Influence of nonesterified fatty acids and lysolecithins on thyroxine binding to thyroxine-binding globulin and transthyretin.

The hydrolysis of lecithin by phospholipase produces equimolar amounts of nonesterified fatty acids (NEFAs) and lysolecithin. In this study, we have evaluated the effect of lysolecithins and NEFAs on thyroid hormone binding by examining their interactions with thyroxine-binding globulin (TBG)(serum 1:10,000 dilution) and purified transthyretin (TTR). Unsaturated NEFAs (palmitoleic, oleic, linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acid) inhibited [125I]T4 binding to TBG. Their affinities, relative to unlabeled T4, ranged from 0.005 to 0.0016%, except for oleic acid with relative affinity of < 0.0005%. Saturated NEFAs, lauric, myristic, palmitic, and stearic acid were inactive. After purification by high-performance liquid chromatography, 1-oleoyl and 2-oleoyl lysolecithin displaced [125I]T4 from TBG with an affinity of 0.0006 and 0.0005%, respectively. On a molar basis, this affinity was approximately 10-fold lower than arachidonic acid, the most potent NEFA in inhibiting T4 binding to TBG in this assay system. Of all the NEFAs tested, only arachidonic acid inhibited [125I]T4 binding to TTR, with an affinity relative to unlabeled T4 of 0.49%. 1-Oleoyl, 1-palmitoyl, and 1-stearoyl lysolecithin were without effect on TTR binding. The T4-displacing effects of NEFAs are markedly attenuated by their extensive binding to albumin. Using purified [14C]NEFA preparations and heptane partitioning, the mean unbound percentages of linoleic, eicosapentaenoic, and docosahexaenoic acid in undiluted normal human serum were 0.00099, 0.0050, and 0.0042%, respectively (n = 3). In view of the very high degree of albumin binding of NEFAs, studies in diluted serum will grossly overestimate their competitor potency. The affinities of lysolecithins for the T4 binding sites of TBG and TTR are lower than those of NEFAs and depend on the fatty acid component. Lysolecithins are unlikely to influence plasma protein binding of T4 during critical illness.

A naturally occurring furan fatty acid enhances drug inhibition of thyroxine binding in serum.

We studied the thyroxine (T4)-displacing effects of a naturally occurring, highly albumin-bound furanoid acid that accumulates in serum in renal failure to concentrations in excess of 0.2 mmol/L. This substance, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), has been shown to displace acidic drugs from albumin binding. The effects of CMPF on ligand binding were assessed in the following systems: (1) T4 binding to T4-binding globulin (TBG) and transthyretin (TTR), (2) T4 binding in undiluted serum, (3) T4-displacing potency of fenclofenac, furosemide, diflunisal, and aspirin in undiluted serum, (4) serum binding of [14C]-drug preparations, and (5) serum binding of [14C]-oleic acid. CMPF had a minor direct effect on T4 binding to TBG comparable in relative affinity to that of aspirin, ie, almost 7 orders of magnitude less than T4 itself. CMPF alone at a concentration of 0.3 mmol/L, which produced only a 10% to 14% increase in free T4 augmented the T4-displacing effects of high therapeutic concentrations of the various drugs in undiluted serum as follows: furosemide by 180%, fenclofenac by 160%, diflunisal by 130%, and aspirin by 40%. In the presence of fenclofenac, increments of CMPF from 0.075 to 0.3 mmol/L progressively augmented the T4-displacing effect of this drug, associated with a progressive increase in its calculated free concentration. CMPF also inhibited the binding of [14C]-oleic acid, suggesting that in some situations CMPF could also indirectly influence thyroid hormone binding by increasing the unbound concentration of nonesterified fatty acids (NEFA), as previously described.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of the unbound fraction of long chain nonesterified fatty acids (NEFA) in serum: methodological considerations.

