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Staphylococcal alpha-toxin induced ventilation-perfusion mismatch in isolated blood-free perfused rabbit lungs.

Gas exchange conditions in blood-free perfused isolated rabbit lungs were assessed by the use of the multiple inert gas elimination technique. Under baseline conditions, unimodal narrow distribution of perfusion and ventilation to midrange-ventilation-perfusion (VA/Q) areas was noted. Intravascular challenge with staphylococcal alpha-toxin caused a rapid increase in pulmonary arterial pressure (to > 40 mmHg within approximately 15 min) and delayed-onset (> 10-15 min) lung edema formation, with unaltered ventilation pressures. The vasoconstrictor response was paralleled by a progressive, severe leftward shift of perfusion to areas with low-VA/Q ratios, accompanied by a minor fraction of shunt flow. At pulmonary arterial pressures > 40 mmHg, extreme VA/Q mismatch with near absence of perfusate flow to midrange-VA/Q areas was registered. Vasoconstrictor response and VA/Q mismatch, but not the progressive edema formation, were virtually completely suppressed in lungs pretreated with acetylsalicylic acid or the thromboxane receptor antagonist BM 13505. Moreover, "rescue" application of BM 13505 after onset of alpha-toxin-induced pressor response and gas exchange abnormalities completely reversed pressure elevation and loss of VA/Q matching. We conclude that the marked vasoconstrictor response to staphylococcal alpha-toxin is paralleled by severe VA/Q mismatch with predominant perfusion of low-VA/Q areas independent of lung edema formation. Pressor response and VA/Q mismatch, but not vascular leakage, are suppressed by thromboxane inhibition.

Animals↗

Clinical renal preservation by cryoperfusion with an albumin perfusate: renal perfusion with albumin.

Eighty-six human kidneys have been preserved by cryoperfusion with an albumin-based perfusate for five to 50 hours prior to transplantation. Sixty-three of the kidneys were transplanted. The overall immediate function rate was 72% and was 100% (34/34) for kidneys with no warm ischemic damage transplanted into recipients without hypotension or prior sensitivity. The overall actuarial one-month kidney survival rate was 87%, the three-month survival was 73%, and the one-year survival rate was 65%. No kidney was discarded because of poor perfusion. Perfusion data, including flow, dastolic pressure, perfusion time, and lactate concentration were not predictive of immediate renal function. Light, electron, and immunofluorescence microscopic study of biopsy specimens showed no evidence of perfusion or immunologic damage to the kidneys. Perfusion of transplantable kidneys with albumin provides reliable preservation for up to 50 hours without producing either structural or immunologic damage to the organ.

Albumins↗

The rate of percutaneous permeation of xylene, measured using the "perfused pig ear" model, is dependent on the effective protein concentration in the perfusing medium.

In order to study the dermal permeation of compounds through the skin, an in vitro model was developed which utilized pig ears perfused with autologous pig blood (de Lange, J., van Eck, P., Elliott, G. R., de Kort, W. L. A. M., and Wolthuis, O. L. (1992). J. Pharmacol. Toxicol. Methods 27, 71-77). In the present article we investigated to what extent the rate of permeation of xylene through pig ear skin is dependent on the perfusion medium used. Pig ears were exposed to xylene (10 cm2 area) for a 4-hr period (30 degrees C, relative humidity of 40-60%) and the perfusate was analyzed for xylene using gas chromatography. The rates of permeation of xylene for whole blood, blood depleted of white blood cells, and a buffer containing 4.5% albumin were similar (+/- 300 ng/min/cm2). The rate of penetration was fivefold higher when pig plasma was used and ninefold lower when albumin was excluded from the buffer. Using the buffer, we found that the rate of permeation of xylene was proportional to flow (constant protein concentration) and protein concentration (constant flow). Our data demonstrate that the measured permeation rate for xylene is, to a large degree, dependent on the effective protein concentration (mg/min) passing through the ear. Differences in this parameter could explain the variations in rates of permeation found using the different perfusion media. To avoid problems associated with the choice of receptor fluid for permeation experiments, we suggest that full blood remains the vehicle of choice, although the practical perfusion period is limited to about 6 hr. If longer perfusion periods are required, then it should be possible to reproduce results obtained with whole blood by choosing an appropriate buffer.

