Regional acetate kinetics and oxidation in human volunteers

B. Mittendorfer1, L. S. Sidossis1,3, E. Walser2, D. L. Chinkes1, and R. R. Wolfe1,3

Departments of 1 Surgery and 2 Radiology, University of Texas Medical Branch, and 3 Metabolism Unit, Shriners Burns Institute, Galveston, Texas 77550

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

We have used a 3-h primed continuous infusion of [1,2-13C]acetate in five fasted (24 h) volunteers to quantify splanchnic and leg acetate metabolism (protocol 1). Fractional extraction of acetate by both tissues was high (~70%), and simultaneous uptake and release of acetate were observed. Labeled carbon recovery in CO2 was 37.9 ± 2.3% at the whole body level, 37.7 ± 1.5% across the splanchnic bed, and 37.3 ± 2.9% across the leg. Furthermore, we calculated whole body labeled carbon recovery during 15 h of [1,2-13C]acetate infusion in three volunteers (protocol 2). Whole body acetate carbon recovery in CO2 was significantly higher (66.7 ± 4.5%) after 15 h of tracer infusion than after 3 h. We conclude that acetate is rapidly taken up by the leg and splanchnic tissues and that the percent recovery of CO2 from the oxidation of acetate is heavily dependent on the length of acetate tracer infusion. In the postabsorptive state, labeled carbon recovery from acetate across the leg and the splanchnic region is similar to the whole body CO2 recovery.

fatty acids; liver; muscle; acetate correction factor

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

DETERMINATION of the rate of recovery of labeled CO2 from labeled acetate allows the calculation of a correction factor for use in the estimation of plasma fatty acid oxidation (13). The present studies were performed to determine whole body and regional (splanchnic and leg) acetate kinetics and oxidation in normal human volunteers after 3 h of infusion of labeled acetate (protocol 1) to obtain "acetate correction factors" across the splanchnic region and the leg and to compare those values with the whole body values.

Metabolic pathways differ among various tissues, so it is possible that the carbon label recoveries differ as well. For example, a rapid rate of gluconeogenesis might cause exchange of acetate label via the oxaloacetate pool in the liver but not in muscle. Regional differences in recovery could be obscured at the whole body level. It is therefore of importance to determine the recoveries of 13CO2 from the oxidation of labeled acetate at the regional level, not only for the determination of acetate oxidation by different tissues, but also to verify the validity of the acetate correction factor for the calculation of whole body free fatty acid oxidation using carbon-labeled free fatty acids. Because the length of tracer infusion influences carbon label recovery (17), we also determined acetate carbon recovery in CO2 in the whole body after 15 h of labeled acetate infusion (protocol 2).

The major sites of acetate uptake and release in humans are still not known, and the role of acetate in intermediary metabolism is unclear. Acetate is formed endogenously from acetyl-CoA by two enzymes, acetyl-CoA synthetase and acetyl-CoA hydrolase. Both are present in the cytosol and the mitochondrial fraction of different tissues in mammals (6). In rat and sheep, for example, acetate is produced by liver and heart slices from pyruvate or fatty acid precursors. Liver mitochondrial fractions do not form acetate from either substrate but instead convert acetate into acetoacetate (6). Heart mitochondrial fractions, on the other hand, form acetate from pyruvate or fatty acid precursors (6).

It has long been believed that the liver is the primary site of acetate metabolism in humans. However, acetate is utilized by peripheral tissues in humans, as evidenced by the fall in acetate concentration between arterial and venous blood in the forearm in fasting subjects (4, 8, 9). Furthermore, it has recently been proposed that there is considerable oxidation of [2-14C]acetate to 14CO2 in muscle (12).

