Methionine Metabolism

BASIC SCIENCE

AND

CLINICAL APPLICATIONS

                                                                                                                                                                                

OF

THE ESSENTIAL AMINO ACID METHIONINE

         

                                                                         Written by

                                                                      Robert Bayless

                                                                                                                                           In                                           In collaboration with:

            

                                                                   Earl Matthew, M. D.

                                                           ALL RIGHTS RESERVED

           

                                                                            2005

                               

                                                                     SUMMARY                                                       

            Methionine, one of eight essential amino acids, participates in many biochemical reactions in the body.  These include protein synthesis, transmethylation, DNA/RNA synthesis, genetic expression, myelin formation, lipid metabolism, and after conversion of demethylated methionine (homocysteine) to cysteine, sulfhydral and disulfide contributions to protein structure and enzyme function, glutathione-related anti-oxidant functions, taurine formation, and the synthesis, metabolism, and excretion of the metabolic end product, sulfate.  The importance of methionine to the human body is demonstrated by the fact that the intestines absorb it first from a mixture of the eight essential amino acids, and at a higher percentage (98%) than any of the others (Adibi, 1967).

           

            Methionine is the major source of both methyl (CH3) groups and sulfur in the body.  It is converted to S‑adenosyl‑methionine (SAM), the primary methyl donor, which after demethylation yields S‑adenosyl‑homocysteine (SAH). A deficiency of methionine inhibits SAM formation, which may lead to hypomethylation of various substrates, such as neuronal damage due to hypomethylation of myelin. Other consequen­ces of hypomethylation may include cardiomyopathy and improper lipid metabolism. The criticality of methylation reactions is expressed by the fact that under various stressors, the metabolism of homocysteine to cysteine is inhibited, preserving methionine for protein synthesis and methylation at the expense of sulfhydration.  Additional dietary methionine may serve to (1) optimize methylation reactions, pre­venting hypomethylation of DNA, reduce neuronal and cardiac damage, restore proper fat utili­zation, and (2) increase cysteine supplies to facilitate structural protein formation, increase glutathione synthesis for anti-oxidant protection, optimize taurine formation for bile synthesis, increase sulfate availability for collagen and articular cartilage formation, and sulfate conjugation and excretion of otherwise toxic compounds.

Infants need 42-49 mg/kg/day of methionine for positive nitrogen balance; young adults need 13 mg/kg/day, while the requirement for the elderly increases back to 42 mg/kg/day, for unknown reasons.  Since food processing techniques such as bleaching, acid or alkali extraction, oxidation by unsaturated fats or sugars, and heat-related denaturation renders approximately one third of the methionine in raw food unavailable for human nutrition, a marginal methionine deficiency may develop as a person ages.  90 grams of unprocessed animal protein supplies 3 grams of methionine per day (43 mg/kg for a 70 kg man).   Loss of available methionine through processing, reduction of protein intake (dieting or poverty) or increased need based on pregnancy, aging, trauma or infection may further marginalize methionine nutrition.  Methionine deficiency may express itself in various guises, depending on the individual environment and heredity.  During marginal methionine intake, vitamins B12 and folate serve to remethylate homocysteine back to methionine, providing an avenue for endogenous synthesis of this essential amino acid.  Simultaneous B12 or folate deficiencies exacerbate a methionine deficiency, as illustrated by the well-known association of demyelination and megaloblastic anemia during deficiencies of these vitamins. Since the body continues to excrete cysteine and sulfate during food deprivation, depletion of structural protein-related cysteine following methionine deficiency may also occur over time, as well as depletion of sulfate stores, leading to loss of critical functions.

Albumin, at 42 grams/liter in serum, is the major repository of SH groups in the body.   As SH groups are the first line of antioxi­dant defense, maintaining normal levels of albumin becomes criti­cal in attempts to keep antioxidant and reducing capacity ele­vated in the body.  Since cysteine is the primary source for SH groups and cysteine is synthesized from methionine, supplemental methionine may help maintain serum albumin levels.

            The following discussion traces the metabolic pathway of methionine through the body, in the order in which it occurs naturally.  Since methionine may impact disease states in various stages of its metabolism, reference to a given disease may occur more than once.

                                    PATHWAYS OF METHIONINE METABOLISM

A.  Methionine Metabolism

 

            The essential amino acid methionine has several points of control in its’ metabolic cycle (Storch, 1990).  First, it is either incorporated into protein, or activated to S‑adenosyl‑methionine (SAM), the primary methyl donor in the human, which after demethylation yields S‑adenosyl‑homocysteine (SAH), and then homocysteine. Homocysteine is the second point of control.   If dietary me­thionine intake is deficient (less than 1 gram/day), the bulk of homocysteine is re-methylated back to methionine, with the methyl group ob­tained from serine through the oxidation of glucose. Vitamins B2, B6, B12, and folate are all necessary for the enzymatic movement of methyl groups from glu­cose to methionine by way of serine (Bridgers, 1965).  Dietary deficiency of methionine and/or the necessary B vitamins can lead to a deficiency of SAM, and thus to a deficiency of methyl groups for methylation reactions, as well as a deficiency of cysteine.  If methio­nine intake is adequate (1‑3 grams/day), about half of homo­cysteine remethylates to methionine, while the remainder conden­ses with serine to irreversibly form cystathionine, which then forms cysteine.  Cysteine, the primary source of sulfhydrals in the body, has several important functions.  It provides the internal structure for proteins; it can metabolize to glutathione; or it can metabolize to hypotaurine, and then taurine.  Cysteine is also metabo­lized to sulfate, the primary endpoint of methionine metabolism, and excreted in the urine. Sulfate is 2/3 the molecular weight of methionine, so that 3 grams/day of methionine results in the urinary excretion of 2 grams of sulfate.  Urinary test strips can be used to measure sulfate levels, and monitor methionine intake and bio-availability. If methionine intake is in excess of need (more than 3 grams/day) then excess methyl groups are transmethylated to sarcosine and oxidized, while the sulfur of homocysteine conden­ses with serine, forming cysteine, which the kidneys ultimately excrete as sulfate. The liver can also obtain methyl groups from choline, by way of betaine, to remethylate homocysteine back to methionine.  This pathway is relatively minor, and is not B vitamin dependent.

            1.  Protein Synthesis

            The inability of humans to remain in positive nitrogen balance on a diet with all known nutrients except methionine illustrates the essentiality of methionine.  As discussed below, limited intake of methionine inhibits protein synthesis at the DNA level by depriving mTHF of the homocysteine necessary for demethylation.  Once satisfaction of the essential transmethylation reactions occurs, additional methionine diverts to protein synthesis.   Interestingly, the transfer RNA must be methionylated before protein synthesis initiates (Cooper, 1981).  Then, adequate methionine must be available for incorporation into the protein itself.  This provides three points where the presence of methionine regulates the rate of protein synthesis.  The WHO sets the amount of methionine and cystine necessary for a normal adult to maintain a positive nitrogen balance at 13 mg/kg/day (Young, 1994). Supply by methionine of all the methyl groups used by the adult in one day requires 42 mg/kg/day (Mudd, 1980).  Infants require 42-49 mg/kg/day of methionine for nitrogen balance because of their high rates of protein synthesis (Snyderman, 1964), as do older adults (Tuttle, 1965), but for unknown reasons. 

            2. Transmethylation

            The most important of the various functions involving methionine constitutes the use of methyl groups (CH3) supplied by S-Adenosyl-Methionine (SAM) in a vast array of biochemical reactions.  In the metabolic economy, methyl groups follow only Adenosine Tri Phosphate (ATP) in importance.  Since the enzyme SAM Synthase produces SAM by condensing ATP and methionine, proper levels of SAM depend on adequate supplies of methionine.   Total daily SAM production in a normal adult averages 6 to 8 grams per day (Mato, 1994).  Donation of the methyl group to various receptors from SAM results in S-adenosyl-homocysteine (SAH) formation.  SAH acts as an inhibitor of SAM methylation reactions unless removal by further enzymatic action occurs.  SAH hydrolyzes to homocysteine.  Homocysteine must then be removed, or SAH degradation is inhibited, which then inhibits methylation reactions.  Excess intracellular homocysteine is exported to the plasma so that plasma homocysteine levels represent the amount of this amino acid in transit from the site of production to the site of catabolism (Finkelstein, 1998), primarily the kidneys.  Thus, the elevation of plasma homocysteine indicates a functional intracellular methylation blockade (Schatz, 1981).  Yi (2000) showed that as plasma SAH levels increased, so did plasma homocysteine levels, while lymphocyte DNA methylation decreased.  This suggests that plasma homocysteine levels may serve as an inverse indicator of the methylation state of the body.

            Several investigators have studied the generation and utilization of methyl groups in humans.  Mudd and Poole (1975) used oral methionine, cystine, and choline to determine methyl balance in normal subjects.  Storch (1990) used radiolabeled methionine orally to determine the same thing.  They both found total methyl usage to approximate 3.6 mg/kg/24 hr, or 250-mg/70 kg/day.  Since 1/10 of methionine is CH3, and since about 500 mg of methionine is required for protein synthesis each day (Storch, 1988), and if all the daily requirement of methyl groups is obtained as methionine, then the total daily intake of methionine should be about 3 grams. If methionine intake is inadequate, cells can make methyl groups from glucose, and remethylate homocysteine back to methionine.  However, the vitamins B2, B6, B12, and folate must be present in sufficient amounts to allow this to occur.  In this situation, the catabolism of homocysteine is inhibited, and cysteine formation suffers.  Urinary sulfate excretion is also inhibited, which the dipstick method can measure. Inhibition of methylation can have wide-ranging effects, depending on the cell type or metabolic function involved. 

Methylated products include:

           

Guanidoacetate       --->                Creatine       (140 mg/CH3/day)

Phosphatidylethanolamine            Phosphatidylcholine  (Lecithin) (45-90 mg CH3/day)

Phosphatidylethanolamine            Phosphatidylcholine (Myelin maintenance and repair)

Norepinephrine                            Epinephrine

DNA cytosine                               5-methyl-cytosine

Acetylserotonin                            Melatonin

Histamine                                     Methyl-histamine

Protein-Lysine                              Carnitine

           

                        a) Creatine synthesis.

            Creatine/phosphocreatine is a methylated compound, that serves as a high-energy storage medium in heart, muscle, and brain.  The liver and kidneys synthesize creatine by methylating guanidinoacetate, using SAM as the methyl donor.  Creatine spontaneously decomposes to creatinine, which is excreted in the urine at a rate proportional to lean muscle mass.  In strict vegetarians, daily creatinine excretion averages 15 mg/kg/day, or about 1 gram per day, which means that approximately 140 mg of methyl groups are lost each day, and must be replaced.  This represents about 56% of the total methyl consumption of normals per day.   Creatine synthesis is obligatory, and not affected by methionine or folate restriction (Jacob, 1995).  However, oral creatine supplements inhibit creatine biosynthesis, so that methyl groups otherwise lost in the urine are spared for other uses (Persky, 2001). 