Animals↗

The isolated perfused rabbit ovary-a model for studies of ovarian function. Ultrastructure after perfusion with different media.

In the present investigation the ultrastructure of isolated rabbit ovaries, perfused with different media for various time periods, was studied. The steroid hormone production by the perfused ovary was also determined. Perfusion with Medium 199 results in prominent interstitial ovarian oedema which increases with perfusion time. Even after the addition of 6-10% Dextran T40, oedema appears in the interstitial tissue of the ovary. Perfusion solutions with osmotically active colloid particles of large molecular size (Dextran T70; average molecular weight 70,000 and bovine serum albumin), cause less distortion in the ovarian structure, and ultrastructurally the ovarian tissues appear essentially the same as in the control ovaries. The results indicate that the perfused rabbit ovary, under strictly controlled conditions, can be used as an experimental model for studies of various aspects of ovarian function, including follicular rupture.

Animals↗

Physiological pharmacokinetics of solutes in the isolated perfused rat hindlimb: characterization of the physiology with changing perfusate flow, protein content, and temperature using statistical moment analysis.

Distribution of Evans Blue (EB), sucrose, and water into the isolated perfused rat hindlimb was studied under various conditions using the multiple indicator dilution (MID) technique. Statistical moment analysis of the outflow profiles for the EB, sucrose, and water were used to define the vascular, extravascular, and total water spaces, respectively. The varied perfusion conditions included albumin content (2, 4.7, and 7%), temperature (25, 37, and 42 C), perfusate flow rate (2, 4, 8, and 12 ml/min) and the presence/absence of red blood cells. The range of studies undertaken were chosen to represent the variety of conditions used in the preparation of both isolated animal and human limbs, the latter being particularly important in cytotoxic therapy for recurrent malignant melanoma. The distribution volumes of EB, sucrose, and water were dependent on the flow rate and the albumin content of perfusate. The normalized variances (CV2) of the markers were of the following order: sucrose (2.18) > water (1.58) > EB (0.68), indicating that some disequilibrium occurs during the capillary exchange of water and sucrose. It is suggested that a Krebs-Henseleit buffer containing 2% BSA is a suitable perfusate for most studies of the isolated rat hindlimb perfusion. The effect of albumin concentration manifests itself only at higher flows.

Animals↗

Influence of pulsatile perfusion upon renin release from the isolated perfused rat kidney.

It is well established that renin release from the juxtaglomerular epithelioid cells in the media of the afferent arteriole strongly depends on the mean renal perfusion pressure, whereas a possible influence of the pulsation of blood pressure on renin release has only occasionally been investigated, and the results are contradictory. Such an influence on renin release cannot be excluded because pulsation is known to modulate arterial baroreceptors and vascular tone in some resistance vessels. In the isolated perfused rat kidney, we found a pulsation amplitude-dependent inhibition of renin release that could be blocked either by vasodilatation or by calcium channel blockade. The inhibition occurred at perfusion pressures between 85 and 125 mm Hg. The underlying pulsation pressure-sensitive mechanism has to be ascribed integrating properties, because a constant-flow pressure rise to the "systolic" value of pulsatile perfusion resulted in virtually the same inhibition of renin release. Moreover, a reduced urine flow during pulsatile perfusion provides evidence for preglomerular constriction under these conditions. It is concluded that, besides pathological changes of renal perfusion pressure, variations of the pulse amplitudes, e.g. resulting from renal artery stenosis or atherosclerosis, may also influence renin release and contribute to renovascular hypertension.

Animals↗

The isolated perfused porcine liver: assessment of viability during and after six hours of perfusion.