Long-chain nonesterified fatty acids (NEFA) are extensively bound to albumin; knowledge of their unbound concentrations is important in evaluating the numerous biologic effects attributed to these compounds. We measured the unbound fraction of five long-chain NEFA in serum using the equilibrium partition of 14C-NEFA between heptane and aqueous phases. Commercial 14C-NEFA preparations gave non-linear estimates of unbound fraction with serum dilution, consistent with the presence of polar tracer impurities, but 14C-NEFA purified by alkaline ethanol extraction gave an approximately linear relationship between unbound fraction and serum dilution over a 4096-fold range of dilution, provided that pH of the aqueous phase remained stable. Mean unbound percentages were: myristic acid 0.0066, linolenic acid 0.0019, arachidonic acid 0.0017, oleic acid 0.00078 and palmitic acid 0.00061. These data suggest that some previous studies appear to have overestimated the free fraction of long-chain NEFA at physiological albumin concentrations by at least one order of magnitude.

Isolation of opiate receptor ligands in coffee.

1. We have reported previously that instant coffee contains ligands for opiate receptors with characteristics similar to those of opiate antagonists. 2. A concentrate of receptor-active ligands from instant coffee was prepared by serial treatments involving Amberlite XAD-2, flash chromatography and gel permeation chromatography. 3. Examination of the final concentrate by GC-MS showed the presence of a number of isomeric (iso)feruloylquinic acid lactones. 4. It is suggested that the synthesis and biological testing of each quinide isomer will establish which is responsible for the opiate receptor activity of instant coffee.

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells.

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.

Non-isotopic spectrophotometric determination of the unbound fraction of drugs in serum.

A spectrophotometric method to measure the free fraction of highly-bound drugs in serum has been established for a range of non-steroidal anti-inflammatory drugs (NSAIDs) and for frusemide. Spectrophotometry is used to measure fractional transit of drug from a large volume of dialysate to a small volume of serum during dialysis to equilibrium. The method, which depends on the principle that drug transit from dialysate to serum is proportional to serum binding, requires neither isotopic drug preparations nor specific drug assays, is independent of extraction efficiency from the dialysate and requires no measurements from the serum compartment. Estimates of percent unbound fraction (% UF) for aspirin (6.0 +/- 0.9%), phenylbutazone (0.9 +/- 0.2%), and frusemide (1.8 +/- 0.2%) were comparable with those obtained with 14C drug preparations. Values for % UF were determined for eleven additional NSAIDs. The method was valid for a four-fold change in serum: dialysate ratio. Kinetics of frusemide binding to serum were comparable using [14C]frusemide and the test method. This technique may have general application in establishing the % UF for substances that are extensively bound to serum proteins and for identifying sera that show abnormal binding.

Interaction of furosemide with serum thyroxine-binding sites: in vivo and in vitro studies and comparison with other inhibitors.

The diuretic furosemide inhibits serum protein binding of T4 in equilibrium dialysis, dextran-charcoal, and competitive ligand binding separation systems and displaces [125I]T4 from isolated preparations of T4-binding globulin (TBG), prealbumin, and albumin. Equilibrium dialysis studies of undiluted normal serum showed that about 10 micrograms/ml furosemide increased the free T4 and free T3 fractions. Displacement occurred at lower drug concentrations in sera with subnormal albumin and TBG levels. Binding of [14C]furosemide to TBG was inhibited by unlabeled T4, suggesting that furosemide and T4 share a common binding site. A single oral dose of 500 mg furosemide given to five patients maintained on peritoneal dialysis increased the percentage of charcoal uptake of [125I]T4 (using serum diluted 1:10) from 4.1 +/- 1.0 (+/- SE) to 10.8 +/- 4.3 (P less than 0.01) after 2 h, while decreasing total T3 from 75 +/- 5 to 56 +/- 13 ng/dl (P less than 0.01) and total T4 from 6.7 +/- 0.9 to 4.8 +/- 0.8 micrograms/dl (P less than 0.01) after 5 h. Various ligands inhibited [125I]T4 binding to serum proteins in the following relative molar relationship: T4, 1; furosemide, 1.5 X 10(3); fenclofenac, 2 X 10(4); mefenamic acid. 2.5 X 10(4); diphenylhydantoin, 4 X 10[4); ethacrynic acid, 10(5); heparin 5 X 10(5); 2-hydroxybenzoylglycine, 10(6); and sodium salicylate, 1.5 X 10(6). These studies demonstrate that furosemide competes for T4-binding sites on TBG, prealbumin, and albumin, so that a single high dose can acutely lower total T4 and T3 levels. The drug is much more potent on a molar basis than other drug inhibitors of T4 binding, but at normal therapeutic concentrations, furosemide is unlikely to decrease serum T4 or T3. However, high doses, diminished renal clearance, hypoalbuminemia, and low TBG accentuate its T4- and T3-lowering effect. Hence, furosemide should be considered a possible cause of low thyroid hormone levels in patients with critical illness. The significance of this drug in reports of impaired hormone and drug binding in renal failure requires further assessment.

