Reduction in Blood Culture Contamination Through Use of Initial Specimen Diversion Device.

BACKGROUND: Blood culture contamination is a clinically significant problem that results in patient harm and excess cost. METHODS: In a prospective, controlled trial at an academic center Emergency Department, a device that diverts and sequesters the initial 1.5-2 mL portion of blood (which presumably carries contaminating skin cells and microbes) was tested against standard phlebotomy procedures in patients requiring blood cultures due to clinical suspicion of serious infection. RESULTS: In sum, 971 subjects granted informed consent and were enrolled resulting in 904 nonduplicative subjects with 1808 blood cultures. Blood culture contamination was significantly reduced through use of the initial specimen diversion device™ (ISDD) compared to standard procedure: (2/904 [0.22%] ISDD vs 16/904 [1.78%] standard practice, P = .001). Sensitivity was not compromised: true bacteremia was noted in 65/904 (7.2%) ISDD vs 69/904 (7.6%) standard procedure, P = .41. No needlestick injuries or potential bloodborne pathogen exposures were reported. The monthly rate of blood culture contamination for all nurse-drawn and phlebotomist-drawn blood cultures was modeled using Poisson regression to compare the 12-month intervention period to the 6 month before and after periods. Phlebotomists (used the ISDD) experienced a significant decrease in blood culture contamination while the nurses (did not use the ISDD) did not. In sum, 73% of phlebotomists completed a post-study anonymous survey and widespread user satisfaction was noted. CONCLUSIONS: Use of the ISDD was associated with a significant decrease in blood culture contamination in patients undergoing blood cultures in an Emergency Department setting. CLINICAL TRIALS REGISTRATION: NCT02102087.

Disinfection of vascular catheter connectors that are protected by antiseptic caps is unnecessary.

OBJECTIVE: Determination of whether vascular catheter disinfecting antiseptic-containing caps alone are effective at decreasing microbial colonization of connectors compared to antiseptic-containing caps plus a 5-second alcohol manual disinfection. SETTING: The study was conducted in a 718-bed, tertiary-care, academic hospital. PATIENTS: A convenience sample of adult patients across intensive care units and acute care wards with peripheral and central venous catheters covered with antiseptic-containing caps. METHODS: Quality improvement study completed over 5 days. The standard-of-care group consisted of catheter connectors with antiseptic-containing caps cleaned with a 5-second alcohol wipe scrub prior to culture. The comparison group consisted of catheter connectors with antiseptic-containing caps without a 5-second alcohol wipe scrub prior to culture. The connectors were pressed directly onto blood agar plates and incubated. Plates were assessed for growth after 48-72 hours. RESULTS: In total, 356 catheter connectors were cultured: 165 in the standard-of-care group, 165 in the comparison group, and 26 catheters connectors without an antiseptic-containing cap, which were designated as controls. Overall, 18 catheter connectors (5.06%) yielded microbial growth. Of the 18 connectors with microbial growth, 2 (1.21%) were from the comparison group, 1 (0.61%) was from the standard-of-care group, and 15 were controls without an antiseptic-containing cap. CONCLUSIONS: Bacterial colonization rates were similar between the catheter connectors cultured with antiseptic-containing caps alone and catheter connectors with antiseptic-containing caps cultured after a 5-second scrub with alcohol. This finding suggests that the use of antiseptic-containing caps precludes the need for additional disinfection.

Fetal demise secondary to massive rhabdomyoma in the early second trimester of pregnancy.

Rhabdomyoma is the most common cardiac tumor in fetuses, often associated with tuberous sclerosis complex and usually diagnosed in the third trimester of pregnancy, with a benign course in the majority of cases. The hemodynamic impact of cardiac tumor depends on the location and size of the mass and the presence of dysrhythmia (4). Fetal cardiac rhabdomyoma accounts for less than 10% of fetal demise cases (1). This report presents a case of massive cardiac rhabdomyoma filling the entire right heart with pericardial extension, leading to hydrops and subsequent fetal death in the early second trimester of pregnancy.

In-vacuum Faraday isolation remote tuning.

