old age group than in any other age group older than 31 years. The SAM level in children and youth younger than 18 years was not evaluated.
A previous report indicated global DNA hypomethylation in older individuals compared with a younger population. The data indicated that age-related variations in the global DNA methylation profile of leukocytes might be modulated by the daily intake of carbohydrates, lipids, vitamin B6, and magnesium and associated with serum protein levels. These nutritional factors may cause the SAM level to decrease greatly due to metabolic changes that occur as people age, contributing to the hypomethylation observed in the elderly population.
Men and women have different hormone levels and considerably different hormone compositions, and thus their metabolisms differ. Liver cells express estrogen receptors that are related to development, lipid metabolism and insulin sensitivity, hepatic carcinogenesis, protection from drug-induced toxicity and fertility. In addition, estrogens can modulate growth hormone activity in the liver by acting centrally (pituitary growth hormone) and by modulating the GHR-JAK2-STAT5 pathway . Age and gender affect metabolic syndrome and metabolic disorders in all-cause and cardiovascular disease-related mortality . The Met cycle may be related to the pathways indicated above, and further investigations may elucidate the gender-dependent SAM level. A recent clinical trial assessing the efficacies of SAM for the treatment of depression in female and male patients indicated that gender might impact the antidepressant efficacy of SAM, with a greater therapeutic effect observed in males  only. The average lower level of SAM in males than in females may contribute to the underlying mechanisms.
SAM and MI are reduced in disease
Results of t test in diseases from different studies
High blood pressure
High blood pressure
High blood pressure
Chronic liver diseases
Chronic liver diseases
Chronic liver diseases
In a controlled study conducted at Xiangya Hospital, SAM levels were measured in patients with severe liver conditions such as acute viral hepatitis, cirrhosis, and liver failure. Patients who received SAM as a liver-protecting medicine were excluded from the study. We observed that SAM levels and the MI in different types of liver diseases, including hepatitis, carcinoma, cirrhosis and liver failure, were very different from those in the normal population. These findings were striking. Patients with hepatitis were diagnosed with acute severe viral hepatitis (B and C). Patients with cirrhosis were also in an advanced stage requiring aggressive treatments in the hospital. Liver failure indicated that liver functions could no longer be compensated, and laboratory results revealed a severe loss of liver function. In hepatitis, 19.57% of the 46 samples had SAM levels higher than 120 nM, in contrast to 95% of normal samples; 4.35% had SAM levels greater than 240 nM, in contrast to 69.14% of the normal samples; and 94.87% of the 46 samples had an MI less than 0.5, in contrast to 8.64% of the normal samples. Among patients with hepatocyte carcinoma, 4 of 14 (28.57%) exhibited SAM levels greater than 120 nM, 7.14% had SAM levels greater than 240 nM, and 85.71% had an MI less than 0.5. Among patients with cirrhosis, 3 of 20 (15%) exhibited SAM levels greater than 120 nM, 5% had SAM levels greater than 240 nM, and 89.47% had an MI of less than 0.5. Finally, all 19 patients with liver failure had SAM levels less than 120 nM and an MI of less than 0.5. These results demonstrate that SAM levels and MI values were significantly reduced compared with those in the normal controls; thus, SAM levels could be used as a biomarker to assist in the diagnosis of liver diseases.
SAM levels and MI values in brain diseases
Diseases (case #)
% of SAM > 240 nM
% of SAM > 120 nM
% of MI > 2
% of MI > 1
% of MI < 0.5
Normal plasma (310)
Normal serum (81)
Cerebral hemorrhage (10), embolism (6), infarction (4)
Parkinson’s disease (10), depression (10)
Table 6 Study C shows the t-test results for other diseases versus normal serum controls. The decreases in SAM in cerebrovascular diseases, kidney diseases, chronic liver diseases, and respiratory diseases were not significant compared with the normal controls, whereas the increases in SAH in this diseases were significant. The levels of SAM decreased significantly in diabetes, high blood pressure, heart diseases and inflammation. However, the decreases in the MI were all extremely significant for all analyzed diseases, which suggests that the MI is a better biomarker for these diseases.