Whole body oxidation of acetate has been suggested to account for up to 10% of energy expenditure (16), and it has been proposed that acetate serves to redistribute oxidizable substrate throughout the body (11), especially under conditions of caloric deprivation (1). To clarify some of these aspects, we quantified whole body and regional acetate kinetics after 3 h of labeled acetate infusion (protocol 1).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Experimental Design

Eight healthy volunteers (7 male, 1 female, age 29 ± 2 yr, weight 76 ± 5 kg) were recruited to participate in the studies, which were approved by the Institutional Review Board and the General Clinical Research Center of the University of Texas Medical Branch in Galveston. After informed consent had been obtained, all subjects were given a comprehensive physical examination and were considered in good health at the time of the study. Two experimental protocols were performed. Each volunteer participated in one of the protocols. In protocol 1, regional and whole body acetate kinetics were quantified during a 3-h infusion of [1,2-13C]acetate at the end of 24 h of fasting. In protocol 2, whole body recovery of acetate carbon in CO2 was determined at the end of a 15-h infusion of [1,2-13C]acetate.

Protocol 1 (n = 5). The subjects were given a standardized meal in the evening of day 1 and were then fasted (24 h) until the end of the tracer infusion the following day (day 2). The next morning (day 2), a catheter for tracer infusion was placed in a peripheral vein and sampling catheters were placed in a femoral artery and a femoral vein as described in Procedures. After baseline blood samples were collected (6:00 PM, day 2), a primed (45 µmol/kg) continuous (1.5 µmol · kg-1 · min-1) infusion of [1,2-13C]acetate (Cambridge Isotope Laboratories, Andover, MA) was started and maintained for 3 h with a calibrated Harvard syringe pump (Harvard Apparatus, Natick, MA). At the beginning of the study, a 150-µmol/kg NaH13CO3 bolus was given to prime the bicarbonate pool. The exact tracer infusion rates were determined by measurement of concentration of the infusion mixture.

Breath samples were taken every 30 min for the initial 150 min of tracer infusion and every 10 min for the remaining 30 min. During the last 20 min of isotope infusion, blood samples (2 ml each) from the artery and the femoral and hepatic veins were taken simultaneously with the breath samples at 10-min intervals. Whole body oxygen consumption (VO2) and carbon dioxide production (VCO2) were measured at 140-160 min of tracer infusion by means of indirect calorimetry. Four blood samples for the determination of blood flow (see Procedures) were obtained at 105, 110, 115, and 120 min after the start of tracer infusion.

Protocol 2 (n = 3). Before the start of the tracer infusion, each subject was given a standardized meal on day 1 and was then fasted (15 h) until the end of the tracer infusion the next day (day 2). After dinner (9:00 PM) a catheter for tracer infusion was placed in a forearm vein. After baseline blood and breath samples were collected, a primed (15 µmol/kg) continuous infusion (0.5 µmol · kg-1 · min-1) of [1,2-13C]acetate (Cambridge Isotope Laboratories) was started and maintained for 15 h by use of a calibrated Harvard syringe pump (Harvard Apparatus). The exact tracer infusion rates were determined by measurement of concentration of the infusion mixture.

Breath samples were taken every hour for 14 h and every 10 min during the last hour of tracer infusion. VO2 and VCO2 were measured during a 20-min period 2 h before the end of tracer infusion.

Procedures

Catheter placement. On the morning of the study, volunteers were brought to a vascular radiology suite at the University of Texas Medical Branch, where the right groin was prepared and draped in a sterile fashion. A lead glove was placed over the genitalia before the procedure. After patient preparation, the right common femoral vein was punctured, and a 6-Fr sheath was placed. Through this sheath, a straight 5-Fr catheter with several side holes near its tip was manipulated into the right or middle hepatic vein. This catheterization was performed by using a deflecting-tip 0.035" guidewire within the straight catheter. After the catheter was positioned into the hepatic vein, a digital venogram was performed to verify placement, and both the sheath and catheter were infused with heparinized saline to maintain patency. The position of the catheter was confirmed again by a plain view abdominal X ray immediately after the end of the study. A short, straight 4-Fr catheter was then placed retrograde into the right common femoral artery, and it too was connected to a pressurized flush setup. After both catheters and the sheath had been sutured in place, a sterile transparent dressing was used to cover the vascular entry sites.

