Development of a novel mouth model as an alternative tool to test the effectiveness of an in vivo EPR dosimetry system.

In a large-scale radiation event, thousands may be exposed to unknown amounts of radiation, some of which may be life-threatening without immediate attention. In such situations, a method to quickly and reliably estimate dose would help medical responders triage victims to receive life-saving care. We developed such a method using electron paramagnetic resonance (EPR) to make in vivo measurements of the maxillary incisors. This report provides evidence that the use of in vitro studies can provide data that are fully representative of the measurements made in vivo. This is necessary because, in order to systematically test and improve the reliability and accuracy of the dose estimates made with our EPR dosimetry system, it is important to conduct controlled studies in vitro using irradiated human teeth. Therefore, it is imperative to validate whether our in vitro models adequately simulate the measurements made in vivo, which are intended to help guide decisions on triage after a radiation event. Using a healthy volunteer with a dentition gap that allows using a partial denture, human teeth were serially irradiated in vitro and then, using a partial denture, placed in the volunteer's mouth for measurements. We compared dose estimates made using in vivo measurements made in the volunteer's mouth to measurements made on the same teeth in our complex mouth model that simulates electromagnetic and anatomic properties of the mouth. Our results demonstrate that this mouth model can be used in in vitro studies to develop the system because these measurements appropriately model in vivo conditions.

Evolution and Optimization of Tooth Models for Testing In Vivo EPR Tooth Dosimetry.

Testing and verification are an integral part of any cycle to design, manufacture and improve a novel device intended for use in humans. In the case of testing Dartmouth's electron paramagnetic resonance (EPR) in vivo tooth dosimetry device, in vitro studies are needed throughout its development to test its performance, i.e. to verify its current capability for assessing dose in individuals potentially exposed to ionizing radiation. Since the EPR device uses the enamel of human teeth to assess dose, models that include human teeth have been an integral mechanism to carry out in vitro studies during development and testing its ability to meet performance standards for its ultimate intended in vivo use. As the instrument improves over time, new demands for in vitro studies change as well. This paper describes the tooth models used to perform in vitro studies and their evolution to meet the changing demands for testing in vivo EPR tooth dosimetry.

Tooth Retrospective Dosimetry Using Electron Paramagnetic Resonance: Influence of Irradiated Dental Composites.

In the aftermath of a major radiological accident, the medical management of overexposed individuals will rely on the determination of the dose of ionizing radiations absorbed by the victims. Because people in the general population do not possess conventional dosimeters, after the fact dose reconstruction methods are needed. Free radicals are induced by radiations in the tooth enamel of victims, in direct proportion to dose, and can be quantified using Electron Paramagnetic Resonance (EPR) spectrometry, a technique that was demonstrated to be very appropriate for mass triage. The presence of dimethacrylate based restorations on teeth can interfere with the dosimetric signal from the enamel, as free radicals could also be induced in the various composites used. The aim of the present study was to screen irradiated composites for a possible radiation-induced EPR signal, to characterize it, and evaluate a possible interference with the dosimetric signal of the enamel. We investigated the most common commercial composites, and experimental compositions, for a possible class effect. The effect of the dose was studied between 10 Gy and 100 Gy using high sensitivity X-band spectrometer. The influence of this radiation-induced signal from the composite on the dosimetric signal of the enamel was also investigated using a clinical L-Band EPR spectrometer, specifically developed in the EPR center at Dartmouth College. In X-band, a radiation-induced signal was observed for high doses (25-100 Gy); it was rapidly decaying, and not detected after only 24 h post irradiation. At 10 Gy, the signal was in most cases not measurable in the commercial composites tested, with the exception of 3 composites showing a significant intensity. In L-band study, only one irradiated commercial composite influenced significantly the dosimetric signal of the tooth, with an overestimation about 30%. In conclusion, the presence of the radiation-induced signal from dental composites should not significantly influence the dosimetry for early dose assessment.

In vivo EPR tooth dosimetry for triage after a radiation event involving large populations.

