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Keywords:

  • alcohol;
  • epigenome stability and cancer;
  • folate;
  • folate malabsorption;
  • folate transport;
  • proton coupled folate transporter;
  • reduced folate carrier

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Folates are members of the B-class of vitamins, which are required for the synthesis of purines and pyrimidines, and for the methylation of essential biological substances, including phospholipids, DNA, and neurotransmitters. Folates cannot be synthesized de novo by mammals; hence, an efficient intestinal absorption process is required. Intestinal folate transport is carrier-mediated, pH-dependent and electroneutral, with similar affinity for oxidized and reduced folic acid derivatives. The various transporters, i.e. reduced folate carrier, proton-coupled folate transporter, folate-binding protein, and organic anion transporters, are involved in the folate transport process in various tissues. Any impairment in uptake of folate can lead to a state of folate deficiency, the most prevalent vitamin deficiency in world, affecting 10% of the population in the USA. Such impairments in folate transport occur in a variety of conditions, including chronic use of ethanol, some inborn hereditary disorders, and certain diseases. Among these, ethanol ingestion has been the major contributor to folate deficiency. Ethanol-associated folate deficiency can develop because of dietary inadequacy, intestinal malabsorption, altered hepatobiliary metabolism, enhanced colonic metabolism, and increased renal excretion. Ethanol reduces the intestinal and renal uptake of folate by altering the binding and transport kinetics of folate transport systems. Also, ethanol reduces the expression of folate transporters in both intestine and kidney, and this might be a contributing factor for folate malabsorption, leading to folate deficiency. The maintenance of intracellular folate homeostasis is essential for the one-carbon transfer reactions necessary for DNA synthesis and biological methylation reactions. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, in chromosomal modifications, and in the development of mutations. Ethanol, a toxin that is consumed regularly, has been found to affect the methylation of DNA. In addition to its effect on DNA methylation due to folate deficiency, ethanol could directly exert its effect through its interaction with one-carbon metabolism, impairment of methyl group synthesis, and affecting the enzymes regulating the synthesis of S-adenosylmethionine, the primary methyl group donor for most biological methylation reactions. Thus, ethanol plays an important role in the pathogenesis of several diseases through its potential ability to modulate the methylation of biological molecules. This review discusses the underlying mechanism of folate malabsorption in alcoholism, the mechanism of methylation-associated silencing of genes, and how the interaction between ethanol and folate deficiency affects the methylation of genes, thereby modulating epigenome stability and the risk of cancer.


Abbreviations
5,10-CH2-THF

5,10-methylenetetrahydrofolate

5-methyl-THF

5-methyltetrahydrofolate

BBM

brush border membrane

BBMV

brush border membrane vesicle

BHMT

betaine–homocysteine methyltransferase

BLM

basolateral membrane

BLMV

basolateral membrane vesicle

FBP

folate-binding protein

FR

folate receptor

GCPII

glutamate carboxypeptidase II

MTHFR

methylenetetrahydrofolate reductase

MTX

methotrexate

OAT

organic anion transporter

PCFT

proton-coupled folate transporter

PKA

protein kinase A

RBC

red blood cell

RFC

reduced folate carrier

SAH

S-adenosylhomocysteine

SAM

S-adenosylmethionine

SLC19

solute carrier 19

TMD

transmembrane domain

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Water-soluble vitamins represent a group of structurally and functionally unrelated compounds that share the common feature of being essential for normal health and wellbeing. These micronutrients play critical roles in maintaining the normal metabolic, energy, differentiation and growth status of mammalian cells. Because humans and other mammals either cannot synthesize these compounds or synthesize insufficient amounts, they must obtain them from exogenous sources via intestinal absorption. Thus, the intestine plays an important role in maintaining and regulating normal body homeostasis of these micronutrients, and impairment of intestinal absorption of these compounds can lead to states of vitamin deficiency. Folates are a family of molecules based on folic acid (pteorylglutamic acid). The name is derived from the Latin word ‘folium’, meaning leaf, and indicates the main dietary source, green-leafed vegetables. Folates are members of the B-complex vitamins, which are required for the synthesis of nucleotide precursors, and the methylation of a wide variety of essential biological substances, including phospholipids, proteins, DNA, and neurotransmitters [1]. Folate, as a cofactor in one-carbon transfer, is an important nutrient factor that may modulate the development of cancers. Maintenance of intracellular folate homeostasis is essential for the one-carbon transfer reactions necessary for DNA synthesis and biological methylation reactions [2]. DNA methylation is an important epigenetic determinant in gene expression, in the maintenance of DNA integrity and stability, in chromosomal modifications, and in the development of mutations. As mammals are unable to synthesize folate de novo, the requirements must be satisfied from nutritional sources. Folates are hydrophilic anionic molecules that can only minimally traverse biological membranes by simple diffusion [3]. Therefore, it is not surprising that sophisticated membrane transport systems have been evolved for folate transport. These transport systems are vital at two levels: for absorption of folate in the intestine [4] and for reabsorption in the proximal renal tubules [5]; and for internalization through the plasma membrane of various proliferative and nonproliferative tissues [6]. The transport systems responsible for uptake of folate in these tissues include reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), folate-binding protein (FBP), and organic anion transporters (OATs) [7]. In contrast to the ubiquitous expression of RFC and PCFT, FBP and OATs have restricted patterns of tissue expression. Any impairment in these folate transport systems or folate metabolism might lead to a state of folate deficiency, the most prevalent vitamin deficiency throughout the world [8]. Such impairment can occur in a variety of conditions, including chronic use of ethanol, intestinal resection, and drug interactions [9,10]. Ethanol-associated folate deficiency can develop because of folate malabsorption, derangement of hepatobiliary metabolism, and/or increased renal excretion of folate [9,11].

