Decreased Protein Binding Research Articles

ERK and p38 MAPK‐regulated peroxiredoxin 6 localization to lamellar bodies in MLE 12 cells

Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (aiPLA2) activities, participates in lung surfactant phospholipid turnover and protects lung epithelium against oxidative stress. Prdx6 has been localized by immunocytochemistry and sub‐cellular fractionation to cytosol and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization determines the balance between the 2 activities. Using a GFP‐Prdx6 fusion protein, we show that ERK and p38 MAPK regulate Prdx6 localization to organellar structures compatible with lamellar bodies in the MLE12 mouse type II alveolar epithelial cell line. Treatment of GFP‐Prdx6 transfected MLE12 cells with PD98059 and SB202190, inhibitors of ERK and p38 MAPK, respectively, abolished Prdx6 organellar targeting; a JNK kinase inhibitor had no effect. Using mutagenesis, we show that Prdx6 organellar localization is determined by the 31–40 amino acid region which contains a phospholipid binding sequence GDSWG. Serine to alanine (S32A) mutation within this sequence abolished targeting and decreased protein binding to phospholipids as determined by FRET analysis. These studies suggest that Prdx6 targeting to acidic organelles and its consequent PLA2 activity may be determined by MAPK‐regulated lipid binding to a specific Prdx6 amino acid sequence. [HL019737]

Decrease in protein binding and its effect on toxicokinetics (TK) / toxicodynamics (TD) of diclofenac and propranolol in pregnant rats

Plasma protein binding is an important factor for the kinetics of drugs and how they act since it is the first step in drug distribution. Physiological changes in pregnancy, which include plasma composition, can affect drug binding and subsequent drug response. In the present study, we investigated the toxicokinetics (TK) and/or toxicodynamics (TD) of diclofenac and propranolol comparing pregnant Sparague-Dawley (SD) rats with non-pregnant SD rats in terms of protein binding and drug distribution. Diclofenac and propranolol are reported to bind to albumin and alpha(1)-acid glycoprotein (AGP), respectively. After a single administration of diclofenac, the area under plasma concentration-time curve (AUC) based on free diclofenac in pregnant rats was 3.9 times higher than that in non-pregnant rats. This difference is considered to be due to a lower concentration of serum albumin and a higher concentration of non-esterified fatty acid (NEFA) that inhibits drug binding to albumin, in pregnant rats. In histopathological examination, more severe gastrointestinal toxicity was observed in pregnant rats at 24 hr after dosing. This severe toxicity was likely to be correlated with the higher AUC. With respect to propranolol, the difference of the AUC based on free propranolol was not clear although the concentration of serum AGP was lower in pregnant rats. However, the binding analysis data suggested a difference of protein binding at a lower propranolol concentration range. Consequently, lowered serum proteins and increased NEFA in pregnant rats can lead to low protein binding, subsequent increase in free drug concentrations, and eventual increase in the TD of drugs.

Effects of Fluoride on the Interactions between Amelogenin and Apatite Crystals

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogenin-modulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

Transient cerebral hypoperfusion enhances intraarterial carmustine deposition into brain tissue

We hypothesized that bolus injections of lipid soluble chemotherapeutic drugs during transient cerebral hypoperfusion could significantly boost regional drug delivery. In the first two groups of New Zealand White rabbits we measured brain tissue carmustine concentrations after intravenous infusion, intraarterial infusion with normal perfusion, and after intraarterial injections during transient cerebral hypoperfusion. In the third group of animals we assessed the safety of the technique by assessing electroencephalographic changes for 6 h after flow arrest carmustine administration and subsequent histological examination. The brain tissue carmustine concentrations were fivefold to sevenfold higher when the drug was injected during cerebral hypoperfusion compared to a conventional intracarotid infusion (68.4 +/- 24.5 vs. 14.2 +/- 8.3 microg/g, n = 5 each, respectively, P < 0.0001). The brain tissue carmustine concentrations (y) were a linear function of the bolus dose (x) injected during cerebral hypoperfusion, y = 10.4 x x - 21 (R = 0.84, P < 0.001). Stable EEGs were recorded several hours after flow arrest carmustine exposure and histological examinations did not reveal any gross evidence of cerebral injury. Transient cerebral hypoperfusion during intraarterial bolus injection of carmustine significantly increases drug delivery. Clinical techniques that decrease CBF, such as, transient arterial occlusion by balloon tipped catheters, hyperventilation, hypothermia, induced hypotension, or transient circulatory arrest, could enhance intraarterial drug delivery to the brain. We believe that the mechanisms for improved drug delivery is the decrease in drug dilution by reduced or absent blood flow, decreased protein binding and a longer time for high concentrations of free drugs to transit through the blood brain barrier.

