Phenylalanine kinetics in healthy volunteers and liver cirrhotics: implications for the phenylalanine breath test

I. Tugtekin1, U. Wachter1, E. Barth1, H. Weidenbach2, D. A. Wagner3, G. Adler2, M. Georgieff1, P. Radermacher1, and J. A. Vogt1

1 Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Universitätsklinik für Anästhesiologie Ulm, und 2 Abteilung Innere Medizin I, Medizinische Universitätsklinik und Poliklinik Ulm, 89070 Ulm, Germany; and 3 Metabolic Solutions, Nashua, New Hampshire 03063


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Expired 13CO2 recovery from an oral L-[1-13C]phenylalanine ([13C]Phe) dose has been used to quantify liver function. This parameter, however, does not depend solely on liver function but also on total CO2 production, Phe turnover, and initial tracer distribution. Therefore, we evaluated the impact of these factors on breath test values. Nine ethyl-toxic cirrhotic patients and nine control subjects received intravenously 2 mg/kg of [13C]Phe, and breath and blood samples were collected over 4 h. CO2 production was measured by indirect calorimetry. The exhaled 13CO2 enrichments were analyzed by isotope ratio mass spectrometry and the [13C]Phe and L-[1-13C]tyrosine enrichments by gas chromatography-mass spectrometry. The cumulative 13CO2 recovery was significantly lower in cirrhotic patients (7 vs. 12%; P < 0.01), in part due to lower total CO2 production rates. Phe turnover in cirrhotic patients was significantly lower (33 vs. 44 µmol · kg-1 · h-1; P < 0.05). When these extrahepatic factors were considered in the calculation of the Phe oxidation rate, the intergroup differences were even more pronounced (3 vs. 7 µmol · kg-1 · h-1) than those for 13CO2 recovery data. Also, the Phe-to-Tyr conversion rate, another indicator of Phe oxidation, was significantly reduced (0.7 vs. 3.0 µmol · kg-1 · h-1).

cirrhosis; stable isotopes; 13CO2 recovery; phenylalanine breath test


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

13co2 breath tests are a simple clinical method for quantifying liver function (2), based only on hepatic oxidation of a 13C-labeled tracer. One possible liver breath test substrate is L-[1-13C]phenylalanine ([13C]Phe), which is converted to L-[1-13C]tyrosine ([13C]Tyr) and decarboxylated exclusively in the liver. Because impaired liver function may limit Phe catabolism, and thereby 13CO2 production, reduced 13CO2 release in the expired breath is used as an indicator for a disturbed Phe oxidation (4, 10, 12, 13). In fact, Burke et al. (4) showed that the severity of liver cirrhosis correlates with the suppression of 13CO2 recovery after an oral Phe load. The basis of the phenylalanine breath test to quantitate liver function is that 13CO2 recovery reflects Phe oxidation within the liver. However, three important nonhepatic effects must be considered when accounting for the kinetic and metabolic fate of the tracer from its administration to the final 13CO2 release in the expired breath.

1) Total CO2 production rate. It dilutes the 13CO2 label produced by the liver and is required for calculation of 13CO2 recovery from 13CO2 enrichment. To simplify the test conditions for a rapid clinical evaluation, most authors use estimated CO2 production rates. Because these might vary between cirrhotic and volunteer subjects, they could cause a bias for intergroup comparisons.

2) Phe turnover. Before oxidation, the tracer is diluted by Phe turnover, which equals the release of unlabeled phenylalanine by protein breakdown. Therefore, the 13CO2 recovery depends on a combination of protein breakdown and oxidation rates (28). With increasing Phe turnover rate, the 13C precursor is more diluted, and its enrichment becomes smaller. Consequently, if Phe turnover is increased, as shown by Tessari et al. (23) for cirrhotic patients, the [13C]Phe enrichment becomes smaller, thereby leading to a diminished 13CO2 recovery rate. Because of this potential mathematical coupling between Phe turnover and oxidation rate, a correct interpretation of 13CO2 recovery data as a method to quantify liver function requires estimates of Phe turnover. Previous studies indicate both increased (18, 21-23) and decreased Phe turnover rates (11) in cirrhosis. Thus published data concerning Phe turnover are not consistent. It is unclear whether Phe turnover affects 13CO2 recovery.

3) Initial distribution processes. Clinical breath tests utilize a bolus administration of tracer. In a previous bolus study, we found a sharp peak in the 13CO2 recovery kinetic curve of healthy volunteers in contrast to a plateau for cirrhotic subjects (24). A larger distribution space in cirrhotic patients may absorb the bolus and lead to a less pronounced peak and lower precursor enrichment values. This results in reduced 13CO2 recovery values, which mimic decreased oxidation.