Folate homeostasis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Folates are highly lipophobic bivalent anions that can only minimally traverse biological membranes by simple diffusion. Therefore, their internalization through mammalian cell plasma membranes must occur by means of a mediated process [12]. The mechanism of folate transport in the small intestine is a subject of considerable interest, because mammals require the ingestion and absorption of preformed folates to meet their needs for one-carbon moieties to sustain key biosynthetic reactions [1,13]. Intestinal folate transport shows the characteristics of a carrier-mediated process [1]. Similar carrier-mediated mechanisms have been identified in human colonocytes, which are capable of absorbing some of the folates that are synthesized by normal microflora of the large intestine [14]. Although the contribution of the latter source of vitamins to overall host nutrition is not clear and requires further investigation, it is highly likely that it does contribute to the cellular homeostasis of folate in localized colonocytes [15].

Complex dietary folates are hydrolyzed at the intestinal brush border membrane (BBM) by an enzyme called glutamate carboxypeptidase II (GCPII) or folate hydrolase (also referred to as folylpoly-γ-glutamate carboxypeptidase) [16]. After the hydrolytic digestion of dietary polyglutamyl folates, the intestinal transport of their monoglutamyl derivatives takes place. Each process occurs mainly at the brush border of the duodenum and upper jejunum [17]. GCPII, which is active in the glycosylated form, is an exopeptidase that cleaves polyglutamyl folates in a stepwise fashion to the final monoglutamate derivatives at a pH optimum of 6.5 and with a Km of 0.6 μm [18]. Monoglutamyl folates, including folic acid, are believed to be transferred across the human intestinal brush border membrane by RFC, which functions at pH 5.0 with a Km in the same range as that of GCPII [19]. However in addition to RFC, the recently identified PCFT has been found to play an important role in folic acid absorption at acidic pH [20]. Once the folate is inside the cell, folylpolyglutamate synthetase adds glutamate residues to the single glutamyl residue at the C-terminus. This increases its size and prevents its loss from the cell via the folate export pumps [21]. After intracellular modifications, reduced and methylated folate, i.e. 5-methyltetrahydrofolate (5-methyl-THF), crosses the basolateral enterocyte membrane by an active anion exchange mechanism, and enters the portal circulation via the submucosa [22]. From the portal circulation, the folates (mainly as 5-methyl-THF) are transported to the liver, the main storage organ of the body, by specialized transporters [23]. The liver basolateral membrane (BLM) also shows the characteristics of a carrier-mediated process for folate uptake, which is sodium-dependent and ouabian-sensitive [24]. Once transported into hepatocytes, the folate monoglutamates must be converted to their polyglutamate forms by folylpoly-γ-glutamate synthetase (EC 3.2.17), the latter being preferentially retained in cells and increases the (polyglutamated folate) affinity of folate-dependent enzymes [25]. The enzyme folylpoly-γ-glutamate synthetase can effectively add up to eight l-glutamate residues to folate monoglutamate through γ-carboxypeptide linkage [26]. Following the release of folate monoglutamate by folylpoly-γ-glutamate carboxypeptidase, folates are secreted into the bile and are transported to the small intestine, where they are reabsorbed [27]. As well as the liver, the pancreas also stores folate; it is the second richest store, and therefore may have a significant role in folate homeostasis. The uptake of folate in this tissue is pH-dependent, energy-dependent, sodium-independent, and temperature-dependent [1]. The kidneys also play an important role in the regulation of folate homeostasis, through the uptake of folates across their membranes [28]. Circulating folate is filtered in the glomeruli, is extensively reabsorbed within the nephron, and enters the renal vascular circulation. In addition to RFC, which is generally involved in folate transport in various tissues, the kidneys contain a high-affinity FBP, which has been shown to be concentrated in the BBMs of proximal tubule cells [29]. Moreover, the renal OAT may also play a role in nonspecific folate transport across the apical membranes of kidneys. Urinary folate excretion is regulated more than 95% at the kidney brush border membrane [30], maintaining physiological folate homeostasis (Fig. 1).

image

Figure 1.  Folate homeostasis: (1) dietary folate; (2) conversion of dietary the polyglutamate form of folate to the monoglutamate form in the intestinal lumen. Absorption across the BBM and BLM. (3) Transport to the liver and other tissues, involving FBPs and RFC. (4) Intracellular conversion to polyglutamates for biochemical functions and storage. (5) Reconversion to monoglutamates, which may be redistributed. (6) Salvage of folates from senescent cells for reutilization. (7) Return of the monoglutamated form to the liver involving, FBPs and RFC. (8) Transport of 5-methyl-THF into the enterohepatic cycle for distribution to tissues. (9) Renal tubular reabsorption of folate involving FBPs, PCFT, RFC and OATs. (10) Excretion of small amounts of folate.

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Folate cycle

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Folic acid is essential for nucleic acid synthesis and methylation of DNA, proteins, phospholipids, and neurotransmitters, thus regulating their function. In the activated methyl cycle, folate as N5-methyl-THF supplies a methyl group for the conversion of homocysteine to methionine by the enzyme methionine synthase. Thereafter, methionine adenosyl transferase catalyzes the addition of ATP to methionine for the generation of S-adenosylmethionine (SAM), which acts as the universal donor in the methylation reactions (Fig. 2).

image

Figure 2.  Folate metabolic pathway. DHF, dihydrofolate; DHFR, dihydrofolate reductase; THF, tetrahydrofolate; SHMT, serine hydroxymethyltransferase; MS, methionine synthase; Ts, thymidylate synthase.

