QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lamers, Y. Articles by Gregory, J. F. Search for Related Content PubMed PubMed Citation Articles by Lamers, Y. Articles by Gregory, J. F., III

---

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Glycine Turnover and Decarboxylation Rate Quantified in Healthy Men and Women Using Primed, Constant Infusions of [1,2-¹³C₂]Glycine and [²H₃]Leucine^1,2

³ Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences; ⁴ Division of Endocrinology and Metabolism, Department of Medicine; and ⁵ Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32611-0370

^* To whom correspondence should be addressed. E-mail: jfgy{at}ufl.edu.

	ABSTRACT

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
Glycine plays several roles in human metabolism, e.g. as a 1-carbon donor, in purine synthesis, and as a component of glutathione. Glycine is decarboxylated via the glycine cleavage system (GCS) that yields concurrent generation of a 1-carbon unit as 5,10-methylenetetrahydrofolate (methyleneTHF). Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of glycine and serine, another 1-carbon donor. The quantitative role of glycine in human 1-carbon metabolism has received little attention. The aim of this protocol was to quantify whole body glycine flux, glycine to serine flux, and rate of glycine cleavage in humans. A primed, constant infusion with 9.26 µmol·kg^–1·h^–1 [1,2-¹³C₂]glycine and 1.87 µmol·kg^–1·h^–1 [²H₃]leucine was used to quantify the kinetic behavior of glycine in young, healthy volunteers (n = 5) in a fed state. The isotopic enrichment of infused tracers and metabolic products in plasma, as well as breath ¹³CO₂ enrichment, were determined for use in kinetic analysis. Serine synthesis by direct conversion from glycine via SHMT occurred at 193 ± 28 µmol·kg^–1·h^–1 (mean ± SEM), which comprised 41% of the 463 ± 55 µmol·kg^–1·h^–1 total glycine flux. Nearly one-half (46%) of the glycine-to-serine conversion occurred using GCS-derived methyleneTHF 1-carbon units. Based on breath ¹³CO₂ measurement, glycine decarboxylation (190 ± 41 µmol·kg^–1·h^–1) accounted for 39 ± 6% of whole body glycine flux. This study is the first to our knowledge to quantify human glycine cleavage and glycine-to-serine SHMT kinetics. GCS is responsible for a substantial proportion of whole body glycine flux and constitutes a major route for the generation of 1-carbon units.

	Introduction

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

Through the mitochondrial glycine cleavage system (GCS),⁶ a 4-protein enzymatic complex, glycine is catabolized to CO₂, ammonia, and a 1-carbon unit in the form of 5,10-methylenetetrahydrofolate (methyleneTHF) (3). Mutations affecting genes encoding the components of the GCS can cause reduced catalytic activity of the enzyme complex (4). Such loss-of-function mutations cause accumulation of glycine to pathological levels responsible for the conditions of nonketotic hyperglycinemia or glycine encephalopathy (1,4). Typical assays for GCS activity in vitro involve incubation with [carboxyl-¹⁴C]glycine and measurement of the released ¹⁴CO₂ (5).

Glycine is an important precursor of serine. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible formation of serine from glycine and a 1-carbon unit donated by methyleneTHF. Because GCS forms methyleneTHF, GCS and SHMT are able to synthesize serine in a concerted manner (6,7). Both amino acids, glycine and serine, are gluconeogeneic via pyruvate (1) and serve as 1-carbon donors. Serine was shown to be the main source of 1-carbon units for regeneration of methionine from homocysteine (8) and, thus, S-adenosylmethionine, the primary agent used in biological methylation reactions.

Tracers labeled with stable isotopes facilitate the study of in vivo kinetics in metabolism. [¹⁵N]glycine was the first amino acid tracer used in kinetic research to investigate whole body protein turnover (9,10). [¹⁵N]glycine remains the most widely used tracer for this purpose (9,11,12). Human tracer protocols using [¹⁵N]glycine have been designed to investigate glycine nitrogen metabolism (13), de novo glycine synthesis (14), dietary effects on glycine metabolism (15,16), and lipoprotein metabolism (17). With the use of [¹³C₁]glycine, Cetin et al. (18) investigated placental glycine transport.

