THYROID Volume 2, Number 1, 1992 Mary Ann Liebert, Inc., Publishers

Transport of the Thyroid Hormones

Across the Feline Gut Wall

MARGUERITE T. HAYS, LICHI HSU, and SHIOCHI KOHATSU

ABSTRACT Intestinal absorption of radioiothyroxine (T4*) and of radiotriiodothyronine (T3*) was studied in normal cats 6 for T3*). Absorption was localized by a series of studies comparing the time-activity (n 5 for T4* and data for plasma and fecal T4* or T3* radioactivity after i.v. administration with data obtained in the same cats after administration by the oral, jejunal, midileal, and cecal routes. Data were analyzed using a multicompart¬ mental model to account for longitudinal gut transit, metabolic loss, and reversible and irreversible binding, as well as absorption. The relative importance of the gut as a site for T4* or T3* residence at steady state was determined from T4* (n = 6) and T3* (n 3) content of gut wall and contents at the end of tracer infusion to Secretion of from and the general pool into the various parts of the gut was calculated from steady state. T4* T3* the steady-state data by fitting them to the model using its steady-state parameters. Secretion parameters were adjusted in this fitting process, which established the rate and sites of T4* and T3* secretion into the gut. Overall, feline absorption of T4* and T3* is low compared with absorption in human subjects. Gentamicin treatment enhanced T3* absorption, but general wasting (weight loss associated with time under study) had no effect on either T4* or T3* absorption. The most important site for absorption of both T4* and T3* is the jejunum-upper ileum bowel segment. However, when the mass of gut wall in each segment is taken into account, absorption rate is highest at the duodenum and decreases distally. Secretion of both T4* and T3* is most active into the duodenum, presumably largely through the bile. In addition, there is active secretion of both T4* and T3* into the ileum and of T4* into the colon. These studies indicate that T4* and T3* absorption and secretion balance to make the gut an important locale for both hormones. Because of the cat's relatively low T4 and T3 absorption rates, much of the gut contents are destined for excretion. However, an active secretion-reabsorption loop for both T4* and T3* is present. =

=

=

Where absorption and secretion of T4 and T3 occur in the intestine is not well established, although absorption has been shown to occur at all small and large intestinal levels in the rat (8,9) and to be more prominent in the proximal small intestine in the human (10). The series of experiments presented here were undertaken to try to answer this question, by applying mathematical modeling techniques combined with tracer T4 and T, instillations in the feline gut to establish the locales of T4 and T3 absorption. We also present preliminary evidence about the sites and extent of secretion of T4 and T, into the gut, derived from analysis of steady-state distribution data.

INTRODUCTION

I

NTESTINAL absorption OF the thyroid HORMONES

thyrox-

and triiodothyronine (T3) is known to be incomvariable and (3). T4 and T, are also known to be seplete creted into the gut, through the bile (4) and, by recent reports, through direct secretion from the mesenteric capillary bed (5). The importance of a hepatoenteric T4 circulation has been debated for many years, and recent work in the rat shows the gut to be an important thyroid hormone absorption site (6,7). ine

(T4)

Department of Veterans Affairs Medical Center, Palo Alto. California. Departments of Radiology. Medicine, and Surgery, University of California at Davis School of Medicine, Sacramento. California, and Department of Surgery, Stanford University School of Medicine, Stanford. California. Portions of this work have been presented previously in abstract form ( 1.2). 45

HAYS ET AL.

46

MATERIALS AND METHODS

Preparation of tracers Stable T, and 3,5-T2 were radioiodinated by the chloramine method with l25I, l3,I, or l23l iodide to produce carrier-free radioiodinated T4 and T3 (T4* and T,*). After radioiodination, the T4* and T3* were purified by HPLC, using a method previously reported from this laboratory (11). The fraction of HPLC eluate containing T4* or T3* was evaporated to dryness and then reconstituted in 1% human serum albumin in normal saline. The dose administered in each gut instillation study, determined by weight, was held close to 1 mL, with an albumin content of IO mg, an amount previously shown to stabilize T4 for oral administration to humans without affecting absorption (12).

Animal

subjects

Six domestic short-hair cats were studied in the thyroid hormone instillation studies. One of these and also 5 similar cats were subjects of the infusion pump study. Cats were housed in an accredited animal facility, and their care was supervised by a veterinarian. This study was approved by the local animal studies subcommittee.

Plasma volume

measurement

Plasma volume was estimated at least 5 days after preparatory surgery by the dilution method from extrapolation to zero time of plasma dye concentrations 10, 20, and 30 mins after i.v. injection of Evans blue dye. The plasma volume assumed for each experimental session was adjusted for changes in the cat's body weight, using the weight on the date ofthat session. Plasma volume was 26.3 ± 6.6 mL/kg body weight, with a range of 18.2 to 39.0.

Radiothyronine gut

instillation studies

Preparatory surgery. Cats to be subjects of the instillation studies (3 male, 3 female) were operated on under halothane anesthesia at least 1 week before the experimental studies were begun. All were splenectomized to stabilize blood volume. Each received a cannula in the upper right atrium through a jugular vein (for blood sampling and i.v. administrations) and an instillation cannula in the upper small bowel, the tip placed to deliver a dose approximately at the junction of the duodenum and the jejunum. In addition, 5 cats each had a cannula placed to deliver a dose into the middle ileum, 1 cat had a cannula placed in the middle jejunum (instead of the middle ileum), and 2 cats received cecal cannulas. Postoperatively, all cats received gen¬ tamicin, 5 mg/kg i.m. daily for 3 days. After recovery from surgery, all of the cats appeared to be healthy and ate well. However, as time went on, subcutaneous infections developed around the cecal cannulas in the 2 cats that had them and, in 3 other cats, around the deal cannulas. These infections were treated by local drainage and i.m. gentamicin. No cat was studied while obviously systemically ill from these infections, but some studies were carried out during gentamicin

therapy.