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Second, folate in the form of 5,10-methylenetetrahydrofolate (5,10-CH2-THF) donates a methyl group to uracil, converting it to thymidine, which is used for DNA synthesis and repair. Because deoxyribonucleotides are the substrates for the polymerases involved in DNA replication and repair, the fidelity of DNA synthesis is critically dependent on the correct balance and availability of deoxynucleotides [31]. An enzyme that shunts methyl groups in one-carbon metabolism is methylenetetrahydrofolate reductase (MTHFR). MTHFR irreversibly converts 5,10-CH2-THF to 5-methyl-THF, which then donates a methyl group to homocysteine to produce methionine. Individuals who are severely deficient in MTHFR activity because of a germline mutation have excessive amounts of homocysteine in the blood and urine, and develop severe mental retardation and vascular diseases. Furthermore, the common polymorphism associated with MTHFR (C677T) has been linked to increased risk for certain cancers, such as endometrial cancer, breast cancer, ovarian cancer, esophageal cancer, and gastric cancer, and decreased risk for others, such as leukemia and colorectal cancer [32]. The cancer risk associated with MTHFR polymorphisms may be modulated by folate intake. When folate intake is sufficient, individuals carrying the variant MTHFR genotypes may have a decreased risk, because enhanced genomic integrity is achieved as a result of the greater availability of the MTHFR substrate 5,10-CH2-THF for conversion of uracil to thymidine, and hence DNA synthesis [33]. Importantly MTHFR polymorphism and ethanol intake have been associated with colorectal cancers [34,35].

Folic acid absorption and transport – features and mechanisms

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

The absorption of dietary folate monoglutamates involves a specific, carrier-mediated system [36]. This system is highly pH-dependent, with higher uptake at acidic pH than at neutral or alkaline pH [37]. Studies with purified intestinal BBM vesicles (BBMVs) in the presence of metabolic and membrane transport inhibitors have also shown that the folate uptake system is concentration dependent, electroneutral, and inhibited by the anion transport inhibitors, and has a similar affinity for reduced (e.g. 5-methyl-THF) and oxidized (e.g. folic acid) folate derivatives [22]. These observations have led to the conclusion that a folate/OH exchanger or folate/H+ cotransport is involved in intestinal folate uptake process [22]. The fact that the intestinal folate uptake process has similar affinities for reduced and oxidized folate derivatives is unique to the gut, and is not true for the widely investigated folate uptake system of mouse leukemia cells. In the latter cell type, the folate uptake system has a preference for reduced over oxidized folate derivatives, and hence is referred to as RFC [38]. Concerning the mechanism of folate exit from the enterocytes, i.e. transport across the basolateral membrane, studies with purified intestinal BLM membrane vesicles (BLMVs) have shown the involvement of a specific carrier-mediated system that appears to be shared by oxidized and reduced folate derivatives and is anion-sensitive [38]. Studies have demonstrated the requirement for RFC, but not for FBP, for folic acid transport in the jejunum [39]. As well as RFC, PCFT was recently reported to be responsible for transport of folate in the intestine [20]. The PCFT activity showed the characteristics of pH dependence and of having similar affinities for reduced and oxidized folates. The PCFT-mediated transport was reduced by ionophores that dissipated the transmembrane proton gradient [20]. PCFT expression was also found in the liver (the organ that stores maximum folate), kidney, and placenta [20]. In addition to its activity in the tissues with acidic pH environments, PCFT also mediates folate uptake at neutral pH, owing to its high level of expression and the fact that PCFT has considerable residual activity for reduced folates even at neutral pH [20]. The mechanism of absorption of dietary folate has been intensively examined over the past two decades at the tissue, cellular, subcellular and, more recently, molecular levels. Balamurugan & Said [2] have reported that the intestinal folate absorption process is ontogenically regulated. This was observed to involve the initial step in intestinal folate uptake, i.e. transport across the BBM, and it is mediated though a decrease in the Vmax of the folate uptake process [40]. Parallel declines in intestinal RFC protein and mRNA levels and in the rate of RFC transcription were also observed with maturation, indicating the involvement of a transcriptional mechanism in the ontogenic regulation of the intestinal folate uptake process.

Subsequent studies have shown that the characteristics displayed by RFC depend on the cell context, and this could be attributed to differences in membrane composition, cell-specific post-translational modifications, and/or involvement of cell-specific accessory proteins that modulate RFC activity in the different cell types [41]. Moreover, the intensity of the RFC message along the vertical axis of the intestine was shown to be significantly higher in mature epithelial cells of the villus tip than in the immature epithelial cells of the crypt [19,42]. Mutations in human RFC have been reported in two siblings with folate malabsorption syndrome [43]. These patients were found to respond favorably to oral pharmacological (but not physiological) doses of folinic acid [44]. This inhibition of folate uptake was found to be the result of malfunctioning of the folate uptake system itself, and not impairment in the expression of human RFC at the cell membrane [43].

Folate uptake in the native intestine is considerably higher at acidic buffered pH than at neutral or alkaline pH [22,36].There are two schools of thought regarding the mechanism of folate absorption at low pH. One school proposed that RFC is responsible for folate transport in different tissues. It was suggested that low-pH transport activity of RFC is due to cell-specific or tissue specific post-translational modification of RFC, which leads to differences in the pH optimum and structural specificity [43,45]. Studies have found that transfection of RFC in rat intestinal IEC-6 cells increases folate transport at pH 5.5 without causing any increase in uptake at pH 7.4 [43]. Moreover, the observed increase in expression of RFC at acidic pH in intestinal BBMs was associated with an increase in folate uptake in rodents fed a folate-deficient diet [2,46]. On the other hand, it was recently proposed that low-pH folate transport activity in the intestine must be accounted for by genetically distinct transporter, PCFT [20]. The studies have found that PCFT has similar affinities for reduced and oxidized folates, in contrast to RFC, which shows different affinities for oxidized and reduced folates [47]. PCFT was also ubiquitously expressed, and there was an increase in the expression of PCFT in rodents fed a folate-deficient diet [48]. Thus, a better understanding of the regulation of folate transport by RFC and PCFT should provide new insights into the regulation of folate transport.

