| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Biocybernetics Laboratory, Departments of Computer Science and Medicine, University of California, Los Angeles, California 90095-1596
Address all correspondence and requests for reprints to: Prof. Joseph J. DiStefano III, University of California, 4531 Boelter Hall, Los Angeles, California 90095-1596. E-mail: joed{at}cs.ucla.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
D1 and D2 activities are strongly correlated with thyroid hormone status. D2 activity is increased in hypothyroidism (2, 11) or hypothyroxinemia of iodine deficiency (12) and decreased in hyperthyroidism (13); D1 activity is decreased in hypothyroidism and increased in hyperthyroidism (2, 11). Each deiodinase also responds to thyroidal status differently in different tissues, with D2 tissues having a tighter T3 homeostasis than D1 tissues, suggesting tissue-specific deiodinase regulation of local thyroid hormone concentrations (14).
Quantitatively, the D1 pathway has long been assumed to be the major one for peripheral deiodination of T4 to T3 in euthyroid rats, with D2 considered the main pathway for extrathyroidal T3 generation when the enzyme levels are sufficiently elevated and/or when D1 activity is depressed (6). However, the relative contributions of D1 and D2 to whole body T3 production are still not well established, the question having been rarely addressed on a whole body basis. Three reports, published more than 20 yr ago, provided estimates of the contribution of PTU-insensitive (D2) pathways to T3 production from T4 ranging from 23–44% (15, 16, 17), consistent with the idea of D1 dominance. Two, more recent reports provide estimates ranging from 22–65% (18, 19). All were obtained using noncompartmental analysis of plasma-borne data only, an indirect approach that can be unreliable in comparative studies (20). We have revisited this problem more directly and precisely, as described below, to quantify the contributions of each of the D1 and D2 pathways to whole body extrathyroidal steady state T3 production in vivo.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
9.21 Ci/mg) for radioiodination were
purchased from New England Nuclear (Boston, MA). Tracer
131I-labeled 3,5,3'-T3 (T3**;
3680 mCi/mg) and [125I]T4
(T4*; 3000 mCi/mg), each labeled in the outer ring, were
synthesized from 3,5-diiodothyronine and T3, respectively,
and T4* was purified on HPLC as previously
described (19). T3** was purified on Sephadex LH-20
(Sigma Chemical Co.), using ethyl acetate-MeOH-2N
NH4OH as eluent, as described below. I* and I**
contaminations of T3** and stock solutions were less than
2% and less than 5%, respectively, and were accounted for in all
results. All counting of 125I and 131I
radioactivity was performed in a dual channel counter (model 1197,
Tracor, Austin, TX), with appropriate corrections for counting volume
and density differences, and isotopic cross-over. BSA,
2-N-propyl-6-thiouracil (PTU), iopanoic acid, Sephadex G-25,
and other reagents were purchased from Sigma Chemical Co.,
and Alzet Osmotic Minipumps (model 2001) were purchased from Alza
Corp. (Palo Alto, CA). A combination of 0.1 ml/100 g BW Ketaject
(ketamine HCl, Phoenix Pharmaceuticals, Inc., St. Joseph,
MO) and 0.1 ml/100 g BW Anased (xylazine, Lloyd Laboratory, Shenandoah,
IA) were used as anesthetics. PTU pellets (150 mg/7 days) and 0.1
N NaOH placebo pellets manufactured to special order were
purchased from Innovative Research of America (Sarasota,
FL). Evaporations were conducted under vacuum in a rotary evaporator
(model SVC-100H, Savant, Hicksville, NY). All HPLC analyses were
performed using a gradient HPLC system (model 2150-G1,
LKB/Pharmacia, Piscataway, NJ) with a reverse phase
3-µm analytical ODS column (Bodman), UV detector
(LKB/Pharmacia), and fraction collector (LKB
SuperRak) for radioactive samples collected 15 drops/tube. The mobile
phase was acetonitrile-H2O (40:60; 0.1% phosphoric acid)
run at 1.5 ml/min.
Animals and care
Animal studies were approved by the UCLA Chancellor’s Animal
Research Committee. Sprague-Dawley male euthyroid and surgically
thyroidectomized (TX) rats purchased from Harlan Sprague-Dawley
(Indianapolis, IN), 250–350 g, were housed in individual metabolic
cages and maintained at 21–25 C on a 12-h light, 12-h dark schedule
and fed tap water and Wayne Lab Blox ad libitum.