Blood flow. Blood flow was determined 60 min before the end of the study using a constant infusion of indocyanine green dissolved in 0.9% saline. The dye was infused through the femoral artery catheter at the rate of 0.5 mg/min for 55 min during the 2nd hour of protocol 1, and blood samples were taken at 40, 45, 50, and 55 min after the start of dye infusion simultaneously from the hepatic vein, the femoral vein, and a peripheral vein. The concentrations of the dye in the infusate and in serum samples were determined using a spectrophotometer set at 805 nm. Leg plasma flow was determined by dividing the infusion rate of the dye by the concentration difference of the dye in femoral venous and peripheral venous serum. The peripheral venous concentration of the dye was subtracted from the concentration in the femoral vein to account for recycling of the dye into the artery. Splanchnic plasma flow was determined by dividing the infusion rate of the dye by the concentration difference in arterious and hepatic venous serum. Leg and splanchnic blood flows were then calculated by dividing the plasma flow by 1 minus the hematocrit. With this method, we have found that the SE of the mean is generally within ±3% of the mean values.

Analysis of samples. Analytic procedures for breath samples have been previously described (14). Briefly, breath samples were collected by inflation of an anesthesia bag and then transferred to Vacutainers. 13CO2 enrichment was then determined by isotope ratio mass spectrometry (IRMS; SIRA Series II, VG Isogas, Middlewich, Cheshire, UK, with an HP 3392A integrator). A Sensor Medics 2900 Metabolic Cart (Thermodex Instruments, Pittsburgh, PA) was used to determine expired VCO2. All blood samples for CO2 analysis were collected into prechilled tubes containing heparin sodium. Blood CO2 concentration was measured immediately using a 965 Ciba Corning CO2 analyzer, and the remaining blood samples were kept frozen until further analysis. Blood samples for acetate analysis were collected in iced heparinized tubes and centrifuged immediately at 4°C. Plasma was separated and stored at -70°C. For the determination of blood CO2 enrichment, 5-10 µl phosphoric acid, 85%, were added to 1 ml blood in a sealed tube to release the CO2 into the headspace of the tube. The headspace represents free CO2 and CO2 bound to bicarbonate. The 13C-to-12C ratio of the CO2 in the headspace was then determined by IRMS. Plasma acetate concentration and enrichment were determined by gas chromatography-mass spectrometry (GC-MS; Hewlett Packard, Palo Alto, CA) using a recently described method (10). Briefly, a known amount of [1,2-13C-2,2,2-2H3]acetate was added to 200 µl plasma. After direct derivatization with difluoroaniline, the samples were extracted with ethyl acetate, dried over N2, and redissolved in 70 µl ethyl acetate. One microliter was injected into the GC-MS, and isotopic enrichment was determined using electron impact ionization. Ions at mass-to-charge ratio (m/z) 171, 173, and 176, representing the molecular ions of unlabeled and enriched acetyl derivatives, respectively, were selectively monitored, and their corresponding peaks were integrated. The ion at m/z 173 results from the tracer infusion, and m/z 176 represents the internal standard that we used for the calculation of plasma acetate concentration.