In-vacuum Faraday isolators (FIs) are used in gravitational wave interferometers to prevent the disturbance caused by light reflected back to the input port from the interferometer itself. The efficiency of the optical isolation is becoming more critical with the increase of laser input power. An in-vacuum FI, used in a gravitational wave experiment (Virgo), has a 20 mm clear aperture and is illuminated by an almost 20 W incoming beam, having a diameter of about 5 mm. When going in vacuum at 10(-6) mbar, a degradation of the isolation exceeding 10 dB was observed. A remotely controlled system using a motorized lambda=2 waveplate inserted between the first polarizer and the Faraday rotator has proven its capability to restore the optical isolation to a value close to the one set up in air.

Microbial colonization of intravascular catheter connectors in hospitalized patients.

BACKGROUND: Central line-associated bloodstream infections may be due to catheter connector colonization and intraluminal migration of pathogens. We assessed the colonization of the split septum catheter connector system, and subsequently the luer lock catheter connector system. METHODS: This was a prospective, 2 phase, quality improvement study at a tertiary referral center. Each phase of the study was performed over 3 consecutive days in hospitalized patients receiving an active infusion; first with a split septum lever lock connector and second with a luer lock connector and alcohol port protector. The connectors were inoculated onto blood agar plates and incubated. Plates were assessed for microbial growth after 48-72 hours. RESULTS: In phase I, 98 (41.9%) of 234 split septum connectors yielded microbial growth. In phase II, 56 (23.1%) of 243 luer lock connectors yielded microbial growth. In phase II only, there was a significant increased rate of contamination in peripheral catheters compared with all other catheters, and the rate of contamination on the acute care wards was significantly higher when compared with the intensive care units. CONCLUSIONS: Bacterial colonization of the lever lock system was unacceptably high among all catheter types and hospital locations. Transition to luer lock catheter connectors and alcohol port protectors decreased the colonization; however, colonization still remained substantial. Causation of colonization cannot be determined with these results.

In-vacuum optical isolation changes by heating in a Faraday isolator.

We describe a model evaluating changes in the optical isolation of a Faraday isolator when passing from air to vacuum in terms of different thermal effects in the crystal. The changes are particularly significant in the crystal thermal lensing (refraction index and thermal expansion) and in its Verdet constant and can be ascribed to the less efficient convection cooling of the magneto-optic crystal of the Faraday isolator. An isolation decrease by a factor of 10 is experimentally observed in a Faraday isolator that is used in a gravitational wave experiment (Virgo) with a 10 W input laser when going from air to vacuum. A finite element model simulation reproduces with a great accuracy the experimental data measured on Virgo and on a test bench. A first set of measurements of the thermal lensing has been used to characterize the losses of the crystal, which depend on the sample. The isolation factor measured on Virgo confirms the simulation model and the absorption losses of 0.0016 +/- 0.0002/cm for the TGG magneto-optic crystal used in the Faraday isolator.

The distribution kinetics of triiodothyronine: studies of euthyroid subjects with decreased plasma thyroxine-binding globulin and patients with Graves' disease.

The kinetics of distribution of 3,3',5-triiodo-L-thyronine (T(3)) have been studied employing both a single-injection and a continuous infusion of T(3-) (131)I. External monitoring of radioactivity in the liver during the infusion permitted estimation of the hepatic distribution volume (V(H)) and the one-way hepatic clearance (C(H)) of the hormone. Among 10 euthyroid control subjects, V(H) averaged 2.07 liters +/-0.50 (SD), and the mean value for C(H), 231 ml of plasma per min (+/-64). In three euthyroid men whose plasma showed decreased binding capacity by thyroxine-binding globulin (TBG) abnormally high V(H) and C(H) values were found, the increase in C(H) being proportional to the decrease in binding activity by plasma proteins. Among all 13 subjects, there was a high correlation (+ 0.86) between C(H) and the proportion of free hormone in plasma, measured in vitro. In four patients with hyperthyroid Graves' disease V(H) ranged from 3.2 to 4.2 liters and C(H) was elevated in every case, averaging 989 ml/min. The increase in C(H) in this group was out of proportion to the elevation of free hormone fraction in plasma. Seven patients who were either euthyroid or hypothyroid after treatment of Graves' disease showed a slight but significant increase in C(H) compared with the euthyroid controls without Graves' disease. The percentage of free hormone in the plasma of the treated group was normal or low and therefore could not explain the persistent elevation in unidirectional hepatic clearance observed. The rate of accumulation of labeled T(3) in the tissues of the thigh during the interval from 10 to 60 min of the sustaining infusion of tracer was slow compared to the rate of equilibration in the liver and did not differ significantly among the various groups studied. These latter findings suggest that in slowly equilibrating tissues such as the thigh the kinetics of T(3) distribution are relatively insensitive to alterations in hormone-binding activity by plasma proteins.

