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The iv administration of heparin causes an increase in the plasma free T4 concentration, as determined by equilibrium dialysis. The mechanism and physiological consequences of this action of heparin are unknown. To explore the possibility that the heparin-induced increase in plasma free T4 is an in vitro artifact due to generation of FFA during equilibrium dialysis, we studied plasma samples from 10 subjects treated with iv heparin. In plasma from 4 of these subjects, free T4 concentrations measured by equilibrium dialysis did not increase above baseline values after heparin administration. In incubations performed in parallel with the equilibrium dialysis measurements, FFA concentrations in these plasma samples were found to increase, but in no subject did they exceed 2.5 meq/L after incubation. In contrast, in plasma from the other 6 subjects treated with heparin, free T4 concentrations rose markedly (by 130-520%) above baseline values after heparin administration. In all of these postheparin plasma samples, FFA concentrations were less than 2.8 meq/L before incubation, but rose during incubation by 80-270% to more than 3.8 meq/L. Treatment of these plasma samples with protamine to inhibit lipoprotein lipase and with specific antiserum to inhibit hepatic triglyceride lipase before equilibrium dialysis or incubation prevented, in parallel, the heparin-induced increases in FFA and free T4 concentrations. From these findings we conclude that the heparin-induced increase in free T4 is usually an in vitro artifact, and that most subjects receiving heparin have a normal plasma free T4 concentration in vivo. We also conclude that this in vitro artifact may account for many of the findings that led to the postulate of an inhibitor of T4 binding to plasma and intracellular proteins in heparin-treated patients and perhaps in patients with nonthyroid illness as well.

To investigate the mechanism by which T3 in plasma enters hepatic cells, we measured rate constants for the uptake of unbound (free) T3 by the perfused rat liver, for the hepatic uptake of T3 from serum, and for the spontaneous dissociation of T3 from its plasma binding proteins. Quantitative autoradiography of liver lobules after perfusion with [125I]T3 in protein-free buffer indicated a high apparent rate constant for removal of T3 from the sinusoids; its minimum estimate was 2.4 +/- 0.2 sec-1. The single pass extraction of T3 in human serum by the perfused rat liver was 31.6 +/- 4.5% at the supraphysiological flow rate of 3 ml/min/g liver (sinusoidal transit time, approximately 3 sec). Sixty percent of the T3 in this serum dissociated spontaneously from its binding proteins in 3 sec, as determined by a rapid filtration assay. Based on these data, we conclude that the pool of free T3 in plasma turns over very rapidly in vivo and probably accounts for the entire hepatic uptake of T3 from plasma. Using additional data on the rate constant for cellular metabolism of T3 obtained from values reported in the literature, a previously published general mathematical model of ligand transport was applied to all of these data, yielding the following conclusions for the physiological state. 1) Metabolism, not uptake, is rate limiting to removal of T3 from plasma by the liver. 2) Intracellular T3 is in virtual equilibrium with the free T3 pool in plasma. 3) Intracellular T3 concentrations reflect the concentration of free T3 in plasma, as predicted by the free hormone hypothesis. It is shown mathematically that these conclusions are independent of whether a gradient exists between extra- and intracellular T3 concentrations, and that they would still hold even if the tissue uptake of T3 occurred by a mechanism that acted directly on the plasma protein-bound pool of T3.