Isolated liver perfusion was developed for the study of liver physiology and preservation. The recent development of new perfusion devices and appropriate liver preservation solutions prompted us to reconsider liver perfusion for the specific purpose of evaluating viability in terms of biochemical changes, paying special attention to modifications in the histological ultrastructure. Twenty-two isolated pig livers were perfused with autologous blood. Arterio-portal perfusions were carried out using an extracorporeal perfusion circuit with a hollow fibre membrane oxygenator. Four groups of pig livers were studied using three different liver flushing solutions [Ringer's lactate, ELOHES, and University of Wisconsin (UW)] and two different oxygenation modalities. Liver function tests and histological studies were done. Our results revealed that a high partial oxygen pressure (PO2) level was deleterious to the ultrastructural elements of hepatocytes, in particular to the mitochondria. It was also associated with deficient metabolic performance, i.e., poor bile production and lack of aerobic metabolism. Normal blood gas values could be obtained with the use of air for liver oxygenation. Flushing of the liver with Ringer's lactate or a macromolecular solution such as ELOHES was associated with severe liver cell injuries, as reflected by a marked rise in liver enzymes and histological lesions. Satisfactory results were obtained when UW solution was used for liver harvesting. We conclude that an appropriate liver preservation solution, normal blood gas values, and normal physiological arterio-portal pressure and blood flow are essential for appropriate liver function with preservation of liver architecture and of hepatocyte ultrastructures. Total bilirubin in bile and Factor V are sensitive indicators of good liver function.

Animals↗

Diagnosis of perfusion abnormality of the pulmonary artery in Takayasu's arteritis using contrast-enhanced MR perfusion imaging.

To determine the clinical efficacy of contrast-enhanced magnetic resonance (MR) perfusion imaging in the diagnosis of perfusion abnormality in the pulmonary artery (PA) in Takayasu's arteritis (TA). Twenty-one patients were evaluated. Pulmonary MR perfusion images were acquired using a 2-dimensional (2D) fast spoiled gradient echo sequence with single-slice technique (TR/TE, 5.3/1.3; flip angle, 30 degrees; receiver bandwidth, 31.2 kHz/pixel; acquisition time, 0.7 s; and total acquisition time, 49 s). Seventy continuous subtracted MR images were evaluated, and the presence of perfusion abnormality was determined in lobe-based (n=126) and patient-based (n=21) analyses. Sensitivity, specificity, positive predictive value (PPV), and negative predictive values (NPV) were calculated using perfusion scintigraphy as a standard reference. For lobe-based analysis, sensitivity was 91.7-95.8%, specificity was 92.2-93.7%, and PPV and NPV were 73.3-76.7% and 97.9-99.0%, respectively. For patient-based analyses, sensitivity was 100%, specificity was 72.7%, and PPV and NPV were 76.9% and 100%, respectively. Kappa values for each analysis were between 0.78-1.00. In conclusion, MR perfusion imaging appears to be a valuable, noninvasive method to estimate PA involvement in patients with TA.

Adult↗

Alteration of cerebral perfusion in patients with idiopathic normal pressure hydrocephalus measured by 3D perfusion weighted magnetic resonance imaging.

OBJECTIVE: It is controversial whether alteration of cerebral perfusion plays an important role in the pathophysiology of patients with idiopathic normal pressure hydrocephalus (NPH) and can help to predict the outcome after shunt surgery. MATERIALS AND METHODS: 28 patients with suspected NPH were examined clinically (Homburg Hydrocephalus Scale, walking test, incontinence protocol) and by 3D dynamic susceptibility based perfusion weighted magnetic resonance imaging (PWI-MRI) before and after cerebrospinal fluid release (spinal tap test, STT). The perfusion parameters (negative integral (NI), time of arrival (T0), time to peak (TTP), mean transit time, and the difference TTP-T0 were analysed. RESULTS: Three different groups of patients were identified preoperatively: In group 1 seven patients showed an increase in the cerebral perfusion and a clinical improvement after STT. The second group (9 patients) also revealed an increase of the cerebral perfusion, but no significant alteration of the clinical assessment could be found. In the third group neither the cerebral perfusion nor the clinical assessment changed. 14 of the 16 patients (group 1 and 2) were examined three months after shunt placement. 11 patients showed a good or excellent result, 2 patients revealed a fair assessment, and only 1 patient had transiently improved. No patient was downgraded after shunting. In the patient group 1 and 2 the NI increased significantly (effect size: 34%), whereas in group 3 no significant alteration of NI was observed. CONCLUSION: PWI-MRI improves the prediction of outcome after shunt placement in patients with NPH and can offer new insights into the pathophysiology.

Adult↗

Cocaine-induced changes in perfusion pressure and bile flow in perfused rat livers.

