Methionine and Cysteine Kinetics at Different Intakes of Methionine and Cystine in Elderly Men and Women1–3

Methionine and cysteine kinetics at different intakes of methionine and cystine in elderly men and women1–3

Naomi K Fukagawa, Yong-Ming Yu, and Vernon R Young

See corresponding editorial on page 224. Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 ABSTRACT Earlier nitrogen balance studies led to the con- requirement for methionine. On the other hand, recent kinetic clusion that requirements for methionine in older individuals are studies by Fereday et al (6) imply that the protein requirements much higher than those in younger adults. Hence, we examined of healthy elderly, at least as assessed from tracer leucine bal- the kinetics of whole-body methionine, cysteine, and leucine ance studies, may not be any higher than those of younger adults. metabolism postabsorptively using a continuous intravenous Indeed, there is a great deal of uncertainty about the protein and 2 13 2 infusion of L-[C H3,1- C]methionine, L-[ H3]leucine, and [3,3- amino acid requirements of elderly subjects and the effects of 2 H2]cysteine in 12 elderly men (n = 5) and women (n = 7) given aging (7Ð9); therefore, further relevant studies in this age group as a 3-h infusion after a 12-h fast (study 1) and in 8 elderly men are highly desirable. In a review published previously (8), we (n = 4) and women (n = 4) as an 8-h infusion according to a 3-h concluded that amino acid requirements are similar in healthy fasted, 5-h fed protocol (study 2) for 6 d. Before tracer infusion, younger and older subjects. each of 3 L-amino acid diets supplying the following nominal, This article presents the results of 2 studies of plasma methion- but known, amounts (mgákgϪ1 ádϪ1) of methionine and cystine, ine and cysteine kinetics. Because no studies have been reported respectively, were used in study 2: diet 1: 13 and 0; diet 3: 6.5 on methionine kinetics in the elderly, the ﬁrst study was carried and 5.2; and diet 5: 6.5 and 21. Studies 1 and 2 gave values for out to obtain initial data on the dynamic status of whole-body plasma methionine flux that agreed with the leucine flux data, methionine and cysteine metabolism postabsorptively in elderly which, in turn, also appeared to be comparable with findings in men and women and, in particular, its relation to plasma leucine healthy younger adults. In study 2, methionine oxidation rates kinetics. There are more extensive data on leucine kinetics in the were the same across all diets in the fasted state and the same elderly (10, 11) and so we considered it important to begin our with diets 1 and 3 in the fed state but lower with diet 5, suggest- investigation of sulfur amino acid metabolism in this age group. ing a modest sparing effect of dietary cystine on methionine oxi- In addition, we thought that this study would provide an addi- dation. Estimated daily methionine balance was at equilibrium tional means of judging the suitability of the correction factor for diet 1 and negative (significantly different from zero, that we used previously (1, 12, 13) and in this study (study 2) to P<0.05) with diets 3 and 5. The results were evaluated against estimate methionine oxidation. We examined the dynamic status our previous findings in younger adults. Am J Clin Nutr of methionine metabolism at different intakes of methionine and 1998;68:380Ð8. cystine in healthy elderly subjects.

KEY WORDS Balance study, amino acid requirements, kinetics, oxidation, protein turnover, methionine, leucine, cys- SUBJECTS AND METHODS tine, elderly Subjects Two separate studies were conducted. In study 1 (postabsorp- INTRODUCTION tive), 5 elderly men and 7 elderly women in generally good We studied plasma methionine and cysteine kinetics previously in healthy young adult men and women and found little evi- 1 From the University of Vermont, the Department of Medicine and the dence for a sparing effect of dietary cystine on the requirement Clinical Research Center, Burlington; the Laboratory of Human Nutrition, for dietary methionine, other than at an extremely low methion- School of Science and the Clinical Research Center, Massachusetts Institute ine intake (1Ð3). However, nitrogen balance studies by Tuttle et of Technology, Cambridge; and the Shriners Burns Institute, Boston. 2 al (4) led them to suggest that methionine and lysine require- Supported by NIH grants AG 00599, DK15856, DK42101, RR00088, and RR00109, and by grants from the Shriners Hospital for Crippled Chil- ments in older individuals are much higher than those estab- dren. Ajinomoto Inc, Teaneck, NJ, donated the amino acids. lished, with similar procedures, in young adults. Hence, in com- 3 Address reprint requests to NK Fukagawa, University of Vermont, Col- parison with the young, it is possible that older individuals lege of Medicine, Given Building, Room C-207, Burlington, VT 05405-0068. would also have higher requirements for the sulfur amino acids E-mail: [email protected]. as judged from methionine tracer balance studies (5) and might Received September 19, 1997. be more sensitive to a sparing effect of dietary cystine on the Accepted for publication February 23, 1998.

TABLE 1 for blood sampling. The isotope tracers were given as a primed, Composition of the L-amino acid mixtures used (expressed as nominal continuous intravenous infusion, as described previously (1). intakes) to study methionine and cysteine kinetics in elderly subjects The bicarbonate pool was primed with 0.8 ␮mol [13C]sodium Diet bicarbonate/kg (99 atom%; Cambridge Isotope Laboratories, Amino acid 1 3 5 Inc) before the continuous isotope infusion began. The known mean infusion rates for methionine, cysteine, and leucine were mgákgϪ1 ádϪ1 1.7, 0.7, and 5.1 ␮molákgϪ1 áhϪ1, respectively. Blood and L-Methionine 13 6.5 6.5 expired breath samples were obtained at timed intervals L-Cystine 0 5.2 20.9 between 120 and 180 min of the tracer infusion for determina- L-Tryptophan 6.0 6.0 6.0 tion of plasma methionine, cysteine, leucine, and ␣-ketoiso- L-Threonine 15.0 15.0 15.0 caproic acid (KIC) isotopic enrichments and for 13C enrichment L-Isoleucine 23.0 23.0 23.0 in expired carbon dioxide. Total carbon dioxide production and L-Leucine 40.0 40.0 40.0 L-LysineáHCl 30.0 30.0 30.0 oxygen consumption were measured with a ventilated-hood sys-

