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Supplement: 3rd Amino Acid Workshop

Advancing Age and Other Factors Influencing the Balance between Amino Acid Requirements and Toxicity^1,2

Department of Medicine, General Clinical Research Center, University of Vermont College of Medicine, Burlington, VT 05405

³ To whom correspondence should be addressed. E-mail: Naomi.Fukagawa{at}uvm.edu.

	ABSTRACT

TOP ABSTRACT LITERATURE CITED

 
As the average human lifespan increases, so does the recognition that advancing age is associated with changes in nutrient intake and requirements as a consequence of biological, social, and pathological factors. Studies show that whereas protein requirements may not differ significantly between younger and older adults, the adaptive mechanisms and responses to nutritional or pathological stressors may differ and alter the balance between requirement and toxicity of specific amino acids (AAs). As an individual gets older, cardiovascular disease and cancer become the leading causes of morbidity and mortality. Advancing age is also associated with changes in appetite, food intake, and physical activity, all of which can influence protein and AA metabolism. The sulfur amino acids (SAAs) methionine and cysteine recently attracted attention because of their pivotal roles in methyl group metabolism and maintenance of the cellular redox state. Methionine, an indispensable AA, is important for methylation reactions and as a precursor for cysteine, which is the rate-limiting AA for glutathione (GSH) synthesis. On one hand, high intake levels or blood concentrations of methionine are associated with adverse consequences such as hyperhomocysteinemia and endothelial dysfunction, which are risk factors for cardiovascular disease. On the other hand, methionine deficiency is reported to lower the threshold of chemical-induced toxicity and play a role in carcinogenesis. Therefore, it is evident that understanding the biological significance of the interrelationship between SAAs, GSH, and methyl group metabolism is key to determining optimal dietary intakes of SAAs in older individuals.

KEY WORDS: • aging • sulfur amino acids • glutathione • methyl groups • methionine • cysteine

Everyone is aware of the increasing population of individuals over the age of 50 as well as the significant changes in body composition (decreased lean body mass), energy utilization and requirements (reduced requirements and apparently decreased energy expenditure), and physical activity (generally reduced) that occur with advancing age. Yet when it comes to protein and amino acid (AA)⁴ requirements, recommendations for individuals over age 50 are largely based on extrapolation from nitrogen-balance studies in young men. Investigators have attempted to more precisely determine AA requirements for different populations using stable isotope methodologies. In 1997, Morais et al. (1) reported that although there were no significant age- or sex-related effects on rates of nonmuscle lean-tissue protein breakdown (i.e., organs and bones), there was a reduction in the contribution by muscle to whole-body protein catabolism in older men and women. Furthermore, others have reported that elderly men have higher rates of leucine extraction by the gut or liver or both during the fed state, which leads to lower peripheral availability of this indispensable amino acid (IAA) than in young men (2). Work by Millward and colleagues (3,4) led to their conclusion that the 1985 Food and Agriculture Organization/World Health Organization/United Nations University (FAO/WHO/UNU) estimates for protein (0.75 g·kg^–1·d^–1) and AA requirements (Table 1) are reasonable, because there was no evidence of a change in protein utilization efficiency with age, and protein requirements were actually lower as a reflection of the reduction in fat-free mass. Campbell et al. (6) used stable isotope probes, nitrogen balance, and computerized tomography scans to assess mid-thigh muscle area and determine the protein needs of older men and women. In contrast to the findings of Millward et al. (3,4), they concluded that the recommended daily allowance for protein might not be adequate for older individuals (6). These investigators proposed a higher safe protein intake of 1–1.25 g·kg^–1·d^–1. Despite these seemingly contradictory conclusions, the sum of the evidence suggests that because of the increased morbidity and disease burden on older persons, a rational and safe recommendation is a protein intake no lower than 0.75 g·kg^–1·d^–1 and most likely to approximate 1 g of good-quality protein·kg^–1·d^–1 (5). Although Young and Borgonha (5) note that further research into protein requirements for the older age group are desirable, it does not seem that nitrogen requirements at different life stages or in generally healthy individuals currently arouse strong debate or major controversy. This is not necessarily the case for specific AA requirements. This is especially true in those instances wherein the adaptive mechanisms and responses to nutritional or pathological stressors may differ with age and thereby influence or alter the balance between requirement and toxicity of specific AAs. Some recent estimates of adult requirements for specific AAs are summarized (Table 1). Whereas the Massachusetts Institute of Technology (MIT) estimates are generally higher than those proposed by the 1985 FAO/WHO/UNU and 1999 Millward estimates, both MIT and Millward's group support a slightly higher content of sulfur amino acids (SAAs) per gram of protein.