Studies on the regulation of intestinal folate absorption by intracellular protein kinase-mediated pathways have also been attempted [46,49]. The consensus sequences for the phosphorylation sites of protein kinase C and protein kinase A (PKA) have been shown to exist in folate transporters [19,50]. Studies with cultured IEC-6 epithelial cells [49] and the human colonic epithelial cell line NCM460 [51] suggested that folate transport is regulated through protein tyrosine kinase and cAMP-mediated pathways, but the mechanisms through which these pathways exert their regulatory effects on intestinal folate uptake under physiological conditions and in various pathological conditions are not clear. With the use of various modulators (Fig. 3) of the signaling pathways, folate transport in viable intestinal epithelial cells isolated from the rat intestine was found to be under the regulation of cAMP-dependent PKA [52].

image

Figure 3.  Effect of positive and negative modulators of signaling pathways involving PKA and PKC on [3H]folic acid (0.5 μm) uptake in viable isolated intestinal epithelial cells. The isolated cells were incubated for 30 min the in presence of different modulators before uptake studies. The PK inhibitor is an inhibitor of cAMP-dependent protein kinases, atropine and dibutyryl cAMP are an inhibitor and an activator, respectively, of cAMP levels, and chelerythrine chloride and phorbol-12-myristate-13-acetate are an inhibitor and an activator, respectively, of PKC. Each data point is the mean ± standard deviation of 12 independent observations. ***P < 0.001 versus control; #< 0.001, ###P < 0.001 versus none.

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Using purified colonic apical membrane vesicles isolated from the colon of human organ donors and human-derived, nontransformed colonic epithelial NCM460 cells, recent studies have shown that folate uptake in the colon is efficient and occurs via a specific, carrier-mediated, pH-dependent, anion-sensitive and electroneutral mechanism similar to that found in the small intestine [14,51]. The identification of an efficient carrier-mediated mechanism for folate uptake in the human colon further suggests that this source of folate may contribute to the body’s folate homeostasis, or to cellular homeostasis of the local colonocytes [53]. Such findings may also lead to a better understanding of the causes of localized folate deficiency described in colonic epithelia that are believed to be associated with premalignant changes in the colonic mucosa. The kidneys also play an important role in the regulation of folate homeostasis through the uptake of folates across their membranes [28]. The renal uptake of folate involves glomerular filtration followed by tubular reabsorption, preventing urinary excretion of folate from the body. In the kidneys, the folate uptake mechanism involves the binding of folate to FBP localized to BBMs [54,55] and transport by means of RFC, which was found to be more concentrated in BLMs [56]. Moreover PCFT has also been found to play an important role in the transport of folate in the kidneys [57].

Similar systems are involved in the transmembrane movement of folates within the choroid plexus, placenta, and various subcellular organelles, such as lysosomes and mitochondria [58]. Studies have shown the operation of a carrier-mediated system within the mitochondrial membrane for internalizing 5-formyltetrahydrofolate. After internalization, this folate is required for the metabolism of glycine in this organelle. This system is acid pH-dependent, and exhibits relatively high saturability (Km 2–8 μm) for 5-substituted tetrahydrofolates but less so for folic acid and methotrexate [59]. The localization of human RFC to the mitochondrial membrane is a novel finding, and it suggests a role for the mitochondrial membrane in the transport of folates [60]. In addition, similar systems for folate transport have been found in plasma membrane systems identified in nonabsorptive normal cells, i.e. fibroblasts, erythroid cells [6], and hepatocytes [61]. Inward transport of folate compounds occurs in fibroblasts and erythroid cells in rabbit and human erythrocytes by a system that is similar to the one-carbon, reduced-folate system in that it exhibits the same relative preferences among folate compounds. Interestingly, RFC was also detected in plasma membranes on the apical surface of axons and dendrites [62] and on the apical membranes of cells lining the spinal canal [63].

Thus, the overwhelming evidence is that RFC is ubiquitously expressed in tissues. Intricate mechanisms for controlling patterns of RFC expression and function in response to diverse tissue environments have been suggested [64]; however, their evaluation in nonabsorptive tissues is only just beginning, and demands extensive research. Moreover, much of the folate is bound tightly to enzymes involved in its use, indicating that there is not an excess of this cofactor and that its cellular availability is protected as well as being strictly regulated [65].

Structural features of folate transporters

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

RFC

RFC is a typical member of the major facilitator superfamily located on human chromosome 21 (21q22.2–q22.3). The human RFC gene is ubiquitously and differentially expressed in normal human tissues [66]. Multiple variants of human RFC (driven by multiple promoters) have been identified, with variant I being the prominent form expressed in the intestine [67]. Transcript heterogeneity of human RFC results from the use of multiple promoters and variable splicing of alternative upstream exons [40,68,69]. The GC-rich 5′-flanking regions of the genomic DNA upstream of the alternative exon 1a contain many putative transcription factor-binding sites for transcriptionally involved proteins such as MZF1, AP1, AP2, and SP1. In addition, the 5′-flanking region of exon 1b contains a direct repeat sequence [69–71].

RFC is a member of the solute carrier 19 (SLC19) family of transporters [72]. Among the SLC19 family, SLC19A1 transports folates; it was first cloned in 1994, and is now referred to as RFC. Besides RFC, no other member of the SLC19 family has been identified that transports folates, and other than some members of the organic anion family (e.g. solute carrier 21), no other facilitative carriers have been confirmed to transport folates in mammalian cells [73].

PCFT

PCFT is localized to chromosomes 17, 11 and 10 in humans, mouse and rat respectively. The gene consists of five exons; the coding region of exon 5 is very short. On the basis of hydropathy analysis, there are 12 transmembrane domains (TMDs), with a large loop separating six of these domains, directed, along with the N-terminus and C-terminus, to the cytoplasm. The large predicted external loop between the first and second TMDs contains two predicted glycosylation sites. The other loops between the TMDs are quite short [74]. Human PCFT shares 91% similarity and 87% identity with its mouse and rat counterparts, respectively, and rat PCFT has 459 amino acids, 11 putative membrane-spanning domains, and a molecular mass of 50 kDa [48].

