Gender differences in tuberculosis treatment outcomes: a post hoc analysis of the REMoxTB study.

BACKGROUND: In the REMoxTB study of 4-month treatment-shortening regimens containing moxifloxacin compared to the standard 6-month regimen for tuberculosis, the proportion of unfavourable outcomes for women was similar in all study arms, but men had more frequent unfavourable outcomes (bacteriologically or clinically defined failure or relapse within 18 months after randomisation) on the shortened moxifloxacin-containing regimens. The reason for this gender disparity in treatment outcome is poorly understood. METHODS: The gender differences in baseline variables were calculated, as was time to smear and culture conversion and Kaplan-Meier plots were constructed. In post hoc exploratory analyses, multivariable logistic regression modelling and an observed case analysis were used to explore factors associated with both gender and unfavourable treatment outcome. RESULTS: The per-protocol population included 472/1548 (30%) women. Women were younger and had lower rates of cavitation, smoking and weight (all p < 0.05) and higher prevalence of HIV (10% vs 6%, p = 0.001). They received higher doses (mg/kg) than men of rifampicin, isoniazid, pyrazinamide and moxifloxacin (p ≤ 0.005). There was no difference in baseline smear grading or mycobacterial growth indicator tube (MGIT) time to positivity. Women converted to negative cultures more quickly than men on Lowenstein-Jensen (HR 1.14, p = 0.008) and MGIT media (HR 1.19, p < 0.001). In men, the presence of cavitation, positive HIV status, higher age, lower BMI and 'ever smoked' were independently associated with unfavourable treatment outcome. In women, only 'ever smoked' was independently associated with unfavourable treatment outcome. Only for cavitation was there a gender difference in treatment outcomes by regimen; their outcome in the 4-month arms was significantly poorer compared to the 6-month treatment arm (p < 0.001). Women, with or without cavities, and men without cavities had a similar outcome on all treatment arms (p = 0.218, 0.224 and 0.689 respectively). For all other covariate subgroups, there were no differences in treatment effects for men or women. CONCLUSIONS: Gender differences in TB treatment responses for the shorter regimens in the REMoxTB study may be explained by poor outcomes in men with cavitation on the moxifloxacin-containing regimens. We observed that women with cavities, or without, on the 4-month moxifloxacin regimens had similar outcomes to all patients on the standard 6-month treatment. The biological reasons for this difference are poorly understood and require further exploration.

Pretreatment chest x-ray severity and its relation to bacterial burden in smear positive pulmonary tuberculosis.

BACKGROUND: Chest radiographs are used for diagnosis and severity assessment in tuberculosis (TB). The extent of disease as determined by smear grade and cavitation as a binary measure can predict 2-month smear results, but little has been done to determine whether radiological severity reflects the bacterial burden at diagnosis. METHODS: Pre-treatment chest x-rays from 1837 participants with smear-positive pulmonary TB enrolled into the REMoxTB trial (Gillespie et al., N Engl J Med 371:1577-87, 2014) were retrospectively reviewed. Two clinicians blinded to clinical details using the Ralph scoring system performed separate readings. An independent reader reviewed discrepant results for quality assessment and cavity presence. Cavitation presence was plotted against time to positivity (TTP) of sputum liquid cultures (MGIT 960). The Wilcoxon rank sum test was performed to calculate the difference in average TTP for these groups. The average lung field affected was compared to log 10 TTP by linear regression. Baseline markers of disease severity and patient characteristics were added in univariable regression analysis against radiological severity and a multivariable regression model was created to explore their relationship. RESULTS: For 1354 participants, the median TTP was 117 h (4.88 days), being 26 h longer (95% CI 16-30, p < 0.001) in patients without cavitation compared to those with cavitation. The median percentage of lung-field affected was 18.1% (IQR 11.3-28.8%). For every 10-fold increase in TTP, the area of lung field affected decreased by 11.4%. Multivariable models showed that serum albumin decreased significantly as the percentage of lung field area increased in both those with and without cavitation. In addition, BMI and logged TTP had a small but significant effect in those with cavitation and the number of severe TB symptoms in the non-cavitation group also had a small effect, whilst other factors found to be significant on univariable analysis lost this effect in the model. CONCLUSIONS: The radiological severity of disease on chest x-ray prior to treatment in smear positive pulmonary TB patients is weakly associated with the bacterial burden. When compared against other variables at diagnosis, this effect is lost in those without cavitation. Radiological severity does reflect the overall disease severity in smear positive pulmonary TB, but we suggest that clinicians should be cautious in over-interpreting the significance of radiological disease extent at diagnosis.