Cocaine produces hepatotoxicity. To study the acute effect of cocaine on the liver, we used the isolated, single-pass perfused rat liver. When perfusion pressure was measured in a constant flow system, a 15-min infusion of cocaine (1.47 mM) increased perfusion pressure (136 +/- 15%), decreased bile flow (61 +/- 5%), and decreased oxygen uptake (82 +/- 5%). The vasoconstriction was concentration-dependent and reversible. The pressure increase elicited by cocaine was not inhibited by the alpha-receptor antagonists phentolamine, prazosin, or yohimbine. These antagonists did inhibit phenylephrine-induced increases in perfusion pressure. Neither serotonin at concentrations up to 1 mM nor lidocaine or procaine in concentrations equimolar to cocaine increased the perfusion pressure. Indomethacin (5 microM), SKF-525A, and chloramphenicol also failed to block vasoconstriction induced by cocaine. High concentrations of cocaine were cholestatic, while concentrations lower than 0.6 mM were choleretic. These results indicate that cocaine-induced vasoconstriction in the liver is not mediated by alpha-receptor activation or prostaglandins and does not require metabolic activation of cocaine. The acute effects of cocaine in the perfused liver are vascular (vasoconstriction) and functional (alteration in bile formation).

Anesthetics, Local↗

Effects of perfusion flow rate, prostaglandin F2 alpha, phenylephrine, and serotonin on isolated, perfused brains of spontaneously hypertensive rats.

Effects of perfusion flow rate and three vasoconstrictors, phenylephrine, prostaglandin F2 alpha (PGF2 alpha) and serotonin, on isolated, perfused brain preparations of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY) were investigated. The basal perfusion pressure of the cerebral vascular beds at a flow rate of 2.5 ml/min was 48 +/- 3 mm Hg (n = 11) in SHR and 32 +/- 2 mm Hg (n = 12) in WKY (P less than 0.005). The perfusion pressures at all flow rates tested (2.5-6.5 ml/min) in SHR were significantly greater than those in WKY. Concentration-perfusion pressure curves for the vasoconstrictors showed that the brain vascular bed was much more reactive to serotonin compared with phenylephrine and PGF2 alpha. EC50 values (-logM) for serotonin in the perfused brains of SHR and WKY were 7.0 +/- 0.06 (n = 10) and 6.5 +/- 0.06 (n = 11), respectively (P less than 0.01). There were no differences in EC50 values for phenylephrine or PGF2 alpha between SHR and WKY. Exogenous serotonin and phenylephrine caused significantly greater maximal vasoconstrictor responses in SHR compared with WKY, while the pressor response to PGF2 alpha was very weak and no significant difference between SHR and WKY preparations was observed. These results indicate that cerebral vascular beds in SHR exhibit higher cerebrovascular resistance than those in WKY. and that reactivity and sensitivity to serotonin and reactivity to phenylephrine in SHR rats are enhanced to a greater extent compared to WKY.

Animals↗

Effect of concentration of perfusing free fatty acid on arterial lipid synthesis in perfused normal and atherosclerotic rabbit aortas.

Normal and atherosclerotic rabbit aortas were perfused at physiological pressure for 1 hour with media containing various concentrations of [3H]oleic acid, between 0.5 and 2.0 mmoles/l, complexed to a fixed concentration 40 g/l of bovine serum albumin (BSA). The mass of free fatty acid (FFA), which entered the arterial wall and was subsequently utilised for lipid synthesis, was calculated from the measured specific activities of FFA in the perfusates. In normal tissue, at all concentrations of FFA in the perfusate, the highest rates of utilisation of perfusate FFA for arterial lipid synthesis were for phospholipids (PL) and triglycerides (TG), with only about 2% in cholesteryl esters (CE). In atherosclerotic tissue, at both low and high concentrations of perfusate FFA, about 25% of fatty acid entering arterial lipids was in CE. When the concentration of FFA in the perfusion medium was raised, the mass of FFA from the medium that was incorporated in the total arterial lipids, increased in both normal and atherosclerotic tissue. The increase was due in normal tissue, to significant increases in incorporation into FFA, lecithin (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), TG and CE, whilst in atherosclerotic tissue it was due to increased incorporation into PC, PI, TG and CE. The results suggest that raised concentrations of FFA in blood may increase the rate of synthesis of lipids in normal and atherosclerotic tissue and thus exacerbate the accumulation of certain lipids such as cholesteryl esters, in fatty streak lesions of atherosclerosis.