Folate transport by PCFT shows pH-dependent, sodium-independent characteristics, with higher affinity for 5-methyl-THF [20]. Beyond its role in mediating intestinal folate absorption, PCFT may also play role in folate receptor (FR)-mediated endocytosis [74]. In this process, folates bind to FRs on the cell surface, which are then internalized in endocytic vesicles. Upon acidification of vesicles within the cytoplasm, folates are released from the receptors and exit the vesicles. The mechanism of export from the vesicles has not been clarified; PCFT, if present in the vesicle membrane, may represent the mechanism of folate export driven by the very high transvesicular pH gradient [74]. As well as in the intestine, PCFT mRNA expression is prominent in the liver, an organ that possesses and stores a large amount of folate, along with the kidney, colon, spleen, and placenta [48], confirming its role in folate homeostasis.

FRs or FBPs

The term FR refers to a family of high-affinity FBPs encoded by three distinct genes, designated α, β, and γ, localized to chromosome 11q13.3–q13.5 [75]. The mammalian FR or FBP is a single polypeptide that binds folic acid with a relatively high affinity (Km ∼ 1 nm) and with a stoichiometry of 1 : 1 [76]. In addition, the receptor also binds the major circulating folate coenzyme, 5-methyl-THF and various antifolate drugs. Multiple isoforms of FR have been identified from human (hFRα, hFRβ and hFRγ) and murine (mFRα and mFRβ) sources, and they share amino acid sequence identities of 68–79%, including 16 cysteine residues, which are all conserved [77]. Among FRα, FRβ, and FRγ, FRα and FRβ are glycosylphosphatidylinositol-anchored proteins, whereas FRγ is secretory [78]. Glycosylphosphatidylinositol-linked membrane FRs have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs [79].

Mechanistically, folate uptake into the cytosol by membrane-bound FR is believed to involve an endocytotic process [80,81]. A recent study showed that megalin, a large, multiligand endocytic receptor and a member of the low-density lipoprotein receptor family, is able to bind and mediate cellular uptake of FR [82]. However, on the basis of studies in MA104 monkey kidney epithelial cells, another uptake process, termed potocytosis, was proposed [83]. This involves the recycling of clusters of FRs in membrane-associated vesicular structures, termed caveolae, between the cell surface and acidic endocytic compartments, with release of ligand into the cytosol [77].

OATs

In the early 1990s, members of a new family of facilitative carriers (solute carrier 21) began to be cloned that transport organic anions in epithelial tissues. Many of these carriers transport folates. Rat OAT-K1, expressed in liver and kidney, transports methotrexate (MTX) with higher affinity than for a variety of other organic anions, including other folates, bromosulfophthalein, taurocholate, and probenecid [84]. Rat OAT-K2, expressed in kidney tubules, also transports MTX and folic acid with apparently comparable affinities, but with a lower affinity than for taurocholate [85]. The OAT-K2 cDNA had an ORF encoding a 498 amino acid protein (calculated molecular mass of 55 kDa) that shows 91% identity with the rat kidney-specific OAT-K1 [86]. Both transporters are localized to the apical BBM of renal tubular epithelial cells and appear to be involved in the reabsorption of folates from the glomerular filtrate [7]. Human OAT2, which is within the OAT1–5 group, is expressed in liver and kidney, and transports MTX and a spectrum of other organic anions. Human OAT3 protein is expressed primarily in the kidney (basolateral membrane), and transports MTX (sodium-independent). Hence, these OATs, along with others not yet identified or fully characterized, probably play an important role in the transport of folates in the kidney, biliary tract, and some tumors [86].

Influence of alcoholism on the membrane transport and bioavailability of folate

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Folate deficiencies can occur for many reasons, including reduced folate intake, increased metabolism and/or increased requirements, malabsorption, and genetic defects [87]. Congenital errors in folate metabolism can be related either to defective transport of folate through various cells, or to defective intracellular utilization of folate, because of some enzyme deficiencies [88]. Folate malabsorption can be at the level of impaired intestinal absorption and renal tubular reabsorption. Such impairments occur in a variety of conditions, including chronic ethanol consumption, intestinal diseases, and renal malfunction.

Ethanol must be considered as one of the most important toxins consumed regularly and in large quantities by humans [89]. Since ethanol intake has steadily increased during the last two decades, alcoholism has become one of the major health problems worldwide. Among the various other ethanol-associated disorders, folate deficiency is commonly associated with chronic alcoholism, because the processes involved in the absorption, transport and intracellular metabolism of this cofactor are complex and are quite susceptible to the cellular microenvironment [90]. Niacin, thiamine and pyridoxine (vitamin B6) deficiencies have also been confirmed in alcoholics, but ethanol-induced folate deficiency has been extensively studied.

It is clear from the available literature that ethanol-induced folate deficiency is caused, in part, by intestinal malabsorption of folate [91]. Three decades ago, Halsted et al. [92,93] demonstrated that individuals who abuse ethanol on a regular basis malabsorb folate. The absorption of folic acid by the jejunum and cecum of ethanol-fed rats was found to be decreased [94]. There was a significant decrease in folate absorption and hepatic folate content in alcoholic monkeys [95,96]. In vitro studies on the effect of ethanol on the absorption of folate in the rat proximal jejunum suggested that the malabsorption of folate could be related to the effect of ethanol on the acid microclimate [91]. However, there is no in vivo study to support such a finding. The reduced intestinal transport of folate in a chronic alcoholism model was found to be associated with a reduction in folic acid binding to intestinal BBM, with a significant alteration in Bmax, SH group status, and divalent and monovalent cation dependency [97]. The deregulated kinetic characteristics of the folate uptake process (Table 1) might contribute to intestinal folate malabsorption during alcoholism [98]. The increased Km and lower Vmax values of transport activity were associated with lower uptake capability corresponding to villus tip, mid-villus and crypt base cells across the vertical axis of the intestine during alcoholism [99]. Moreover decreased RFC mRNA levels were observed in the primary absorptive surface of jejunal tissue (Fig. 4), with parallel changes in RFC protein levels at the brush border as well as at the BLM all across the crypt–villus axis (Figs 4 and 5) [42]. These changes in functional activity of the membrane transport system could not be related to the general loss of intestinal architecture, but has been attributed to the specific effect of ethanol ingestion on the folate transport system.