                        b).  Myelin formation

            Myelin consists of myelin basic protein and various lipids.  Schwann cells synthesize peripheral nerve myelin sheaths, while oligodendroglia cells form and maintain myelin sheaths in the central nervous system.  The liver forms the fatty acid lipids used in myelin synthesis, and the blood transports them to the appropriate cells.   Phosphatidylcholine (PC) or lecithin, quantitatively the most important phospholipid in the body, appears in plasma at a concentration of about 2 grams/liter.   PC is a mixture of the diglycerides of stearic, palmitic, and oleic acids, linked to the choline ester of phosphoric acid.   The liver can form PC in two ways.    In rats, approximately 60-80% of PC derives from the CDP-choline pathway (CT), which uses preformed choline obtained from the diet.  In a lesser pathway, tri-methylation of phosphatidyl-ethanolamine by SAM to form PC by the enzyme Phosphatidyl-Ethanolamine Methyltransferase (PEMT) occurs.  A SAM deficiency inhibits this second pathway.  The myelin sheath also contains sulpholipids, which may act as the structural stabilizer for it.   The formation of sulfated lipids involves the transfer of activated sulfate to the lipid by PAPS (Farooqui, 1978) (see below).   Myelin turns over with a half-life of a year, while myelin basic protein has a half-life of one month.    Myelin synthesis occurs at a high rate early in life, and then maintenance and repair continue for the life of the person.

           

            Kim (1994) shows that a severe folate deficiency in rats causes an 80% reduction in hepatic SAM levels and an equal decrease in hepatic choline and phosphocholine.  Thus, the maintenance of adequate choline for myelin synthesis depends on the proper functioning of the PEMT pathway as well as the CT pathway.   If a dietary choline deficiency exists, extrapolating from rat data, the human liver may make up to 2.5 grams of PC per day using PEMT (Wise, 1965).   In the rat brain, PEMT contributes only 1% of the total PC formed (Cui, 1996).  However, myelin basic protein formation depends on adequate supplies of methionine for protein synthesis to proceed (Gould, 1987).  Thus, proper synthesis and repair of myelin sheaths requires the presence of adequate amounts of methionine.

            3.  DNA Synthesis

            DNA synthesis depends on a timely supply of purine and pyrimidine bases.  Intracellular tetrahydrofolate (THF) occupies a central role in the movement of carbon groups from glucose into both purines and pyrimidines.  The primary plasma transport vehicle for THF from the gut to cells is 5-methyl-THF (mTHF).  For a cell to retain THF, demethylation of mTHF must occur.  The B12-dependent enzyme Homocysteine-THF Methyltransferase performs the dual function of reforming methionine from homocysteine as well as demethylating mTHF, which the cell can then retain to participate in the formation of DNA.   Thus, a deficiency of vitamin B12, folate, or methionine can inhibit DNA synthesis.  A methionine deficiency results in a homocysteine deficiency, which means mTHF cannot be demethylated.  A B12 deficiency also means mTHF demethylation inhibition and folate deficiency likewise inhibits mTHF formation.   The results of this interdigitation of methionine, B12, and folate illustrate the primary importance of transmethylation reactions.  As methionine levels fall, intracellular THF levels fall, which inhibits DNA synthesis.  This results in an inhibition of protein synthesis, curtailing a major demand for methionine.  At the same time, the decline of methionine levels inhibits the condensation of homocysteine with serine to form cysteine, which further conserves homocysteine for remethylation to methionine, while starving the body for cysteine. This leaves the remaining methionine for SAM synthesis, and protects transmethylation reaction products, as shown above.

            4.  Transcription regulation

            Each mammalian cell contains the entire genome sequence of DNA.  Regulators of expression of gene activity appropriate for each cell type include methylation of Cytosine-phosphate-Guanine base pairs (CpG) by the S-adenosyl-methionine-dependent enzyme DNA methyltransferase.  A given cell transmits its methylation pattern to its progeny.  A methylation deficiency may allow hypomethylation of daughter cells.  Hypomethylation of DNA (when compared to that of a “normal” cell) is necessary but not sufficient to allow inappropriate expression of otherwise silent genes, some of which may be detrimental to the functioning of the cell.  Examples include:

                        a. Oncogenes.  

                        Genes whose products can change the cell into a malignant one are called oncogenes.  Hypomethylation allows the expression of otherwise suppressed genes.  If the randomly hypomethylated gene codes for malignant growth, cancer results (Balmain, 1995).  Rats fed a methyl-deficient diet developed hypomethylated hepatic DNA within one week of beginning the diet (Wainfan, 1989).  Nicotine reduces guinea pig lung tissue SAM levels 15-fold after 21 days of s.c. injection (Godin, 1986).  This may contribute to cigarette carcinogenesis.  Chronic ethanol feeding to rats causes a decrease in hepatic SAM levels (Barak, 1986, 1993).  This may explain the increased risk of colorectal adenoma associated with chronic alcohol abuse (Giovanucci, 1993).   Diets low in methyl groups allow spontaneous cancers to develop in animals, so a methyl-rich diet may prevent some types of cancers in humans (Hoffman, 1986).  Kim (1997) found that folate deficiency caused DNA strand breaks and hypomethylation within the p53 tumor suppressor gene in rats.  Since alterations within this gene have been implicated in greater than 50% of human cancers, folate deficiency, and thus methyl deficiency, may have an important impact on nutrition-related cancers.

                        b. Latent virus genes.  

                        Latent viruses, including HIV, randomly infect DNA.  Should the viral RNA land on a CpG sequence, or island, and then methylation of the island prevents transcription and expression of the virus.  Induced hypomethylation allows the virus to replicate (Bednarik, 1993).  The supplemental consumption of methionine and B vitamins by HIV-positive individuals may suppress HIV replication.

                        c. Senescence genes. 

                        Stable, inheritable methylation patterns of DNA appropriate to a given cell type are necessary for proper cell functioning over time.  Should DNA hypomethylation occur, otherwise silenced genes may be expressed, and if the gene in question codes for otherwise inappropriate products, cell dysfunction may occur, perhaps leading to cell death (Cooney, 1993).   Supplemental methionine and B vitamins may slow this process.

            5.  Methylation deficiencies and effects

                        a. Megaloblastic Anemia

            Deficiency of vitamin B12, due to poor absorption of the vitamin caused by a lack of intrinsic factor, produces megaloblastic anemia, and over time, demyelination of peripheral nerves, and later of the spinal cord.  Dietary deficiency of folate produces only megaloblastic anemia.  Scott (1981 a, b) demonstrated in monkeys that blocking the B12-dependent enzyme Methionine Synthetase with nitrous oxide caused degeneration of the spinal cord, and that simultaneous administration of methionine blocked most of the degeneration.  They surmised that a methionine deficiency directly causes the B12-related demyelination seen in megaloblastic anemia.   Administration of folate alone for the treatment of megaloblastic anemia, when the anemia is due to vitamin B12 deficiency, worsens the demyelination because folate increases consumption of the limited stores of methionine for protein synthesis due to increased mitosis, decreasing further the methionine available for the slower process of myelin formation and repair.  Patients suffering from megaloblastic anemia secondary to a B12 deficiency worsened when treated with methionine alone (Rundles, 1958).  However, patients with these anemias may show additional benefit by supplementation with methionine in addition to the usual B12 and folate.

B12 deficiency results in the inability to demethylate methylTHF, causing inhibition of DNA synthesis.  Megaloblastic blood cells result.

B12 deficiency also leads to methionine deficiency and demyelination results. 

Folate deficiency leads to inhibition of DNA synthesis, and only megaloblastic blood cells result.

                        b.  Brain injury repair

                                    1.  Physical trauma

                                    Most head-injury patients exhibit hyperglycemia upon admission, and the higher the peak 24-hour serum glucose level following injury, the worse the neurologic outcome (Young, 1989).     If the elevation of blood glucose following injury involves damage to the hypothalamic membranes, then perhaps membrane repair through supplemented methyl supply can reduce neurological damage. The regeneration of neuronal cells relies in part on adequate supplies of S-adenosyl-methionine (Cestaro, 1994).   The gas nitrous oxide inhibits the vitamin B12-dependent remethylation of homocysteine, which leads to a methionine deficiency, which leads to demyelination of neuronal cells.   Supplemental methionine can block this nitrous oxide effect on demyelination, as shown in pigs (Weir, 1988).  Stacy (1992) describes two cases of nitrous oxide-related neuropathy in humans.  One of these patients received supplemental dietary methionine, which seemed to speed recovery.  Thus, methionine, vitamin B12, and folate may show benefit in recovery from physical brain injury.

           

                        c.   Alzheimer’s damage: prevention and repair

                                    1.   Oxidative events play a role in brain damage typical of Alzheimer’s Disease.  Reactive microglia are associated with neuritic plaque formation in AD (MacKenzie, 1995).  Tumor Necrosis Factor, which triggers Nitric Oxide release (Rockett, 1992) is significantly elevated in the serum of AD patients when compared to controls (Fillit, 1991).  Nitric Oxide Synthase activity is significantly higher in brain microvessels of AD patients, compared to controls.  Erythrocyte glutathione peroxidase is significantly lower in AD patients, compared to controls (Jeandel, 1989).  Finally, rheumatoid arthritics, on anti-inflammatories for much of their lives, has 1/7 the expected incidence of AD for their cohort (McGeer, 1990).  Providing additional methionine to early-stage Alzheimer’s patients may decrease oxidative damage to brain tissue by providing additional sulfhydryl groups to bind excess NO, as well as increase glutathione peroxidase synthesis for increased antioxidant protection.

                                    2. Since AD patients exhibit significantly lower SAM levels in cerebrospinal fluid (Bottiglieri, 1990), and a decrease of 67 to 85% in selected brain tissues (Morrison, 1996) methylation reactions may be inhibited.  Morrison did not find a decrease in SAM in brain tissue from Parkinson’s Disease patients, suggesting that the decrease in methylation is specific to Alzheimer’s, and not simply a characteristic of chronic neurodegeneration.  McCaddon (1998) demonstrated a highly significant elevation of serum total homocysteine in patients with AD.  Griffith (1969) speculated that memory storage may occur on the DNA of nerve cells, through methylation of bases, and predicted an association of memory disturbances with a deficiency of methionine.  Riggs (1996) found lower concentrations of B12 and folate, and higher concentrations of homocysteine associated with poorer spatial copying skills on a cognitive test. During learning experiments in mice, brain uptake of radiolabeled sulfur from methionine increased significantly (Hershkowitz, 1975).   Thus, supplemental methionine and B vitamins will supply additional methyl and sulfhydryl groups necessary for the proper functioning of learning and memory systems, and perhaps delay the memory loss typical of Alzheimer’s Disease.  If onset of AD could be delayed by 5 years, half of people who would otherwise suffer from the disease would die of other causes, and not have to suffer the consequences of memory loss.

                        d. HIV Dementia

            HIV infection of the brain frequently results in AIDS Dementia Complex.  Autopsy of the brains of patients with ADC reveals myelopathy, similar to that of vitamin B12-related sub-acute combined degeneration.  Analysis of CSF levels of SAM shows a significant lowering compared to controls, while levels of s-adenosyl-homocysteine, a SAM-blocker, were significantly elevated (Keating, 1991).  Hortin (1994) found plasma levels of methionine 1/3 below normal and cystine 2/3 below normal in blood analyses from 20 HIV-positive people.  Chamberlin (1996) found that a SAM deficiency caused demyelination of the brain.  These data indicate limited availability of methionine for myelin and myelin basic protein synthesis, perhaps resulting in myelopathy and dementia.  The myelopathy may be exhibited by impotence and urinary urgency and frequency, both of which depend on signal transmission from the brain to the genital region by way of the spinal cord.  Di Rocco (1998) hypothesized that HIV-related myelopathy may be secondary to a methionine deficiency.  Therefore, they gave 12 HIV-positive persons who exhibited clinical signs of progressive myelopathy 6 grams of l-methionine a day for 6 months.  9 completed the trial, and 7 of them showed improvement in Central Conduction Time, a measure of spinal cord signal transmission time.  This was assumed to show increased myelination of degenerated nerve tissue.