SAM and inflammation
Interesting results were obtained for the levels of SAM, SAH and MI under conditions of inflammation (Table 6 Study C). The levels of SAM were significantly reduced in inflammation conditions, whereas the increase in SAH in inflammation conditions was not significant. The diseases included in this inflammation group were mostly digestive, vascular and other benign inflammatory conditions. Common features of the group were chronic and widespread inflammatory responses. Thus, SAM appears to play a larger role in these conditions than SAH. This phenomenon is likely related to the important role of SAM in inflammatory processes in which internal strong anti-inflammatory factors such as spermine or spermidine or MTA are generated as a result of SAM aminopropylation. Under certain pathological conditions, SAM aminopropylation might be blocked or deficient due to reduced SAM levels, resulting in defective anti-inflammatory functions of SAM and uncontrolled inflammatory reactions. Accordingly, supplementation with SAM would, in most cases, control inflammation . In North America, SAM pills are sold over-the-counter for the relief of joint pain or the treatment of osteoarthritis. The present study provided a foundation for the use of SAM for controlling inflammation.
Ranges of the MI
Ranges and averages of MI values in different diseases
Range of MI
SAM and the MI as disease biomarkers
If the antibodies used in the assays are specific, the MI measured by immunoassays carries greater weight than the MI measured based on free SAM and SAH alone. Because the free and complex forms of SAM and SAH are subject to enormous dynamics, a method that evaluates the complete profiles has obvious advantages over simply examining one subset. SAM, SAH and the MI were previously used as biomarkers for diagnosing acute rejection after renal transplantation and nephrotoxicity events . The present work and other studies investigating Pneumocystis carinii pneumonia (PCP) in patients with HIV infection or other immunocompromised conditions  suggest that the measurement of SAM levels by cELISA could be an effective tool to diagnose the occurrence of PCP in immunocompromised conditions.
We evaluated SAM, SAH and MI levels in liver diseases in three different scenarios: (a) hepatocyte cancers (Table 6 Study A), (b) acute or severe liver diseases described in the liver disease section above, and (c) chronic liver disorders (Table 6 Study C). The results from (b) indicate that SAM and the MI can be used to identify severe liver diseases based on the significant decreases in these parameters. The cutoff value could be set between 120 nM and 240 nM. Because SAM is actively generated and metabolized in the liver, deterioration of liver function will significantly affect SAM metabolism. The results from (c) indicate that a non-significant decrease in SAM might be related to milder and chronic liver conditions in which SAM might be compensated as part of liver functional compensation over time. The results from (a) demonstrate a significant decrease in SAM in liver cancer patients but no significant increase in SAH levels, suggesting differences in the disruption of the Met cycle between carcinogenesis and other pathological processes. The level of SAM in hepatic carcinoma was decreased significantly, in contrast to a report by Li . The transient high levels of SAM were caused by the release of intracellular SAM from cells into the bloodstream (confirmed by immunohistochemistry staining of SAM in cancer cells, data not shown) at some stage of cancer progression. Because SAM release is only significant in certain stages of cancer development, after this release is complete, the elevated SAM levels in the bloodstream will not be maintained, contributing to the large ranges of SAM levels observed for all cancer types Our samples were collected from various stages of cancers, and therefore wide ranges of MI values were observed in the cancer samples. Table 8 also shows that the average MI for liver cancers was much higher than that for other diseases, indicating that in some liver cancers, the levels of SAM were high, which is consistent with Li’s report. In conclusion, SAM is a biomarker of the stages or severity of liver diseases.
Although we did not exhaustively examine all disease types, the cases observed in the present study were derived from a variety of diseases, although some diseases were combined for analysis due to the small number of samples. The significant reduction of the observed in many disease types suggest that MI could serve as a useful biomarker for the evaluation of an individual’s health status. Furthermore, the diseased group exhibited significantly lower SAM and MI levels and significantly elevated SAH levels compared to healthy people. MI can thus serve as a health status indicator. We believe the major use of the MI as a health marker would involve monitoring changes in the MI of an individual over time. MI can be used as a screening biomarker to detect an unfavorable health condition in humans, although further examinations are warranted for a final disease diagnosis. Further clinical investigations in different races and segments of the population stratified by age, gender, diet, medication or even genetic background are needed. The MI will likely be useful for aiding the detection of disease conditions in healthy populations and be included in annual health examination panels.