Calculations

In protocol 1, VCO2, plasma acetate, and blood CO2 concentrations and enrichments at 160, 170, and 180 min after the beginning of the labeled acetate infusion were averaged for each sampling site for use in subsequent calculations. In protocol 2, the values for breath CO2 enrichment and VCO2 obtained at 14:40, 14:50, and 15:00 h after the start of the tracer infusion were averaged for the calculation of acetate kinetics. The following equations were used for rate of appearance of acetate, whole body acetate carbon recovery, organ fractional extraction, uptake and release of acetate, percent acetate taken up by the tissues that was oxidized, and net balance across the tissues
Rate of appearance of acetate (R<SUB>a</SUB>) = F/ E (1)
Whole body acetate carbon recovery 
= Breath E<SC>co</SC><SUB>2</SUB> ⋅ <A><AC>V</AC><AC>˙</AC></A><SC>co</SC><SUB>2</SUB> / F (2)
Regional acetate carbon recovery = {[(E<SC>co</SC><SUB>2</SUB> ⋅ C<SC>co</SC><SUB>2</SUB>)<SUB>a</SUB> 
− (E<SC>co</SC><SUB>2</SUB> ⋅ C<SC>co</SC><SUB>2</SUB>)<SUB>v</SUB>]/2}/[(E<SUB>acct</SUB> ⋅ C<SUB>acet</SUB> )<SUB>a</SUB> − (E<SUB>acet</SUB> ⋅ C<SUB>acet</SUB> )<SUB>v</SUB>] (3)
Acetate fractional extraction (fe) = [(E<SUB>acet</SUB> ⋅ C<SUB>acet</SUB> )<SUB>a</SUB> 
− (E<SUB>acet</SUB> ⋅ C<SUB>acet</SUB> )<SUB>v</SUB>]/(E<SUB>acet</SUB> ⋅ C<SUB>acet</SUB> )<SUB>a</SUB> (4)
Regional acetate uptake = fe ⋅ (C<SUB>acet</SUB> )<SUB>a</SUB> ⋅ PF/BW (5)
Regional acetate release
 = uptake + [(C<SUB>acet</SUB> )<SUB>v</SUB> − (C<SUB>acet</SUB> )<SUB>a</SUB>] ⋅ PF/BW (6)
Net balance = (C<SUB>acet</SUB> )<SUB>a</SUB> − (C<SUB>acet</SUB> )<SUB>v</SUB> (7)
where F is the tracer infusion rate, and ECO2 and CCO2 stand for the isotopic enrichment and the concentration of CO2, respectively. Eacet is the isotopic enrichment of acetate in plasma, and Cacet is the concentration of acetate. Subscript a indicates artery, subscript v indicates vein. PF is plasma flow, and BW is body weight. Fractional extraction is abbreviated as fe. Regional acetate carbon label recovery (Eq. 3) is the release of carbon-labeled CO2 (labeled CO2 arteriovenous balance) by the tissue divided by the uptake of acetate tracer (labeled acetate arteriovenous balance) by that tissue. The labeled CO2 arteriovenous balance is divided by 2 because the oxidation of 1 mole of [1,2-13C]acetate produces 2 moles of 13CO2. Statistical comparisons were made using Student's t-test. Data are expressed as means ± SD.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References


Protocol 1 


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Steady states for plasma acetate enrichment, breath CO2 enrichment, and blood CO2 enrichment were achieved within 90-120 min after the start of tracer infusion and were maintained until the end of the study period (Fig. 1). Average breath CO2 enrichment at steady state was 0.0088 ± 0.0007 (tracer/tracee ratio), and average VCO2 was 130.1 ± 15.6 µmol · kg-1 · min-1. Average arterial and venous plasma acetate concentrations and enrichments, as well as blood CO2 concentrations and enrichments, are presented in Table 1. Average splanchnic blood flow was 1.097 ± 0.195 l/min; average leg blood flow was 0.467 ± 0.036 l/min.


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Fig. 1.   Breath CO2 enrichment during 3 h of [1,2-13C]acetate infusion (protocol 1) from one volunteer.

                              
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Table 1.   Enrichment and concentration of plasma acetate and blood CO2 after 3 h of [1,2-13C]acetate infusion (protocol 1)

Labeled carbon recovery as CO2 across the splanchnic region and the leg was not different from whole body carbon label recovery (Table 2). Whole body acetate Ra, clearance, and fractional extraction by the tissues are presented in Table 2.