Further studies on neomycin and experimental hypertension.

Acute glucocorticoid (corticosterone) hypertension in the rate is significantly attenuated by neomycin administration (Honour 1981), as is ACTH-induced hypertension is the same species (Honour & Kent 1981) presumably by altering gut bacterial steroid metabolism. The effect on blood pressure of oral neomycin administration was therefore examined in hypertension resulting from administration of a variety of glucocorticoid hormones. Neomycin significantly attenuated the elevation of blood pressure following ACTH or corticosterone, as previously shown, or prednisolone 1 mg/day; it did not affect blood pressure in rats given dexamethasone 0.2 mg/day, or cortisol 4 mg twice daily. Since the differential effect of neomycin on hypertension parallels neither pressor nor glucocorticoid activity of the administered steroids we propose that it reflects different patterns of enterohepatic handling and metabolism.

High concentrations of furosemide inhibit serum binding of thyroxine.

Serum samples taken from four patients who had low serum T4 concentrations (less than 2 micrograms/dl) during severe non-thyroidal illness were found to contain a heat-stable, dialyzable inhibitor of 125I T4 binding to plasma proteins. Inhibitory activity coincided with high dose furosemide treatment for oliguric renal failure. Inhibition was proportional to the serum furosemide concentration and the effect was reproduced in vitro by addition of furosemide to normal serum. The inhibitory effect diminished with serum dilution while maintaining the same relative concentration of furosemide. A time-course study in one patient demonstrated a close temporal relationship between high serum concentrations of furosemide and subnormal T4, associated with T3 resin uptake values compatible with increased occupancy of T4-binding globulin by a competitor. These findings demonstrate that furosemide in high concentrations can inhibit T4 binding in plasma and may be a factor contributing to the development of the low T4 state in critical illness.

Coffee contains potent opiate receptor binding activity.

Opiate receptor-active peptide fragments (exorphins) have been identified recently in casein and gluten hydrolysates, and morphine has been found in bovine and human milk. To determine whether similar peptides or alkaloids occur in other foodstuffs, we have screened potential sources using a rat brain homogenate assay to detect opiate receptor activity. We report here that instant coffee powders from a variety of manufacturers compete with tritiated naloxone for binding to opiate receptors in the rat brain membrane preparations, with no significant difference between normal and decaffeinated coffee. The receptor binding activity resembles that seen with opiate antagonists, in that there was no change in the half-maximal effective dose (ED50) in the presence of 100 mM Na+; on bioassay, the activity was similarly shown to be antagonistic and specific for opiate-induced inhibition of twitch. Preliminary characterization of the activity reveals that it has a molecular weight (MW) in the range 1,000-3,500, is heat-stable, ether-extractable, not modified by enzymatic digestion with papain, and clearly separable from caffeine and morphine on TLC. As its concentration in an average cup of coffee is five times the ED50, these data suggest that drinking coffee may be followed by effects mediated via opiate receptors, as well as effects of caffeine.

Panax ginseng and Eleuthrococcus senticosus extracts--in vitro studies on binding to steroid receptors.

We have examined the binding of 44% saponin from Panax ginseng, and extracts from Eluthrococcus senticosus (Siberian ginseng) to classical steroid receptors in vitro. Both extracts had demonstrable affinity for progestin, mineralocorticoid and glucocorticoid receptors; the Siberian ginseng also bound to estrogen receptors. Highest affinity binding was to glucocorticoid receptors, with an approximate Ki of 8 x 10(-6) M for Panax ginseng. Such interactions may explain the reported glucocorticoid-like effects of ginseng in vivo.

