Folate status modulates the induction of hepatic glycine N-methyltransferase and homocysteine metabolism in diabetic rats

A diabetic state induces the activity and abundance of glycine N-methyltransferase (GNMT), a key protein in the regulation of folate, methyl group, and homocysteine metabolism. Because the folate-dependent one-carbon pool is a source of methyl groups and 5-methyltetrahydrofolate allosterically inhibits GNMT, the aim of this study was to determine whether folate status has an impact on the interaction between diabetes and methyl group metabolism. Rats were fed a diet containing deficient (0 ppm), adequate (2 ppm), or supplemental (8 ppm) folate for 30 days, after which diabetes was initiated in one-half of the rats by streptozotocin treatment. The activities of GNMT, phosphatidylethanolamine N-methyltransferase (PEMT), and betaine-homocysteine S-methyltransferase (BHMT) were increased about twofold in diabetic rat liver; folate deficiency resulted in the greatest elevation in GNMT activity. The abundance of GNMT protein and mRNA, as well as BHMT mRNA, was also elevated in diabetic rats. The marked hyperhomocysteinemia in folate-deficient rats was attenuated by streptozotocin, likely due in part to increased BHMT expression. These results indicate that a diabetic state profoundly modulates methyl group, choline, and homocysteine metabolism, and folate status may play a role in the extent of these alterations. Moreover, the upregulation of BHMT and PEMT may indicate an increased choline requirement in the diabetic rat.     

Insulin administration abrogates perturbation of methyl group and homocysteine metabolism in streptozotocin-treated type 1 diabetic rats

Modifications in methyl group and homocysteine metabolism are associated with a number of pathologies, including vascular disease, cancer, and neural tube defects. A diabetic state is known to alter both methyl group and homocysteine metabolism, and glycine N-methyltransferase (GNMT) is a major regulatory protein that controls the supply and utilization of methyl groups. We have shown previously that diabetes induces GNMT expression and reduces plasma homocysteine pools by stimulating both its catabolism and folate-independent remethylation. This study was conducted to determine whether insulin plays a role in the control of homocysteine concentrations and GNMT as well as other key regulatory proteins. Male Sprague-Dawley rats were randomly assigned to one of three groups: control, streptozotocin (STZ)-induced diabetic (60 mg/kg body wt), and insulin-treated diabetic (1.0 U bid). After 5 days, rats were anesthetized (ketamine-xylazine) for procurement of blood and tissues. A 1.5-fold elevation in hepatic GNMT activity and hypohomocysteinemia in diabetic rats was completely prevented by insulin treatment. Additionally, diabetes-mediated alterations in methionine synthase, phosphatidylethanolamine N-methyltransferase, and DNA methylation were also prevented by insulin. We hypothesize that the concentration of blood glucose may represent a regulatory signal to modify GNMT and homocysteine. In support of this, blood glucose concentrations were negatively correlated with total plasma homocysteine (r = -0.75, P < 0.001) and positively correlated with GNMT activity (r = 0.77, P < 0.001). Future research will focus on further elucidating the role of glucose or insulin as a signal for regulating homocysteine and methyl group metabolism.     

Folate deprivation reduces homocysteine remethylation in a human intestinal epithelial cell culture model: role of serine in one-carbon donation

Little is known about homocysteine metabolism in intestine. To address this question, we investigated homocysteine metabolism under conditions of folate adequacy and folate deprivation in the Caco-2 cell line, a model of human intestinal mucosal cells. Caco-2 cells were cultured in media enriched with [3-(13)C]serine and [U-(13)C(5)]methionine tracers, and enrichments of intracellular free amino acid pools of these amino acids as well as homocysteine, cystathionine, and cysteine were measured by using gas chromatography/mass spectrometry. Homocysteine transsulfuration plus folate-dependent and total remethylation were quantified from these amino acid enrichments. Homocysteine remethylation accounted for 19% of the intracellular free methionine pool in cells cultured with supplemental folate, and nearly all one-carbon units used for remethylation originated from the three carbon of serine via folate-dependent remethylation. Labeling of cystathionine and cysteine indicated the presence of a complete transsulfuration pathway in Caco-2 cells, and this pathway produced 13% of the intracellular free cysteine pool. Appearance of labeled homocysteine and cystathionine in culture medium suggests export of these metabolites from intestinal cells. Remethylation was reduced by one-third in folate-restricted cell cultures (P < 0.001), and only approximately 50% of the one-carbon units used for remethylation originated from the three carbon of serine under these conditions. In conclusion, the three carbon of serine is the primary source of one-carbon units used for homocysteine remethylation in folate-supplemented Caco-2 cell cultures. Remethylation is reduced as a result of folate restriction in this mucosal cell model, and one-carbon sources other than the three carbon of serine contribute to remethylation under this condition.     

