In vivo 19F NMR chemical-shift imaging of Ancistrocladus species

Summary ¹⁹F nuclear magnetic resonance (NMR) imaging and¹⁹F NMR chemical-shift imaging (¹⁹F CSI) have been used to localize fluorinated compounds administered to stems ofAncistrocladus heyneanus andA. abbreviatus for the elucidation of biosynthetic pathways in living plants. This first application of¹⁹F CSI on plants proved CSI to be a valuable technique for mapping fluorinated molecules in plants. Exemplarily using trifluoroacetate as a model compound allowed to select appropriate feeding methods and to optimize both concentration and duration of the application to the plant. The time course of the uptake and distribution of trifluoroacetate was monitored by both¹⁹F imaging and¹⁹F CSI. Fluorinated metabolites formed by uptake of 3-fluoro-3-deoxy-D-glucose were detected with¹⁹F CSI.Abbreviations 3-FDG 3-fluoro-3-deoxy-D-glucose - CSI chemicalshift imaging - NMR nuclear magnetic resonance - SNR signal-to-noise ratio - TFA trifluoroacetate Dedicated to Professor Manfred Christi on the occasion of his 60th birthday     

In vivo metabolism of 3-deoxy-3-fluoro-D-glucose

Recent studies have demonstrated that 3-deoxy-3-fluoro-D-glucose (3-FG) is metabolized to 3-deoxy-3-fluoro-D-sorbitol (3-FS), via aldose reductase, and 3-deoxy-3-fluoro-D-fructose (3-FF), via the sorbitol dehydrogenase reaction with 3-FS, in rat cerebral tissue (Kwee, I. L., Nakada, T., and Card, P. J. (1987) J. Neurochem. 49, 428-433). However, the biochemistry of 3-FG in other mammalian organs has not been investigated making the application of 3-FG as a metabolic tracer uncertain. To address this issue we investigated 3-FG metabolism and distribution in isolated cell lines and in rabbit tissues in vivo with 19F NMR and gas chromatography-mass spectrometry. In general, the production of 3-FS is well correlated with the known distribution of aldose reductase in all the systems studied. Further metabolism of 3-FS to 3-FF was verified to occur in cerebral tissue. Surprisingly, two new fluorinated compounds were found in the liver and kidney cortex. These compounds are identified as 3-deoxy-3-fluoro-D-gluconic acid, which is produced via glucose dehydrogenase activity on 3-FG, and 3-deoxy-3-fluoro-D-gluconate-6-phosphate. Based on enzyme studies, it is argued that the 3-deoxy-3-fluoro-D-gluconate-6-phosphate is derived directly from 3-deoxy-3-fluoro-D-gluconic acid and not as a product of pentose phosphate activity. Direct oxidation and reduction are the major metabolic routes of 3-FG, not metabolism through glycolysis or the pentose phosphate shunt. Thus, 3-FG metabolism coupled with 19F NMR appears to be very useful for monitoring aldose reductase and glucose dehydrogenase activity in vivo.     

19F NMR quantitation of lens aldose reductase activity using 3-deoxy-3-fluoro-D-glucose

In the present study we have determined the kinetics of 3-deoxy-3-fluoro-D-glucose (3-FG) as a substrate for the aldose reductase reaction in vitro. In addition, we compared the 3-deoxy-3-fluoro-sorbitol (3-FS) production rates from 3-FG in the intact lens using 19F NMR with conventional aldose reductase determinations in extracts from the same lenses. The affinity of in vitro aldose reductase for 3-FG was approximately 20 times greater (9.3 mM) than that for glucose (188 mM). An excellent correlation between the rate of 3-FS production in the intact canine lens, determined with 19F NMR, and extracted aldose reductase activity was observed. The relatively high affinity of aldose reductase for 3-FG and the correlation of 3-FS production with enzyme activity in the intact lens suggests that 3-FS production from 3-FG detected by 19F NMR could provide an accurate noninvasive determination of aldose reductase activity in the eye lens.     