In the present study, monoclonal antibodies against SAM and SAH were characterized, and associated immunoassays were established. This preliminary investigation indicated that SAM, SAH and MI are good indicators of general health. In the absence of factors that may alter the value of the MI, such as special diets and medications, a significant reduction of the MI compared with an individual’s baseline MI reference profile may suggest unfavorable conditions or diseases and warrant a visit to the doctor.
XH directed all the experimental and research, designed the experiments, and performed data analysis, data integration, manuscript writing and proof-reading. HR and HG performed the generation of the monoclonal antibodies. HL, YZ and JX involved in the characterization of antibodies and establishment of cELISA; performed the sample measurement, figures creation, data integration and statistics. YH, WL, MQ, CY and LG carried out the sample collection and sorting. IA performed literature search, manuscript writing and proof-reading.
We acknowledge Dr. Huaitian Liu and Sydney Wong for their kindly contribution to statistical analysis and interpretation.
The author(s) declare that they have no competing interests.
Availability of data and materials
The datasets during and/or analyzed during the current study are available from the corresponding author on reasonable request. A part of data generated or analyzed during this study are included in this published article and its supplementary information file.
Ethics approval and consent to participate
The studies were approved by The Ethics Committee of Hunan SkyWorld Biotechnologies. Informed consent, written or verbal, was obtained from all participants.
Hunan Changsha Economic and Developmental Technology Zone Administrative Committee funded the studies and personnel.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Obeid J, Jung J, Falk J, Herrmann W, Geisel J, Friesenhahn-Ochs B, Lammert F, Fassbender K, Kostopoulos P. Serum vitamin B12 not reflecting vitamin B12 status in patients with type 2 diabetes. Biochimie. 2013;95:1056–61.View ArticlePubMedGoogle Scholar
- Shea TB. Effects of dietary supplementation with N-acetyl cysteine, acetyl-l-carnitine and S-adenosyl methionine on cognitive performance and aggression in normal mice and mice expressing human ApoE4. NeuroMol Med. 2007;9(3):264–9.View ArticleGoogle Scholar
- di Padova C. S-adenosylmethionine in the treatment of osteoarthritis. Review of the clinical studies. Am J Med. 1987;83(supp 5A):60–5.View ArticlePubMedGoogle Scholar
- Tavoni A, Vitali C. Bombardieri, Pasero GS. Evaluation of S-adenosylmethionine in primary fibromyalgia. A double-blind crossover study. Am J Med. 1987;83(suppl 5A):107–10.View ArticlePubMedGoogle Scholar
- Alsayed R, Quobailia FAL, Srour S, Geisel J, Obeid R. Elevated dimethylglycine in blood of children with congenital heart defects and their mother. Metab Clin Exp. 2013;62:1074–80.View ArticlePubMedGoogle Scholar
- Dayal S, Bottiglieri T, Arning E, Maeda N, Malinow MR, Sigmund CD, et al. Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionineb-synthase-deficient mice. Circ Res. 2001;88:1203–9.View ArticlePubMedGoogle Scholar
- Lu SC, Mato JM. S-adenosylmethionine in liver health, injury, and cancer. Physiol Rev. 2012;92:1515–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Lu SC, Mato JM. Role of methionine adenosyltransferase and S-adenosylmethionine in alcohol-associated liver cancer. Alcohol. 2005;35:227–34.View ArticlePubMedGoogle Scholar
- Coppedè F. The potential of epigenetic therapies in neurodegenerative diseases. Front Genet. 2014;5:1–8.Google Scholar
- Guerra-Shinohara EM, Morita OE, Peres S, Pagliusi RA, Sampaio NLF, D’Almeida V, et al. Low ratio of S-adenosylmethionine to S-adenosylhomocysteine is associated with vitamin deficiency in Brazilian pregnant women and newborns. Am J Clin Nutr. 2004;80:1312–21.PubMedGoogle Scholar