Using pharmacokinetics to predict the effects of pregnancy and maternal–infant transfer of drugs during lactation

Knowledge of pharmacokinetics and the use of a mechanistic-based approach can improve our ability to predict the effects of pregnancy for medications when data are limited. Despite the many physiological changes that occur during pregnancy that could theoretically affect absorption, bioavailability does not appear to be altered. Decreased albumin and α1-acid glycoprotein concentrations during pregnancy will result in decreased protein binding for highly bound drugs. For drugs metabolised by the liver, this can result in misinterpretation of total plasma concentrations of low extraction ratio drugs and overdosing of high extraction ratio drugs administered by non-oral routes. Renal clearance and the activity of the CYP isozymes, CYP3A4, 2D6 and 2C9, and uridine 5′-diphosphate glucuronosyltransferase are increased during pregnancy. In contrast, CYP1A2 and 2C19 activity is decreased. The dose of a drug an infant receives during breastfeeding is dependent on the amount excreted into the breast milk, the daily volume of milk ingested and the average plasma concentration of the mother. The lipophilicity, protein binding and ionisation properties of a drug will determine how much is excreted into the breast milk. The milk to plasma concentration ratio has large inter- and intrasubject variability and is often not known. In contrast, protein binding is usually known. An extensive literature review was done to identify case reports including infant concentrations from breast-fed infants exposed to maternal drugs. For drugs that were at least 85% protein bound, measurable concentrations of drug in the infant did not occur if there was no placental exposure immediately prior to or during delivery. Knowledge of the protein binding properties of a drug can provide a quick and easy tool to estimate exposure of an infant to medication from breastfeeding.

Aldosterone Receptor Antagonists and Cardiovascular Disease: Do We Need a Change of the Guard?

Aldosterone is a mineralocorticoid primarily produced in the zona glomerulosa of the adrenal gland. For many years, aldosterone (Aldo) was thought to have its sole site of action in the kidney, where it regulated sodium excretion and potassium reabsorption. It is now known that Aldo is produced in cardiovascular tissues, and has been implicated in the development of ventricular hypertrophy and cardiac fibrosis. The precise mechanisms whereby Aldo acts in cardiac tissues are diverse. It was assumed that Aldo production could be limited by angiotensin-converting enzyme (ACE) inhibition, but serial measurements during therapy reveal only a transient decrease in Aldo levels. Moreover, the effects of Aldo on cardiac tissues occur even when angiotensin II (Ang II) has been suppressed or eliminated. Multiple investigators have examined effects of Aldo receptor blockade in human subjects and various animal models using the two Aldo receptor antagonists (ARAs), spironolactone and eplerenone. Major clinical trials involving spironolactone (RALES) and eplerenone (EPHESUS) ARAs have shown significant benefits in the treatment of congestive heart failure (CHF). In RALES, patients with New York Heart Association (NYHA) Class III or IV systolic heart failure treated with spironolactone had a 30% relative risk decrease in mortality. Although spironolactone is an effective competitive inhibitor of the mineralocorticoid receptor (MR), progestational and antiandrogenic side effects limit its use in some patients. Eplerenone, a more selective ARA, lacks these undesirable side effects. Although eplerenone is 20-fold less potent at the MR, it demonstrates efficacy similar to spironolactone, possibly due to decreased protein binding. Eplerenone has fewer side effects than spironolactone, which has been attributed to the low cross-reactivity with androgen and progesterone receptors. In EPHESUS, patients with left ventricular systolic dysfunction [Ejection Fraction (EF) <40%] and CHF following an acute myocardial infarction (AMI), were treated with eplerenone, resulting in a 17% reduction in cardiovascular mortality. However, these studies were limited in that diastolic function was not evaluated, although approximately 1/2 of CHF is due to diastolic dysfunction alone. To date, neither ARA has been studied for the treatment of diastolic dysfunction in a major clinical trial. However, numerous animal studies employing ARAs have shown a decrease in cardiac hypertrophy and fibrosis, indicating the potential benefits of these agents in the treatment of diastolic heart failure. In this review, we discuss possible underlying mechanisms responsible for Aldo effects on cardiovascular function and compare the beneficial effects of spironolactone and eplerenone in the treatment of heart disease.

