MODELING IN PHYSIOLOGY
Protein kinetics determined in vivo with a multiple-tracer, single-sample protocol: application to lactase synthesis

Mary A. Dudley1, Douglas G. Burrin1, Linda J. Wykes2, Gianna Toffolo3, Claudio Cobelli3, Buford L. Nichols1, Judy Rosenberger1, Farook Jahoor1, and Peter J. Reeds1

1 United States Department of Agriculture/Agricultural Research Service Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, Texas 77030; 2 School of Dietetics and Human Nutrition, McGill University, Montreal, Quebec, Canada H9X 3V9; and 3 Department of Electronics and Informatics, University of Padua, Padua, Italy 35131

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 ± 1.1%/min, 17.4 ± 11 %/min, and 0.089 ± 0.02 %/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.

brush border; compartmental modeling; fractional synthesis rate; glycoprotein; isotopomer; in vivo protein synthesis; lactase phlorizin hydrolase

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

LACTASE PHLORIZIN HYDROLASE (LPH), the enzyme responsible for the digestion of the milk sugar lactose, is a type 1 glycoprotein found in the brush-border (BB) membrane of small intestinal enterocytes (20). In principle, the regulation of BB LPH abundance can involve regulation at the level of mRNA abundance (15, 18, 19, 27), as well as at the levels of the two glycosylation and terminal proteolytic steps in its posttranslational processing (3, 6, 8, 26). Thus LPH is not only of vital physiological importance, but its synthesis can be viewed as a particularly appropriate general model for the synthesis of membrane glycoproteins.

To fully analyze the regulation of LPH (or any glycoprotein) synthesis, LPH mRNA abundance as well as the rates of translation and posttranslational modification must be determined. Measurement of LPH mRNA requires only a single, small piece of tissue (26). However, the fractional synthesis rates (FSR) of LPH precursors and the mature enzyme differ by more than an order of magnitude (8, 9), so that their simultaneous determination requires measurement for <60 min for precursor LPH proteins and >4 h for BB LPH.

To properly quantify individual steps during in vivo posttranslational LPH polypeptide synthesis, the conventional approach entails the infusion of a tracer amino acid so that the blood and mucosal free amino acid pools are quickly brought to isotopic equilibrium. The tracer-to-tracee ratios of the precursor and mature BB LPH polypeptides are then determined in tissue samples obtained at appropriately timed intervals during the infusion, and the kinetics of synthesis can be estimated reliably (8, 9). Such studies are routinely carried out in laboratory rodents, using protocols in which multiple animals are killed at intervals throughout the infusion (9). However, using multiple animals for each time point introduces interanimal statistical variation. Even in larger animals, in which the removal of multiple tissue samples is feasible (8), ethics demand that the experiment be performed under anesthesia. This procedure is in principle undesirable for no other reason than the likelihood that anesthesia alters LPH synthesis. Clearly, though, obtaining multiple tissue samples is impossible in humans. Therefore, there is a need for an approach to the measurement of the dynamics of the LPH system that requires only small, single tissue samples.

Selected ion-monitoring gas chromatography-mass spectroscopy (GCMS) has the ability to quantify different isotopomers of the same tracer amino acid in a single analysis (1, 2, 5). For the present study we reasoned that, provided a sufficient number of isotopomers of the same amino acid could be infused in an overlapping fashion, complex kinetic analysis could be performed with labeling data obtained from a single tissue. In other words, multiple isotopomers could be substituted for multiple time points. This study describes the principles of this new method and its validation in a case study in which six amino acid tracers were used to define the kinetics of precursor and BB LPH synthesis in a single tissue sample obtained at the end of the infusion. Pigs were used for the study because they are an apposite model of human gastrointestinal function and development, the focus of our interest (26). However, this approach could be readily applied to many other complex systems and should be particularly useful in the analysis of protein labeling kinetics in human subjects.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Animals. The protocol for these studies was approved by the Animal Care and Use Committee of Baylor College of Medicine and was carried out in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals [DHHS Publication No. NIH 85-23, Revised 1985, Office of Science and Health Reports, Bethesda, MD 20892].

Six 5-day-old piglets were purchased from a colony of standard commercial pigs at Texas A & M University (College Station, TX). The following day, catheters were implanted in the jugular vein and carotid artery (10). The animals were allowed to recover from the surgery for 3 days, and after an overnight fast, each conscious, catheterized piglet received infusions of four stable isotopomers of phenylalanine and two of leucine via the jugular vein catheter. During the infusion, blood samples were collected from the arterial catheter at timed intervals. The pigs were killed at the end of the infusion, and tissue was collected as previously described (10).

