Phosphatidylethanolamine N-methyltransferase Research Articles

Phosphatidylcholine containing docosahexaenoic acid (DHA) as a marker for in vivo phospatidylethanolamine methyltransferase: implications for brain development

Phosphatidylethanolamine methyltransferase (PEMT), which sequentially methylates phosphatidylethanolamine to phosphatidylcholine (PtdCho), is the body’s only pathway for de novo synthesis of choline. Mice lacking functional PEMT (PEMT −/−) quickly become choline deficient and require dietary choline for survival. PEMT −/− mice also have a substantial loss of PtdCho-DHA in liver and plasma, regardless of dietary choline intake. Therefore, we propose plasma PtdCho-DHA to be a useful, non-invasive marker of in vivo PEMT activity in humans. To test this, we used gas chromatography to measure PtdCho-DHA in plasma samples obtained from adult men and women as well as in primary human hepatocytes. PEMT activity using [3H]S-adenosylmethionine (a substrate for PEMT) was also measured in the hepatocytes. To date, plasma PtdCho-DHA was elevated in pre-menopausal women as compared to post-menopausal women and men, consistent with the ability of estrogen to induce PEMT (p<0.01). Furthermore, women with a single nucleotide polymorphism (SNP) in the PEMT gene that possibly decreases PEMT expression were found to have correspondingly less plasma PtdCho-DHA (p<0.05). With corresponding data in hepatocytes, PtdCho-DHA shows promise as a marker of in vivo PEMT activity in humans and could help with identifying individuals that may be at risk of choline deficiency. A deficiency in PEMT has also been shown to alter brain development in mice and loss of PtdCho-DHA may be partially responsible for these effects. Ongoing investigations explore the importance of PtdCho-DHA in fetal brain development.

Phosphatidylethanolamine N -methyltransferase ( PEMT ) gene expression is induced by estrogen in human and mouse primary hepatocytes

Phosphatidylethanolamine N -methyltransferase ( PEMT ) gene expression is induced by estrogen in human and mouse primary hepatocytes

Gene response elements, genetic polymorphisms and epigenetics influence the human dietary requirement for choline

Recent progress in the understanding of the human dietary requirement for choline highlights the importance of genetic variation and epigenetics in human nutrient requirements. Choline is a major dietary source of methyl-groups (one of choline's metabolites, betaine, participates in the methylation of homocysteine to form methionine); also choline is needed for the biosynthesis of cell membranes, bioactive phospholipids and the neurotransmitter acetylcholine. A recommended dietary intake for choline in humans was set in 1998, and a portion of the choline requirement can be met via endogenous de novo synthesis of phosphatidylcholine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. Though many foods contain choline, many humans do not get enough in their diets. When deprived of dietary choline, most adult men and postmenopausal women developed signs of organ dysfunction (fatty liver, liver or muscle cell damage, and reduces the capacity to handle a methionine load, resulting in elevated homocysteine). However, only a portion of premenopausal women developed such problems. The difference in requirement occurs because estrogen induces expression of the PEMT gene and allows premenopausal women to make more of their needed choline endogenously. In addition, there is significant variation in the dietary requirement for choline that can be explained by common polymorphisms in genes of choline and folate metabolism. Choline is critical during fetal development, when it alters DNA methylation and thereby influences neural precursor cell proliferation and apoptosis. This results in long term alterations in brain structure and function, specifically memory function.

