High Concentrations Of Methionine Research Articles

Genetic and biochemical differences in populations bred for extremes in maize grain methionine concentration

BackgroundMethionine is an important nutrient in animal feed and several approaches have been developed to increase methionine concentration in maize (Zea mays L.) grain. One approach is through traditional breeding using recurrent selection. Using divergent selection, genetically related populations with extreme differences in grain methionine content were produced. In order to better understand the molecular mechanisms controlling grain methionine content, we examined seed proteins, transcript levels of candidate genes, and genotypes of these populations.ResultsTwo populations were selected for high or low methionine concentration for eight generations and 40 and 56% differences between the high and low populations in grain methionine concentration were observed. Mean values between the high and low methionine populations differed by greater than 1.5 standard deviations in some cycles of selection. Other amino acids and total protein concentration exhibited much smaller changes. In an effort to understand the molecular mechanisms that contribute to these differences, we compared transcript levels of candidate genes encoding high methionine seed storage proteins involved in sulfur assimilation or methionine biosynthesis. In combination, we also explored the genetic mechanisms at the SNP level through implementation of an association analysis. Significant differences in methionine-rich seed storage protein genes were observed in comparisons of high and low methionine populations, while transcripts of seed storage proteins lacking high levels of methionine were unchanged. Seed storage protein levels were consistent with transcript levels. Two genes involved in sulfur assimilation, Cys2 and CgS1 showed substantial differences in allele frequencies when two selected populations were compared to the starting populations. Major genes identified across cycles of selection by a high-stringency association analysis included dzs18, wx, dzs10, and zp27.ConclusionsWe hypothesize that transcriptional changes alter sink strength by altering the levels of methionine-rich seed storage proteins. To meet the altered need for sulfur, a cysteine-rich seed storage protein is altered while sulfur assimilation and methionine biosynthesis throughput is changed by selection for certain alleles of Cys2 and CgS1.

Stability of DNA Methylation Patterns in Mouse Spermatogonia Under Conditions of MTHFR Deficiency and Methionine Supplementation1

Little is known about the conditions contributing to the stability of DNA methylation patterns in male germ cells. Altered folate pathway enzyme activity and methyl donor supply are two clinically significant factors that can affect the methylation of DNA. 5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key folate pathway enzyme involved in providing methyl groups from dietary folate for DNA methylation. Mice heterozygous for a targeted mutation in the Mthfr gene (Mthfr(+/-)) are a good model for humans homozygous for the MTHFR 677C>T polymorphism, which is found in 10% of the population and is associated with decreased MTHFR activity and infertility. High-dose folic acid is administered as an empirical treatment for male infertility. Here, we examined MTHFR expression in developing male germ cells and evaluated DNA methylation patterns and effects of a range of methionine concentrations in spermatogonia from Mthfr(+/-) as compared to wild-type, Mthfr(+/+) mice. MTHFR was expressed in prospermatogonia and spermatogonia at times of DNA methylation acquisition in the male germline; its expression was also found in early spermatocytes and Sertoli cells. DNA methylation patterns were similar at imprinted genes and intergenic sites across chromosome 9 in neonatal Mthfr(+/+) and Mthfr(+/-) spermatogonia. Using spermatogonia from Mthfr(+/+) and Mthfr(+/-) mice in the spermatogonial stem cell (SSC) culture system, we examined the stability of DNA methylation patterns and determined effects of low or high methionine concentrations. No differences were detected between early and late passages, suggesting that DNA methylation patterns are generally stable in culture. Twenty-fold normal concentrations of methionine resulted in an overall increase in the levels of DNA methylation across chromosome 9, suggesting that DNA methylation can be perturbed in culture. Mthfr(+/-) cells showed a significantly increased variance of DNA methylation at multiple loci across chromosome 9 compared to Mthfr(+/+) cells when cultured with 0.25- to 2-fold normal methionine concentrations. Taken together, our results indicate that DNA methylation patterns in undifferentiated spermatogonia, including SSCs, are relatively stable in culture over time under conditions of altered methionine and MTHFR levels.