- Bell KM, Potkin SG, Carreon D, Plon L. S-adenosylmethionine blood levels in major depression: changes with drug treatment. Acta Neurol Scand Suppl. 1994;154:15–8.View ArticlePubMedGoogle Scholar
- Goodnick PJ, Sandoval R. Psychotropic treatment of chronic fatigue syndrome and related disorders. J Clin Psychiatry. 1993;54:13–20.PubMedGoogle Scholar
- Seivwright C, Macnab JC, Adams RL. S-adenosylmethionine metabolism in herpes simplex virus type 2-infected cells. J Gen Virol. 1993;7:1405–7.View ArticleGoogle Scholar
- Shea TB, Chan A. S-adenosyl methionine: a natural therapeutic agent effective against multiple hallmarks and risk factors associated with Alzheimer’s disease. J Alzheimers Dis. 2008;13:67–70.PubMedGoogle Scholar
- Skelly M, Hoffman J, Fabbri M, Holzman RS, Clarkson AB Jr, Merali S. S-adenosylmethionine concentrations in diagnosis of Pneumocystis carinii Pneumonia. Lancet. 2003;361:1267–8.View ArticlePubMedGoogle Scholar
- Padova CD. S-adenosyl-l-methionine in the treatment of osteoarthritis: review of the clinical studies. Am J Med. 1987;83:60–5.View ArticlePubMedGoogle Scholar
- Kagan BL, Sultzer DL, Rosenlicht N, Gerner RH. Oral S-adenosylmethionine in depression: a randomized, double blind, placebo-controlled trial. Am J Psychiatry. 1990;147:591–5.View ArticlePubMedGoogle Scholar
- Bottiglieri T, Godfrey P, Flynn T, Carney MW, Toone BK, Reynolds EH. Cerebrospinal fluid S-adenosyl-l-methionine in depression and dementia: effects of treatment with parenteral and oral S-adenosyl-l-methionine. J Neurol Neurosurg Psychiatry. 1990;53:1096–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Bottiglieri T, Hyland K, Reynolds EH. The clinical potential of admetionine (S-adenosyl-l-methioinine) in neurological disorders. Drugs. 1994;48:137–52.View ArticlePubMedGoogle Scholar
- Morrison LD, Smith DD, Kish SJ. Brain S-adenosylmethionine levels are severely decreased in Alzheimer’s disease. J Neurochem. 1996;67:1328–31.View ArticlePubMedGoogle Scholar
- Cheng H, Gomes-Trolin C, Aquilonius SM, Steinberg A, Löfberg C, Ekblom J, et al. Levels of l-methionine S-adenosyltransferase activity in erythrocytes and concentrations of S-adenosylmethionine and S-adenosylhomocysteine in whole blood of patients with Parkinson’s disease. Exp Neurol. 1997;145:580–5.View ArticlePubMedGoogle Scholar
- Mischoulon D, Fava M. Role of S-adenosyl-l-methionine in the treatment of depression: a review of the evidence. Am J Clin Nutr. 2002;76(5):1158S–61S.PubMedGoogle Scholar
- Anstee QM, Day CP. S-adenosylmethionine (SAMe) therapy in liver disease: a review of current evidence and clinical utility. J Hepatol. 2012;57:1097–109.View ArticlePubMedGoogle Scholar
- Almasio P, Bortolini M, Pagliaro L, Coltorti M. Role of S-adenosyl-l-methionine in the treatment of intrahepatic cholestasis. Drugs. 1990;40:111–23.View ArticlePubMedGoogle Scholar
- Bressa GM. S-adenosyl-l-methionine (SAMe) as antidepressant: meta-analysis of clinical studies. Acta Neurol Scand Suppl. 1994;154:7–14.View ArticlePubMedGoogle Scholar
- Yokoyama S, Sells MA, Reddy TV, Lombardi B. Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat. Cancer Res. 1985;45:2834–42.PubMedGoogle Scholar
- Rogers AE. Methyl donors in the diet and responses to chemical carcinogens. Am J Clin Nutr. 1995;61:659S–65S.PubMedGoogle Scholar
- Poirier LA, Wilson MJ, Shivapurkar N. Carcinogenesis and DNA hypomethylation in methyl-deficient animals. Exp Biol Med. 1986;12:151–61.Google Scholar
- Pogribny IP, Miller BJ, James SJ. Alterations in hepatic p53 gene methylation patterns during tumor progression with folate/methyl deficiency in the rat. Cancer Lett. 1997;115:31–8.View ArticlePubMedGoogle Scholar