Experimental design. The infusion protocol that we have used for this study takes advantage of the fact that multiple stable isotopomers of an amino acid can be obtained and that selected ion monitoring of the mass spectrum allows the independent and simultaneous quantitation of their tracer-to-tracee ratios. The isotopomers are infused in an overlapping fashion such that each additional isotopomer is infused for a progressively shorter period of time. All infusions end at the same time, at which point a single tissue sample is obtained. The relative isotopic enrichment of each tracer in the sample is therefore directly analogous to the conventional approach of a single tracer and multiple samples.

Figure 1, A and B, illustrates the principle of the overlapping infusion design, using five different isotopomers of the same tracer amino acid. The various tracers are infused for 180, 90, 60, 30, and 15 min (Fig. 1A), with all infusions ending at the same time. In the single sample taken at the end of the combined infusion, the tracer-to-tracee ratio of the second tracer represents that of the first isotopomer as though it had been infused for only 90 min. Likewise, the tracer-to-tracee ratios of the third, fourth, and fifth isotopomers represent the tracer-to-tracee ratio of the first isotopomer as though it had been infused for 60, 30, and 15 min. The tracer-to-tracee ratio of each isotopomer in each LPH precursor and the mature BB LPH polypeptide in the single tissue sample obtained at the end of the infusion can therefore be used to construct a curve of the kinetics of labeling of each protein (Fig. 1B).


View larger version (13K):
[in this window]
[in a new window]
 


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 1.   A: theoretical labeling pattern for 5 phenylalanine isotopomers in a hypothetical protein after an overlapping infusion. X-axis represents time elapsed from start of infusion of the first isotope. B: theoretical tracer-to-tracee ratios with time of a hypothetical protein calculated from tracer-to-tracee ratio of each phenylalanine isotopomer in the sample at the end of the infusion. X-axis represents time elapsed from start of infusion of individual isotopomers.

Infusion study. The animals received a total of six stable amino acid isotopomers: two leucine tracers ([5,5,5,2H3]- and [13C1]leucine) and four phenylalanine tracers ([13C1]-, ring [2H5]-, ring [13C6]-, and [2H8]phenylalanine). The tracers were purchased from Cambridge Isotope Laboratories (Andover, MA). All were the L-form of the amino acid and were checked for chemical and isotopomeric purity.

Figure 2 depicts an infusion protocol representative of that used for the study. The total infusion time was 360 min, with infusions of different tracers beginning at timed intervals from 0 to 345 min. All the pigs received the leucine tracers for 4.5 h ([13C1]leucine) or 6 h ([2H3]leucine). These tracers were used to define additional points on the BB LPH labeling curve. [13C6]phenylalanine was infused for 60 min in all the animals. To properly define the kinetics of labeling of the LPH precursor polypeptides, [13C1]phenylalanine was infused for 180 min in three of the animals and for 230 min in the other three animals, [2H5]phenylalanine was infused for 90 min in three animals and for 120 min in the other three animals, and [2H8]phenylalanine was infused for 15 min in three animals and for 30 min in the other three animals. [13C1]leucine, [2H3]leucine, and [2H8]phenylalanine were infused at 20 µmol · kg-1 · h-1. [13C1]-, [13C6]-, and [2H5]phenylalanine were infused at a rate of 10 µmol · kg-1 · h-1. The volume infusion rate was 1 ml · kg-1 · h-1.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Representative infusion protocol.

Arterial blood samples were taken at frequent intervals throughout the infusion and were consistently taken immediately before and 10 min after a new tracer was added to the infusate (Fig. 2). These samples were particularly important because plasma is the only pool in which we can directly test the validity of the single-sample, multiple-tracer overlapping infusion by comparing its results with those obtained with the conventional multiple-sample, single-tracer infusion.

Analyses. The immunoisolation and purification of LPH polypeptides using the hybridoma PBB3/7/3/2 have been described previously (8, 10, 13, 24). The preparation of plasma and mucosal free amino acid pools and LPH polypeptides for GCMS analysis has also been described previously (10, 13). GCMS was performed with n-propyl ester heptafluorobutyramide derivatives as previously described, using methane negative chemical ionization, with helium as the carrier gas on a Hewlett Packard 5988A SC linked to an HP 5890 H quadrupole gas chromatograph. One-microliter samples were injected using a 30:1 split onto a silica-based DB5 capillary column (30 m × 0.2 mm, 1-µm film thickness; J & W Scientific, Folsom, CA). Chromatography was effected with a linear temperature gradient (80-250°C at 10°C/min). The isotope ratios were calculated using ions at a mass-to-charge ratio (m/z) of 349, 350, and 352 for the isotopomers of leucine and m/z of 383, 384, 388, 389, and 391 for the isotopomers of phenylalanine.