                        e. Multiple Sclerosis

            Periodic destruction and regrowth of CNS myelin characterize multiple sclerosis (Prineas, 1993).  Chamberlin (1996) showed that persons deficient in Methionine Adenosyl-Transferase activity suffered brain demyelination, probably by interfering with myelin phospholipid synthesis.  During the periods of remission, when myelin regrowth occurs, MS patients may benefit from supplementation of dietary precursors for endogenous synthesis of myelin, including methionine, B vitamins, choline, and sulfate.

B.  Membranes, Methylation, and Calcium Transport

            The methylation of membrane phospholipids affects calcium transport into cells.   During the membrane methylation process, three methyl groups, obtained from SAM, are sequentially added to phosphatidyl-ethanolamine by the enzyme PEMT, creating phosphatidyl-choline (lecithin).  Hirata (1980) shows that rat mast cells increase calcium uptake following membrane methylation, leading to histamine release.  Panagia (1986) shows that the addition of SAM to rat sarcolemmal membrane preparations increased the activity of the Ca-pump necessary for the efflux of calcium from the cell.   Schanne (1979) demonstrates that elevation of cytosolic calcium leads to cell death.  Thus, methionine deficiency could cause a methyl deficiency, leading to disruption of calcium transport.  Various effects may be expected, depending on the cell type affected.

            1.  Cardiomyopathy

                        a. Calcium Transport. 

                       

                        The etiology of cardiomyopathy remains unknown (Olsen, 1992).  Calcium transport, both into and out of the myocardial cell, intimately relates to contractile function.  Gupta (1988) investigates the effect of methylation on contractile functions of rat myocardium.  They show that methionine can increase the developed force of the perfused heart.  They also demonstrated a linear relationship between the increase in developed force and the increase in methyl group incorporation into heart phospholipids.  Finally, they show that methionine stimulates Ca-pump activity (which leads to a reduction of intracellular calcium).

                        b. Calmodulin

                       

                        Homeostasis of calcium metabolism in the heart includes the maintenance of a 20,000-fold concentration gradient for calcium across the myocardial membrane (Clapham, 1995).  Calcium influx, as shown above, involves methylation of the sarcolemma and the sarcoplasmic reticulum.  Calcium efflux uses the Calcium pump and includes calmodulin as the carrier protein to bind calcium during transport.   Calmodulin, the primary intracellular calcium regulatory protein, is trimethylated at the lysine position by SAM.  Causes of cardiomyopathy may include calmodulin deficiencies.  Jeck (1994) found a significant decrease in calmodulin mRNA in heart tissue in patients with end-stage heart failure.  They did not measure heart calmodulin levels.  Muzulu (1994) found a 21% decrease in calmodulin-stimulated calcium pump activity in RBC membranes from Type II diabetics, compared to controls.  This may contribute to diabetic cardiomyopathy.  Methionine and B vitamins may normalize calmodulin synthesis in congestive heart failure, as measured by myocardial membrane calmodulin levels, and cardiac calmodulin mRNA levels.

                        c. Carnitine and Ethanol.

                        The liver synthesizes carnitine, beginning with trimethyl-lysine (TML).  TML is produced by the post-translational methylation of protein-bound lysine, with the three methyl groups obtained from SAM.  Given the relative paucity of proteins methylated at the lysine position (calmodulin, histones, and cytochrome c) calmodulin is probably the primary precursor protein for carnitine synthesis (Paik, 1980). Carnitine provides an essential co-factor in the transfer of long-chain fatty acids into the mitochondria.  Carnitine deficiency inhibits myocardial fatty acid oxidation.   Chawla (1985) shows that plasma levels of total carnitine (free carnitine plus fatty acylcarnitine esters) in patients deprived of methionine and choline fell significantly.  Regitz (1990) demonstrated a significant reduction of total carnitine in myocardial tissues from CHF patients.  However, plasma total carnitine levels were elevated, indicating a defect in myocardial carnitine uptake rather than in hepatic synthesis (Rebouche, 1983).   The hallmark impact of ethanol ingestion on the heart is a loss of contractility (Bing, 1978).   Dodd (1987) showed that the combination of ethanol and a methionine/choline deficient diet led to a significant fall in total myocardial carnitine levels in rats.  Trimble (1993) showed that chronic ethanol feeding to rats caused a significant decline in hepatic SAM levels and a rise in SAH levels.  This would definitely interfere with SAM-dependent methylation reactions, such as synthesis of calmodulin, and carnitine, and interfere with calcium transport because of deficient methylation of the myocardium, each contributing to loss of heart muscle contractility.  The use of methionine, choline, the B-vitamins, and carnitine by itself, may show benefits in alcoholic as well as other cardiomyopathies.

                        d. Carnitine and Remodeling

                        Following the onset of cardiomyopathy, remodeling may occur as a compensatory enlargement following an increase in cardiac workload.  Iliceto (1995) reports a 36-center study in Italy found supplemental carnitine to cause a significant reduction in left ventricular remodeling, post-infarct, compared to placebo.  This suggests a further study of the effects of methionine, B vitamins, and carnitine on remodeling in cardiomyopathy.

                        e. Nitric Oxide.

                        Additional factors affect cardiac function.  Winlaw (Lancet, 1994) shows that CHF patients have twice the levels of plasma nitrate (the oxidized product of nitric oxide), compared to controls.  They postulate that increased nitric oxide synthase activity leads to increased production of nitric oxide, which increases the relaxation of cardiac muscle.  Stamler (1992) demonstrates that nitric oxide forms a stable, bio-active adduct with the functional sulfhydryl group of albumin.  The overproduction of nitric oxide, or a sulfhydryl deficiency, may interfere with this system, causing inappropriate dilation of heart vessels. 

                        f.   Folate.

                       

                        Brody (1969) demonstrated that 10 sequentially admitted patients with CHF all had abnormally low serum folate levels.  B12 levels were normal in all patients.   All 10 also exhibited megaloblastosis.  This deficiency might have been due to the use of diuretics, which may decrease intestinal absorption and increase renal excretion of folate.  Certainly, folate deficiency will decrease the remethylation of homocysteine, and interfere with methylation reactions as shown above.

                        g. Selenium. 

                        Dietary selenium deficiency increases the incidence of CHF in rural China (Johnson, 1981).  Glutathione peroxidase (GP), the primary antioxidant enzyme in blood, exists in two forms, one of which is selenium-dependent.  GP helps prevent oxidative damage in erythrocytes, and may also protect myocytes as well.  The increase in nitric oxide discussed above would have additional adverse effects if the reduction in amounts of antioxidant enzymes occurred as well.  However, Tho (1987) did not find a reduction in erythrocyte GP content obtained from CHF patients, although SOD levels were lower than normals.  Since methionine serves as a precursor for glutathione peroxidase synthesis, supplemental oral methionine, and selenium may show benefit in congestive heart failure, both by normalizing membrane methylation and scavenging excess oxidants, including nitric oxide.

            2.  Pancreatitis

            In 1959, Kahn demonstrated the mechanism by which ethionine, a methionine antagonist, causes acute pancreatitis.  In the rat pancreas, ethionine caused a 50% reduction in phospholipid phosphorus, indicating an inhibition of membrane synthesis due to a methyl-supply deficiency.  The decrease in membrane methylation causes a decrease in permeability, which leads to an increase in proteolytic enzyme activity within the pancreas, leading to acute pancreatitis.  Kahn reports that simultaneous administration of methionine prevents these effects.  Since normal pancreatic exocrine secretion in the humans varies between 1 and 4 liters of pancreatic juice per day, interference with normal membrane permeability quickly causes the pancreas to swell.  Balaghi (1995) reports that rats fed a folate-deficient diet for 5 weeks exhibited a significant reduction in pancreatic amylase secretion, probably due to methyl deficiency.  Ward (1995) postulates that interference with Calcium transport mechanisms, either into or out of the acinar cells, triggers acute pancreatitis.  Ishiguro (1992) demonstrated that inhibition of calmodulin activity in cultured rat acinar cells reduced amylase release.  Uden (1990) reports a 20-week, double-blind clinical trial using the vitamins A, C, and E, selenium, and 2 grams per day of methionine in 20 patients with chronic recurrent pancreatitis.  Methionine was chosen because patients with chronic pancreatitis exhibit deranged sulfur metabolism (Martensson, 1986).  Six patients had an attack while on placebo, but none while on active treatment. Thus, methionine and B vitamins may be of benefit in the treatment of pancreatitis by increasing methyl supply, thus normalizing membrane methylation, calmodulin activity, calcium transport, and ultimately amylase transport.

                        a. Alcohol-induced Pancreatitis

                        Major effects of long-term ingestion of alcohol include pancreatitis.  Barak (1993) shows that alcohol feeding significantly reduces hepatic activity of the folate-dependent enzyme Homocysteine-mTHF methyltransferase in rats.  In addition, patients with alcohol-related cirrhosis have 56% of the activity of S-adenosyl-methionine synthetase, compared to controls (Duce, 1988).  The activity of the SAM-dependent enzyme Phospholipid Methyltransferase was less than half that of normals.  Since the pancreas exhibits the highest uptake of methyl-labeled methionine per gram of organ weight in the mouse (Comar, 1976), the poor eating habits of alcoholics, combined with a block of methionine resynthesis, SAM formation, and SAM-dependent methylation reactions, would cause acute pancreatitis due to a methylation deficiency as shown above.  Thus, alcoholics suffering an attack of acute pancreatitis may benefit from alcohol restriction, and adequate refeeding, along with methionine and B vitamin supplementation.

                        b. Alcohol and the Salivary Gland

                        Morphologically, the pancreas and the parotid glands are related.  Chronic alcoholics exhibit reduced salivary flow rates, as well as decreased amylase secretion (Dutta, 1992).  Deenmamode (1993) describes alcohol-induced parotid enlargement, as well as increased serum amylase content in patients with alcoholic liver disease.  The enlargement, decreased secretion rate, and entry of amylase into the blood mimic the symptoms of acute pancreatitis.  In rats, feeding alcohol resulted in a significant decrease in sodium and an increase in the potassium content of saliva ((Maier, 1986).  However, Syrota (1983) found no significant difference in parotid gland uptake of radiolabeled methionine between 11 normal subjects and 9 alcoholics.  If alcoholic parotid glands were methionine deficient, they should have shown increased uptake.  Nevertheless, methionine and B vitamins may have a beneficial impact on parotid gland function in alcoholics similar to the effects in pancreatitis.

                        c. Drug-induced Pancreatitis

                        Dideoxyinosine (ddI) is an effective HIV anti-viral.  However, it can cause acute pancreatitis in a significant portion of recipients (Maxson, 1992).   Badr (1991) reports that ddI stimulates hepatic glycolysis in the perfused rat liver and that the stimulation was probably dependent on the ddI-induced mobilization of intracellular calcium stores.  This effect on calcium in hepatocytes may explain the effects on the pancreatic acinar cells.  Boobis (1990) demonstrates the elevation of intracellular calcium by toxic doses of acetaminophen causes cell death because the co-administration of a calcium-specific chelator prevents the lethal effect.   Thus methionine, an effective antidote for acetaminophen overdose (Vale, 1981), may show benefit in the prevention of ddI-induced pancreatitis.