A study of the interaction between ropranolol and NSAIDs in protein binding by gel filtration method.

Drug protein binding phenomena can lead to some interesting drug-drug interactions when one drug displaces another in the binding site. Studies of protein binding are conducted by several methods including equilibrium dialysis, ultra-filtration and chromatographic methods. Gel filtration is a simple chromatographic method in protein binding studies. Propranolol binds to plasma proteins by 90%-95% in circulation system and other drugs with high protein binding may displace it. In this study protein binding of propranolol has been studied using gel filtration to Bovine Serum Albumin (BSA) alone and in the presence of Acetyl salicylic acid (ASA), Indomethacin and mefenamic acid has been studied using gel filtration method. The results indicated that ASA decreased protein binding of propranolol by 20% to BSA and other drugs did not displace propranolol from the binding site. Therefore, ASA may alter pharmacological effects of propranolol.

Pharmacokinetic/Pharmacodynamic Modeling of Anesthetics in Children

Modeling the pharmacokinetics and pharmacodynamics of anesthetics in children is performed as a response to the clinical need for safe and efficacious administration of drugs with a low therapeutic index. Rates and concentrations of these drugs, which are the primary parameters used by anesthesiologists, depend on physiologic parameters that are markedly affected by development. Volatile anesthetics have been used for >50 years in pediatric patients. The pharmacokinetics of inhalation agents are context sensitive, but little difference between age groups has been described. These agents are not only eliminated unchanged by the lung but they are also metabolized by the liver. Halothane has Michaelis-Menten kinetics, with up to 40% of the administered dose metabolized by the liver. For volatile anesthetics, the effect measured is the minimum alveolar concentration (MAC) that leads to movement of the limb in response to skin incision in 50% of the patients studied. The MAC is higher in infants than in children and adults. Infants aged 6 months have a MAC 1.5-1.8 times the MAC observed in adults aged 40 years. Children have a greater clearance and volume of distribution of propofol than adults. In order to achieve similar plasma concentrations, children require three times the initial dose used in adults. In adults, an increased sensitivity to propofol has been demonstrated with aging, but nothing is known about the effects in children. However, it is clear that equipotent doses of propofol induce marked deleterious hemodynamic effects in infants compared with children. Regional anesthesia is used in pediatrics, both in combination with general anesthesia during surgery or alone for postoperative analgesia. A marked decrease in protein binding has been described in infants. In the postoperative period, a rapid increase in binding because of inflammation decreases the free fraction, but the free drug concentration remains constant because of the resulting decrease in total clearance. A low clearance because of liver function immaturity has been observed during the first year(s) of life for bupivacaine and ropivacaine. Pharmacodynamic interactions between general anesthesia and regional anesthesia need to be modeled. This is one of the future tasks for pharmacokineticists. Methods such as the Dixon up-and-down allocation and the isobolographic technique are promising in this field.

Pharmacokinetics of the Active Metabolites of Ethyl Loflazepate in Elderly Patients Who Died of Asphyxia Associated with Benzodiazepine-Related Toxicity