Role of transmethylation reactions in alcoholic liver disease

Alcoholic liver disease is a major health care problem worldwide. Findings from many laboratories, including ours, have demonstrated that ethanol feeding impairs several of the many steps involved in methionine metabolism. Ethanol consumption predominantly results in a decrease in the hepatocyte level of S-adenosylmethionine and the increases in two toxic metabolites, homocysteine and S-adenosylhomocysteine. These changes, in turn, result in serious functional consequences which include decreases in essential methylation reactions via inhibition of various methyltransferases. Of particular interest to our laboratory is the inhibition of three important enzymes, phosphatidylethanolamine methyltransferase, isoprenylcysteine carboxyl methyltransferase and protein L-isoaspartate methyltransferase. Decreased activity of these enzymes results in increased fat deposition, increased apoptosis and increased accumulation of damaged proteins-all of which are hallmark features of alcoholic liver injury. Of all the therapeutic modalities available, betaine has been shown to be the safest, least expensive and most effective in attenuating ethanol-induced liver injury. Betaine, by virtue of aiding in the remethylation of homocysteine, removes both toxic metabolites (homocysteine and S-adenosylhomocysteine), restores S-adenosylmethionine level, and reverses steatosis, apoptosis and damaged proteins accumulation. In conclusion, betaine appears to be a promising therapeutic agent in relieving the methylation and other defects associated with alcoholic abuse.

Methyl balance and transmethylation fluxes in humans

Various questions have been raised about labile methyl balance and total transmethylation fluxes, and further discussion has been encouraged. This report reviews and discusses some of the relevant evidence now available. The fact that, if needed, labile methyl balance is maintained by methylneogenesis appears to be established, but several aspects of transmethylation remain uncertain: definitive measurements of the rate of total transmethylation in humans of both sexes on various diets and at various ages; the extent to which synthesis of phosphatidylcholine has been underestimated; and the relative contributions of the 2 pathways for the formation of sarcosine (ie, N-methylglycine). The available evidence indicates that the quantitatively most important pathways for S-adenosylmethionine–dependent transmethylation in mammals are the syntheses of creatine by guanidinoacetate methyltransferase, of phosphatidylcholine by phosphatidylethanolamine methyltransferase, and of sarcosine by glycine N-methyltransferase. Data presented in this report show that S-adenosylmethionine and methionine accumulate abnormally in the plasma of humans with glycine N-methyltransferase deficiency but not of those with guanidinoacetate N-methyltransferase deficiency or in the plasma or livers of mice devoid of phosphatidylethanolamine N-methyltransferase activity. The absence of such accumulations in the latter 2 conditions may be due to removal of S-adenosylmethionine by synthesis of sarcosine. Steps that may help clarify the remaining issues include the determination of the relative rates of synthesis of sarcosine, creatine, and phosphatidylcholine by rapid measurement of the rates of radiolabel incorporation into these compounds from l-[methyl-3H]methionine administered intraportally to an experimental animal; clarification of the intracellular hepatic isotope enrichment value during stable-isotope infusion studies to enhance the certainty of methyl flux estimates during such studies; and definitive measurement of the dietary betaine intake from various diets.

Lipid Homeostasis and Lipoprotein Secretion in Niemann-Pick C1-deficient Hepatocytes

Niemann-Pick C (NPC) disease is a fatal inherited disorder characterized by an accumulation of cholesterol and other lipids in late endosomes/lysosomes. Although this disease is considered to be primarily a neurodegenerative disorder, many NPC patients suffer from liver disease. We have investigated alterations that occur in hepatic lipid homeostasis using primary hepatocytes isolated from NPC1-deficient mice. The cholesterol content of Npc1(-/-) hepatocytes was 5-fold higher than that of Npc1(+/+) hepatocytes; phospholipids and cholesteryl esters also accumulated. In contrast, the triacylglycerol content of Npc1(-/-) hepatocytes was 50% lower than of Npc1(+/+) hepatocytes. We hypothesized that the cholesterol sequestration induced by NPC1 deficiency might inhibit very low density lipoprotein secretion. However, this process was enhanced by NPC1 deficiency and the secreted particles were enriched in cholesteryl esters. We investigated the mechanisms responsible for these changes. The synthesis of phosphatidylcholine, cholesteryl esters, and cholesterol in hepatocytes was increased by NPC1 deficiency and the amount of the mature form of sterol response element-binding protein-1 was also increased. These observations indicate that the enhanced secretion of lipoproteins from NPC1-deficient hepatocytes is due, at least in part, to increased lipid synthesis.