Noninvasive In Vivo Demonstration of 2-Fluoro-2-Deoxy-d-Glucose Metabolism Beyond the Hexokinase Reaction in Rat Brain by 19F Nuclear Magnetic Resonance Spectroscopy

The metabolism of 2-fluoro-2-deoxy-D-glucose (FDG) in vivo was observed noninvasively in rat brain using 19F nuclear magnetic resonance (NMR) spectroscopy following an intravenous injection of FDG (400 mg/kg). At 3 h after infusion, four resonances with discrete chemical shifts were resolved. Chemical shift analysis of these resonances suggested the chemical identity of two of the resonances to be FDG and/or FDG-6-phosphate and 2-fluoro-2-deoxy-delta-phosphogluconolactone and/or 2-fluoro-2-deoxy-6-phosphogluconate. The chemical identities of the other two resonances remain to be elucidated. The present study indicates that the metabolism of FDG in vivo is more extensive than is previously recognized and demonstrates the feasibility of using 19F NMR spectroscopy to follow the 19F-containing metabolites of FDG in vivo.     

Metabolism of Hydroxylated and Fluorinated Benzoates by Syntrophus aciditrophicus and Detection of a Fluorodiene Metabolite

Transformations of 2-hydroxybenzoate and fluorobenzoate isomers were investigated in the strictly anaerobic Syntrophus aciditrophicus to gain insight into the initial steps of the metabolism of aromatic acids. 2-Hydroxybenzoate was metabolized to methane and acetate by S. aciditrophicus and Methanospirillum hungatei cocultures and reduced to cyclohexane carboxylate by pure cultures of S. aciditrophicus when grown in the presence of crotonate. Under both conditions, transient accumulation of benzoate but not phenol was observed, indicating that dehydroxylation occurred prior to ring reduction. Pure cultures of S. aciditrophicus reductively dehalogenated 3-fluorobenzoate with the stoichiometric accumulation of benzoate and fluorine. 3-Fluorobenzoate-degrading cultures produced a metabolite that had a fragmentation pattern almost identical to that of the trimethylsilyl (TMS) derivative of 3-fluorobenzoate but with a mass increase of 2 units. When cells were incubated with deuterated water, this metabolite had a mass increase of 3 or 4 units relative to the TMS derivative of 3-fluorobenzoate. ¹⁹F nuclear magnetic resonance spectroscopy (¹⁹F NMR) detected a metabolite in fluorobenzoate-degrading cultures with two double bonds, either 1-carboxyl-3-fluoro-2,6-cyclohexadiene or 1-carboxyl-3-fluoro-3,6-cyclohexadiene. The mass spectral and NMR data are consistent with the addition of two hydrogen or deuterium atoms to 3-fluorobenzoate, forming a 3-fluorocyclohexadiene metabolite. The production of a diene metabolite provides evidence that S. aciditrophicus contains dearomatizing reductase that uses two electrons to dearomatize the aromatic ring.     

Direct F NMR observation of the conformational selection of optically active rotamers of the antifolate compound fluoronitropyrimethamine bound to the enzyme dihydrofolate reductase

The molecular basis of the binding of the lipophilic antifolate compound fluoronitropyrimethamine [2,4-diamino-5-(4-fluoro-3-nitrophenyl)-6-ethylpyrimidine] to its target enzyme dihydrofolate reductase has been investigated using a combination of ¹⁹F NMR spectroscopy and molecular mechanical calculations. ¹⁹F NMR reveals the presence of two different conformational states for the fluoronitropyrimethamine-Lactobacillus casei enzyme complex. MM2 molecular mechanical calculations predict restricted rotation about the C5-C1′ bond of the ligand and this gives rise to two slowly interconverting rotamers which are an enantiomeric pair. The results of ¹⁹F NMR spectroscopy reveal that both these isomers bind to the enzyme, with different affinities. There is no detectable interconversion of the bound rotamers themselves on the NMR timescale. The effect of the addition of co-enzyme to the sample is to reverse the preference the enzyme has for each rotamer.     