All of these factors dilute the 13C label either before or after oxidation. The quantification of these dilution steps allows one to assess their impact on 13CO2 recovery and Phe oxidation. Therefore, we simultaneously performed a 13CO2 Phe breath test and an intravenous bolus technique to determine the processes that dilute the 13C tracer by measuring Phe turnover, conversion to Tyr, and total 13CO2 recovery with noncompartmental analysis. We also present a non-steady-state analysis based on a three-compartment Phe model, in which we considered the interaction between labeled and unlabeled Phe as a second estimate for Phe turnover and distribution spaces.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Subjects

Nine patients with anamnestically and histologically proven ethyl-toxic liver cirrhosis (54 ± 2 yr, 74 ± 4 kg body wt, 171 ± 3 cm height, and a Child-Pugh score >= 8) were studied. Table 1 shows the individual clinical and laboratory parameters used to calculate the Child-Pugh score. All patients had esophageal varices and a negative hepatitis serology. Patients with disturbed kidney function, cholinesterase activity above a threshold value of 3,200 U/l, or phenylketonuria were excluded. All pharmacological therapy was discontinued in the evening before the study day. Patients were investigated after a hospital stay of >= 3 days and adaptation to a weight-maintaining diet (~2,000 kcal/day) providing ~50% calories as carbohydrate, 20% as protein, and 30% as lipid. The control group consisted of nine healthy men (27 ± 1 yr, 73 ± 3 kg body wt, and 179 ± 3 cm height). Healthy subjects were asked to keep their usual weight-maintaining diet and avoid excessive use of alcohol, nicotine, and caffeine for 3 days. Each subject was free of metabolic, gastrointestinal, cardiovascular, neurological, or infectious disorders as verified by medical history, physical examination, and a laboratory screening test for the following parameters: aspartate aminotransferase, alanine aminotransferase, prothombin time, bilirubin, cholinesterase activity, and albumin. For two patients, measured enrichment values were not available, due either to problems during plasma sampling or to interferences in the chromatographic elution curve caused by unidentified chemical compounds. The result of the noncompartmental analysis is based on seven cirrhotic patients and nine control subjects. All subjects were instructed of the purpose, benefits, and risks of the study and gave their written consent in accordance with the protocol approved by the ethics committee of the university hospital.

                              
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Table 1.   Biochemical and clinical parameters of the liver cirrhotic patients

Isotopes

L-[1-13C]phenylalanine ([13C]Phe) and L-[1-13C]tyrosine ([13C]Tyr), all labeled >99 atom percent excess and with a comparable isotope distribution, were purchased from MassTrace (Woburn, MA). Chemical and isotopic purity was determined by gas chromatography-mass spectrometry (GC-MS). A sterile solution of [13C]Phe was prepared by the university hospital pharmacy with aseptic techniques. The solutions were tested for bacteria and pyrogen before use.

Experimental Design

The protocol started at 8:00 AM after an overnight fast. Ambient temperature was always maintained between 22 and 24°C to avoid discomfort to subjects. Polyethylene catheters were placed into a forearm vein for isotope administration, and a dorsal vein of the contralateral hand was used for blood withdrawal. This hand was wrapped around by a cuff heated at 65°C to obtain arterialized blood samples (1). Before isotope administration, total CO2 production was determined for 20 min via a flow-through hood and indirect calorimeter (DeltaTrac; Datex, Helsinki, Finland), with the subjects resting in the supine position. Baseline blood and duplicate breath samples were initially collected before dosing for the determination of the natural enrichment. Thereafter, 2 mg/kg of [13C]Phe (iv) were administered. Duplicate blood and breath samples were collected at 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, 60, 90, 120, 150, 180, 210, and 240 min after bolus administration. Blood samples were centrifuged at 4°C for 10 min at 3,000 rpm. Aliquots of plasma were stored at -20°C for analysis. Samples of expired breath for 13CO2 analysis were collected in breath collection tubes (Labco; Buckinghamshire, UK).

Analytical Methods

Plasma concentrations of unlabeled Phe (tracee) were determined as the tert-butyldimethylsilyl derivative (6) by GC-MS (5890/5970 System, Hewlett Packard, Palo Alto, CA) with [2H5]Phe as an internal standard. The ion pair at mass-to-charge ratio (m/z) 466 and 471 was measured. The [13C]Phe and [13C]Tyr labeling was determined with a methyl chloroformate derivative of Phe and Tyr (26). Mass fragmentation with electron impact ionization produced fragments of the Phe and Tyr derivatives, which lost the derivatized amino group but retained the carboxyl-labeling position. The ions, recorded in the selected ion monitoring (SIM) mode, ranged from m/z 161 to 164 for Phe and from m/z 236 to 239 for Tyr. Their signal ratios were converted to tracer/tracee ratios (also denoted as enrichment) by use of calibration graphs based on the properties of pure tracer and tracee (25). The tracer concentration values were determined from the product of the tracer/tracee ratio and concentration of tracee. The 13CO2 enrichment in the breath samples was measured by isotope ratio mass spectrometry with Europa Scientific 20/20 at Metabolic Solutions (Nashua, NH).

