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Elevated serum S-adenosylhomocysteine in cobalamin-deficient elderly and response to treatment^1,2,3

¹ From the Department of Medicine, University of Colorado Health Sciences Center, Denver, CO (RHA and SPS), and the Department of Foods and Nutrition, University of Georgia, Athens, GA (MAJ and ETD)

² Supported by grant no. GEO2000-01327 from the US Department of Agriculture National Research Initiative Competitive Grants Program (to MAJ), grant no. GEO00916 from the University of Georgia Agricultural Experiment Station (to MAJ), a grant from the Northeast Georgia Area Agency on Aging (to MAJ), and grant no. AG-09834 from the National Institute on Aging (to SPS).

³ Address reprint requests to SP Stabler, Box B170, 4200 East 9th Avenue, Denver, CO 80262. E-mail: sally.stabler{at}UCHSC.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Background: S-Adenosylmethionine (SAM)–dependent methylation reactions produce S-adenosylhomocysteine (SAH), the precursor of homocysteine, which has been associated with adverse events when it is elevated.

Objective: We studied a cohort of elderly with a high prevalence of cobalamin deficiency to determine whether SAH, SAM, or their ratio was abnormal; whether they correlated with other markers of vitamin deficiency; and whether they changed with cobalamin therapy.

Design: A convenience sample of elderly attending nutrition centers was enrolled for baseline demographic, biochemical, and nutritional assessments. Methylmalonic acid (MMA), total homocysteine, and other metabolites were measured by using gas chromatography–mass spectrometry. Serum SAM and SAH were measured by using stable-isotope-dilution liquid chromatography–mass spectrometry. Subjects found to have elevated serum MMA were treated with oral cyanocobalamin tablets (1000 µg/d) for 3 mo. Subjects with normal MMA were randomly assigned to 1 of 3 dosage groups: 0, 25, or 100 µg cyanocobalamin/d.

Results: The 149 elderly subjects had a mean age of 76.3 y; 81% were female, and 30% were African American. Serum MMA concentrations were elevated in 30% and SAH concentrations were elevated in 64% of the cohort. Those with elevated MMA concentrations had higher SAH and SAM concentrations. High-dose oral cobalamin lowered SAH, MMA, and total homocysteine concentrations significantly, although subjects with creatinine concentrations >109 umol/L had higher posttreatment SAH than did those with lower creatinine.

Conclusions: Elevated serum SAH concentrations are common in elderly and are strongly influenced by both renal status and cobalamin deficiency. These elevated concentrations can be lowered with high-dose oral cobalamin therapy.

Key Words: Methylmalonic acid • vitamin B-12 • total homocysteine • folate • S-adenosylmethionine

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Hyperhomocysteinemia is associated with adverse events such as mortality (1, 2), cardiovascular events (1, 3), stroke (3, 4), and cognitive disorders (5, 6), including Alzheimer disease (7). The underlying pathophysiology of the deleterious effects of hyperhomocysteinemia is not clear. It is possible that elevated total homocysteine (tHcy) is simply a marker of underlying vitamin deficiency or that the compound itself actually causes abnormal methylation status or other toxicity (8).

Homocysteine is methylated to form methionine, which is the precursor of S-adenosylmethionine (SAM), the physiologic methylator of creatine, phospholipids, neurotransmitters, and methylated DNA (9). S-Adenosylhomocysteine (SAH) is a product of these transmethylation reactions, which can be hydrolyzed to form homocysteine and adenosine (9). SAH is an inhibitor of many transmethylation reactions (10), and it is thought that maintenance of the appropriate ratio of SAM to SAH methylation (SAM:SAH) is important for health (11). Several studies have explored differences in SAM and SAH concentrations between healthy subjects and subjects with Alzheimer disease; results have been mixed (12, 13).

