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Biomedical subjects

C M Mendel

Publications and source records attributed to C M Mendel.

51 records · Page 3Linked to original sources
PubMed1988

Cell-surface binding sites for high density lipoproteins do not mediate efflux of cholesterol from human fibroblasts in tissue culture.

The present investigation was designed to test the hypothesis that binding sites for high density lipoproteins (HDL3) on cell surfaces of peripheral tissues mediate cholesterol efflux from these cells. This hypothesis had been formulated to explain two observations: 1) HDL3 binding to peripheral cells and HDL3-mediated cholesterol efflux from these cells had both been found to saturate at similar unbound (free) HDL3 concentrations; and 2) both of these processes had been found to be similarly "up-regulated" by loading the cells with cholesterol. In the present study, however, we found that the "specific" binding of HDL3 to cholesterol-loaded human fibroblasts was saturated at a free HDL3 concentration of approximately 20 micrograms protein/ml, whereas efflux of cholesterol from these cells to HDL3 did not "saturate" even at a free HDL3 concentration of 2000 micrograms protein/ml. In addition, we found that the increase in cholesterol efflux caused by loading the fibroblasts with cholesterol was no greater when the acceptor particles were HDL3 than when albumin or phospholipid vesicles served as acceptors, despite a marked increase in HDL3 binding to these cells. Because HDL3 binding to these cells and HDL3-mediated cholesterol efflux from these cells do not saturate at similar free HDL3 concentrations, and because the cholesterol-induced increase in HDL3 binding is not accompanied by a similar increase in cholesterol efflux that is specific for HDL3, we conclude that the described HDL3 binding sites on human fibroblasts do not mediate cholesterol efflux.

Carrier Proteins↗
PubMed1987

Binding of lipoproteins to inert materials revisited with computer-assisted analysis.

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.

Culture Techniques↗
PubMed1987

Thyroid hormone-binding proteins in plasma facilitate uniform distribution of thyroxine within tissues: a perfused rat liver study.

We used autoradiography to test the hypothesis that a major function of thyroid hormone-binding proteins in plasma is to ensure uniform distribution of thyroid hormones among cells of a given tissue. The distribution of [125I]T4 within rat hepatic lobules was determined after its single pass perfusion through the portal vein in solutions containing or lacking thyroid hormone-binding proteins. These proteins included thyroid hormone-binding globulin, thyroid hormone-binding prealbumin, and albumin. In the absence of these proteins, virtually all of the perfused T4 was taken up by the periportal cells, and subsequent perfusion with protein-free solution did not cause redistribution of this T4. In the presence of these proteins, in contrast, the perfused T4 was taken up uniformly by all cells within the lobule. Albumin alone was sufficient to ensure uniform cellular uptake of T4. However, variation of oleic acid concentrations within the physiological range markedly influenced the concentration of free T4 in a solution of 4% human serum albumin, but not in human serum. These results indicate that uniform distribution of T4 within tissues requires circulating thyroid hormone-binding proteins, and that the specific binding proteins, thyroid hormone-binding globulin and thyroid hormone-binding prealbumin, are required to ensure nonfluctuating circulating concentrations of free T4 in vivo. Other hormone-binding proteins in plasma and some transport proteins may function similarly.

Animals↗
PubMed1987

Mechanism of the heparin-induced increase in the concentration of free thyroxine in plasma.

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.

Fatty Acids, Nonesterified↗
PubMed1986

A trial of amitriptyline and fluphenazine in the treatment of painful diabetic neuropathy.

We conducted a double-blind, placebo-controlled, crossover study of the effectiveness of amitriptyline and fluphenazine in alleviating the pain of diabetic peripheral neuropathy in six diabetic patients. Pain was evaluated by the patients with a graphic rating scale. A placebo response was found, but no additional effect of amitriptyline and fluphenazine was seen. Although the statistical power of this study was low, these data, when combined with a reevaluation of previous trials of amitriptyline and fluphenazine in the treatment of painful diabetic neuropathy, indicate that there is no justification for the use of these agents in the treatment of painful neuropathy outside of large, controlled clinical trials. Depression as a possible cause of this condition should not go unnoted or untreated.

Adult↗
PubMed1986

Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver.

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.

Animals↗
PubMed1986

The hepatic sinusoid is not well-stirred: estimation of the degree of axial mixing by analysis of lobular concentration gradients formed during uptake of thyroxine by the perfused rat liver.

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)

Animals↗
PubMed1986

Effect of free fatty acids on the concentration of free thyroxine in human serum: the role of albumin.

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.

Fatty Acids, Nonesterified↗
PubMed1985

'Non-specific' binding. The problem, and a solution.

The concept of 'non-specific' binding, as it relates to studies of the binding of hormones to their receptors, is reviewed. It is concluded that the most widely used operational definition, namely binding that is not displaceable by an excess of unlabelled ligand, is often inaccurate, resulting either in overestimation of the number of high-affinity receptors and underestimation of the affinity of a given hormone for its receptor, or in a curvilinear Scatchard plot suggesting (artifactually) the presence of negative co-operativity or multiple classes of binding sites. The general use of an alternative approach to non-specific binding, in which the non-specific component is assessed from an analysis of total binding, is advocated. The superiority of this approach is illustrated with data on the binding of high-density lipoproteins to their receptors.

Animals↗
PubMed1985

The effect of ligand heterogeneity on the Scatchard plot. Particular relevance to lipoprotein binding analysis.

Computer simulations of equilibrium binding studies of a mixture of two labeled ligands binding competitively to a single class of identical and independent sites (receptors) were performed to investigate how ligand heterogeneity affects the observed data in such studies. The simulated data are presented in Scatchard plots. Ligand heterogeneity was generally found to be indistinguishable from the case of a homogeneous ligand when usual experimental conditions applied (that is, Scatchard plots of the data were straight lines). Some factors that increased the probability of recognizing heterogeneity in the system were identified, however. These are 1) a large difference between the dissociation constants of the two ligands, 2) a high concentration of receptors relative to the dissociation constant of the higher-affinity ligand, 3) a high concentration of the lower-affinity ligand relative to that of the higher-affinity ligand, 4) a high specific activity of the lower-affinity ligand relative to that of the higher-affinity ligand, and 5) lack of experimental error. When ligand heterogeneity (under certain conditions) did cause curvilinearity in the Scatchard plot, the curve formed was always concave-downwards. Thus, ligand heterogeneity may occasionally mimic positive cooperativity, but never mimics negative cooperativity or multiple classes of binding sites. Implications of these findings for equilibrium binding studies involving lipoproteins (which are generally isolated as heterogeneous mixtures of particles) are discussed in detail. These findings are also relevant to equilibrium binding studies using ligands which are mixtures of stereoisomers or which contain chemical or radiochemical impurities.

Computers↗
PubMed1984

Red blood cell thyroxine in nonthyroid illness and in heparin-treated patients.

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.

Erythrocytes↗
PubMed1984

Thyroxine distribution and metabolism in familial dysalbuminemic hyperthyroxinemia.

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.

Adult↗
PubMedDate not supplied

The free hormone hypothesis. Distinction from the free hormone transport hypothesis.

I. The free hormone transport hypothesis: distinction from the free hormone hypothesis. II. The free hormone hypothesis: importance of the rate-limiting step in tissue uptake. III. Explicit consideration of multiple plasma hormone-binding proteins in transport models. IV. Diffusion barriers in transport models. V. Functions of plasma hormone-binding proteins. VI. Conclusions and areas for future investigation.

Animals↗