Critical kinetic studies were performed in two TX rat groups; all rats
were replaced with both T4 (0.9 µg/day·100 g BW) and
T3 (0.15 µg/day·100 g BW). T4 was included
in the pump with T4* tracer; T3 was included
with T3** in a second pump (see below). These infusion
rates were found to produce euthyroid concentrations in tissues as well
as plasma of TX rats (21). The first group (n = 7) also had
chronically implanted 150-mg PTU pellets (TXR-PTU), and the second
(n = 4) had implanted 0.1-N NaOH placebo pellets
(TXR-EU). A third group of euthyroid intact rats (n = 4) was used
as the controls (EU). Two additional euthyroid intact rats, one with a
PTU pellet and the other with a placebo pellet implanted, were used in
a pilot study to test for in vivo 5'-D activity (see
below).
All TX rats were studied 3 weeks after thyroidectomy. Potassium iodide (0.1%) was added to the drinking water of all rats 2 days before the start of the experiment and throughout the infusion period to inhibit (dilute) radiolabeled iodide uptake by the thyroid. TX rats also received 3% calcium lactate in their drinking water. Food and water intake, feces and urine production, and body weight were measured daily, and the animals were also monitored continually for overall health status. On the day of minipump implantation (day 0), a 0.5-ml sample of blood was collected via translumbar vena cava puncture for measurement of hematocrits and endogenous plasma T3 and T4 concentrations.
Pump preparation and implantation days -1 and 0
Infusates were prepared as described previously (20, 22, 23). In
brief, the purified tracers T3** and T4* were
evaporated to dryness and reconstituted in an aqueous solution of 20
mM NaOH, 50 mM Na2CO3,
and 2% BSA. Minipumps were filled with about 200 µl of this infusate
(200 µCi/7 days·pump of T3** in one pump, and about
0.7–1 mCi/7 days·pump of T4* in a second pump), equal to
approximately 2.5 ng/day·100 g BW T3 and about 10
ng/day·100 g BW T4. These infusion rates represented
small perturbations of endogenous production rates of T3
and T4 in all groups (<1% for T3 and <2%
for T4 in controls, and <3% for T3 and <10%
for T4 in TX rats). The radiochemical purity of infusate
samples collected after filling (day -1) and immediately before
implantation (day 0) were determined by chromatographic analysis (see
below).
Rats were anesthetized ip, and both minipumps were implanted sc between the shoulders. The skin was closed with wound clips, and the rat was treated with topical and minimal im (0.03 mg kanamycin sulfate) antibiotics to prevent infection. The animals were then placed in individual metabolic cages for the duration of the study.
Day 7 operations
The rats were anesthetized, and 3–6 ml blood were collected in
heparinized syringes by cardiac puncture through the thoracic cage.
This provided final hematocrits, sera for RIAs, steady state
concentrations of total 125I and 131I in
plasma, and, after chromatography, plasma concentrations of labeled
T3** (denoted Cp3** or Cp4*) as
well as their metabolites. Pumps were removed, and fur was shaved to
ease homogenization, which results in no loss of iodothyronines, as fur
radioactivity is virtually all I* or I** (20, 23). Residual pump
contents were chromatographed to obtain day 7 infusate spectra.
Homogenization, extraction, and chromatography
Whole rat carcass was frozen in liquid nitrogen, rapidly crushed
in a mill, and transferred immediately into a blender with dry ice to
produce a fine tissue powder. A 3-fold dilution (vol/wt) of extractant
(95% ethanol, 10 mM PTU, 10 mM iopanoic acid,
and 1% NH4OH) was added to the tissue powder, stirred, and
stored at -30 C overnight for CO2 sublimation (20, 23).
Approximately 4.5-g aliquots of homogenate were transferred into each
of eight preweighed glass tubes, followed by vigorous vortexing for 2
min. Total homogenate radioactivity in each tube was measured and
divided by the tube tissue weight to obtain the homogenate
concentration of total 125I (denoted CH125*)
and 131I (denoted CH131**), which was then
corrected for isotopic crossover, and all were averaged. Homogenate
tubes were centrifuged at 2500 rpm for 20 min at 4 C, and supernatants
were combined. After two additional extractions, providing more than
90% recoveries of total radioactivity, supernatants were frozen at
-30 C until chromatography.