The SAM-dependent methylations are largely intracellular reactions, and it is not clear whether an assay of SAM and SAH in serum or plasma will reflect the intracellular status of methionine metabolism. Studies in normal volunteers have shown that most serum SAM and a large proportion of SAH are cleared by the kidneys (12). Therefore, subjects with impaired renal function will have build-up of these compounds in the blood (14-17), which may explain the relation between elevated SAH and vascular disease that was described elsewhere (17-20). However, it was also shown that, in inborn errors of metabolism, hypermethioninemia, or both (21, 22) and in severe cobalamin deficiency (23), elevated serum SAM or SAH concentrations change in response to methionine restriction or vitamin treatments. Thus, to some extent, the serum values probably do reflect intracellular methionine metabolism. To determine whether elevated values for SAH correlated with vitamin-related variables or poor cognition, we studied the determinants of serum SAM and SAH and their relations with hyperhomocysteinemia, renal insufficiency, impaired cognition, and other clinical characteristics in an ambulatory elderly population from rural Georgia. Evidence of cobalamin deficiency, renal insufficiency, or both was found in a large proportion of the subjects. Oral cobalamin at various doses was given to assess the changes in SAH and other metabolites in response to treatment. An elderly cohort was chosen to assess these relations because of the likelihood of finding persons with hyperhomocysteinemia due to vitamin deficiency, renal insufficiency, or both and of finding persons with impaired cognition so that relations could be explored.

	SUBJECTS AND METHODS

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Subjects
Subjects were recruited from the Older Americans Act Nutrition Program at 6 senior centers in northeast Georgia, by sending a letter describing the study to the directors of the centers in Clarke, Barrow, Morgan, Jackson, Franklin, and Newton counties (24). The directors and other employees of the centers recruited clients of the centers for the study. All interviews and procedures were performed at the senior centers.

Baseline measurements of demographic, biochemical, and clinical variables were taken for all subjects, who were then treated with oral cobalamin supplements or placebo for 3 mo. Measurements were repeated after treatment. Subjects were asked about their multivitamin use at the baseline assessment. Participants were not excluded for taking nutritional supplements, including those supplements containing cobalamin. The only exclusion criterion was medical treatment for cobalamin deficiency by injection or high-dose oral administration. At baseline, 149 participants were enrolled in the study.

Written informed consent was obtained from each participant. The institutional review boards on human subjects of the Georgia Department of Human Resources, the University of Georgia, the University of Colorado, and the Athens Community Council on Aging approved the questionnaires and procedures used in this study.

Baseline assessment
Participants were not asked to fast before blood collection because of their advanced age and possible frailty. Venous blood samples were stored on ice for 1–4 h before separation of serum for assays of SAM, SAH, total homocysteine (tHcy), methylmalonic acid (MMA), cystathionine, and methylcitric acid. The serum was frozen at –20 °C and sent, frozen on dry ice, by overnight delivery to the University of Colorado for analysis of the metabolites. Serum MMA, tHcy, methylcitric acid, and cystathionine concentrations were measured by using stable-isotope-dilution capillary gas chromatography–mass spectrometry (25, 26). SAM and SAH concentrations were measured by using stable-isotope-dilution liquid chromatography–mass spectrometry as described previously (14). Despite immediate chilling of samples, some assays resulted in uninterpretable chromatography for SAM, and values were available for a lower number of samples, which is indicated in the tables as applicable. The previously determined normal ranges were 73–271 nmol/L for MMA (25), 5.4–13.9 µmol/L for tHcy, 44–342 nmol/L for cystathionine (27), and 60–228 nmol/L for methylcitric acid (25). Normal ranges, determined in 48 healthy controls aged 22–59 y, were 71–168 nmol/L for SAM, 8–26 nmol/L for SAH, and 4.4–12.4 for SAM:SAH (14).

Automated complete blood counts and serum creatinine concentrations were assayed by a local clinical laboratory (SmithKline-Beecham Clinical Laboratories, Atlanta, GA). Sera for folate and cobalamin analyses were frozen at –70 °C in cryogenic vials with minimal airspace and sent by overnight delivery for analysis with a radioassay (Quantaphase II Vitamin B-12/Folate Radioassay; Bio-Rad, Richmond, CA) at the Centers for Disease Control and Prevention (Atlanta, GA) (28). Serum pepsinogen 1 was measured with the use of a kit (SORIN/Bio-medica kit P2560; INCSTAR Corporation, Stillwater, MN). Serum pepsinogen 1 was used as an indirect index of atrophic gastritis (29). Participants were considered to have mild atrophic gastritis if their pepsinogen 1 concentrations were between 10 and 60 µg/L and to have severe atrophic gastritis if their pepsinogen 1 concentrations were <10 µg/L (29).