The management of radiation injuries following a catastrophic event where large numbers of people may have been exposed to life-threatening doses of ionizing radiation will rely critically on the availability and use of suitable biodosimetry methods. In vivo electron paramagnetic resonance (EPR) tooth dosimetry has a number of valuable and unique characteristics and capabilities that may help enable effective triage. We have produced a prototype of a deployable EPR tooth dosimeter and tested it in several in vitro and in vivo studies to characterize the performance and utility at the state of the art. This report focuses on recent advances in the technology, which strengthen the evidence that in vivo EPR tooth dosimetry can provide practical, accurate, and rapid measurements in the context of its intended use to help triage victims in the event of an improvised nuclear device. These advances provide evidence that the signal is stable, accurate to within 0.5 Gy, and can be successfully carried out in vivo. The stability over time of the radiation-induced EPR signal from whole teeth was measured to confirm its long-term stability and better characterize signal behavior in the hours following irradiation. Dosimetry measurements were taken for five pairs of natural human upper central incisors mounted within a simple anatomic mouth model that demonstrates the ability to achieve 0.5 Gy standard error of inverse dose prediction. An assessment of the use of intact upper incisors for dose estimation and screening was performed with volunteer subjects who have not been exposed to significant levels of ionizing radiation and patients who have undergone total body irradiation as part of bone marrow transplant procedures. Based on these and previous evaluations of the performance and use of the in vivo tooth dosimetry system, it is concluded that this system could be a very valuable resource to aid in the management of a massive radiological event.

Real-time monitoring of ischemic and contralateral brain pO2 during stroke by variable length multisite resonators.

PURPOSE: Electron paramagnetic resonance (EPR) oximetry using variable length multi-probe implantable resonator (IR), was used to investigate the temporal changes in the ischemic and contralateral brain pO2 during stroke in rats. MATERIAL AND METHODS: The EPR signal to noise ratio (S/N) of the IR with four sensor loops at a depth of up to 11 mm were compared with direct implantation of lithium phthalocyanine (LiPc, oximetry probe) deposits in vitro. These IRs were used to follow the temporal changes in pO2 at two sites in each hemisphere during ischemia induced by left middle cerebral artery occlusion (MCAO) in rats breathing 30% O2 or 100% O2. RESULTS: The S/N ratios of the IRs were significantly greater than the LiPc deposits. A similar pO2 at two sites in each hemisphere prior to the onset of ischemia was observed in rats breathing 30% O2. However, a significant decline in the pO2 of the left cortex and striatum occurred during ischemia, but no change in the pO2 of the contralateral brain was observed. A significant increase in the pO2 of only the contralateral non-ischemic brain was observed in the rats breathing 100% O2. No significant difference in the infarct volume was evident between the animals breathing 30% O2 or 100% O2 during ischemia. CONCLUSIONS: EPR oximetry with IRs can repeatedly assess temporal changes in the brain pO2 at four sites simultaneously during stroke. This oximetry approach can be used to test and develop interventions to rescue ischemic tissue by modulating cerebral pO2 during stroke.

Acyl-coenzyme A:cholesterol acyltransferase 1 - significance of single-nucleotide polymorphism at residue 526 and the role of Pro347 near the fifth transmembrane domain.

Acyl-coenzyme A:cholesterol acyltransferases (ACATs), which are members of the membrane-bound O-acyltransferase family, catalyze the conversion of cholesterol to cholesteryl esters. Mammals have two isoenzymes: ACAT1 and ACAT2. Both enzymes are drug targets for treating human diseases. ACAT1 is present in various cell types. It contains nine transmembrane domains (TMDs), with the active site His460 located within TMD7, and the active site Asn421 located within the fourth large cytoplasmic loop. In human ACAT1, a single-nucleotide polymorphism exists for residue 526: the codon is either CAG for Gln, or CGG for Arg. Gln526/Arg526 is present within the C-terminal loop. Its biochemical significance is unknown. In addition, within the C-terminal half of ACAT1, numerous residues conserved with those of ACAT2 are present; the functions of these conserved residues are largely unknown. Here, we performed single-substitution mutagenesis experiments to investigate the roles of individual residues present in the C-terminal loop, including Gln526/Arg526, and the eight conserved Pro residues located near/in various TMDs. The results show that the enzyme activity of ACAT1 with Gln526 is less active than that of ACAT1 with Arg526 by 40%. In addition, several residues in the C-terminal loop are important for maintaining proper ACAT1 protein stability. Other results show that Pro347 plays an important role in modulating enzyme catalysis. Overall, our results imply that the CAG/CGG polymorphism can be utilized to perform ACAT1 activity/human disease susceptibility studies, and that Pro347 located near TMD5 plays an important role in modulating enzyme catalysis.

Electron paramagnetic resonance dosimetry for a large-scale radiation incident.