Postoperatively, the cats were housed in metabolic cages. Sodium iodide, 1 mg i.v., was given daily to suppress thyroid uptake of the radioiodide resulting from T4* orT3* metabolism. Experimental protocol. Instillation studies, begun at least 7 days after surgery, were spaced at least 5 days apart to ensure essentially complete disappearance of the tracers before a new study began. In these studies, tracers were administered in pairs, T4 orT, labeled with I being given by a different route than that labeled with ,2SI. In 3 cats, the i.v. T4, and in 2 cats the i.v. T3, were labeled with l23I, given in a triple-isotope experiment. When feasible, the jejunal route was used repeatedly, paired with the other routes. These repeat jejunal studies served to normalize the data for biologic variation, as will be described. Each session began with administration of the experimental tracers to the awake, calm cat. By gentle handling, it was possible to carry out the studies with no apparent discomfort to the animal. Oral doses were given through a short pharyngeal tube, with induction of swallowing by stroking the anterior neck. Intravenous doses were given through the jugular cannula, and a sample of the cat's own blood was used to flush the cannula. (In pilot studies, comparison of subsequent blood samples obtained from this cannula with those obtained from a separate venipuncture site showed no significant difference in tracer content.) Jejunal, deal, and cecal doses were given through the respective indwelling cannulas. One cat received only T3* studies. The other 5 had the full set of T4* and T3* studies, except that only 2 cats had cecal can¬ nulas. In 1 cat, the second intestinal cannula tip was found at necropsy to be in the middle jejunum instead of the middle ileum. His results for instillation in that cannula are reported separately. Sample processing. Blood samples were obtained through the jugularcannulaat2, 5. 10, 15, 30, and45 min and 1.5. 3. 6, and 24 h after administration of the tracers. When the session did not include an i.v. administration, the 2 min and 5 min samples were omitted. Plasma samples were aliquoted and counted for total radioac¬ tivity in a gamma scintillation counter. Percent dose per milliliter of plasma was calculated from comparison with a standard made from a dilution of the injected material. This was multi¬ plied by the plasma volume to determine the percent dose in the plasma pool.

To separate radioiodide and radioiodoproteins from radioiodothyronines in the plasma samples, 1 mL CI8 Sep-Pak cartridges (Waters and Associates, Milford, MA) were used in a modification of a method shared with us by Drs. Carol Spencer and Jonathan Lo Presti (personal communication). Each car¬ tridge was activated with 5 mL 100% methanol, followed by 3 mL 0.1% trifluoroacetic acid (TFA) in water. Plasma samples,

NaOH, 20 µ /mL of 0.5-1.2 mL. were alkalinized with 5 plasma, and then passed through the Sep-Pak columns. Radioiodides were washed from each column with 1 mL 0.1% TFA in water, followed by 2.0 mL 20 mM sodium acetate to acidify the column. The radioiodothyronines were then eluted from the column with 2.0 mL acetonitrile containing 0.1% TFA. The iodide-iodoprotein and iodothyronine eluates, as well as the Sep-Pak columns themselves, were counted for I, I, and, if applicable, l23I activity, and the iodothyronine fractions were calculated. The results, together with total plasma activity rate and plasma volume, were combined to yield a time-activity curve for the percent dose of the tracer iodothyronine present in

T4

, ABSORPTION

AND

AND SECRETION

the plasma volume. A combined value for yield of the Sep-Pak method and for the radioactive purity of the dose was assessed in each session for each cat by Sep-Pak separation of a control plasma sample spiked with the administered tracers and ana¬ lyzed as an experimental sample. Plasma values for each experimental session were corrected by dividing them by the iodothyronine fraction of the total counts in this spiked sample for that particular tracer and session. When recently repurified T3* orT4* were added to nonradioactive cat plasma and analyzed by this method, 92.2 ± 3.2% was in the radioiodothyronine fraction, 5.3 ± 2.1% was in the iodide-iodoprotein fraction, and 2.4 ± 1.5% adhered to the column. The radiothyronine fraction in the spiked samples of this experiment ranged from 77% to 96% for T3 and from 71 % to 95% for T4, the balance consisting of the combination of radiochemical impurities and incomplete yield. Pilot HPLC studies confirmed that almost all of the first elution fraction in the experimental samples was in the form of iodide. Feces were collected for 5-7 days after each dose. They were homogenized, weighed, and aliquoted, and the aliquots were counted for total radioactivity. From these observations, we calculated a cumulative time-activity curve for fecal excretion. Quantitation ofradioactivity. I25I, l3i I, and, where used, 12,I activities were measured in a three-channel gamma spectrome¬ ter. Crossover of 131I onto 125I (9-10%) was assessed during each counting session and was corrected for. When l23I was used, the two-way crossover of it with l25I and l3,I was measured and corrected for. Preliminary data analysis: Calculation of global absorption. To examine global T4* or T3* absorption after each tracer instillation, all of the separate plasma T4* or T3* curves were fitted to multiexponential models using the SAAM 30 modeling program (13,14). Initially, the data after i.v. injection of T4* or T3* to a given cat were fitted to a three-exponential curve. Then, holding the parameters from that fit constant but using an adjustable scaling multiplier and a fourth, input, exponential, the data for the oral, jejunal, deal, and cecal instillation studies were modeled. Finally, all parameters forT4* orT3* in a given cat were adjusted to all ofthat cat's data sets. The rationale for this approach is based on the assumption that systemic T4* or T3* kinetics are identical regardless of route of tracer adminis¬ tration. If the sum of the entire time-activity curve for an i.v. study is q¡v and those for the corresponding oral, jejunal, deal, and cecal studies are qpo, qje, q^, qce, respectively, then

C,e

q,v

qp„ (t) qje (t)

=

=

M +

Kpoqiv

C2e C4e

(t)

klt +

C,e M

-x4t

-

C5e"-M

(t) -

q„(t)

=

KMqlv (t) -

qcc (t)

=

Kccqiv (t)

C6e" C7e~

-

These multiexponential equations were then integrated to obtain the total area under each time-activity curve (q¡). In some instances, to achieve a reasonable fit of the data from the gut instillation studies to the simple equations used, it was necessary to unweight one or more early data points. When this was done, the part of that curve with unweighted points was integrated manually, interpolating linearly between observations.