The data on the ion abundances of the respective amino acid isotopomers were converted to tracer-to-tracee ratios by the matrix method (1, 2, 5). The calculation involved the insertion into a matrix of the isotopic enrichments of unenriched material as determined by the elemental composition of the heptafluorobutyramide derivative and of the tracer material as measured directly. Due account was taken of the influence of the mass + 1, mass + 2, etc., isotopomers on the enrichment of isotopes of lesser mass. The software that enables this calculation can be obtained from Dr. D. L. Hachey at the Children's Nutrition Research Center. The tracer-to-tracee ratios were then normalized to a constant infusion rate (10 µmol tracer · kg-1 · h-1), and the leucine data were normalized to the phenylalanine data by taking account of the differences in the relative molar fluxes of leucine and phenylalanine as predicted by their relative contributions to body protein (14, 25). This method was chosen for ease of data presentation. However, the protein-labeling kinetics are based on precursor and product data, so that the normalization procedure has no bearing on the calculation of the final protein kinetic parameters.

Kinetic analysis. Linear, time-invariant compartmental modeling is appropriate to describe LPH tracer kinetics, since the tracee system is in steady state (4). The model (8) is shown in Fig. 3. It assumes a monocompartmental description for plasma and mucosal free amino acids and for the three LPH polypeptides, with unidirectional fluxes from plasma to mucosal free amino acids and thence to the high mannose precursor (proLPHh), the complex glycosylated precursor (proLPHc), and mature BB LPH.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Compartmental model for lactase phlorizin hydrolase (LPH) polypeptide synthesis. BB, brush border; proLPHh, high-mannose LPH precursor, proLPHc, complex glycosylated LPH precursor.

The plasma free amino acid submodel consists of a single compartment having the tracer infusion as known input. For this system, the mass balance equation for the plasma free amino acid tracer mass, mp, is
<FR><NU>d<IT>m</IT><SUB>p</SUB>(<IT>t</IT>)</NU><DE>d<IT>t</IT></DE></FR> = −<IT>k</IT><SUB>p</SUB> · <IT>m</IT><SUB>p</SUB>(<IT>t</IT>) + u (1)
where kp (min-1) is the plasma free amino acid fractional turnover rate, u is the infusion rate, and t is time (min). Dividing both terms of the equation by the plasma free amino acid tracee mass, Mp, yields the equation for the tracer-to-tracee ratio (Zp)
<FR><NU>dZ<SUB>p</SUB></NU><DE>d<IT>t</IT></DE></FR> = −<IT>k</IT><SUB>p</SUB> · Z<SUB>p</SUB>(<IT>t</IT>) + <IT>A</IT> (2)
where A is a constant.

The mucosal free amino acid, proLPHh, proLPHc, and BB LPH submodels are described by four precursor-product models, e.g., for the mucosal free amino acid submodel, the plasma free amino acid pool is the precursor and the mucosal free amino acid pool is the product. The mass balance equation for the tracer is
<FR><NU>d<IT>m</IT><SUB>B</SUB>(<IT>t</IT>)</NU><DE>d<IT>t</IT></DE></FR> = <IT>k</IT><SUB>1</SUB> · <IT>m</IT><SUB><IT>A</IT></SUB>(<IT>t</IT>) − <IT>k</IT><SUB>2</SUB> · <IT>m</IT><SUB>B</SUB>(<IT>t</IT>) (3)
where mA and mB denote the precursor and product tracer mass, respectively, k1 (min-1) is the rate constant describing transfer from A to B, and k2 (min-1) is the fractional turnover rate of B. By dividing both sides of Eq. 3 by the tracee product mass (MB), we obtain the equation for the tracer-to-tracee ratio (ZB)
<FR><NU>dZ<SUB><IT>B</IT></SUB>(<IT>t</IT>)</NU><DE>d<IT>t</IT></DE></FR> = <FR><NU><IT>k</IT><SUB>1</SUB><IT>M</IT><SUB><IT>A</IT></SUB></NU><DE><IT>M<SUB>B</SUB></IT></DE></FR> · Z<SUB><IT>A</IT></SUB>(<IT>t</IT>) − <IT>k</IT><SUB>2</SUB> · Z<SUB><IT>B</IT></SUB>(<IT>t</IT>) = FSR · Z<SUB><IT>A</IT></SUB>(<IT>t</IT>) − <IT>k</IT><SUB>2</SUB> · Z<SUB><IT>B</IT></SUB>(<IT>t</IT>) (4)
where ZA and ZB denote the tracer-to-tracee ratios of precursor and product, respectively, MA is the precursor tracee mass, and FSR (min-1) denotes the fractional synthesis rate of the product, i.e., the rate at which the product is synthesized from the precursor per unit of product.