Muscle free amino acid profiles are related to differences in skeletal muscle growth between single and twin ovine fetuses near term

Twin sheep fetuses have reduced skeletal muscle weight near birth relative to singles as a result of restricted muscle hypertrophy. Intracellular free amino acids (FAA) are reported to regulate metabolic pathways which control muscle protein accretion, whereby reduced intracellular content of specific FAA may reduce their activation and therefore, muscle hypertrophy. The aim of this study was to determine whether differences in muscle weight between singleton and twin fetuses, under different maternal conditions is associated with reduced concentration of specific FAA. The FAA content in the semitendinosus muscle (ST) in singleton and twin fetuses (rank) at 140 days of gestation from heavy (H) or light (L) ewes fed ad libitum (A) or maintenance (M) level of nutrition was measured. Muscle weight was reduced in twin fetuses compared to singletons in all groups. Reduced concentrations of leucine, threonine and valine, but higher concentrations of methionine, ornithine, lysine and serine were found in twin fetuses compared to singletons. Maternal size and nutrition interaction with rank resulted in reduced glutamine in twins from HM-ewes (H-ewes under M nutrition) compared to their singleton counterparts. Maternal weight interaction with pregnancy rank reduced the concentration of arginine in twins, with a larger effect on H-ewes compared with L-ewes. Maternal size interaction with pregnancy rank resulted in twins from M-ewes to have lower alanine, while twins from A-ewes had lower aspartic acid concentration compared to singletons. The ST muscle weight was positively correlated only with arginine concentration after taking into account rank, size and nutrition. The present results indicate that reduced concentrations of specific intracellular FAA, such as arginine, leucine, valine, glutamine, which are known to play a role in muscle growth, could be acting as limiting factors for muscle hypertrophy in twin fetuses during late gestation. Ewe size and nutrition can influence the concentration of specific FAA in muscle and should be considered in any intervention plan to improve twin fetal muscle growth.

Effects of dietary concentrations of methionine on growth performance and oxidative status of broiler chickens with different hatching weight

1. A study was conducted to evaluate the effects of two hatching weight (HW) levels and two dietary concentrations of methionine on the growth performance and oxidative status of broilers. Male Arbor Acres chickens were divided into two groups on their HW (low and high HW, H and L). Each HW group was then distributed into two subgroups, of similar HW, receiving either low or high dietary concentrations of methionine (4.9 g methionine/kg, LM; 5.9 g methionine/kg, HM). Thus, all day-old birds were distributed into 4 treatments (H-LM, H-HM, L-LM, L-HM) × 6 replicates × 10 birds for 21 d. 2. Broilers with high HW were heavier than those with low HW during the 21 d assay, which appeared to result from increased body weight gain rather than improved feed conversion efficiency. A higher dietary concentration of methionine (5.9 g/kg) improved growth performance of broilers with low HW in terms of body weight gain and feed conversion ratio. 3. Broilers with different HW had similar antioxidant status both in serum and liver. 4. Broilers given a diet containing 5.9 g/kg methionine had enhanced serum superoxide dismutase (SOD) activity and decreased hepatic malondialdehyde (MDA) content at day 7. 5. Broilers given a diet containing 5.9 g/kg methionine had a higher hepatic reduced glutathione (GSH):glutathione disulphide (GSSG) ratio than those given a diet containing 4.9 g/kg methionine at day 21. High dietary methionine concentration reduced hepatic GSH content and glutathione peroxidase (GPX) activity of broilers with high HW at day 7 and at day 21, respectively, but increased hepatic GSH content of broilers with low HW at day 7. 6. Although broilers with different HW had similar oxidative status as indicated by several parameters in blood and liver, HW can have positive effects on the subsequent growth performance of broilers, and a higher dietary methionine concentration (5.9 g/kg) can improve growth performance and antioxidant status in broilers exhibiting low HW.

The Effects of Methionine Acquisition and Synthesis on Streptococcus Pneumoniae Growth and Virulence