L-Phenylalanine 25.9 25.9 25.9 tem (Delta-Trac Metabolic Monitor; Sensor Medics Corp, Yorba Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 L-Tyrosine 13.0 13.0 13.0 Linda, CA) as described previously (1). L-Valine 20.0 20.0 20.0 Study 2 L-Histidine HCláH2O 12.0 12.0 12.0 L-ArginineáHCl 73.5 73.5 73.5 The diets used in this study were semisynthetic and the pro- L-Alanine 124.4 124.4 122.7 tein component was supplied as a mixture of L-amino acids. The L-Aspartic acid 185.8 185.8 183.4 major proportion of energy intake, other than that supplied by L-Glutamic acid 205.4 205.4 202.6 the amino acid mixture and beet sucrose, was provided as pro- Glycine 104.8 104.8 103.4 tein-free, wheat-starch cookies. Vitamins were supplied as a L-Proline 160.6 160.6 158.5 L-Serine 146.7 146.7 144.7 daily supplement to meet recommended dietary allowances (14) Total amino acids 1198.4 1197.1 1200.1 and safe intakes. Macromineral supplements provided sodium, Total nitrogen intake 160 160 160 potassium, calcium, and phosphate at amounts within the range (mg NákgϪ1 ádϪ1) of recommended dietary allowances (14). A trace mineral mixture, provided as a capsule, supplied magnesium and those trace minerals not present in the vitamin and mineral supplements. health, as assessed by a medical history, physical examination, Choline was added as a supplement to provide an intake of 500 and routine blood and urine tests, participated. Their mean mg/d. The basic diet was described previously (1). (±SD) age was 74 ± 2 y (range: 66Ð92 y) and they weighed Three diets based on different formulations of the L-amino 68 ± 11 kg (range: 49Ð85 kg). None were taking medications and acid mixture were used (Table 1). They are designated as diets all consumed regular “house” diets supplying ≥ 1 g 1, 3, and 5 because they were the same as those used in our pre- proteinákgϪ1 ádϪ1 and energy estimated to be adequate for vious studies in young adults (1). Each diet was offered to the weight maintenance. Subjects were studied as inpatients in the subjects in random order and each began after 7Ð14 d of a break Clinical Research Center (CRC) of the Massachusetts Institute between the diets, during which subjects ate usual foods. The of Technology (MIT). Written informed consent was obtained in control, amino acid mixture supplied 13.2 mg methio- accordance with the protocol approved by the MIT Committee nineákgϪ1 ádϪ1 (87 ␮molákgϪ1 ádϪ1) and no cystine (diet 1) dur- on the Use of Humans as Experimental Subjects and the CRC ing the 6 d before the tracer studies. This amino acid mixture was Scientific Advisory Committee. similar to that used in a previous tracer-metabolic study (1) but In study 2 (which involved methionine and cystine intakes), 8 it supplied a somewhat lower daily intake of methionine than we normal-weight (64 ± 3 kg), elderly men (n = 4) and women had used in our initial series of studies of methionine kinetics (3, (n = 4) aged 79 ± 7 y (range: 71Ð92 y) participated. These sub- 12, 13). However, methionine (in the absence of dietary cystine) jects were also in generally good health, as assessed by a thor- and the other indispensable amino acids were supplied in ough medical history, physical examination, and routine labora- amounts we suggested previously to be sufficient to meet mean tory tests of blood and urine. Again, each subject signed a con- requirements for total sulfur and other indispensable amino acids sent form after reviewing the purpose of the study and the nature in healthy young adults (13). Dispensable (nonessential) amino and potential hazards of the protocol with the investigator and acids were adjusted slightly between the various diets, supplying was paid for his or her participation in the experiment. The pro- variable amounts of methionine and cystine to achieve an isoni- tocol and consent forms were evaluated and approved by the trogenous intake from all 3 diets. MIT Committee on the Use of Humans as Experimental Subjects The taste of the amino acid mixture was improved by serving and the CRC Scientific Advisory Committee. it with an equal weight of beet sucrose and a flavoring agent (Vivonex flavor packets; Norwich Eaton Pharmaceuticals, Nor- Study designs wich, NY). Beet sucrose was used to avoid possible changes in the background isotopic enrichment of expired 13CO in subjects Study 1 2 between the postabsorptive (fasted) and the fed states when the Subjects were studied after consuming a weight-maintaining tracer studies were being conducted. Each diet was consumed for diet consisting of usual foods for 3 d. After a 12-h overnight 6 d before the isotope-tracer study began in the early morning of fast, intravenous catheters were placed for administration of L- the seventh day. 2 13 2 2 [C H3,1-C]methionine, L-[ H3]leucine, and L-[3,3- H2]cys- The major features of this 8-h (3-h fast, 5-h fed states) stable- teine (Cambridge Isotope Laboratories, Inc, Woburn, MA) and isotope-tracer infusion protocol were described previously (12, 382 FUKAGAWA ET AL

13). Subjects were admitted to the infusion room in the CRC at rium with the free form in plasma. However, residual free Ϸ0630. Intravenous lines were inserted and baseline samples of cyst(e)ine, which we analyzed as cysteine, gives a reproducible blood and breath were collected. Intravenous priming doses of isotope abundance when repeated measurements are made over the methionine, cysteine, and leucine tracers and of [13C]sodium many months. bicarbonate were then administered; continuous infusions of Isotopic enrichments were measured by electron-impact ion- each tracer began immediately after the priming doses were ization with gas chromatographyÐmass spectrometry (HP 5890 given. Samples of blood and expired breath for determination of Series II and HP 5988A; Hewlett-Packard, Palo Alto, CA). 13 13 2 methionine, cysteine, and carbon dioxide isotopic enrichments Methionine, [1- C]methionine, and [1- C, methyl- H3]methio- were collected between 2 and 3 h after the infusion began and nine were monitored at a mass-to-charge ratio (m/z) of 320, 321, 2 again between 6 and 8 h after the infusion began. The rates of and 324, respectively. Cysteine and [ H2]cysteine were moni- carbon dioxide production and 13C excretion in expired breath tored at m/z 406 and 408, respectively. The isotopic enrichment were also made at these same time intervals. of the experimental samples was determined by multivariate Three hours after the infusion began, subjects were given their spectral deconvolution (17) by using the observed abundances of

diets as equal meals at a rate equivalent to one-twelfth the total known tracer-tracee combinations, with molar ratios from 0 to Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 daily amino acid and energy intakes per hour for the final 5 h of 0.1 as standards. The validation standards were analyzed before the infusion. The diet was provided as cookies and the appropri- and after each set of unknowns to adjust for variations in instru- ate L-amino acid mixture. The infusates of the tracers were pre- ment response. Enrichments of plasma leucine and KIC were 2 pared from sterile powders of high chemical purity (99%), high determined as described previously (18, 19). Plasma [ H3]KIC optical purity, and high isotopic enrichment. All of the methion- enrichment was measured in study 2 only. ine kinetic studies were performed with a dual tracer, L-[methyl- 2 13 Experimental model H3, 1- C]methionine (Tracer Technologies, Inc, Somerville, 2 MA). The cysteine tracer was L-[3,3- H2]cysteineáHCl (Cam- Flux (Q) rates of methionine carboxyl (Qc) and methyl (Qm), bridge Isotope Laboratories) and the leucine tracer was the same when specifically referring to measurements with the [13C]car- 2 as that used in study 1. The bicarbonate pool was primed (1.7 boxyl and [ H3]methyl tracers, respectively, were calculated as ␮ 13 mol/kg ) with a sterile solution of [ C]sodium bicarbonate described previously (12). Briefly, however, Qc and Qm were cal- (99% atoms percent excess; Tracer Technologies, Inc) containing culated as follows: 25 g sodium bicarbonate/L. The priming doses of labeled Ϸ ϫ Ϫ methionine, cysteine, and leucine tracers were equivalent to 1 Qm = Itr [(Etr/E4) 1] (1) ϫ the amount of the tracers infused each hour. Target constant ϫ Ϫ infusion rates for the methionine, cysteine, and leucine tracers Qc = Itr [(Etr/(E1 + E4) 1] (2) were 0.9, 1.2, and 4.8 ␮molákgϪ1 áhϪ1, respectively.