The concentration of FFA in normal human plasma in vivo generally ranges between 0.2 and 0.7 meq/liter; slightly higher concentrations have occasionally been reported in patients who are seriously ill. To determine whether such FFA concentrations may increase the concentration of free T4 in serum, we added increasing amounts of oleic acid to pooled normal human serum (with known FFA content) and measured free T4 by equilibrium dialysis. Total FFA up to 3 meq/liter in normal serum, representing an FFA to albumin molar ratio of about 5:1, had little or no effect on the free T4 concentration, while higher FFA concentrations progressively increased free T4. This same molar ratio of FFA to albumin had to be exceeded to cause a significant increase in the free T4 concentration in diluted serum and in serum from patients with nonthyroid illness. Serum from which more than 95% of the albumin had been removed by chromatography with Affi-Gel blue was much more sensitive to the effects of FFA on free T4. This enhanced sensitivity was reversed by readdition of albumin to the serum, and the addition of albumin to normal serum resulted in diminished effects of FFA on free T4. These results indicate the following: physiological concentrations of FFA do not significantly increase the free T4 concentration in normal human serum; when FFA reach supraphysiological concentrations in serum (in vitro) and the higher affinity FFA-binding sites on albumin become saturated (apparently at an FFA to albumin molar ratio of approximately 5:1), the excess FFA interact with other serum proteins, including thyroid hormone-binding globulin, and thereby increase the free T4 concentration; the concentration of albumin (or other FFA binders) must be considered when evaluating the observed effects of FFA. To explore the relevance of these findings to the hypothesis that FFA may inhibit the binding of T4 to plasma proteins in patients with nonthyroid illness, we measured plasma FFA concentrations in 11 severely ill patients hospitalized in the intensive care unit. We found a mean plasma FFA concentration of 0.45 +/- 0.11 (+/- SEM) mEq/liter and a mean serum albumin concentration of 2.39 +/- 0.29 g/dl in these patients. Their mean plasma FFA to albumin molar ratio was 1.53 +/- 0.41. Since the FFA to albumin molar ratio must exceed about approximately 5:1 before a significant increase in the serum free T4 concentration occurs, these results suggest that FFA do not commonly influence the circulating free T4 concentration in vivo, even in severely ill patients.

The mechanism by which cortisol in plasma enters hepatic cells was investigated using the isolated perfused rat liver. To determine whether hepatic uptake of cortisol from serum can be accounted for entirely by the pool of unbound (free) cortisol, we compared observed uptake rates with the equilibrium-free fraction of cortisol in serum and the rates of dissociation of cortisol from its serum binding proteins (determined using a rapid filtration assay based on transfer of [3H] cortisol to dextran-coated charcoal). More than 95% of the cortisol in both human and rat serum dissociated spontaneously from its binding proteins within 5 sec at 37 C. The fractional unidirectional hepatic uptakes of cortisol from pooled human serum and pooled rat serum were 59.4 +/- 5.4% and 59.5 +/- 1.0% (mean +/- SE), respectively, at the physiological flow rate of 1 ml/min.g liver. The corresponding free cortisol fractions in these sera were 4.53 +/- 0.15% and 8.16 +/- 0.23%, respectively. The fractional unidirectional hepatic uptake of cortisol from protein-free buffer averaged 99.9% (n = 5) at a flow rate of 3 ml/min.g liver. By calculating the appropriate rate constants and applying the Kety-Renkin-Crone equation to the above data, it can be shown that all of the cortisol taken up from serum by the perfused rat liver can be accounted for by the pool of free cortisol, which turns over very rapidly. The physiological significance of this finding is discussed in terms of a general mathematical model of hormone transport that delineates the conditions under which the free hormone hypothesis is and is not valid.

A number of studies conducted in the last decade showed that saturable ('specific') binding, by itself, does not necessarily imply biological significance. That is, biological ligands were shown to bind to inert materials as well as to biological receptors in a saturable manner. In these studies specific binding was operationally defined as binding that was displaceable by excess concentrations of unlabeled ligand. This method of measuring specific binding is now no longer considered optimal. To investigate whether optimal (computer-assisted) techniques of measuring specific binding--namely, nonlinear least-squares curve fitting of total binding data, with mathematical separation of the total binding into its various components--might ensure biological significance of measured specific binding, we studied the binding of high-density lipoproteins (HDL3) to tissue culture dishes as an example of binding without biological significance. This binding closely followed the paradigm of a ligand interacting with a class of homogeneous, saturable sites and with a class of relatively unsaturable sites, just as it would have if the HDL3 were interacting with an unpurified biological receptor. This finding indicates that computer-assisted analysis, while most accurately describing binding data, nevertheless does not ensure that measured specific binding has biological significance. Saturability is such a nonselective feature of equilibrium binding data that it should probably no longer be considered one of the criteria for deciding whether or not a defined binding site is a receptor.

Investigations we carried out in man and animals in order to study the relationships between the electrical and the mechanical activities of the intestine, and to precise the role of BER in the regulation of intestinal motility. In the dog, an intestinal movements transducer allowed to establish that spiking activities were concomitant with circular contractions, and that these two phenomena were rhythmed by the slow waves. Under basal conditions, the longitudinal movements occurred independently; under stimulation longitudinal movements occurred independently; under stimulation longitudinal relaxations were observed to correspond to circular contractions. In man, the post-laparotomy paralytic ileus provoked abnormalities in the BER which seemed to be related to a disturbance of the coordination of the motility, resulting in the absence of intraluminal propulsion. Administration of coherin, a postpituitary extract, was followed by a regularization of the BER and by a decrease of paralytic ileus duration. This confirms the important role of the BER in the myogenic regulation of the intestinal motility.