Bacterial pathogens need to acquire nutrients from the host, but for many nutrients their importance during infection remain poorly understood. We have investigated the importance of methionine acquisition and synthesis for Streptococcus pneumoniae growth and virulence using strains with gene deletions affecting a putative methionine ABC transporter lipoprotein (Sp_0149, metQ) and/or methionine biosynthesis enzymes (Sp_0585 - Sp_0586, metE and metF). Immunoblot analysis confirmed MetQ was a lipoprotein and present in all S. pneumoniae strains investigated. However, vaccination with MetQ did not prevent fatal S. pneumoniae infection in mice despite stimulating a strong specific IgG response. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry demonstrated that MetQ has both a high affinity and specificity for L-methionine with a KD of ∼25 nM, and a ΔmetQ strain had reduced uptake of C14-methionine. Growth of the ΔmetQ/ΔmetEF strain was greatly impaired in chemically defined medium containing low concentrations of methionine and in blood but was partially restored by addition of high concentrations of exogenous methionine. Mixed infection models showed no attenuation of the ΔmetQ, ΔmetEF and ΔmetQ/ΔmetEF strains in their ability to colonise the mouse nasopharnyx. In a mouse model of systemic infection although significant infection was established in all mice, there were reduced spleen bacterial CFU after infection with the ΔmetQ/ΔmetEF strain compared to the wild-type strain. These data demonstrate that Sp_0149 encodes a high affinity methionine ABC transporter lipoprotein and that Sp_0585 – Sp_0586 are likely to be required for methionine synthesis. Although Sp_0149 and Sp_0585-Sp_0586 make a contribution towards full virulence, neither was essential for S. pneumoniae survival during infection.

Pulmonary delivery of d-methionine is associated with an increase in ALCAR and glutathione in cochlear fluids

In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [14C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [14C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.

Epigenetic effects of transmethylation micronutrients on gene expression in T cells from patients with active lupus. (116.12)

Abstract T cell DNA demethylation contributes to the development of human lupus. However, the mechanisms causing T cell DNA demethylation in lupus patients are incompletely understood. The replication of DNA methylation patterns during mitosis depends on DNA methyltransferase (Dnmt) enzymatic activity and the methyl donor S-adenosylmethionine (SAM), and is inhibited by S-adenosylhomocysteine (SAH). SAM levels are determined by various dietary nutrients including folate and methionine, and previous work demonstrates that patients with active lupus patients have decreased Dnmt activity. We hypothesized that lupus patients will be more susceptible to environmental changes in folate and methionine due to their already low levels of T cell Dnmt activity compared to controls, resulting in aberrant overexpression of genes normally suppressed by DNA methylation. We cultured PHA-stimulated lupus T cells in media containing physiologic high and low concentrations of folate or methionine, and compared effects on methylation sensitive gene expression (CD11a, CD70, KIR and perforin) by flow cytometry. Lupus T cells cultured in lower concentrations of folate and methionine overexpress CD70, KIR and perforin compared to those cultured in standard RPMI 1640 media, relative to control T cells. These results suggest that folate and methionine may contribute to abnormal T cell gene expression in lupus, and that dietary supplementation may have a beneficial effect.

High dietary concentrations of methionine reduce the selenium content, glutathione peroxidase activity and oxidative stability of chicken meat

Three experiments (EXP) were conducted using two hundred seventy male chicks Ross 308 in each (90 cockerels per treatment; 3 replications, 30 chickens per pen) for 42, 38 and 35 days. The basal diets (treatment 1) for three EXPs contained the identical ingredients, and the concentration of selenium (Se), methionine (Met) and total sulphur amino acids (TSAA) in the control diet was 0.11 mg/kg, 5.65 g per kg and 9.25 g/kg, respectively. Chicks in dietary treatment 2 were fed a basal diet supplemented with 0.3 mg/kg of Se (EXP 1, 2 and 3) and in dietary treatment 3 they were fed a basal diet with the addition of 0.3 mg/kg of Se and 1 g/kg (EXP 1 and 2) or 2.5 g/kg of dl-methionine (EXP 3). In EXP 1, sodium selenite and in EXP 2 and 3 Se-enriched yeast were used as sources of supplemental selenium, respectively. The results indicated that the addition of Se or Se and Met into the diet of broilers did not significantly affect the final live weight of chickens and the feed consumption. Moreover, the concentration of Met (P = 0.004), Cys (P = 0.01) and tyrosine (P < 0.001) in breast muscle increased with an increase in dietary Met content, and the isoleucine concentration decreased (P < 0.001). Moreover, the addition of inorganic and organic sources of Se increased the Se content of breast meat (P < 0.001). On the other hand, the addition of Met decreased the concentration of Se (P < 0.001) in breast meat and reduced glutathione peroxidase activity and oxidative stability of raw breast muscle (P = 0.019, P < 0.001) and breast meat stored for 3 days (P = 0.016, P = 0.006) in EXP 2 and 3.