47 The absorption fraction enterai dose as

Absorption

fraction

=

was

then calculated for each oral

or

q¡/q¡v

where q¡ refers to a particular oral or enterai administration and qiv refers to the i.v. administration ofthat particular iodothyro¬ nine and that particular cat. It should be pointed out that this formula calculates a lower bound for absorption, as there is no estimate included of absorbed T4 or T3 that may be resecreted into the gut without entering the central circulation (7). This caveat applies also to all other absorption calculations to be presented. In each case, absorption means net absorption. Normalization of data. When multiple jejunal instillation studies were done, each paired with an oral, i.v., or deal study, they provided estimates of variations in absorption, at least from the jejunal site. In all, replicate studies were available for 19 jejunal instillations, 12 of T4* and 7 of T3*. Deviation of these sessions from the mean was 28.4 ± 16.4% (mean ± SD). In order to reduce session-to-session variability, when these mul¬ tiple jejunal studies were available, the plasma time-activity curves for the other routes in those sessions were normalized to the cat's mean global absorption of T4* or T3* after jejunal instillation. A normalization factor, that cat's mean jejunal global absorption fraction divided by its jejunal global absorp¬ tion fraction in the particular session, was applied to all samples from that session. These normalized data values were used in calculation of the mean data time-activity curves and also in the multicompartmental model fits but not in the analysis of the effects of time and of gentamicin treatment on global absorption. In these normalized data sets, all of the jejunal data points were combined as if they were all observations in a single experimen¬ tal session. In calculating the mean data for the model fit, all of each cat's mean normalized jejunal data were treated as a single data set. Subcutaneous infusion pump studies. To begin the infusions, an osmotic infusion capsule (Alza, Inc.) containing approxi¬ mately 100 µc T4*, T,*, or both was placed subcutaneously between the cat's scapulae under ketamine anesthesia. A fine polyethylene cannula was attached to the pump outlet so as to deliver the tracers into loose fatty tissue. T4* and T3* in the plasma and total radioactivity in the urine and feces were measured over the subsequent 6 days (7 days in 1 cat), at the end of which time the cat was killed and necropsied. At necropsy, the gastrointestinal tract was separated into segments corresponding to those studied in the instillation experiments. Gut contents were separated gently from mucosa by scraping with a scalpel blade. The blood plasma, the liver, and the gut segments and contents below the stomach were extracted in acid ethanol as described by DiStefano et al. (7), and the extracts were then lyophilized, redissolved in 40:60 acetonitrile/water, and analyzed by HPLC. Total radioactivity in these tissues and also in the thyroid and other tissues was measured as described previously. In the extracted tissues, the fraction present as T4* orT3* was multiplied by the total activity to yield the total T4* or T3* activity present. Data are expressed as the total T4* or T3* present in the organ or its contents, as a fraction of total plasma T4* or T,* at steady state (the mean of the final 3 days' plasma content). The 6 cats whose data are presented here received infusions of both T4* and T,*, but final activity levels of the in 3 of

[l31IjT3

HAYS ET AL.

48 were too low for statistical validity (because of laboratory problems that delayed tissue analysis). Hence, although T4* data are presented from 6 cats, 4 with l25I label and 2 with l31l label, T3* data are presented from only 3 cats, 2 with l2,I label and 1 with l3,I label. In addition to these 6 cats, infusion pump studies were attempted as terminal studies in 4 other cats that were subjects of the instillation studies. In these cats, plasma, urine, and fecal tracer activity did not stabilize, indicating that steady state had not been achieved. At necropsy, we found

plasma after cecal instillation,

and most of the cecal dose was recovered in the feces. Plasma appearance was greatest after jejunal instillation. After oral administration, there was a delay in plasma appearance and the peak plasma levels were less than after jejunal instillation, indicating that gastric absorption does not occur and that there is some diversion of the oral dose. (One cat, whose other absorption studies were typical of the group, had no increment in plasma T4* activity after the oral dose of T4*. It was assumed that he had somehow failed to swallow the dose, and his oral T4* instillation data were omitted from the summaries presented here. ) Also omitted from the summary are the instillations into the midjejunum in the cat whose second cannula was placed there instead of the midileum. His T4* study was similar to his upper jejunal T4* study, whereas his T3* study showed much less absorption. The complete series of studies for an individual cat took 5-10 weeks after surgery for the cats with both T4* and T3* studies and 4 weeks for the cat with only T3* studies. During this time, all cats lost weight. Most also developed localized subcutaneous infections around the deal or cecal cannula sites. These infec¬ tions were treated as needed with drainage and with gentamicin. The cats did not become febrile and did not show evidence of pain. Studies were not done while an infection was acute, but often the cat was still taking gentamicin when a session began. The question arises whether these infections themselves or their treatment or the gradual inanition observed withjime affected the absorption studies.

them

fibrous tissue around the outlet of the infusion pump. To correct this problem in the final 6 infusion pump studies (using the last of the instillation study cats and 5 fresh cats, the 6 cats whose results are reported here), care was taken to deliver the infusion dose into loose subcutaneous tissue through a small cannula attached to the pump. The data from these 6 cats showed stable plasma, urine, and fecal radioactivity levels during the final 3 days of the infusion.

RESULTS Instillation

experiments

The normalized plasma time-activity data after instillation of T4* and T,* at different sites are presented in Table 1 together with their global absorption as calculated by the curve summa¬ tion method described. The mean plasma data also appear as the data points in Figure 1. Very little radioactivity entered the

Table 1. Plasma Values

SD) (% Dose Plasma Volume) After T4:

(mean

and

Minimum Global Absorption Values

Route Time

(h)

0.03 0.08 0.17 0.25 0.50 0.75 1.5 3 6 24 Global

Oral 77.5 67.5 52.9 46.2 37.7 33.2 25.6 25.8 17.9 6.2

± ± ±

± ± ± ± ± ± ±

24.9 15.6 12.0 14.3 14.8 8.1 7.1 3.5 2.3 5.4

absorption {%)

0.0 0.0 0.1 0.5 1.2 1.7 2.3 0.6 10.5

0.0 0.0 0.2 0.8 2.2 2.0 2.8 0.5 10.8 Route

Time (h) 0.03 0.08 0.17 0.25 0.50 0.75 1.5 3 6 24 Global

Oral 26.71 11.58 8.91 8.01 6.06 4.79 3.75 2.17 1.61 0.78

absorption (%)