                              
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Table 2.   Whole body and regional acetate kinetics and labeled carbon recovery from acetate after 3 h of [1,2-13C]acetate infusion (protocol 1)

Isotopic data indicated there was simultaneous uptake and release of acetate across both the splanchnic region (Table 3) and the leg (Table 4). The absolute amount of uptake of acetate by both the leg and the splanchnic region was strongly correlated with the delivery of acetate to the tissue (Pearson's R > 0.9, P < 0.001) (Fig. 2). Net acetate uptake by the leg was observed in all subjects. In contrast, net acetate release was observed in some subjects across the splanchnic bed, whereas other subjects had net uptake. Individual and mean values are presented in Table 3 and Table 4.

                              
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Table 3.   Splanchnic acetate uptake and release in 5 volunteers (protocol 1)

                              
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Table 4.   Leg acetate uptake and release in 5 volunteers (protocol 1)


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Fig. 2.   Acetate uptake by tissues as a function of delivery (blood flow to tissue × blood acetate concentration).

Protocol 2 


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Breath CO2 enrichment increased with time (Fig. 3). Percent acetate carbon recovery was significantly higher (P < 0.05) at 15 h (66.7 ± 4.5%) after the start of tracer infusion than at 3 h.


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Fig. 3.   Breath CO2 enrichment during 15 h of [1,2-13C]acetate infusion (protocol 2).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Interpretation of acetate kinetics, as with lactate kinetics (3), is complicated by the likelihood that isotopic exchange occurs within intracellular pools. However, the fact that the isotopic exchange reactions involving acetate should be the same as for acetyl-CoA makes acetate a useful substrate for kinetic studies of substrate oxidation by use of tracers. In fact, labeled CO2 recovery from labeled acetate infusion can be used as a correction factor for the estimation of plasma fatty acid oxidation (13). Briefly, the acetate correction factor accounts for label fixation that might occur at any step between the entrance of labeled acetyl-CoA into the tricarboxylic acid (TCA) cycle until the recovery of label in breath CO2. This unique characteristic makes the acetate correction factor preferable to the bicarbonate correction factor for correcting isotopic estimates of plasma fatty acid oxidation (13).

At the end of 3 h of labeled acetate infusion (protocol 1), we calculated whole body recovery of acetate carbon in CO2 to be ~40%. This value differs from earlier reports in which acetate recoveries of 90% (16), 70% (9), and 50% (13) have been obtained using [1-14C]- or [1-13C]acetate in the postabsorptive state. The lower recovery of labeled carbon atoms from acetate in our study can be explained by the use of doubly labeled [1,2-13C]acetate as opposed to singly labeled acetate, and also by the relatively short infusion time. As previously reported, the recovery of label from the two position of acetate is less than recovery from the one position (17). This is because the two position has a much greater opportunity to participate in exchange reactions in the TCA cycle before it is excreted as CO2. Thus doubly labeled acetate will result in lower recoveries over the same period of time than acetate labeled only in the one position. Furthermore, recovery increases with length of infusion (17). This finding is evidenced by the data from our 15-h infusion study (protocol 2) and follows from the expectation that labeled carbon atoms that participate in exchange reactions will eventually recycle and go to CO2. Therefore, the value of label recovery calculated in protocol 1 represents an underestimate of the final labeled carbon recovery, because a true steady state was not achieved within 3 h of tracer infusion. Rather, a "pseudo"-plateau was achieved at a value somewhat less than the ultimate true plateau. However, the same is true for labeled carbons from fatty acid tracers as well. Thus the appropriate acetate correction factor for use in isotopic estimates of plasma fatty acid oxidation is obtained when labeled acetate is infused over the same period of time as are the labeled fatty acids.

Acetate carbon recoveries across the leg and the splanchnic region were not different from whole body acetate recoveries. Therefore, whole body acetate recovery can be used to correct substrate oxidation estimates across the leg and splanchnic region in the postabsorptive state. However, regional differences could be obscured at the whole body level under other conditions. In lipogenic states, for example, some acetate may be incorporated into newly synthesized fatty acids (5). Thus a significant amount of labeled acetate may be lost before the possible exchange with the bicarbonate and/or glutamate/glutamine pool. During lipogenesis, the application of the acetate correction factor may therefore overestimate substrate oxidation (13), because the incorporation of acetate into fatty acids occurs before the TCA cycle. The extent of overestimation depends on the rate of de novo fatty acid synthesis.