All sample extracts were chromatographed on both Sephadex G-25 (LPLC) and HPLC. For HPLC, extracts were evaporated with unlabeled T3 (4 µg) and T4 (5 µg) to facilitate accurate quantification of radioactive components, reconstituted in mobile phase (acetonitrile-H2O, 40:60; 0.1% phosphoric acid), and centrifuged for 10 min at 14,000 rpm. Supernatants were syringe-filtered (0.2 µm) before injection onto HPLC. For LPLC, duplicate tissue extracts were first evaporated in vacuum and reconstituted in 1 M NaOH; unextracted plasma samples were chromatographed in duplicate as previously described (22). This aqueous system distinctly separates all labeled components of interest, including labeled iodoprotein (in the void volume), iodide, T1, T2, T3, rT3, and T4.
T3** Sephadex LH-20 purification
We adapted a separation method reported by Williams and
co-workers (24). Sephadex LH-20 was equilibrated in ethyl
acetate-methanol-2 N NH4OH (400:100:40,
vol/vol/vol) eluent for 24 h, and the fines were discarded
by decantation. Swollen gel was transferred into an 0.8 x 26-cm
glass column (Bio-Rad), with glass-wool fitted at the bottom, and
packed with the same eluent to a column height of 23 cm for 3 h at
a flow rate of 0.8 ml/min. The tracer sample was applied to the column,
and 1-ml fractions were collected and counted to determine the elution
patterns of T3** and T4**. Duplicate fractions
under the T3** peaks were chromatographed on LPLC and HPLC
to verify the purity of the T3** peak.
Total blood T3 and
T4 pools
Total blood hormone pools are needed because red blood cell
(RBC)-associated pools of T3 and T4 are
substantial in euthyroid rats (20). The ratios of RBC to plasma tracer
T3** and T4* concentrations (K3 
CRBC3**/Cp3** and K4
CRBC4*/Cp4*) are needed to compute blood
T3** and T4* concentrations and pool sizes
using Eq III below. The rationale and methodology for these
measurements are described fully in the same reference.
In vitro assay for in vivo 5'-D activity
The completeness of type I 5'-deiodinase (D1) inhibition
in vivo was ascertained in vitro in liver and
kidney homogenates in a pilot study with two additional euthyroid
intact rats. These animals were treated identically as in the critical
study groups, with 150-mg PTU or placebo (0.1 N NaOH)
pellets implanted sc for 7 days. On day 7, the rats were anesthetized
and killed, and liver and kidneys were dissected out and immediately
frozen in liquid N2. Liver and kidneys were homogenized in
3 vol 0.01 M Tris-HCl, 0.25 M sucrose, and 1
mM EDTA, pH 7.4, buffer (25) and centrifuged at 1100
x g for 15 min. Labeled rT3*, containing less
than 4% free I* contaminant, was used as the deiodinase substrate.
Supernatants (0.1 ml S1) and blanks in homogenization
buffer (n = 6 each) were supplemented with about 0.1 µCi (0.12
nM) rT3*, then treated with 0.1 M
potassium phosphate, 1 mM EDTA, and 1 mM
dithiothreitol (DTT), pH 7.0 (1:3 vol/vol), and incubated at 37 C for 5
min. In our pilot studies, we also verified that 1 mM DTT
was optimum (25), using 0, 1, 5, 10, and 20 mM DTT in
similarly prepared samples; 0 and 1 mM DTT gave no
activity, and 5 mM and above reactivated
in vitro samples inhibited by PTU in vivo. The
reaction was stopped with 0.5 ml 33% horse plasma, then centrifuged at
1500 x g for 10 min. We used horse plasma instead of
BSA because our pilot studies showed that horse plasma protein binding
with rT3* was complete, whereas it was not with BSA,
especially for kidney samples. Tissue supernatants (S2) and
blanks were chromatographed directly on Sephadex G-25 to quantify the
fractions of I*, T2*, and rT3* in
the samples.