We describe here a new steady-state tracer protocol to quantify whole body glycine flux and glycine to serine flux after primed, constant infusion of [1,2-¹³C₂]glycine and [²H₃]leucine in healthy male and female volunteers. [²H₃]Leucine was included to determine any nutritional effects on protein turnover when this protocol is applied to studies with nutritional interventions (8,19). In addition to analysis of plasma from serial blood samples collected during the infusion, we also measured breath ¹³CO₂ enrichment (20) to quantify the flux of glycine decarboxylation through the GCS and estimated the concurrent glycine-based generation of 1-carbon units. Through the use of this protocol, we examined GCS flux, the rate of glycine-to-serine conversion and the fraction of this conversion specifically using a glycine-derived 1-carbon unit in healthy young adults. Overall, these findings demonstrate the utility of this protocol and provide new insight into quantitative aspects of human glycine and 1-carbon metabolism.

	Methods

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
Materials

[1,2-¹³C₂]Glycine and L-[5,5,5-²H₃]leucine were purchased from Cambridge Isotopes Laboratories. Isotope solutions were prepared in isotonic saline, filter sterilized, and analyzed to ensure lack of pyrogenicity and microbial contamination.

Human subjects

Healthy adult male and nonpregnant female subjects (20–40 y old) were recruited and met the following inclusion criteria: no history of gastrointestinal surgery, abnormal kidney, or thyroid function, or any other chronic disease; no smoking or chronic drug use or alcoholism; no vitamin, amino acid, or protein supplementation; no chronic consumption of a high-protein diet; and a BMI of <28 kg/m². Medical history, dietary habits, and demographic data were assessed by a questionnaire. Adequate nutritional status for folate and vitamins B-12 and B-6 was defined as serum folate >7 nmol/L, serum vitamin B-12 >200 pmol/L, and plasma pyridoxal 5'-phosphate (PLP) >30 nmol/L, respectively, and plasma total homocysteine concentration <12 µmol/L. For determination of general health, subjects were screened by standard clinical measures for normal hematological pattern, blood chemistry, and thyroid status and underwent a physical examination. All subjects gave written informed consent. The University of Florida Institutional Review Board and the General Clinical Research Center (GCRC) Scientific Advisory Committee approved this protocol.

Dietary treatment

All meals were prepared by the Bionutrition Unit of the GCRC. Prior to the infusion day, subjects consumed nutritionally adequate meals with standardized composition for 2 d to minimize dietary variation immediately prior to the study.

Analytical methods

    Screening measurements. Serum folate and vitamin B-12 were analyzed with the use of a commercial chemiluminescence-based assay (Elecsys, Roche Diagnostics). Plasma PLP concentration was measured as the semicarbazone-derivative by reverse-phase HPLC with fluorescence detection (21). Plasma total homocysteine concentration was measured as the ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate derivative by reverse-phase HPLC with fluorescence detection (22).

    GC-MS analysis of amino acid isotopic enrichment. Plasma free amino acids were isolated, derivatized, and analyzed as previously described (8). The N-heptafluorobutyramide-n-propyl ester derivatives were dried, dissolved in ethyl acetate, and stored at –20°C until analysis. Isotopic enrichment was determined by negative chemical ionization-GC/MS with the use of a Thermo-Finnigan DSQ GC-MS and a 30-m poly (5% diphenyl and 95% dimethylsiloxane) fused silica capillary column (Equity 5, Supelco). The relative abundance of specific ions was determined by selected-ion monitoring at the following mass/charge ratios: glycine (293–295); serine (519–522); leucine (349–352); and methionine (367–372). Isotopic enrichments are expressed as molar ratios (mol % excess) of labeled to nonlabeled isotopomers after correction for the natural abundance of stable isotopes essentially as performed by Storch et al. (23). Natural abundance measured for glycine M+2, leucine M+3, serine M+1, and serine M+2 was 1.2, 0.2, 17, and 2.6 mol % excess, respectively.

    Breath CO₂. For determination of the isotopic enrichment of breath ¹³CO₂, samples were collected in Exetainer tubes provided by Metabolic Solutions and shipped to Metabolic Solutions for isotope ratio-MS analysis. Total CO₂ production rate (VCO₂) was determined with the use of a metabolic cart (TrueMax 2400; ParvoMedics). Measurements were taken at 30-s intervals for 5 min until 4 consecutive time points differed by no more than ± 0.01 L/min.