            3.  Drug-induced Neuropathy

            The nucleoside analog HIV-reverse transcriptase inhibitors azidothymidine (AZT), dideoxyinosine (ddI), dideoxycytidine (ddC), and dideoxydidehydrothymidine (D4T) all exhibit toxic side effects in addition to their intended effects on a key enzyme in the infection cycle of the AIDS virus.  ddI, D4T, and ddC cause painful peripheral neuropathy in a significant portion of recipients (Simpson, 1995), which resolves following discontinuation of the drugs.  These side effects limit the usefulness of otherwise effective tools in the fight against AIDS.  An examination of the mechanisms involved in the induction of these side effects may reveal a method to improve the toxicity profile of these drugs.

            Chen (1991) demonstrates a reduction of mitochondrial DNA (mtDNA) in CEM cells in culture by nucleoside analogs, in the order of ddC > D4T > AZT > ddI.   Since mitochondrial defects are associated with neuropathy, they speculate that this may be the mechanism by which nucleoside analogs induce neuropathy in a significant portion of HIV-positive patients who receive the drugs. 

            On the other hand, Feldman (1992) shows that ddC induces neuropathy in the rabbit through myelin splitting and demyelination of axons.  Werth (1994) discusses the effects of ddC on mitochondrial calcium in neurons.  They feel ddC may be an effective tool to study the role of mitochondria in calcium homeostasis and neuropathy.  If these are the actual mechanisms, then the combination of B vitamins and methionine with nucleoside analogs may provide a way to improve the toxicity profile associated with these anti-virals by restoring myelin as shown above.

C.   Lipid Metabolism

                        a) Lipid metabolism in the body has several aspects:

                                    1.   Phosphatidylcholine (lecithin) has three sequential functions in lipid metabolism.  First, during digestion, the liver secretes bile, composed of bile salts, bile fluid, and lecithin, into the intestinal lumen.  Bile partially hydrolyzes dietary lipids, which are then taken up and delivered to the liver, which reassembles them into lipoproteins.  Second, the liver conjugates lipoproteins with lecithin, forming a water-soluble fatty molecule, known as VLDL lipoproteins, or phospholipids, which are secreted into the plasma.  This allows the plasma to transport lipids without the formation of vessel-clogging droplets. Third, the liver also secretes lecithin by itself directly into the plasma, where it appears at a concentration of 2 grams/ liter. 

            The liver can produce lecithin in two ways.   In bile lecithin formation 70% of choline is obtained pre-formed from the diet using the CDP-choline pathway (CT), with the remainder synthesized by the tri-methylation of phosphatidyl-ethanolamine by the enzyme Phosphatidyl-Ethanolamine Methyl Transferase (PEMT) with SAM as the methyl donor.  A SAM deficiency inhibits this second pathway.   In fatty acid and plasma lecithin formation, 70% is formed endogenously by methylation, with 30% coming from preformed dietary choline. In methyl deficiency, fatty buildup in the liver ensues because the packaging of lipoproteins with lecithin, and their secretion, cannot occur.  Rat studies show that of the eight essential amino acids, only me­thionine deficiency leads to fatty liver.  For this reason, methionine is known as a lipotrope or fat lover. Thus the digestion and uptake, and subsequent packaging and export of lipids are all methionine dependent.

            Bile lecithin formation averages 1-2 grams per day, as does average fatty acid and plasma lecithin synthesis.  Thus, the consumption of methyl groups for the synthesis of lecithin averages 45-90 mg per day in a normal person.  This represents 18-36% of the total daily methyl usage.   An inhibition of methionine availability has deleterious effects on both total lecithin formation and the character of the lecithin fractions (Lyman, 1974).   Thus, a methylation deficiency could cause an energy deficit, as an inhibition of fatty acid metabolism occurs.

                                    2.    All cells have membranes, which they must maintain.  Nerve cells synthesize, maintain, repair, and degrade their outer covering, called myelin.  The phospholipid portion of membranes and myelin is obtained from plasma lecithin.

                       a) Synthesis, maintenance, and repair of myelin.  

                                    Myelin consists of myelin basic protein and various lipids.  Schwann cells synthesize peripheral nerve myelin sheaths, while oligodendroglia cells form and maintain myelin sheaths in the central nervous system.  The liver forms the lecithin used in myelin synthesis, which the blood then transports to the appropriate cells. Turnover of membrane-associated lipids averages 2-3 days in men. A lecithin deficiency can thus affect membrane and myelin integrity throughout the body. In addition, membrane function can be altered by the methylation of phosphatidylethanolamine to phosphatidylcholine in each cell.   The myelin sheath also contains sulpholipids, which may act as the structural stabilizer for it.   The formation of sulfated lipids involves the transfer of activated sulfate to the lipid by the enzyme PAPS (Farooqui, 1978). Thus, methionine is crucial to proper membrane synthesis and function.

            Kim (1994) shows that a severe folate deficiency in rats causes an 80% reduction in hepatic SAM levels and an equal decrease in hepatic choline and phosphocholine.  Thus, the maintenance of adequate choline for myelin synthesis depends on the proper functioning of the PEMT pathway as well as the CT pathway.   If a dietary choline deficiency exists, extrapolating from rat data, the human liver may make up to 2.5 grams of PC per day using PEMT (Wise, 1965).   In the rat brain, PEMT contributes only 1% of the total PC formed (Cui, 1996).  However, myelin basic protein formation depends on adequate supplies of methionine for protein synthesis to proceed (Gould, 1987).

D.   Prevention of Neural Tube Defects During Pregnancy

            A clinical trial has shown that maternal supplementation with folic acid significantly reduces the incidence of neural tube defects in newborns (Wald, 1991).  However, the mechanism by which this occurs has not yet been demonstrated.  Mills (1995) found that mothers of babies with NTD had significantly lower plasma levels of folate and correspondingly higher levels of plasma homocysteine.  Vitamin B12 levels were normal.  Ubbink, (1995) speculates that the primary defect is a dietary deficiency of methionine, coupled with a dietary folate deficiency.  The methionine deficiency means fetal cells must depend on the folate-dependent remethylation of homocysteine by the enzyme methionine synthase to supply the methionine needed for SAM synthesis.  SAM then supplies the methyl groups necessary for, among other reactions, the manufacture of myelin and myelin basic protein, both used in neural tube formation.  In addition, the plasma transfer form of folate is 5-methyl-tetrahydrofolate.  This must be demethylated before it can be retained and utilized by the cell.  The remethylation of homocysteine simultaneously demethylates methyl-THF, so that folate can then perform its intracellular function of promoting DNA synthesis so necessary for fetal growth.   Thus, Ubbink holds that folate supplementation serves to remedy both a folate deficiency and a methionine deficiency and that the elevation of plasma homocysteine  He recommends vitamin B12 as well as folate supplementation for pregnant women, in order to prevent masking megaloblastic anemia.    VanAerts (1994) shows that high levels of homocysteine reduce the incidence of neural tube defects in 9.5-day rat embryos, as did methionine.  They speculate that the NTDs associated with elevated homocysteine in humans is due to a methionine deficiency leading to a methylation deficiency, and not due to elevated homocysteine per se.  Scott (Lancet, 1981) in a discussion of pernicious anemia, states that if folic acid is to be given to large portions of a population to prevent disease, methionine should also be given to prevent masking a possible B12 deficiency.  Taken together, these results indicate that when either B12, folate, or methionine is taken supplementally, the other two should be co-administered.  Vitamins B2 and B6 are also involved in the methionine metabolism cycle and should be consumed with the others.   Since a folate deficiency can lead to a choline deficiency (Kim, 1994), and since fetal development requires increased synthesis of myelin, choline supplementation during pregnancy should provide additional substrate for nerve development.

E.  Folate, Homocysteine, and Premature Vascular Disease

            Plasma folate status and plasma homocysteine levels display an inverse relationship (Jacob, 1994), as do plasma homocysteine levels and lymphocyte DNA methylation states (Yi, 2000). Low plasma folates and elevated plasma homocysteine significantly correlate with an increased risk of premature vascular disease of the heart, brain, and peripheral vascular bed (Miller, 1994).   Israelsson (1988) found a high proportion of men with low conventional risk factors who suffered myocardial infarction exhibited moderate hyperhomocysteinemia.  Nygard (1997) found that plasma homocysteine levels directly correlated with increasing coffee and cigarette consumption.  While the exact mechanism by which mild fasting hyperhomocysteinemia (>16 umol/L) induces vascular damage remains elusive, Olszewski (1993) points to the oxidative modification of LDL cholesterol as one culprit.  Stamler (1993) demonstrates that nitric oxide reacts with homocysteine to form S-nitroso-homocysteine and that the complex has vasodilator effects, while the oxidative properties of the parent homocysteine are eliminated.  They propose that in normal persons, NO inactivates homocysteine, and a homocysteine excess overwhelms this process, allowing the toxic properties of homocysteine to manifest themselves.   Whether homocysteine itself is toxic, or the pathogenic effects of mild hyperhomocysteinemia are due to a methyl deficiency, or both, the reduction of plasma homocysteine levels becomes a prime therapeutic objective.  As Jacob (1994) shows in a study of ten healthy men, plasma homocysteine levels do not exhibit a correlation with methionine intake, but with plasma folate status.  Ubbink (1994) discusses the relationship between vitamin status and plasma homocysteine and says that 400 mcg of folate/day should maintain homocysteine levels below the desirable 14 umol/L threshold.       

            Miller (1994) proposes that folate and/or B12 deficiency allows homocysteine elevation because inhibition of homocysteine remethylation occurs, leading to a methionine deficiency and thus a SAM deficiency.  Since the homocysteine-serine condensation to the cystathionine pathway is inhibited by a SAM deficiency, the removal of homocysteine to cysteine does not occur.  Also, in humans, the betaine-homocysteine remethylation pathway is inhibited when methionine intake is low, to conserve the demethylation of the methyl-THF pathway (Scott, 1981).   All of this presupposes a methionine deficiency.  The end result of a combination of folate, B12, and methionine deficiency is an accumulation of homocysteine and possible induction of atherosclerosis.  Van den Berg (1994) found 23% of 309 patients with arterial disease to exhibit hyperhomocysteinemia following methionine loading, and that 12 weeks of treatment with 5 mg/day of folate and 250 mg/day of vitamin B6 normalized the post-load values in all but 3 patients, who responded to additional betaine.  Thus, adequate intake of methionine and the vitamins B6, B12, and folate are necessary to prevent homocysteine accumulation and the possibility of homocysteinemic atherosclerosis.