- Cooney CA, Wise CK, Poirier LA, Ali SF. Methamphetamine treatment affects blood and liver S-adenosylmethionine (SAM) in mice: correlation with dopamine depletion in the striatum. Ann NY Acad Sci. 1998;844:191–200.View ArticlePubMedGoogle Scholar
- Perna AF, Ingrosso D, Zappia V, Galletti P, Capasso G, De Santo NG. Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine. J Clin Invest. 1993;91:2497–503.View ArticlePubMedGoogle Scholar
- Suma Y, Matsumoto C, Sawai Y, Tsukada K. A sensitive assay method for the measurement of S-adenosylmethionine in tissue. J Biochem. 1984;96:679–82.PubMedGoogle Scholar
- Mudd SH. Activation of methionine for transmethylation. VI. Enzyme-bound tripolyphosphate as an intermediate in the reaction catalyzed by the methionine-activating enzyme of Baker’s yeast. J Biol Chem. 1963;238:2156–63.PubMedGoogle Scholar
- Loehrer FM, Angst CP, Brunner FP, Haefeli WE, Fowler B. Evidence for disturbed S-adenosylmethionine: s-adenosylhomocysteine ratio in patients with end-stage renal failure: a cause for disturbed methylation reactions? Nephrol Dial Transplant. 1998;13:656–61.View ArticlePubMedGoogle Scholar
- Klepacki J, Brunner N, Schmitz V, Klawitter J, Christians U, Klawitter J. Development and validation of an LC-MS/MS assay for the quantification of the transmethylation pathway intermediates S-adenosylmethionine and S-adenosylhomocysteine in human plasma. Clin Chim Acta. 2013;421:91–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Borman S. Heated dispute over analytical method. 2015. http://cen.acs.org/articles/93/i42/Heated-Dispute-Over-Analytical-Method.html. Accessed 23 Oct 2015.
- Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–7.View ArticlePubMedGoogle Scholar
- Wagner C, Koury MJ. S-adenosylhomocysteine: a better indicator of vascular disease than homocysteine? Am J Clin Nutr. 2007;86:1581–5.PubMedGoogle Scholar
- Mirecki-Garrido MD, Guerra B, Mateos-Díaz C, Jiménez-Monzón R, Díaz-Chico N, Díaz-Chico JC, et al. The influence of estrogens on the biological and therapeutic actions of growth hormone in the liver. Pharmaceuticals. 2012;5:758–78.View ArticlePubMedPubMed CentralGoogle Scholar
- Wang WS, Wahlqvist ML, Hsu CC, Chang HY, Chang WC, Chen CC. Age- and gender-specific population attributable risks of metabolic disorders on all-cause and cardiovascular mortality in Taiwan. BMC Public Health. 2012;12:111.View ArticlePubMedPubMed CentralGoogle Scholar
- Sarris J, Price LH, Carpenter LL, Tyrka AR, Ng CH, Papakostas GI, Jaeger A, Fava M, Mischoulon D. Is S-adenosyl Methionine (SAMe) for depression only effective in males? A re-analysis of data from a randomized clinical trial. Pharmacopsychiatry. 2015;48(4–5):141–4.PubMedGoogle Scholar
- Najm WI, Reinsch S, Hoehler F, Tobis JS, Harvey PW. S-adenosyl methionine (SAMe) versus celecoxib for the treatment of osteoarthritis symptoms: a double-blind cross-over trial. BMC Musculoskelet Disord. 2004;5:6–20.View ArticlePubMedPubMed CentralGoogle Scholar
- Greenberg AK, Rimal B, Felner K, Zafar S, Hung J, Eylers E, et al. S-adenosylmethionine as a biomarker for the early detection of lung cancer. Chest. 2007;132:1247–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Klepacki J, Brunner N, Schmitz V, Klawitter J, Christians U, Klawitter J. Development and validation of an LC-MS/MS assay for the quantification of the trans-methylation pathway intermediates S-adenosylmehtionine and S-adenosylhomocysteine in human plasma. Clin Chim Acta. 2013;421:91–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Skelly MJ, Holzman RS, Meral S. S-adenosylmethionine levels in the diagnosis of Pneumocystis carinii Pneumonia in patients with HIV pnfection. Clin Inf Dis. 2008;46:467–71.View ArticleGoogle Scholar
- Li T, Yu G, Guo T, Qi H, Bing Y, Xiao Y, et al. The plasma S-adenosylmethionine level is associated with the severity of Hepatitis B-related liver disease. Medicine. 2015;94:1–8.Google Scholar