Infusion protocol

Subjects were admitted to the GCRC on the evening before the infusion protocol and consumed no food and drinks, except water, between 2100 and the first blood draw (Fig. 1). On the morning of the infusion, an angiocatheter was inserted in the antecubital vein of each arm, 1 for the tracer infusion and 1 for blood collection. Fasting blood samples were taken 2 h before infusion (at 0700) for measurement of plasma PLP, total homocysteine concentrations, and background isotopic enrichment of amino acids. Infusions were initiated at 0900 with a 5-min, 20-mL priming dose that delivered 9.26 µmol/kg [1,2-¹³C₂]glycine and 1.87 µmol/kg [5,5,5-²H₃]leucine, along with a simultaneous priming dose of 2.15 µmol/kg of NaH¹³C0₃. The 9-h constant infusion followed immediately after the priming dose and delivered 20 mL/h infusion solution that contained 9.26 µmol/kg [1,2-¹³C₂]glycine and 1.87 µmol/kg [5,5,5-²H₃]leucine.

View larger version (9K): [in this window] [in a new window]   FIGURE 1  [13C2]Glycine tracer infusion protocol.

To measure ¹³CO₂ production, breath samples were collected into Exetainer tubes at 0, 1, 2, 3, 4, 5, and 6 h of infusion. Measurements of VCO₂ were conducted at 0, 2, 4, 6, and 8 h of infusion. The subjects received a nutritive formula hourly starting 2 h before infusion to maintain a fed state (23). This formula provided a balanced pattern of amino acids at a rate based on requirements of 0.8 g protein·kg^–1·d^–1, which equals an hourly protein dose of 0.03 g/kg with 5.23 and 5.44 kJ·kg^–1·d^–1 (1.25 and 1.30 kcal·kg^–1·d^–1) for women and men, respectively. The formula further provided an adequate energy intake according to the requirements of 126 and 130 kJ·kg^–1·d^–1 (30 and 31 kcal·kg^–1·d^–1) for women and men, respectively.

Kinetic principles and analysis

The tracer model is based on the same general principle as our previous studies of the role of serine in 1-carbon metabolism (8,19,24), analogous to that of Schalinske and Steele (25). The combined use of [1,2-¹³C₂]glycine and [²H₃]leucine permitted the determination of the kinetics of whole body glycine turnover, the conversion of glycine to serine, the rate of glycine decarboxylation, and its role as a source of 1-carbon units in 1-carbon metabolism. [²H₃]Leucine is included to evaluate any nutritional effects on protein turnover in applications involving dietary interventions (8,19). As the glycine tracer is decarboxylated and catabolized via the GCS, the original glycine 2-carbon ¹³C-labeled carbon is transferred to tetrahydrofolate (THF) to yield methyleneTHF. The major focus of this protocol was the determination of the quantitative aspects of glycine decarboxylation, glycine-to-serine interconversion by SHMT (as monitored by the formation of [¹³C₂]serine, and the contribution of glycine-derived 1-carbon units (as methyleneTHF) in serine formation via SHMT (as indicated by the formation of [¹³C₁]serine) (Fig. 2). We also examined in this protocol the use of glycine-derived 1-carbon units for homocysteine remethylation. In this process, the 1-carbon unit of methyleneTHF is reduced by methyleneTHF reductase to yield 5-methylTHF, then transferred to homocysteine via methionine synthase to generate methionine (or [methyl-¹³C₁]methionine if derived in labeled form from the glycine tracer) (8,24). The rate of remethylation of homocysteine through methionine synthase using a 1-carbon unit derived from the glycine tracer (via GCS) provides a measure of the in vivo rate of this pathway.

View larger version (8K): [in this window] [in a new window]   FIGURE 2  Schematic of carbon flow from infused [13C2]glycine to give singly or doubly labeled serine. This is the basis of differentiating whether labeled serine is generated directly from glycine via SHMT (lower pathway yielding [13C2]serine) or via SHMT using a glycine-derived methyleneTHF (upper pathway yielding [13C1]serine).

(Eq. 1)

In this equation, E is the enrichment at time t (h), while E_f and k are the enrichment at infinity (i.e. Ep) and rate constant (h^–1) from the fitted curve, respectively (26). Data were fit to a single exponential regression equation using the "exponential rise to maximum" function of SigmaPlot 2002 (Version 8.02; SPSS).

Steady-state kinetics of amino acid tracers were calculated using standard equations (12), including correction for overestimation of intracellular enrichment from plasma enrichment data (12,23,26), as discussed below. The flux of an amino acid is the rate of appearance of that amino acid from endogenous production (de novo synthesis and protein breakdown), absorption, and the tracer infusion, and is calculated from the Ep of the corresponding amino acid tracer. Specifically, the flux of leucine (Q_Leu) in the plasma pool is calculated as:

(Eq. 2)

where I_Leu is the [²H₃]leucine infusion rate, E_Leu is the enrichment of the [²H₃]leucine tracer, and Ep_Leu is the Ep of [²H₃]leucine in plasma. The Ep of plasma leucine was not corrected for overestimation of intracellular enrichment, consistent with previous studies using leucine flux as a relative indicator of protein turnover (19,24,27).