Calculations and Statistical Analysis

For 13CO2 recovery, let VCO2 denote the total CO2 production (ml/min) and R1,R2 the 13CO2/12CO2 ratio for the ith measurement point and the prebolus value. The expired per minute 13CO2 release for the time interval i, denoted as V13cio2, was calculated as
<A><AC>V</AC><AC>˙</AC></A><SUP>13</SUP><SC>c</SC><SUB><IT>i</IT></SUB><SC>o</SC><SUB>2</SUB><IT>=</IT><FR><NU>(R<SUB><IT>i</IT></SUB><IT>−</IT>R<SUB>0</SUB>)<IT>+</IT>(R<SUB><IT>i+</IT>1</SUB><IT>−</IT>R<SUB>0</SUB>)</NU><DE>2</DE></FR><IT>×</IT><A><AC>V</AC><AC>˙</AC></A><SC>co</SC><SUB>2</SUB> (1)
The release per time interval was calculated as V13CiO2 (ti+1 - ti) and the cumulative 13CO2 release as the sum of all release per time interval values. The 13CO2 percent recovery per time interval was defined as the ratio of expired 13C to the 13C content of the bolus. Their sum over all intervals gives the cumulative percent 13CO2 recovery.

Noncompartmental Analysis

For Phe turnover, let Q denote Phe turnover in µmol · kg-1 · h-1 and int <UP><SUB>0</SUB><SUP>∞</SUP></UP> EP dt the area under the Phe plasma enrichment (expressed as tracer/tracee ratio) curve of the [13C]Phe tracer. The Phe turnover can be calculated according to the following equation
Q<SUB>P</SUB><IT>=</IT><FR><NU>bolus (&mgr;mol/kg)</NU><DE><LIM><OP>∫</OP><LL>0</LL><UL><IT>∞</IT></UL></LIM> E<SUB>P</SUB> d<IT>t</IT></DE></FR> (2)
For the oxidation rate, the absolute Phe oxidation rate (Qox) can be calculated, according to Wolfe (28), from the product of turnover and percent 13C recovery
Q<SUB>ox</SUB><IT>=</IT>(percent<SUP> 13</SUP>C recovery) <FR><NU><IT>Q</IT><SUB>p</SUB></NU><DE><IT>f<SUB>R</SUB></IT></DE></FR> (3)
where fR describes the fraction of the produced 13CO2 that is released by breath. Following the calculations of Marchini et al. (14), we assumed this fraction to be 0.71.

Under steady state, Phe turnover equals the sum of Phe oxidation and its use for protein synthesis (Qs); hence, Eq. 3 can be transformed to
(percent<SUP> 13</SUP>C recovery) (4)

<IT>=f<SUB>r</SUB> </IT><FR><NU><IT>Q</IT><SUB>ox</SUB></NU><DE><IT>Q</IT><SUB>p</SUB></DE></FR><IT>=f<SUB>r</SUB> </IT><FR><NU><IT>Q</IT><SUB>ox</SUB></NU><DE><IT>Q</IT><SUB>S</SUB><IT>+Q</IT><SUB>ox</SUB></DE></FR><IT>=f<SUB>r</SUB> </IT><FR><NU><IT>Q</IT><SUB>ox</SUB><IT>/Q</IT><SUB>S</SUB></NU><DE><IT>Q</IT><SUB>ox</SUB><IT>/Q</IT><SUB>S</SUB><IT>+</IT>1</DE></FR>
In this equation, 13CO2 recovery depends on the ratio of oxidation to use for synthesis.

To assess the conversion rate from [13C]Phe to [13C]Tyr (Qpt), we used the equation developed by Short et al. (20), which is based on a constant relation between the contents of Phe and Tyr in average body proteins and equals
Q<SUB>pt</SUB><IT>=f</IT><SUB>tp</SUB><IT>Q</IT><SUB>P</SUB> <FENCE> <FENCE><FR><NU><LIM><OP>∫</OP><LL>0</LL><UL><IT>∞</IT></UL></LIM> E<SUB>P</SUB></NU><DE><LIM><OP>∫</OP><LL>0</LL><UL><IT>∞</IT></UL></LIM> E<SUB>&tgr;</SUB></DE></FR><IT>−</IT>1</FENCE></FENCE> (5)
where int <UP><SUB>0</SUB><SUP>∞</SUP></UP> Etau denotes the area under the [13C]Tyr enrichment curve. The enrichment used in Eq. 5 reflects tracer/tracee ratios and not the atom percent excess (APE) enrichment definition, which allows a simpler notation of the equation. On the basis of the data of Short et al. (20), the Tyr/Phe ratio ftp was taken to be 0.64.

