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Biomedical subjects

C M Mendel

Publications and source records attributed to C M Mendel.

At least 19 recordsLinked to original sources
PubMed1992

Thyroxine (T4) transport and distribution in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from transthyretin.

To test whether plasma transthyretin (TTR) might play a specific direct role in the transfer of T4 from the plasma to tissues, in vivo kinetic studies were performed in control rats and in rats treated with EMD 21388, a synthetic flavonoid that displaces T4 from TTR. The plasma disappearance curves of simultaneously injected [125I]T4 and [131I]albumin were analyzed to determine the rate constant for the transfer of T4 from the extracellular compartment to the rapidly exchangeable intracellular compartment (KE) and the steady state distribution ratio of T4 between the rapidly exchangeable intracellular compartment and the extracellular compartment (Imax/Emin). When rats were injected ip with EMD 21388 (2 mumol/100 g BW), the free T4 fraction in serum increased approximately 8-fold. This was due to displacement of T4 from TTR, as assessed by electrophoresis of serum proteins in the presence of [125I]T4. Concomitantly, both KE and Imax/Emin increased 6-fold in the treated rats. These results fail to confirm a major specific role for TTR in the transfer of T4 from the plasma to tissues. Instead, they are consistent with both the free hormone transport hypothesis and the free hormone hypothesis in this setting.

Animals↗
PubMed1992

Interconversion between apolipoprotein A-I-containing lipoproteins of pre-beta and alpha electrophoretic mobilities.

Apolipoprotein (apo) A-I-containing lipoproteins can be separated into two subfractions, pre-beta HDL and alpha HDL (high density lipoproteins), based on differences in their electrophoretic mobility. In this report we present results indicating that these two subfractions are metabolically linked. When plasma was incubated for 2 h at 37 degrees C, apoA-I mass with pre-beta electrophoretic mobility disappeared. This shift in apoA-I mass to alpha electrophoretic mobility was blocked by the addition of either 1.4 mM DTNB or 10 mM menthol to the plasma prior to incubation, suggesting that lecithin:cholesterol acyltransferase (LCAT) activity was involved. There was no change in the electrophoretic mobility of either pre-beta HDL or alpha HDL when they were incubated with cholesterol-loaded fibroblasts. However, after exposure to the fibroblasts, the cholesterol content of the pre-beta HDL did increase approximately sixfold, suggesting that pre-beta HDL can associate with appreciable amounts of cellular cholesterol. Pre-beta HDL-like particles appear to be generated by the incubation of alpha HDL with cholesteryl ester transfer protein (CETP) and either very low density lipoproteins (VLDL) or low density lipoproteins (LDL). This generation of pre-beta HDL-like particles was documented both by immunoelectrophoresis and by molecular sieve chromatography. Based on these findings, we propose a cyclical model in which 1) apoA-I mass moves from pre-beta HDL to alpha HDL in connection with the action of LCAT and the generation of cholesteryl esters within the HDL, and 2) apoA-I moves from alpha HDL to pre-beta HDL in connection with the action of CETP and the movement of cholesteryl esters out of the HDL. Additionally, we propose that the relative plasma concentrations of pre-beta HDL and alpha HDL reflect the movement of cholesteryl esters through the HDL. Conditions that result in the accumulation of HDL cholesteryl esters will be associated with low concentrations of pre-beta HDL, whereas conditions that result in the depletion of HDL cholesteryl esters will be associated with elevated concentrations of pre-beta HDL. This postulate is consistent with published findings in patients with hypertriglyceridemia and LCAT deficiency.

Apolipoprotein A-I↗
PubMed1991

Inability to detect an inhibitor of thyroxine-serum protein binding in sera from patients with nonthyroid illness.