We determined the pharmacokinetics of ethyl loflazepate (Lof) in elderly patients who died of benzodiazepine-related toxicity. Three elderly patients with body mass indexes of less than 17 kg/m2 died of asphyxia after having taken maintenance doses of Lof for 2 to 3 weeks. We measured serum concentrations of the active metabolites of Lof using gas chromatography-mass spectrometry and a benzodiazepine receptor assay to determine the pharmacokinetics of each. On admission, the serum concentrations of the active metabolites of Lof ([Lofl) were 256 ng/mL, 425 ng/mL, and 177 ng/mL in cases 1, 2, and 3, respectively. Serum benzodiazepine-receptor binding activities, expressed as diazepam equivalent concentrations ([Bz]), were 1800 ng/mL, 2200 ng/mL, and 1500 ng/mL. The T1/2(beta) of [Lof] were 124 and 121 h in cases 1 and 2 and the T1/2(beta) of [Bz] were 75 and 87 h. The distribution volume in the elderly was reduced due to a small lipid compartment, and total drug clearance was decreased due to the decline in liver and kidney function. These changes did not prolong T1/2(beta) but did increase plasma concentrations of active metabolites, especially in case 2, and a slight decrease in protein binding increased the amount of free active metabolites greatly.

Pyridoxine (vitamin B6) neurotoxicity: enhancement by protein‐deficient diet

Large doses of pyridoxine cause injury to the primary sensory neurons in trigeminal and dorsal root ganglia of animals and patients subjected to megavitamin therapy. The increased hazard to subjects with reduced renal excretory function has been explored previously. In the present work, the neurotoxicity of pyridoxine for rats was found to be increased by dietary protein deficiency. A mere 3 or 7 days of pretreatment with either of two protein-deficient diets were sufficient to accelerate and intensify the clinical neurological signs and histological lesions from pyridoxine injections. These results are caused, at least in part, by loss of body weight, decreased protein binding in serum and decreased consumption of water and decreased volume of urine, which reduce the urinary losses of the toxicant. The vitamers related to pyridoxine (pyridoxal, pyridoxamine) and the coenzyme (pyridoxal 5-phosphate) did not cause clinical signs or lesions similar to those produced by pyridoxine even when injected in maximum tolerated doses. Neither a protein-deficient diet nor bilateral nephrectomy changed the results with the vitamers.

The Id2 Transcriptional Repressor Is Induced by Follicle-stimulating Hormone and cAMP

Id (inhibitor of DNA binding/differentiation) proteins repress differentiation and promote cell division by dimerizing with and inhibiting the action of basic helix-loop-helix transcription factors including those that bind to E-box motifs. Of the four characterized Id proteins, only Id2 is found in the nucleus of Sertoli cells that support the development of spermatozoa in the testis. Differential display analysis of rat primary Sertoli cell mRNA identified Id2 as being inducible by forskolin, a stimulator of cAMP production. Northern blot analysis confirmed that Id2 mRNA expression peaked in Sertoli cells 6-12 h after stimulation with forskolin or follicle-stimulating hormone (FSH), the major physiological stimulator of cAMP in Sertoli cells. Similarly, Id2 promoter activity in Sertoli cells was induced after forskolin or FSH stimulation as well as by overexpression of protein kinase A. Forskolin induction of the Id2 promoter required sequences located between positions -122 and -82. Protein(s) of 40-45 kDa were found to bind two activated transcription factor/cAMP-response element-like sites and a GATA motif within the regulatory region. The induction of the Id2 gene by FSH corresponded with a decrease in protein binding to an E-box consensus motif and decreased E-box-mediated transcription. Together, these findings raise the possibility that FSH-mediated induction of Id2 and resultant inhibition of basic helix-loop-helix transcription factor-regulated genes in Sertoli cells may contribute to the regulation of spermatogenesis.

Micronutrients: Too Much or Not Enough?

Micronutrients (ie, vitamins and trace elements) are considered essential for human metabolism and should be delivered to all patients receiving enteral nutrition (EN) and parenteral nutrition (PN) support. Despite incorporation into EN products or the commercial availability of parenteral multiple trace element and multivitamin preparations, episodes of deficiency and toxicity continue to occur in patients receiving specialized nutrition support. Although the micronutrient content of these preparations is based upon expert consensus recommendations to meet the demands of most patients, acute and chronic conditions may account for individual variations in requirements. Evolving research shows that micronutrient status is affected by multiple factors, such as the acute phase response, causing redistribution and changes in plasma micronutrient concentrations. Serum C-reactive protein (CRP), a positive acute phase protein, is the most important marker of the acute phase response. CRP activates the classic complement pathway and acts as an opsonin involved in the clearance of microorganisms. During the acute phase response, as reflected by an increased CRP, plasma concentrations of the trace elements iron and zinc fall as a result of decreased protein binding and transfer into other body compartments. In contrast, serum copper rises because of an increased production and release of the major copper-binding plasma protein, ceruloplasmin, from the liver during inflammatory conditions. Micronutrient requirements are frequently

Priorities in perioperative geriatrics.