Aldose reductase regulates miR-200a-3p/141-3p to coordinate Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling in renal mesangial cells and the renal cortex of diabetic mice

Aberrant regulation in oxidative stress, fibrogenesis, and the epithelial–mesenchymal transition (EMT) in renal cells under hyperglycemic conditions contributes significantly to the onset and progression of diabetic nephropathy. The mechanisms underlying these hyperglycemia-induced dysregulations, however, have not been clearly elucidated. Herein, we report that aldose reductase is capable of regulating the expression of miR-200a-3p/141-3p negatively in renal mesangial cells. MiR-200a-3p/141-3p, in turn, act to target Keap1, Tgfβ2, fibronectin, and Zeb2 directly and regulate Tgfβ1 and Nrf2 indirectly under high-glucose conditions, resulting in profound dysregulations in Keap1–Nrf2, Tgfβ1/2, and Zeb1/2 signaling. In vivo in streptozotocin-induced diabetic mice, we found that aldose reductase deficiency caused significant elevations in miR-200a-3p/141-3p in the renal cortex, which were accompanied by a significant downregulation of Keap1, Tgfβ1/2, and fibronectin but significant upregulation of Nrf2. Moreover, in vivo administration of inhibitors of miR-200a-3p in diabetic animals significantly exacerbated cortical and glomerular fibrogenesis and increased urinary albumin excretion, tightly linking dysregulated miR-200a-3p with the development of diabetic nephropathy. Collectively, our results reveal a novel mechanism whereby hyperglycemia induces aldose reductase to regulate renal expression of miR-200a-3p/141-3p to coordinately control hyperglycemia-induced renal oxidative stress, fibrogenesis, and the EMT. Our novel findings also suggest that inhibition of aldose reductase and in vivo renal cortical restoration of miR-200a-3p/141-3p or their combination are very promising avenues for the development of therapeutic strategies or drugs against diabetic nephropathy.     

Metabolism of the 2-oxoaldehyde methylglyoxal by aldose reductase and by glyoxalase-I: roles for glutathione in both enzymes and implications for diabetic complications

Numerous physiological aldehydes besides glucose are substrates of aldose reductase, the first enzyme of the polyol pathway which has been implicated in the etiology of diabetic complications. The 2-oxoaldehyde methylglyoxal is a preferred substrate of aldose reductase but is also the main physiological substrate of the glutathione-dependent glyoxalase system. Aldose reductase catalyzes the reduction of methylglyoxal efficiently (k_cat=142 min⁻¹ and k_cat/K_m=1.8×10⁷ M⁻¹ min⁻¹). In the presence of physiological concentrations of glutathione, methylglyoxal is significantly converted into the hemithioacetal, which is the actual substrate of glyoxalase-I. However, in the presence of glutathione, the efficiency of reduction of methylglyoxal, catalyzed by aldose reductase, also increases. In addition, the site of reduction switches from the aldehyde to the ketone carbonyl. Thus, glutathione converts aldose reductase from an aldehyde reductase to a ketone reductase with methylglyoxal as substrate. The relative importance of aldose reductase and glyoxalase-I in the metabolic disposal of methylglyoxal is highly dependent upon the concentration of glutathione, owing to the non-catalytic pre-enzymatic reaction between methylglyoxal and glutathione.     

α-Trifluoromethyl-β-Ureido-Propionic Acid (F3MUPA): A New Metabolite of Trifluridine (F3TdR)

Abstract

The metabolism of 5-trifluro-2′-deoxythymidine (trifluridine; F₃TdR) in male BALB/c mice bearing EMT-6 tumors has been investigated using ¹9F NMR spectroscopy. We previously (Tandon et al, 1992) reported the detection and identification of 5,6-dihydro-5-trifluorothymine (DHF₃T, 3) and 5,6-dihydroxy-5-trifluorothymine (DOHF₃T) as new metabolites of F₃TdR in mice urine. Further exploration of the metabolism of trifluridine has led to the identification of α-trifluoromethyl-β-ureido-propionic acid (F₃MUPA, 4) as a previously unreported metabolite in the urine of F₃TdR treated mice. Authentic F₃MUPA was obtained by synthesis via an established route. A comparison of chemical shift and the H-F coupling constant of an authentic sample, with the ¹⁹F signal from urine, indicated the presence of F₃MUPA in murine urine. Mixing crude urine with authentic F₃MUPA resulted in the enhancement of the corresponding fluorine signal without affecting or introducing others, thereby confirming the presence of F₃MUPA as a urinary metabolite.     