Sera from 111 patients hospitalized on acute-care wards (including 32 in the intensive care unit) were examined for the possible presence of inhibitors of thyroxine (T4)-serum protein binding in an assay employing equilibrium dialysis. In 38 of these sera, the unbound (free) T4 fraction was 50% or more higher than the free T4 fraction in a pool of normal sera. From the free T4 fraction in each of the 111 serum samples and the free T4 fraction in the pool of normal sera, the predicted free T4 fractions in mixtures (1:1) of each of these sera with the normal pool were calculated (assuming the absence of binding inhibitors) from the appropriate mass action equations. It was reasoned that a free T4 fraction in any mixture that exceeded this predicted value would indicate the possible presence of a binding inhibitor. (The normal pool was selected for having a low serum triglyceride concentration, to minimize in vitro generation of free fatty acids.) However, for the 111 serum samples studied, the free T4 fraction in the mixture exceeded the upper 95% confidence limit of this predicted value in only one case, and then just barely. Thus, evidence for an inhibitor of T4-serum protein binding in sera from patients with nonthyroid illness could not be found. Twenty-eight of the serum samples were also examined in a similar assay that employed ultrafiltration of undiluted serum instead of equilibrium dialysis. Evidence for an inhibitor of T4-serum protein binding similarly could not be found. Because part of the reason for postulating the existence of such a binding inhibitor has been the performance of the triiodothyronine (T3) resin uptake test in patients with nonthyroid illness, an alternative explanation for this phenomenon was sought. When thyroid hormone-binding globulin (TBG) was desialylated by treatment with neuraminidase, its avidity for T4 was markedly decreased, but its avidity for T3 was unchanged. Thus, if desialylated TBG circulates in patients with nonthyroid illness as previously reported, it could explain not only the low serum T4 concentrations despite near normal immunoreactive TBG concentrations, but also the poor performance of the T3 resin uptake test (where T4 binding capacity is overestimated) in these patients.

Blood Proteins↗
PubMed1991

Uptake of corticosterone by the perfused rat liver.

The mechanism of hepatic uptake of corticosterone from plasma was investigated in the isolated perfused rat liver using an indicator-dilution method. The hepatic influx rate constant for free corticosterone was determined from measurements of the rate of hepatic uptake of corticosterone from protein-free buffer. The rate of hepatic uptake of corticosterone from pooled normal rat serum was then measured. A general model of hormone transport that does not assume that hormone-protein complexes remain at equilibrium during transit through the hepatic sinusoids was used to ask whether this observed rate of uptake could be accounted for by a pool of free corticosterone that turns over very rapidly. Parameter values used in this analysis included the measured concentrations of albumin and corticosteroid-binding globulin in the serum, literature values for the rate constants describing the interactions of corticosterone with these proteins, and the value of the hepatic influx rate constant for free corticosterone determined in the present study. The rate of hepatic uptake of corticosterone from rat serum that we observed was very similar to the rate of uptake predicted by this model to occur via the pool of free corticosterone.

Animals↗
PubMed1991

Free fatty acids do not influence the concentrations of free steroid hormones in serum under physiological conditions.

In several recent studies it has been suggested that FFA may influence the concentrations of unbound steroid hormones in serum, but the experimental design of these studies has been questioned. We have reexamined the effects of oleic acid on the unbound concentrations of several steroid hormones in serum, including cortisol, testosterone, and estradiol. The results demonstrate that under physiological conditions, oleic acid does not affect the unbound concentrations of these hormones when assays are carried out with whole serum.

Estradiol↗
PubMed1990

Rates of dissociation of steroid and thyroid hormones from human serum albumin.

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure rates of dissociation of steroid and thyroid hormones from human serum albumin. Modification of a previously described assay allowed measurements at 1-s intervals. Nevertheless, this still permitted only minimum estimates of the dissociation rate constants. The hormones studied were thyroxine, 3,5,3'-triiodothyronine, cortisol, corticosterone, testosterone, dihydrotestosterone, estradiol, progesterone, and aldosterone. The apparent dissociation rate constant of the thyroxine-albumin complex at 37 degrees C was 1.3 +/- 0.2 s-1 (t 1/2, 0.5 s). The apparent dissociation rate constants of the other hormone-albumin complexes at 37 degrees C generally exceeded 2 s-1 (t 1/2 less than 0.35 s). Apparent dissociation rate constants at 4 degrees C were only slightly lower. These findings indicate that steroid and thyroid hormones dissociate from albumin rapidly compared with the 1-s capillary transit times that characterize many tissues.