Hepatic phosphatidylethanolamine N-methyltransferase expression is increased in diabetic rats.

Phosphatidylcholine is an essential phospholipid that is synthesized by 2 different pathways, the CDP-choline pathway and the methylation of phosphatidylethanolamine by phosphatidylethanolamine N-methyltransferase (PEMT). Recent studies have suggested that PEMT is an important consumer of methyl groups from S-adenosylmethionine (SAM) and is a major determinant of homocysteine pools. Diabetes and all-trans-retinoic acid (ATRA) have been shown to alter the activities of several enzymes involved in methyl group metabolism. Thus, we investigated how diabetes and ATRA, individually and together, affect SAM-dependent phospholipid methylation. Rats received a single injection of streptozotocin (60 mg/kg body wt) or vehicle followed by administration of ATRA (30 mumol/kg body wt) or vehicle for 5 d. The hepatic activity of PEMT increased 50% in both diabetic rat groups, whereas administration of ATRA was without effect. In diabetic rats, plasma total homocysteine decreased 30-35% in all treatment groups as compared with the control group. Thus, alterations in the activity of PEMT were not directly correlated to changes in homocysteine concentrations. Moreover, treatment of diabetic rats with insulin prevented the increase in PEMT activity and abundance. Because these observations support an increased need for SAM-dependent phosphatidylcholine synthesis, this may also indicate an increased choline requirement in diabetes.

Betaine attenuates alcoholic steatosis by restoring phosphatidylcholine generation via the phosphatidylethanolamine methyltransferase pathway

Previous studies in our laboratory implicated ethanol-induced decreases in hepatocellular S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH) ratios in lowering the activity of phosphatidylethanolamine methyltransferase (PEMT), which is associated with the generation of steatosis. Further in vitro studies showed that betaine supplementation could correct these alterations in the ratio as well as attenuate alcoholic steatosis. Therefore, we sought to determine whether the protective effect of betaine is via its effect on PEMT activity. Male Wistar rats were fed the Lieber DeCarli control or ethanol diet with or without 1% betaine supplementation for 4 weeks. We observed that ethanol feeding resulted in decreased phosphatidylcholine (PC) production by a PEMT-catalyzed reaction. Betaine supplementation corrected the ethanol-induced decrease in hepatic SAM:SAH ratios and by normalizing PC production via the PEMT-mediated pathway, significantly reduced fatty infiltration associated with ethanol consumption. This restoration of hepatocellular SAM:SAH ratio by betaine supplementation was associated with increases in the activity, enzyme mass and gene expression of the enzyme, betaine homocysteine methyltransferase (BHMT), that remethylates homocysteine. Betaine, by virtue of promoting an alternate remethylation pathway, restores SAM:SAH ratios that, in turn, correct the defective cellular methylation reaction catalyzed by PEMT resulting in protection against the generation of alcoholic steatosis.

People with fatty liver are more likely to have the PEMT rs7946 SNP, yet populations with the mutant allele do not have fatty liver