Biomarkers Related to One-Carbon Metabolism as Potential Risk Factors for Distal Colorectal Adenomas

Efficient one-carbon metabolism, which requires adequate supply of methyl group donors and B-vitamins, may protect against colorectal carcinogenesis. However, plasma folate and vitamins B2 and B12 have inconsistently been associated with colorectal cancer risk, and there have been no previous studies relating plasma concentrations of methionine, choline, and betaine to this outcome. This study comprised 10,601 individuals, 50 to 64 years of age, participating in the Norwegian Colorectal Cancer Prevention (NORCCAP) screening study. Using logistic regression analyses, we crosssectionally investigated associations between distal colorectal adenoma occurrence-potential precursor lesions of colorectal carcinomas-and plasma concentrations of methyl group donors and B-vitamins, and polymorphisms of genes related to one-carbon metabolism. Screening revealed 1,809 subjects (17.1%) with at least one adenoma. The occurrence of high-risk adenomas (observed in 421 subjects) was inversely associated with plasma concentrations of methionine (highest versus lowest quartile: odds ratio (OR) = 0.61; 95% confidence interval (CI) = 0.45-0.83), betaine: OR = 0.74; 95% CI = 0.54-1.02, the vitamin B2 form flavin-mononucleotide (FMN): OR = 0.65; 95% CI = 0.49-0.88, and the vitamin B6 form pyridoxal 5'-phosphate (PLP): OR = 0.69; 95% CI = 0.51-0.95, but not with folate, choline, vitamin B12 concentrations, or with the studied polymorphisms. High methionine concentration in combination with high vitamin B2 or B6 concentrations was associated with lower occurrence of high-risk adenomas compared with these factors individually. High plasma concentrations of methionine and betaine, and vitamins B2 and B6 may reduce risk of developing colorectal adenomas. In addition to B-vitamins, methyl group donors such as methionine and betaine may play a role in colorectal carcinogenesis.

Amino acid composition of xylem and phloem sap varies in canola genotypes differing in nitrogen- and sulfur-use efficiency

There is little information on amino acid composition of xylem and phloem sap in canola genotypes differing in use efficiency of nitrogen (N) and/or sulfur (S). Four canola genotypes with either high or low N- and/or S-use efficiency indices were grown in a glasshouse using deficient or adequate N and/or S supply. Xylem sap was obtained by vacuum extraction 46 days after sowing. Phloem sap was collected via bleeding from the petioles of the maturing siliques 120 days after sowing. Among the genotypes, Wesway (N-use efficient) had the highest concentrations of total N, glutamine (235 mmol/L), and asparagine (14 mmol/L) in the xylem sap in the vegetative stage. In contrast, genotype Westar (classified previously as N-use inefficient) had the highest concentrations of most amino acids in the phloem sap among genotypes grown at deficient N supply. Compared with the other tested genotypes, Surpass 402 CL (S-use efficient) had a higher concentration of S, and Karoo (S-use inefficient) had higher concentrations of glutamate and methionine, in the xylem sap in the vegetative stage. Similarly, Karoo had a higher concentration of methionine under adequate S supply and of glutamate under deficient S in the phloem sap at maturity. In conclusion, in canola phloem sap, the most important amino acid for N transport is glutamine, and for S transport methionine and the glutathione-precursor glutamate. It does not appear there is a relationship between differential N- or S-use efficiency of canola genotypes and the concentration of N- or S-transporting amino acids in the phloem sap.

Methylenetetrahydrofolate Reductase Activity Is Involved in the Plasma Membrane Redox System Required for Pigment Biosynthesis in Filamentous Fungi

Methylenetetrahydrofolate reductases (MTHFRs) play a key role in biosynthesis of methionine and S-adenosyl-l-methionine (SAM) via the recharging methionine biosynthetic pathway. Analysis of 32 complete fungal genomes showed that fungi were unique among eukaryotes by having two MTHFRs, MET12 and MET13. The MET12 type contained an additional conserved sequence motif compared to the sequences of MET13 and MTHFRs from other eukaryotes and bacteria. Targeted gene replacement of either of the two MTHFR encoding genes in Fusarium graminearum showed that they were essential for survival but could be rescued by exogenous methionine. The F. graminearum strain with a mutation of MET12 (FgDeltaMET12) displayed a delay in the production of the mycelium pigment aurofusarin and instead accumulated nor-rubrofusarin and rubrofusarin. High methionine concentrations or prolonged incubation eventually led to production of aurofusarin in the MET12 mutant. This suggested that the chemotype was caused by a lack of SAM units for the methylation of nor-rubrofusarin to yield rubrofusarin, thereby imposing a rate-limiting step in aurofusarin biosynthesis. The FgDeltaMET13 mutant, however, remained aurofusarin deficient at all tested methionine concentrations and instead accumulated nor-rubrofusarin and rubrofusarin. Analysis of MET13 mutants in F. graminearum and Aspergillus nidulans showed that both lacked extracellular reduction potential and were unable to complete mycelium pigment biosynthesis. These results are the first to show that MET13, in addition to its function in methionine biosynthesis, is required for the generation of the extracellular reduction potential necessary for pigment production in filamentous fungi.