Synthesis and CNS Activity of Some Fluorine Containing 3-Indolylglyoxamides and Tryptamines

A number of new 5-/6-fluoro-2-substitutedaryl-3-indolylglyoxamides and their corresponding tryptamines have been synthesized. 5-/6-Fluoro-2-arylindoles, prepared by Fischer indole synthesis on treatment with oxalyl chloride and subsequent reaction with secondary amines, gave 5-/6-fluoro-2-aryl-3-indolylglyoxamides. The indolylglyoxamides were reduced with lithium aluminium hydride to yield corresponding tryptamines. All the synthesized compounds have been characterized by IR, PMR and ¹⁹F NMR spectral studies. CNS activity of some representative compounds has also been evaluated.     

Determination of glucose exchange rates and permeability of erythrocyte membrane in preeclampsia and subsequent oxidative stress-related protein damage using dynamic-<Superscript>19</Superscript>F-NMR

The cause of the pregnancy condition preeclampsia (PE) is thought to be endothelial dysfunction caused by oxidative stress. As abnormal glucose tolerance has also been associated with PE, we use a fluorinated-mimic of this metabolite to establish whether any oxidative damage to lipids and proteins in the erythrocyte membrane has increased cell membrane permeability. Data were acquired using ¹⁹F Dynamic-NMR (DNMR) to measure exchange of 3-fluoro-3-deoxyglucose (3-FDG) across the membrane of erythrocytes from 10 pregnant women (5 healthy control women, and 5 from women suffering from PE). Magnetisation transfer was measured using the 1D selective inversion and 2D EXSY pulse sequences, over a range of time delays. Integrated intensities from these experiments were used in matrix diagonalisation to estimate the values of the rate constants of exchange and membrane permeability. No significant differences were observed for the rate of exchange of 3-FDG and membrane permeability between healthy pregnant women and those suffering from PE, leading us to conclude that no oxidative damage had occurred at this carrier-protein site in the membrane.     

Structural Studies of Bcl-xL/ligand Complexes using 19F NMR

Fluorine atoms are often incorporated into drug molecules as part of the lead optimization process in order to improve affinity or modify undesirable metabolic and pharmacokinetic profiles. From an NMR perspective, the abundance of fluorinated drug leads provides an exploitable niche for structural studies using ¹⁹F NMR in the drug discovery process. As ¹⁹F has no interfering background signal from biological sources, ¹⁹F NMR studies of fluorinated drugs bound to their protein receptors can yield easily interpretable and unambiguous structural constraints. ¹⁹F can also be selectively incorporated into proteins to obtain additional constraints for structural studies. Despite these advantages, ¹⁹F NMR has rarely been exploited for structural studies due to its broad lines in macromolecules and their ligand complexes, leading to weak signals in ¹H/¹⁹F heteronuclear NOE experiments. Here we demonstrate several different experimental strategies that use ¹⁹F NMR to obtain ligand–protein structural constraints for ligands bound to the anti-apoptotic protein Bcl-xL, a drug target for anti-cancer therapy. These examples indicate the applicability of these methods to typical structural problems encountered in the drug development process.     

Polyfluorinated bis-styrylbenzenes as amyloid-β plaque binding ligands

Detection of cerebral β-amyloid (Aβ) by targeted contrast agents remains of great interest to aid the in vivo diagnosis of Alzheimer’s disease (AD). Bis-styrylbenzenes have been previously reported as potential Aβ imaging agents. To further explore their potency as ¹⁹F MRI contrast agents we synthetized several novel fluorinated bis-styrylbenzenes and studied their fluorescent properties and amyloid-β binding characteristics. The compounds showed a high affinity for Aβ plaques on murine and human brain sections. Interestingly, competitive binding experiments demonstrated that they bound to a different binding site than chrysamine G. Despite their high logP values, many bis-styrylbenzenes were able to enter the brain and label murine amyloid in vivo. Unfortunately initial post-mortem ¹⁹F NMR studies showed that these compounds as yet do not warrant further MRI studies due to the reduction of the ¹⁹F signal in the environment of the brain.     