We reported that individuals with nonalcoholic fatty liver were significantly more likely to have a single nucleotide polymorphism (SNP) in the gene encoding for phosphatidylethanolamine N-methyltransferase (PEMT). This SNP (rs7946, +5465 G. → A) leads to a V175M substitution and is a loss of function mutation (1). Drs. Romeo, Cohen, and Hobbs conducted an interesting study further investigating our observation. They asked whether individuals with the mutant allele (A) were more likely to have fatty liver, and concluded that this is not the case. At first glance, the two reports appear to be arriving at opposite conclusions. Populations with fatty liver are more likely to have the mutant allele (Song et al.), yet populations with the mutant allele do not, on average, have fatty liver (Romeo et al.). However, there is an important difference in the questions being asked. When the underlying mechanisms are considered, it is apparent that both observations can be true. PEMT catalyzes de novo synthesis of phosphatidylcholine in liver (2, 3). Loss of function of this enzyme can result in fatty liver because phosphatidylcholine is required for hepatic secretion of triacylglycerol in very low density lipoproteins (VLDL) (4–6). Thus, the rs7946 SNP in PEMT slows the rate of removal of fat from the liver. If the rate of production of triacylglycerol exceeds the rate of removal, fatty liver occurs. It is possible that under normal circumstances the rate of triacylglycerol synthesis is slow enough that even the mutant PEMT activity is sufficient to sustain adequate export of this fat. Only under conditions of rapid triacylglycerol production (i.e., excess calorie intake) would the defect in PEMT become rate-limiting and cause fatty liver. Thus, the rs7946 SNP in PEMT could be necessary but not sufficient to cause fatty liver. If this were the case, then individuals with fatty liver would be more likely to have the PEMT SNP, but not every individual with the PEMT SNP would have fatty liver. Romeo et al. report data from 2349 subjects. We studied only 87 subjects. Romeo included many more African American and Hispanic subjects, and in these two populations a third are wild type, a third heterozygous for the rs7946 SNP, and a third homozygous for the rs7946 SNP. This is very different from the distribution in Caucasians. Our study included mainly Caucasian subjects, so it is most appropriate to compare our results with those observed in Romeo's Caucasian subjects. These results (Table 1) are remarkably similar. To ascertain whether the A allele is over-represented in subjects with fatty liver, we can use the Fisher's Exact Test (7) (Table 2). We conclude that the study of Romeo et al., in a very large population, supports our finding that the rs7946 SNP in PEMT is more common in Caucasians with fatty liver. The Romeo et al. study provides important additional information: 1) Caucasians have a different distribution of this SNP than do Hispanics and African Americans; 2) having this SNP is not sufficient to cause fatty liver, as many individuals with the SNP have normal liver fat. It is likely that this SNP slows export of fat from liver, and results in fatty liver only when triacylglycerol synthesis is accelerated.

Phosphatidylethanolamine &lt;I&gt;N&lt;/I&gt;-Methyltransferase and Regulation of Homocysteine

Homocysteine is a non-protein, sulfur-containing amino acid derived from methionine metabolism and S-adenosylmethionine (SAM)-dependent transmethylation. Elevations in plasma homocysteine have been associated with increased risk of cardiovascular disease, atherosclerosis, birth defects, Alzheimer's disease, and osteoporosis. Of the many known methyltransferases that utilize SAM, phosphatidylethanolamine N-methyltransferase has recently received much attention for its possible role in the regulation of homocysteine.

PhosphatidylethanolamineN-Methyltransferase and Regulation of Homocysteine

Homocysteine is a non-protein, sulfur-containing amino acid derived from methionine metabolism and S-adenosylmethionine (SAM)-dependent transmethylation. Elevations in plasma homocysteine have been associated with increased risk of cardiovascular disease, atherosclerosis, birth defects, Alzheimer's disease, and osteoporosis. Of the many known methyltransferases that utilize SAM, phosphatidylethanolamine N-methyltransferase has recently received much attention for its possible role in the regulation of homocysteine.

A metabolic link between S-adenosylhomocysteine and polyunsaturated fatty acid metabolism in Alzheimer's disease