The Orientation-Memory-Concentration (OMC) test (30), which is a brief cognitive measurement tool, was administered to all participants at baseline and after treatment. This instrument has 6 questions: the current month, year, and time of day; phrases to be repeated; counting backwards; and naming the months of the year in reverse order. A score of 8 indicated normal cognition or minimal impairment, 9–19 indicated moderate impairment, and 20 indicated severe impairment. The correlations of MMA and homocysteine with detailed neurocognitive screening were previously reported in a subgroup of the current cohort (31). The OMC test had shown a correlation of poor scores with elevated MMA and tHcy in another cohort from a Georgia senior center (24). Thus we hypothesized that elevated SAH concentrations or abnormal SAM:SAH may also correlate with poor cognition.

Treatment phase
Tablets of identical appearance containing 0, 25, 100, or 1000 µg cyanocobalamin were used (Sunstar Pharmaceutical Inc, Elgin, IL). After baseline testing, the participants were assigned to a treatment group. Those with serum MMA concentrations >271 nmol/L (n = 45) were offered daily high-dose oral cobalamin in 1000-µg tablets. For reasons of subject safety and to ensure treatment efficacy, the phlebotomist and the research coordinator ensured that participants with elevated MMA were aware of their potential for cobalamin deficiency. Other researchers and staff did not have access to information about the MMA status of the participants. In addition, the physicians of the participants with elevated MMA were notified in writing by the phlebotomist and research coordinator. Therefore, the intervention in those with elevated MMA was neither randomized nor blinded.

The 104 subjects with MMA 271 nmol/L were invited to participate in a randomized, double-blind, placebo-controlled trial comparing 3 doses of cobalamin (0, 25, and 100 µg/d). After 3 mo of supplementation, 132 participants completed a follow-up examination involving the same methods for phlebotomy and cognitive screening as were used at baseline. Compliance was monitored by calculating pill counts and, for the high-dose groups, by evaluating the follow-up serum cobalamin concentration.

The group taking 0 and the group taking 1000 µg cobalamin/d continued the treatment for another 6 mo and underwent a second round of cognitive testing 9 mo after the initiation of supplementation.

Statistical analysis
Statistical analysis was performed by using SAS software (version 8.2; SAS Institute, Cary, NC) and SPSS software (version 13.0; SPSS Inc, Chicago, IL). P < 0.05 was considered significant. Data were log transformed to approximate normal distributions when necessary. Levene's test for equality of variance was employed, and the t value for unequal variance was used when appropriate. For baseline values, cross-sectional analyses including t tests for continuous variables and chi-square analyses for dichotomous variables were used to examine differences between subjects with and subjects without elevated MMA. Multiple stepwise regression analyses were used to evaluate the independent effects of variables on SAM, SAH, SAM:SAH, tHcy, and OMC test score. The independent variables were age, sex, race, creatinine, MMA, tHcy, cystathionine, methylcitric acid, folate, cobalamin, pepsinogen, OMC test score, SAM, SAH, and SAM:SAH. However, SAM and SAH were not used in models for SAM:SAH, and vice versa. Paired t tests were used to analyze posttreatment changes in the subjects receiving the 1000-µg/d dose. The pretreatment and posttreatment values for the randomly assigned dose groups (ie, 0, 25, or 100 µg) were evaluated by using analysis of variance, and changes were compared by using general linear models and least-significant-difference tests.