If A is the sole precursor of B, then k1MA represents the production rate of B. At steady state this is equal to its turnover rate (k2MB), and FSR = k2. In the general case, where B can be produced from sources other than A, then k1MA <=  k2MB and FSR <=  k2. Thus the ratio FSR/k2 quantifies the contribution of the precursor to the product turnover, e.g., for the mucosal free amino acid submodel it measures the contribution of the plasma free amino acid pool to the mucosal free amino acid turnover. By multiplying FSR/k2 of each submodel between plasma and each LPH polypeptide, the contribution of plasma free amino acids to that LPH polypeptide turnover can also be quantified. FSR/k2 also provides a model prediction of the ratio between the plateau isotopic enrichments ZA and ZB, since under tracer steady-state conditions, Eq. 4 becomes
<FR><NU>dZ<SUB><IT>B</IT></SUB>(<IT>t</IT>)</NU><DE>d<IT>t</IT></DE></FR> = 0 = FSR · Z<SUP>ss</SUP><SUB><IT>A</IT></SUB> − <IT>k</IT><SUB>2</SUB> · Z<SUP>ss</SUP><SUB><IT>B</IT></SUB> (5)
and thus
<FR><NU>FSR</NU><DE><IT>k</IT><SUB>2</SUB></DE></FR> = <FR><NU>Z<SUP>ss</SUP><SUB><IT>B</IT></SUB></NU><DE>Z<SUP>ss</SUP><SUB><IT>A</IT></SUB></DE></FR> (6)
where the superscript ss indicates the plateau value of Z.

Parameters kp and A were estimated by fitting Eq. 1 to the plasma free amino acid tracer-to-tracee data. Parameter estimation was performed twice, first by applying the conventional approach to a single tracer in multiple blood samples and then using the novel approach of basing the calculation only on the multiple tracers in a single, terminal blood sample. In the latter case, u represents the constant infusion rate to which the data were normalized.

For each precursor product submodel, parameters FSR and k2 were estimated by fitting Eq. 4 to the product tracer-to-tracee ratio, assuming the precursor tracer-to-tracee ratio as the known input. Model identifications were performed using weighted nonlinear least squares as implemented in the SAAMII software (SAAM Institute, University of Washington, Seattle, WA). Weights were chosen optimally, i.e., equal to the inverse of the variance of the measurement errors, which were assumed to be independent, Gaussian with zero mean and with a constant coefficient of variation. The respective coefficients of variation for the measurements of the tracer-to-tracee ratios were 16% for [13C1]leucine and [13C1]phenylalanine, 8% for [2H3]leucine, 6% for [2H5]phenylalanine and [13C6]phenylalanine, and 3% for [2H8]phenylalanine. Data are expressed as means ± SD.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Figure 4 depicts the time course of labeling of the plasma free amino acid pools in two representative animals (animals 1 and 5). The figure compares the tracer-to-tracee ratios of [2H3]leucine and [13C1]phenylalanine in plasma samples obtained at multiple time points during the infusion with the values of all six tracers determined in the blood sample taken at the end of the infusion. In animal 1, the tracers were infused for 15, 60, 90, 180, 270, and 360 min; in animal 5, the infusion times were 30, 60, 120, 230, 270, and 360 min. Plasma tracer-to-tracee ratios rose rapidly and by 60 min were at isotopic steady state. Table 1 shows the estimates of plasma amino acid kinetics calculated with multiple samples and a single tracer as well as with multiple tracers in the single, terminal sample. The rate constants derived from the two approaches (5.2 ± 1.1% and 5.1 ± 1.6% per minute) were very similar. It is notable that the coefficient of variation of the estimate from multiple samples was uniformly higher than that derived from the six isotopomer values, presumably because the former includes all sources of variation whereas the latter accommodates only the known analytical (mass spectrometric) error.