Table 1.   The Km and Vmax values of folate transport across intestinal and renal absorptive surfaces in control and ethanol-fed rats. Units for Km and Vmax are μm and pmol/30 s·mg−1 protein, respectively. Rats were fed ethanol at 1 g·kg−1 body weight for 3 months. The uptake of [3H]folic acid was studied at different substrate concentrations (0.125–2.0 μm), and the kinetic constants were determined by Lineweaver–Burke plots. Each data point is the mean ± standard deviation of three separate uptake determinations carried out in duplicate.
TissuePreparationsKinetic constantsControlEthanol
  1. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

IntestineBBMKm Vmax0.90 ± 0.08 100 ± 5.601.53 ± 0.09** 83 ± 3.65*
BLMKm Vmax1.42 ± 0.13 250 ± 20.52.0 ± 0.21** 167 ± 15.75***
KidneyBBMKm Vmax0.33 ± 0.03 143 ± 5.10.50 ± 0.02** 125 ± 3.9**
BLMKm Vmax0.07 ± 0.001 50 ± 5.30.14 ± 0.002*** 20 ± 2.6***
image

Figure 4.  (A, B) Western blot analysis of (A) intestinal BBMVs (lanes 1 and 2) and BLMVs (lanes 3 and 4), and (B) renal BBMVs (lanes 1 and 2) and BLMVs (lanes 3 and 4), respectively, using antibody against RFC (65 kDa). Lanes 1 and 3, control; lanes 2 and 4, ethanol. (C, D) RT-PCR analysis of RFC (489 bp) and β-actin (588 bp) in the intestine (lanes 1 and 2 control; lanes 3–5, ethanol) (C) and the kidney (lanes 1–3, control; lanes 4 and 5 ethanol) (D). (E) RT-PCR analysis of FBP (370 bp) and β-actin (588 bp) in the kidney (lanes 1–3, control; lanes 4 and 5 ethanol).

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image

Figure 5.  Immunohistochemical analysis of rat jejunal sections exposed to antibodies against RFC, showing the relative localization and distribution pattern of RFC protein (as depicted by brown counterstaining of hematoxylin) along the intestinal absorptive axis. The figures (× 450) shown are representative of each group: (A) control; (B) ethanol.

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In contrast to the BBM surface, the neutral carrier-mediated saturable folate transport at the basolateral surface showed sodium and ATP independence. Ethanol ingestion results in potassium and ATP dependence, besides affecting the status of S–S linkage of the folate transport system. Importantly, chronic ethanol ingestion reduced folate exit across the basolateral surface by a mechanism involving decreased affinity of RFC for folate and a decrease in the number of transporter molecules on the surface [42]. Another factor contributing to the lower bioavailability of dietary folate that accompanies ethanol ingestion is increased renal excretion. Both acute (4 h) and chronic (12 weeks) ethanol ingestion seem to increase the loss of folate in the urine [100]. Our recent findings showed that ethanol exerts its effect by altering the binding and kinetic characteristics (Table 1) of folate transport at both the BBM and the BLM of the kidney [98]. In addition to this, the downregulation of RFC and FBP genes (Fig. 4) in the renal absorptive and conservative surfaces, respectively, along with that of RFC in the intestine might play a role in the observed reduced folate transport efficiency in the kidney and intestine during alcoholism [98], and result in low red blood cell (RBC) and serum folate levels. The role of PCFT and OATs in chronic ethanol-induced reduced folate transport in the absorptive epithelia has not been explored as yet.

In addition, a significant decrease in folate uptake in intestinal epithelial cells in the presence of inhibitors of cAMP-dependent protein kinase implied a role of protein kinase in the efficiency of the transport process (Fig. 3). The best defined target of cAMP is PKA, which in turn mediates most of the physiological effects of cAMP in eukaryotes. Moreover, dibutyryl cAMP, a compound that increases the intracellular cAMP levels, resulted in a significant increase in folic acid uptake, with 2.0-fold higher increase in the ethanol-fed group than in the control group. This suggested that cAMP may be an important regulatory component of signaling cascade that is affected in chronic alcoholism [52]. Moreover, atropine, which causes decreased intracellular cAMP levels by inhibiting the activity of adenylyl cyclase, inhibited folic acid transport in both groups of rats, but the decrease was less (51%) in the case of alcoholic rats (Fig. 3) [52]. Importantly, many groups have demonstrated, in models of chronic alcoholism, that both the initial deconjugation and the subsequent transport of monoglutamic folate are impaired in alcoholic individuals [101]. In alcoholic pigs, loss of deconjugation activity at the brush border was observed [102,103], as was the partial loss of both FBPs that coordinately affect the transmembrane transport of folate [104]. Current data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account, in part, for tissue-specific differences in folate accumulation [13].