Aldosterone↗
PubMed1990

Rates of dissociation of sex steroid hormones from human sex hormone-binding globulin: a reassessment.

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure the rates of dissociation of dihydrotestosterone (DHT), testosterone (T), and estradiol (E2) from their binding proteins in human serum at 37 degrees C. Because measurements were begun after each hormone had fully (greater than 99%) dissociated from albumin, the observed rates of dissociation correspond to the rates of dissociation of the sex hormone-binding globulin (SHBG)-hormone complexes. The dissociation rate constants of the hormone-SHBG complexes were determined to be 0.016 +/- 0.001, 0.056 +/- 0.002, and 0.083 +/- 0.003 s-1 for DHT, T, and E2, respectively, corresponding to half-times of dissociation (t1/2) of 43, 12 and 8.4 s, respectively. The physiological significance of these findings can best be appreciated by comparing these t1/2 s with the capillary and sinusoidal transit times of various tissues (less than 1 s to approximately 10 s).

Dihydrotestosterone↗
PubMed1990

Thyroxine uptake by perfused rat liver. No evidence for facilitation by five different thyroxine-binding proteins.

Rates of hepatic uptake of thyroxine (T4) from dilute solutions of five different plasma T4-binding proteins were measured in the isolated perfused rat liver using an indicator dilution method. For each protein, this rate was compared with the rate of spontaneous dissociation of the T4-protein complex measured in vitro. Proteins studied were human T4-binding globulin (TBG), human T4-binding prealbumin (TBPA), human albumin, rat TBPA, and human albumin isolated from subjects with familial dysalbuminemic hyperthyroxinemia. For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.

Animals↗
PubMed1989

Distribution of lipid-binding regions in human apolipoprotein B-100.

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.

Apolipoprotein B-100↗
PubMed1989

Modeling thyroxine transport to liver: rejection of the "enhanced dissociation" hypothesis as applied to thyroxine.

Three models for the hepatic uptake of thyoxine (T4) from human plasma were considered: 1) uptake occurs exclusively via the pool of free T4 after spontaneous dissociation of T4-plasma-protein complexes, 2) uptake occurs primarily via the pool of bound T4 by the interaction of one or more binding proteins with the cell-surface membrane, and 3) uptake occurs primarily by "enhanced dissociation" of T4 from one or more of its binding proteins within the sinusoids. Each of these models was examined in relation to well-accepted unidirectional uptake and steady-state kinetics data that indicate that 1) between 4 and 24% of the T4 in normal human serum is taken up unidirectionally by the liver in a single pass, and 2) the in vivo disposal rate of T4 is unaffected by primary changes in the plasma concentration of thyroid hormone-binding globulin. Both analytical and numerical techniques were used. The first two models were found to be compatible with both the steady-state kinetics data and the unidirectional uptake data, given certain assumptions in each of the models. Although theoretically distinguishable on the basis of unidirectional uptake data, uncertainty over the true uptake (influx) rate constant for free T4 prevented resolution between these two models. In contrast, the third model, that of enhanced dissociation [W. M. Pardridge, Am. J. Physiol. 252 (Endocrinol. Metab. 15): E157-E164, 1987], was found, as currently formulated with respect to T4, to be incompatible with both the steady-state kinetics data and the unidirectional uptake data.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Thyroxine transport and distribution in Nagase analbuminemic rats.