Compartmental Analysis

The compartmental model was developed to calculate Phe distribution volume and to consider a potential mass effect of the tracer. The model is based on the structure shown in Fig. 1. It consists of a central compartment, which is the plasma sampling site and assumed to be the major site of Phe metabolism. This compartment is the entry point for both the intravenous tracer bolus and the endogenous production and is linked to two other compartments that serve as distribution spaces. The exchange rates between the compartments are assumed to be proportional to the size of the pool from which they arise. The disposal from the central compartment consists mainly of Phe utilization for protein synthesis and, to a smaller extent, Phe oxidation. An increase in the Phe plasma concentration values in response to a bolus is unlikely to cause a proportional increase in protein synthesis. Tracer and tracee compete for disposal and, as a consequence, the unlabeled concentration increases. To simulate this mass effect, we separated disposal into one component with a constant flow rate Qconst and a second component that is a linear function of the precursor concentration, just like the exchange processes. When applied to the metabolism of labeled and unlabeled Phe, this model predicts that the constant flow carries both labeled and unlabeled Phe in amounts proportional to their concentration relationship in the central pool. Immediately after bolus administration, labeled Phe and unlabeled Phe have about equal concentrations; hence, they are equally removed by a constant flow. At that time point, the absolute removal rate for unlabeled material is about one-half of its basal rate; consequently, unlabeled PHE accumulates. By fitting the response curves to actual measurements, we can determine the rate coefficients and pool sizes of endogenous Phe. These variables can then be used to estimate the concentration-dependent and -independent disposal rates. The sum of the constant and concentration-dependent disposal rates equals the turnover rate if the metabolic system is at steady state. This turnover rate can be compared with the corresponding estimate derived from noncompartmental analysis. Details of the compartmental analysis are described in the APPENDIX. The data were sufficiently described by the model when the difference between model prediction and measurement could be explained by measurement error alone.


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Fig. 1.   Structure of the 3-compartment model, adapted, to describe the decay kinetics after an intravenous phenylalanine (Phe) tracer bolus.

Statistical Analysis and Data Presentation

All data are presented as means ± SE, except for the individual data presentation in Figs. 2 and 5. Results between the different groups were compared by nonparametric statistics with the Wilcoxon rank test for significance levels of P < 0.01 or P < 0.05. 


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Fig. 2.   Individual, cumulative 13C recovery in breath shown as a percentage of administered 13C dose. A: calculation based on an estimated total CO2 production of 210 ml/min. B: calculation based on measured individual total CO2 production values. Data were collected from 9 control and 9 cirrhotic subjects.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Cirrhotic patients had significantly higher prebolus Phe plasma concentrations than healthy controls (respectively, 80 ± 9 vs. 57 ± 3 µmol/l, P < 0.05). The Phe tracer bolus increased the Phe plasma concentrations by ~10% in the control and 17% in the cirrhotic group within the 1st h of the experiment. Mean Phe concentration was constant for the rest of the experiment.

Our initial evaluation used a simple calculation of the cumulative 13CO2 recovery over 240 min from 13CO2 enrichments in the expired air, with the assumption of a total CO2 production (VCO2) of 210 ml/min for both groups. We obtained the cumulative recovery values shown in Fig. 2A. However, the individually measured VCO2 values, shown in Table 2, were significantly reduced in cirrhotic patients (171 ± 11 vs. 214 ± 10 ml/min, P < 0.05). The cumulative 13CO2 recovery as calculated from individual VCO2 values is shown in Fig. 2B, which were significantly lower in cirrhotic patients (6.6 ± 0.4 vs. 12.3 ± 0.7, P < 0.01). In contrast to the recovery values calculated with a mean estimated VCO2, there was no intergroup overlap when the measured VCO2 rates were used. Figure 3 shows the 13CO2 recovery per minute after the intravenous tracer dose. The recovery curve peaked at ~15 min in control subjects, whereas this peak did not exist in the cirrhotic group. In the later phase of the experiment, ~150 min after bolus administration, the recovery curves are comparable. Figure 4, A and B, shows mean plasma tracer enrichment values after the intravenous [13C]Phe bolus. The average enrichment values were higher in the cirrhotic group. Phe turnover rates were significantly lower in the cirrhotic group than in healthy control subjects (33 ± 3.6 vs. 43 ± 1.6 µmol · kg-1 · h-1, P < 0.05). Individual values are shown in Table 2. Figure 4, C-D, shows the response curves for the [13C]Tyr tracer enrichment, which depends on hepatic conversion of [13C]Phe to [13C]Tyr. The 13CO2 recovery per minute curve (Fig. 2) and the [13C]Tyr plasma enrichment peaked at ~15 min after bolus application in control subjects, whereas this peak did not exist in the cirrhotic group.