With possibilities for radiation terrorism and intensified concerns about nuclear accidents since the recent Fukushima Daiichi event, the potential exposure of large numbers of individuals to radiation that could lead to acute clinical effects has become a major concern. For the medical community to cope with such an event and avoid overwhelming the medical care system, it is essential to identify not only individuals who have received clinically significant exposures and need medical intervention but also those who do not need treatment. The ability of electron paramagnetic resonance to measure radiation-induced paramagnetic species, which persist in certain tissues (e.g., teeth, fingernails, toenails, bone, and hair), has led to this technique becoming a prominent method for screening significantly exposed individuals. Although the technical requirements needed to develop this method for effective application in a radiation event are daunting, remarkable progress has been made. In collaboration with General Electric and through funding committed by the Biomedical Advanced Research and Development Authority, electron paramagnetic resonance tooth dosimetry of the upper incisors is being developed to become a Food and Drug Administration-approved and manufacturable device designed to carry out triage for a threshold dose of 2 Gy. Significant progress has also been made in the development of electron paramagnetic resonance nail dosimetry based on measurements of nails in situ under point-of-care conditions, and in the near future this may become a second field-ready technique. Based on recent progress in measurements of nail clippings, it is anticipated that this technique may be implementable at remotely located laboratories to provide additional information when the measurements of dose on-site need to be supplemented. The authors conclude that electron paramagnetic resonance dosimetry is likely to be a useful part of triage for a large-scale radiation incident.

Dynamic changes in oxygenation of intracranial tumor and contralateral brain during tumor growth and carbogen breathing: a multisite EPR oximetry with implantable resonators.

INTRODUCTION: Several techniques currently exist for measuring tissue oxygen; however technical difficulties have limited their usefulness and general application. We report a recently developed electron paramagnetic resonance (EPR) oximetry approach with multiple probe implantable resonators (IRs) that allow repeated measurements of oxygen in tissue at depths of greater than 10mm. METHODS: The EPR signal to noise (S/N) ratio of two probe IRs was compared with that of LiPc deposits. The feasibility of intracranial tissue pO(2) measurements by EPR oximetry using IRs was tested in normal rats and rats bearing intracerebral F98 tumors. The dynamic changes in the tissue pO(2) were assessed during repeated hyperoxia with carbogen breathing. RESULTS: A 6-10 times increase in the S/N ratio was observed with IRs as compared to LiPc deposits. The mean brain pO(2) of normal rats was stable and increased significantly during carbogen inhalation in experiments repeated for 3months. The pO(2) of F98 glioma declined gradually, while the pO(2) of contralateral brain essentially remained the same. Although a significant increase in the glioma pO(2) was observed during carbogen inhalation, this effect declined in experiments repeated over days. CONCLUSION: EPR oximetry with IRs provides a significant increase in S/N ratio. The ability to repeatedly assess orthotopic glioma pO(2) is likely to play a vital role in understanding the dynamics of tissue pO(2) during tumor growth and therapies designed to modulate tumor hypoxia. This information could then be used to optimize chemoradiation by scheduling treatments at times of increased glioma oxygenation.

A Deployable In Vivo EPR Tooth Dosimeter for Triage After a Radiation Event Involving Large Populations.

In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators.Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately five minutes, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care.

Synergistic combination of hyperoxygenation and radiotherapy by repeated assessments of tumor pO2 with EPR oximetry.

The effect of hyperoxygenation with carbogen (95% O(2) + 5% CO(2)) inhalation on RIF-1 tumor pO(2 )and its consequence on growth inhibition with fractionated radiotherapy is reported. The temporal changes in the tumor pO(2) were assessed by in vivo Electron Paramagnetic Resonance (EPR) oximetry in mice breathing 30% O(2) or carbogen and the tumors were irradiated with 4 Gy/day for 5 consecutive days; a protocol that emulates the clinical application of carbogen. The RIF-1 tumors were hypoxic with a tissue pO(2) of 5-9 mmHg. Carbogen (CB) breathing significantly increased tumor pO(2), with a maximum increase at 22.9-31.2 min on days 1-5, however, the magnitude of increase in pO(2) declined on day 5. Radiotherapy during carbogen inhalation (CB/RT) resulted in a significant tumor growth inhibition from day 3 to day 6 as compared to 30%O(2)/RT and carbogen (CB/Sham RT) groups. The results provide unambiguous quantitative information on the effect of carbogen inhalation on tumor pO(2) over the course of 5 days. Tumor growth inhibition in the CB/RT group confirms that the tumor oxygenation with carbogen was radiobiologically significant. Repeated tumor pO(2) measurements by EPR oximetry can provide temporal information that could be used to improve therapeutic outcomes by scheduling doses at times of improved tumor oxygenation.