Glycine flux (Q_Gly) was calculated from plasma [¹³C₂]glycine enrichment after correcting for the overestimation of the intracellular [¹³C₂]glycine enrichment that occurs when the plasma Ep of the glycine tracer is used. This prediction of intracellular [¹³C₂]glycine enrichment (Ep'_Gly) was accomplished by multiplying the observed plasma [¹³C₂]glycine enrichment by a correction factor of 0.4 derived from previous glycine tracer infusion studies in humans (17,28).

(Eq. 3)

Flux values for labeled metabolic products, i.e. serine M+1 and serine M+2, are estimated assuming a serine flux of Q_Ser = 271 µmol·kg^–1·h^–1 (8).

(Eq. 4)

The synthesis rates of metabolic products derived from the infused tracers were calculated from the flux and the Ep of the infused tracer after correction for intracellular isotopic dilution (8). This serine flux reported by Davis et al. (8) was determined after correction for intracellular serine enrichment with the value 0.4 derived from previous serine tracer studies (19,27). The rate of serine M+1 synthesis, which was indicative of serine synthesis using a glycine-derived 1-carbon unit, was thus calculated as:

(Eq. 5)

In analogous fashion, the rate of serine M+2 synthesis, reflective of direct conversion of glycine to serine, was:

(Eq. 6)

The rate of production of ¹³CO₂ provided a direct measurement of the whole body flux of the decarboxylation of the glycine tracer. This was measured in standard fashion as in amino acid oxidation studies (14,23). In this procedure, the rate of ¹³CO₂ release (V¹³CO₂, in units of µmol·h^–1·kg body weight^–1) and the rate of glycine catabolism via decarboxylation (C_Gly, µmol·h^–1·kg body weight^–1) were calculated as follows:

(Eq. 7)

where E¹³CO₂ is breath CO₂ enrichment plateau and 0.81 is the assumed fraction of CO₂ release from the body pool of bicarbonate and W is body weight (14).

(Eq. 8)

where Ep'_Gly is the Ep of plasma [¹³C₂]glycine corrected for intracellular overestimation and Ei_Gly is the enrichment of the infused glycine tracer.

The fraction of glycine flux occurring via glycine decarboxylation was calculated as:

(Eq. 9)

All data are presented as means ± SEM.

	Results

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
The subjects (3 male and 2 female, age 21–28 y) had a BMI < 25 kg/m². Their serum folate, vitamin B-12, and plasma PLP concentrations were in the normal range and their plasma total homocysteine (<12 µmol/L) indicated normal 1-carbon metabolism.

After primed, constant infusion with 9.26 µmol·kg^–1·h^–1 [¹³C₂]glycine and 1.87 µmol·kg^–1·h^–1 [²H₃]leucine, plasma enrichments of the infused stable isotope-labeled amino acids were measured in all subjects (Fig. 3). The Ep and flux values derived from the infused glycine and leucine tracers and the glycine-derived metabolic products (Fig. 2) are shown in Table 1. Using the data of [¹³C₂]serine enrichment, the mean (± SEM) rate of serine synthesis directly from [¹³C₂]glycine via SHMT was 193 ± 28 µmol·kg^–1·h^–1 comprising 41% of the 463 ± 55 µmol·kg^–1·h^–1 total glycine flux. The rate of serine synthesis via GCS and SHMT using a glycine cleavage-derived 1-carbon unit via SHMT (a labeled 1-carbon unit coupled to an unlabeled glycine to yield [¹³C₁]serine, i.e. serine M+1) was 88 ± 13 µmol·kg^–1·h^–1 and thus contributed to 46% of the glycine-to-serine conversion. The enrichment of M+3 serine from coupling of a glycine-derived ¹³C 1-carbon unit with the infused [¹³C₂]glycine was below detection limits (data not shown). Under the conditions of this protocol, the enrichment of [¹³C₁]methionine (i.e. M+1) was typically <0.2 mol % excess, which was below the limit of precise measurement.

View larger version (9K): [in this window] [in a new window]   FIGURE 3  Plasma enrichment of infused amino acids (A) and metabolic products (B) in healthy men and women during primed, constant infusion with 9.26 µmol·kg–1·h–1 [13C2]glycine and 1.87 µmol·kg–1·h–1 [2H3]leucine. Values are means ± SEM, n = 5.