± ±

10.76 3.05 2.23 2.36 2.64 1.49 1.94 1.07 0.75 0.32

T3*

Administration

and

Their

of administration of'T4* Jejunal 1.3 1.3 1.8 2.4 2.5 2.5 2.9 3.3 2.6 0.9 16.2

±

Heal

Cecal

0.8

± 1.0 ± 1.4

1.4 ± 1.2 1.6 ± 1.1 1.8 ± 1.0 1.9 ± 1.1 1.8 ± 1.0 1.3 ± 0.7 0.9 ± 0.5 0.4 ± 0.3 4.3 ± 1.2

± ±

2.1 1.6 ± 1.2 ± 1.2 ± 1.6 ± 1.1 ± 0.3 ± 7.8

of administration of

Jejunal 0.33 0.03 0.34 ± 0.12 0.28 ± 0.28 0.49 ± 0.21 0.56 ± 0.36 0.65 ± 0.31 0.80 ± 0.42 0.52 ± 0.23 0.37 ± 0.21 0.19 ± 0.16 21.9 ± 13.6

0.04 0.08 0.11 0.10 0.11 0.07 1.3

*

Heal

Cecal

±

0.16 0.17 0.24 0.27 0.41 0.40 0.32 0.15 16.8

± 0.14

± 0.10 ±0.16 ± 0.17 ± 0.21 ± 0.17 ±0.15 ±0.11 ± 7.4

0.21 0.27 0.31 0.26 0.21 0.19 0.11 0.06 7.4

0.16 0.29 0.30 0.23 0.21 0.18 0.09 0.06 6.6

0.006 0.011 0.014 0.014 0.012 0.003 0.4

T4

AND

, ABSORPTION AND SECRETION

To explore this issue, the global T4* or T3* absorption for each study was first transformed to a fraction of the mean global absorption for that instillation site. These fractions were then examined in comparison with two indices, whether the cat was receiving gentamicin on the study date and the postoperative day of the study. Eleven of the T3* studies were done while the cat was taking gentamicin and 11 while it was not. Mean absorption on gentamicin was significantly increased to 1.3 ± 0.7timesthe group mean (p < 0.05). Four T4* studies were done on genta¬ micin, 17 off. For T4*, absorption on gentamicin was 1.2 ± 1.1 of that off gentamicin (not significant). These results (Fig. 2) suggest that antibiotic treatment enhances T3*, and possibly T4*, absorption. On the other hand, the time since the initial

100.0

49 surgery, used as an index of general decline, had no effect on the relative absorption rate (Fig. 3).

Infusion pump experiments Table 2 presents the steady-state data for the 6 cats infused with T4* and the 3 cats infused with T,*, expressed as the ratio of organ T4* or T3* activity to that in the cat's plasma volume. There was considerable variability among the cats, as shown by the relatively large standard deviations. The liver contained 82% as much T4* and 73% as much T3* as did the plasma volume. Assuming the kidneys (which were not extracted) to have the same chemical distribution of tracer as does the liver, the kidneys contained 23% as much T4* and 43% as much T3* as did the plasma volume. The gut wall contained about 6% of plasma T4* content and about 26% of plasma T3* content. The gut contents were an important site for T4 and T3, with T4* and T3* content both increasing distally. In all cases, total thyroidal radioactivity at necropsy was less than 3% of whole body radioactivity. The major metabolite present in the tissues at steady state was iodide, which accounted, after T4* infusion, for 43 ± 3% and, after T3* infusion, for 54 ± 8% of the total plasma radioactiv¬ ity. Iodide after T4* andT3*, respectively, was 55% and 58% in the liver, 64 ± 4% and 54 ± 12% in the gut wall, and 36 ± 15% and 27 ± 12% in the gut contents. Jhe iodide fraction in the gut contents decreased distally after both T4* and

T3*.

The gallbladder bile at equilibrium (obtained in 5 of the T4* and 2 of the T3* studies) showed a full spectrum of metabolites. After T4*, 17 ± 9% was present as iodide, 14 ± 11% as pre-T2 compounds (such as T2 glucuronide), 24 ± 19% as T2, 12 ± 8% as T3 glucuronide, 16 ± 20% as T3, 2 ± 2% as rT3

3

Oral

FIG. 1.

Time-activity

curves

(mean data) for T4* (a) and T3*

(b) present in the plasma volume after instillation of T4* orT3*, respectively, by various routes. The cecal T3* curve is omitted,

it is entirely below the scale shown. Observed values are shown as discrete symbols. The lines represent fits to the model shown in Figure 3. Standard deviations for the observed data are presented in Table 1. as

On

Gentamycin

Off Gentamycin

Jejunal

T3 T4 o

·

*

Heal

Scatter plot of the global absorption values for individual sessions normalized to mean absorption for that particular route of administration (oral, jejunal, or deal) for sessions initiated while the cat was off or on gentamicin therapy.

FIG. 2.

50

HAYS ET AL.

The

2.0

multicompartmental model fit

FIG. 3. Scatter plot of the global absorption values for individual sessions normalized to mean absorption for that particular route of administration (oral, jejunal, or deal) as a function of time. The solid line shows the linear regression fit, demonstrating no significant variation with time.