                              
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Table 2.   Individual total CO2 production and phenylalanine turnover rates estimated from a noncompartmental and a 3-compartmental analysis



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Fig. 3.   Time course of the per minute 13C recovery in the breath shown as a percentage of administered 13C dose after an iv bolus of 2 mg/kg [13C]Phe. Values are means ± SE of 9 healthy volunteers and 9 ethyl-toxic cirrhotic patients..



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Fig. 4.   Response curves of plasma enrichment values for [13C]Phe in control subjects (A) and cirrhotic patients (B). C: [13C-]Tyr plasma enrichments. Data are mean values ± SE for 8 control subjects and 7 cirrhotic patients.

Using Phe turnover and 13CO2 recovery values, we calculated the individual Phe oxidation rate (Fig. 5A) by use of Eq. 3. We obtained oxidation rates of 3.0 ± 0.4 µmol · kg-1 · h-1 for cirrhotic patients (P < 0.01) and 7.5 ± 0.6 µmol · kg-1 · h-1 for the control group. Figure 5B shows the individual Phe-to-Tyr conversion rates, as calculated from Eq. 5, which were significantly lower in the cirrhotic group (0.7 ± 0.3 vs. 3.0 ± 0.4 µmol · kg-1 · h-1, P < 0.05). The Phe-to-Tyr conversion rate is less than one-half of the corresponding oxidation rate.


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Fig. 5.   A: individual Phe oxidation rates, as calculated from Eq. 3. B: individual Phe-to-Tyr conversion rates, as calculated from Eq. 4. Data were collected from 8 control subjects and 7 cirrhotic patients.

The [13C]Phe plasma enrichment curves were transformed to [13C]Phe concentrations and analyzed with a three-compartmental model, as shown in Fig. 1. The model was fitted to the individual [13C]Phe plasma concentration curves with consideration of a relative change in the tracee concentration of 10% for control subjects and 17% for cirrhotic patients within 30 min after bolus application. The difference between measured and predicted tracer concentration values was, on average, ~5% of the measured value, except for one healthy control, for whom the shape of the [13C]Phe plasma concentration curve showed an atypical peak shape that could not be explained with the proposed compartmental model. Hence, the compartmental analysis is based on seven cirrhotic patients and eight control subjects. The turnover rates determined by the compartmental model are shown in Table 2 and are comparable with the noncompartmental turnover rates.

Table 3 shows all kinetic parameters derived from this compartmental analysis. In control subjects, the tracee pool size was 5.5 µmol/kg in the central compartment. On the basis of an average Phe concentration of 50 µmol/l, we calculated a distribution volume of 100 ml/kg for the central compartment. This corresponds to approximately 7-8 liters for the whole body. In contrast, the pool size of the central compartment was significantly lower in the cirrhotic group, concomitant with a significantly lower distribution volume of ~50 ml/kg. All other calculated kinetic parameters were comparable between the two groups. In a simulation approach described in the APPENDIX, we estimated how a random error of 5% in the measurement of the tracer/tracee ratios and random error in the determination of the change in the tracee concentration (10 or 30% of actual change value) would affect the precision in the determination of the turnover rate and other parameters. The last two columns of Table 3 express the precision as the standard error in the determination. All parameters can be determined with sufficient precision, which implies that the model parameters can be reliably determined on the basis of the experimental data set.

                              
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Table 3.   Parameter values determined for the 3-compartment model


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Phenylalanine is mainly catabolized in the liver, and thus a reduced oxidation might be used as a marker for liver failure. Accordingly, Burke et al. (4) used a simple Phe breath test to differentiate the various stages of liver cirrhosis. However, to facilitate test conditions, they did not consider pathophysiological changes in total CO2 production or Phe turnover rate as reasons for Phe oxidation differences between healthy control subjects and liver patients.

It was the aim of this study to evaluate the impact of tracer kinetics and total CO2 production on the 13CO2 recovery results in both normal subjects and liver patients.

Most investigators have avoided individual measurements of total CO2 production rate, for simplicity, and have assumed equal rates for healthy volunteers and cirrhotic patients (4). By contrast, we found a reduced total CO2 production rate in cirrhotic patients, which led to a lower 13CO2 recovery compared with healthy volunteers. Thus assuming equal total CO2 production rates for cirrhotic patients and healthy volunteers underestimates the difference in the 13CO2 recovery between these groups (Fig. 3). A further interpretation for the differences for total CO2 production between volunteers and cirrhotic patients is difficult, because other organs besides the liver and other oxidative processes produce CO2.

We also investigated the dilution of the tracer by Phe turnover before oxidation as a second process affecting the CO2 recovery rate. In the steady state, turnover reflects Phe release by protein breakdown, which is equal to the sum of oxidation and protein synthesis. When this equivalence is considered, Eq. 3, which describes 13CO2 recovery as a function of tracer dilution by turnover and oxidation, can be transformed to Eq. 4, in which the 13CO2 recovery depends solely on the partitioning of Phe for protein synthesis and oxidation. Equation 3 calculates the Phe oxidation rate after determining 13CO2 recovery and Phe turnover, whereas Equation 4 provides a more intuitive physiological understanding of the 13CO2 recovery. Therefore, our results can be interpreted from different points of view.