Pretomanid with bedaquiline and linezolid for drug-resistant TB: a comparison of prospective cohorts.

BACKGROUND: There are no data comparing the 6-9 month oral three-drug Nix regimen (bedaquiline, pretomanid and linezolid [BPaL]) to conventional regimens containing bedaquiline (B, BDQ) and linezolid (L, LZD).METHODS: Six-month post end-of-treatment outcomes were compared between Nix-TB (n = 109) and 102 prospectively recruited extensively drug-resistant TB patients who received an ˜18-month BDQ-based regimen (median of 8 drugs). A subset of patients received BDQ and LZD (n = 86), and a subgroup of these (n = 75) served as individually matched controls in a pairwise comparison to determine differences in regimen efficacy.RESULTS: Favourable outcomes (%) were significantly better with BPaL than with the B-L-based combination regimen (98/109, 89.9% vs. 56/86, 65.1%; adjusted relative risk ratio [aRRR] 1.35; P < 0.001) and in the matched pairwise analysis (67/75, 89.3% vs. 48/75, 64.0%; aRRR 1.39; P = 0.001), despite significantly higher baseline bacterial load and prior second-line drug exposure in the BPaL cohort. Time to culture conversion (P < 0.001), time to unfavourable outcome (P < 0.01) and time to death (P < 0.03) were significantly better or lower with BPaL than the B-L-based combinations.CONCLUSION: The BPaL regimen (and hence substitution of multiple other drugs by pretomanid and/or higher starting-dose LZD) may improve outcomes in drug-resistant TB patients with poor prognostic features. However, prospective controlled studies are required to definitively answer this question.

A partially randomised trial of pretomanid, moxifloxacin and pyrazinamide for pulmonary TB.

BACKGROUND: Treatment for TB is lengthy and toxic, and new regimens are needed.METHODS: Participants with pulmonary drug-susceptible TB (DS-TB) were randomised to receive: 200 mg pretomanid (Pa, PMD) daily, 400 mg moxifloxacin (M) and 1500 mg pyrazinamide (Z) for 6 months (6Pa200MZ) or 4 months (4Pa200MZ); 100 mg pretomanid daily for 4 months in the same combination (4Pa100MZ); or standard DS-TB treatment for 6 months. The primary outcome was treatment failure or relapse at 12 months post-randomisation. The non-inferiority margin for between-group differences was 12.0%. Recruitment was paused following three deaths and not resumed.RESULTS: Respectively 4/47 (8.5%), 11/57 (19.3%), 14/52 (26.9%) and 1/53 (1.9%) DS-TB outcomes were unfavourable in patients on 6Pa200MZ, 4Pa200MZ, 4Pa100MZ and controls. There was a 6.6% (95% CI -2.2% to 15.4%) difference per protocol and 9.9% (95%CI -4.1% to 23.9%) modified intention-to-treat difference in unfavourable responses between the control and 6Pa200MZ arms. Grade 3+ adverse events affected 68/203 (33.5%) receiving experimental regimens, and 19/68 (27.9%) on control. Ten of 203 (4.9%) participants on experimental arms and 2/68 (2.9%) controls died.CONCLUSION: PaMZ regimens did not achieve non-inferiority in this under-powered trial. An ongoing evaluation of PMD remains a priority.

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.

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.

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.

Familial ligand-defective apolipoprotein B. Identification of a new mutation that decreases LDL receptor binding affinity.

Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by single-strand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-->Cys mutation, caused by a C-->T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age- and sex-adjusted TC of 240 +/- 14 mg/dl and LDL-C of 169 +/- 10 mg/dl. This compares with eight unaffected family members with age- and sex-adjusted TC of 185 +/- 12 mg/dl and LDL-C of 124 +/- 12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-->Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys3531 LDL particles bound with 27% of normal affinity. Deduced haplotypes using 10 apoB gene markers showed the Arg3531-->Cys alleles to be different in the two kindreds and indicates that the mutations arose independently. The Arg3531-->Cys mutation is the second reported cause of familial ligand-defective apoB.

TB Alliance regimen development for multidrug-resistant tuberculosis.

The recent approval of new tuberculosis (TB) drugs raises hope for new and more effective anti-tuberculosis treatment regimens. The Global Alliance for TB Drug Development (TB Alliance) is committed to ensuring that new anti-tuberculosis drugs fulfill the needs of patients, their families and the local health services that serve the communities. Here we present highlights of the TB Alliance's pipeline of regimen development, with novel regimens for patients with drug-susceptible, multidrug-resistant and extensively drug-resistant TB. The ongoing clinical trials (STAND, NC-005, Nix-TB and LIN-CL001) are outlined and their rationale and goals presented.

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.

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.

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.

Efficacy and safety of sibutramine in obese white and African American patients with hypertension: a 1-year, double-blind, placebo-controlled, multicenter trial.

BACKGROUND: Obesity is a highly prevalent medical condition and is commonly accompanied by hypertension. This study assessed the efficacy and safety of treatment with sibutramine hydrochloride for promoting and maintaining weight loss in obese patients with controlled hypertension, including a subset analysis of African American patients. PATIENTS AND METHODS: Obese patients with a body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) between 27 and 40 and a history of hypertension controlled with a calcium channel blocker (with or without concomitant thiazide diuretic treatment) were randomized to receive sibutramine (n = 150) or placebo (n = 74) with minimal behavioral intervention for 52 weeks. African Americans constituted 36% of enrolled patients. Efficacy assessments were body weight and related parameters (BMI and waist and hip circumferences), metabolic parameters (serum levels of triglycerides, high-density lipoprotein cholesterol [HDL-C], low-density lipoprotein cholesterol [LDL-C], total cholesterol, glucose, and uric acid), and quality-of-life measures. Safety assessments included recording of blood pressure, pulse rate, adverse events, and reasons for discontinuation. RESULTS: For patients receiving sibutramine, weight loss occurred during the first 6 months of the trial and was maintained to the end of the 12-month treatment period. Among patients receiving sibutramine, 40.1% lost 5% or more of body weight (5% responders) and 13.4% lost 10% or more of body weight (10% responders) compared with 8.7% and 4.3% of patients in the placebo group, respectively (P<.05). Changes in body weight were similar among African Americans and whites. Sibutramine-induced weight loss was associated with significant improvements in serum levels of triglycerides, HDL-C, glucose, and uric acid. Waist circumference and quality-of-life measures also improved significantly in patients receiving sibutramine. Sibutramine-treated patients had small but statistically significant mean increases in diastolic blood pressure (2.0 mm Hg) and pulse rate (4.9 beats/min) compared with placebo-treated patients (-1.3 mm Hg and 0.0 beats/min; P<.05); these changes were similar among African Americans and whites. Most adverse events were mild to moderate in severity and transient. The most common adverse event resulting in discontinuation among patients receiving sibutramine was hypertension (5.3% of patients receiving sibutramine vs 1.4% of patients receiving placebo). CONCLUSIONS: In obese patients with controlled hypertension, sibutramine was an effective and well-tolerated treatment for weight loss and maintenance. Sibutramine-induced weight loss resulted in improvements in serum levels of triglycerides, HDL-C, uric acid, and glucose, and in waist circumference and quality-of-life measures. Blood pressure and heart rate increased by a small amount. Efficacy and safety profiles for sibutramine among African American and white obese patients with controlled hypertension were similar.

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.

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.

Uptake of 3,5,3'-triiodothyronine by the perfused rat liver: return to the free hormone hypothesis.

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.

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.

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.

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.

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.