Modeling strategy. To localize intestinal absorption and secretion of T4* and T3*, a multicompartmental model was developed and applied using the SAAM 30 modeling program (13,14). Initially, the data from each session for each cat (adjusted as described) were modeled separately. First, the cat's i.v. data were fitted to a three-compartment mammilary model with all exit from the fast exchange compartment, as shown at left in Figure 4. [This model form was chosen because it is simple and identifiable and because the partition of metabolism (exit) between the slow and fast exchange compartments is not of importance to the present study.] Once the parameters for this systemic T4* or T3* model were established for a given cat, they were held constant in the modeling of the gut instillation studies. Next, the data after cecal tracer instillation were modeled. Time-activity curves for tracer appearance in the plasma and in the feces were fitted to various model forms. The model form shown in the lower right segment of Figure 4 was most compatible with the data. As only 2 cats received cecal instilla¬ tions and absorption of the cecal dose was minimal, the param¬ eters for model fit to the mean data from the 2 cats were fixed and used in all of the subsequent model forms. The data from deal instillation was fitted to several model forms, assuming that all unabsorbed activity leading the deal segment enters the cecal submodel. The model form that served best to fit the individual cats' data is shown in the deal section of Figure 4. Next, the data from jejunal instillation were treated in the same way, holding that cat's deal and the mean cecal

and its glucuronide, 3 ± 2% as T4 glucuronide, 8 ± 3% as T4 and 3 ± 4% as post-T4 compounds, such as tetrac. After T3*, the distribution was 6% iodide, 9% pre-T2, 31% T2, 4% T3 glucuronide, 34% T3, and 16% post-T, compounds, such as triac.

setting up this part of the model, we assumed that no absorption occurred in the stomach. We also assumed, based on our human studies (10), that the diversion of radioactivity from the absorption pathway (needed to fit both plasma and fecal data) and also the loss pathway occurs at the level of the stomach

1.5

to

.5

4·

*

3

4

Oral

·

Jejunal Heal

D

_

20

Days

50

40

30 After

60

70

Preparatory Surgery

Table 2. Steady-State Distribution of T4* After Osmotic Pump Infusion

Liver Gallbladder bile Gut wall Duodenum Jejunum and upper ileum Lower ileum Colon Gut contents Duodenum Jejunum and upper ileum Lower ileum Colon "Ratio of the tissue T4* volume (mean ± SD).

or

T3*

Tj* activity

T4* activity =

and

6

3

n

=

0.82 ± 0.41" 0.04 ± 0.02

0.73 0.06

± ±

0.02

0.008 ± 0.005 0.024 ±0.010

0.042 0.095

± ±

0.038 0.044

0.019 ± 0.013 0.013 ±0.005

0.051 0.068

±

0.050 0.034

0.003 ± 0.003 0.025 ± 0.025

0.017 ± 0.018 0.113 ± 0.130

0.058 ± 0.062 0.996 ± 0.387

0.284 ± 0.283 3.194 ±1.287

T3*

to

the total

'

T4*

or

T,*

±

in the

0.36

plasma

processing parameters constant. Finally, the data from oral administration were superimposed. In

rather than the duodenum. After the necessary model form was established (Fig. 4) and shown to apply both to T4* and to T3* and, at least approxi¬ mately, to all of the individual cats, the mean data were fitted to this model form. Primary attention was paid to fitting of the plasma data, but the shapes of the mean fecal appearance curves also were used in determining the rate of longitudinal intestinal transport. Once the parameters for absorption, exchange, and transport were established, the steady-state gut T4* and T3* model solutions (the M values calculated by SAAM) were adjusted to fit the mean activities observed in the summed gut wall and contents at the end of the constant infusion study (Table 3). This fitting yielded the rate constants shown in Figure 4 for the mean rates of transport into the gut from the systemic T4* or

T3* pools. Finally, all parameters in the model in Figure 4, with the exception of those for the individual cats' central T4* and T3* systems and for their T4* and T3* absorptions, were fixed from

the fit of the mean data. The data for the individual cats were then refitted to this model to determine the individual absorption

parameters. Results of the model fits. Figure 4 presents the final model form resulting from these modeling procedures and the param-

T4 AND ,

ABSORPTION AND SECRETION

51

Stomach 167 h

I

sio«

I

Upper Colon

2.37 h

Lower Colon 1.71 h

FIG. 4. The multicompartmental model used to estimate T4* and T3* absorption and secretion at different intestinal sites. The left section represents systemic T4* or T3* distribution and disposal outside of the gut. The arrows connecting this section with the gastrointestinal model (within rectangles) represent secretion into the gut and absorption from the gut. The gastrointestinal model is divided horizontally into the various gut segments evaluated. The vertical segments represent (left to right) the absorptive mucosal sites, the gut contents sites in exchange with the absorptive sites, and the irreversibly bound T4* or T3* within the gut. The rate constants between horizontal segments represent longitudinal gut transit. The other rate constants shown (Roman type forT4 and italics for T3) are those that were held constant in the final fit of data from the individual cats. The parameters identified as k values are listed in Table 2.

that were fixed after solution of the mean data. Table 4 shows those parameters that were adjusted in final fitting of the data of the individual cats. Percent of T4* or T3* absorption from a segment of the model was calculated as the percent of the dose entering that bowel segment times the ratio of the absorption rate from that segment to the total exit from that segment. As exchange holding compartments (presumably luminal bowel contents not in con¬ tact with the absorptive surfaces) were needed for fitting the eters

jejunal, deal, and cecal instillation data (compartments 21, 22, and 23 in Fig. 4), the clearance values (Rs) from the SAAM model solutions were used in this calculation rather than the rate constants themselves. In assessing the absorption from a site distal to that where the dose was administered, removal at the more proximal sites was taken into account. Table 5 presents the results of the calculation of local absorption, both for the individual cats and for the mean data. Of note is the importance of the gastric bypass, reducing overall absorption after oral

HAYS ET AL.

52 Table 3. Steady-State Contents in

Gastrointestinal Segments of Model Figure 4"

of

Fraction

of total plasma iodothyronine

content

present

in gut segment

3*

r/ Duodenum and upper ileum Lower ileum Colon

Jejunum

Observed

Model fit

Observed

0.010 0.049 0.077 1.009

0.010 0.052

0.060 0.208 0.334 3.262

0.077 1.009

Model

fit

0.049 0.245 0.311 3.287

"The observed data presented are for the combined gut wall and contents. The model fit values presented are the sums of the steady-slate contents of all of the compartments in the respective submodel. Both are presented as the fraction of the total steady-state plasma content represented by the activity in the particular bowel

segment.

administration, and the relatively small impact of duodenal absorption. However, when the weight of the gut wall in the segment studied is taken into account (Table 6), absorption by the duodenum is shown to be quite active. The balance between transport of T4* and T3* into and out of the gut in this model's steady state is presented in Table 7. For bothT4* andT,*, the largest secretion is into the duodenum, and much of this probably is biliary secretion rather than secretion