Between blood sampling, the intravenous lines were kept open where Itr and Etr are the infusion rate and the enrichment of the 13 2 with a slow drip of sterile physiologic saline. Arterialized venous tracer [1- C, methyl- H3]methionine, respectively, and E1 and E4 blood (collected by using a hand-warming device) and expired air are the isotopic plateau plasma enrichments of [1-13C]methion- 13 2 samples for determination of isotopic enrichment in carbon diox- ine and [1- C, methyl- H3]methionine, respectively, from plas- ide and measurements of total carbon dioxide production were ma samples obtained during the last hour of each (fasted and fed) collected at timed intervals as described previously (1). metabolic phase.

Equations 3 and 4 below relate Qm and Qc rates to their indi- Measurement of isotope enrichments of cysteine, vidual components. These calculations and the assumptions ␣ methionine, leucine, and -ketoisocaproic acid in plasma involved were discussed previously (12). In steady state condi- We described previously, in detail, treatment of blood and tions, Q = the sum of inputs = the sum of outputs: expired air samples for determination of isotopic enrichment, 13 measurement of total CO2 production, and analysis of plasma Qm = I + B + RM = S + TM (3) free methionine and cysteine (1). Briefly, N-methyl-N-(tert- butyldimethylsilyl) trifluoroacetamide (Pierce Chemical Co, Qc = I + B = S + TS (4) Rockford, IL) was used to form the tert-butyldimethylsilyl deriv- atives of these amino acids. Ethanethiol was also used in the where I is dietary intake, B is plasma methionine appearance via derivatization mixture to convert cystine to cysteine and to serve tissue protein breakdown, RM is methionine appearance from as an antioxidant, which greatly increased the yield of the deriva- remethylation of homocysteine, S is methionine plasma disap- tized cysteine. pearance via nonoxidative catabolism (assumed to be protein Note that the cysteine bound to protein and dipeptides would synthesis), TM is transmethylation (rate of conversion of methio- not be recovered in this assay because ethanethiol was added nine to homocysteine), and TS is transsulfuration, which we after the free amino acids had been extracted from the plasma. assume to be equivalent to whole-body methionine oxidation. The cysteine isotope enrichments reflect, therefore, the com- Thus, the following 2 equations can be derived from equations 3 bined free cysteine and cystine in plasma (ie, total free plasma and 4 as follows: cysteine). It is also relevant that Wiley et al (15) found that con- Ϫ centrations of free and bound plasma cysteine tracked each other RM = Qm Qc (5) after a methionine load in patients with homocysteinemia, and Malloy et al (16) showed that within 1 wk of storage, the major- TM = RM + TS (6) ity of plasma free cyst(e)ine is reduced via binding to plasma proteins and that the protein-bound cyst(e)ine is in rapid equilib- The TS rate was calculated as follows: METHIONINE KINETICS 383

á 13 ϫ 13 Ϫ TS = V CO2 [(1/[ C]methionine pool enrichment) With method 1, the amount of tracer given per hour is extrap- (1/[13C]methionine tracer enrichment)] (7) olated for the entire 12-h the fasted and fed states. With method 2, only the amount of tracer actually given (for 3 or 5 h in fasted á 13 13 where V CO2 is the rate of C output in expired air. Qcys is cal- and fed states, respectively) is included in the calculation of bal- culated as for methionine flux: ance for the respective 12-h fasted and fed states. With both methods, however, the assumption is made that the periods of ϫ Ϫ Qcys = Itr [(Etr/E2) 1] (8) measurement of methionine oxidation reflect the mean rates throughout each 12-h metabolic condition. From studies with L- where E2 is the plateau plasma enrichment of cysteine. leucine (5, 24) and perhaps also for phenylalanine (25, 26), this As in previous studies (1, 12, 13), a correction factor was used may well be a reasonable assumption, although it is to be recog- for the plasma intracellular gradient in the methionine and cys- nized that the overall temporal 24-h pattern of amino acid oxida- teine tracer enrichment. Hence, we assumed that the intracellular tion, including that presumably for methionine, depends on the enrichment of tracer methionine and cysteine was 80% of the dietary intake of the amino acid. In their recent study of leucine

measured plasma enrichment of the relevant labeled species. We oxidation in young adult and elderly subjects, Fereday et al (6) Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 applied this correction in our earlier studies on sulfur amino acid made similar assumptions to estimate daily leucine balance. In kinetics (1, 12) and found that it gave a determination of methio- addition, for the feeding conditions used in the present study, we nine oxidation that was entirely consistent with the anticipated assumed that there was complete absorption of methionine. rate for a methionine intake that was generous and that whole- However, if this was not the case and if the absorbed methionine body methionine equilibrium could be expected. As discussed did not mix with the intravenously administered labeled methio- below, the results obtained in study 1 supported the use of this nine tracer, it is possible that the actual rate of methionine oxi- correction factor. dation would have been somewhat higher than determined. The Some of the 13C label of methionine that is liberated during the values reported below for methionine balance are really more ␣ 13 oxidative decarboxylation of -ketobutyric acid as CO2 is also positive or less negative than they actually should be. retained by the body and it is necessary to correct for this reten- tion. The recovery of 13C in breath after intravenous and intragas- Statistical methods tric infusions of [13C]bicarbonate was determined to be 70% and Data were analyzed by two-way repeated-measures analysis 74% for the postabsorptive state and 82% and 79% for the fed of variance with a subject ϫ diet ϫ metabolic condition factori- state, respectively, when based on short-term bicarbonate-infu- al design. Dependent variables were methionine and cysteine sion studies (20). Hence, correction factors of 70% and 80% for kinetics and independent variables (repeated measures) were diet the postabsorptive and fed states, respectively, were used in the and metabolic state (fasted and fed). Significant differences calculation of methionine oxidation under the fasted (postabsorp- between mean values for methionine and cysteine kinetics (flux tive) and fed conditions. In study 1, leucine fluxes were based and oxidation) among the diets within fasted and fed states were both on determination of plasma leucine enrichment (QL) and on determined from one-way repeated-measures analysis of vari- ␣ -KIC labeling (QKIC) as described previously (18Ð22). ance followed post hoc by pairwise comparisons among means by using the Student-Neuman-Keuls test. Data are presented as Determination of methionine balance means ± SDs. An ␣ level <0.05 was considered statistically Daily body methionine kinetic balance was determined by significant. All statistical analyses were run by using SAS soft- using 2 methods as follows (23): ware (SAS Institute Inc, Cary, NC).