Animals↗

Effects of particle size and perfusate composition on the uptake of colloidal gold by the rabbit thoracic aorta perfused in situ.

The influence has been investigated of particle size on the uptake of radioactive gold colloid by the rabbit thoracic aorta perfused in situ. Particles ranging in diameter from 14 nm to 40 nm were suspended in 0.9% NaCl and infused either at a pressure of 15 mm Hg for times of between 2 1/2 and 60 min or at pressure of between 15 and 160 mm Hg for 5 min. Uptake by the whole intima-media increased with perfusion time and hydrostatic pressure but did not depend on particle size. Radioactive assay of serial sections across the aortic wall also showed that particle size did not influence the distribution of tracer. An effect of perfusate composition on uptake was demonstrated in further experiments in which particles either 14 or 40 nm in diameter were suspended in pooled rabbit serum and infused at pressures of between 15 and 140 mm Hg for 5 min. Uptake and transmural distribution were again independent of particle size, but uptake was 4-5-fold less than when the particles were perfused in saline. Under all perfusion conditions radioactivity fell steeply across the intima and then rose gradually across the media and adventitia. Radioactivity in the outer media and adventitia increased with perfusion time but little change could be detected in intimal activity. In transmission electron micrographs, particles in the intima were not seen to penetrate the internal elastic lamella and in the outer media particles remained extracellular and did not enter collagen bundles. Autoradiographs showed that particles in the intima were uniformly distributed around the circumference of the vessel but in the outer media and adventitia particles usually clustered close to the vasa vasorum.

Animals↗

The effect of norepinephrine on intestinal transport and perfusion pressure in the isolated perfused rabbit ileum.

Norepinephrine (NE) is an autonomic neurotransmitter and a potent vasoactive agent, with putative effects on intestinal absorption and secretion. To characterize the effects of NE on intestinal water and ion transport, independent of changes in intestinal blood flow, we studied the effects of three doses of NE (0.1, 0.4, and 2.0 microgram/min) on the isolated, vascularly perfused rabbit ileum at a constant vascular perfusion rate. Twenty-centimeter segments of New Zealand white rabbit distal ileum (n = 21) were vascularly perfused at a fixed rate of 1.5 ml/min using a modified Krebs buffer solution (37 degrees C) containing washed human red blood cells with a hematocrit of 15-20%. The intestinal lumen was perfused at 2 ml/min with a warm buffered isotonic electrolyte solution containing 10 microCi [14C]PEG as a nonabsorbable volume marker. Net fluxes of H2O, Na+, and Cl- were calculated during 20-min basal, NE infusion, and recovery periods. Perfusion pressure was monitored continuously. NE caused statistically significant graded increases in vascular resistance, as reflected by perfusion pressure, with increasing doses. NE stimulated absorption of H2O, Na+, and Cl- with a less distinct response to increasing dose. These data suggest that the separate effects of NE on absorption and hemodynamics may be mediated through different pathways or different receptors in the intestine.

Animals↗

High-performance liquid chromatographic assay for the measurement of melphalan and its hydrolysis products in perfusate and plasma and melphalan in tissues from human and rat isolated limb perfusions.

A sensitive, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of melphalan and its hydrolysis products in samples from the isolated perfusion of human and rat limbs. Samples of perfusate, plasma and tissue were analysed, following methanol precipitation, using a phenyl column and fluorescence detection. Dansyl-arginine (38 micrograms ml-1) was employed as the internal standard. Good resolution was observed allowing quantitation of melphalan, monohydroxymelphalan (MOH) and dihydroxymelphalan (DOH) in perfusate and plasma were all 100 +/- 10%. The recovery of melphalan in tissue was 93.5%. A linear response was demonstrated for melphalan in the concentration range 1.8 - 56.8 micrograms ml-1, for DOH in the concentration range 0.5 - 30.0 micrograms ml-1 and for MOH in the range 1.4-25.1 micrograms ml-1, in perfusate and plasma. The lower limits of quantitation of melphalan, MOH and DOH in perfusate and plasma were 1.4, 2.4 and 1.2 ng on column, respectively, and 7.2 ng of melphalan on column in tissue. Intra-assay coefficients of variation (C.V.) for melphalan, MOH and DOH, at low and high concentrations were all less than 5% and the inter-assay C.V.s were less than 9%. An ultra-filtration study to determine the protein binding of melphalan and the hydrolysis products showed that the unbound fractions (fu) of melphalan in buffer containing dextran and bovine serum albumin were 0.873 and 0.521, respectively. The assay was used to quantitate melphalan and its hydrolysis products in samples from isolated perfusions in the human limb and rat hindlimb.