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin-bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550). To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values. Evaluation of the plasma disappearance curves of simultaneously injected 125I-T4 and 131I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51 +/- 21 ng/min, mean +/- SD) and in the control rats (54 +/- 15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13 +/- 0.53 vs. 1.22 +/- 0.36 micrograms). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9 +/- 3.3%, n = 3, vs. 11.4 +/- 3.4%). When greater than 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3 +/- 2.1%, n = 5, vs. 15.2 +/- 2.5%). These data provide the first valid experimental test of the enhanced dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.

Animals↗
PubMed1989

The free hormone hypothesis: a physiologically based mathematical model.

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.

Animals↗
PubMed1989

Uptake of cortisol by the perfused rat liver: validity of the free hormone hypothesis applied to cortisol.

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.

Animals↗
PubMed1989

Conservation of free but not total or non-sex-hormone-binding-globulin-bound testosterone in serum from Nagase analbuminemic rats.

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.

Animals↗
PubMed1988

Radiation inactivation of binding sites for high-density lipoproteins in human liver membranes.

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.

5'-Nucleotidase↗
PubMed1988

Radiation inactivation of binding sites for high density lipoproteins in human fibroblast membranes.

Radiation inactivation and target analysis were used to determine the molecular mass of the binding sites for high density lipoproteins (HDL) on membranes prepared from human fibroblasts. These membrane binding sites shared characteristics with the previously described HDL binding sites on whole fibroblasts in tissue culture. They exhibited the same affinity for HDL, the same ligand specificity, and the same sensitivity to proteolytic agents. They were also up-regulated by cholesterol loading of the cells. Kinetics of HDL dissociation from membrane binding sites could not be described by a single exponential function, indicating that HDL probably bind to multiple classes of sites on fibroblast membranes. After exposure to ionizing radiation, these sites decreased in number as an apparent single exponential function of radiation dose, corresponding to an average molecular mass of 16,000 +/- 1,000 Da, which is smaller than any known cell-surface receptor protein. These data indicate that HDL binding sites on fibroblast membranes are not "classical" receptors in that they are kinetically heterogeneous and small in molecular mass.

Binding Sites↗
PubMed1988

Uptake of thyroxine by the perfused rat liver: implications for the free hormone hypothesis.

To investigate the mechanism by which thyroxine (T4) in plasma enters hepatic cells, we measured the rate constants for uptake of free T4 by the perfused rat liver and for dissociation of T4 from its plasma binding proteins. Quantitative autoradiography of liver lobules after perfusion with [125I]T4 indicated an apparent rat constant for removal of free T4 from the sinusoids of at least 1.1 +/- 0.2 s-1. Single-pass extraction of T4 from human serum was 10.6 +/- 1.7% at physiological flow rates (1 ml.min-1.g liver-1). Rate constants for dissociation of T4 from plasma binding proteins at 37 degrees C (determined by rapid filtration) were 0.017 +/- 0.002 s-1 for human thyroid hormone-binding globulin, 0.080 +/- 0.015 s-1 for human thyroid hormone-binding prealbumin, and greater than 0.5 s-1 for human albumin. To investigate the factors that determine the concentration of T4 within hepatic cells, we analyzed the above data together with data reported in the literature on the equilibrium-binding constants and the rate constant for cellular metabolism of T4. Analysis of all of these data using a previously published mathematical model leads to the following conclusions for the physiological state: 1) metabolism, not uptake, is rate limiting to removal of T4 from plasma by the liver; 2) binding equilibrium is present in the intrahepatic plasma; 3) intracellular T4 is in equilibrium with the free T4 pool in plasma (and maintenance of this equilibrium may be an important function of plasma thyroid hormone-binding proteins); and 4) the concentration of T4 within the liver is proportional to the concentration of free T4 in the plasma. Our data do not allow us to determine definitively whether hepatic uptake of T4 occurs only from the free T4 pool in plasma or also from the protein-bound pool by interaction of one or more of the binding proteins with the liver cell. However, mathematical analysis indicates that this distinction is irrelevant to steady-state intracellular hormone concentrations when equilibrium exists between the plasma and cytosolic pools of hormone.

Animals↗