Total Serum Homocysteine consists of:         

Free Homocysteine (10%)

Homocysteine-homocysteine mixed disulfide

Homocysteine-cysteine mixed disulfide

Homocysteine-protein mixed disulfide

   (The protein involved is mostly albumin)

Normal Ranges of Total Serum Homocysteine:

            10 umol/L: Normal means (fasting)

            5.4 to 16.2 umol/L (± 2 S.D.): Normal range (fasting)

            4.1 to 21.3 umol/L (± 3 S.D.): Normal range (fasting)

            ³ 16.3 umol/L:  Mild Hyperhomocysteinemia

            24.3 ±  7.1 umol/L:  Renal failure

           

            No change: 1 gram methionine/day for one month in 16 normals

             9 umol/L: post typical meal in normals

            27 umol/L: post 3 gram methionine load in normals

            37 umol/L: post 7 gram methionine load in normals

           

            58.3 ± 37.7 umol/L:  In Megaloblastic anemia due to folate deficiency

            87.3 ± 50.1 umol/L:  In Pernicious anemia due to B12 deficiency

            50 to 300+ umol/L:   In Homocysteinurics

F.   Type II Diabetes and Methionine

            Adequate methionine intake ensures the presence of sufficient homocysteine to ensure demethylation of 5-methyl-tetrahydrofolate for normal DNA synthesis.  As plasma levels of methionine rise, hepatic levels of S-adenosyl-methionine rise due to increased synthesis.  Increased SAM levels activate the vitamin B6-dependent enzyme cystathionine synthase (CS), which catalyzes the condensation of homocysteine and serine to irreversibly form cystathionine, which cleaves to form cysteine.  Thus, the formation of cysteine depends on adequate intake of methionine, and the vitamins B2, B6, B12, and folate (Finkelstein, 1990).  The functioning of CS depends on adequate supplies of homocysteine serine and vitamin B6.    Serine is an enzymatic product of glucose and is elevated in the urine of adult diabetics, while glycine, the demethylated product of serine, is decreased (Sasaki, 1988).  Munshi (1996) found hyperhomocysteinemia in 7 of 18 Type II diabetics following a methionine load.  Ubbink (1995) demonstrated that a vitamin supplement of B6, B12, and folate could significantly reduce plasma homocysteine in normal men.  Hardwick (1970) found that large oral doses of methionine drastically reduced blood glucose in guinea pigs.  Sprince (1969) showed that i.p. injection of homocysteine in rats produced convulsions and that prior injection of serine could prevent this effect.  Supplemental methionine and B vitamins may serve to lower blood glucose levels by increasing serine, and thus glucose, disposal. 

            a. Methylation deficiency.

            Various reports illustrate a methylation deficiency in diabetes.  Dyer (1988) found a 25% reduction in brain SAM levels in genetically diabetic rats.  Tashiro (1983) found an increase in phosphatidylethanolamine methyltransferase activity in diabetic rat brains, which probably reflects the decrease in SAM levels.  Inhibition of membrane phospholipid methylation may account for the peripheral neuropathy and demyelination typical of diabetes (Duchen, 1980).  Ganguly (1984) found a significant decrease in radiolabeled methyl incorporation into rat cardiac sarcolemma following the induction of diabetes.  This decreased methylation may contribute to cardiac dysfunction.  Glucose stimulated methylation of rat pancreatic islet membranes before insulin release (Kowluru, 1984) Application of a methylation inhibitor decreased insulin release in a similar system (Laychock, 1984).  A methylation deficiency may partially account for the delayed insulin response following a meal found in Type II diabetics (Kelley, 1994) by both inhibiting neural transmission of a hypothalamic signal to the pancreas and by inhibiting insulin release from the pancreas in the presence of increased blood glucose.  Muzulu (1994) found a 21% reduction in calmodulin-stimulated calcium pump activity in Type II diabetics when compared to controls.  Calmodulin, a tri-methylated protein, maintains normal intra-cellular calcium levels.  A deficiency in calcium export leads to increased intracellular osmotic pressure, increased platelet rigidity, and decreased microcirculation, all of which may contribute to the vascular complications typical of Type II diabetes.  A methylation deficiency may inhibit calmodulin synthesis and activity.

            b. Chromium

           

            Chromium, an essential trace element, functions with insulin to promote normal glucose tolerance in man.  Chromium deficiency results in insulin resistance.  Chromium-deficient patients with Type II diabetes respond to increased consumption of chromium, especially when administered as chromium picolinate.  Chromium supplementation lowered glycosylated hemoglobin and lipid fraction components such as LDL cholesterol and apolipoprotein B (Evans, 1989).  Combining chromium picolinate with methionine and B vitamins may provide additional glucose-lowering benefits.

            c. Gestational Diabetes

            Insulin resistance and hyperglycemia are typical in human pregnancy, are increased in gestational diabetes, and resolve following delivery (Ryan, 1985).  This process decreases maternal glucose consumption and provides maximal glucose for fetal development (Johnson, 1996).  Serine and glycine are excreted in large amounts during pregnancy (Hytten, 1972), indicating the elevation in DNA synthesis required by fetal growth.  Methionine is needed both for protein synthesis, and to provide the homocysteine to allow the demethylation of 5-methyl-tetrahydrofolate required for DNA synthesis.  During pregnancy, plasma homocysteine drops to about 50% of normal (Andersson, 1992).  The fetus extracts homocysteine from the umbilical vein (Malinow, 1998), probably to provide a way to maximally demethylate mTHF for the elevated DNA synthesis necessary for fetal growth.  Since urinary methionine excretion is also elevated, meeting maternal and fetal needs requires additional methionine during pregnancy.  Supplemental methionine, choline, and B vitamins may normalize blood glucose and decrease the incidence of neural tube defects, as shown above.

G.  Cysteine and Sulfhydral Groups

            When the presence of sufficient methionine satisfies the needs of transmethylation, DNA, protein, and myelin reactions, excess homocysteine exits the methionine cycle through the transsulfuration pathway.  This B6-dependent pathway, activated by elevated SAM levels, forms cystathionine from homocysteine and serine, with cysteine being the final product.  The bulk of Cystathionine Synthetase (CS) activity lies in the liver and kidneys.    Essentially, the sulfur of methionine forms the sulfhydryl (SH) of cysteine, which various enzymes use as active sites, and supplies structural integrity for proteins.  Thus, a limited methionine intake can result in a sulfhydryl deficiency and inhibit enzyme activity and protein formation.  In addition, surgical stress can inhibit the cystathionine condensation pathway (Vina, 1992), as can sepsis (Malmezat, 1998).  Sulfhydryl deficiency effects include:

            a. Total Parenteral Nutrition

               Stegink (1972) showed that i.v. administration of Total Parenteral Nutrition (TPN) to eight healthy subjects caused plasma cystine (cysteine-cysteine disulfide) concentrations to drop to 1/5 of normal in 12 hours, and remain there until the feeding stopped.  Oral administration of the same TPN solution caused plasma cystine concentrations to fall to 3/5 of normal, and return to normal on a regular diet.  Since TPN contains methionine but not cysteine, Stegink surmised that systemic administration of methionine bypasses the liver and gut and that peripheral tissues cannot metabolize methionine to cysteine.  However, peripheral tissues can degrade methionine through the transamination pathway, but Blom (1989) demonstrates the quantitative unimportance of this pathway in normal adults.

               Chawal (1985) compared the effects of normal diet, oral TPN, and i.v. TPN containing methionine but not cysteine in normals, cirrhotics, and undernourished noncirrhotic patients.  They found abnormally low plasma cystine and protein-bound cysteine in the undernourished non cirrhotics, which indicated that when methionine delivery bypasses the liver and gut, it renders the transsulfuration pathway from methionine to cysteine inoperable.    Since protein breakdown continues even during inhibition of protein synthesis, as urinary cysteine loss during fasting demonstrates (Martensson, 1982), one can expect the inability to form cysteine during TPN to cause muscle wasting.   Choline and phosphocholine levels also fell, further illustrating the dependence of myelin formation on adequate methionine intake.

               Selberg (1995) found that AIDS patients on TPN displayed lowered plasma methionine and plasma cystine 1/5 of normal.  This deficiency occurred despite the addition of methionine and cysteine to the TPN solution.  In a similar study, Vinton found plasma taurine levels reduced to 40% of normal levels in patients receiving TPN even though the solutions delivered about 2 grams of methionine per day.  Clearly, systemic administration could not overcome the need by the liver to be supplied with sulfur amino acids through the portal circulation.

               Oral administration of methionine during TPN may overcome the shortcomings inherent in the systemic administration of amino acid solutions.  Since methionine absorption occurs early in the small intestine, the presence of a short small bowel due to resection should not prevent methionine uptake (Canolty, 1975).  Measurement of serum albumin provides a simple method to track protein synthetic rates before and during oral methionine administration.

            b. Microalbuminuria.

           

              The onset of microalbuminuria typifies Type II diabetes.  This onset correlates with the development of renal disease, nephrotic syndrome, and renal insufficiency.  Hayashi (1990) shows that the charge state of albumin differs between normals and both Type II diabetics with microalbuminuria and those with nephrotic syndrome.  A methionine/cysteine deficiency may allow albumin to fragment, and the kidney may preferentially excrete these fragments.  Examination of protein from blood and urine for protein fragments before and after methionine supplementation may show alteration of these fragments, and thus expose a methionine/cysteine deficiency in diabetes. 

            c. Arthritis

            Albumin composes 60% of total plasma proteins.  Albumin functions include the regulation of osmotic pressure and the transport of fatty acids.  Albumin with one free SH group available for reaction with plasma constituents is called mercaptalbumin.   The free sulfhydryl of albumin can form a disulfide bridge with cysteine, glutathione, or homocysteine, and is called nonmercaptalbumin.   Two albumin molecules can form a disulfide bridge, are called macroglobulins, and compose the other 40% of plasma proteins.    In normal plasma, 60-70% of total albumin is unoxidized, or mercaptalbumin, and the other 30-40% is the oxidized nonmercaptalbumin.  This ratio of free to oxidized albumin defines the redox state of the organism, and in normal plasma is maintained at about 2 to 1. A fall of this ratio to 1 to 1 or less is associated with several disease states, including rheumatoid arthritis (Thomas, 1975). Given the quantity of albumin in plasma, albumin represents a significant portion of the total antioxidant capacity of plasma (Halliwell, 1990).   A change in the free SH content of plasma can occur in four ways:

                        1.  Reduction in overall albumin levels.  This can occur by interference with albumin synthesis, or by increased albumin breakdown.  Plasma albumin comprises an accurate indicator of protein intake and is sensitive to essential amino acid intake, including methionine.  A nutritional deficit leading to a reduction in overall plasma albumin, and thus in free sulfhydryls, does not necessarily mean a change in the ratio of free to oxidized albumin but may indicate a decrease in the overall antioxidant capacity of plasma.

                        2.  Increase in mixed disulfide content.  An increase in cysteine binding to the free SH of albumin results in mixed disulfide formation.  Thomas (1975) found such an increase in rheumatoid arthritics, as have many other researchers.  Whether the fall in the ratio of free to oxidized SH groups in plasma is related to a cause or simply an effect in arthritis remains to be demonstrated.

                        3.  Increase in macroglobulin formation.  An increase in macroglobulin formation will decrease free SH groups.   Rheumatoid factor, a macroglobulin produced in rheumatoid arthritis, is associated with a decrease in serum-free sulfhydryl content (Lorber, 1964).    

                        4.  Increased oxidative attack of albumin.    Hyperactivity of immune cells, especially polymorphonuclear leukocytes, characterizes rheumatoid arthritis.  These cells produce a wide range of oxidative reactants, including peroxide, hypochlorous acid, and nitric oxide.  Albumin acts as a sacrificial antioxidant when these oxidizers appear in plasma, and damaged albumin is quickly removed from circulation (Halliwell, 1990).  This process may partially account for the decrease in free SH groups typical of RA.

            Given the important position of albumin as a plasma antioxidant, and the demonstrable decrease of free SH groups in arthritis, sulfhydryl replacement therapy to treat arthritis makes sense.  In addition, the continuation of cysteine loss during fasting (Martensson, 1982) indicates that a methionine deficiency will lead to a cysteine deficiency, further exacerbating sulfhydryl depletion.  The use of methionine and the B vitamins along with the measurement of the ratio of free to total sulfhydryls should reveal any benefit from this therapy.