There is evidence that vascular risk factors contribute to the pathology of Alzheimer's disease. Increased concentrations of circulating homocysteine are associated with vascular risk factors and Alzheimer's disease but the mechanisms involved are unclear. Homocysteine inhibits the hydrolysis of S-adenosylhomocysteine (SAH) which is a product inhibitor of S-adenosylmethionine (SAM) dependent methyltransferase reactions. It has been shown previously that SAH inhibits phosphatidylethanolamine N-methyltransferase (PEMT) in the liver. The activity of PEMT in the liver plays an important role in the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) and the delivery of essential polyunsaturated fatty acids (PUFAs) to peripheral tissues. In the present study, the plasma concentrations of SAH, SAM and homocysteine and the erythrocyte composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and their respective polyunsaturated fatty acid concentrations were determined in 26 patients with Alzheimer's disease and compared to those in 29 healthy control subjects. There was a significant increase in the plasma concentrations of SAH ( p < 0.001) and homocysteine ( p < 0.001) and a significant increase in the plasma concentrations of SAM ( p < 0.001) in the Alzheimer's patients. A significant positive correlation was found between the plasma concentrations of SAH and homocysteine ( r = 0.738, p < 0.001). There was a negative correlation between the plasma concentrations of homocysteine and the ratio of SAM/SAH ( r = −0.637, p < 0.01). There was a significant decrease in the erythrocyte content of PC ( p < 0.001) and an increase in the erythrocyte content of PE ( p < 0.001) in the Alzheimer's patients. Plasma SAH concentrations were negatively related to erythrocyte PC concentrations ( r = −0.286, p < 0.01) and positively related to erythrocyte PE concentrations ( r = 0.429, p < 0.001). The erythrocyte PC from Alzheimer's patients had a significant depletion of docosahexaenoic acid (DHA) ( p < 0.001) while there was no significant difference in the DHA content of erythrocyte PE. There was a significant negative correlation between plasma SAH and the DHA composition of erythrocyte PC ( r = −0.271, p < 0.001). This data may reflect the inhibition of hepatic PEMT activity by SAH in Alzheimer's disease. The decreased mobilization of DHA from the liver into plasma and peripheral tissues may increases the risk of atherosclerosis and stroke leading to chronic cerebral hypoperfusion. The evidence suggests that a metabolic link between the increased production of SAH and phospholipid metabolism may contribute to cerebrovascular and neurodegenerative changes in Alzheimer's disease.

Folate status modulates the induction of hepatic glycineN-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.

Common genetic polymorphisms affect the human requirement for the nutrient choline

Humans eating diets deficient in the essential nutrient choline can develop organ dysfunction. We hypothesized that common single nucleotide polymorphisms (SNPs) in genes involved in choline metabolism influence the dietary requirement of this nutrient. Fifty-seven humans were fed a low choline diet until they developed organ dysfunction or for up to 42 days. We tested DNA SNPs for allelic association with susceptibility to developing organ dysfunction associated with choline deficiency. We identified an SNP in the promoter region of the phosphatidylethanolamine N-methyltransferase gene (PEMT; -744 G-->C; rs12325817) for which 18 of 23 carriers of the C allele (78%) developed organ dysfunction when fed a low choline diet (odds ratio 25, P=0.002). The first of two SNPs in the coding region of the choline dehydrogenase gene (CHDH; +318 A-->C; rs9001) had a protective effect on susceptibility to choline deficiency, while a second CHDH variant (+432 G-->T; rs12676) was associated with increased susceptibility to choline deficiency. A SNP in the PEMT coding region (+5465 G-->A; rs7946) and a betaine:homocysteine methyltransferase (BHMT) SNP (+742 G-->A; rs3733890) were not associated with susceptibility to choline deficiency. Identification of common polymorphisms that affect dietary requirements for choline could enable us to identify individuals for whom we need to assure adequate dietary choline intake.

Inactivation of PEMT2 in hepatocytes initiated by DENA in fasted/refed rats

Phosphatidylethanolamine N-methyltransferase (PEMT) is the enzyme that converts phosphatidylethanolamine (PE) into phosphatidylcholine. We have previously shown that PEMT suppressed hepatoma growth by triggering apoptosis. We investigate whether PEMT controlled cell death and cell proliferation triggered by fasting/refeeding and whether it is a marker of early preneoplastic lesions. The induction of programmed cell death and suppression of cell proliferation by fasting were associated with enhanced PEMT expression and activity, and with a decrease in CTP:phosphocholine cytidylyltransferase expression. Refeeding returned the liver growth and expression of CTP:phosphocholine cytidylyltransferase to control levels, while the expression of PEMT decreased to below control values. After DENA administration, PEMT protein, evaluated by Western blotting, slightly increased, but it remained below control levels. The treatment with 20 mg/kg DENA to refed rats induced the appearance of initiated hepatocytes that were negative for PEMT expression. Present findings indicate that PEMT is a novel tumour marker for early liver preneoplastic lesions.