Our results indicate a reduction in both Phe turnover rate and 13CO2 recovery in cirrhotic patients. According to Eq. 3, this means a massive reduction of the oxidation rate in cirrhotic patients. Physiologically, the reduced turnover means a lower release of Phe by protein breakdown and therefore leads to a higher precursor enrichment for oxidation. However, this does not necessarily mean that the 13CO2 recovery is increased if the oxidation step per se is reduced. Therefore, it is important not to use Phe turnover as an isolated factor to interpret 13CO2 recovery data.

According to Eq. 4, 13CO2 recovery reflects the relationship in the use of Phe between oxidation and protein synthesis. For cirrhotic patients, a reduced 13CO2 recovery would mean a shift in the partitioning toward synthesis at the expense of oxidation. By use of our experimental results, the ratio between oxidation and synthesis was twice as high in healthy volunteers, indicating a clear oxidative defect in cirrhotic patients. Phe oxidation data obtained for the control subjects are in the range of the previously reported data of Marchini et al. (14).

We measured the Phe-to-Tyr conversion rate as another indicator for Phe oxidation. Values for the control subjects were less than those reported by Short et al. (20) (3.0 vs. 3.9 µmol · kg-1 · h-1) and less than one-half of the Phe oxidation rates for both groups. This is surprising, because most of the tracer released as 13CO2 is derived from the Phe-to-Tyr conversion within the liver. A possible explanation is that only a fraction of the converted Tyr reaches the circulation. The enzymes for both the Phe-to-Tyr conversion and Tyr decarboxylation are membrane bound, which should channel a substantial fraction of newly synthesized Tyr directly into decarboxylation (19).

The Phe-to-Tyr conversion in cirrhotic patients is ~25% of the control value, whereas the oxidation accounts for 40%. In contrast to the Phe-to-Tyr conversion, the calculated Phe oxidation rate depends on the 13CO2 metabolism, which also includes 13CO2 fixation by hepatic gluconeogenesis and urea production. A reduced fixation in the cirrhotic patients might cause the smaller intergroup difference in the oxidation rate. This would imply that the intergroup difference for oxidation was underestimated. However, any interpretation beyond that is difficult, because the determination of the Phe-to-Tyr conversion is subject to a large measurement error.

The kinetic profiles of the 13CO2 recovery per minute and the enrichment of the conversion product [13C]Tyr, as shown in Fig. 4C, correspond to each other. Common for both processes is the Phe tracer dilution, Phe tracer uptake by the liver, and Phe hydroxylation. Because the difference in plasma Phe enrichment between these two groups is not parallel to the results of 13CO2 recovery, the rate of Phe tracer uptake and its subsequent hydroxylation are assumed to be of rate-limiting importance. Hence, this dynamic response to the bolus qualitatively supports the notion of a reduced hepatic oxidation.

A third interfering factor for 13CO2 breath tests might be the initial tracer distribution over the various body spaces, as these distribution volumes can vary from healthy volunteers to cirrhotic patients. We have found intergroup differences for the kinetic curves of the 13CO2 recovery per minute (Fig. 3). The control curves peak within 10 min after bolus administration, whereas the corresponding responses for patients approximated a plateau at significantly lower levels between 15 and 60 min after bolus. By contrast, the later "wash-out" parts of the curves were identical. This plateau was present in all individual response curves and therefore is not due to an artifact of averaging peaks with maximal values at different time points. It could be argued that cirrhotic patients might have a higher distribution volume for Phe, resulting in a less pronounced peak in the observed 13CO2 recovery and a lower enrichment in the plasma Phe tracer, the precursor for oxidation. To exclude this possibility, we calculated the Phe distribution volume by a three-compartment model. The results of the compartmental modeling do not support this alternative interpretation: the determined central Phe body pools, in fact, were significantly lower in the cirrhotic group, with equal exchange rates between the central and the two peripheral pools. The different peak shapes in cirrhotic patients might be explained by a serious reduction of liver cell mass, the activity of the phenylalanine hydroxylase within the liver, or hepatic Phe uptake.

It is noteworthy that we found a reduced Phe turnover rate in cirrhotic patients, whereas other groups have reported just the opposite (18, 22, 23). This divergence could refer to differences either of the methodological approach or the investigated patient population. First, we used a bolus, non-steady-state kinetic analysis, whereas the other investigators applied a primed constant infusion approach. The turnover rates derived from our noncompartmental and three-compartmental approaches closely match the estimates from steady-state primed constant infusion studies for the control subjects as reported by Marchini et al. (14), Matthews et al. (15), and Tessari and colleagues (22, 23). With both analytical methods, ignoring and considering potential mass effects of the tracer, we have found a lower Phe turnover for cirrhotic patients. Because our approaches deliver comparable Phe turnover rates for the control subjects, we expect that the cirrhotic values reflect reality and are not a methodological artifact.