T he aging of the baby-boom population and the decreases in adult mortality seen in the last few decades will dramatically increase the age of Americans between 2010 and 2030. During that time, the population older than age 65 yr is expected to grow by 75%, whereas between 1995 and 2050, the cumulative growth of the population older than 85 yr is expected to exceed 400% (1). Furthermore, it has been reported that the increased demand for surgery in this population may exceed the rate of population growth (2). The implications of an aging population for the practice of anesthesiology are profound. Age-related changes in physiology and pharmacology can affect every aspect of perioperative care. The changes in surgical demographics will compel the anesthesiologist to become familiar with the physiology and clinical care of the aged. This review will serve as an introduction. First, some of the physiologic changes that occur with aging will be presented. Second, the preoperative assessment of the older surgical patient will be discussed. Third, some of the research related to intraoperative management of the geriatric surgical patient will be described. In the fourth section, we will discuss some geriatric-specific issues related to postoperative management.

Adsorption of immunoglobulin G to plasma-co-polymer surfaces of acrylic acid and 1,7-octadiene

Functionalised thin films of copolymers of acrylic acid and 1,7-octadiene have been deposited by plasma-co-polymerisation to create a range of surfaces of well-defined chemistry, containing carboxyl functional groups. X-Ray photoelectron spectroscopy (XPS) has been used to characterise the deposits. Adsorption of immunoglobulin G from single solution has been studied by indirect, antibody detection (ELISA), and by direct, radio-labelling and fluorescent detection methods. In addition, XPS analysis of the surface adsorbed protein was also performed. Direct measurement techniques showed an apparent decrease in protein binding on carboxylic acid functionalised surfaces, whilst antibody detection showed an increase in binding with increased carboxylic acid content of the surface. This difference has been ascribed to orientational/conformational and/or packing differences of the proteins on the different surfaces, leading to an increased biological (“functional”) activity on the carboxylic acid functionalised surfaces. Adsorption of IgG from serum has also been probed by ELISA. An increase in IgG binding with surface carboxylic acid content was measured, indicating a similar effect when IgG is adsorbed from a complex protein mixture.

Effect of guanidino modification and proline substitution on the in vitro stability and blood–brain barrier permeability of endomorphin II

Endomorphin II (ENDII), an endogenous ligand for the μ‐opioid receptor, was investigated as a possible analgesic with fewer side effects than morphine. To improve CNS entry of END II, structural modification was also examined to determine whether Pro4 substitution and cationization affected physico‐chemical characteristics, blood–brain barrier (BBB) transport, and analgesic profile. END II and its Pro4‐substituted analog, Morphiceptin (MOR), were cationized by guanidino (GU)‐addition. MOR was seven times less lipophilic than END II, whereas GU‐addition decreased lipophilicity of both peptides. MOR did not affect in vitro BBB permeability; however, GU‐addition increased permeability of MOR by 31%. MOR decreased protein binding by 23% compared to END II, whereas GU‐addition increased protein binding of both peptides by 71 and 113%, respectively. MOR increased brain t1/2 compared to END II. GU‐addition significantly increased t1/2 of MOR and END II in both brain (sixfold and 10‐fold, respectively) and serum (over 10‐fold). Pro4‐substitution and GU‐addition enhanced the in vivo analgesia profiles of i.v. administered END II and MOR, but decreased i.c.v. analgesia profiles. This study demonstrates Pro4‐substitution decreases protein binding and enhances brain stability while cationization enhances both brain and serum stability with variable effects on BBB permeability. The analgesic profiles show that both Pro4‐substitution and cationization enhance i.v. analgesia and thus, are promising structural modifications for the development of successful opioid drugs. © 2002 Wiley‐Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2140–2149, 2002

TNFalpha decreases alphaMHC expression by a NO mediated pathway: role of E-box transcription factors for cardiomyocyte specific gene regulation.