Methylation of CpG dinucleotides in the Helicobacter pylori genome at a higher methionine concentration

Methylation of CpG dinucleotides in the Helicobacter pylori genome at a higher methionine concentration

Recurrent Selection to Alter Grain Methionine Concentration and Improve Nutritional Value of Maize

Methionine is an essential amino acid that is limiting in maize‐ (Zea mays L.) based diets. The objective of this work was to determine whether we could alter grain methionine concentration in random‐mated maize populations by mass selection for methionine concentration using a microbial assay. In one study, we developed two populations by selecting for high or low methionine concentration (HM or LM, respectively) for three generations starting from the random‐mated population BS11. Grain from these populations was used to formulate diets for a feeding trial in which 15 rats were fed HM grain and 15 rats were fed LM grain. Rats on the HM diet had a 0.018 higher feed efficiency (g gain/g feed) than rats on the LM diet. In a second study, we performed three cycles of selection for high or low methionine concentration starting with two random‐mated populations, BS11 and BS31. We evaluated each cycle of selection in a field trial with two replications in each of two years. Methionine concentration was significantly correlated with the cycle of selection, changing on average 0.004 g methionine/100 g grain per cycle. Kernel mass, %N, oil, protein, starch, tryptophan, and lysine concentration did not exhibit significant correlations with cycle of selection. We conclude that recurrent selection for grain methionine concentration using a microbial assay is an effective method to alter methionine content.

Methionine inhibits cellular growth dependent on the p53 status of cells

Background Methionine, an essential amino acid, is important for normal growth and development, as it is required for both protein and polyamine synthesis as well as in methylation reactions. It has been reported that high concentrations of methionine inhibit cellular growth and gene expression in the human breast tumor-derived MCF-7 cells. These effects are thought to be mediated by the modulation of p53. However, the generalizability of this observation and the precise role of p53 in methionine-induced growth suppression needs to be determined. Methods To determine if the inhibition of cell growth by methionine applies to other cell lines and to characterize further the role of p53 in methionine-induced growth suppression, we have assessed the effects of methionine on cellular growth and proliferation and p53 expression in cells expressing native p53, eg, breast cancer MCF-7 cells and prostate cancer LNCaP cells, and also in cells expressing a mutated (point) form of p53, eg, prostate cancer DU-145 cells. These cell lines were treated with varying concentrations of l-methionine. The effects of l-methionine on cell growth were assyed by using cell viability assays and immunostaining for Ki-67, a cell proliferation marker. The effects of methionine on p53 expression were assessed by using reverse transcriptase–polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. The role of p53 in l-methionine–mediated growth suppression was evaluated using short-interference RNA for p53 (siRNA-p53), immunoprecipitation, and direct DNA sequencing. Results We demonstrated that methionine at a concentration of 1 to 5 mg/mL inhibited the growth of both MCF-7 and LNCaP cells. In association with the inhibition of growth, methionine also inhibited native p53 expression at the mRNA and protein levels, respectively. Furthermore, transfection with siRNA-p53, to knock down p53 expression, increased cell growth and proliferation of the LNCaP cells even when they were exposed to methionine. In contrast, the same treatment did not diminish growth or proliferation of the DU-145 cells. Also, the expression of mutated p53 at the mRNA or protein levels was not altered. Conclusion Our results extend a prior observation to other cell lines and demonstrate that high concentrations of methionine suppress the expression of native but not mutated p53. These inhibitory effects on cellular growth are, in part, due to inhibition of cellular proliferation probably via a p53-dependent pathway.