Ethanol and folate in epigenome stability and cancer development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Epigenetics can be defined as a stable alteration in gene expression potential without any change in gene sequence that takes place during development and cell proliferation [105]. Epigenetic mechanisms include DNA methylation, modification of histones and incorporation of histone variants, and ATP-dependent chromatin remodeling, execution of RNA interference and nonprotein-coding RNAs. DNA methylation is a crucial epigenetic modification of the genome that is involved in regulating many cellular processes. Biochemically, DNA methylation is a covalent chemical modification of the fifth carbon position of the pyrimidine ring of cytosines in CpG dinucleotides. As DNA is composed of four bases, there are 16 possible dinucleotide combinations, and therefore the CpG dinucleotides should occur with a frequency of approximately 6% [106]. However, the actual frequency is only 5–10% of that predicted. This CpG suppression may be related to the hypermutability of methylated cytosine. Such a process causes C[RIGHTWARDS ARROW]G to T[RIGHTWARDS ARROW]A transitions, and results in strong suppression of the CpG methyl acceptor site in human DNA. However, CpG islands, which are regions of more than 500 bp with a high GC content, occurring on average every 100 kb, have been conserved during evolution, because they are normally kept free of methylation [107]. These stretches of DNA are located within the promoter or first exon of about 40% of mammalian genes, and when methylated they cause stable heritable transcriptional silencing [108]. Importantly, aberrant methylation of CpG islands is a hallmark of a vast number of human diseases, including cancer and immune system dysregulation [109,110].

Excessive ethanol intake has a multifaceted impact on the bioavailability and subsequent metabolism of folate and, more broadly, on one-carbon metabolism as a whole [102]. The significant decrease in folate levels can explain the observed jejunal genomic hypomethylation observed in these studies [111]. Moreover, decreases in RBC and serum folate levels (Table 2), which are indicators of folate status, were also observed in such studies [111]. In addition to its effect on one-carbon metabolism and DNA methylation due to diminished folate levels, several mechanisms have been suggested by which ethanol could directly interact with one-carbon metabolism and DNA methylation [112]. Chronic ethanol consumption: (a) affects the intake, absorption and subsequent metabolism of B vitamins involved in hepatic transmethylation reactions, namely folate and pyridoxal 5-phosphate (vitamin B6), resulting in impaired methyl group synthesis and transfer [113]; (b) reduces the activity of methionine synthetase, which remethylates homocysteine to methionine with methyltetrahydrofolate as the methyl donor [114,115]; (c) decreases the levels of glutathione, a reductive tripeptide, which is synthesized from homocysteine via trans-sulfuration in the liver, and thereby enhances the susceptibility of the liver to ethanol-related peroxidative damage [115,116]; and (d) inhibits the activity of DNA methylase, which transfers methyl groups to DNA in rats [117].

Table 2.   RBC and serum folate levels in control and ethanol-fed rats. Rats were fed ethanol at 1 g·kg−1 body weight for 3 months. Folate estimations in RBC and serum were determined by microtiter plate assay using Lactobacillus casei. Values are means ± standard deviation (n = 8).
GroupFolate (μg·L−1)
RBCSerum
  1. ***P < 0.001 versus control.

Control950 ± 29.8449.64 ± 5.29
Ethanol-fed624 ± 49.73***33.71 ± 4.95***

Importantly, modifications of the degree of hepatic and intestinal (Fig. 6) DNA methylation have also been observed in experimental models of chronic alcoholism [42,118]. In fact, ethanol has a marked impact on methylation capacity, as reflected by decreased levels of SAM, an important methyl group donor, and increased levels of S-adenosylhomocysteine (SAH), resulting in a decrease in the SAM/SAH ratio [113]. In a rodent model of alcoholism, several weeks of ethanol ingestion led to a significant reduction in tissue SAM, an increase in SAH, a halving of the SAM/SAH ratio, and a substantial degree of genomic DNA hypomethylation in the colonic mucosa [118,119]. Recent studies have found that smoking and ethanol have strong influences on the methylation levels of distinct genes in lung cancer [120]. Other studies have found that ethanol causes different site-specific methylation and acetylation of histones; for example, ethanol caused an increase in H3K9 acetylation, but decreased the methylation at the same lysine residue. Concurrently, methylation at H3 lysine 4 increased. Through a series of chromatin immunoprecipitation experiments, it became clear that H3K4 methylation by ethanol was associated with upregulation of genes (e.g. the Adh and glutathione S-transferase-yc2 genes), whereas methylation at H3K9 was associated with downregulation of genes (the l-serine dehydratase and cytP450 2c11 genes) [121]. Although hypermethylation would seem to be a paradoxical effect of factors that diminish the capacity for biological methylation, there is evidence that folate inadequacy may also have such an effect and that it may act in concert with excess ethanol ingestion. In an epidemiological examination of human colorectal cancers, hypermethylation of a battery of relevant tumor suppressor and DNA repair genes was shown to be related to diets that are low in folate and high in ethanol [107,122]. It is well established that dietary depletion of lipotropes, including methionine, choline, betaine, SAM, and folate, leads to hypomethylation of oncogenes such as c-Ha-ras, c-Ki-ras and c-fos and to DNA strand breaks, all of which are associated with an increased incidence of cancers in rats [123,124]. In an experimental model of folate deficiency, there was an increase in the SAH/SAM ratio in the colon, associated with genomic DNA hypomethylation (Figs 6 and 7), which may be a risk factor for colorectal cancer. Furthermore, low-methionine, low-folate diets and ethanol increase the risk for colorectal cancer in men [125,126]. Low folate levels have been found to increase p53 exon strand breaks, and induce changes in the wnt–APC pathway and in genes involved in cell adhesion, migration and invasion, which may underlie the observed relationship between folate status and cancer risk [127]. Importantly, changes in the degree of methylation of cytosine are frequently encountered in human cancers, but their relevance as epigenetic factors in carcinogenesis is only partially understood [128]. Recent reports suggested that the perturbed one-carbon transfer reaction associated with folate depletion resulted in transformation of normal colonic epithelial to neoplastic cells [129]. As well as this, a diet deficient in folate and choline increased the expression of insulin-like growth factor Hi9, leading to prostate cancer [130].

image

Figure 6.  [3H]SAM incorporated (μm·μg−1 DNA) as an index of (A) jejunal genomic DNA methylation in control and ethanol-fed rats and (B) colonic genomic DNA methylation in control and rats fed a folate-deficient diet. The [3H]methyl incorporation is inversely proportional to the endogenous unmethylated cytosines in DNA. Values are means ± standard deviations (n = 8). ***P < 0.001 versus control.