View larger version (34K): [in this window] [in a new window]   FIGURE 1  Major components of the methionine cycle are transmethylation (TM), remethylation (RM), transsulfuration (TS), and methionine incorporation into or release from body proteins. In TM, the initial step is catalyzed by MAT, whereby methionine forms SAM that goes on to transfer the methyl group to various methyl group acceptors using several different methyltransferases (MTs) to yield SAH. SAH is then hydrolyzed to form HCY. In the TS pathway, HCY is condensed with serine to form cystathionine via CBS. Cystathionine can then be hydrolyzed to form cysteine and -ketobutyrate via cystathionine -lyase (CL), which is also known as SDH. HCY can also be remethylated to methionine by accepting a methyl group from 5-methyltetrahydrofolate or betaine, which is catalyzed by MS or BMT, respectively.

Methionine, an IAA, is converted to S-adenosylmethionine (SAM) by methionine adenosyl transferase (MAT) (Fig. 1). SAM is the methyl donor in many important transmethylation reactions that lead to the formation of the short-lived compound S-adenosylhomocysteine (SAH). A vital role of SAM is to coordinate regulation of remethylation and transsulfuration. SAM acts as an allosteric inhibitor of 5,10-methylenetetrahydrofolate reductase (MTHFR), which is crucial for 5-methyltetrahydrofolate synthesis and as an activator of cystathionine ß-synthase (CBS) at micromolar concentrations (Fig. 1). As the primary source of SAM, methionine and a number of vitamins and micronutrients influence DNA synthesis and/or repair and the expression of genes (11). Through an effect on genome integrity and alteration of DNA methylation, methyl group availability provides a link between SAAs, DNA synthesis, and modulation of gene expression (Table 3).

In a recent review, Ross (21) noted that global DNA hypomethylation occurs as a consequence of inadequate intake of methionine, choline, or folate and was also found to be associated with deficiencies of trace metals such as zinc and selenium. Methionine deficiency has been associated with DNA fragmentation and strand breaks (22). In animals, methionine depletion lowers the threshold of chemical-induced toxicity, which suggests that this may be a significant factor in carcinogenesis (22). In human studies, a consistent relationship between folate status and genomic DNA methylation in lymphocytes was reported (23). In contrast with methylation reactions, the process of demethylation is less well understood but is assumed to be a passive process. A mammalian gene that codes for a protein that catalyzes demethylation has been identified (24), but several attempts to characterize demethylase activity have yielded variable results (21). DNA methylation clearly enhances the ability of cells to regulate and package genetic material. Epigenetic changes may be potential biomarkers for risk assessment and detection of early disease.

An important but yet-unanswered question is the extent to which dietary intake of methionine influences DNA methylation. Understanding the relationship between methionine and SAM intake and methylation reactions becomes relevant when considering the potential for toxicity associated with methionine excess. An important question to be answered is the degree to which manipulation of the diet (e.g., methyl supplementation with methionine, betaine, folic acid, and vitamin B-12) can alter methylation patterns that promote health or disease. In addition, bioactive food components other than essential nutrients, e.g., flavonoids or phytoestrogens, might influence DNA methylation processes (21).

SAAs and cardiovascular disease

Methionine is reported to protect against atherosclerotic lesions induced by dietary fats in rats. Seneviratne et al. (25) reported that methionine supplementation in the drinking water of spontaneously hypertensive rats increased myocardial antioxidant (GSH peroxidase, superoxide dismutase, and catalase) activity and mRNA levels. They concluded that methionine supplementation improved endogenous antioxidant reserves in heart and suggested that the methionine-related increase in antioxidant enzyme activities and mRNA levels could account for the protective effects. However, the mechanisms whereby methionine induces these changes are unclear.

