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
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ABSTRACT |
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 |
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.
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METHODS |
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).

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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.
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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.
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.

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Fig. 3.
Compartmental model for lactase phlorizin hydrolase (LPH) polypeptide
synthesis. BB, brush border;
proLPHh, high-mannose LPH
precursor, proLPHc, complex
glycosylated LPH precursor.
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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
|
(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)
|
(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
|
(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)
|
(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
|
(5)
|
and
thus
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(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 |
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.

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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.
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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
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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%.

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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. ,
[2H8]phenylalanine
(n = 3); ,
[13C6]phenylalanine
(n = 6); ,
[2H5]phenylalanine
(n = 3); ,
[13C1]phenylalanine
(n = 3); ,
[13C1]leucine
(n = 6); ,
[2H3]leucine
(n = 6).
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Table 2.
Fractional turnover rates of plasma and mucosal free AA, synthesis
rates of LPH polypeptides, and plasma free AA contributions for 6 animals
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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 |
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.
 |
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