Ketogenesis should not interfere with the acetate correction factor, because ketone bodies are oxidized in all tissues (e.g., liver, leg) rather than "stored." The only significant storage pool for ketone bodies is the plasma. Therefore, as long as the plasma concentration of ketones is not changing during the time course of the experiment, we can be confident that our assumption regarding ketone oxidation is reasonable.

The plasma acetate concentrations in our study (Table 1) compare well with other studies in human subjects (4, 7-9, 15, 16). Also, our data indicating simultaneous uptake and release of acetate across the splanchnic region and the leg are consistent with previous studies carried out in perfused rat livers (10) and in dogs (1, 2). The observation is analogous to the situation with lactate (3), and the interpretation of this observation is difficult. Nonetheless, simultaneous uptake and release indicate rapid intracellular exchange of acetate with the intracellular acetyl-CoA pool.

In response to the tracer infusion, we observed an increase in plasma acetate concentration that corresponded to the infusion rate (i.e., the contribution of infused tracer to the observed concentration). Hence, the endogenous Ra was likely not affected by the rapid infusion rate used in protocol 1.

The absolute amount of acetate uptake by both the leg and the splanchnic region was strongly correlated with the delivery of acetate to the tissue (Fig. 2). This is similar to earlier studies in the sheep (6) and in humans (16) in which uptake was observed when arterial acetate concentrations were high and release was observed when arterial concentrations of acetate were low. Acetate uptake presumably occurs by simple diffusion, and thus exchange of acetate between the plasma and the tissue depends only on the concentration gradient.

Net uptake of acetate across the leg occurred in all our studies. The same has been proposed previously across the forearm in human volunteers in the postabsorptive state (4, 8). However, in another study in human volunteers (16), net release as well as uptake of acetate from the leg was reported. Across the splanchnic region, which represents the gut and the liver, we found net uptake in some subjects and net release in other subjects. Data from dogs (1) showed that the liver was the main site of acetate uptake, whereas the intestine was the major source of acetate release in the postabsorptive state.

We are unable to account for the whole body Ra of acetate by the net rate of release from the leg and the splanchnic region. However, any acetate that is released by the gut into the portal vein and is subsequently taken up by the liver was not accounted for by our whole body Ra value. Such unlabeled acetate does not appear in the systemic circulation. Consequently, it will not "dilute" the acetate tracer in the systemic circulation. The kidneys and adipose tissue are an unlikely source of Ra acetate, because net acetate uptake by the kidneys has been reported in dogs (1) and by the adipose tissue in humans (4). Therefore, the best candidate for systemic acetate appears to be the gut.

In summary, acetate is rapidly metabolized in human subjects by the liver as well as by peripheral tissues (leg). This notion is supported by the high fractional extraction of acetate from the plasma. Oxidation of labeled acetate results in similar labeled carbon recoveries across the leg, the splanchnic region, and the whole body during fasting. Therefore, we conclude that whole body acetate recoveries can be used to correct substrate oxidation by these two tissues, as suggested previously for the whole body (13).

    ACKNOWLEDGEMENTS

The authors appreciate the help of the nursing staff of the General Clinical Research Center (GCRC) in the performance of the experiments and thank Dr. Y. Zheng for excellent technical assistance.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-34817-12 (R. R. Wolfe) and DK-51969 (L. S. Sidossis), Shriners Hospital Grant 8490 (R. R. Wolfe), and GCRC Grant 00073.

Address for reprint requests: R. R. Wolfe, Metabolism Dept., Shriners Burns Institute, 815 Market St., Galveston, TX 77550.

Received 14 August 1997; accepted in final form 16 February 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Endocrinol Metab 274(6):E978-E983
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