Method 1 = 12-h fasted methionine balance (FaB) + 12-h fed methionine balance (FeB) (9) RESULTS

With method 1, balances were calculated as follows: Study 1 Ð ± Plasma fluxes (x SD) for methionine as based on Qm and Qc FaB = (i Ϫ O) ϫ 12 + prime (10) labels, with use of the correction noted in the Methods section, for the 12 fasted subjects were 19.5 ± 2.8 and 16.2 ± 2.4 and ␮molákgϪ1 áhϪ1, respectively (Table 2). There were no significant differences between the mean values for the 5 men and 7 FeB = (i + diet Ϫ O) ϫ 12 (11) women and so we combined the results for both sexes. Cysteine flux was Ϸ2.5 times that found for methionine (Table 2). Mean With method 2, balances were calculated as follows: leucine fluxes based on plasma leucine and KIC enrichments, respectively, were 76.9 and 98.6 ␮molákgϪ1 áhϪ1. The ratio of FaB = 3 ϫ (i Ϫ O) Ϫ 9 ϫ (O) + prime (12) the enrichment of plasma KIC to leucine was 0.79 ± 0.11 in these fasted, elderly subjects. and If it can be assumed that the concentrations of methionine, cysteine, and leucine in mixed body proteins are Ϸ120, 206, and FeB = 5 ϫ (i + diet Ϫ O) + 7 ϫ (diet Ϫ O)(13) 603 ␮mol/g protein (27), respectively, then we could predict methionine and cysteine fluxes from the leucine kinetic data and where “diet” is dietary methionine intake (␮molákgϪ1 áhϪ1), vice versa, and we did; the results are summarized in Table 2. “prime” is the primer dose of methionine (␮mol/kg), and i is the There was reasonable agreement between measured and predict- ␮ Ϫ1 Ϫ1 continuous tracer dose of methionine ( molákg áh ). ed Qc values when the former was based on directly measured 384 FUKAGAWA ET AL

TABLE 2 found that the use of this correction factor permitted determina- Methionine, cysteine, and leucine kinetics in elderly men and women tion of methionine oxidation at a rate anticipated for a generous studied in the postabsorptive state1 methionine intake, where an equilibrium in body methionine bal- Amino acid Measured flux Predicted fluxes (from) ance would be reasonably expected (3, 12). The methionine and leucine flux data generated from study 1, therefore, were useful ␮molákgϪ1 áhϪ1 in the further interpretation of the isotopic data obtained from 2 ± ± Methionine (Qc) 13.1 1.9 15.3 2.4 (QL) study 2, as presented below. Methionine (Q )3 16.2 ± 2.4 19.6 ± 1.8 (Q ) c KIC Leucine flux in fasted, elderly subjects, when based on plas- ± Methionine (Qm) 19.5 2.8 — ± ± 2 ma KIC enrichment, amounted to a daily turnover equivalent of Cysteine 35.6 8.8 22.5 3.2 (Qc) Ϫ1 Ϫ1 ± ± 2 3.9 g proteinákg ád . The measured cysteine flux exceeded, Leucine (QL) 76.9 12.3 66 9.8 (Qc) ± ± 3 by Ϸ40%, that which would be predicted from the turnover of Leucine (QKIC) 98.6 9.1 81.4 12.1 (Qc) 1 Ð ± leucine and body protein alone. This apparent discrepancy is x SD; n = 12. Qc, flux rate of methionine carboxyl; Qm, flux rate of ␣ considered in the Discussion section. methionine methyl; QL, plasma leucine enrichment; QKIC, -ketoisocaproate labeling. Study 2 Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 2 Values estimated from measured plasma enrichments of the [methyl- 2 13 13 H3,1- C]- and [1- C]methionine species, without correction. For the 4 men and 4 women who participated in this study, 3 Values corrected for 80% factor (see Methods). resting metabolic rate was slightly lower in the women (5103 ± 218 kJ/d, or 1215 ± 52 kcal/d) than in the men (5796 ± 210 kJ/d, or 1380 ± 50 kcal/d) (P<0.06). Energy expen- plasma enrichments and the latter was predicted from QL. How- diture during feeding increased by 14% in women and 10% in ever, because the rate of whole-body and tissue protein synthesis men to 5846 ± 143 kJ/d (1392 ± 34 kcal/d) and 6363 ± 164 kJ/d appears to be best determined from the measured enrichment of (1515 ± 39 kcal/d), respectively (P<0.01). plasma KIC (28Ð30), an additional comparison is given in Table Methionine and cysteine kinetics 2. Methionine flux estimated with use of the 80% correction factor was determined to be 16.2 ± 2.4 ␮molákgϪ1 áhϪ1 whereas the The tracer-infusion protocol permitted achievement of a steady predicted value with use of the QKIC determination was state level of isotopic enrichment of plasma methionine and cys- 19.6 ± 1.8 ␮molákgϪ1 áhϪ1. Hence, in both cases the experimen- teine as we found previously in young adults (1, 12). Thus, val- tal value for methionine flux was somewhat below the predicted ues for the last hour of the fasted and fed periods are summarized estimate. This may suggest that the gradient between the isotopic in Table 3. As expected, rates of carbon dioxide production enrichment of plasma and intracellular methionine is somewhat increased during feeding (Table 3). From these isotopic data, esti- greater than that for leucine. The leucine gradient is reflected by mates of methionine, cysteine, and leucine fluxes were generated the ratio of plasma KIC to leucine enrichment (19). On the other for the different dietary groups and are presented in Table 4. Qm hand, the extent to which precise comparisons can be made is increased significantly during the fed period with diets 1 and 3 limited by the assumption regarding the relative concentrations but not with diet 5, with which cystine was consumed in excess. of methionine and leucine in the proteins contributing to methio- Turnover of Qc did not differ between the fed and fasted states nine and leucine fluxes. However, because, as stated above, plas- with these various methionine intakes. Finally, for all 3 diets, cys- ma KIC flux is judged to be an acceptable basis for estimating teine flux declined during the fed period although it did not differ whole-body protein turnover, we consider it best to assume for significantly among the different diets. the present purposes that the intracellular enrichment of the rel- Plasma leucine (QKIC) flux during the fasted state was similar evant isotopomers of methionine is Ϸ80% of the measured plas- across all diet groups, with the mean group values during the ma enrichment, from the comparison of the values given in Table fasted and fed states ranging from 85 to 92 ␮molákgϪ1 áhϪ1.

2. In addition, the agreement between the measured QKIC and the Fluxes during the fed state were similar and not significantly dif- predicted flux from Qc and between the measured Qc in study 2 ferent from those determined during the fasted state. This was as and the Qc predicted from QKIC (see below) supports the use of expected on the basis of results from our previous studies (5), the 80% correction factor just as it did in our earlier studies on with the observation that the isotopic enrichment of plasma KIC sulfur amino acid kinetics (3, 12). In this previous study we and, therefore, flux showed little change with feeding when the

TABLE 3 ␣ 13 Plasma isotopic enrichment of methionine, cysteine, and -ketoisocaproic acid (KIC) and of CO2 enrichment of expired air and carbon dioxide production in elderly men and women receiving different intakes of methionine and cystine: study 21 Diet 1 Diet 3 Diet 5 Plasma enrichment Fasted Fed Fasted Fed Fasted Fed [2H]Methyl-methionine2 6.71 ± 1.98 5.67 ± 1.19 5.00 ± 0.93 4.70 ± 0.33 5.70 ± 1.61 5.49 ± 1.19 [1-13C]Methionine2 1.36 ± 0.68 1.26 ± 0.68 1.09 ± 0.62 1.05 ± 0.54 1.17 ± 0.34 1.32 ± 0.59 [2H]Cysteine2 2.34 ± 0.25 3.65 ± 0.34 2.17 ± 0.40 3.25 ± 0.51 2.45 ± 0.48 2.92 ± 0.48 2 2 ± ± ± ± ± ± [ H3]KIC 5.31 0.89 5.27 0.78 5.22 0.81 4.98 0.54 5.40 0.97 5.12 0.58 13 ϫ 3 ± ± ± ± ± ± CO2 (APE 10 ) 2.90 1.10 2.60 1.40 2.80 0.50 1.90 0.50 3.10 0.70 1.50 0.40 Carbon dioxide production (mL/min) 147 ± 13 184 ± 9 153 ± 13 186 ± 10 149 ± 15 188 ± 14 1 Ðx ± SD; n = 8. APE, atoms percent excess. 2 Molar ratio (%). METHIONINE KINETICS 385