The Sulfur-Containing Amino Acids: An Overview

Methionine, cysteine, homocysteine, and taurine are the 4 common sulfur-containing amino acids, but only the first 2 are incorporated into proteins. Sulfur belongs to the same group in the periodic table as oxygen but is much less electronegative. This difference accounts for some of the distinctive properties of the sulfur-containing amino acids. Methionine is the initiating amino acid in the synthesis of virtually all eukaryotic proteins; N-formylmethionine serves the same function in prokaryotes. Within proteins, many of the methionine residues are buried in the hydrophobic core, but some, which are exposed, are susceptible to oxidative damage. Cysteine, by virtue of its ability to form disulfide bonds, plays a crucial role in protein structure and in protein-folding pathways. Methionine metabolism begins with its activation to S-adenosylmethionine. This is a cofactor of extraordinary versatility, playing roles in methyl group transfer, 5'-deoxyadenosyl group transfer, polyamine synthesis, ethylene synthesis in plants, and many others. In animals, the great bulk of S-adenosylmethionine is used in methylation reactions. S-Adenosylhomocysteine, which is a product of these methyltransferases, gives rise to homocysteine. Homocysteine may be remethylated to methionine or converted to cysteine by the transsulfuration pathway. Methionine may also be metabolized by a transamination pathway. This pathway, which is significant only at high methionine concentrations, produces a number of toxic endproducts. Cysteine may be converted to such important products as glutathione and taurine. Taurine is present in many tissues at higher concentrations than any of the other amino acids. It is an essential nutrient for cats.

Effects of citrin deficiency in the perinatal period: feasibility of newborn mass screening for citrin deficiency.

Deficiency of citrin due to mutations of the SLC25A13 gene causes adult-onset type II citrullinemia (CTLN2) and one type of neonatal intrahepatic cholestasis (NICCD). About half of the NICCD patients are detected based on high galactose, phenylalanine, and/or methionine concentrations on newborn mass screening (NMS). To clarify the perinatal and neonatal effects and the inconsistent results on NMS, we examined aminograms, the levels of bile acids and galactose in dried blood spots for NMS from 20 patients with NICCD. Birth weight was low for gestational age (-1.4 +/- 0.7 SD). Affected fetuses may have suffered intrauterine citrin deficiency. The first abnormality detected after birth was citrullinemia, and 19 of 20 patients had citrulline levels higher than +2 SD of controls. Tyrosine, phenylalanine, methionine, galactose, and bile acids were less affected than citrulline on d 5 after birth. Galactose and bile acids levels were increased at 1 mo in comparison with d 5 after birth due to impairment of the cytosolic NADH reducing-equivalent supply into mitochondria of hepatocytes. Patients with negative findings on NMS had low levels of total 20 amino acids. Citrulline/serine, citrulline /leucine plus isoleucine, and citrulline/total amino acids ratios, controlled for the confounding effect of low amount of total amino acids, were higher in all patients than +2 SD, +2 SD, and +3 SD of controls, respectively. NMS for citrin deficiency (frequency of homozygote with SLC25A13 mutation: 1/10,000-1/38,000 in East Asia) will be useful for clarification of the clinical course, treatment, and prevention of this disease.

Advancing Age and Other Factors Influencing the Balance between Amino Acid Requirements and Toxicity

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

Prevention by methionine of dichloroacetic acid-induced liver cancer and DNA hypomethylation in mice.

Dichloroacetic acid (DCA) is a liver carcinogen that induces DNA hypomethylation in mouse liver. To test the involvement of DNA hypomethylation in the carcinogenic activity of DCA, we determined the effect of methionine on both activities. Female B6C3F1 mice were administered 3.2 g/l DCA in their drinking water and 0, 4.0, and 8.0 g/kg methionine in their diet. Mice were sacrificed after 8 and 44 weeks of exposure. After 8 weeks of exposure, DCA increased the liver/body weight ratio and caused DNA hypomethylation, glycogen accumulation, and peroxisome proliferation. Methionine prevented completely the DNA hypomethylation, reduced by only 25% the glycogen accumulation, and did not alter the increased liver/body weight ratio and the proliferation of peroxisomes induced by DCA. After 44 weeks of exposure, DCA induced foci of altered hepatocytes and hepatocellular adenomas. The multiplicity of foci of altered hepatocytes/mouse was increased from 2.41 +/- 0.38 to 3.40 +/- 0.46 by 4.0 g/kg methionine and decreased to 0.94 +/- 0.24 by 8.0 g/kg methionine, suggesting that methionine slowed the progression of foci to tumors. The low and high concentrations of methionine reduced the multiplicity of liver tumors/mouse from 1.28 +/- 0.31 to 0.167 +/- 0.093 and 0.028 +/- 0.028 (i.e., by 87 and 98%, respectively). Thus, the prevention of liver tumors by methionine was associated with its prevention of DNA hypomethylation, indicating that DNA hypomethylation was critical for the carcinogenic activity of DCA.