The leucine flux (102 ± 7 µmol·kg^–1·h^–1) was 20% higher than that observed in previous studies from this laboratory (8,23), which reflected greater intake of dietary amino acids in the nutritive formula used in this protocol.

	Discussion

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 
    Sources of ¹³CO₂. This is the first study to our knowledge to quantify human GCS. Using ¹³CO₂ enrichment to enable the calculation of glycine decarboxylation, glycine cleavage accounted for 39% of the whole body glycine flux in these healthy men and women as determined by primed, constant infusion with [¹³C₂]glycine. Although the greatest portion of CO₂ release from glycine occurs from glycine decarboxylase activity of the GCS, we acknowledge that CO₂ also might be generated by several other processes. That both carbon atoms of glycine ultimately can yield CO₂ was demonstrated in studies of patients with inherited enzymatic defects of GCS. In hepatocytes of patients with hyperglycinemia, CO₂ formation could be detected after incubation with labeled glycine even though these patients have little or no enzyme activity of GCS (3).

In healthy individuals, CO₂ can be formed from glycine through pathways of folate or pyruvate metabolism in addition to decarboxylation in the GCS. One-carbon units derived from the 2-carbon of glycine are transferred to THF forming methyleneTHF, which can be enzymatically oxidized to 10-formylTHF by methyleneTHF dehydrogenase. 10-Formyltetrahydrofolate dehydrogenase converts 10-formylTHF to THF and CO₂, which appears to be a mechanism of achieving a regeneration of THF and a means of regulating the 10-formylTHF pool (29). Recent mathematical modeling predicts that 10-formylTHF dehydrogenase would contribute to CO₂ generation at a rate of 30% of the rate of GCS in hepatic metabolism (30). Thus, the reaction catalyzed by 10-formylTHF dehydrogenase might contribute partially to ¹³CO₂ formation after [¹³C₂]glycine infusion. However, as glycine is the direct substrate for GCS, we proposed that CO₂ formation through the GCS is likely to be substantially faster and quantitatively greater than that from 10-formylTHF dehydrogenase.

Glycine and serine are interconvertible through SHMT, and serine dehydratase transforms serine to pyruvate that enters the tricarboxylic acid cycle either as oxaloacetate or acetyl-CoA. The decarboxylation of pyruvate by pyruvate dehydrogenase to acetyl-CoA and CO₂ also could contribute to ¹³CO₂ formation in this protocol. However, the activity of serine dehydratase is lower in humans than other mammalian species (31) and, thus, would contribute minimally to CO₂ formation from glycine. The report that pyruvate oxidation rate was only 10 µmol·kg^–1·h^–1 in healthy subjects (32) is consistent with our assumption that generation of CO₂ from glycine by pyruvate oxidation is far less than that from the GCS. An alternative glycine tracer study is planned to quantify the CO₂ production rate while distinguishing between its formation by GCS and 10-formylTHF dehydrogenase reactions.

    Glycine and human 1-carbon metabolism. During glycine decarboxylation, 1 molecule of methyleneTHF is formed per molecule of CO₂ in the GCS. Despite the potential overestimation of GCS flux in this study, the glycine cleavage rate of 190 µmol·kg^–1·h^–1 observed in this study implies a high rate of methyleneTHF formation from glycine. MethyleneTHF is used for serine synthesis via SHMT, formation of 10-formylTHF or 5-methylTHF, and the formation of various metabolic products derived from these folates (serine, thymidylate, methionine, and purines). In the current protocol, the rate of serine synthesis using a glycine-derived methyleneTHF was 88 µmol·kg^–1·h^–1. Previous studies in this laboratory and others have shown that homocysteine remethylation flux is in the range of only 2–8 µmol·kg^–1·h^–1 (24). Overall, these data suggest a very substantial flow of carbon units from glycine into other aspects of 1-carbon metabolism such as purine and thymidylate synthesis.

The use of [¹³C₂]glycine in this protocol has allowed independent assessment of serine synthesis by SHMT and the fraction of that synthesis specifically using a glycine-derived methyleneTHF. Under the conditions of this protocol, serine formation by SHMT accounted for 41% of whole body glycine flux. Kalhan et al. (33) showed that serine contributes to 15–20% of the plasma glycine pool in pregnant and nonpregnant women. A similar ratio of glycine to serine and serine to glycine formation was observed in fetal lamb hepatocytes (6). The net flux through cytosolic SHMT mainly occurs in the glycine to serine direction (29,34,35). However, increased cellular glycine concentrations cause a reversal of the mitochondrial SHMT from its steady-state direction of serine to glycine formation (30,35) and yield net formation of serine. Serine synthesis is important because of its role as a substrate in the synthesis of glucose via pyruvate, protein, phosphatidylserine, cystathionine, and neuromodulators (36). Serine also serves as the main 1-carbon donor for remethylation of homocysteine to methionine (8), although remethylation accounts for a small part in the whole body serine flux.