H.  Glutathione

            Synthesis of glutathione, a tripeptide composed of glutamate, glycine, and cysteine, occurs primarily in the liver, although all cells make it.  Cysteine, existing in the lowest concentration of the three constituents, is the rate-limiting component so the supply of glutathione depends on adequate methionine intake to ensure activation of the homocysteine-serine condensation pathway (Bianchi, 2000).  Since cysteine easily forms the insoluble disulfide cystine, glutathione acts as an oxidation-blocked form of cysteine, allowing it to move through blood without reacting with sulfhydryls already present.  Thus the liver synthesizes glutathione using cysteine obtained from methionine and then exports glutathione to the blood for delivery elsewhere in the body.   Glutathione does not pass through cell membranes intact, but is broken down into its three parts; they are imported and then reassembled into glutathione as needed.    In man, plasma levels of glutathione average 5-10 um/l, while intracellular levels range from 500 to 10,000 um/l (Meister, 1984).

            Mosharov (2000) has demonstrated that the two homocysteine-utilizing enzymes, methionine synthase and cystathionine b-synthase, have reciprocal sensitivity to oxidization.  Under experimental oxidizing conditions, methionine synthase is inhibited, and cystathionine b-synthase is activated.  This may serve as a physiological control mechanism, so that as glutathione is consumed by oxidative stress, increased flux through the transulfuration pathway occurs, leading to an increase in the precursors of glutathione.  These results also suggest that an increase in plasma levels of homocysteine may not only indicate a decrease in methyl availability, but an increase in the oxidative load experienced by the body.

                        Glutathione has many functions, including:

                        1.  The Oxidation-Reduction System and Reducing Equivalents        

                 

            The Oxidation-Reduction Potential (Redox Potential) of a system refers to the measurement of the relative tendency of a substance to acquire or donate an electron.  The term “oxidation” refers to the loss of one or more electrons, and the term “reduction” refers to the gain of one or more electrons.  Mitochondria metabolize oxygen and glucose, and using oxidation-reduction reactions in the electron transport chain produce Oxidizing Equivalents in the form of ATP, and Reducing Equivalents in the form of glutathione, either of which cells can use as an energy source.  Some of the glucose moves through the pentose phosphate shunt, which supplies electrons for reductive functions (Reducing Equivalents).  Nicotinamide Adenine Dinucleotide (NAD+) is reduced, forming NAD (H), which can then reduce Nicotinamide Adenine Dinucleotide Phosphate (NADP+), forming NADP (H).   The production of ATP and Reducing Equivalents are uncoupled in mitochondria, and cells can respond to an increased oxidative load by increasing the production of Reducing Equivalents without increasing ATP production (Sullivan, 1983, 1984).   NADP (H) can then transfer an electron to Glutathione Disulfide Reductase, a riboflavin-dependent intracellular enzyme that then reduces glutathione disulfide back to glutathione.  Glutathione provides the primary means by which cells store and transport labile Reducing Equivalents (electrons).  Since glutathione can reduce oxidized ascorbate, and ascorbate can reduce oxidized vitamin E (Kehrer, 1994), the primary source of Reducing Equivalents in the body flows from aerobic oxidation of glucose by mitochondria through glutathione.

                    Flow of Mitochondrial Reducing Equivalents

 Diet --->Tocopheral (Vit. E)-->----+ e ------->--------Tocopheral Radical  

                         |                                                                 |
               Dehydroascorbate --<----+ e -------<--------Ascorbate((Vit.C) <---Diet                          |                                                                 |

               Glutathione --------->----+ e -------->--------Glutathione Disulfide

                         |                                                                 |                                                                      |                            + e                                |

                         |--------<--- Glutathione Reductase ---<----|

                         |                     (B2 dependent)                    |      ­

                         |                                                                 |

                   2 NADP+                                                  2 NADP (H+)  

                         |                                                                 |                     ­

                         ---->------------------------------------>------          

                                     Mitochondrial Membrane

                       ------------------------------------------------------

                               |      Electron Transport Chain ­      |

                               >------------------------------------>

             Glucose-6-Phosphate Dehydrogenase (Pentose Phosphate Shunt)

                          Generates Protons for Reducing Equivalents

             Beutler, (1969) demonstrates that administration of 5 mg/day of riboflavin to normal subjects maximized their red cell glutathione disulfide reductase activity, while the MDR of 1.5 mg/day did not.  Taniguchi (1983) showed that rats on a riboflavin-deficient diet exhibited reduced liver glutathione content, and reduced glutathione disulfide reductase activity.

           

             The importance of maintenance of the proper redox state in the cell becomes apparent when one realizes that the conformation of proteins depends on structural linkages provided by disulfide bridges and that the reactivity of cysteine-containing enzymes depends on the supply of electrons to maintain them in the reduced state.  In addition, cysteine-containing proteins in membranes alter their transport properties depending on their redox state (Shapiro, 1972).  Ammon (1973) shows that isolated rat pancreatic cell release of insulin was dependent on NADP (H) production from glucose.  Inhibition of NADP (H) production inhibited insulin secretion.  Langhans (1985) found that feeding behavior in rats was inversely related to the generation of Reducing Equivalents in hepatic mitochondria.  Sun (1990) reviews the induction of carcinogenesis by free radicals, and the role of antioxidant enzymes and Reducing Equivalents in the removal of oxidizing species before they can damage DNA. 

                        2.  Oxidative damage prevention.

             Normal biological processes result in the production and utilization of aggressive oxygen species.   This necessitated the development of antioxidant defenses to control the use and direct the activity of these oxidants.   Ease of oxidation typifies sulfhydryls, and several antioxidant defense systems rely on adequate supplies of sulfhydryls for proper operation, including glutathione, the predominate intracellular antioxidant, and glutathione peroxidase, the predominate extracellular antioxidant.   A disease such as arthritis, which involves the increased production of oxidative species (Grabowski, 1996), or aging, which exhibits decreased production of antioxidants, each may respond to elevation of antioxidants and antioxidant precursors, such as methionine and the vitamins C, E, and beta carotene, selenium and zinc (Ames, 1993).

                        3.  Prevention of acetaminophen toxicity

            Hepatic glutathione depletion results from acetaminophen overdose, and i.v. administration of a large dose of methionine to mice prevents this depletion (Miners, 1984).    Since a calcium chelator can prevent cell death in vitro from an otherwise toxic dose of acetaminophen, calcium overload probably causes cytotoxicity, while glutathione synthesized from methionine probably acts as a chelator of calcium and prevents loss of viability (Boobis, 1990).  Vale (1981) reports that none of 96 acetaminophen overdose patients died if they received 2.5 grams of methionine every four hours for twelve hours when the therapy was initiated within 10 hours of the overdose.  Use of methionine and the B vitamins to ensure maximum glutathione production should provide adequate treatment of acetaminophen overdose.

                        4.  Inhibition of HIV Transcription.

            Infection by HIV generates a chronic inflammatory state in the human immune system, and the oxidative stress results in, among several effects, depletion in the primary extracellular and intracellular reducing agent, glutathione.  Since N-acetyl-cysteine, a glutathione prodrug, inhibits HIV replication in stimulated cell lines (Roederer, 1991) several researchers have tried giving NAC to HIV-positive patients, to little effect (Kalayjian, 1994; Jarstrand, 1994; Witschi, 1995).  However, a recent report by Herzenberg (1997), well-known NAC researchers, shows the association between poor patient survival rates and low glutathione levels in CD4 T cells, and that NAC supplementation in such patients doubled their chances to survive 2 years, compared to those who did not take NAC.   Since HIV-positives exhibit significant reductions in plasma methionine and cysteine (Hortin, 1994), supplementation with methionine and B vitamins should correct these deficiencies, allow maximization of plasma and cellular antioxidant defense systems, provide sulfhydryls for albumin and antibody synthesis, and supply methyl groups to suppress HIV transcription as shown above.

                        5.  Hepatitis C

            Barbaro (1999) studied blood and liver levels of glutathione in 130 Hepatitis C patients.  They found significant reductions in both versus controls. Increased oxidative stress and depleted glutathione may lead to interference with mitochondrial function and accelerated hepatocytosis.  Bianchi (2000) showed oral methionine caused a significant increase in blood glutathione in six healthy controls. 

            Hourigan (1999) found fatty liver (hepatic steatosis) in 61% of 148 Hepatitis C patients they studied.  Hoyumpa (1975) discusses the biochemistry of fatty liver.  Gabuzda (1956) discloses that methionine and the methionine derivative choline are called lipotropes because they prevent fatty liver in experimental animals. It seems possible that a dietary methionine deficiency may lead to both a decline of hepatic and blood levels of glutathione as well as hepatic accumulation of fat as seen in Hepatitis C infection.  Oral methionine and B vitamins should be tried in a study of Hepatitis C.

                        6.  Protection of S-Adenosyl-Methionine Synthetase

            S-adenosyl-methionine is formed from methionine and ATP by S-adenosyl-methionine synthetase.  Glutathione depletion causes a SAM synthetase as well as SAM depletion in rat liver (Corrales, 1991).  This inactivation probably occurs because of free radical interaction with SAM synthetase, such as nitric oxide (NO) (Ruiz, 1998) which glutathione would otherwise prevent (Corrales, 1990) (Avila, 1998).

                        7.  Inhibition of Nitrate Tolerance.

                                    a. Abrams (1991) discusses the mechanism of action of nitroglycerin, and nitrate tolerance, in vascular smooth muscle relaxation.  Nitroglycerin converts to nitric oxide (NO) in the cardiac endothelium, forms S-nitrosothiols (SNO) at the plasma membrane of smooth muscle cells, and catalyzes the conversion of GTP to cGMP, leading to muscle relaxation.  This same mechanism operates normally in that the enzymatic synthesis of NO from arginine in the endothelium creates an SNO, which relaxes cardiac smooth muscle (Stamler, 1992).  Both the normal and chemically induced NO rely on adequate amounts of cellular sulfhydryls to properly bind and create SNO, a relatively stable form of NO.  Thus, several researchers have suggested a sulfhydryl deficiency to explain the tolerance typical of chronic nitroglycerin administration (Abrams, 1991).  Levy (1988) administered nitroglycerin i.v. with and without methionine to 15 patients with suspected coronary artery disease, and found a significant decrease in the amount of nitroglycerin necessary to maintain a 10% decrease in mean arterial pressure (MAP) when combined with methionine.  This indicates that methionine decreased the tolerance to nitroglycerin which otherwise occurred.  I.v. methionine alone had no effect on MAP.   Since the periphery converts i.v. methionine to cysteine poorly (Stegink, 1972), oral administration of methionine and B vitamins should improve results. Ogawa (1985) found a significant reduction in plasma sulfur amino acids (methionine, cystine, and taurine) in hypertensives.   Sander (1995) showed that blocking NO synthase with methyl-arginine caused hypertension in rats.  Hishikawa (1993) demonstrated that administration of i.v. arginine to ten patients with hypertension caused a significant increase in NO production and a significant decrease in MAP.  Since oral administration provides a better test of the sulfhydryl-repleting effects of methionine, a trial of nitroglycerin oral methionine and B-vitamins in hypertension seems warranted.

                                     b. Myocardial remodeling, including hypertrophied myocytes and increased interstitial collagen, can occur as a result of infarct, cardiomyopathy, or hypertension, each of which causes an increase in cardiac workload, leading to a compensatory enlargement (Anversa, 1992; Goldstein, 1994).  Angiotensin Converting Enzyme inhibitors (ACE inhibitors) prevent remodeling after infarct by both interfering with the growth-stimulating effects of Angiotensin II and also by lowering overall cardiac load (Johnston, 1993).  McDonald (1993) showed that i.v. nitroglycerin prevented cardiac remodeling following experimental infarction in dogs.  Numaguchi (1995) demonstrated that inhibition of NO synthesis in rats caused hypertension and myocardial remodeling.  Iliceto (1995) reports that a 36-center study in Italy of the effects of carnitine on cardiac remodeling post-infarct showed a significant reduction in left ventricular remodeling, as measured by a reduction in the increase of left ventricular volumes compared to placebo.  Jugdutt (1996) suggests a long-term study of the effects on postinfarct mortality of the combination of ACE inhibitors and nitroglycerin or other NO donors.  ACE inhibitors, nitroglycerin, arginine, carnitine, methionine, and B vitamins include candidates for such a trial.  