TABLE 4 TABLE 5 Methionine, cysteine, and leucine (calculated by using KIC enrichment) Methionine oxidation (TS), rate of transmethylation (TM), and rate fluxes at different intakes of methionine and cysteine during the tracer of homocysteine remethylation (RM) at different intakes of methionine period in elderly men and women1 and cystine in elderly men and women1 Diet 1 Diet 3 Diet 5 Diet 1 Diet 3 Diet 5 Methionine intake ␮molákgϪ1 áhϪ1 Ϫ1 Ϫ1 (mgákg ád ) 13.12 6.6 6.6 TS ␮ Ϫ1 Ϫ1 2 ( molákg ád ) 87.6 44.1 44.1 Fasted 3.82 ± 2.03 4.99 ± 1.38 4.92 ± 2.05 Cysteine intake ± ± 2 ± 2 Ϫ1 Ϫ1 Fed 3.57 2.26 3.74 0.78 2.99 1.15 (mgákg ád ) 0.04 5.3 21.1 TM ␮ Ϫ1 Ϫ1 2 ( molákg ád ) 0.3 43.9 174.6 Fasted 7.85 ± 2.31 9.56 ± 2.073 9.48 ± 4.15 ␮ Ϫ1 Ϫ1 Leucine ( molákg áh ) Fed 9.03 ± 4.19 9.86 ± 3.57 8.15 ± 3.04 ± 3 ± ± Fasted 87 13 88 12 85 13 RM Fed 87 ± 12 92 ± 10 89 ± 10 ± ± ± Fasted 4.03 1.62 4.57 1.28 4.57 2.28 Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 Methionine (Q ) (␮molákgϪ1 áhϪ1) m Fed 4.95 ± 1.98 5.58 ± 3.08 5.09 ± 2.72 Fasted 19.6 ± 3.4 22.1 ± 4.6 19.9 ± 5.3 1 Ð ± Fed 22.9 ± 3.44 23.8 ± 5.64 20.2 ± 4.4 x SD; n =8. 2 ␮ Ϫ1 Ϫ1 Significantly different from fasted, P <0.05 (repeated-measures Methionine (Qc) ( molákg áh ) Fasted 15.5 ± 3.0 17.5 ± 4.7 15.4 ± 3.4 ANOVA and Student-Neuman-Keuls test). 3 Fed 17.9 ± 3.0 18.2 ± 3.6 15.1 ± 2.7 Significantly different from diet 1, P <0.05 (repeated-measures Cysteine (␮molákgϪ1 áhϪ1) ANOVA and Student-Neuman-Keuls test). Fasted 49.7 ± 5.3 54.4 ± 9.2 48.3 ± 8.8 Fed 31.3 ± 3.15 35.8 ± 5.85 39.9 ± 6.66 ence from zero) and were significantly below those achieved 1 n = 8. Qm, flux rate of methionine methyl; Qc, flux rate of methionine with diet 1 (P<0.01). Although the difference in balances during carboxyl. the fed state and for the 24-h day between diets 1 and 5 (low 2 Includes tracer. methionine plus cystine) were more highly significant than were 3 Ð x ± SD. the differences between diets 1 and 3, there was no significant 4-6 4 Significantly different from fasted (paired t test): P < 0.02, difference between diets 3 and 5 regardless of low or high cys- 5P<0.001, 6P<0.01. tine amounts. Thus, a possible sparing effect of a high cystine intake is suggested from these data but is not proven nor dis- leucine intake, which approximated the requirement recom- proven. mended by the FAO/WHO (31), was essentially identical to that in the present study. This contrasts with the substantial changes that occur with meals supplying a more generous intake leucine DISCUSSION (24). Also, with use of the approach outlined above (ie, that in which approximations are made based on the content of amino Study 1 Ϸ ␮ Ϫ1 Ϫ1 acids in protein), a mean QKIC of 90 molákg áh would Although we were interested primarily in the relation between give a predicted QC value, determined by using the 80% correc- plasma leucine and methionine kinetics, a comment about tion factor, of Ϸ18 ␮molákgϪ1 áhϪ1. This, again, is in good leucine kinetics per se in both experiments might be useful. agreement with the experimentally derived values found for each Leucine flux derived from measurement of plasma KIC enrich- diet group (Table 4). ment in the 12 elderly subjects was Ϸ90Ð100 ␮molákgϪ1 áhϪ1, Estimates of methionine oxidation, transmethylation, and which is equivalent to a whole-body protein breakdown rate of homocysteine remethylation at the different intakes of methion- 3.6Ð4 gákgϪ1 ádϪ1. This rate is comparable with values reported ine and cystine are summarized in Table 5. Methionine oxidation previously by us (32Ð34) and others (35, 36) in studies of elder- declined significantly during the fed period when diets 3 and 5, ly subjects in which leucine tracers were used. Furthermore, which contained cysteine, were consumed and the rate appeared when compared with data obtained in younger adults (10, 11), to be lower (P = 0.08) with diet 5 than with diet 1. Thus, in ref- the rate of whole-body protein turnover does not appear to erence to our earlier findings in younger adults (1), the addition change markedly with age. With advancing old age, overall evi- of cystine appeared to modestly reduce methionine oxidation dence suggests to us that age-related changes in whole-body pro- during feeding in the elderly subjects. tein turnover are probably relatively small. We recognize that Estimates of fasted, fed, and 24-h or daily methionine bal- there does appear to be a decrease in the contribution made by ances made with methods 1 and 2 are presented in Table 6. skeletal muscle to whole-body protein turnover (35Ð37) and that Although considerable variation in balance values existed among albumin synthesis seems to be less responsive to a change in pro- the different subjects, there were significant differences, with tein intake in the elderly than in the young (Ϸ<40 y) (38). How- respect to metabolic state and methionine and cystine intakes, ever, these changes may be compensated for by changes in the between group mean values. Thus, with method 1, which would splanchnic region and they are difficult to expose in whole-body be expected to give a more positive or less negative value than amino acid kinetic studies. method 2, all balances were positive during the fed state; how- To our knowledge, there are no other published data on ever, it was only with diet 1, which supplied 13 mg methion- methionine and cysteine kinetics in elderly subjects for purposes ineákgϪ1 ádϪ1 and no cystine, that it was possible to support an of comparison. Hence, an assessment of the present data can overall, positive daily balance. At the lower methionine intakes only be made in relation to the leucine kinetic data, for which (diets 3 and 5), daily balances were negative (P<0.05 for differ- there are, as noted above, data for both elderly and younger 386 FUKAGAWA ET AL