[18F]-Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography of LAPC4-CR Castration-Resistant Prostate Cancer Xenograft Model in Soft Tissue Compartments

Preclinical xenograft models have contributed to advancing our understanding of the molecular basis of prostate cancer and to the development of targeted therapy. However, traditional preclinical in vivo techniques using caliper measurements and survival analysis evaluate the macroscopic tumor behavior, whereas tissue sampling disrupts the microenvironment and cannot be used for longitudinal studies in the same animal. Herein, we present an in vivo study of [¹⁸F]-fluorodeoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT) designed to evaluate the metabolism within the microenvironment of LAPC4-CR, a unique murine model of castration-resistant prostate cancer. Mice bearing LAPC4-CR subcutaneous tumors were administered [¹⁸F]-FDG via intravenous injection. After a 60-minute distribution phase, the mice were imaged on a PET/CT scanner with submillimeter resolution; and the fused PET/CT images were analyzed to evaluate tumor size, location, and metabolism across the cohort of mice. The xenograft tumors showed [¹⁸F]-FDG uptake that was independent of tumor size and was significantly greater than uptake in skeletal muscle and liver in mice (Wilcoxon signed-rank P values of .0002 and .0002, respectively). [¹⁸F]-FDG metabolism of the LAPC4-CR tumors was 2.1 ± 0.8 ID/cm³*wt, with tumor to muscle ratio of 7.4 ± 4.7 and tumor to liver background ratio of 6.7 ± 2.3. Noninvasive molecular imaging techniques such as PET/CT can be used to probe the microenvironment of tumors in vivo. This study showed that [¹⁸F]-FDG-PET/CT could be used to image and assess glucose metabolism of LAPC4-CR xenografts in vivo. Further work can investigate the use of PET/CT to quantify the metabolic response of LAPC4-CR to novel agents and combination therapies using soft tissue and possibly bone compartment xenograft models.     

Introduction of Fluorine Into the C8 Position of Purine Nucleosides

Abstract

We have synthesized 2-amino-6,8-difluoro-9-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)purine (3) from 2-amino-6,8-dichloro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (1) in a two-step procedure. The reaction of 3 with anhydrous ammonia in dry 1,2-dimethoxyethane gave 2,8-diamino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (4) in 64.1% yield. Compound 4 was deaminated with t-butylnitrite in tetrahydrofuran to give 2-amino-6-fluoro-9-(2,3,5-tri-O-acetyl-ß-D-ribofuranosyl)purine (6). The ¹H, ¹⁹F, and ¹³C NMR spectral data were determined and evaluated for each of the compounds.     

Site-specific protein backbone and side-chain NMR chemical shift and relaxation analysis of human vinexin SH3 domain using a genetically encoded 15N/19F-labeled unnatural amino acid

SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for ¹⁵N/¹⁹F-trifluoromethyl-phenylalanine (¹⁵N/¹⁹F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide (¹⁵N)¹H and side-chain ¹⁹F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide (¹⁵N)¹H and side-chain ¹⁹F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes.     

Multiplicity of dog kidney high-Km aldose reductase and conversion mechanism into aldose reductase

• 1.1. High-K_m, aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution.
• 2.2. High-K_m, aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC.
• 3.3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (al-, a2- and a3-forms), one was aldose reductase (a 1-form) and the others were the precursors of aldose reductase (a2- and a3-form).
• 4.4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a 1-form.
• 5.5. The c-form was either directly converted into the al-form, or indirectly into the a2-form followed by the al-form.
• 6.6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Ap, aldose reductase were finally converted into aldose reductase (al-form).