RIAs
Plasma samples were assayed in triplicate for T3 and
T4 concentrations using GammaCoat M[125I]
total T3 and T4 assay kits (Incstar
Corp., Stillwater, MN). Day 0 (before infusions) and day 7 sera
were assayed together after several months of frozen storage. Day 7
samples had less than 0.5% of the amount of RIA radioactivity added at
the time of assay.
Modeling, data, and statistical analyses
The percent T4 to T3 conversion rate
(%CR4–3) computations were based a new experiment design
model, shown in Fig. 1
. This model
explicitly incorporates corrections for small amounts
of T3 and T4 contaminants in the infusates, an
extension of the model used for computing percent conversion in our
earlier work (20, 23). The key equations are summarized below. Most
were derived and justified in our earlier work, and several new ones
incorporate the complexities introduced by including dual contaminant
inputs. All data were analyzed using these equations. This purely
algebraic constant steady state infusion model does not require
simplifying pseudosteady state or other assumptions required by
pulse-dose noncompartmental or compartmental approaches. Results are
reported as the mean ± SD, and significance levels
were determined by unpaired Student’s t test.
|
 | (I) |
From Eq I
, the plasma clearance rates of T3 and
T4 are
 | (II) |
We evaluated the total blood tracer T3** and
T4* pool sizes (Q**blood3
= Qplasma3** +
QRBC3** and
Qblood4** =
Qplasma4* +
QRBC4*) in terms of plasma and RBC
T3** or T4* radioactivity concentrations per
unit mass (cpm/g), hematocrits, blood densities (db =
1.097 ± 0.0048 g/ml; n = 3), and plasma densities
(dp = 1.031 ± 0.03 g/ml; n = 3). The total
T3** pool in blood is:
 | (III) |
CRBC3**/Cp3** is the ratio of
RBC to plasma T3**
concentrations (similar equation for T4*), and
Vp is the plasma volume. The
Qblood4** formula is the same, with
subscript 4 substituted for 3 in Eq III (20, 23).
The steady state total body tracer
T3** and
T4** pool sizes (Qtotal3**,
Qtotal4**) are estimated as the product of the
measured concentration of total 125I and 131I
in the whole body homogenate (CH125**,
CH131**), the fraction measured chromatographically
as T3** or T4* (fH3 or
fH4), and the measured total body mass M, corrected by
compensating for the pool of T3** or T4*
(Qr3** or Qr4**)
in the mass Mr (grams) of blood removed from the animal
before death, i.e.
 | (IV) |
The percentage of whole body T4 converted to
T3 (conversion ratio) can be calculated from whole body
tracer T4*, T3*, and T3**
measurements. However, the derivation is complicated further here by
explicit inclusion of the contaminant infusion rates in Fig. 1
(see Appendix). This yields:
 | (V) |
The absolute conversion rate of T4 into T3
is:
 | (VI) |
 | (VII) |
For comparison purposes, we also computed traditional noncompartmental
values for the conversion ratio:
 | (VIII) |
and VI, where Cp3 and Cp4
are the endogenous steady state plasma T3 and
T4 concentrations. |
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
depicts D1 activity results
in control and PTU-treated liver and kidney samples. The
rT3* in liver and kidney control samples was virtually
completely degraded, whereas the PTU-treated samples were unchanged
compared with blank samples in the assays. These data support our
critical study results, namely that a 150-mg PTU pellet implanted for 7
days effectively blocked D1 activity, at least in major D1 organs liver
and kidneys.
|
summarizes our primary results. Total T4 converted to
T3 (%CR4–3) was reduced 50.1% in the
PTU-treated group (TXR-PTU) compared with that in the untreated group
TXR-EU (P < 0.0005), implying that D1 and D2 each
contribute about half of the T3 production from
T4. Corresponding results for the euthyroid intact group
are also shown in Table 1 (third column) as are plasma
hormone concentrations for all groups on day 7 (second row)
and day 0 (before pump implantations; third row). Steady
state conversion measures are the same for the euthyroid intact (EU)
and thyroidectomized and replaced (TXR-EU) groups (row 1);
plasma hormone concentrations are euthyroid, but somewhat higher in the
TXR-EU group (see Discussion).
|
for TXR-PTU and TXR-EU animals. They
represent a composite, overall whole body picture of the distribution
of T3** and T4* and their primary metabolites
in the steady state. Chromatograms for different rats were similar
within each group. For 131I label (**) in steady state,
whole body extracts contained mainly I** and T3** in both
TXR-EU and TXR-PTU rat groups. For 125I label (*), whole
body extracts contained mainly I*, T3*, rT3*,
and T4* for both TXR-EU and TXR-PTU rat groups and much
smaller T3* fractions in TXR-PTU than in TXR-EU rat groups.