Table 4. Rate Constants Derived from Fitting Model Data from Individual Cats" in Figure 4 Individual cats

(fraction/h) (mean

Absorption parameters T4 k2

SD)

Mean data

(fraction/h)

k2

0.29 2.54 0.40

0.29 1.89 0.24

0.21 2.31 0.30

^2.10 ^2.1 I

0.48 2.07 0.20

0.35 0.83 0.17

0.40 1.68 0.21

4.2 6.6 2.2 1.2 0.58

1.2 3.1 2.1 1.0 0.43

5.1 9.8 2.0 1.1 0.47

T. 2.1 2

Systemic T4

ka.i

k,.2 k.,., T,

±

^0,2

ka.i

k,.2 k,., k,.., ko.2

model

from the mesenteric circulation. Nevertheless, significant secre¬ tion of T4* at more distal sites, which must be mesenteric in oriain, was observed.

DISCUSSION

Projections from the model The model shown in Figure 4 was used as the basis for theoretical projections of the impact of experimental and clinical situations on the dynamics of T4* and T3* absorption. In these projections, the parameters for the mean data solution were used, except when a parameter was altered to mimic the situation

being projected. One type of experiment that has been used by those interested in T4* absorption is to localize absorption by isolating gut segments surgically and to introduce the tracer of interest into that segment (8,9). Absorption is then inferred from the activity remaining in the segment at different time intervals. Figure 5 shows projections of the time-activity curves that would be expected from such an experiment in the cat. In this situation, where the rapid transit through the duodenum and the stomach bypass are inactivated, absorption of both T4* and T3* is greatest from the duodenum. Absorption of T4* from jejunal instillation, as judged by the appearance of absorbed T4* in a receiving compartment, initially exceeds that from the duode¬ num, but the curve rapidly flattens because of the large ex¬ changeable nonabsorptive pool in the jejunum (compartment 21). On the other hand, when the activity remaining in the bowel segment is projected, more T4* remains in the jejunum-upper ileum bowel segment than in the terminal ileum segment. This be attributed to the impact of compartment 21 and, more importantly, to nonspecific loss from compartment 22, the deal

can

37.2 5.8 7.4 0.23 0.29

12.2 2.1 2.9

0.10 0.22

37.4 5.8 6.9 0.26 0.25

"Rale constants shown in Figure 2 were held constant. Shown here are the parameters that were adjusted in the fit of data from each cat's series of experiments.

exchangeable nonabsorptive pool. These projections are similar to the experimental results reported by Chung and Van Middlesworth (9) in intact bowel loops. However, those workers found that in washed bowel loops, the T4* activity remaining in isolated rat gut segments become constant after 1 h. They interpreted their results to indicate irreversible binding of T4* to gut contents. To make our

T4

AND

, ABSORPTION AND SECRETION Table 5. Absorption

(%)

of

53

T4

and

T3

into

Plasma

from

Bowel Absorptive Sites

Tj

T4 Individual n 5

Individual n 6

cats

=

SD

Mean data

3.0 3.5 0.8 0.0 0.4

3.6 4.8 0.4 0.2 9.1

5.8 0.7 0.0 6.0

1.2 ±0.9 0.5 ± 0.0 1.7 ±0.9

mean ±

Oral administration" Duodenum Jejunum-upper ileum Lower ileum Upper colon Total Jejunal adminstration Jejunum-upper ileum Lower ileum Upper colon Total Heal adminstration Lower ileum Upper colon Total Cecal administration Upper colon Oral

model

compatible

3.0 5.4 0.5 0.2 9.1

± ±

9.3 0.8 0.4 10.6

±

± ± ±

±

± ±

mean

SD

±

7.0 8.2 0.9 0.2 16.4:

Mean data

5.0 2.2 1.0 0.0 6.0

6.4 7.4 1.3 0.2

15.3

9.2 0.9 0.4 10.5

17.6 ± 5.8 1.8 ±1.9 0.4 ± 0.0 19.9 ± 6.9

15.4 2.7 0.4 18.5

0.9 0.5 1.4

2.3 ± 2.5 0.5 ± 0.0 2.8 ± 2.4

3.2 0.5 3.7

0.5

0.5

T4 data from cat 4 omitted (see text).

with these observations, it would be

neces¬

sary to connect the exchangeable nonabsorptive compartments with the bypass route. For example, a rate constant to compart¬

ment 32 from compartment 22, (k,2 22), would be added. Our data could be fitted to such a model, but they are insufficient to separate out such a pathway with good identifiability. Simulations (Fig. 6) also were performed to assess the impact on

cats

=

T4* and T3* absorptive patterns of doubling or halving gut Table 6. Absorption

of

transit rate. Since net absorption from a given site is the result of a balance between the absorption rate and the rates of departure from that site, one would expect greater total absorption when transit is slower. Since the most efficient absorption is at the more proximal sites, the plasma activity curves peak earlier when gut transit rates are doubled. Projections of the effects on T4 absorption of anatomic gut alterations sometimes encountered in clinical practice are shown

Orally Administered T4 and Wall Segment

T3

Corrected for Weight

Segmenta! absorption mean

Segmental weight (g) mean

8.9 59.8 13.7 11.2

Duodenum and upper ileum Lower ileum Upper colon

Jejunum

Table 7. Transport

(%

of

data (%)

Duodenum Lower ileum Upper colon Lower colon

upper ileum

Bowel

Segmental absorption mean data (%/g)

± SD

,

,

T4

T,

6.4 10.7 7.2 4.9

3.6 4.8 0.4 0.2

6.4 7.4 1.3 0.2

0.41 0.08 0.03 0.02

0.73 0.12 0.10 0.02

± ± ± ±

Total Plasma T4* or T3*/h) Solution Using Mean Data

from

Steady-State Model

T3

T4

Jejunum and

of

Secretion

Absorption

Secretion

Absorption

4.58 1.89 0.00 1.01 1.01

0.29 0.57 0.05 0.03 0.00

24.80 0.55 0.00 0.00 0.00

2.93 3.45 0.61 0.08 0.00

HAYS ET AL.