19-nor analogs of adrenal steroids: mineralocorticoid and glucocorticoid receptor activity.

The effect of absence of the C-19 methyl group from five adrenal steroids has been studied in terms of their affinity for mineralocorticoid (MR) and glucocorticoid receptors (GR). In MR assays, 19-nordeoxycorticosterone and 19-norprogesterone showed 3-fold higher affinity for MR than did their respective parent steroids; 19-norcortisol had 1.5 times the affinity of cortisol for MR. In contrast, corticosterone and 19-nororticosterone showed equal affinity, and 19-noraldosterone showed less than 1% the MR activity of aldosterone. In GR assays, the absence of the C-19 methyl group from progesterone increased GR affinity 3-fold and deoxycorticosterone affinity 1.5-fold. In contrast, the other 19-nor steroids showed decreased affinity vis à vis their parent compounds (19-norcorticosterone, 30%; 19-norcortisol, 10%; 19-noraldosterone, < 1%). These findings suggest that while the 19-nor analogs of 11-deoxy steroids are consistently more active than their parent steroid, the 19-nor 11-oxygenated adrenal steroids show no predictable pattern of binding for MR or GR.

16Beta-hydroxydehydroepiandrosterone: the dichotomy between renal receptor binding and urinary electrolyte activity.

Excessive production of 16beta-hydroxydehydroepiandrosterone (16beta-OH-DHEA) has been suggested as a cause of low-renin essential hypertension. The mineralocorticoid effect of 16beta-OH-DHEA was reported to be one-fortieth that of aldostereone in the rat bioassay. Using kidney slices from adrenalectomized rats, the affinity of 16beta-OH-DHEA and a series of related compounds for mineralocorticoid receptors has been determined. In studies done at both 4 C and 37 C, the affinity of 16beta-OHDHEA for mineralocorticoid receptors was found to be less than 0.1% that of aldosterone (P less than 0.01). Various related steroids and/or potential metabolites similarly showed negligible affinity for the aldosterone receptor. In addition, In addition, 16beta-OHDHEA showed no significant affinity for renal dexamethasone-binding sites (Type II glucocorticoid receptors), corticosterone-binding sites (Type III glucocorticoid receptors), dihydrotestosterone binding sites, or estradiol binding sites. In in vivo competition experiments, the concurrent administration of 50 mug deoxycorticosterone reduced (3H)aldosterone binding to 20-30% of control levels; 50 mug 16beta-OH-DHEA did not compete for (3H)aldosterone binding sites. In in vivo bioassay electrolyte excretion was found-in contrast to that of aldosterone-to be variable. Within a given group, certain rats reproducibly responded to 16beta-OH-DHEA by sodium retention and kaliuresis; in others no response was observed. In vitro binding studies comparing "responders" with "non-responders" demonstrated that in neither group did 16beta-OH-DHEA have significant affinity for renal mineralocorticoid receptors. Accordingly, the mechanism whereby 16beta-OHDHEA produces changes in urinary electrolyte excretion appears independent of classical mineralocorticoid effector mechanisms. The conditions under which this effect is seen await eludication.

16Beta-hydroxylation of dehydroepiandrosterone sulphate by homogenates of human foetal liver.

The chemical syntheses of the 3-sulphates of 16 alpha-acetoxy-, 16 alpha-hydroxy- and 16 beta-hydroxy-dehydroepiandrosterone as their triethylammonium salts are described preparatory to studies of the metabolism of [7 alpha-3 H]dehydroepiandrosterone sulphate by homogenates of human foetal liver. Five main radioactive products of the incubation were separated by partition chromatography on a Celite column. Two were identified as 16 alpha-and 16 beta-hydroxydehydroepiandrosterone 3-sulphates by crystallization to constant specific radioactivity before and after solvolysis. The yields of the conversion to the two epimers were 24.4% (16 alpha) and 1.8% (16 beta).