     

Biogenic aldehyde metabolism in rat brain. Differential sensitivity of aldehyde reductase isoenzymes to sodium valproate

The effects of inhibitors of aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) on the formation of 3-methoxy-4-hydroxyphenethylene glycol from normetanephrine have been studied in rat brain homogenates. The reaction pathway was shown to be unaffected by several inhibitors of the major (high Km) form of aldehyde reductase such as sodium valproate. Two isoenzymes of aldehyde reductase have been separated and characterized from rat brain. The minor (low Km) isoenzyme is shown to be relatively insensitive to sodium valproate and exhibits a similar inhibitor-sensitivity profile to that obtained for methoxyhydroxyphenethylene glycol formation. The low Km isoenzyme is therefore implicated in catecholamine metabolism. The metabolism of succinic semialdehyde and xylose by rat brain cytosol has also been examined. Aldose metabolism may also be attributed to the action of the low Km reductase, but the existence of a separate succinic semialdehyde reductase is postulated. The possible roles of aldehyde reductases in brain metabolism and the relationship between these enzymes and aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) are discussed.     

<Superscript>1</Superscript>H NMR based metabolomic approach to monitoring of the head and neck cancer treatment toxicity

Introduction

Anticancer treatment results in temporary or permanent toxicity considered as changes in normal tissues and/or involved regions. The net effect is mirrored in morphological, functional and molecular disturbances—thus in a systemic response of the human body. To date, specific NMR biomarkers of radiation therapy toxicity in head and neck squamous cell carcinoma (HNSCC) patients are scarce or even missing.

Objectives

We aimed to investigate molecular processes reflecting acute radiation sequelae (ARS) in HNSCC patients using NMR-based metabolomics of blood serum.

Methods

45 patients with HNSCC were treated with radiotherapy (RT) or chemoradiotherapy (CHRT). Blood samples were collected within a week after RT/CHRT completion. Patients were divided into two classes (of high and low ARS) on the basis of the highest individual ARS value observed during the treatment. ¹H NMR spectra of serum samples were acquired on a Bruker 400.13 MHz spectrometer at 310 K and analyzed using principal component analysis and orthogonal partial least squares discriminant analysis. Additional statistical analyses were performed on quantified metabolites.

Results

1D projections of the J-resolved NMR spectra seem to be of the great potential in the quest for the HNSCC treatment toxicity biomarker. The metabolic features characteristic for high ARS are the increased signals of N-acetyl-glycoprotein and acetate, as well as decrease of choline and the metabolites involved in energy metabolism: branched chain amino acids (BCAAs), alanine, creatinine and carnitine. Furthermore, we observed significant correlations between N-acetyl-glycoprotein and clinical markers of inflammation as well as acetate and a percentage-weight-loss during the treatment. CRP was also negatively correlated with alanine and BCAAs.

Conclusion

NMR-based metabolomics provides relevant biomarkers of RT/CHRT toxicity (ARS) in HNSCC patients. The results indicate at least three concomitant processes related to high ARS: inflammation, altered energy metabolism and disturbed membrane metabolism, and indicate an exciting potential of J-resolved NMR spectroscopy combined with multivariate projection techniques.

     

Structural differences between apolipoprotein E3 and E4 as measured by 19F NMR

The apolipoprotein E family contains three major isoforms (ApoE4, E3, and E2) that are directly involved with lipoprotein metabolism and cholesterol transport. ApoE3 and apoE4 differ in only a single amino acid with an arginine in apoE4 changed to a cysteine at position 112 in apoE3. Yet only apoE4 is recognized as a risk factor for Alzheimer''s disease. Here we used ¹⁹F NMR to examine structural differences between apoE4 and apoE3 and the effect of the C-terminal domain on the N-terminal domain. After incorporation of 5-¹⁹F-tryptophan the 1D ¹⁹F NMR spectra were compared for the N-terminal domain and for the full length proteins. The NMR spectra of the N-terminal region (residues 1–191) are reasonably well resolved while those of the full length wild-type proteins are broad and ill-defined suggesting considerable conformational heterogeneity. At least four of the seven tryptophan residues in the wild type protein appear to be solvent exposed. NMR spectra of the wild-type proteins were compared to apoE containing four mutations in the C-terminal region that gives rise to a monomeric form either of apoE3 under native conditions (Zhang et al., Biochemistry 2007; 46: 10722–10732) or apoE4 in the presence of 1 M urea. For either wild-type or mutant proteins the differences in tryptophan resonances in the N-terminal region of the protein suggest structural differences between apoE3 and apoE4. We conclude that these differences occur both as a consequence of the Arg158Cys mutation and as a consequence of the interaction with the C-terminal domain.