View larger version (22K):
[in this window]
[in a new window]
 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4.   A comparison of plasma free leucine and phenylalanine pool labeling from multiple samples with a single tracer and predicted kinetics from 6 tracers in the single terminal sample. A: animal 1 with high between-sample variation. B: animal 5 with low between-sample variation. Lines are drawn from kinetic parameters derived from the multiple-sample approach.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Plasma free amino acid pool turnover rates estimated from the conventional multiple-sample, single-tracer infusion and the novel single-sample, multiple-tracer infusion

Figure 5 shows the summation of the data from the six animals with the calculated rate constants and plasma free amino acid contributions in Table 2. The equilibration of the mucosal free amino acid pool was rapid (12.7% per minute), and this pool reached steady state between 60 and 90 min. At steady state, the tracer-to-tracee ratio of the mucosal free amino acid pool was on average 77% (Table 2) of the plasma free pool for both leucine and phenylalanine, indicating that the plasma free amino acid contribution to the mucosal free amino acid turnover, shown in Table 2, is 77%.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5.   Composite data from 6 animals for labeling of plasma and mucosal free amino acid pools and of proLPHh, proLPHc, and BB LPH. Lines are drawn to simplify presentation of data. bullet , [2H8]phenylalanine (n = 3); open circle , [13C6]phenylalanine (n = 6); black-triangle, [2H5]phenylalanine (n = 3); triangle , [13C1]phenylalanine (n = 3); square , [13C1]leucine (n = 6); black-square, [2H3]leucine (n = 6).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Fractional turnover rates of plasma and mucosal free AA, synthesis rates of LPH polypeptides, and plasma free AA contributions for 6 animals

After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of the gels, LPH polypeptides that had been immunoisolated from the solubilized, scraped mucosa separated into four bands (8, 10). We have previously identified these as two forms of precursor BB LPH [the high-mannose form, proLPHh (200 kDa), and the complex glycosylated protein, proLPHc (220 kDa)] and two forms of BB LPH (the 160-kDa polypeptide and a dimer of BB LPH with an apparent molecular mass of ~240 kDa) (3, 8, 9, 10). The LPH polypeptides are synthesized in the following order: proLPHh, proLPHc, and the mature BB enzyme (8, 10).

The measured tracer-to-tracee ratios of each amino acid isotopomer in proLPHh, proLPHc, and BB LPH are also shown in Fig. 5. The plasma amino acid fractional turnover rate, the FSR of LPH polypeptides, and the percentage of plasma contributions are presented in Table 2. It is clear from Fig. 5 that the sequence of labeling of LPH polypeptides in these pigs was the same as the sequence we have previously reported (8, 10).

The tracer-to-tracee ratios of proLPHh rose more slowly than the the tracer-to-tracee ratios of the mucosal free pool, and the protein had a FSR of 3.3% per minute. It reached isotopic steady state by 2 h, at which time the tracer-to-tracee ratio of proLPHh was 70% of the steady-state tracer-to-tracee ratio of the mucosal free amino acid pool. The values for leucine and phenylalanine were identical. This indicates that the mucosal free amino acid pool contribution to proLPHh turnover was 70%. As a result, the plasma free phenylalanine and leucine contribution to proLPHh turnover was 54%.

Although not evident from Fig. 5, the model-calculated FSR of proLPHc was very high (17.4% per minute). The steady-state tracer-to-tracee ratios of proLPHc leucine and phenylalanine were not significantly different from the proLPHh values, indicating that proLPHh is the sole precursor of proLPHc. Nominally, the plasma free pool contribution to proLPHc (Table 2) was 52%. It was notable, however, that the coefficient of variation of the FSR of proLPHc (56%) was very high. This was not due to analytical problems but illustrates the fact that if the FSR of a product exceeds that of its precursor, then the product FSR cannot be determined with good precision.

There was a substantial time delay (60-70 min) before tracer was detectable in BB LPH. Thereafter, the tracer-to-tracee ratio of BB LPH and its 240-kDa dimer rose in parallel (data not shown). Neither reached isotopic steady state during the 6-h infusion (Fig. 5), and the model indicated an average FSR of 0.089% per minute.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The accurate in vivo measurement of glycoprotein synthesis is complicated by the cellular and metabolic heterogeneity of the tissue in which the polypeptide is produced and by the complex series of intracellular events that result in the expression of the mature BB protein (7-11). LPH, for example, is synthesized only in the villus enterocyte (13, 17, 20), and the free amino acid pools of these cells may be derived from the arterial circulation, from intracellular proteolysis, or from amino acids transported across the apical membrane from the intestinal lumen. Thus the intracellular free pool from which LPH is formed may be compartmentalized, and its composition will be influenced by whether an animal is fasted or fed and, in fed animals, by the amino acid composition of the diet.