Aside from providing novel experimental data describing novel quantitative aspects of several phases of human 1-carbon metabolism, this and our previous studies (8,24) also yield experimental confirmation of predictions of mathematical modeling (30). These include: 1) glycine is an important source of 1-carbon units to support a high rate of serine synthesis; and 2) homocysteine remethylation from 5-methylTHF via methionine synthase constitutes a small fraction of human 1-carbon metabolic flux, as mentioned above. Our experimental study also suggests that the GCS is an important source of glycine-derived 1-carbon units needed to support the high steady-state fluxes of thymidylate and purine synthesis predicted in mathematical modeling (30).

A single-pool model using plasma amino acid enrichment for calculation of whole body amino acid kinetics often overestimates the whole body amino acid pool. To compensate for such overestimation in the case of plasma glycine enrichment, we employed a correction factor of 0.4 (12,17,28,37). This is based on consensus data from studies using metabolically produced surrogate indicators of intracellular glycine, including urinary hippurate (12) and plasma apolipoprotein B-100 in VLDL (17,28,37), both of which reflect the enrichment of hepatic free glycine pools.

The whole body glycine flux in our healthy male and female volunteers was 463 µmol·kg^–1·h^–1, which is consistent with a report of 458 µmol·kg^–1·h^–1, also determined in the fed state, by Gersovitz et al. (15). Because the flux of an amino acid is the rate of appearance of that amino acid from endogenous production (de novo synthesis and protein breakdown), the tracer infusion, and absorption (12), the intake of amino acids during a tracer infusion increases the amino acid flux. Thus, glycine flux determined in the fasting state was only about one-half of the flux in the fed state (240 µmol·kg^–1·h^–1) as reported by Robert et al. (14). Measurements of glycine metabolism have been shown to be independent of the route of administration (38). Intracellular dilution of glycine enrichment results from glyoxylate metabolism (39), de novo synthesis, protein degradation, and interconversion from serine. Thirty-five percent of systemic flux of glycine occurs from endogenous synthesis (40). Pathways removing glycine from its pool are formation of serine and glutathione, transamination, protein synthesis, and gluconeogenesis.

This infusion protocol provides new quantitative information about human glycine and 1-carbon metabolism. The glycine decarboxylation rate accounted for over one-third of whole body glycine flux. This finding of a high rate of glycine degradation via GCS is supported by the symptoms of hyperglycinemia in patients with enzymatic defects in the glycine decarboxylase (1,4). Nearly one-half of the methyleneTHF formed by GCS was used for serine formation, with a substantial supply of glycine-derived methyleneTHF available to support other requirements in human 1-carbon metabolism. This protocol is being applied in a protocol with additional subjects to investigate glycine kinetics, the sensitivity of glycine metabolism to gender, marginal deficiency of vitamin B-6, and the kinetics of nucleotide and glutathione synthesis.

	FOOTNOTES

 
¹ Supported by NIH grant DK072398 and General Clinical Research Center grant M01-RR00082.

² Author disclosures: Y. Lamers, J. Williamson, L. R. Gilbert, P. W. Stacpoole, and J. F. Gregory III, no conflicts of interest.

⁶ Abbreviations used: Ep, plateau enrichment; GCRC, General Clinical Research Center; GCS, glycine cleavage system; I, infusion rate; methyleneTHF, 5,10-methylenetetrahydrofolate; Q, a measurement of flux; PLP, pyridoxal 5'-phosphate; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; VCO₂, rate of CO₂ production.

Manuscript received 28 August 2007. Initial review completed 6 September 2007. Revision accepted 13 September 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Methods Results Discussion LITERATURE CITED

 

1. Pearl PL, Capp PK, Novotny EJ, Gibson KM. Inherited disorders of neurotransmitters in children and adults. Clin Biochem. 2005;38:1051–8.[Medline]

2. Bailey LB, Gregory JF III. Folate metabolism and requirements. J Nutr. 1999;129:779–82.[Abstract/Free Full Text]

3. Kikuchi G. The glycine cleavage system: composition, reaction mechanism, and physiological significance. Mol Cell Biochem. 1973;1:169–87.[Medline]