54

Bypass of the stomach and duodenum yields a faster T4* absorption than does oral administration. This projection approximates the situation after Billroth 2 gastrectomy surgery. Such patients have been shown to have increased T4* absorption (3). Bypass of the terminal ileum alone has little effect on absorption of oral T4, but bypass of the jejunum is important. These findings are consistent with those of Stone et al. (15), who reported decreased T4* absorption in patients with short bowel conditions. Similar alterations were seen on projec¬ in Figure 7. and greater

tion from the

Problems

T3* model.

of model identifiability

This study is complex, and the model shown combines data for T4* and T3* activities after five separate routes of bolus administration in each cat and also steady-state data from infusion studies in a different group of cats. To evaluate the degree of certainty with which we can accept the configuration

of the model and its rate constants, it is necessary to examine both the identifiability of the model itself and the quality of the data on which it is based. A number of problems with the data were encountered. Because of the cat's limited plasma volume and the need to do chemical analyses of plasma samples, the number of samples collected in each study was limited. In addition, there was marked variation among individual cats. Variability with repeat studies in a given cat was less and was corrected for in the data analysis, as described previously. Fecal data, potentially of great importance in establishing the model, presented problems, even though the feces were col¬ lected in a metabolic cage. Some of the cats became constipated, and this varied among experimental sessions. Also, some radioiodide may contaminate the T4* or T3* in the feces, even though the model assumes that the fecal radioactivity all repre¬ sents unabsorbed T4* orT3*. Radioiodide contamination would tend to exaggerate the importance of the fecal route, whereas

T4

T3

CO

KO

200

290

2

300

Hour· of Incubation

14

Hours of Incubation

co

Duodenum

3

Hours

Hour*

FIG. 5. Simulation of projected absorption from isolated gut segments. Shown are time-activity curves forT4* (a and c) and T3* (b and d) as they would appear in the absorbed pool (a and b) or would remain in the gut segment (c and d). For these simulations, the model from Figure 3 was used, with parameters derived for the mean data (Fig. 3 and Table 2), except that the initial condition was set at 100% in the gut segment of interest, and longitudinal transit out ofthat segment was set to zero.

T4

AND

3

ABSORPTION AND SECRETION

T4, Slow Transit

FIG. 6. Simulation showing the impact of gut transit time on T4* and T3* plasma time-activity curves after oral administra¬ tion. The model parameters are as described in the legend for Figure 4, except that the longitudinal rate constants between gut segments were held to the values shown on Figure 3, to double

those rates (fast transit), orto half those rates (slow transit).

constipation

would tend to undervalue it. Finally, 3 of the cats had their i.v. studies done with [123I]T4* and For those cats, fecal appearance data for the i.v. studies were not useful because of the short l23I physical half-life. We have based the final model (Fig. 4 and Table 4) on the means of the plasma activity levels for all of the cats after each route of tracer administration. Fecal appearance data were used primarily to separate loss pathways (k09, k(122, and k,123),

[1231 3*.

I

2.5

Bypass of Stomach and Duodenum

55 which do not contribute to fecal activity, from the pathways that do contribute to fecal activity. Interpretation of these loss pathways must take into account the uncertainties in the fecal data. The infusion pump data were used primarily to establish the rates of T4* and T3* transport into the gut. However, these data, as well as the fecal appearance data, helped to define the gut transit parameters in the model. Gut transit parameters were based initially on information provided by a veterinary radiolo¬ gist about barium transport in the cat intestine. They were adjusted manually as the model segments took form to fit the shapes of the various fecal appearance curves. Finally, small adjustments were made in fitting the steady-state data. The model for plasma activity after i.v. T4* or T3* is theoretically identifiable (16,17), as it has exit from only one compartment. T4 and T3 are both known to be metabolized in many tissues, including those with slow exchange, so this is an oversimplification. However, it was considered justifiable to use this model form to reduce uncertainties in other portions of the model of more importance to the experimental questions. Similarly, placing the pathways for secretion into the gut from the fast exchange compartment is arbitrary. This compartment was selected because the biliary component of secretion is from the liver and the mesenteric capillary secretion component may be expected to occur with little time lag. We do not believe that either of these assumptions will markedly affect our estimates of T4 or T3 absorption or of the secretion clearance. The establishment of the parameters for each model segment, based on plasma and fecal data for instillation into that segment, was at least partially identifiable, judging by the certainties of parameter fit in the SAAM statistics. However, as many as five parameters (one each for absorption, for nonspecific loss, and for transport to the next level and two for exchange) are needed for each of these fits in addition to the linkages of these segments to model segments established from other data sets. Whether such a model segment is truly identifiable is unknown. Biologic variability is another reason to view cautiously the individual model parameter values presented here. As shown previously, T4 and T3 absorption vary widely between individ¬ ual cats and also in a single cat at different times. We have observed this variability also in human subjects. Nevertheless, the model form shown is well defined because it is necessary to accommodate all of the data sets fitted to it.

Comparison

FIG. 7. Simulations of T4* plasma time-activity curves after oral administration in anatomic alterations of the gut, using model parameters for the mean data, as presented in Figure 3 and Table 2. Bypass of the stomach and duodenum was modeled by introducing the dose into the jejunum instead of the stomach. The other bypasses were modeled by setting absorption from the bypassed site to zero and transit out of it to 10 times per minute.

with human

T4

and

T3 absorption studies

This study shows that in the cat, there is an active bidirectional exchange of T4 and T3 across the bowel wall. The stomach serves as a delay site after oral administration and also as a diversion site for orally administered tracers, with some nonspe¬ cific loss and some irreversible binding to stomach contents that move through the gut and are excreted unchanged in the feces. The duodenum is most active (on a weight-corrected basis) in both absorption and secretion of T4* and T3*. However, the rapid transit of gut contents through the duodenum minimizes its overall impact on T4* and T3* absorption. These observations are compatible with those in the human in our laboratory (10). However, overall T4 and T3 absorption in these cats was much lower than that which we have observed in human subjects

(10,12,18). The

cat was

chosen for these studies because of

HAYS ET AL.