Modulation of methyl group metabolism by streptozotocin-induced diabetes and all-trans-retinoic acid

The hepatic enzyme glycine N-methyltransferase (GNMT) plays a major role in the control of methyl group and homocysteine metabolism. Because disruption of these vital pathways is associated with numerous pathologies, understanding GNMT control is important for evaluating methyl group regulation. Recently, gluconeogenic conditions have been shown to modulate homocysteine metabolism and treatment with glucocorticoids and/or all-trans-retinoic acid (RA)-induced active GNMT protein, thereby leading to methyl group loss. This study was conducted to determine the effect of diabetes, alone and in combination with RA, on GNMT regulation. Diabetes and RA increased GNMT activity 87 and 148%, respectively. Moreover, the induction of GNMT activity by diabetes and RA was reflected in its abundance. Cell culture studies demonstrated that pretreatment with insulin prevented GNMT induction by both RA and dexamethasone. There was a significant decline in homocysteine concentrations in diabetic rats, owing in part to a 38% increase in the abundance of the transsulfuration enzyme cystathionine beta-synthase; treatment of diabetic rats with RA prevented cystathionine beta-synthase induction. A diabetic state also increased the activity of the folate-independent homocysteine remethylation enzyme betaine-homocysteine S-methyltransferase, whereas the activity of the folate-dependent enzyme methionine synthase was diminished 52%. In contrast, RA treatment attenuated the streptozotocin-mediated increase in betaine-homocysteine S-methyltransferase, whereas methionine synthase activity remained diminished. These results indicate that both a diabetic condition and RA treatment have marked effects on the metabolism of methyl groups and homocysteine, a finding that may have significant implications for diabetics and their potential sensitivity to retinoids.     

Nuclear localization of de novo thymidylate biosynthesis pathway is required to prevent uracil accumulation in DNA

Uracil accumulates in DNA as a result of impaired folate-dependent de novo thymidylate biosynthesis, a pathway composed of the enzymes serine hydroxymethyltransferase (SHMT), thymidylate synthase (TYMS), and dihydrofolate reductase. In G1, this pathway is present in the cytoplasm and at S phase undergoes small ubiquitin-like modifier-dependent translocation to the nucleus. It is not known whether this pathway functions in the cytoplasm, nucleus, or both in vivo. SHMT1 generates 5,10-methylenetetrahydrofolate for de novo thymidylate biosynthesis, a limiting step in the pathway, but also tightly binds 5-methyltetrahydrofolate in the cytoplasm, a required cofactor for homocysteine remethylation. Overexpression of SHMT1 in cell cultures inhibits folate-dependent homocysteine remethylation and enhances thymidylate biosynthesis. In this study, the impact of increased Shmt1 expression on folate-mediated one-carbon metabolism was determined in mice that overexpress the Shmt1 cDNA (Shmt1tg+ mice). Compared with wild type mice, Shmt1tg+ mice exhibited elevated SHMT1 and TYMS protein levels in tissues and evidence for impaired homocysteine remethylation but surprisingly exhibited depressed levels of nuclear SHMT1 and TYMS, lower rates of nuclear de novo thymidylate biosynthesis, and a nearly 10-fold increase in uracil content in hepatic nuclear DNA when fed a folate- and choline-deficient diet. These results demonstrate that SHMT1 and TYMS localization to the nucleus is essential to prevent uracil accumulation in nuclear DNA and indicate that SHMT1-mediated nuclear de novo thymidylate synthesis is critical for maintaining DNA integrity.     

Nuclear Enrichment of Folate Cofactors and Methylenetetrahydrofolate Dehydrogenase 1 (MTHFD1) Protect de Novo Thymidylate Biosynthesis during Folate Deficiency

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.     