                        8.  Adriamycin (Doxorubicin) and Sulfhydral Depletion

                        Doxorubicin, an effective antineoplastic drug, causes irreversible cardiomyopathy in 30% of patients who receive more than a 550 mg cumulative dose (Saltiel, 1983).  Saltiel points out that animal studies must be performed in mice, as only mice exhibit both cardiac toxicity and appropriate tumor response to the drug.  Olson (1980) gave Adriamycin to mice and found cardiac glutathione levels significantly reduced.  I.p. injections of N-acetyl-cysteine were able to prevent the fall of glutathione, as well as prevent cardiac damage.  In acute toxicity studies, NAC was able to significantly reduce the number of deaths due to Adriamycin.  The authors speculate that the glutathione and glutathione peroxidase system may be depleted by the excessive free radicals generated by adriamycin (similar to acetaminophen), and that NAC reduces toxicity by both acting as a free radical quencher and by replenishing glutathione and glutathione peroxidase.  If so, then oral methionine and B vitamins should be able to reduce the side effects and extend the effectiveness of a significant tumoricidal agent.

                        9.  Lead chelation.

                        Lead poisoning in rats causes a reduction of hepatic glutathione, while methionine supplementation increases hepatic glutathione while simultaneously increasing fecal excretion of lead (Kachru, 1989).  Methionine and B vitamins may be of value in the treatment of acute lead poisoning in children.

                        10.  Sunlight and skin cancer.

                        The association between sunburn and skin cancer is well known.  Ultraviolet radiation (UV) can directly damage skin cell DNA and also generate free radicals, which can continue to damage cell constituents (Axelrod, 1990).    Conner (1987) demonstrated in mice that UV exposure causes transient depletion of glutathione in the skin.  The loss of this crucial intracellular protection may lead to skin cancer (Slaga, 1995).   Methionine and B vitamins may help prevent the progression from sunburn to skin cancer.

I.  Sulfate

            Assuming satisfaction of the cysteine demand for sulfhydryls, excess cysteine exits through two pathways.   In the minor pathway, hypotaurine and taurine result, with daily synthesis amounting to approximately 100 mg/day in the adult (Irving, 1986), which does not increase in response to additional intake of oral methionine (Block, 1965).   In the major pathway, cysteine oxidizes to cysteine sulfinic acid, and then to pyruvate and sulfite, which further oxidizes to sulfate (Lemann, 1959).  Feeding three grams of methionine to an adult results in two grams of sulfate excreted in the urine within 24 hours of ingestion (Laster, 1965).  Thus, urinary sulfate measurement provides an easy way to monitor methionine intake.  During fasting, urinary sulfate excretion falls, but obligatory losses of about 250 mg/day reflect protein breakdown (Lakshmanan, 1976).   Collagen synthesis depends on adequate supplies of sulfate (Brown, 1965), and feeding a sulfate-deficient diet to rats resulted in significant weakening of aortal breaking strength (Brown, 1965a).   Wound healing in rats increases the rate of sulfur metabolism in wound tissue (Williamson, 1955). Burn patients exhibit a greatly increased need for methionine as measured by sulfate retention following methionine supplementation (Larsson, 1982) (Martensson, 1985).  Mucus and sulpholipid synthesis depends on adequate sulfate availability (Farooqui, 1978).  In addition, since certain drugs require sulfation for detoxification, increased intake of them creates a “methionine drain” by depleting the body of sulfate.   Zezulka (1976) found that supplemental sulfate could partially replace methionine in a nitrogen balance study in humans, providing evidence that sulfate depletion can create a methionine drain.

            1.  Sulfate Conjugation

            Prior to conjugation, the activation of sulfate to phosphoadenosine phosphosulfate (PAPS) in the liver must occur.  PAPS then donates an “active sulfate” to the substrate during conjugation.  Many substances are detoxified and eliminated by this process, including acetaminophen (Farooqui, 1980).   Morris (1983) showed that acetaminophen could deplete serum sulfate and decrease urinary excretion of sulfate in men.  This implies that acetaminophen usage could create a methionine drain by consuming sulfate.

            2.  Inhibition of Calcium Oxalate Stone Formation.

            Renal calculi are comprised of calcium oxalate in 75% of kidney stone patients.  Since the urinary content of calcium and oxalate do not significantly differ between stone formers and non-formers, some other factor affects saturation and thus precipitation.  Since protein intake and renal calculi display a positive correlation (Schuette, 1980), and since calcium and sulfate excretion display a positive correlation (Tschope, 1985) several researchers suggest increased sulfur amino acid intake as the primary culprit in stone formation.  Contradicting this suggestion, Curhan (1997) found little or no association of calcium oxalate stone formation with protein intake in a ten-year study of over 90,000 nurses.  Supplemental calcium, in the form of dairy products, did reduce stone incidence, probably by reducing dietary oxalate absorption.  They did not measure urinary sulfate.  However, Selvam (1991) found that all 12 rats consuming a calculi-producing diet exhibited renal stone formation while doubling dietary methionine for others receiving the same diet prevented the formation of calcium oxalate renal stones in 12 of 12 rats. The addition of methionine changed neither calcium nor oxalate excretion rates when compared to controls.  The sulfate metabolized from the methionine may have inhibited nucleation, and thus stone formation. 

J.   Methionine Losses in Food Processing

             Conversion of methionine in food protein to various forms, which have lesser nutritive value, can occur by a variety of food processing methods. Rats can grow on oxidized methionine (methionine sulfoxide) about half as well as on methionine.  However, Stegink (1986) shows that the administration of methionine sulfoxide to humans does not cause an elevation of plasma methionine levels. This result implies that humans cannot convert methionine sulfoxide to methionine. Breslau (1988) provides evidence for losses of available methionine through processing.   In a study of fifteen subjects, the calculation of total dietary sulfate intake using food tables indicated an average intake of 33 mmol/day for those subjects consuming animal protein.  However, urinary analysis indicated that those same subjects excreted only 20 mmol/day of sulfate.  Similar ratios in the same subjects were obtained during the consumption of a vegetarian diet.  These data indicate that approximately 1/3 of total methionine/cysteine in food protein was unavailable for human nutrition, presumably as a result of processing, as indicated below. Martensson (1982) shows that cysteine excretion continues during fasting so a methionine deficiency will lead to a cysteine deficiency.   Lakshmanan (1976) demonstrated that humans continued to lose sulfate in the urine during a fast, averaging 280 mg/day.  Zezulka (1976) found that dietary sulfate could partially replace the loss of dietary methionine in a nitrogen balance study, implying that the body has a sulfate demand.  D-methionine has no such effect, again demonstrating that it is not available for human nutrition.

            Taken together, the losses of available methionine due to processing, a limitation on the ability to utilize methionine sulfoxide, and continual urinary loss of cysteine as well as sulfate, provide the basis for the existence and effects of a chronic methionine dietary deficiency.

Food processing methods causing methionine oxidation include:

            1:  Flour Bleaching

            Probably the single largest source of methionine conversion results from the bleaching of flour.  Ground wheat is treated with chlorine at a dose of about 3 ounces per 100 pounds to break down some proteins and thus allow a lighter baked pro­duct. While the mechanism of cleavage has not been characterized the methionine peptide bond is known to be particularly sensitive to chlorine. Cyanogen bromide, an oxidant like chlorine, is routinely used to specifically cleave methionine residues in proteins. Young rats fed flour treated with chlorine at 2 ounces per 100 pounds gained 21% less weight than control rats (Cunningham, 1977). Bleached flour, white or wheat, is the predominant source of bread and other baked goods that are sold and consumed in the United States and other "Western" countries.

            2:  Oxidation by Reducing Sugars.

            Methionine readily oxidizes when heated with reducing sugars, such as glucose and fructose.   This process, called the Maillard Reaction, results in browning.  Thus canned fruits containing syrup are likely to contain methionine sulfoxide.  Horn (1968) found the reaction product of dextrose and methionine almost com­pletely unavailable as a nutritional source of methionine in the rat.

            3:  Oxidation by Unsaturated Fat

 

            Mixing of protein and unsaturated fats in the presence of air results in the oxidation of methionine to the sulfoxide.  Relative humidity affects the process somewhat.  As more and more emphasis is placed on reducing cholesterol and saturated animal fat intake, more and more unsaturated fats are used in preparing foods.  Simply mixing unsaturated fat with protein allows oxidation to occur.  A cake mix containing unsaturated fat, sitting in a hot warehouse for several months, will have lost some of its available methionine by this mechanism.  Storage of milk protein (casein) with unsaturated fat for 3 months at 40 degrees C results in about 30% of methionine oxidizing to the sulfoxide (Coq, 1978).

            4.  Destruction by Heat

            Fujimaki (1972) showed that when casein, a dried milk pro­tein, was roasted at 250 degrees C. for 20 minutes, 100% of the methionine and cysteine was destroyed.

            5.   Acid and Alkali Extraction

            Chemical extraction of protein products from their original sources using acid or alkali extraction procedures, such as those used in the processing of soy protein, results in the loss of some methionine.  Fortification with methionine of soy protein extracted in alkali restores positive nitrogen balance in men fed soy protein (Zezulka, 1976).

            6.  Protein Denaturation

            During the process of cooking, some proteins are denatured in such a way that digestive enzymes cannot degrade the protein.  Only about 75% of the methionine content of some types of peas remains available to rats after heating (Sarwar, 1986).

 

K.         Toxicity or Side Effects

            1.  L-Methionine

                Adverse effects of l-methionine do not occur until consumption of excessive levels occurs.  With excessive consumption of l-methionine, the results in rats can vary from slight suppression of food intake, followed by an adaptation and return to normal food intake, to marked food intake suppression. Alterations in tissues can also occur in which the spleen may enlarge and contain increased iron.  The kidneys and livers of rats may also enlarge from excessive ingestion of l-methionine (Benevenga, 1974).  The amounts of methionine necessary to cause these alterations are many times higher than those suggested by this report.  The only physical side effects reported from more reasonable, normal doses (5 and 10 grams/day for 2 months) in humans have been gastric distress and flatulence (Delrieu, 1988).

               Infants:  During the period from about 1950 to 1970 soy-based infant formula for babies with milk intolerance contained dl-methionine.  Supplementation of these products with dl-methionine occurred because the method of extraction of the soy protein destroyed methionine. As of 1979 350,000 infants were being fed soy protein formula. The recommended daily allowance of methionine for infants is four times that for adults.   Assuming that infant formula based on soy protein containing dl‑methionine was used from 1950 to 1970 and that an average of 200,000 infants per year consumed dl‑methionine as it would be given in the ProSoBee formula then a total exposure of 4 million baby years can be estimated as the exposure group for dl‑methionine.  If infants consume about 1 liter per day of the final volume of formula they would receive about 400 mg of dl‑methionine or about 200 mg per day of d‑methionine.  Infants excrete 70‑90% of ingested d‑methionine unchanged in the urine.  No side effects were noted in infants during this time.  The dl-methionine mixture is no longer allowed in baby food as the d-form interferes with the PKU (phenylketonuria) urine test.  During mass screening for phenylketonuria, one infant was discovered with abnormal urine levels, which turned out to result from formula-based dl-methionine intake (Efron, 1969).  In another study, an infant was discovered who had decreased hepatic SAM synthesis.  Blood levels of methionine were 20 to 30 times normal.  This infant was 1 year old and showed no adverse effects of the highly increased blood levels of methionine.  Some breaks were seen in the outer membrane of mitochondria and hyperplasia of the smooth endoplasmic reticulum in the liver was noted (Gaull, 1981). Because d‑methionine was shown to be poorly utilized by infants, and because d‑methionine gives a false positive on the phenylketonuria test for newborns, the use of d‑methionine in infant formula was banned in 1972, and only l‑methionine was added to soy‑based formula thereafter (Stegink, 1971).