4. Hiraga K, Kochi H, Hayasaka K, Kikuchi G, Nyhan WL. Defective glycine cleavage system in nonketotic hyperglycinemia. Occurrence of a less active glycine decarboxylase and an abnormal aminomethyl carrier protein. J Clin Invest. 1981;68:525–34.[Medline]

5. Kure S, Narisawa K, Tada K. Enzymatic diagnosis of nonketotic hyperglycinemia with lymphoblasts. J Pediatr. 1992;120:95–8.[Medline]

6. Narkewicz MR, Thureen PJ, Sauls SD, Tjoa S, Nikolayevsky N, Fennessey PV. Serine and glycine metabolism in hepatocytes from mid gestation fetal lambs. Pediatr Res. 1996;39:1085–90.[Medline]

7. Thureen PJ, Narkewicz MR, Battaglia FC, Tjoa S, Fennessey PV. Pathways of serine and glycine metabolism in primary culture of ovine fetal hepatocytes. Pediatr Res. 1995;38:775–82.[Medline]

8. Davis SR, Stacpoole PW, Williamson J, Kick LS, Quinlivan EP, Coats BS, Shane B, Bailey LB, Gregory JF III. Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor. Am J Physiol Endocrinol Metab 2004;286:E272–9. Erratum in Am J Physiol Endocrinol Metab 2004;286: E674.[Abstract/Free Full Text]

9. Duggleby SL, Waterlow JC. The end-product method of measuring whole-body protein turnover: a review of published results and a comparison with those obtained by leucine infusion. Br J Nutr. 2005;94:141–53.[Medline]

10. Sprinson DB, Rittenberg D. The rate of interaction of the amino acids of the diet with the tissue proteins. J Biol Chem. 1949;180:715–26.[Free Full Text]

11. Jackson AA, Duggleby SL, Grove G. Whole body protein turnover can be measured non-invasively in women using the end product method with [15N]glycine to show changes with the menstrual cycle and pregnancy. Eur J Clin Nutr. 2000;54:329–36.[Medline]

12. Wolfe RR. Radioactive and stable isotope tracers in biomedicine: principles and practice of kinetic analysis. New York: Wiley Liss; 1992.

13. Matthews DE, Conway JM, Young VR, Bier DM. Glycine nitrogen metabolism in man. Metabolism. 1981;30:886–93.[Medline]

14. Robert JJ, Bier DM, Zhao XH, Matthews DE, Young VR. Glucose and insulin effects on the novo amino acid synthesis in young men: studies with stable isotope labeled alanine, glycine, leucine, and lysine. Metabolism. 1982;31:1210–8.[Medline]

15. Gersovitz M, Bier D, Matthews D, Udall J, Munro HN, Young VR. Dynamic aspects of whole body glycine metabolism: influence of protein intake in young adult and elderly males. Metabolism. 1980;29:1087–94.[Medline]

16. Yu YM, Yang RD, Matthews DE, Wen ZM, Burke JF, Bier DM, Young VR. Quantitative aspects of glycine and alanine nitrogen metabolism in postabsorptive young men: effects of level of nitrogen and dispensable amino acid intake. J Nutr. 1985;115:399–410.[Abstract/Free Full Text]

17. Arends J, Schafer G, Schauder P, Bircher J, Bier DM. Comparison of serine and hippurate as precursor equivalents during infusion of [15N]glycine for measurement of fractional synthetic rates of apolipoprotein B of very-low-density lipoprotein. Metabolism. 1995;44:1253–8.[Medline]

18. Cetin I, Marconi AM, Baggiani AM, Buscaglia M, Pardi G, Fennessey PV, Battaglia FC. In vivo placental transport of glycine and leucine in human pregnancies. Pediatr Res. 1995;37:571–5.[Medline]

19. Cuskelly GJ, Stacpoole PW, Williamson J, Baumgartner TG, Gregory JF III. Deficiencies of folate and vitamin B(6) exert distinct effects on homocysteine, serine, and methionine kinetics. Am J Physiol Endocrinol Metab. 2001;281:E1182–90.[Abstract/Free Full Text]

20. Parra MD, Martinez JA. Nutritional aspects of breath testing based on stable isotopes. Nutr Rev. 2006;64:338–47.[Medline]

21. Ubbink JB, Serfontein WJ, de Villiers LS. Stability of pyridoxal-5-phosphate semicarbazone: applications in plasma vitamin B6 analysis and population surveys of vitamin B6 nutritional status. J Chromatogr. 1985;342:277–84.[Medline]