TABLE 6 Methionine balance during the tracer day in elderly subjects given different methionine and cystine intakes Diet 1 Diet 3 Diet 5 Method 1 Methionine intake (mgákgϪ1 ádϪ1) 13.12 6.6 6.6 Cystine intake (mgákgϪ1 ádϪ1) 0.04 5.3 21.1 Methionine balance (␮molákgϪ1 á12 hϪ1) Fasted Ϫ31 ± 25 Ϫ48 ± 16 Ϫ47 ± 25 Fed2 58 ± 253 10 ± 93,4 18 ± 143,5 Daily balance (␮molákgϪ1 ádϪ1)2 27 ± 39 Ϫ38 ± 234 Ϫ29 ± 325 (mgákgϪ1 ádϪ1)2 4.03 ± 5.82 Ϫ5.67 ± 3.434 Ϫ4.33 ± 4.775 Method 2 Methionine intake (mgákgϪ1 ádϪ1) 13.12 6.6 6.6 Cystine intake (mgákgϪ1 ádϪ1) 0.04 5.3 21.1 Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 Methionine balance (␮molákgϪ1 á12 hϪ1) Fasted Ϫ41 ± 24 Ϫ56 ± 16 Ϫ55 ± 24 Fed2 50 ± 263 3 ± 93,4 12 ± 143,5 Daily balance (␮molákgϪ1 ádϪ1)2 9 ± 40 Ϫ53 ± 234 Ϫ43 ± 325 (mgákgϪ1 ádϪ1)2 1.34 ± 5.97 Ϫ7.91 ± 3.434 Ϫ6.42 ± 4.775 1 Ðx ± SD; n =8. 2 Significant difference among diets, P<0.002 (repeated-measures ANOVA). 3 Significantly different from fasted, P<0.001 (repeated-measures ANOVA). 4,5 Significantly different from diet 1 (post hoc comparisons, Student-Neuman-Keuls test): 4 P<0.001, 5 P<0.005.

adults. We found that the estimate of plasma Qc was generally fore, a sparing effect of high dietary cystine in the elderly, consistent with that of plasma QL, given that these fluxes which was not observed in our studies with younger adults (1, reflect the turnover and composition of body proteins in the 2). Thus, aging in humans appears to affect the integrative postabsorptive state (39). Furthermore, whole-body leucine nature of whole-body methionine metabolism, although the flux is thought to be best determined in relation to the isotopic mechanisms and points of biochemical control involved could enrichment of plasma ␣-ketoisocaproate after administration of not be determined from the present studies. labeled leucine (28Ð30). Because this flux is Ϸ20% greater This study provided us with an opportunity to examine the than that based on the isotopic enrichment of leucine and response of whole-body protein synthesis and breakdown to the because the ratio of plasma QL to Qc agrees reasonably well ingestion of meals in elderly subjects. Hence, when whole- with that predicted from the assumed concentrations of these 2 body protein synthesis and breakdown were estimated by using amino acids in mixed body proteins, it follows that the intra- equation 4, feeding caused, or showed, a strong tendency to cellular enrichment of the labeled tracer methionine relative to increases in whole-body protein synthesis of 26 ± 34% that in plasma also would approximate the ratio of plasma (P = 0.06), 18 ± 26% (P = 0.08), and 17 ± 13% (P <0.01) with leucine enrichment to KIC. Thus, it seems that we were justi- diets 1, 3, and 5, respectively. These changes are similar to fied in using the 80% correction factor to estimate whole-body those that we (40) and others (41, 42) reported in younger methionine oxidation. It is possible, however, that whole-body adults, although greater changes due to meal intake were methionine oxidation would be underestimated somewhat if the reported by some investigators (43, 44) in the younger age percentage correction factor is really <80%. group. However, as discussed elsewhere (40, 45), if it had been possible to take the first-pass disappearance of dietary methio- Study 2 nine into account, the changes in protein synthesis with feeding The rate of methionine oxidation during the postabsorptive in the present study would have been somewhat greater and the state was similar after all 3 diets and was not significantly dif- changes possibly more highly significant. Unfortunately, data ferent during the fed state for diets 1 and 3, which supplied on the first-pass splanchnic uptake of methionine in elderly Ϸ13 and 6.5 mg methionine ákgϪ1 ádϪ1, respectively. This con- subjects are not available. trasts with our previous findings in younger adults, in whom The measured cysteine flux did not change in response to the methionine oxidation in the fed state was reduced by Ϸ40% marked increase in cystine intake provided by diet 5. We when methionine intake was reduced from 13 to 6.5 observed previously this same lack of responsiveness of cystine mgákgϪ1 ádϪ1 (1, 2). Hence, in the absence of dietary cystine it flux in young adults (2) and we suggested that it is probably appears that older, compared with younger, individuals may due to the fact that dietary cystine largely disappears in the have a reduced capacity to conserve body methionine when the intestinal wall and liver, where it is likely incorporated into dietary intake is inadequate. Furthermore, in the present exper- glutathione. In addition, the predicted cysteine flux was iment, addition of a relatively high amount of cystine to the markedly lower than the measured flux and, again, we observed low-methionine diet, such that the total sulfur amino acid this in younger adults (1, 2). The reason for this appears to be intake (on a molar basis) was about double that for diet 1, the catabolism of glutathione, which serves as a major source resulted in a reduced rate of methionine oxidation during the of circulating cysteine (46), and we (47) showed that Ϸ50% of fed state compared with that for diet 1. This suggests, there- the plasma cysteine flux was determined by its release from the METHIONINE KINETICS 387 turnover of glutathione. rather than being essentially absent from the diet (3), even Finally, estimations of daily methionine balance were made though cystine intakes were sufficient to bring the total sulfur from the methionine oxidation-intake data. We concluded (Table amino acid intake up to the FAO/WHO/UNU (31) requirement. 6) that in healthy elderly subjects a mean intake of methionine Hence, further research is necessary to determine the minimum that meets the FAO/WHO/UNU (31) recommendation for total ratio of dietary methionine to cystine needed to maintain a sulfur sulfur amino acids, namely 13 mgákgϪ1 ádϪ1, appears to be suf- amino acid balance when there is a limited intake of methionine. ficient to achieve body methionine balance. We drew a similar Meanwhile, it seems prudent to continue to determine definitive- conclusion for younger adults (14); however, this conclusion ly what the adequate intake of sulfur amino acids is for elderly contrasts with the findings of Tuttle et al (4), whose data were subjects, at least in terms of a dietary ratio of methionine to cys- based on nitrogen balance experiments and who concluded that tine that is somewhat >1 (by wt). the methionine requirement increases significantly with aging. However, the conclusions of Tuttle et al should not be considered We thank Gail Langeloh for her assistance in the careful conduct of these definitive for several reasons, including studies.