               Adults:  In a paper co-authored by some of the best researchers on methionine, Gahl (1988) found a 31-year-old male who complained of bad-smelling breath to have a partial defect in hepatic SAM synthase capabilities.  He displayed a 20 to 30-fold increase in blood levels of methionine due to this block.  His breath contained 17-fold elevations of dimethylsulfide.  No other physical abnormalities were found.

               During treatment of the genetic disease Homocystinuria, Smolin (1981) inadvertently elevated the patient’s blood levels of methionine.  During the 10-month treatment period, the blood levels were 10 times normal for 8 patients, and no abnormalities of liver, kidney, or bone marrow function were detected.  Similar treatment of two additional patients for two years resulted in a 10-30-fold elevation of methionine blood levels and no obvious side effects, other than improvement in other disease symptoms.   In a similar study, six grams of betaine per day was given to 10 patients with cystathionine synthase de­ficiency to lower homocysteine through the betaine­: homocysteine remethylation pathway.  While homocysteine was lowered to normal, plasma methionine levels were elevated, in seven cases to more than 30 times normal.  They found no evidence of change in liver, renal, or bone marrow function after two years of such treatment (Wilchen, 1983).

               L-methionine given in conjunction with psychotropic drugs for disease treatment at 20 grams per day for 5 days and 40 grams per day for 2 days resulted in symptoms associated with alcohol intoxication, namely euphoria, sleepiness, and confusion (Wortis, 1963).  They reported no other adverse physiological effects.

               Blom (1989) gave 17 normal individuals 7 grams of l-methionine each to determine the rate of metabolism.   Blood levels of methionine increased to 20 times normal at 4 hours, remained at 14 times normal at 8 hours, fell to 8 times normal at 12 hours, and returned almost to normal at 24 hours.  No adverse reactions were reported.

               When two normal subjects received 9 grams of l-methionine per day on an adequate diet, a reduction of urinary glycine excretion occurred.  No other urinary amino acid changed (Block, 1965).

               L-methionine used to be prescribed for the treatment of liver disease in the amounts of 3 to 9 grams per day.  It was also recommended in the treatment of pancreatitis in the amounts of 4 to 5 grams per day (The Martindale Pharmacopoeia, 1973).

            McAuley (1999) gave 1 gram/day to sixteen healthy volunteers for one month and measured total plasma homocysteine.  No measurable increase occurred.  However, when they gave 7 grams/day for 7 days, total plasma homocysteine more than doubled.  They did not give the homocysteine-reducing compounds.

            Vale (1981) recommends the use of 10 grams of oral l‑methionine (2.5 grams every 4 hours for 12 hours) in the treatment of acetaminophen poisoning.  Acetamino­phen is toxic because it interferes with glutathione synthesis, and methionine can increase hepatic glutathione following acetaminophen depletion.  Methionine can also supply inorganic sulfate, which increases the conversion of acetaminophen to its less toxic sulfate conjugate. The use of methionine also prevented the vomiting typical of all patients treated with n‑acetyl‑cysteine for acetaminophen poisoning.

            To the extent that persons may benefit from returning methionine, glutathione, and glutathione peroxidase to normal levels, methionine is a rational choice to accomplish this objective.  In animal cell studies methionine is equally as effective as cysteine as a precursor for glutathione biosynthesis (Reed, 1977).  Rat fee­ding studies show that methionine can raise liver and muscle glutathione levels (Seligson, 1983).  Ayala (1991) found that in rats fed a low-protein diet, a decrease in dietary methionine leads to a decrease in glutathione.   Bianchi (2000) found that 7 grams of oral l-methionine given to six healthy adults caused blood levels of methionine to increase to 20 times normal in 90 minutes, while plasma glutathione doubled at 2 hours.  All values returned to normal by 24 hours.

               Under some circumstances, methionine administration causes a reduction in blood folate levels (Connor, 1978).   Brattstrom (1988) showed administration of folate at 5 mg per day to be an innocuous means to reduce plasma homocysteine by an average of 52% in normal subjects.  Thus methionine should be supplemented with folate for chronic consumption.

               Vitamin B6 at 2 mg per day prevented excretion of urinary homocysteine following a 3-gram load of l-methionine in six healthy subjects.  21 days of vitamin B6 depletion allowed homocysteine to appear in the urine, and repletion with 2 mg per day of vitamin B6 again caused homocysteine excretion to cease (Park, 1969). Thus methionine should also be supplemented with vitamin B6 when chronically consumed.

               A deficiency of vitamin B2 can lead to a functional deficiency of vitamin B6 (Nutr. Rev. 1977).  Since vitamin B2 is used in the transfer of the serine methyl group to homocysteine, is necessary for proper B6 metabolism, and participates in the movement of Reducing Equivalents by way of Glutathione Reductase, methionine should be supplemented with B2 when chronically consumed.

            Taniguchi (1983) showed that rats on a riboflavin-deficient diet exhibited reduced liver glutathione content, and reduced glutathione disulfide reductase activity.  Beutler, (1969) demonstrates that administration of 5 mg/day of riboflavin (Vitamin B2) to normal human subjects maximized their red cell glutathione disulfide reductase activity, while the MDR of 1.5 mg/day did not.

               While vitamin B12 deficiency is rare, since B12 is used in the remethylation of homocysteine, it should be administered when chronically consuming methionine.   Ubbink (1994) recommends 400 micrograms of B12 a day to effectively reduce plasma homocysteine levels.

               5 grams of l-methionine causes the urine to become more acidic, and in doing so, stimulates the excretion of calcium (Tschope, 1985).  Calcium supplementation of about 20 mg per gram of l-methionine may be needed to compensate for this loss.

            Microencapsulation of methionine tablets can help reduce the possibility of gastric upset, and provide additional time‑release effects for maintenance of even elevation of blood levels.

            At least 80% of ingested l‑methionine sulfur is excreted in the urine as inorganic sulfate, and dipstick test strips are available to measure sulfate.  Urinary sulfate levels partially indicate methionine intake and can be used to back‑calculate methionine intake and metabolic availability.  Typical sulfate excretion levels in men range between 0.6 and 2.0 grams per day.  Three grams of oral l‑methionine result in about 2 grams of urinary sulfate, which in 2 liters of urine becomes about 1000 mg/liter.

            2.  D-Methionine

            Methionine occurs in proteins as the l‑isomer as do all other protein amino acids.  When methionine is made chemically both the natural l‑form and its "mirror image", the d‑analog are produced.  The chemically manufactured racemic mixture of the two forms is the only amino acid mixture allowed by the U.S. Government as a food additive that contains the synthetic d‑form. (The dl‑methionine mixture is no longer allowed in baby food as the d‑form interferes with the PKU [phenylketonu­ria] test).  Both the l‑form of methionine and the d-form are natural compounds found in nature, as shown by the presence of d-amino acid oxidase in animals. Methionine in many circumstances is given to animals or humans as a mixture of the two forms, that is, the synthetic product. Man metabolizes D‑methionine more slowly than natural l‑methionine, and this difference can potentially be used to elevate the blood and tissue levels of a structure that has the antioxidant properties of methionine. Most biochemical systems, and enzymes, in particular, distinguish between chemically related but structurally different molecules.  Thus as much as 40% of d‑methionine is excreted unchanged within 8 hours by humans.    

            The breakdown pathway for d‑methionine is essentially the same as the breakdown (transamination) pathway for excess amounts of the natural l‑methionine.  A specific enzyme, d‑amino acid oxidase, degrades most d‑amino acids.  The degraded form of d‑methionine is identical to the transaminated form of l‑methionine and thus the compounds formed in the path of degradation are the same for the d‑form as it is for the l‑form.  A comparison has been made of the breakdown of metabolites of high doses of l‑methionine with a person having persistently high blood levels of methionine (and no symptoms of toxicity) and concomitant in­creased breakdown through the transamination pathway.  Values of intermediates in the transamination breakdown pathway for normal persons who have consumed 7 grams of l‑methionine range from 100% to 10% of those seen for an individual with a genetic persistent elevated blood methionine (Blom, 1989).  Thus elevated amounts of breakdown components seen in the genetic condition of elevated blood methionine will be generally the same for normal individuals who consume excess d‑methionine because it is metabolized by this same breakdown pathway.

            No references have been found that show whether the d‑form of methionine is incorporated into protein.  Based on the low levels of d‑methionine excreted after 8 hours an upper limit can be set for this potential mechanism of toxicity.  Generally, d‑forms of amino acids are incorporated into protein very infre­quently (about 10 times per million).   An in vitro study (Lemoine, 1968) of the E. coli enzyme Methionyl Transfer RNA Synthetase, which places methionine on the ribosome during protein synthesis, showed d-methionine to be completely inactive, as well as noninhibitory to l-methionine.  It should be noted that while most d‑forms of amino acids are not transported into brain tissues (less than 1% as often as the l‑form) d‑methionine is transported into brain tissue about as efficiently as the natu­ral l‑methionine (Oldendorf, 1973).  This would allow d‑methionine to act ef­fectively as an antioxidant in the brain but it also would allow the d‑form to act in other ways as well.

            Lombardini (1970) found that d-methionine was neither a substrate nor an inhibitor of methionine adenosyltransferase in the formation of S‑adenosyl-l‑methionine by E. coli, yeast, and rat enzyme systems.  If d‑methionine were enzymatically attached to adenosine to form S‑adenosyl-d‑methionine, it is known that methylation reactions would be differentially affected (Oliva, 1980).

            Animal observations dealing with the consumption of d‑­methionine may be of little value in predicting potential long‑term hazards in man because most species examined can convert the d‑form to the l‑form efficiently.  Humans make this conversion poorly and hence derive little if any nutritional benefit from d‑methionine.  Any specific effect of the d‑form would not be apparent in pigs, chickens, or other species that have been consuming significant amounts of d‑methionine because of this difference in metabolism.

L.  Daily Methionine Requirements for Adults.

             Achievement of positive nitrogen balance requires 13 mg/kg/day of methionine (Young, 1994).  However, Mudd (1980) using methyl balance as a criterion, found 42 mg/kg/day of methionine necessary, if all the methyl groups were obtained from methionine.  This is the same amount found necessary for infants (Snyderman, 1964) and elderly men (Tuttle, 1965).   

            42 mg/kg/day for a 70 kg adult equals 3 grams per day of l-methionine.  An increase in the catabolic state of the body, such as occurs following infection, injury, and during pregnancy, causes a further increase in methionine requirements.  Intake by healthy persons of 3 grams per day of methionine results in the excretion of 2 grams per day of sulfate in the urine, allowing measurement of methionine intake by the dipstick method following 24-hour urine collection. 

               

                                      Written by Robert Bayless and Earl Matthew, M. D.                                                                                                           2005              All rights reserved.