	RESULTS

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Baseline variables
One hundred forty-nine elderly subjects entered into the study, of whom 81% were female and 30% were African American. The mean age and biochemical variables are shown in Table 1. Although the mean serum creatinine concentration was normal, values ranged to 418 umol/L. This cohort appeared to have high folate status because the mean serum folate concentration was 46.3 nmol/L and the lowest value was 8.8 nmol/L. The proportions of the subjects who had biochemical values either higher or lower than the 95% CIs that we had found in younger populations are also shown. As expected, higher creatinine, MMA, and tHcy and lower cobalamin were found in a significantly greater percentage of subjects in the current study than in a younger population. However, the most striking change was that the SAH value was higher than our previously determined upper limit in 64% of the subjects, whereas the SAM value was high in only 10% or low in only 10%.

Correlations between variables
The univariate correlations between variables are shown in Table 2. Strong correlations were seen between creatinine, SAH, SAM, tHcy, methylcitric acid, and cystathionine. SAH was strongly correlated with tHcy, cystathionine, and methylcitric acid and more weakly correlated with MMA. However, after adjustment for serum creatinine, SAH was correlated only with SAM, SAM:SAH, and methylcitric acid, although it trended toward correlation with homocysteine (data not shown). SAM was strongly correlated with creatinine, methylcitric acid, tHcy, cystathionine, and SAH. After adjustment for creatinine, the correlation with MMA, methylcitric acid, cystathionine, and SAH remained significant (data not shown). Serum folate was correlated with SAM and SAH, and this correlation remained after adjustment for creatinine. Serum cobalamin was not correlated with SAM, SAH, or SAM:SAH unless adjusted for creatinine, in which case cobalamin was correlated with SAM:SAH ratio. Results of a stepwise ordinary least-squares regression for a number of dependent variables are shown in Table 3. SAM and SAH were mutually predictive. It is interesting that creatinine was a strong predictor of SAH but not of SAM. Methylcitric acid independently affected SAH and tHcy. The OMC test score was predicted by MMA but not by SAM, SAH, SAM:SAH, or tHcy.

Table 4

Table 5

Table 6

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

We studied a mostly female elderly cohort, who in general had very high folate status. The mean tHcy in these elderly was remarkably low at 10.7 umol/L, which is lower than or equivalent to values seen in younger subjects in unfortified populations (32, 33). Despite this relatively low tHcy, the mean SAH concentration was above the range we previously measured in 64% of the subjects. Multivariate analysis in our cohort showed that only creatinine, SAM, cystathionine, and methylcitric acid were independent predictors of SAH, but together they accounted for most of the variability. Because creatine, the precursor of creatinine, is a product of the SAM-to-SAH reaction, it is not surprising that a strong correlation may be found, although creatinine was not an independent predictor of SAM. Methylcitric acid is formed in excess in persons with severe cobalamin deficiency; however, methylcitric acid concentrations are also highly dependent on renal excretion (25), which may explain the relation with SAH in our cohort. It is surprising that tHcy was not a predictor of the SAH value in the regression analysis. Serum cobalamin was not a predictor of SAM or SAH, and serum folate predicted only SAM in the regression model. Other investigations reported similar data (15, 16, 18).

Elevated MMA is frequently found in elderly subjects, and it predicts elevated tHcy. Both metabolites readily decrease with cobalamin therapy, which suggests that depleted vitamin status rather than age is the variable more likely to cause elevated values (24). The subjects with elevated MMA in the current study had the expected higher concentrations of creatinine, tHcy, cystathionine, and methylcitric acid, as well as higher SAM and SAH. We chose to treat all subjects with elevated MMA with high-dose oral cobalamin tablets. As expected, the serum MMA and tHcy fell markedly in these subjects after treatment. In addition, after treatment with high-dose oral cobalamin, serum SAH fell significantly in both groups of subjects: those with high and those with low creatinine. Improved cobalamin status apparently lowers the serum SAH value, possibly as a result of greater tissue methionine synthase activity after treatment. We previously showed that cobalamin-deficient subjects with megaloblastic anemia also had decreases in serum SAH with cobalamin treatment (23). Thus, it appears from our data that the serum SAH concentration is mainly influenced by renal status, but that an effect of cobalamin status is also seen, which is easier to appreciate when renal status is normal. These findings may affect the interpretation of studies of the role of SAH abnormalities in vascular disease, because impaired renal status is extremely common in such populations and is associated with such cardiovascular disease risk factors as hypertension, diabetes, male sex, and age. One can expect that the plasma SAH will be high in such persons with those traits or conditions (15-20).