Interaction between genotype and dietary concentrations of methionine for immune function in commercial broilers

1. Growth, antibody response to sheep red blood cells (SRBC) and resistance to Escherichia coli were measured in broiler female chicks received from 4 (n = 100 in each) commercial genotypes (A, B, C and D) and fed with maize-soybean-deoiled rice bran based diets containing 4 concentrations of methionine (3·91, 4·46, 5·00 and 5·54 g/kg). The diets were fed ad libitum from 1 to 49 d of age. 2. Body weight gain and weight gain/food intake at 2 week intervals, response of broilers to inoculation of 0·5 ml of SRBC (0·5 or 2·5%), 0·1 ml of E. coli (10−4 dilutions) culture, and 100 µg phytohaemogglutinin-P (PHA-P) at 43 d of age were measured. The responses to SRBC and E. coli inoculation were recorded at 5 d post inoculation (PI), while the responses to PHA-P were recorded at 12 and 24 h PI. 3. Genotype by methionine interaction was not significant for body weight gain, but significant differences in weight gain were observed among different genotypes. Variation in methionine concentration did not influence body weight gain or weight gain/food intake at 1 to 14, or 42 d of age. At 28 d of age, chicks fed on the 3·91 g methionine/kg diet weighted significantly less than those on the other methionine concentrations. Genotype by methionine interaction was observed for food efficiency at 0 to 28 d of age but not at other ages. 4. Antibody titres against SRBC and heart and air sac lesion score to E. coli challenge were not influenced by genotype-methionine interaction. Chicks given higher concentrations of methionine had higher antibody titres and greater cutaneous basophilic hypersensitivity (CBH) response than those given low levels of methionine. Also, variation was observed in expression of CBH response to PHA-P among different genotypes. 5. It may be concluded that, although the commercial broiler chicks do not require more than 3·91 g methionine/kg for optimum growth and food efficiency, the immunity in terms of CBH response and antibody production to SRBC increased with the concentration of methionine in the diet in the majority of genotypes, indicating a higher methionine requirement for immunity than for weight gain.

Effects of prolactin administered to a perfused area of the skin of Angora goats.

It is suspected that prolactin may affect mohair growth; therefore, effects of infusing prolactin on mohair growth were investigated using a skin perfusion technique. Seven Angora wethers (average body weight, 30 +/- 3 kg) were implanted bilaterally with silicon catheters into the superficial branches of the deep circumflex iliac artery and vein. For the first 14 d of the experiment, animals were infused (2.4 mL/h) with prolactin (one side) or control (other side) into the deep circumflex iliac arteries. The infusion rate of prolactin was 2.21 mg/d and was calculated to triple prolactin blood concentration in the perfused region. The area of skin supplied by the deep circumflex iliac artery was approximately 240 cm2. Two weeks after the cessation of infusions, 100-cm2 areas within the perfused regions were shorn to determine mohair growth. Greasy and clean mohair production was decreased (P < 0.05) by prolactin compared with control (3.79 vs 4.62 and 3.02 vs 3.67 g/[100 cm2 x 28 d], respectively). Oxygen satura tion in blood hemoglobin from the deep circumflex iliac veins was greater (P < 0.02) on the side infused with prolactin than on the control side (75.1 vs 68.2%). Higher concentrations of methionine, lysine, valine, isoleucine, and leucine were observed in blood of the deep circumflex iliac vein on the side infused with prolactin vs that infused with control (P < 0.05). In conclusion, direct skin infusion with prolactin decreased mohair fiber synthesis by the skin and may have concomitantly lessened oxygen consumption. Thus, effects of increasing prolactin concentration approximately two-fold in the skin on mohair fiber growth may not be limited to simple competition for nutrients between skin and other tissues such as the mammary gland.