Red blood cell T4 concentrations (RBC T4) were measured in 15 normal subjects, 13 patients with hypo- or hyperthyroidism, and 10 patients with elevated or decreased serum thyroid hormone binding. In each case, RBC T4 was compared with the serum concentration of free T4 measured by equilibrium dialysis ( FT4D ). RBC T4 correlated significantly with FT4D in these subjects (r = 0.90; P less than 0.001). The normal range for RBC T4 was 0.27-0.83 ng/ml. RBC T4 was below the normal range in all 8 patients with hypothyroidism and above the normal range in all 5 patients with hyperthyroidism. It was within the normal range in all 4 subjects with absent or low T4-binding globulin (TBG) and in 5 of the 6 subjects with elevated TBG or familial dysalbuminemic hyperthyroxinemia. The sixth subject (increased TBG) had elevated RBC T4 and FT4D . RBC T4 was similarly measured in 10 patients with severe nonthyroid illness (NTI), 5 of whom had decreased serum concentrations of total T4. RBC T4 was normal in 8 of these patients, elevated in 1, and decreased in 1; in comparison, FT4D was normal in 4, elevated in 5, and decreased in 1. Eight patients receiving continuous iv infusions of heparin were also studied because of previously described similarities in the in vitro thyroid tests of heparin-treated and euthyroid sick patients. FT4D was elevated in 7 of the heparin-treated patients, whereas RBC T4 was elevated in only 2. Furthermore, for any given value of FT4D , RBC T4 was lower in heparin-treated patients than in normal subjects, indicating the presence of an inhibitor of cellular T4 binding in these patients. This putative inhibitor, demonstrated by an elevated FT4D to RBC T4 ratio, was present in 6 of the 8 heparin-treated patients and in 5 of the 10 patients with NTI. The findings of this study support the hypothesis that an inhibitor of cellular T4 binding is present in the serum of some patients with NTI and in most heparin-treated individuals.

Two general models have been proposed for predicting the effects of metabolism, protein binding, and plasma flow on the removal of drugs by the liver. These models differ in the degree of plasma mixing assumed to exist within each hepatic sinusoid. The venous equilibrium model treats the sinusoid as a single well-stirred compartment, whereas the sinusoidal model effectively breaks up the sinusoid into a large number of sequentially perfused compartments which do not exchange their contents except through plasma flow. As a consequence, the sinusoidal model, but not the venous equilibrium model, predicts that the concentration of highly extracted drugs will decline as the plasma flows through the hepatic lobule. To determine which of these alternative models best describes the hepatic uptake process, we looked for evidence that concentration gradients are formed during the uptake of [125I]thyroxine by the perfused rat liver. Autoradiography of tissue slices after perfusion of the portal vein at physiologic flow rates with protein-free buffer containing [125I]thyroxine demonstrated a rapid exponential fall in grain density with distance from the portal venule, declining by half for each 8% of the mean length of the sinusoid. Reversing the direction of perfusate flow reversed the direction of the autoradiographic gradients, indicating that they primarily reflect differences in the concentration of thyroxine within the hepatic sinusoids rather than differences in the uptake capacity of portal and central hepatocytes. Analysis of the data using models in which each sinusoid was represented by different numbers of sequentially perfused compartments (1-20) indicated that at least eight compartments were necessary to account for the magnitude of the gradients seen.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied two families with familial dysalbuminemic hyperthyroxinemia (FDH), a recently described entity characterized by marked elevation of serum T4 due to increased binding of T4 to albumin. The seven affected subjects had elevated serum total T4 levels (range, 15.3-25.2 micrograms/dl; normal, 4.5-11.0 micrograms/dl), but normal serum free T4 levels, as measured by equilibrium dialysis. Their serum T3 levels ranged from 1.40-2.46 ng/ml (normal, 0.9-2.0 ng/ml). The proportion of T4 associated with serum albumin was increased approximately 4-fold in the affected subjects, as shown both by reverse flow paper electrophoresis and immunoprecipitation of albumin-bound T4 with antihuman serum albumin. In vivo T4 kinetic studies were performed in the two index subjects to assess the effects of the increased binding of T4 to albumin on the in vivo transport, distribution, and disposal of T4. Compared to values in normal subjects, the MCR of T4 was decreased by about 50%, and its total body (extrathyroidal) pool size was increased by approximately 50%; the T4 production rate was normal. The extracellular T4 pool size was increased by about 100% in the FDH subjects, but the rapidly exchangeable intracellular T4 pool size was normal. The unidirectional T4 clearance rate from plasma into the rapidly exchangeable cellular compartment was reduced by approximately 50%, but the absolute rate of T4 flux from plasma into the cellular compartment was normal. Thus, the in vivo kinetic data indicate that the increased plasma binding of T4 in FDH alters the distribution of T4 in favor of the extracellular compartment, retards the fractional rate of transfer of T4 into cells, and slows the metabolic clearance of T4. However, the absolute rate of T4 flux into the rapidly exchangeable cellular compartment, the intracellular T4 pool size, and the T4 disposal rate are all normal in FDH, consistent with the normal serum concentrations of free T4 and the eumetabolic state of these individuals.