Tracer-derived total and folate-dependent homocysteine remethylation and synthesis rates in humans indicate that serine is the main one-carbon donor

Hyperhomocysteinemia in humans is associated with genetic variants of several enzymes of folate and one-carbon metabolism and deficiencies of folate and vitamins B12 and B6. In each case, hyperhomocysteinemia might be caused by diminished folate-dependent homocysteine remethylation, but this has not been confirmed in vivo. Because published stable isotopic tracer approaches cannot distinguish folate-dependent from folate-independent remethylation, we developed a dual-tracer procedure in which a [U-13C5]-methionine tracer is used in conjunction with a [3-13C]serine tracer to simultaneously measure rates of total and folate-dependent homocysteine remethylation. In young female subjects, plasma [U-13C4]homocysteine enrichment, a surrogate measure of intracellular [U-13C5]methionine enrichment, reached approximately 90% of the plasma [U-13C5]methionine enrichment. Methionine-methyl and -carboxyl group fluxes were in the range of previous reports (approximately 25 and approximately 17 micromol.kg(-1).h(-1), respectively). However, the rate of overall homocysteine remethylation (approximately 8 micromol.kg(-1).h(-1)) was twice that of previous reports, which suggests a larger role for homocysteine remethylation in methionine metabolism than previously thought. By use of estimates of intracellular [3-13C]serine enrichment based on a conservative correction of plasma [3-13C]serine enrichment, serine was calculated to contribute approximately 100% of the methyl groups used for total body homocysteine remethylation under the conditions of this protocol. This contribution represented only a small fraction (approximately 2.8%) of total serine flux. Our dual-tracer procedure is well suited to measure the effects of nutrient deficiencies, genetic polymorphisms, and other metabolic perturbations on homocysteine synthesis and total and folate-dependent homocysteine remethylation.     

Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.     

NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice

Glutathione is the major intracellular redox buffer in the liver and is critical for hepatic detoxification of xenobiotics and other environmental toxins. Hepatic glutathione is also a major systemic store for other organs and thus impacts on pathologies such as Alzheimer's disease, Sickle Cell Anaemia and chronic diseases associated with aging. Glutathione levels are determined in part by the availability of cysteine, generated from homocysteine through the transsulfuration pathway. The partitioning of homocysteine between remethylation and transsulfuration pathways is known to be subject to redox-dependent regulation, but the underlying mechanisms are not known. An association between plasma Hcy and a single nucleotide polymorphism within the NADPH oxidase 4 locus led us to investigate the involvement of this reactive oxygen species- generating enzyme in homocysteine metabolism. Here we demonstrate that NADPH oxidase 4 ablation in mice results in increased flux of homocysteine through the betaine-dependent remethylation pathway to methionine, catalysed by betaine-homocysteine-methyltransferase within the liver. As a consequence NADPH oxidase 4-null mice display significantly lowered plasma homocysteine and the flux of homocysteine through the transsulfuration pathway is reduced, resulting in lower hepatic cysteine and glutathione levels. Mice deficient in NADPH oxidase 4 had markedly increased susceptibility to acetaminophen-induced hepatic injury which could be corrected by administration of N-acetyl cysteine. We thus conclude that under physiological conditions, NADPH oxidase 4-derived reactive oxygen species is a regulator of the partitioning of the metabolic flux of homocysteine, which impacts upon hepatic cysteine and glutathione levels and thereby upon defence against environmental toxins.     

Thymidylate synthase: a novel genetic determinant of plasma homocysteine and folate levels

The thymidylate synthase gene ( TYMS or TS) encodes a tightly regulated enzyme that catalyzes the conversion of deoxyuridylate to thymidylate, and contains a tandem repeat polymorphism that affects expression of the enzyme. We have investigated the relationship between TYMS genotype and plasma concentrations of homocysteine and folate in a cohort of 505 Chinese from Singapore. TYMS 3/3 genotype was associated with reduced plasma folate and, among individuals with low dietary folate intake, with elevated plasma homocysteine levels. These associations were independent of the well-established methylenetetrahydrofolate reductase ( MTHFR) C677T genotype effects on plasma folate and homocysteine levels. Our results suggest that TYMS and MTHFR compete for limiting supplies of folate required for the remethylation of homocysteine. These genetic determinants of plasma folate and homocysteine levels may be useful in identifying individuals at increased risk for cardiovascular disease.     