Impaired methionine synthesis, and therefore deficient synthesis of SAM with a decrease in transmethylation reactions, is a potential underlying cause of pathophysiologic conditions in cobalamin or folate deficiency. However, we previously showed that even severely cobalamin-deficient subjects rarely have low serum methionine (34), and we did not find that SAM was low in subjects with severe cobalamin deficiency anemia (23). The current investigation showed similar findings, in that the SAM was significantly higher in those subjects with elevated MMA than in those without elevated MMA, and no change was seen in SAM after treatment with 1000 µg cobalamin/d. The elevated SAM in the high-dose group may have been due to the impaired renal status of many of those with elevated MMA. Multivitamin use did not affect SAM:SAH or the SAM concentration. However, in multivariate analysis, the serum SAM concentration was modestly affected by folate. In addition, the African American subjects had lower SAM than did the white subjects, but, because the African Americans also had lower serum folate values, this relation may not be completely independent. SAM:SAH was not affected by cobalamin treatment, although it trended lower in subjects with elevated MMA at baseline.

Hyperhomocysteinemia may be a risk factor for cognitive defects and even for the future diagnosis of Alzheimer disease (6, 7). We also previously showed that depression (35) and cognition (24, 31) are related to cobalamin status. We used a screening instrument to test cognition in the current study and found that age, MMA, and race influenced the score, but tHcy, SAM, and SAH did not. Therefore, our data do not provide any documentation of the concept that abnormal methylation ratios play a role in cognitive disorders in elderly—a finding that is similar to that of a recent report of spinal fluid, SAM, and SAH values in patients with dementia (13). Tissue SAM and SAH values may be more revealing but usually will not be available. We also did not find an improvement in cognition score after treatment with high-dose cobalamin, despite the marked lowering of MMA and tHcy in those subjects. However, the measure for cognition reported here may not be sensitive enough to allow detection of changes (31).

Post vivo changes in SAH and SAM in both unseparated and separated blood are significant and seem to be preventable with proper handling. We attempted to overcome these difficulties by placing the phlebotomized blood on ice and preparing serum under cold conditions, followed by continuous freezing of samples until they were assayed. Despite these precautions, some SAM values were uninterpretable because of poor chromatography peaks (see Subjects and Methods). It is possible that artifactual increases in SAH due to less-than-optimal sample handling could explain some of the SAH elevations we saw in this cohort of elderly. If that were the case, however, we would not expect the significant associations seen with other variables (that are not affected by sample handling) or the significant decrease in SAH after cobalamin treatment. Future epidemiologic studies must address these concerns.

In conclusion, we found that the most significant determinants of serum SAH in a cohort of elderly are the serum SAM and creatinine concentrations. Yet, some influence is also due to poor cobalamin status because high-dose cobalamin tablets in subjects both with and without elevated creatinine lowered SAH significantly but much less than it lowered elevated MMA or tHcy. It seems likely that serum SAM and SAH will be less useful than the other cobalamin-dependent metabolites in the diagnosis of deficiency and in correlations with clinical syndromes.

	ACKNOWLEDGMENTS

 
We acknowledge the expert recruitment and interviewing assistance of Nicole A Hawthorne, the technical assistance of Carla Ray and Bev Raab, and the secretarial assistance of Theresa Martinez.

MAJ and ETD were responsible for the design and performance of the clinical aspects of the study; SS and RA were responsible for the development and performance of blood assay procedures; all authors contributed to the writing and editing of the manuscript. Two of the authors (SPS and RHA) and the University of Colorado hold patents on the use of assays for total homocysteine and other metabolites to diagnose vitamin B-12 and folate deficiencies, and a company has been formed at the University of Colorado to perform such assays. The other authors had no personal or financial conflict of interest.

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TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

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