The ratio of phosphatidylcholine to phosphatidylethanolamine influences membrane integrity and steatohepatitis

Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are major phospholipids in mammalian membranes. In liver, PC is synthesized via the choline pathway or by methylation of PE via phosphatidylethanolamine N-methyltransferase (PEMT). Pemt(-/-) mice fed a choline-deficient (CD) diet develop rapid steatohepatitis leading to liver failure. Steatosis is observed in CD mice that lack both PEMT and multiple drug-resistant protein 2 (MDR2), required for PC secretion into bile. We demonstrate that liver failure in CD-Pemt(-/-) mice is due to loss of membrane integrity caused by a decreased PC/PE ratio. The CD-Mdr2(-/-)/Pemt(-/-) mice escape liver failure by maintaining a normal PC/PE ratio. Manipulation of PC/PE levels suggests that this ratio is a key regulator of cell membrane integrity and plays a role in the progression of steatosis into steatohepatitis. The results have clinical implications as patients with nonalcoholic steatohepatitis have a decreased ratio of PC to PE compared to control livers.

Effect of dietary methionine on plasma and liver cholesterol concentrations in rats and expression of hepatic genes involved in cholesterol metabolism

Methionine has been shown to increase plasma cholesterol in animals. In the present study, mechanisms were investigated by which methionine could alter cholesterol metabolism. In the first experiment, forty growing rats were fed four casein-based diets differing in methionine content (2.6, 3.5, 4.5 or 6.0 g/kg) for 14 d. In the second experiment, isolated rat hepatocytes were incubated in media supplemented with 50, 100 or 200 micromol/l methionine. Dietary methionine tended to increase plasma homocysteine concentrations in the rats (P=0.058). A weak positive correlation between circulating homocysteine and plasma cholesterol was observed (R2 0.27, P<0.01). Rats fed 3.5 g/kg or more of methionine had higher concentrations of cholesterol in their plasma, in lipoprotein fractions of density (rho; kg/l) 1.006<rho<1.063 and rho>1.063, and in liver than rats fed 2.6 g/kg methionine. Rats fed 6 g/kg methionine had a higher hepatic expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol-7alpha-hydroxylase than rats fed less methionine. The phosphatidylcholine:phosphatidylethanolamine ratio in rat liver increased with rising dietary methionine concentration; the relative mRNA concentrations of phosphatidylethanolamine N-methyltransferase and cystathionine beta-synthase remained unaffected. Hepatocytes incubated in media supplemented with 100 or 200 micromol/l methionine had a higher cholesterol synthesis than hepatocytes incubated in a medium supplemented with 50 micromol/l methionine; the LDL uptake in hepatocytes was independent of the methionine concentration of the medium. In conclusion, the present study suggests that dietary methionine induces hypercholesterolaemia at least in part via an enhanced hepatic cholesterol synthesis.

A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N‐methyltransferase and host cell‐specific binding

The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.