Adipose Tissue↗

An evaluation of whether duration of perfusion alters vascular responses in the isolated dual-perfused rabbit liver.

The isolated dual-perfused rabbit liver has been used to characterize hepatic arterial vascular receptors. These hepatic arterial responses are reproducible for 2.5 hr. Further studies in relation to the assessment of portal venous responses using this model require longer periods of perfusion. This study was designed to determine if this model is suitable for the assessment of hepatic arterial and portal venous vascular responses over a 5-hr perfusion period. Hepatic arterial responses were consistent to all agents during 5 hr of perfusion. Portal venous responses to the direct smooth muscle acting agents, sodium nitroprusside and adenosine, were constant, but the vasoconstrictor responses to acetylcholine and adenosine 5'-triphosphate (agents that cause an endothelium-independent vasoconstriction and an endothelium-dependent vasodilatation) were potentiated. Coarse measurements of liver function were also performed and suggested that the liver remained viable for the 5 hr of perfusion. These results suggest a decrease in the ability of the endothelium of the portal venous vasculature to respond to vasoactive substances as duration of perfusion increases. The possible reasons for this are discussed.

Acetylcholine↗

Glucagon-like peptide-1 secretion is influenced by perfusate glucose concentration and by a feedback mechanism involving somatostatin in isolated perfused porcine ileum.

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to ingestion of meals. The mechanisms regulating its secretion are not clear, but local somatostatin (SS) restrains GLP-1 secretion. We investigated feedback and substrate regulation of GLP-1 and SS secretion, using isolated perfused porcine ileum (n=17). Effluents were measured for GLP-1 and SS. Perfusion pressure and motility were recorded. Investigated parameters included spontaneous fluctuations, changes in perfusate glucose concentrations (3.5, 5, 11 mM) and addition of insulin (1 nM). We also investigated the effect of proglucagon products, glucagon (10 nM), GLP-1 and GLP-2 (0.1, 1, and 10 nM) on GLP-1 and SS secretion, as well as on glucagon-like peptide-2 (GLP-2), peptide YY (PYY) and GIP secretion, all possible product of L-cells or neighbour cells. Perfusate glucose concentration dose-dependently stimulated GLP-1 secretion (p=0.011). Insulin had no effect. Glucagon weakly stimulated GIP secretion. GLP-1 stimulated SS secretion and motor activity, but inhibited GLP-2, GIP and PYY secretion and perfusion pressure. GLP-2 weakly stimulated SS secretion. We conclude (a) that GLP-1 secretion is influenced by perfusate glucose concentration and (b) that L-cell secretion is feedback regulated by GLP-1 itself, probably via paracrine SS activity.

Animals↗

Phthalate monoesters in perfusate from a dual placenta perfusion system, the placenta tissue and umbilical cord blood.

Fetal exposure to phthalates may be associated with adverse reproductive effects, including cryptorchidism and decreased semen quality. Information about human placental transfer is needed to qualify the hypotheses. A dual recirculating placenta perfusion system to monitor concentrations of eight phthalate monoesters in fetal and maternal perfusates was established. In addition to perfusate background measures of phthalate monoesters, the concentrations in umbilical cord plasma and placenta tissue were measured. Monomethyl phthalate (mMP), monoethyl phthalate (mEP), monobutyl phthalate (mBP), and mono (2-ethyl-hexyl) phthalate (mEHP) were detected in both maternal and fetal perfusate, demonstrating a release of compounds from tissue or blood to perfusates. The distribution of compounds between perfusate, umbilical cord plasma, and tissue was in accordance with the physical-chemical properties of the compounds. Results from the present study of compounds residing in the tissue are essential before studying human transplacental transfer, storage, and metabolism of selected phthalate monoesters.

Adult↗