The free hormone hypothesis states that the biological activity of a given hormone is affected by its unbound (free) rather than protein-bound concentration in the plasma. The fundamental mathematical and physiological principles relating to this hypothesis are reviewed, along with experimental data that shed light on its validity. It is shown that whether or not this hypothesis is likely to be valid for any given hormone will depend largely on which step in the tissue uptake process (plasma flow, dissociation from plasma binding proteins, influx, or intracellular elimination) is rate-limiting to the net tissue uptake of that hormone. It is further shown that the free hormone hypothesis could hold even if tissue uptake of hormone occurred by a mechanism that acted directly on one or more circulating protein-bound pools of hormone. Indeed, many of the data previously interpreted as being inconsistent with the free hormone hypothesis are in fact readily consistent with it when its predictions are fully understood. Nevertheless, the free hormone hypothesis is not likely to be valid for all hormones with respect to all tissues. It is likely to be valid with respect to all tissues for the thyroid hormones, for cortisol, and for the hydroxylated metabolites of vitamin D. For many of the other steroid hormones, however, it is likely to be valid with respect to some tissues, but not with respect to others (in particular, the liver). And for some of the steroid hormones (in particular, progesterone) it may not hold at all.

The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.

Nagase analbuminemic rats have normal reproductive capacity, normal apparent libido, and normal serum concentrations of LH and FSH. Therefore, it is reasonable to assume that intracellular sex steroid hormone concentrations are normal or at least adequate to maintain normal reproductive function in these rats. To test whether intracellular testosterone concentrations in these rats are maintained by the circulating concentration of free or free-plus-weakly-bound testosterone, we measured the concentrations of total testosterone, free testosterone, and non-sex-hormone-binding-globulin-bound testosterone in sera from five adult male Nagase analbuminemic rats and from five age- and sex-matched controls. We found that the analbuminemic rats had markedly decreased serum concentrations of total and non-sex-hormone-binding-globulin-bound testosterone, but normal serum concentrations of free testosterone. These results suggest that intracellular concentrations of testosterone in biologically relevant organs of the rat are maintained by the concentration of free rather than free-plus-weakly-bound testosterone in plasma, in accord with the free hormone hypothesis.

High-density lipoproteins (HDL) are involved in 'reverse cholesterol transport'. Whether or not cell-surface receptors for HDL exist and participate in this process remains controversial, and part of this controversy has centered on the nature of the HDL binding sites. We therefore used radiation inactivation to determine the molecular mass of the HDL binding sites in human liver membranes in situ. These binding sites, which shared all the characteristics of previously described putative HDL receptors, had a molecular mass of less than 10 kDa, indicating that they are probably not proteins. In addition, the binding of HDL to protein-free liposomes was characterized and was found to display the same affinity (KD = 5 micrograms protein/ml approximately 5.10(-8) M) as that to cell membranes, indicating that HDL binding to cell membranes may not require membrane proteins. These observations highlight an important application of radiation inactivation: the ability to demonstrate that something - in this case, a high-molecular-weight protein that accounts for the majority of the HDL binding activity in human liver membranes - is absent.