Preparation of 125-I-labeled human thyroxine-binding alpha globulin and its turnover in normal and hypothyroid subjects.

A protein with the electrophoretic, immunologic, and hormone-binding properties of thyroxine-binding globulin (TBG) has been prepared from human plasma and labeled with radioiodine (125-I) by an enzymatic method of iodination. The [125-I]TBG retained the electrophoretic and immunologic characteristics of unlabeled TBG but exhibited a partial loss of thyroxine-binding activity, as assessed by affinity chromatography. The in vivo behavior of [125I]TBG was studied in six euthyroid subjects (controls) with normal serum levels of TBG as measured both by radioimmunoassay and by determination of maximal T4-binding capacity and in four male patients with untreated primary hyperthyroidism, three of whom had elevated serum TBG. The half-time of the final slope of the plasma disappearance curve averaged 5.0 days plus or minus 1.2 (SD) in the controls and ranged from 3.9 to 109 days in the hypothyroid patients. The distribution volume was similar in the two groups, 6.7 plus or minus 1.3 vs. 7.1 plus or minus 2.1 liters. The catabolic clearance rate averaged 0.99 plus or minus 0.33 liters plasma/24 h in the controls and 0.92 plus or minus 0.46 in the hypothyroids. The absolute turnover rate of TBG, calculated from the catabolic clearance rate multiplied by the serum concentration of radioimmunoassayable TBG, averaged 17.8 plus or minus 2.1 mg/day in the controls and ranged from 14.8 to 33.2 mg/day in the hypothyroids. Among the entire group of subjects there was no correlation between the serum TBG concentration and the absolute turnover rate of TBG.

3,3'-Diiodothyronine production, a major pathway of peripheral iodothyronine metabolism in man.

3,3'-Diiodothyronine (3,3'-T(2)) has been detected in human serum and in thyroglobulin. However, no quantitative assessment of its clearance rate (CR), production rate (PR), or of the importance of extrathyroidal sources of 3,3'-T(2) relative to direct thyroidal secretion is yet available. This study examines these parameters in seven euthyroid subjects, and in eight athyreotic subjects (H) eumetabolic due to thyroxine therapy (HT(4)) (n = 5) or triiodothyronine replacement (HT(3)) (n = 3). A highly specific radioimmunoassay for the measurement of 3,3'-T(2) in whole serum was developed. Serum 3,3'-T(2) concentrations were (mean +/- SD) 6.0+/-1.0 ng/100 ml in 13 normal subjects, 9.0+/-4.6 ng/100 ml in 25 hyperthyroid patients, and 2.7+/-1.1 ng/100 ml in 17 hypothyroid patients. The values in each of the latter two groups were significantly different from normal. 3,3'-T(2) was detected regularly in normal concentrations in 11 hypothyroid patients eumetabolic by treatment with synthetic T(4), in 10 eumetabolic patients suffering from nonthyroidal systemic illness, and in 2 subjects with elevated serum T(4)-binding globulin. The 3,3'-T(2) CR was assessed from data acquired from the (125)I-3,3'-T(2) constant infusion technique. The 3,3'-T(2) PR was calculated from CR and serum concentration of 3,3'-T(2) determined by radio-immunoassay. In the HT(4) subjects the 3,3'-T(2) CR averaged 840+/-377 liters/day and 3,3'-T(2) PR 33.9+/-12.5 mug/day. These results were not significantly different from those in the control group: 3,3'-T(2) CR 628+/-218 liters/day and 3,3'-T(2) PR 39.8+/-19.8 mug/day (all corrected to 70 kg body wt). In addition to 3,3'-T(2) PR, T(3), and reverse triiodothyronine (rT(3)) PR were determined in three of the HT(4) subjects. In each case studied, the 3,3'-T(2) PR was close to the combined triiodothyronine (T(3) + rT(3)) PR. The mean molar ratio of T(2) PR/(T(3) + rT(3)) PR was 1.08+/-0.10. The results obtained in the HT(4) subjects indicate that the production of 3,3'-T(2) is a major route of T(4) metabolism. The combined studies of 3,3'-T(2), T(3) and rT(3) PR in the HT(4) subjects indicate that both T(3) and rT(3) are major precursors of 3,3'-T(2). In the HT(3) subjects, the conversion of T(3) to 3,3'-T(2), determined as the molar ratio of 3,3'-T(2) PR to T(3) PR, ranged from 0.36 to 0.92, providing further evidence that T(3) is a precursor of 3,3'-T(2). From the close agreement between the mean values for 3,3'-T(2) PR in the euthyroid and HT(4) group it is concluded that most, if not all of the 3,3'-T(2) produced in normal humans is derived by extrathyroidal conversion from T(3) and rT(3).