Research over the past 20 years demonstrates the importance of endogenous metabolic processes that lead to the production of reactive oxygen species (ROS) on cellular dysfunction, mutagenesis, and death (26). ROS are implicated in the pathogenesis of a variety of disorders including CVD as well as the general aging process (27). In the cardiovascular system, ROS contribute to cardiac dysfunction and myocardial cell death that result from conditions such as ischemia-reperfusion, catecholamine excess, and adriamycin-induced cardiomyopathy. In the immune system, ROS influence T-cell apoptosis and act as important signaling molecules to influence redox- and oxygen-sensitive transcription (28). By virture of their sulfhydryl groups, SAAs are important in establishing the redox state of cells, and their presence in proteins provides key sites for oxidative modification (26). Although beyond the scope of this review, methionine oxidation and reduction in proteins are also shown to be key in regulation of cell function (29).

HCY, which is a well-recognized nonprotein AA and accepted risk factor for CVD, is the product of the transmethylation of methionine. HCY may be recycled to methionine via two different enzymatic pathways (Fig. 1): 1) methionine synthase (MS), which uses vitamin B-12 as the cofactor and N⁵-methyl-tetrahydrofolate as the methyl donor; and 2) betaine-HCY methyltransferase (BMT), which uses betaine as the methyl donor. Recent work by Zeng et al. (30) presents a mechanism whereby HCY exerts its effects on the progression of atherosclerotic lesions in patients with hyperhomocysteinemia. They found that HCY stimulates the expression and secretion of biologically active monocyte chemoattractant protein-1 and interleukin-8 in aortic endothelial cells and VSMCs. Furthermore, these responses were related to HCY-induced oxidative stress with activation of the redox-sensitive transcription factor nuclear factor-B (NF-B). We previously reported that aortic VSMCs isolated from old rats responded to high glucose concentrations or tumor necrosis factor- with increased binding of NF-B to DNA and reduced VSMC apoptosis compared with responses in VSMCs from young animals (31). This suggests that the age-related differences in the activation of redox-sensitive transcription factors might play a role in the higher prevalence of CVD in older diabetics.

Using the apolipoprotein E–deficient mouse as an animal model for atherosclerosis, dietary supplementation with methionine or HCY was shown to promote early atherosclerosis but not to enhance plaque rupture (32). In fact, there was evidence of plaque fibrosis in supplemented animals, which suggests that extracellular matrix formation may be influenced by the amount of SAAs in the diet. In a study conducted on six normal young women who consumed low-protein diets, Meakins et al. (33) found that the availability of methionine was not limiting for nitrogen utilization. Using the urinary excretion of 5-L-oxoproline as an index of glycine sufficiency, they found that excess methionine competes for available glycine, which suggests that alternate pathways for glycine utilization such as the synthesis of GSH may be compromised. It remains to be determined whether high intakes of methionine by older individuals might lead to an increased risk of HCY-related diseases or contribute to alterations in other methylation reactions.

In early work using L-[1-¹³C, methyl-²H₃]methionine as a tracer, we were unable to detect significant age-related differences in rates of remethylation, transmethylation, or transsulfuration in generally healthy older individuals, but we concluded that a total SAA intake of 13 mg·kg^–1·d^–1 might not be adequate if dietary cysteine accounted for as much as half of the total SAA intake (34). Furthermore, it is unclear whether the presence of common diseases associated with advancing age such as CVD or cancer influences methionine metabolism and SAA requirements. Clearly, more work needs to be done to delineate the effects of changes in the intake of specific AAs on common metabolic pathways as well as on proinflammatory pathways and extracellular matrix remodeling, which ultimately influence the onset or progression of disease.

Regulation of CBS

As described earlier, CBS enzyme activity catalyzes the essentially irreversible metabolism of HCY to cystathionine. This irreversibility removes the constituent sulfur of HCY from the pool and precludes its potential recycling to methionine. The absence of CBS leads to homocystinuria and has been well studied (35); as of 7 January 2004, there were 131 CBS mutations listed in the University of Colorado CBS website (36). But what of more subtle or physiological regulation of the enzyme?

CBS activity in fetal liver measured from human abortus material is reported to be 20% of the value in adult liver (7) and, as measured in stimulated human lymphocytes, apparently declines throughout adult life such that 50% of normal activity is lost by 80 y of age (37). In rats, CBS activity increases sharply at d 20 in utero and subsequently doubles during the first 22 d after birth (38). In postnatal rats, parenteral hydrocortisone administration increased CBS activity in liver by 50% (38), and activity was found to be reduced by 10% in the 2-h period on either side of the onset of darkness (39). It was also shown (40) that reduction in the dietary protein content is accompanied by an 83% reduction in CBS activity in rat liver homogenates. How is such regulation achieved?