1) the experimental L-amino acid diet they used may have con- Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 tained inadequate amounts of folate, vitamins B-6 and B-12, REFERENCES and choline, all of which could affect the metabolism of 1. Hiramatsu T, Fukagawa NK, Marchini JS, et al. Methionine and cys- methionine and homocysteine (for reviews see references 48 tine kinetics at different intakes of cystine in healthy adult men. Am and 49); J Clin Nutr 1994;60:525Ð33. 2) their nitrogen balance data were based on only 6 of the 7 or 8 2. Raguso CA, Ajami AM, Gleason R, Young VR. Effect of cystine d of the experimental diet period, which may not have been intake on methionine kinetics and oxidation determined with oral tracers of methionine and cysteine in healthy adults. Am J Clin Nutr long enough to allow for a sufficient period of adjustment in 1997;66:283Ð92. nitrogen excretion after initiation of the experimental diet; 3. Storch KJ, Wagner DA, Burke JF, Young VR. [1-13C, methyl- and 2 H3]methionine kinetics in humans: methionine conservation and 3) their nitrogen balance data were limited because only 2 or 3 cystine sparing. Am J Physiol 1990;258:E790Ð8. subjects were studied at the various intakes of methionine 4. Tuttle SG, Bassett SH, Griffith WH, Mulcare DB, Swendseid ME. used, except for 1 intake (2.1 g L-methionine/d) at which 4 Further observations on the amino acid requirements of older men. subjects had a mean nitrogen balance of Ϫ0.37 g/d. II. Methionine and lysine. Am J Clin Nutr 1965;16:229Ð31. Thus, it is difficult to judge from their experimental design and 5. El-Khoury AE, Fukagawa NK, Sanchez M, et al. The 24-h pattern the data generated whether the minimum physiologic require- and rate of leucine oxidation, with particular reference to tracer esti- ment for methionine was as high as claimed by them. mates of leucine requirements in healthy adults. Am J Clin Nutr Earlier, we (50, 51) predicted, from estimates of obligatory 1994;59:1012Ð20. 6. Fereday A, Gibson NR, Cox M, Pacy PJ, Millward DJ. Protein oxidative losses based on the amino acid composition of body requirements and ageing: metabolic demand and efficiency of uti- mixed proteins, that the mean requirement for total sulfur amino lization. Br J Nutr 1997;77:685Ð702. Ϸ Ϫ1 Ϫ1 acids in adults would be 16 mgákg ád . Although this 7. Young VR, Gersovitz M, Munro HN. Human aging: protein and approach has been questioned and criticized by UK investigators amino acid metabolism and implications for protein and amino acid (52Ð54), the present data lend additional support for our propo- requirements. In: Moment GB, ed. Nutritional approaches to aging sition. In addition, this estimated methionine requirement is sim- research. Boca Raton, FL: CRC Press Inc, 1982:47Ð81. ilar to the estimate derived by Fereday et al (6) in which the 8. Young VR. Macronutrient needs in the elderly. Nutr Rev metabolic demand, calculated as twice the total postabsorptive 1992;50:454Ð62. losses, is corrected for the efficiency of postprandial amino acid 9. Millward DJ, Roberts SB. Protein requirements of older individuals. utilization. For diet 1, on the basis of the Fereday model, the Nutr Res Rev 1996;9:67Ð87. metabolic demand was 62Ð86 ␮molákgϪ1 ádϪ1 (derived from the 10. Young VR. Amino acid and proteins in relation to nutrition in the elderly. Age Ageing 1990;19(suppl):S10Ð24. balance during a fast or total postabsorptive oxidation, respec- Ϸ 11. Millward DJ, Fereday A, Gibson N, Pacy PJ. Aging, protein require- tively) and postprandial amino acid utilization was 1, resulting ments, and protein turnover. Am J Clin Nutr 1997;66:774Ð86. Ϸ Ϫ1 Ϫ1 in a requirement of 9Ð13 mgákg ád . Hence, in the absence 12. Storch KJ, Wagner DA, Burke JF, Young VR. Quantitative study in of a significant source of dietary cystine, the mean requirement 2 vivo of methionine cycle in humans using [methyl- H3] and [1- for methionine, as judged from these tracer data, indeed does 13C]methionine. Am J Physiol 1988;255:E322Ð31. Ϫ Ϫ appear to be Ϸ13 mgákg 1 ád 1. 13. Young VR, Wagner DA, Burini R, Storch KJ. Methionine kinetics Note, however, that methionine balance was not achieved and balance at the 1985 FAO/WHO/UNU intake requirement in Ϫ1 Ϫ1 2 13 when the intake of methionine was reduced to 6.5 mgákg ád adult men studied with L-[ H2-methyl-1- C]methionine as a tracer. and was still not achieved with the simultaneous addition of Am J Clin Nutr 1991;54:377Ð85. dietary cystine to achieve a total intake of sulfur amino acids that 14. National Research Council. Recommended dietary allowances. 10th exceeded the FAO/WHO/UNU (31) requirement. ed. Washington, DC: National Academy Press, 1989. 15. Wiley VC, Dudman NPB, Wilken DGL. Free and protein-bound Conclusion homocysteine and cysteine in cystathionine ␤ synthase deficiency: interrelations during short- and long-term changes in plasma con- Thus, we conclude that a total sulfur amino acid requirement centrations. Metabolism 1989;38:734Ð9. Ϫ1 Ϫ1 of 13 mgákg ád for the elderly may not be met if dietary cys- 16. Malloy MH, Radsin DK, Gaull GE. A method for measurement of tine accounts for as much as half of the total sulfur amino acid free and bound plasma cyst(e)ine. Anal Biochem 1981;113:407Ð15. intake. Also, in previous studies we were not able to show, using 17. Vogt JA, Chapman TE, Wagner DA, Young VR, Burke JF. Determi- tracer approaches, a major dietary cystine-sparing action on nation of the isotope enrichment of one or a mixture of two stable methionine oxidation when methionine intakes were low (1, 2), labelled tracers of the same compound using the complete iso- 388 FUKAGAWA ET AL