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.

UV mutation signature in tumor suppressor genes involved in skin carcinogenesis in xeroderma pigmentosum patients.

Molecular analysis of p53 and patched (PTCH), two candidate tumor suppressor genes for non-melanocytic skin cancer, was performed in skin tumors from six patients affected by the cancer-prone disease xeroderma pigmentosum (XP). UV-specific p53 mutations were detected at a frequency of 38-50% in all the tumor types analysed, including melanomas. Additional analysis of PTCH mutations in the subset of eight basal call carcinomas (BCC) revealed a very high mutation frequency of this gene (90%) which exceeded that detected in the p53 gene in the same tumors (38%). PTCH mutations were predominantly UV-specific C>T transitions. This mutation pattern is different from that reported in BCC from normal donors where PTCH mutation frequency is 27% and mutations are frequently deletions and insertions. These findings suggest that PTCH mutations represent an earlier event in BCC development than p53 alterations and that the inability of XP patients to repair UV-induced PTCH mutations might significantly contribute to the early and frequent appearance of BCC observed in these patients.

Nanosecond time-resolved emission anisotropy of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid.

The fluorescence emission anisotropy of 1,6-diphenyl-1,3,5-hexatriene in human amniotic fluid has been studied using nanosecond time-resolved emission techniques. These studies demonstrate that the previously reported decrease in the steady state emission anisotropy, [r], with gestational age is due to a change in the rate of rotational motion of the probe. The emission anisotropy decays to a limiting value (r infinity) greater than zero, suggesting a hindered rotation of the probe, and this is independent of gestational age. The decay function for the emission anisotropy of amniotic fluids from 17, 29, 40 and 41 weeks in gestational age can be best expressed as a single exponential plus a constant term, with rotational correlation times varying from 17 ns to 2.2 ns, respectively. The zero time emission anisotropy remains approx. 0.30 for both early and late gestational times.

Distribution of a cross-species melanoma-associated antigen in normal and neoplastic human tissues.

In previous studies the monoclonal antibody (MoAb) M2590 elicited in C57/BL6 mice with the syngeneic melanoma cell line B16 has been shown to recognize a 31K glycoprotein expressed by human melanoma cell lines. The present study has shown that the MoAb M2590 cross-reacts with surgically removed benign and malignant lesions of melanocyte origin. The reactivity pattern of the MoAb M2590 with these lesions is different from that of the anti-high-molecular weight-melanoma associated antigen (HMW-MAA) MoAb 225.28S, of the anti-115K MAA MoAb 345.134S, and of the anti-100K MAA MoAb 376.96S, which were elicited with human melanoma cell lines. In particular, the MoAb M2590 reacts with blue nevi. The MoAb M2590-defined MAA, like other types of MAA, is heterogeneous in lesions removed from different patients, in autologous lesions removed from different anatomic sites, and in cells within a lesion. The distribution of the MoAb M2590-defined MAA in normal tissues and in tumors of nonmelanocyte origin is broader than that of the HMW-MAA, but is similar to that of the 115K MAA and of the 100K MAA. The results of this investigation suggest that immunization with xenogeneic melanoma cells may broaden the range of specificity of antihuman MAA MoAbs and provide information about the phylogenetic evolution of MAAs.