Two conditions must be met for the valid measurement of the in vivo synthesis rates of LPH polypeptides. First, because LPH synthesis is typically measured in immunoisolates of solubilized mucosal membranes, which invariably contain both slowly turning over BB protein as well as the rapidly turning over precursor polypeptides (7-10, 23), the precursor and mature BB polypeptides must be separated. Precursor and BB polypeptides are present within the cell in markedly different amounts, with the BB form of the enzyme generally representing >90% of the immunoisolated protein (8-10, 12). As a result, unless the precursor and mature BB proteins are analyzed separately, the interpretation of in vivo labeling data is quantitatively inaccurate. Analysis of a coprecipitate of precursor and BB hydrolase polypeptides is akin to calculating protein synthesis rates from the labeling of a mixture of free and protein-bound amino acids.

Second, in the case of LPH, ~1 h is required for the translocation of the complex glycosylated precursor from the Golgi apparatus to the BB membrane (6, 7). Thus an infusion protocol must maintain the tissue free amino acid pools at isotopic equilibrium for prolonged periods. Infusion protocols such as the "flooding dose" technique (16, 21), in which large amounts of amino acid tracers are rapidly infused for short periods, are not appropriate for measurement of LPH polypeptide synthesis because these methods do not allow sufficient time for adequate tracer incorporation into the mature BB polypeptide.

Over the past several years, we have developed procedures to accurately measure in vivo BB hydrolase synthesis (8-11). We have used a continuous infusion strategy with either radiolabeled (8, 9) or stable isotopically labeled amino acid (10) specifically designed to bring the high-mannose hydrolase precursor to isotopic equilibrium rapidly and to maintain this steady state for a prolonged period of time. We have also separated and individually analyzed the precursor and BB forms of the enzymes. By analyzing multiple tissue samples taken during the infusion, we could calculate the kinetics of synthesis of the high-mannose precursor relative to the tissue free amino acid pool, whereas the rate constants for the synthesis of the complex glycosylated precursor and the mature BB forms of each enzyme could be estimated relative to the steady-state values of their high-mannose precursor. Using these procedures, we have reported that, in vivo in the rat, BB LPH and sucrase-isomaltase turn over at an approximate rate of 300% per day, a value threefold higher than the value reported using the "flooding dose" technique and combined immunoisolates (15, 23).

The present method is identical in concept and calculation. However, it is radically different in execution, as it uses multiple stable isotope tracers as a substitute for multiple tissue samples. In the 1970s, an analogous but less complex radioisotope method using 14C- and 3H-radiolabeled isotopes of proline and tyrosine was applied to the measurement of procollagen synthesis (25), but the scope of this study was limited by the availability of only two radiotracers. The present, more intensive, method is made possible by the commercial availability of multiple 2H- and 13C-labeled amino acids (six, in the case of phenylalanine) and the ability of selected ion monitoring GCMS to analyze these isotopomers in the same sample. The data from the present study demonstrate that the complex labeling kinetics of LPH precursor and mature BB polypeptides can indeed be accurately measured in a single small intestinal jejunal sample after the overlapping intravenous administration of six stable amino acid isotopomers. The estimate of plasma free pool phenylalanine and leucine turnover calculated from multiple tracers was virtually identical to that determined with the conventional approach. Furthermore, the synthesis rates for LPH polypeptides obtained in the present study compare well with values we have previously reported in pigs continuously infused with radiolabeled or stable isotopes, with tissue samples collected at timed intervals during the infusion (10). The FSR of BB LPH measured in the present study (0.089 ± 0.02% per minute) was similar to that which we previously reported in 4-wk-old piglets (0.069 ± 0.013% per minute) (10). In newborn piglets, we have found the rate constant of synthesis to be somewhat lower (on average 0.036 ± 0.004% per minute) (8). The rate constant for proLPHh synthesis in the present study (3.3 ± 1.1% per minute) was also not significantly different from the value we found in newborn piglets (on average 5.0 ± 1.5% per minute) (8).

The method has two further advantages. First, negative chemical ionization mass spectrometry is a very sensitive method of detecting tracee. Thus, provided the tracer-to-tracee ratio of a given isotopomer can be brought to at least 0.1 mol/100 mol, accurate measurements of protein synthesis can be made on extremely small samples of protein. In a previous study (10), we were able to determine the isotopic enrichment of proLPH and prosucrase-isomaltase isolated from 80- to 100-µg samples of mucosal tissue. Second, the fact that selected ion-monitoring GCMS is able to simultaneously detect multiple isotopomers in the same sample speeds up and simplifies the analytical procedures and reduces problems of between-sample variation.