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image

Figure 7.  Colonic (SAM and SAH) levels in the control and folate-deficient groups. The levels in tissue were determined by using HPLC. Values are means ± standard deviations (n = 8). ***P < 0.001 versus control.

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The blocking of the methionine synthase reaction, as observed in vitamin B12 deficiency or in cases of chronic ethanol ingestion, results in the continuous conversion of 5,10-CH2-THF to 5-methyl-THF, and causes an increasing proportion of cellular folate to be locked in a metabolically unavailable form. This is the basis of the methylfolate trap that occurs in vitamin B12 deficiency, producing a functional folate deficiency [131]. The loss of methylenetetrahydrofolates, methenyltetrahydrofolates, and formyltetrahydrofolates, which occurs when a methylfolate trap develops, deprives the reactions that are responsible for purine and de novo thymidine synthesis of their requisite coenzymes, promoting defective DNA synthesis and repair. Moreover, the precursor of thymidine, i.e. deoxyuridine, becomes incorporated into DNA at those loci where the former should be located. Attempts to repair this defective DNA leave breaks in the phosphodiester backbone of DNA, which are procarcinogenic in nature [132,133]. In the alcoholic pig model, alcoholism led to a substantial increase in DNA strand breaks in the liver, and this effect was increased further by the superimposition of folate depletion [102].

Furthermore, epidemiological observations implicate excess ethanol ingestion as well as low dietary folate intake as risk factors for several cancers of the oral cavity, pharynx, larynx, esophagus, liver, colon, rectum, and breast [126,134,135], and an association is suspected for cancers of the pancreas and lung [119,136]. Moreover, the epidemiological observations also support the concept of a synergistic effect between these two factors, i.e. alcohol and low folate intake [137,138]. Such a relationship is biologically plausible, because ethanol lowers the bioavailability of dietary folate and is known to inhibit selected folate-dependent biochemical reactions [117]. Furthermore, SAM is an allosteric inhibitor of the MTHFR reaction, and in situations in which SAM is reduced (e.g. in chronic ethanol ingestion), the loss of inhibition amplifies the accumulation of folate in the form of 5-methyl-THF. Thus, excessive ethanol intake replicates some of the biochemical features of vitamin B12 deficiency. In addition to its inhibitory effect on methionine synthase, ethanol has been observed to diminish the transcription of several other enzymes central to one-carbon metabolism, such as MTHFR, methionine adenosyltransferase 1A, glycine N-methyltransferase, and SAH hydrolase, as well as their enzyme activity [90,139]. Another pathway exists for the remethylation of homocysteine that is neither folate-dependent nor vitamin B12-dependent nor susceptible to inhibition by ethanol, i.e. betaine–homocysteine methyltransferase (BHMT). BHMT catalyzes the transfer of a methyl group from betaine (trimethylglycine) to homocysteine, resulting in the synthesis of dimethylglycine and methionine [140,141]. The intestinal mucosa contains methionine synthase activity, but intestinal homocysteine remethylation capacity is restricted as compared with the liver and kidney, because of the absence of BHMT [114]. This limitation makes the intestine more susceptible than the liver and kidney to the homocysteine-elevating effects of folate depletion in diverse pathological conditions, such as chronic alcoholism [117].

Concluding remarks and future prospectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References

Taken together, such studies demonstrate that folate homeostasis in an organism is associated with diverse cellular phenomena and is tightly regulated. However, perturbances in folate transport regulation, such as occur during alcoholism, might give rise to an array of pathological conditions in a cell or an organism. Ethanol ingestion leads to a state of folate deficiency, which occurs through its effects on the enzymes involved in folate metabolism and the kinetics of folate uptake in absorptive epithelia, including those of the intestine and kidney. Moreover, the decreased folate uptake was also associated with downregulation of RFC and FBP, suggesting a role of transcriptional and translational regulation of folate transport systems in chronic alcoholism. Currently, there is no information on the regulation of folate transport in the colon (which synthesizes some folate), the liver (the largest storage area in terms of folate content) and the pancreas (the second largest storage area after the liver) during chronic alcoholism. Such information will provide additional insights into whether ethanol exerts its effects on depletion of these folate stores and hence contributes to folate deficiency. Also, folate deficiency may affect gene expression by disturbing DNA methylation patterns or by inducing base substitutions, DNA breaks, and gene amplification. In addition to the role of ethanol in modulating methylation of DNA through its association with folate deficiency, ethanol could directly exert its effect on DNA methylation, by reducing the synthesis of SAM, reducing the activity of methionine synthase, decreasing glutathione levels, and inhibiting the activity of DNA methylase, the important determinants of epigenetic regulation. Thus, ethanol ingestion, either directly or through its association with folate deficiency, modulates DNA methylation, an important epigenetic determinant in gene expression, and in the maintenance of DNA integrity and chromosomal stability, which are implicated in the development of cancer. The mechanisms of folate transport and regulation during malabsorption hold great scope for its evaluation. Such knowledge could ultimately help in the design of effective strategies to optimize the normal body homeostasis of folate, especially in cases of its deficiency and suboptimal levels. Also, there is great potential for the study of epigenetic instability associated with alcoholism and folate deficiency to help with the development of epigenetic therapies such as novel demethylating agents, such as antisense and small interfering RNA, to reverse such effects.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Folate homeostasis
  5. Folate cycle
  6. Folic acid absorption and transport – features and mechanisms
  7. Structural features of folate transporters
  8. Influence of alcoholism on the membrane transport and bioavailability of folate
  9. Ethanol and folate in epigenome stability and cancer development
  10. Concluding remarks and future prospectives
  11. Acknowledgements
  12. References
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