There were no detectable analogs, tracer Triac or Tetrac, in any
chromatograms, which would elute distinctly after the T4
peak in our system.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
Inhibition of D1 by PTU
PTU inhibits D1 uncompetitively and has been commonly used as a
tool for suppressing D1 activity. Peripheral T4 to
T3 conversion was inhibited by PTU in rats (15, 16, 17, 18, 19) and in
humans (27), with an apparent insignificant effect on D2 production of
T3 (25). The questions for us were: how much PTU is needed
to completely block D1 without other significant side-effects, and how
should it be delivered? In pilot studies, we found that implanted PTU
pellets (150 mg, for 7 days) effectively blocked D1 enzyme activity, at
least in liver and kidneys, when we tested several conditions and PTU
treatments. The constant infusions of PTU from the implanted pellets,
rather than intermittent dosing (16, 17, 26, 28) or dependence on the
animal’s water drinking or eating habits (15, 18, 19), also seemed to
be a more reliable way to deliver the PTU.
New experiment design model
Our experiment design model is based in part on the well
established idea, at least in the rat, that 5'-monodeiodination of
T4 to T3 occurs in tissues and not in blood.
Conversion Eq V was derived from this model in terms of measurable
whole body and blood radioactively labeled hormone; unlabeled
endogenous blood levels are not needed in any computations. Eq V also
incorporates corrections for small [125I]T3
or [131I]T4 contaminants in the infusates, as
it can be very difficult to eliminate them completely. Ignoring
contaminants can have significant effects on results in pathological
states (20, 23), as noted below for this study.
From Eq V, the percent whole body conversion rate (conversion ratio)
was %CR4–3 = 22.7% in intact EU rats and 23.4% in
TXR-EU rats, the latter in excellent agreement with previously
published results (18, 19, 20, 22, 23). In contrast, with D1 blocked in our
TXR-PTU group, the %CR4–3 was reduced 50%
(P < 0.0005) relative to that in the EU and TXR-EU
groups, based on Eq V. For this pathological state, if we had ignored
the effects of contaminants, we would have erroneously computed that
the D2 pathway contributes 64% instead of 50% of T3
production.
Contribution of D1 vs. D2 To whole body T3
sources
We found four reports providing estimates approximating the
contribution of D2 pathways to whole body T3 production,
three published more than 20 yr ago (15, 16, 17) and one published recently
by Veronikis and co-workers (18). A fifth study with similar goals and
design (19) provided enough data to make noncompartmental calculations,
as with all the others. Estimates ranged widely, from 23–44% in Refs.
15, 16, 17 to 22% in Ref. 19 to 65% in Ref. 18 . Recent increasing
interest in local tissue T3 regulation, especially via D2
pathways, motivated us to revisit this problem, using more direct,
contemporary approaches and knowledge. Instead of intermittent,
periodic replacement treatment with injected T4 only, as in
Refs. 15, 16, 17 , or infusion of T4 only as in Refs. 18, 19 ,
we constantly infused both T4 and T3 for steady
state replacement at rates recently reported as physiological for the
whole animal, in tissues as well as blood (21). Lack of T3
replacement in the study reported in Ref. 18 , which otherwise was
performed the most similarly to our own, could account for the high
(65%) value they reported. A hypothyroid T3 state in
tissues (21) should increase the activity of the D2 pathway. However,
the indirect noncompartmental formula used by this group could also
have been problematic.