A glycine-to-arginine substitution in the triple-helical domain of type VII collagen in a family with dominant dystrophic epidermolysis bullosa.

We recently demonstrated strong genetic linkage between the type VII collagen gene (COL7A1) and both the dominant and recessive forms of dystrophic epidermolysis bullosa. In this study, we searched for mutations in dominant dystrophic epidermolysis bullosa using polymerase chain reaction amplification of segments of COL7A1, followed by heteroduplex analysis. Examination of the polymerase chain reaction corresponding to exon 73 revealed a heteroduplex resulting from a G-to-A transition at nucleotide 6127 in the triple-helical domain of COL7A1, which converted a glycine residue to an arginine (G2043R). The dominant dystrophic epidermolysis bullosa phenotype in this family probably arose because of a dominant negative effect of this mutation in COL7A1, resulting in the formation of structurally abnormal anchoring fibrils.

Glucose and insulin reverse the effects of fasting on 3,5,3'-triiodothyronine neogenesis in primary cultures of rat hepatocytes.

The cellular mechanisms by which carbohydrate refeeding reverses the effect of fasting on T3 metabolism were studied in primary cultures of hepatocytes (24 h) harvested from 48-h fasted rats. Net T3 neogenesis (T3 generated from T4) in the fasted hepatocyte preparations (9.2 +/- 0.9 pmol/min X 100 mg protein) was significantly less (P less than 0.001) than that in hepatocyte cultures derived from 72-h glucose-fed rats (41 +/- 0.8 pmol/min X 100 mg protein). Preincubation (18 h) with either glucose (2.5-10 mM) or insulin (10-500 nM) significantly increased the fasted hepatocyte T3 levels to 28 +/- 0.6 and 22 +/- 1.3 pmol/min X 100 mg protein, respectively. Furthermore, incubation with both of these agents demonstrated a greater effect on hepatic T3 neogenesis than with either alone. Fasted hepatocyte T3 neogenesis was enhanced by enrichment with dithiothreitol (5 mM), but the T3 generation remained significantly less than that in cells exposed to glucose or insulin. Studies with glucose analogs demonstrated that preincubation with 2-deoxyglucose (5 mM) significantly increased (P less than 0.001) hepatocyte T3 neogenesis, but 3-O-methylglucose (5 mM) had no effect. In contrast, the insulin-mimetic compounds Concanavalin-A or spermine did not stimulate T3 neogenesis in the fasted hepatocyte cultures. Thus, rat hepatocytes sustained in primary culture for 24 h retain the T3 metabolic characteristics of the intact animal. Glucose and insulin reverse the effect of fasting on hepatocyte T3 neogenesis. The additive response to glucose and insulin suggests that T3 neogenesis is modulated through different mechanisms. The replication of the glucose effect by 2-deoxyglucose and the inability of dithiothreitol to reverse the effect of fasting on hepatocyte T4 5'-deiodinase activity suggest that neither intermediates in the glycolytic pathway nor thiol cofactors mediate the glucose effect. Thus, the restoration of liver T3 metabolism consequent to carbohydrate refeeding of the fasted rat may be mediated by the glucose and insulin responses.

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 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.

Effects of dexamethasone on kinetics and distribution of triiodothyronine in the rat.