CBS in humans is a homotetramer of 63-kDa subunits that binds the substrates HCY and serine (41). It has two cofactors: pyridoxal-5'-phosphate (vitamin B-6), which is essential and sufficient for catalysis; and heme, which is not. It is also regulated by SAM. The heme (iron protoporphyrin IX) moiety is bound at the NH₂ terminus with the axial ligands being cysteine 52 and histidine 65 (42). Mutations of these residues affect heme content, which in turn reduces CBS activity (43). Evidence from nuclear magnetic resonance spectroscopy suggests that changes in the state of heme oxidation are sensed by the phosphorus nucleus of the pyridoxal-5'-phosphate cofactor (44). Reduction of CBS leads to a twofold decrease in activity (45), whereas oxidizing conditions lead to a twofold increase in activity (46). These observations led to the hypothesis that heme acts as a redox sensor, and by monitoring these changes, heme modifies CBS activity. [Parenthetically, oxidation of cobalamin, with its related corrin rings that contain cobalt, inactivates methionine synthase for which it serves as a cofactor (10,47); thus, oxidation could coordinately increase disposal of HCY through CBS and decrease its remethylation through MS.]

Because CBS mutants exist where both heme content and enzyme activity are decreased (43), it is possible that CBS activity is regulated by the availability of heme, especially when CBS content is increased by de novo synthesis. Acute and catastrophic heme deficiency can occur during crises in porphyria patients (48), but less dramatic alterations can occur under more physiological conditions. For example, many different kinds of stress can lead to rapid induction of heme oxygenase I (HOI), which yields the products carbon monoxide (vasoactive and probably a gaseous neurotransmitter), iron (which is rapidly bound by ferritin), and biliverdin and bilirubin (potent antioxidants) (49). The result of HOI activity is a decreased concentration of heme that could affect the catalytic activity of CBS. There is precedent for this kind of regulation in the hepatic enzyme tryptophan pyrrolase. The greater the saturation of this enzyme with heme, the greater is its activity (50). Deficiency of heme as occurs in porphyria leads to reduced pyrrolase activity, higher tryptophan concentrations in the blood, higher tryptophan uptake rates into the brain, and increased serotonin levels, which have been implicated in mediating the neuropathology of porphyria (51).

If such a mechanism were operative, it might influence the flux of HCY down different pathways and help to regulate the balance between cysteine synthesis and methionine resynthesis. In the case of oxidant stress and resulting induction of HOI, the potential increase in oxidant activation of CBS and cysteine production could be offset by a heme-dependent decrease in CBS activity and less cysteine production. If so, this could compromise de novo synthesis of the major endogenous antioxidant GSH, the production of which might be desirable during times of oxidant stress.

GSH metabolism in aging

The final enzyme in the pathway converting methionine to cysteine (Fig. 1) is cystathionine -lyase, which is also known as serine-threonine dehydratase (SDH). Induction of SDH to oxidize excess HCY consequently results in the increased oxidation of threonine, which is accompanied by a reduction in circulating levels of threonine (52). With potentially sizeable amounts of HCY being produced when either methionine intake or cysteine demand is increased (e.g., when dietary cysteine is limited), HCY levels may rise and lead to diseases as discussed above. Moreover, if the activities of CBS and SDH are altered, downstream pathways (namely, cysteine availability for GSH synthesis) may be compromised. Rees and Hay (53) reported that SDH activity is reduced when animals are fed protein-deficient diets. This may imply that age-related changes in dietary protein intake not only affect SAA availability but also influence the activity of enzymes key to endogenous SAA regulation.