Our main result, that 50.1% of T3 production from T4 is attributable to D2 and the other half to D1 pathways, is based on direct, whole body measures. Our data also permit estimation of this percentage using the same noncompartmental formula used by all previous groups, Eq VIII above, which depends solely on (indirect) plasma clearance rates and endogenous hormone levels in plasma. This calculation suggests that only 23% of T3 production from T4 would be attributable to D2, a value at the low end of the estimates reported previously (15, 16, 17, 19). The problems and ambiguities inherent in using noncompartmental methods for comparative studies have been noted repeatedly in our earlier work (20, 29). They were especially significant in demonstrating a conversion rate estimate anomaly in fasting rats (20), where we found an increased conversion ratio by direct measurement and the opposite by noncompartmental analysis of our data.
As it turned out, in our TXR rat groups, the use of replacement dosages reported to induce tissue euthyroidism (21) appears to have overcompensated somewhat, based on comparisons of the day 7 steady state plasma T3 and T4 concentrations achieved. Day 7 values are indeed well within the euthyroid range, and conversion rates are the same in the TXR-EU and intact EU groups; but day 7 hormone concentrations are greater than corresponding values in the intact EU group. Day 0 RIA values indicate that the TXR groups were initially severely hypothyroid, but some residual thyroid tissue probably remained after surgery, probably accounting for the nonnegligible values measured after 3 weeks, on day 0 of the study. However, we do not believe that this was the cause of the higher T3 and T4 concentrations on day 7. The exogenous replacement dosages would inhibit any secretion from residual thyroid tissue via feedback inhibition of TSH. The question remains, then, as to how overcompensation might affect our primary result, that half of T3 production from T4 is attributable to D2. At some level, it might have the effect of decreasing D2 activity, and therefore, 50% would underestimate the contribution, if there were an effect. Fifty percent might then be considered a lower bound.
Another possible complicating factor, which could bias our results in the other direction, is additional effects of PTU on D1. D1 not only promotes T4 to T3 conversion by 5'-deiodination, but it is also involved in the clearance of T3 and T4 by 5-deiodination of their sulfated analogs, and this, in turn, is inhibited by PTU (30, 31). If this effect were significant in our study treatment, it might result in an increase in plasma and/or tissue levels of T3 (and T3* tracer), because conjugation is, in general, reversible. This would bias our results toward overestimation of the contribution of D2 to T3 production from T4. In fact, in contrast with the lack of effect of PTU on T3 clearance reported previously (30), our data provide some evidence for reduced steady state clearance of T3 in the PTU-treated group: the fractional elimination rate of T3, k3 (h-1), was about a third less, although the difference was not statistically significant. This, then, would act in the direction of reducing the possible effects of overreplacement discussed above, thereby reducing the probability of bias in our 50% estimate.
Until recently, the D1 pathway was considered to be the major one for peripheral T3 production from T4 in euthyroid rats, with the D2 pathway thought to contribute most extrathyroidal T3 only when enzyme levels were sufficiently elevated and/or liver and kidney D1 activity was depressed (6). Our results imply equal importance for the D2 pathway in the euthyroid condition, consistent with the preferential substrate ordering of D2 for T4, compared with that of D1, which favors deactivating pathways (rT3) more strongly. Our results are also consistent with recent findings of substantial D2 activity in human skeletal muscle, heart, and/or thyroid tissue by several groups (8, 9). Similar attempts at finding significant D2 activity in rodent skeletal muscle have been unsuccessful to date (10), suggesting possible species differences. Alternatively, muscle deiodinase might be heterogeneously distributed and highly localized, possibly making detection and isolation of the enzyme more difficult in small muscle samples. These issues remain to be investigated.
|
Appendix |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
T3* conversion in the pump over 7 days.
We call these inputs IR4* and IR3*. On day 7,
IR3* ranged from 10–13% of total *
infused, for all rats studied. The simultaneous and distinct exogenous
input of differently labeled tracer T3
(denoted T3** may also be contaminated with some
T4**, from unavoidable double-labeling and possibly
incomplete purification. These inputs are denoted IR3** and
IR4**. In our study, IR4** was eventually
eliminated by more fastidious control over iodination and
chromatography parameters.
It is useful to separate the model for the steady state into two parts
for analysis, as in Fig. 1, even though both parts (experiments) are
done simultaneously. We assume in both that rate constants
k3 and kCR (min-1) remain
constant, based on a small-perturbation experiment overall.