topomer distribution of an ion fragment; theory and application to in substrate availability. Am J Physiol 1989;256:E288Ð94. vivo human tracer studies. Biol Mass Spectrom 1993;22:600Ð12. 35. Welle S, Thornton C, Jozefowicz R, Statt M. Myofibrillar protein 18. Matthews DE, Motil KJ, Rohrbaugh D, Burke JF,Young VR, Bier DM. synthesis in young and old men. Am J Physiol 1993;264:E693Ð8. Measurement of leucine metabolism in man from a primed, continuous 36. Yarasheski KE, Zachwieja JJ, Bier DM. Acute effects of resistance infusion of L-[1-13C]-leucine. Am J Physiol 1980;238: E473Ð9. exercise on muscle protein synthesis rate in young and elderly men 19. Matthews DE, Schwarz HP,Yang RD, Motil KJ, Young VR, Bier DM. and women. Am J Physiol 1993;265:E210Ð4. Relationship of plasma leucine and ␣-ketoisocaproate during a L-[1- 37. Uauy R, Winterer JC, Bilmazes C, et al. The changing pattern of 13C]leucine infusion in man: a method for measuring human intracel- whole body protein metabolism in aging humans. J Gerontol lular leucine tracer enrichment. Metabolism 1982;31:1105Ð12. 1978;33:663Ð71. 20. Hoerr RA, Yu YM, Wagner DA, Burke JF, Young VR. Recovery of 38. Gersovitz M, Munro HN, Udall J, Young VR. Albumin synthesis in 13 13 C in breath from NaH CO3 infused by gut and vein: effect of young and elderly subjects using a new stable isotope methodology: feeding. Am J Physiol 1989;257:E426Ð38. response to level of protein intake. Metabolism 1980;29:1075Ð86. 21. Hoerr RA, Matthews DE, Bier DM, Young VR. Leucine kinetics 39. Bier DM. Intrinsically difficult problems: the kinetics of body pro- 2 13 from [ H3]- and [ C]leucine infused simultaneously by gut and teins and amino acids. Diabetes Metab Rev 1989;5:111Ð32. vein. Am J Physiol 1991;260:E111Ð7. 40. Forslund AH, Hambraeus L, Olsson RM, El-Khoury AE, Young VR. Downloaded from https://academic.oup.com/ajcn/article/68/2/380/4648746 by guest on 27 September 2021 22. Cortiella J, Matthews DE, Hoerr RA, Bier DM, Young VR. Leucine The 24h status of whole body leucine and urea kinetics at “normal” and kinetics at graded intakes in young men: quantitative fate of dietary “high” intakes, with exercise in healthy adults. Am J Physiol (in press). leucine. Am J Clin Nutr 1988;48:998Ð1009. 41. Pacy PJ, Price GM, Halliday D, Quevedo MR, Millward DJ. Nitro- 23. Marchini JS, Cortiella J, Hiramatsu T, Chapman TE, Young VR. gen homeostasis in man. 2. The diurnal responses of protein syn- Requirements for indispensable amino acids in adult humans: thesis, degradation and amino acid oxidation to diets with increas- longer-term amino acid kinetic study with support for the adequacy ing protein intakes. Clin Sci Lond 1994;86:103Ð18. of the Massachusetts Institute of Technology amino acid require- 42. Gibson NR, Fereday A, Cox M, Halliday D, Pacy PJ, Millward DJ. ment pattern. Am J Clin Nutr 1993;58:670Ð83. Influences of dietary energy and protein on leucine kinetics during 24. El-Khoury AE, Fukagawa NK, Sanchez M, et al. Validation of the feeding in healthy adults. Am J Physiol 1996;270:E282Ð91. tracer-balance concept with reference to leucine: 24-h intravenous 43. Tessari P, Zanetti M, Barazzoni R, Vettore M, Michielan F. Mecha- tracer studies with L-[1-13C]leucine and [15N-15N]urea. Am J Clin Nutr 1994;59:1000Ð11. nisms of postprandial protein accretion in human skeletal muscle. 25. Sanchez M, El-Khoury AE, Castillo L, Chapman TE, Young VR. Insight from leucine and phenylalanine forearm kinetics. J Clin Phenylalanine and tyrosine kinetics in young men throughout a con- Invest 1996;98:1361Ð71. tinuous 24-h period, at a low phenylalanine intake. Am J Clin Nutr 44. Cayol M, Boirie Y, Rambourdin F, et al. Influence of protein intake 1995;61:555Ð70. on whole body and splanchnic leucine kinetics in humans. Am J 26. Sanchez M, El-Khoury AE, Castillo L, et al. Twenty-fourÐhour Physiol 1997;272:E584Ð91. intravenous and oral tracer studies with L-[1Ð13C]phenylalanine and 45. El-Khoury AE, Sánchez M, Fukagawa NK, Young VR. Whole body 2 13 L-[3,3Ð H2]tyrosine at a tyrosine-free, generous phenylalanine protein synthesis in healthy adult humans: CO2 technique vs intake in adults. Am J Clin Nutr 1996;63:532Ð45. plasma precursor approach. Am J Physiol 1995;268:E174Ð84. 27. Stegink LD, Bell EF, Daabees TT, Andersen DW, Zike WL, Filer LJ 46. Liberman MW, Wiseman AL, Shi Z-Z, et al. Growth retardation and Jr. Factors influencing utilization of glycine, glutamate and aspar- cysteine deficiency in ␥-glutamyl transpeptidase-deficient mice. tate in clinical products. In: Blackburn GL, Grant JP, Young VR, Proc Natl Acad Sci U S A 1996;93:7923Ð6. eds. Amino acids. Metabolism and medical applications. Boston: 47. Fukagawa NK, Ajami AM, Young VR. Plasma methionine and cys- John Wright PSG Inc, 1983:123Ð46. teine kinetics in response to an intravenous glutathione infusion in 28. Matthews DE, Rennie MJ, Bier DM. A model for leucine metabo- adult humans. Am J Physiol 1996;270:E209Ð14. lism in the human. In: Waterlow JC, Stephen JML, eds. Nitrogen 48. Graham J, Refsum H, Rosenberg JH, Ueland PM, eds. Homocys- metabolism: man. London: Applied Science Publishers, teine metabolism: from basic science to clinical medicine. Dor- 1981:117Ð23. drecht, Netherlands: Kluwer Academic Publishers, 1997. 29. Matthews DE. Leucine as a tracer for studying protein metabolism 49. Zeisel SH, daCosta K-A, Franklin PD, et al. Choline, an essential in humans. In: Schauder P, Wahren J, Paoletti R, Bernadini R, nutrient for humans. FASEB J 1991;5:2093Ð8. Rinetti M, eds. Branched-chain amino acids: biochemistry, phys- 50. Young VR, Bier DM, Pellett PL. A theoretical basis for increasing iopathology, and clinical science. New York: Raven Press, Ltd, current estimates of the amino acid requirements in adult man, with 1992:55Ð61. experimental support. Am J Clin Nutr 1989;50:80Ð92. 30. Bier DM. Whole body protein kinetic measurements. In: Nair KS, 51. Young VR, El-Khoury AE. Can amino acid requirements for nutri- ed. Protein metabolism in diabetes mellitus. London: Smith-Gordon tional maintenance be approximated from the amino acid composi- Co, Ltd, 1992:61Ð8. tion of body mixed proteins? Proc Natl Acad Sci U S A 31. FAO/WHO/UNU. Energy and protein requirements. Report of a joint FAO/WHO/UNU Expert Consultation. World Health Organ 1995;92:300Ð4. Tech Rep Ser 1985;724. 52. Waterlow JC. The requirements of adult man for indispensable 32. Robert JJ, Cummins JC, Wolfe RR, et al. Quantitative aspects of amino acids. Eur J Clin Nutr 1996;50(suppl 1):S151Ð79. glucose production and metabolism in healthy elderly subjects. Dia- 53. Millward DJ, Price GM, Pacy PJH, Halliday D. Maintenance pro- betes 1982;31:203Ð11. tein requirements: the need for conceptual re-evaluation. Proc Nutr 33. Fukagawa NK, Minaker KL, Rowe JW, Matthews DE, Bier DM, Soc 1990;49:473Ð87. Young VR. Glucose and amino acid metabolism in aging man: dif- 54. Fuller MF, Garlick PJ. Human amino acid requirements: can the ferential effects of insulin. Metabolism 1988;37:371Ð7. controversy be resolved? Annu Rev Nutr 1994;14:217Ð41. 34. Fukagawa NK, Minaker KL, Young VR, Matthews DE, Bier DM, Rowe JW. Leucine metabolism in aging man: effect of insulin and