GSH, a tripeptide that is present in high concentrations in all mammalian cells, is the body's major endogenous antioxidant. It plays a vital role in detoxification reactions and protection of cells from the toxic effects of oxidants. Maintenance of body GSH stores is a complex, integrated phenomenon, and there is a resurgence of interest in potential interventions that may modulate GSH levels in the whole body and in specific tissues and cells. Advancing age, which is known to be associated with increased oxidative stress, is also reported to be associated with low GSH concentrations (54). In addition, aging is associated with an increased prevalence of impaired glucose tolerance and diabetes mellitus, and the latter is accompanied by lower GSH concentrations (55). The mechanisms that could be responsible for a compromised GSH status include decreased synthesis and/or increased utilization relative to synthetic capacity. In the case of GSH, utilization includes pathways in which oxidized GSH can be salvaged and reutilized as well as pathways that lead to irreversible loss such as conjugation reactions, excretion, or degradation. Thus, an inadequate supply of precursor AAs, defects in the biosynthetic pathways, or increased utilization leading to net loss can all ultimately lead to lower body-GSH stores. Ideally, one would like to measure in vivo rates of both GSH synthesis and utilization. Unfortunately, the multitude of pathways that consume GSH as well as variation among different tissues make it impossible to have meaningful simultaneous measurements of utilization by all of the different pathways in humans. Nevertheless, an important area for investigation is the impact of SAA availability for GSH synthesis when an older individual is compromised by disease or inadequate protein intake. Under these conditions, selective supplementation may be necessary to sustain defense mechanisms and prevent deterioration of cell function and overall health.

Summary

Age per se and associated age-related disorders act in concert to influence AA requirements and safe intake levels of specific AAs. The answer to the question of how to assess SAA adequacy and whether supplementation is necessary or approaches toxicity remains elusive. With respect to SAAs, a potential approach is to determine the patterns of DNA methylation of genes associated with specific conditions. Because methylation of CpG islands induces inhibition of expression, the relationship of gene expression in an SAA surfeit condition may differ from the deficiency state. As another example of using DNA methylation as an index of nutrient-gene interactions, individuals homozygous for the MTHFR C677T polymorphism were found to have a lower degree of genomic DNA methylation in peripheral blood monocytes compared with the CC wild-type persons (⁵⁶). The quantitative and highly specific liquid chromatography/electrospray ionization–mass spectrometry assay showed that genomic DNA methylation correlated directly with folate status and inversely with plasma HCY concentration. Extending these types of approaches to specific genes that are associated with age-related disease is an exciting prospect.

It is also possible that indirect measures of physiological function will provide us with an index for SAA adequacy or toxicity in vivo. Cardiologists have used noninvasive means to assess endothelial function as reflected by vascular reactivity and concomitant changes in blood flow (57). The assessment of dynamic changes in forearm blood flow may be used as an endpoint in determining the effects of an acute stimulus such as exercise, nitric oxide infusion, or methionine load. Additional perturbation of the system involved in methionine-HCY–methyl group metabolism may be caused by administration of creatine, which shifts the balance between the pathways as demonstrated in an animal model of uremia (58). In that study, creatine supplementation was found to lower plasma HCY concentrations in uremic animals.

Other potential biomarkers for SAA inadequacy or excess are protein or signaling products present in cells or blood that reflect oxidative damage or abnormal methylation as a consequence of an individual's SAA status. Davis recently commented on the use of exfoliated cells from target tissues to determine responses to bioactive food components (59). One can easily envision this approach combined with microarray analysis, proteomics, and in vivo studies to aid us in the evaluation of SAA metabolism. The toxicity of methionine and its related products needs further investigation, especially in relation to the exacerbation of diseases associated with advancing age.

	FOOTNOTES

 
¹ Presented at the conference "The Third Workshop on the Assessment of Adequate Intake of Dietary Amino Acids" held October 23–24, 2003 in Nice, France. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Vernon R. Young, Yuzo Hayashi, Luc Cynober, and Motoni Kadowaki. Conference proceedings were published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Vernon R. Young, Dennis M. Bier, Luc Cynober, Yuzo Hayashi, and Motoni Kadowaki.

² This work was supported by the National Institutes of Health (grants AG021106, AG00947, and MOIRR00109).

⁴ Abbreviations used: AA, amino acid; BMT, betaine-homocysteine methyltransferase; CBS, cystathionine ß-synthase; CpG, cytosine residue 5' to a guanosine residue; CVD, cardiovascular disease; ER, estrogen receptor-; FAO/WHO/UNU, Food and Agriculture Organization/World Health Organization/United Nations University; GSH, glutathione; HCY, homocysteine; HOI, heme oxygenase I; IAA, indispensable amino acid; MAT, methionine adenosyl transferase; MS, methionine synthase; MIT, Massachusetts Institute of Technology; MTHFR, 5,10-methylenetetrahydrofolate reductase; NF-B, nuclear factor-B; ROS, reactive oxygen species; SAA, sulfur amino acid; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SDH, serine-threonine dehydratase; VSMC, vascular smooth muscle cell.

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