Our patients were, on average, ~20 yr older than the control subjects, which might explain the turnover differences compared with other reports. The oxidation and turnover rates for control subjects, however, agree well with the results for older control subjects in other studies (21). In addition, Fukagawa et al. (9) could not find any age dependency in the basal protein turnover rate as measured by a leucine tracer. Similar findings were reported by Morrison et al. (16). This might also hold for the Phe turnover rate, because it was shown in healthy subjects that the basal turnover rate of an essential amino acid depends on the protein turnover rate (3). However, for subjects older than 60 yr, Welle et al. (27) found a reduced protein turnover, which was linked to a reduced lean body mass. Although our patients were >= 10 yr younger, we cannot exclude such an age effect in our study.

Finally, the discrepancy between the present and previous studies may be due to different disease states of the investigated patient populations. In two subsequent studies, Tessari and colleagues (22, 23) investigated Phe kinetics in 24 patients (by Child-Pugh scores 17 Child A, 6 Child B, and 1 Child C patients), including just 9 with ethyl-toxic genesis. In contrast, we studied only ethyl-toxic patients who had signs of a reduced hepatic synthetic capacity. Compared with the studies of Tessari and colleagues, our patients were in a more advanced stage of their disease, and the etiology of the illness from our patient population was more homogeneous. Fernandez-Rodriguez et al. (8) reported that an advanced stage of liver cirrhosis was associated with lower plasma IGF-I levels. These low levels of an important anabolic agent might lead to an increased net catabolism. Dichi et al. (7) showed that cirrhotic patients in the Child-Pugh classes B and C have a higher fasting net protein catabolism than aged-matched controls. The increased catabolism is reflected in a reduced body protein content: the proportion of patients with a muscle mass index below the 5th percentile and with low values of visceral proteins increases with the severity of liver disease (5). The progressive loss of muscle mass could lead to a reduced amino acid turnover from protein breakdown. This effect might contribute to the differences in Phe turnover between cirrhotic patients in our study and those of previous studies, as our patients were in a more advanced disease state.

A reduced Phe turnover leads to a diminished [13C]Phe dilution, and thus to a higher enrichment of the precursor for the oxidation. As a consequence, this effect contributes to an overestimation of 13CO2 recovery. Neglecting a potential association of advanced disease state with lower Phe turnover could cause a systematic bias in the interpretation of 13CO2 recovery data.

In summary, we investigated metabolic and kinetic parameters that influence the 13CO2 recovery of an intravenous [1-13C]Phe breath test by assessing Phe oxidation as an indicator for liver function. By assuming the same estimated total CO2 production rates for control subjects and cirrhotic patients, we obtained average cirrhotic 13CO2 recovery values that were 68% of the control values. However, the individual total CO2 production rates were lower in cirrhotic patients. Using the measured lower production rate yielded cirrhotic recovery values that were 54% of the controls, with no overlap between the groups. The 13CO2 recovery values also depended on the Phe tracer dilution before oxidation. This dilution is modified by Phe turnover and, correspondingly, we found a significantly lower Phe turnover in cirrhotic patients. When the impact of the Phe tracer dilution on the 13CO2 recovery was considered, we obtained oxidation rates for cirrhotic patients that were 40% of the control values. This difference should be <40%, if we consider that the Phe-to-Tyr conversion rates for cirrhotic patients were 25% of their control values. We found no indication that the dynamic distribution of the Phe tracer could lead to an underestimation of the 13CO2 recovery. Total CO2 production and Phe turnover do not reflect actual liver metabolism but are influenced by chronic liver disease. Neglecting these factors led in the present case to a significant underestimation of the 13CO2 recovery differences between healthy volunteers and liver-cirrhotic patients. For a beginning cirrhosis, Phe turnover and total CO2 production could be increased and cause an overestimation of the differences in oxidation. Hence, our results are difficult to generalize. In view of this uncertainty, we would recommend for the clinical setting individual measurements of total CO2 production, although this complicates the test procedure.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