Phosphatidylethanolamine N‐methyltransferase (PEMT) gene polymorphisms and risk of spina bifida

Recently, our group demonstrated a reduction in risk of neural tube defects (NTDs) in infants of mothers who had elevated intakes of dietary choline [Shaw et al., 2004]. We hypothesized that disturbance in choline metabolism that are secondary to either a choline deficiency, or choline pathway genes mutations, may represent risk factors for NTDs. Evidence to further support this hypothesis is derived from in vitro studies. These studies showed that inhibition of choline uptake and metabolism during murine neurulation result in growth retardation and developmental defects, including an increase in the prevalence of NTDs in mouse embryos [Fisher et al., 2001, 2002]. Choline or its derivatives, such as acetylcholine, betaine, phosphocholine, phosphatidylcholine (PtdCho), and sphingomyelin, are nutrients critical to many cellular processes. These include the maintenance of the structural integrity of cell membranes, phospholipid biosynthesis, cholinergic neurotransmission, transmembrane signaling, and lipid-cholesterol transport and metabolism. Choline is also one of the major sources of methyl groups in the diet and has essential roles in methyl-metabolism [Zeisel and Blusztajn, 1994]. The phosphatidylethanolamine N-methyltransferase (PEMT, EC 2.1.1.17) pathway contributes approximately one-third of the synthesis of PtdCho, which is the most abundant mammalian phospholipids [Walkey et al., 1999; Reo et al., 2002]. In the absence of dietary supplementation, the PEMT pathway provides the only de novo biosynthesis of choline. This reaction requires S-adenosylmethionine (SAM) as the methyl donor [Bremer and Greenberg, 1961], generating S-adenosylhomocysteine (SAH), which is subsequently hydrolyzed yielding adenosine and homocysteine (Hcy) [Finkelstein, 1998]. In mice, PEMT expression enhances not only plasma Hcy levels, but also Hcy secretion from hepatocytes [Schneider and Vance, 1978]. Hyperhomocysteinemia has been identified as a risk factor for NTDs [Rosenquist and Finnell, 2001]. Knowing the regulatory role of PEMT gene in choline metabolism and ultimately in determining Hcy levels, we examined two non-synonymous SNPs, rs7946 (Met212Val) and rs897453 (Val95Ile). We investigated whether PEMT gene variants would disturb PEMT function, rendering affected individuals more susceptible to spina bifida due to their increased dietary requirements for choline and the dysregulation of Hcy homeostasis. Our analytic strategy investigated potential spina bifida risks associated with PEMT gene polymorphisms in a population-based case-control study.

Insights into phosphatidylcholine biosynthesis and function from gene targeting in mice

The biosynthesis of phosphatidylcholine (PC) via choline was elucidated by Kennedy in the 1950s. In 1960, Bremer described a second pathway in liver catalyzed by phosphatidylethanolamine (PE) N-methyltransferase (PEMT). Research in the 1980s revealed regulatory features of PC biosynthesis and most enzymes involved were purified. The genes and cDNAs were cloned in the 1990s that allowed for generation of knockout mice. Pemt−/ − mice fed a chow diet have no phenotype. When fed a choline-deficient diet, the mice die from liver failure after 3 days. We bred Pemt−/ − mice with mice that lacked MDR2, a protein that exports PC into bile. Strikingly, Pemt−/ −/Mdr2−/ − mice fed the choline-deficient diet survived for 90 days. Pemt−/ −/Mdr2−/ −, but not Pemt−/ −, mice efficiently recycle choline and maintain a normal PC/PE ratio in liver. In Pemt−/ − mice, the hepatic PC/PE ratio decreased to below 1 and caused permeabilization of the cell. Pemt−/ − compared to Pemt+/+mice fed a high fat/cholesterol diet had decreased levels of lipids in plasma. PEMT deficiency resulted in decreased secretion of very low density lipoproteins (VLDL). When Pemt−/ − mice were bred with atherosclerosis-prone mice, the lack of PEMT resulted in striking protection from atherosclerosis. Mice with a knockout of hepatic CTP:phosphocholine cytidylyltransferase α survive and maintain normal levels of PC. However, plasma lipid levels are 50% lower than wildtype mice and VLDL secretion from liver is decreased by 50%. It is unlikely that in the 1960s Drs. Kennedy or Bremer would have anticipated the development of knockout mice and the role of PC biosynthesis in liver failure or development of atherosclerosis.