Methylation demand: a key determinant of homocysteine metabolism

Elevated plasma homocysteine is a risk factor for cardiovascular disease and Alzheimer's disease. To understand the factors that determine the plasma homocysteine level it is necessary to appreciate the processes that produce homocysteine and those that remove it. Homocysteine is produced as a result of methylation reactions. Of the many methyltransferases, two are, normally, of the greatest quantitative importance. These are guanidinoacetate methyltransferase (that produces creatine) and phosphatidylethanolamine N-methyltransferase (that produces phosphatidylcholine). In addition, methylation of DOPA in patients with Parkinson's disease leads to increased homocysteine production. Homocysteine is removed either by its irreversible conversion to cysteine (transsulfuration) or by remethylation to methionine. There are two separate remethylation reactions, catalyzed by betaine:homocysteine methyltransferase and methionine synthase, respectively. The reactions that remove homocysteine are very sensitive to B vitamin status as both the transsulfuration enzymes contain pyridoxal phosphate, while methionine synthase contains cobalamin and receives its methyl group from the folic acid one-carbon pool. There are also important genetic influences on homocysteine metabolism.     

Genetic and nutritional deficiencies in folate metabolism influence tumorigenicity in Apcmin/+ mice

Epidemiological studies indicate that adequate dietary folate is protective against colon cancer, although mechanisms remain largely elusive. We investigated the effects of genetic disruptions of folate transport and metabolism and of dietary folate deficiency in a mouse model of colon cancer, the Apc(min/+) mouse. Apc(min/+) mice with heterozygous knockout of the gene for reduced folate carrier 1 (Rfc1(+/-)) developed significantly fewer adenomas compared to Rfc1(+/+)Apc(min/+) mice [30.3+/-4.6 vs. 60.4+/-9.4 on a control diet (CD) and 42.6+/-4.4 vs. 55.8+/-7.6 on a folate-deficient diet, respectively]. Rfc1(+/-)Apc(min/+) mice also carried a lower tumor load, an indicator of tumor size as well as of tumor number. In contrast, there were no differences in adenoma formation between Apc(min/+) mice carrying a knockout allele for methionine synthase (Mtr(+/-)), an enzyme that catalyzes folate-dependent homocysteine remethylation, and Mtr(+/+)Apc(min/+) mice. However, in both Mtr groups of mice, dietary folate deficiency significantly increased adenoma number (from 32.3+/-3.8 on a CD to 48.1+/-4.2 on a folate-deficient diet), increased plasma homocysteine, decreased global DNA methylation in preneoplastic intestines and increased apoptosis in tissues. There were no genotype-associated differences in these parameters in the Rfc1 group, suggesting that the protection conferred by Rfc1 deficiency is carried out through a different mechanism. In conclusion, genetic and nutritional disturbances in folate metabolism can have distinct influences on tumorigenesis in Apc(min/+) mice; altered levels of homocysteine, global DNA methylation and apoptosis may contribute mechanistically to dietary influence.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Differences in folate-protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate-GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate-recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.     

Analysis of pathological defects in methionine metabolism using a simple mathematical model

Derangements in methionine metabolism are a hallmark of cancers and homocystinuria, an inborn error of metabolism. In this study, the metabolic consequences of the pathological changes associated with the key pathway enzymes, methionine adenosyl transferase (MAT), glycine N-methyl transferase (GNMT) and cystathionine beta-synthase (CBS) as well as an activation of polyamine metabolism, were analyzed using a simple mathematical model describing methionine metabolism in liver. The model predicts that the mere loss of allosteric regulation of CBS by adenosylmethionine (AdoMet) leads to an increase in homocysteine concentration. This is consistent with the experimental data on the corresponding genetic defects, which specifically impair allosteric activation but not basal enzyme activity. Application of the characteristics of transformed hepatocytes to our model, i.e., substitution of the MAT I/III isozyme by MAT II, loss of GNMT activity and activation of polyamine biosynthesis, leads to the prediction of a significantly different dependence of methionine metabolism on methionine concentrations. The theoretical predictions were found to be in good agreement with experimental data obtained with the human hepatoma cell line, HepG2.     

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4–8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [¹³C₁]methionine and [²H₃-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 μg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70–80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.