Compartmental Analysis

The compartmental model is based on the structure shown in Fig. 1. Compartment 1 is linked to two other compartments, which serve as distribution spaces. The amount of Phe tracer (µmol/kg) in the compartments is denoted as x1 for the central compartment and x2 and x3 for the two distribution spaces. The amount of endogenous, unlabeled Phe is denoted as X1, X2, and X3. The exchange rates between the compartments are described as linear functions of the donor pool, i.e., the flow from compartments 3 to 1 is k13x3 for labeled and k13X3 for unlabeled Phe. Both the intravenous tracer bolus and the endogenous production feed into the central compartment. The loss from this compartment is separated into one component with a constant flow rate Qconst and a second component, klinearx1, which is a linear function of the precursor concentration just like the exchange processes. The constant loss carries both labeled and unlabeled Phe. The relative proportion of labeled to unlabeled material of this flow equals that in compartment 1, the donor site for this flow. The relative proportion of the labeled material is x1/(x1 + X1), and the loss of labeled material via the constant flow equals Qconstx1/(x1 + X1). Conversely, the loss of unlabeled material is QconstX1/(x1 + X1). For a single pool, we can define the change per time unit as the difference between all flows entering the pool and all flows leaving the pool. These considerations are the basis for the following equations to describe the changes in the pool sizes in response to a tracer bolus
<AR><R><C><FR><NU>d<IT>x</IT><SUB>1</SUB></NU><DE>d<IT>t</IT></DE></FR><IT>=</IT></C><C>−(<IT>k</IT><SUB>21</SUB><IT>+k</IT><SUB>31</SUB>)<IT>x</IT><SUB>1</SUB><IT>−Q<SUB>const</SUB> </IT><FR><NU><IT>x</IT><SUB>1</SUB></NU><DE><IT>x</IT><SUB>1</SUB><IT>+X</IT><SUB>1</SUB></DE></FR><IT>−k<SUB>linear</SUB>x</IT><SUB>1</SUB><IT>+k</IT><SUB>12</SUB><IT>x</IT><SUB>2</SUB><IT>+k</IT><SUB>13</SUB><IT>x</IT><SUB>3</SUB></C></R><R><C><FR><NU>d<IT>x</IT><SUB>2</SUB></NU><DE>d<IT>t</IT></DE></FR><IT>=</IT></C><C>−<IT>k</IT><SUB>12</SUB><IT>x</IT><SUB>2</SUB><IT>+k</IT><SUB>21</SUB><IT>x</IT><SUB>1</SUB></C></R><R><C><FR><NU>d<IT>x</IT><SUB>3</SUB></NU><DE>d<IT>t</IT></DE></FR><IT>=</IT></C><C>−<IT>k</IT><SUB>13</SUB><IT>x</IT><SUB>3</SUB><IT>+k</IT><SUB>31</SUB><IT>x</IT><SUB>1</SUB></C></R></AR> (A1)
A corresponding system of equations holds for endogenous, unlabeled Phe in the compartments X1, X2, and X3. Unlike the tracer, the tracee constantly feeds at the central compartment (rate Ra), which is included in the corresponding equation. The losses via Qconst depend on both the labeled and unlabeled pools, which link the tracer response to that of the unlabeled compound. This approach deviates from standard compartmental modeling; nevertheless, the equations describing the changes of the tracer and tracee systems can be solved simultaneously by use of numerical approaches (see Press et al., Ref. 17, chapter 15). Thus the time course of the tracer and tracee pools can be calculated, and the ratio x1/X1 can be estimated for each measurement point. These estimated [13C]Phe tracer/tracee ratios, together with the time profile of the concentration of unlabeled Phe, can be fitted to the corresponding measurements by use of nonlinear regression (Press et al., chapter 14). We expressed the mean concentration profile for both groups as a percentage of the prebolus values and used these mean relative changes at 30 and 240 min after the bolus as measurements. The fitting procedure allows us to determine the pool sizes and rate coefficients X1, X2, X3, k21, k31, Qconst, and klinear. The precision in the determination of these parameters depends on the measurement error. To assess this impact, we used a Monte Carlo approach (Press et al., chapter 14.5) and simulated 50 variations from a enrichment curve, presenting the response of a typical healthy control subject. In each simulation, this curve was randomly disturbed by an average measurement error of 5% for a single measurement point. The relative changes in the Phe tracee concentrations were disturbed at two error levels of 10 and 30% of the actual change value. From each of these 50 curves, we determined the six unknown parameters x1 to klinear and obtained 50 different determinations for x1 to klinear. Their standard deviation was taken as a measure for the precision in the parameter determination. To assess the remaining coefficients, we assumed that the Phe metabolism of the host is at steady state for the prestudy condition. This implies zero net balances between the compartments for endogenous Phe, or k21X1 k12X2; k31X1 = k13X3. These equations can be solved for k12 and k13; i.e., k12 = k21X1/X2 and k13 = k31X1/X3.


    ACKNOWLEDGEMENTS

This study was supported by a research grant from the university medical school at Ulm (P.328).


    FOOTNOTES

Address for reprint requests and other correspondence: J. A. Vogt, Sektion APV, Universitätsklinik für Anästhesiologie Ulm, Parkstrasse 11, 89070 Ulm, Germany (E-Mail: Josef.Vogt{at}medizin.uni-ulm.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

August 20, 2002;10.1152/ajpendo.00311.2001

Received 20 July 2001; accepted in final form 26 July 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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Am J Physiol Endocrinol Metab 283(6):E1223-E1231
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