Physically-based biodosimetry using in vivo EPR of teeth in patients undergoing total body irradiation.

PURPOSE: The ability to estimate individual exposures to radiation following a large attack or incident has been identified as a necessity for rational and effective emergency medical response. In vivo electron paramagnetic resonance (EPR) spectroscopy of tooth enamel has been developed to meet this need. MATERIALS AND METHODS: A novel transportable EPR spectrometer, developed to facilitate tooth dosimetry in an emergency response setting, was used to measure upper incisors in a model system, in unirradiated subjects, and in patients who had received total body doses of 2 Gy. RESULTS: A linear dose response was observed in the model system. A statistically significant increase in the intensity of the radiation-induced EPR signal was observed in irradiated versus unirradiated subjects, with an estimated standard error of dose prediction of 0.9 ± 0.3 Gy. CONCLUSIONS: These results demonstrate the current ability of in vivo EPR tooth dosimetry to distinguish between subjects who have not been irradiated and those who have received exposures that place them at risk for acute radiation syndrome. Procedural and technical developments to further increase the precision of dose estimation and ensure reliable operation in the emergency setting are underway. With these developments EPR tooth dosimetry is likely to be a valuable resource for triage following potential radiation exposure of a large population.

Cerebral oxygenation of the cortex and striatum following normobaric hyperoxia and mild hypoxia in rats by EPR oximetry using multi-probe implantable resonators.

Multi-site electron paramagnetic resonance (EPR) oximetry, using multi-probe implantable resonators, was used to measure the partial pressure of oxygen (pO(2)) in the brains of rats following normobaric hyperoxia and mild hypoxia. The cerebral tissue pO(2) was measured simultaneously in the cerebral cortex and striatum in the same rats before, during, and after normobaric hyperoxia and mild hypoxia challenges. The mean baseline tissue pO(2) values were not significantly different between the cortex and striatum.During 30 min of 100% O(2) inhalation, a statistically significant increase in tissue pO(2) of all four sites was observed, however, the tissue pO(2) of the striatum area was significantly higher than in the forelimb area of the cortex. Brain pO(2) significantly decreased from the baseline value during 15 min of 15% O(2) challenge.No differences in the recovery of the cerebral cortex and striatum pO(2) were observed when the rats were allowed to breathe 30% O(2). It appears that EPR oximetry using implantable resonators can provide information on pO(2) under the experimental conditions needed for such a study. The levels of pO(2) that occurred in these experiments are readily resolvable by multi-site EPR oximetry with multi-probe resonators. In addition, the ability to simultaneously measure the pO(2) in several areas of the brain provides important information that could potentially help differentiate the pO(2) changes that can occur due to global or local mechanisms.

Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy.

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane-bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl-CoA binds to ACAT1 with K(d) = 1.9 µM and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl-CoA elicits a similar spectrum change but much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3ß-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.

The view from the trenches: part 2-technical considerations for EPR screening.

There is growing awareness of the need for methodologies that can be used retrospectively to provide the biodosimetry needed to carry out screening and triage immediately after an event in which large numbers of people have potentially received clinically significant doses of ionizing radiation. The general approach to developing such methodologies has been a technology centric one, often ignoring the system integrations considerations that are key to their effective use. In this study an integrative approach for the evaluation and development of a physical biodosimetry technology was applied based on in vivo electron paramagnetic resonance (EPR) dosimetry. The EPR measurements are based on physical changes in tissues whose magnitudes are not affected by the factors that can confound biologically-based assessments. In this study the use of a pilot simulation exercise to evaluate an experimental EPR system and gather stakeholders' feedback early on in the development process is described. The exercise involved: ten non-irradiated participants, representatives from a local fire department; Department of Homeland Security certified exercise evaluators, EPR experts, physicians; and a human factors engineer. Stakeholders were in agreement that the EPR technology in its current state of development could be deployed for the screening of mass casualties. Furthermore, stakeholders' recommendations will be prioritized and incorporated in future developments of the EPR technique. While the results of this exercise were aimed specifically at providing feedback for the development of EPR dosimetry for screening mass casualties, the methods and lessons learned are likely to be applicable to other biodosimetric methods.