Studies were designed to examine the effects of the glucocorticoid dexamethasone (Dex) on the distribution and turnover of T3 separately from its effects on the pituitary and thyroid. Male Sprague-Dawley rats (200-250 g) were surgically thyroidectomized and given a replacement dose of T4 (1.6 micrograms/day X 100 g BW) throughout the experiment via a sc implanted osmotic minipump. Six or 7 days after starting the T4 infusion, each animal was given [125I]T3 by constant infusion (via a second minipump) for 5 or 6 days and, during the final 5 days, either Dex (0.15 mg/day X 100 g) or saline in a third minipump. Methanol extracts of serum and tissues removed at the end of the infusion were analyzed for [125I]T3 concentration by high performance liquid chromatography. The MCR, computed from the infusion rate of tracer and the serum concentration of [125I]T3 at the end of the infusion, averaged 25.7 +/- 1.3 (+/-SE) ml/h X 100 g in the controls and 15.1 +/- 2.6 in the Dex-treated rats. Serum T3 (RIA) concentrations were similar in the two groups. The plasma T3 production rate was decreased from 9.51 +/- 1.14 ng/h X 100 g in controls to 5.13 +/- 1.16 in the Dex-treated animals. The fraction of administered T4 converted to T3 was reduced from 0.21 to 0.11 by Dex treatment. Tissue to serum (T/S) [125I]T3 concentration ratios were significantly decreased by Dex to approximately 50% of the control value in each of the tissues sampled (liver, kidney, and skeletal muscle). The reduction in the T/S ratio could not be attributed to an increase in the net serum binding of T3; in fact, serum hormone binding was diminished by Dex treatment. The distribution data indicate that net tissue binding of T3 in these organs is reduced to an even greater extent than is serum binding of T3. The glucocorticoid-induced fall in T3 MCR could be accounted for by the decrease in T/S ratios, the latter being a measure of T3 distribution volume in the tissues studied. The rate of T4 5'-deiodination in vitro was diminished in homogenates of livers from Dex-treated animals when the incubation was performed with and without added thiol as cofactor, indicating that the hepatic level of active T4 5'-deiodinase is reduced by Dex. Thus, Dex causes multiple alterations in T3 metabolism. Total body T3 production from T4 in extrathyroid sites, and in the liver in particular, is reduced.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Carbohydrate reactivation of thyroxine 5'-deiodinase (type II) in cultured mouse neuroblastoma cells is dependent upon new protein synthesis.

The T3 concentration in brain predominantly reflects local production from T4 rather than T3 uptake from the circulating pool. We recently demonstrated that rat brain T3 content is increased by glucose feeding compared to chow feeding. One possible mechanism for this effect is an increase in brain T4 5'-deiodinase (5'-D) activity. Our recent preliminary studies of neuroblastoma (NB) cells demonstrate that renewal of RPMI-1640 medium stimulates T4 5'-D type II (NB T4 5'-D II) activity in these cells. The present studies were performed to determine the mechanism of this response. Studies were performed on NB cells supported in thyroid hormone-depleted (deficient) medium. This approach increased NB T4 5'-DII activity 4-fold compared to that in thyroid hormone-replete medium. Medium renewal further stimulated enzyme activity (7- to 9-fold; maximum at 6 h) in each group. The difference between the hypothyroid group and control was sustained over a 24-h period. Subsequent studies demonstrated that glucose (11 mM) was the specific medium ingredient mediating the medium renewal response. A progressive increase in NB T4 5'-DII activity was noted over 8 h during RPMI-1640 salt plus glucose (11 mM) incubation. This was equivalent to the effect of complete medium containing glucose (11 mM). Coincubation with insulin (10(-7)-10(-9) M) did not modify the enzyme response to glucose. In addition, fructose (10 mM) had a similar effect on enzyme activity. Glycerol and essential and nonessential amino acids also modestly increased NB T4 5'-DII activity compared to that in the control group (P less than 0.01). Actinomycin-D (1 microM), cycloheximide (100 microM), and puromycin (100 microM) significantly (P less than 0.001) decreased the glucose effect on T4 5'-DII by 5-, 9-, and 17-fold, respectively, after 6 h of incubation. In addition, puromycin (10-200 microM) inhibited both NB T4 5'-DII activity and [3H]amino acid incorporation during incubation in glucose. There was a significant correlation between these parameters (r = 0.8; P less than 0.001). The enzyme activity decay curves in the glucose-activated and control groups subsequent to puromycin (100 microM) addition at 8 h were parallel. The fractional turnover rate was 13%/h in the controls and 11%/h in the glucose groups. The calculated enzyme production rate was significantly higher (P less than 0.005) in the glucose group compared to that in the control group (17.4 vs. 6.8 fmol/mg protein.h).(ABSTRACT TRUNCATED AT 400 WORDS)