22. Pfeiffer CM, Huff DL, Gunter EW. Rapid and accurate HPLC assay for plasma total homocysteine and cysteine in a clinical laboratory setting. Clin Chem. 1999;45:290–2.[Free Full Text]

23. Storch KJ, Wagner DA, Burke JF, Young VR. Quantitative study in vivo of methionine cycle in humans using [methyl-2H3]- and [1–13C]methionine. Am J Physiol. 1988;255:E322–31.[Medline]

24. Davis SR, Scheer JB, Quinlivan EP, Coats BS, Stacpoole PW, Gregory JF III. Dietary vitamin B-6 restriction does not alter rates of homocysteine remethylation or synthesis in healthy young women and men. Am J Clin Nutr. 2005;81:648–55.[Abstract/Free Full Text]

25. Schalinske KL, Steele RD. Quantitation of carbon flow through the hepatic folate-dependent one-carbon pool in rats. Arch Biochem Biophys. 1989;271:49–55.[Medline]

26. MacCoss MJ, Fukagawa NK, Matthews DE. Measurement of intracellular sulfur amino acid metabolism in humans. Am J Physiol Endocrinol Metab. 2001;280:E947–55.[Abstract/Free Full Text]

27. Gregory JF III, Cuskelly GJ, Shane B, Toth JP, Baumgartner TG, Stacpoole PW. Primed, constant infusion with [2H3]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism. Am J Clin Nutr. 2000;72:1535–41.[Abstract/Free Full Text]

28. Cryer DR, Matsushima T, Marsh JB, Yudkoff M, Coates PM, Cortner JA. Direct measurement of apolipoprotein B synthesis in human very low density lipoprotein using stable isotopes and mass spectrometry. J Lipid Res. 1986;27:508–16.[Abstract]

29. Garcia-Martinez LF, Appling DR. Characterization of the folate-dependent mitochondrial oxidation of carbon 3 of serine. Biochemistry. 1993;32:4671–6.[Medline]

30. Nijhout HF, Reed MC, Lam SL, Shane B, Gregory JF III, Ulrich CM. In silico experimentation with a model of hepatic mitochondrial folate metabolism. Theor Biol Med Model. 2006;3:40.[Medline]

31. Xue HH, Sakaguchi T, Fujie M, Ogawa H, Ichiyama A. Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase. J Biol Chem. 1999;274:16028–33.[Abstract/Free Full Text]

32. Shangraw RE, Jahoor F. Mechanism of dichloroacetate-induced hypolactatemia in humans with or without cirrhosis. Metabolism. 2004;53:1087–94.[Medline]

33. Kalhan SC, Gruca LL, Parimi PS, O'Brien A, Dierker L, Burkett E. Serine metabolism in human pregnancy. Am J Physiol Endocrinol Metab. 2003;284:E733–40.[Abstract/Free Full Text]

34. Lin BF, Kim JS, Hsu JC, Osborne C, Lowe K, Garrow T, Shane B. Molecular biology in nutrition research: modeling of folate metabolism. Adv Food Nutr Res. 1996;40:95–106.[Medline]

35. Kastanos EK, Woldman YY, Appling DR. Role of mitochondrial and cytoplasmic serine hydroxymethyltransferase isozymes in de novo purine synthesis in Saccharomyces cerevisiae. Biochemistry. 1997;36:14956–64.[Medline]

36. de Koning TJ, Snell K, Duran M, Berger R, Poll-The BT, Surtees R. L-serine in disease and development. Biochem J. 2003;371:653–61.[Medline]

37. Arends J, Chiu F, Bier DM. Analysis of plasma hippurate in humans using gas chromatography-mass spectrometry: concentration and incorporation of infused [¹⁵N]glycine. Anal Biochem. 1990;191:401–10.[Medline]

38. Stein TP, Settle RG, Albina JA, Dempsey DT, Melnick G. Metabolism of nonessential 15N-labeled amino acids and the measurement of human whole-body protein synthesis rates. J Nutr. 1986;116:1651–9.[Abstract/Free Full Text]

39. Rowsell EV, Snell K, Carnie JA, Al-Tai AH. Liver-L-alanine-glyoxylate and L-serine-pyruvate aminotransferase activities: an apparent association with gluconeogenesis. Biochem J. 1969;115:1071–3.[Medline]

40. Reeds PJ. Dispensable and indispensable amino acids for humans. J Nutr. 2000;130:S1835–40.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lamers, Y. Articles by Gregory, J. F. Search for Related Content PubMed PubMed Citation Articles by Lamers, Y. Articles by Gregory, J. F., III