We need to solve for CR4-3* (and/or CR4-3**),
obtainable by writing four independent mass-flux balance equations from
the two models. From Fig. 1, we have:
 | (A-I) |
 | (A-II) |
 | (A-III) |
 | (A-IV) |
 | (A-V) |
 | (A-VI) |
 | (A-VII) |
 | (A-VIII) |
Qtotal -
Qblood, where Qtotal and Qblood are
the masses of tracer measured in whole-body (carcass) and blood,
respectively. Thus, (A-VIII) becomes (A-IX), as shown below.
Now if Qblood3 << Qtotal3 for each
tracer, we can approximate (A-IX) well by (A-X):
 | (A-IX) |
 | (A-X) |
 | (A-XI) |
Qtotal - Qblood.
 | (A-XII) |
|
Footnotes |
|---|
2 For T3, only a minimum value of true
PCR3 of endogenous T3
(PCR3min) is available from a feasible tracer
study such as this one (29 ). The true PCR3 can be estimated
only if all of the multiple tissue sources of T3 can be
traced simultaneously.
Received March 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAppendixReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  O. Gumieniak, T. S. Perlstein, J. S. Williams, P. N. Hopkins, N. J. Brown, B. A. Raby, and G. H. Williams Ala92 Type 2 Deiodinase Allele Increases Risk for the Development of Hypertension Hypertension, March 1, 2007; 49(3): 461 - 466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Schneider, S. N. Fiering, B. Thai, S.-y. Wu, E. St. Germain, A. F. Parlow, D. L. St. Germain, and V. A. Galton Targeted Disruption of the Type 1 Selenodeiodinase Gene (Dio1) Results in Marked Changes in Thyroid Hormone Economy in Mice Endocrinology, January 1, 2006; 147(1): 580 - 589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Kohrle, F. Jakob, B. Contempre, and J. E. Dumont Selenium, the Thyroid, and the Endocrine System Endocr. Rev., December 1, 2005; 26(7): 944 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. G J M Kuiper, W. Klootwijk, and T. J Visser Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast J. Mol. Endocrinol., June 1, 2005; 34(3): 865 - 878. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. J. M. Kuiper, F. Wassen, W. Klootwijk, H. van Toor, E. Kaptein, and T. J. Visser Molecular Basis for the Substrate Selectivity of Cat Type I Iodothyronine Deiodinase Endocrinology, December 1, 2003; 144(12): 5411 - 5421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. T. Nguyen, K. A. Mol, and J. J. DiStefano III Thyroid hormone production rates in rat liver and intestine in vivo: a novel graph theory and experimental solution Am J Physiol Endocrinol Metab, July 1, 2003; 285(1): E171 - E181. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. C. Lisboa, K. J. Oliveira, A. Cabanelas, T. M. Ortiga-Carvalho, and C. C. Pazos-Moura Acute cold exposure, leptin, and somatostatin analog (octreotide) modulate thyroid 5'-deiodinase activity Am J Physiol Endocrinol Metab, June 1, 2003; 284(6): E1172 - E1176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. J. M. Kuiper, W. Klootwijk, and T. J. Visser Substitution of Cysteine for a Conserved Alanine Residue in the Catalytic Center of Type II Iodothyronine Deiodinase Alters Interaction with Reducing Cofactor Endocrinology, April 1, 2002; 143(4): 1190 - 1198. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Bianco, D. Salvatore, B. Gereben, M. J. Berry, and P. R. Larsen Biochemistry, Cellular and Molecular Biology, and Physiological Roles of the Iodothyronine Selenodeiodinases Endocr. Rev., February 1, 2002; 23(1): 38 - 89. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Schneider, S. N. Fiering, S. E. Pallud, A. F. Parlow, D. L. St. Germain, and V. A. Galton Targeted Disruption of the Type 2 Selenodeiodinase Gene (DIO2) Results in a Phenotype of Pituitary Resistance to T4 Mol. Endocrinol., December 1, 2001; 15(12): 2137 - 2148. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Amma, A. Campos-Barros, Z. Wang, B. Vennström, and D. Forrest Distinct Tissue-Specific Roles for Thyroid Hormone Receptors {beta} and {{alpha}}1 in Regulation of Type 1 Deiodinase Expression Mol. Endocrinol., March 1, 2001; 15(3): 467 - 475. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