56

interest in this animal as a spontaneous model for toxic thyroid nodules (19-24). Feline hyperthyroidism has become rather common in elderly cats, and they often are treated with surgical excision or radioiodine. Postreatmcnt hypothyroidism requires replacement T4 therapy, and the usual feline dose is 20-30 µg/kg daily. On a weight basis, this translates to 1.5-2 mg/day in a human subject. Feline T4 production rate as a function of weight is sixfold that in the human (24), but, even so, part of the cat's increased oral T4 requirement is most likely due to decreased efficiency of absorption compared with that in healthy human beings. Whether this low absorption rate limits extrapo¬ lation of these findings to an understanding of human T4 and T3 absorption is unknown. The fact that feline T3* (and possibly T4*) absorption are enhanced by gentamicin therapy is compatible with the observa¬ tions that T4* absorption in the rat is enhanced when the bowel is washed or in the germ-free state (9). It also fits with a single clinical observation (3) of enhanced T4* absorption in a patient with neomycin-induced enteritis, which was causing clinical malabsorption of other substances. On the other hand, general debility with weight loss, as it occurred with time in these cats, had no effect on T4* or T3* absorption. We have observed in human studies that "hospital controls," patients ill with unrelated problems, had somewhat lower T4 absorption than did healthy normal subjects (3). More clinical studies are needed to define what effects, if any, nonthyroidal illness has on T4 and T3 absorption.

6. DiStefano JJ 1988 Excretion, metabolism and enterohepatic circu¬ lation pathways and their role in overall thyroid hormone regulation in the rat. Am Zool 28:373-388. 7. DiStefano JJ. Sternlicht M, Harris DR 1988 Rat enterohepatic circulation and intestinal distribution of enterally infused thyroid hormones. Endocrinology 123:2526-2539. 8. Chung SJ. Van Middlesworth L 1964 Absorption of thyroxine from the small intestine of rats. Endocrinology 74:694-700. 9. Chung SJ. Van Middlesworth L 1967 Absorption of thyroxine from the intestine of rats. Am J Physiol 212:97-100. 10. Hays MT 1991 Localization of human thyroxine absorption. Thy¬ roid 1:241-248. 11. Hays MT, Hsu L 1987 Preparation and separation of the gluc¬ uronide and sulfate conjugates of thyroxine and triiodothyronine. Endocrine Res 13:215-228. 12. Hays MT 1968 Absorption of oral thyroxine in man. J Clin Endocrinol Metab 28:749-756. 13. Berman M. Shahn E, Weiss MR 1962 The routine fitting of kinetic data to models: A mathematical formalism for digital computers. Biophys J 2:275-287. 14. Berman M, Beltz WF, Greif PC. Chabay R. Boston RC 1983 Consam: User's Guide. Department of Health and Human Services, Washington, DC. 15. Stone E. Leiter LA. Lambert JR. Silverberg JDH. Jeejeebhoy KN, Burrow GN 1984 L-Thyroxine absorption in patients with short bowel. J Clin Endocrinol Metab 59:139-141. 16. DiStefano JJ, Jang M, MaloneTK, Broutman M 1982 Comprehen¬ sive kinetics of triiodothyronine production, distribution, and me¬ tabolism in blood and tissue pools of the rat using optimized 17.

ACKNOWLEDGMENTS The authors thank Rosemary Broome, D.V.M., Evelyn Hilbert, and the staff of the PAVAMC Veterinary Medical Facility for skilled and humane care of the cats and for assistance during the surgical procedures. Joseph DiStefano III, Ph.D., provided detailed advice in setting up the tissue extraction methods and also exchanged preliminary information about related studies in his laboratory. Carole Spencer, Ph.D., and Jonathan LoPresti, M.D., provided details about the method used for Sep-Pak extraction of plasma samples. This work was supported by DVA intramural research funds

(Program 821).

REFERENCES 1.

2.

3.

Hays MT, Kohatsu S, Hsu G 1988 Gastrointestinal distribution and disposal of triiodothyronine in the cat. Am Thyroid Assoc T-67. Hays MT, Kohatsu S, Hsu G 1989 Locale of thyroid hormone absorption in the cat. Am Thyroid Assoc T-38. Hays MT 1988 Thyroid hormone and the gut. Endocrine Res

14:203-224. 1956 Enterohepatic circulation of thyroxine in humans. Clin Sci 15:551-555. 5. DiStefano JJ 1991 Biliary duxes and relative uptake dynamics of T, and T, from blood to rat intestines, liver and kidneys in vivo. Tenth International Thyroid Conference.

4.

Myant NB

18.

blood-sampling protocols. Endocrinology 110:198-213. Jacquez JA 1985 Compartmental Analysis in Biology and Medi¬ cine. The University of Michigan Press. Ann Arbor. Hays MT 1970 Absorption of triiodothyronine in man. J Clin

Endocrinol Metab 30:675-677. 19. Jones BR, Johnstone AC 1980 Hyperthyroidism in an aged cat. Zealand Vet J 29:70-72. 20. Holzworth J, Theran P, Carpenter JL, Harpster NK, Todoroff RJ 1980 Hyperthyroidism in the cat: Ten cases. JAVMA 176: 345-353. 21. McMillan FD, Sherding RG 1981 Feline hyperthyroidism. 11:25-32. 22. Peterson ME, Kintzer PP. Cavanaugh PG. Fox PR, Ferguson DC. Johnson GF, Becker DV 1983 Feline hyperthyroidism: Pretreat¬ ment clinical and laboratory evaluation of 131 cases. JAVMA 183:103-110. 23. Turrel JM. Feldman EC. Hays M. Horno!" W 1984 Radioactive iodine therapy in cats with hyperthyroidism. JAVMA 184:554-559. 24. Hays MT, Broome MR, Turrel JM 1988 A multicompartmental model for iodide, thyroxine, and triiodothyronine metabolism in normal and spontaneously hyperthyroid cats. Endocrinology 122:2444-2461.

Address reprint requests to: T. Hays, M.D. (151) DVA Medical Center 3801 Miranda Avenue Palo Alto, CA 94304

Marguerite