Because the method allows precise estimates of the tracer-to-tracee ratios of precursors and their products, it allows more detailed modeling of the data. For example, it is possible to determine the errors of the estimates of the kinetic parameters of interest on a per-animal basis. In addition, the modeling approach permits estimates (together with their errors) of the steady-state tracer-to-tracee ratios in each of the pools, thereby allowing further investigation of kinetic heterogeneity within the system. For example, the present data suggest that the steady-state tracer-to-tracee ratios in the bulked free phenylalanine and leucine pools of the mucosa do not define the tracer-to-tracee ratios of the pool of amino acids used in LPH synthesis. Thus, given that the animals were fasted, it could be argued that there is some degree of channeling of amino acids derived from mucosal intracellular proteolysis into the protein synthetic pool.

We believe this overlapping multiple isotopomer protocol has great potential for studies in humans, from whom multiple tissue samples generally cannot be obtained. By virtue of the infusion protocol, it is possible to determine the rate of increase of blood and tissue free amino acid pool tracer-to-tracee ratios and their steady-state values as well as the tracer-to-tracee ratio of an individual protein in a tissue sample collected at the end of the infusion. Although in this study we have applied the technique to the measurement of LPH kinetics, by choosing appropriate periods of labeling the approach can be applied to many other systems of tissue protein synthesis and processing, such as the plasma lipoprotein system.

    ACKNOWLEDGEMENTS

We are grateful to Leslie Loddeke for editorial assistance and to Adam Gillum for assistance with the illustrations. This project of the United States Dept. of Agriculture/Agricultural Research Service (USDA/ARS) Children's Nutrition Research Center, Dept. of Pediatrics, Baylor College of Medicine and Texas Children's Hospital, Houston, TX, has been funded in part with federal funds from the USDA/ARS under cooperative agreement numbers 58-625001-003 and 96-35206-3903.

    FOOTNOTES

The contents of this publication do not necessarily reflect the views or policies of the United States Dept. of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States government.

Address for reprint requests: M. A. Dudley, Dept. of Pharmacology and Physiology, New Jersey School of Medicine and Dentistry, 185 South Orange Ave., Newark, NJ 07103.

Received 26 March 1997; accepted in final form 1 December 1997.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Berthold, H. K., D. L. Hachey, P. J. Reeds, and P. D. Klein. Uniformly labelled algal protein used to determine amino acid essentiality in vivo. Proc. Natl. Acad. Sci. USA 88: 8091-8095, 1991[Abstract].

2.   Brauman, J. Least squares analysis and simplification of multi-isotope mass spectra. Anal. Chem. 38: 607-610, 1966.

3.   Burrin, D. G., M. A. Dudley, P. J. Reeds, R. J. Shulman, S. Perkinson, and J. Rosenberger. Feeding colostrum rapidly alters enzymatic activity and the relative isoform abundance of jejunal lactase in neonatal pigs. J. Nutr. 124: 2350-2357, 1994.

4.   Carson, E. R., C. Cobelli, and L. Finkelstein. The Mathematical Modeling of Metabolic and Endocrine Systems. New York: Wiley, 1983.

5.   Culea, M., and D. L. Hachey. Determination of multiply labeled serine and glycine isotopomers in human plasma by isotope dilution negative-ion chemical ionization mass spectrometry. Mass Spectrom. 9: 655-659, 1995.

6.   Danielsen, E. M. Post-translational suppression of expression of intestinal brush border enzymes by fructose. J. Biol. Chem. 264: 13726-13729, 1989[Abstract/Free Full Text].

7.   Danielsen, E. M., G. M. Cowell, O. Noren, and H. Sjostrom. Biosynthesis of microvillar proteins. Biochem. J. 221: 1-14, 1984[Medline].

8.   Dudley, M. A., D. G. Burrin, A. Quaroni, J. Rosenberger, G. Cook, B. L. Nichols, and P. J. Reeds. In vivo lactase phlorizin hydrolase turnover in water-fed and colostrum-fed newborn pigs. Biochem. J. 320: 735-743, 1996[Medline].

9.   Dudley, M. A., D. L. Hachey, A. Quaroni, T. W. Hutchens, B. L. Nichols, J. Rosenberger, J. S. Perkinson, G. Cook, and P. J. Reeds. In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat. J. Biol. Chem. 268: 13609-13616, 1993[Abstract/Free Full Text].