Tumor necrosis factor alpha(TNFalpha) is thought to play a key role in the pathogenesis of cardiac failure. In the myocardium, TNFalpha enhances the expression of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) has been shown to affect beta-agonist-dependent cardiac contractility and relaxation. It is not clear, however, whether TNFalpha mediated NO release has sustained cardiac effects, by altering expression of cardiomyocyte specific genes such as alpha-myosin heavy chain (alphaMHC). Neonatal rat ventricular cardiomyocytes (CM) were stimulated with TNFalpha and/or the NOS inhibitor nitro-L-arginine (L-NNA). Protein binding to the E-box enhancer element in the alphaMHC promoter was evaluated by electrophoretic mobility shift assay (EMSA) and transcriptional activity of the E-box consensus motif was determined by luciferase assay. mRNA levels of the endogenous alphaMHC gene were assessed by RT-PCR. In vivo studies were performed in transgenic mice with cardiac specific over-expression of TNFalpha. CM treated with TNFalpha exhibited decreased levels of alphaMHC transcripts (69 +/- 8% of control), the effect of TNFalpha was reversed by L-NNA (94 +/- 14% of control). As shown by EMSA, TNFalpha reduced protein binding to the alphaMHC E-box enhancer motif via NO dependent pathways. Addition of the NO-donor sodium nitroprusside (SNP) to CM nuclear extracts dose dependently disrupted protein binding to the alphaMHC E-box. Furthermore, exposure of CM to TNFalpha or SNP decreased transcription from an E-box luciferase-reporter construct (TNFalpha: 74 +/- 12%; SNP 250 microM: 72 +/- 10%; SNP 500 microM: 66 +/- 11% of control). In myocardial tissue of TNFalpha transgenic mice, increased nitrotyrosine staining, decreased protein binding to the alphaMHC E-box motif and reduced expression of alphaMHC (62 +/- 26%) were observed. The present study shows that TNFalpha reduces alphaMHC transcript levels in cardiomyocytes. Our data obtained in cultured CM and in TNFalpha transgenic mice support the notion that TNFalpha exerts these effects by NO and E-box dependent mechanisms in vitro and possibly in vivo.

Induction of 78 kD glucose-regulated protein (GRP78) expression and redox-regulated transcription factor activity by lead and mercury in C6 rat glioma cells.

Lead (Pb) and mercury (Hg) are widespread environmental contaminants that induce prominent neural toxicity. Although the brain is not the major Pb and Hg depot in the body, these metals preferentially accumulate in astroglia to exert toxic effects. In this study, we examined the effects of Pb acetate and HgCl(2) on the expression of GRP78, a molecular chaperone in the endoplasmic reticulum (ER) that may provide cytoprotection in response to cellular stresses in the C6 rat glioma cell line. We also evaluated the DNA binding activities of several redox-regulated transcription factors in metal-treated cells. Our results showed that mRNA levels of GRP78 were up-regulated by Pb and Hg at 0.1 and 1 micro M, but down-regulated at higher concentrations (10 micro M). GRP78 protein levels increased in a concentration- and time-dependent manner in Pb and/or Hg-treated cells. Pb increased protein binding to the GST- Upsilon a antioxidant/electrophile response element (ARE/EpRE) and to the NF- kappaB consensus binding sequence of the cytomegalovirus 2 (CMB2) promoter, but decreased protein binding to the Ha-ras ARE/EpRE or to the c-fos 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE). In contrast, Hg activated DNA binding by all redox-regulated transcription factors. These studies shed some light on the molecular mechanisms of Pb and Hg toxicity in C6 rat glioma cells and suggest that GRP78 and oxidative stress may participate in the neurotoxic response to these metals.

Ceftriaxone pharmacokinetics during iatrogenic hydroxyethyl starch-induced hypoalbuminemia: a model to explore the effects of decreased protein binding capacity on highly bound drugs.