Development of in vivo tooth EPR for individual radiation dose estimation and screening.

The development of in vivo EPR has made it feasible to perform tooth dosimetry measurements in situ, greatly expanding the potential for using this approach for immediate screening after radiation exposures. The ability of in vivo tooth dosimetry to provide estimates of absorbed dose has been established through a series of experiments using unirradiated volunteers with specifically irradiated molar teeth placed in situ within gaps in their dentition and in natural canine teeth of patients who have completed courses of radiation therapy for head and neck cancers. Multiple measurements in patients who have received radiation therapy demonstrate the expected heterogeneous dose distributions. Dose-response curves have been generated using both populations and, using the current methodology and instrument, the standard error of prediction based on single 4.5-min measurements is approximately 1.5 Gy for inserted molar teeth and between 2.0 and 2.5 Gy in the more irregularly shaped canine teeth. Averaging of independent measurements can reduce this error significantly to values near 1 Gy. Developments to reduce these errors are underway, focusing on geometric optimization of the resonators, detector positioning techniques, and optimal data averaging approaches. In summary, it seems plausible that the EPR dosimetry techniques will have an important role in retrospective dosimetry for exposures involving large numbers of individuals.

In Vivo EPR For Dosimetry.

As a result of terrorism, accident, or war, populations potentially can be exposed to doses of ionizing radiation that could cause direct clinical effects within days or weeks. There is a critical need to determine the magnitude of the exposure to individuals so that those with significant risk have appropriate procedures initiated immediately, while those without a significant probability of acute effects can be reassured and removed from the need for further consideration in the medical/emergency system. In many of the plausible scenarios there is an urgent need to make the determination very soon after the event and while the subject is still present. In vivo EPR measurements of radiation-induced changes in the enamel of teeth is a method, perhaps the only such method, which can differentiate among doses sufficiently for classifying individuals into categories for treatment with sufficient accuracy to facilitate decisions on medical treatment. In its current state, the in vivo EPR dosimeter can provide estimates of absorbed dose with an error approximately +/- 50 cGy over the range of interest for acute biological effects of radiation, assuming repeated measurements of the tooth in the mouth of the subject. The time required for acquisition, the lower limit, and the precision are expected to improve, with improvements in the resonator and the algorithm for acquiring and calculating the dose. The magnet system that is currently used, while potentially deployable, is somewhat large and heavy, requiring that it be mounted on a small truck or trailer. Several smaller magnets, including an intraoral magnet are under development, which would extend the ease of use of this technique.

Experimental Procedures for Sensitive and Reproducible In Situ EPR Tooth Dosimetry.

In vivo electron paramagnetic resonance (EPR) tooth dosimetry provides a means for non-invasive retrospective assessment of personal radiation exposure. While there is a clear need for such capabilities following radiation accidents, the most pressing need for the development of this technology is the heightened likelihood of terrorist events or nuclear conflicts. This technique will enable such measurements to be made at the site of an incident, while the subject is present, to assist emergency personnel as they perform triage for the affected population. At Dartmouth Medical School this development is currently being tested with normal volunteers with irradiated teeth placed in their mouths and with patients who have undergone radiation therapy. Here we describe progress in practical procedures to provide accurate and reproducible in vivo dose estimates.

Human acyl-CoA:cholesterol acyltransferase (ACAT) and its potential as a target for pharmaceutical intervention against atherosclerosis.

Acyl-CoA:cholesterol acyltransferase (ACAT) catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty-acyl-coenzyme A. At the single-cell level, ACAT serves as a regulator of intracellular cholesterol homeostasis. In addition, ACAT supplies cholesteryl esters for lipoprotein assembly in the liver and small intestine. Under pathological conditions, the accumulation of cholesteryl esters produced by ACAT in macrophages contributes to foam cell formation, a hallmark of the early stage of atherosclerosis. Several reviews addressing various aspects of ACAT and ACAT inhibitors are available. This review briefly outlines the current knowledge on the biochemical properties of human ACATs, and then focuses on discussing the merit of ACAT as a drug target for pharmaceutical interventions against atherosclerosis.

Human acyl-coenzyme A:cholesterol acyltransferase 1 (acat1) sequences located in two different chromosomes (7 and 1) are required to produce a novel ACAT1 isoenzyme with additional sequence at the N terminus.

A rare form of human ACAT1 mRNA, containing the optional long 5'-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7. To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5'-untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1(ORF). The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.