10.   Dudley, M., F. Jahoor, D. Burrin, and P. Reeds. Brush-border disaccharidase synthesis in infant pigs measured in vivo with [2H3]leucine. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G1128-G1134, 1994[Abstract/Free Full Text].

11.   Dudley, M. A., B. L. Nichols, J. Rosenberger, J. S. Perkinson, and P. J. Reeds. Feeding status affects in vivo prosucrase-isomaltase processing in rat jejunum. J. Nutr. 122: 528-534, 1992[Medline].

12.   Dudley, M. A., R. J. Shulman, P. J. Reeds, J. N. Rosenberger, M. Putman, P. K. Johnston, J. S. Perkinson, and B. L. Nichols. Developmental changes in lactase-phlorizin hydrolase precursor isoforms in the rat. J. Pediatr. Gastroenterol. Nutr. 15: 260-269, 1992[Medline].

13.   Dudley, M. A., L. Wykes, A. W. Dudley, Jr., M. Fiorotto, D. G. Burrin, J. Rosenberger, F. Jahoor, and P. J. Reeds. Lactase phlorizin hydrolase synthesis is decreased in protein-malnourished pigs. J. Nutr. 127: 687-693, 1997[Abstract/Free Full Text].

14.   Elliott, R. F., G. W. V. Noot, R. L. Gilbreath, and H. Fisher. Effect of dietary protein level on composition changes in sow colostrum and milk. J. Anim. Sci. 32: 1128-1137, 1971[Medline].

15.   Goda, T., H. Yasutake, Y. Suzuki, S. Takase, and O. Koldovsky. Diet-induced changes in gene expression of lactase in rat jejunum. Am. J. Physiol. 268 (Gastrointest. Liver Physiol. 31): G1066-G1073, 1995[Abstract/Free Full Text].

16.   Garlick, P. J., M. A. McNurlan, and V. R. Preedy. A rapid and convenient technique for measuring the rate of protein synthesis in tissues by injection of [3H]phenylalanine. Biochem. J. 192: 719-723, 1980[Medline].

17.   Henning, S. J. Functional development of the gastrointestinal tract. In: Physiology of the Gastrointestinal Tract (2nd ed.), edited by L. R. Johnson. New York: Raven, 1987, p. 285-300.

18.   Hodin, R. A., S. M. Chamberlain, and S. Meng. The pattern of rat intestinal brush-border enzyme gene expression changes with epithelial growth state. Am. J. Physiol. 269 (Cell Physiol. 38): C385-C391, 1995[Abstract/Free Full Text].

19.   Holt, P. R., and R. N. DuBois. In vivo immediate early gene expression induced in intestinal and colonic mucosa by feeding. FEBS Lett. 287: 102-104, 1991[Medline].

20.   Mauri, L., M. Rossi, V. Raia, V. Garipoli, L. A. Hughes, D. Swallow, O. Noren, H. Sjostrom, and S. Auricchio. Mosaic regulation of lactase in human adult-type hypolactasia. Gastroenterology 107: 54-60, 1994[Medline].

21.   McNurlan, M. A., A. M. Tomkins, and P. J. Garlick. The effect of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem. J. 178: 373-379, 1979[Medline].

23.   Olsen, W. A., E. Perchellet, and R. L. Malinowsky. Intestinal mucosa in diabetes: synthesis of total proteins and sucrase-isomaltase. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G788-G793, 1986[Medline].

24.   Quaroni, A., and E. J. Isselbacher. Study of intestinal cell differentiation with monoclonal antibodies to intestine cell surface components. Dev. Biol. 111: 267-279, 1985[Medline].

25.   Robins, S. P. Metabolism of rabbit skin collagen. Differences in the apparent turnover rates of type-I- and type-III-collagen precursors determined by constant intravenous infusion of labelled amino acids. Biochem. J. 181: 75-82, 1979[Medline].

26.   Shulman, R. J., S. J. Henning, and B. L. Nichols. The miniature pig as an animal model for the study of intestinal enzyme development. Pediatr. Res. 23: 311-315, 1988[Abstract].

27.   Shulman, R. J., D. R. Tivey, I. Sunitha, M. A. Dudley, and S. J. Henning. Effect of oral insulin on lactase activity, mRNA and posttranscriptional processing in the newborn pig. J. Pediatr. Gastroenterol. Nutr. 14: 166-172, 1992[Medline].


AJP Gastroint Liver Physiol 274(3):G591-G598
0193-1857/98 $5.00 Copyright © 1998 the American Physiological Society