Although various drugs used by anesthesiologists highly bind to plasma proteins, the impact of iatrogenically induced hypoproteinemia on their pharmacologic effects has never been investigated. The authors determined the pharmacokinetics of ceftriaxone, a cephalosporin that binds strongly to albumin in postsurgical patients with hydroxyethyl starch-induced hypoalbuminemia. Eleven hypoalbuminemic (serum albumin < 25 g/l) patients and age (+/- 5 yr)-, sex-, and body surface area (+/- 10%)-matched healthy volunteers received a 2-g ceftriaxone dose infused over a 15-min period. Fourteen venous blood samples were collected during the 24-h study period. Free ceftriaxone concentrations were determined by ultrafiltration. Antibiotic concentrations in plasma and ultrafiltrate were measured by ion-paired reversed-phase chromatography. The pharmacokinetic parameters derived from total and free antibiotic concentrations were determined using a noncompartmental method. Data are expressed as median and range. The pharmacokinetic parameters derived from total ceftriaxone concentrations were similar for the two groups, except for the median corrected volume of distribution at steady state, which was increased (P = 0.05) to 0.18 l/kg (range, 0. 11-0.29 l/kg) in patients, compared with 0.15 l/kg (range, 0.13-0.22 l/kg) in volunteers. The area under the free ceftriaxone concentration-time curve was twice as high in patients as in volunteers (median 192, range 114-301 vs. median 122, range 84-169 h. mg-1. l-1;P = 0.03). Moreover, the free ceftriaxone concentration remained more than 4 mg/l during more time in patients (median, 16. 7; range, 12.6-21.4 vs. median, 11.1; range, 6.0-19.0 h; P = 0.03). Compared with healthy volunteers, patients with iatrogenic hypoalbuminemia have higher free ceftriaxone concentrations during the 24 h after antibiotic administration. This modification increases drug distribution into extravascular space and may enhance effectiveness.

Usefulness of thyroxine and free thyroxine filter paper measurements in neonatal screening for congenital hypothyroidism of preterm babies.

Low thyroxine (T4) with normal thyroid stimulating hormone (TSH) is a well known condition in preterm (PT) infants. The establishment of T4 and freeT4 (FT4) values in filter paper dried blood spots in PT could provide useful information in the neonatal period. To study T4 and FT4 levels in dried blood filter paper samples of PT and full term (FT) babies. We measured T4 by fluoroimmunoassay (FIA) DELFIA and TSH by IFMA DELFIA (Wallac Inc Turku, Finland) in 193 PT (26 to 37 weeks of gestational age (GA)) in samples from the first and second week of life and in 153 FT babies in the first week of life. In 131 PT and 31 FT we determined FT4 in filter paper blood spots using FIA (Alonso Fernandez J). Infants were grouped according to GA. There was a significant difference in T4 between PT and FT (p < 0.001). The lowest T4 levels were at 26 to 29 weeks GA. T4 values were lower in the second week. FT4 in PT up to 35 weeks GA, during the first week, was significantly different with FT infants (G1 to G3 p < 0.01, G4 p < 0.05). FT4 values in the first and second weeks of life did not vary. T4 values were significantly lower in PT than in FT neonates, increasing with GA. PT infants had low T4 with normal FT4 values. This could suggest a decreased thyroxine binding globulin (TBG) or decreased protein binding and/or an adaptative mechanism that would not require therapeutical intervention.

Pharmacokinetics of mycophenolic acid in renal insufficiency.

Mycophenolate mofetil (MMF) is now widely used in solid organ transplantation. MMF is rapidly converted to its active form, mycophenolic acid (MPA), upon reaching the systemic circulation. MPA is metabolized to its glucuronide metabolite, mycophenolic acid glucuronide (MPAG), by glucoronyl transferases in the liver and possibly elsewhere. MPAG is then excreted by the kidney. MPA is extensively and avidly bound to serum albumin. Previous studies have demonstrated that it is only the free (non-protein-bound) fraction of MPA that is available to exert its action. In vivo and in vitro studies demonstrate that renal insufficiency decreases the protein binding of MPA and increases free MPA concentrations. This decrease in protein binding seems to be caused both by the uremic state itself and by competition with the retained metabolite MPAG. The disposition of MPA in patients with severe renal impairment may be significantly affected by this change in protein binding.