Potential advantages of using synchrotron X-ray based techniques in pediatric research.

Synchrotron radiation (SR), which combines extremely high intensity, high collimation, tunability, and continuous energy spectrum, allows the development of advanced X-ray based techniques that are becoming a uniquely useful tool in life science research, along providing exciting opportunities in biomedical imaging and radiotherapy. This review summarize emerging techniques and their potential to greatly enhance the exploration of dynamical biological process occurring across various spatial and temporal regimes, from whole body physiology, down to the location of individual chemical species within single cells. In recent years pediatric research and clinic practice have started to profit from these new opportunities, particularly by extending the diagnostic and therapeutic capabilities of these X-ray based techniques. In diagnosis, technical advances in DEI and KES imaging modalities have been demonstrated as particularly valuable for children and women since SR allows dose minimization, with significant reductions compared to conventional approaches. However, the greatest expectations are in the field of SR based radiotherapy, increasingly studies are demonstrating SR radiotherapy provides improved chances of recovery; this is especially the case for pediatric patients. In addition, we report on the applicability of advanced X-ray microscopy techniques that offer exceptional spatial and quantitative resolution in elemental detection. These techniques, which are useful for in vitro studies, will be particularly advantageous where investigators seek deeper understanding of diseases where mismetabolism of metals, either physiological important (i.e. Cu, Zn) or outright toxic (i.e. Pb), underlies pathogenesis.

New concepts in bilirubin encephalopathy.

Revised concepts of bilirubin encephalopathy have been revealed by studies of bilirubin toxicity in cultured CNS cells and in congenitally jaundiced Gunn rats. Bilirubin neurotoxicity is related to the unbound (free) fraction of unconjugated bilirubin (Bf), of which the dominant species at physiological pH is the protonated diacid, which can passively diffuse across cell membranes. As the binding affinity of plasma albumin for bilirubin decreases strikingly as albumin concentration increases, previously reported Bf values were underestimated. Newer diagnostic tests can detect reversible neurotoxicity before permanent damage occurs from precipitation of bilirubin (kernicterus). Early toxicity can occur at Bf only modestly above aqueous saturation and affects astrocytes and neurons, causing mitochondrial damage, resulting in impaired energy metabolism and apoptosis, plus cell-membrane perturbation, which causes enzyme leakage and hampers transport of neurotransmitters. The concentrations of unbound bilirubin in the cerebro-spinal fluid and CNS cells are probably limited mainly by active export of bilirubin back into plasma, mediated by ABC transporters present in the brain capillary endothelium and choroid plexus epithelium. Intracellular bilirubin levels may be diminished also by oxidation, conjugation and binding to cytosolic proteins. These new concepts may explain the varied susceptibility of neonates to develop encephalopathy at any given plasma bilirubin level and the selective distribution of CNS lesions in bilirubin encephalopathy. They also can suggest better strategies for predicting, preventing and treating this syndrome.

Evidence for carrier-mediated transport of unconjugated bilirubin across plasma membrane vesicles from human placental trophoblast.

Unconjugated bilirubin (UCB) is currently believed to cross the placenta only by passive diffusion. To assess whether carrier-mediated transport might be involved, the uptake of [(3)H]-UCB by basal (bTPM) and apical (aTPM) plasma membrane vesicles from human placental trophoblast at term was investigated. In both types of vesicles, the uptake of [(3)H]-UCB into an osmotically sensitive space was temperature-dependent, independent of the presence of Na(+), and not affected by changes in membrane potential. The uptake of [(3)H]-UCB by aTPM, but not bTPM, was activated by ATP hydrolysis and inhibited by vanadate. Thus, the exact contribution of both inside out and right-side out bTPM to UCB uptake could not be distinguished, while only inverted aTPM were expected to carry out ATP-dependent UCB uptake. In bTPM and aTPM, uptake of free (unbound) [(3)H]-UCB (B(f)) consisted of a dominant, saturable, presumably carrier-mediated process and a diffusional component that became predominant only at B(f) near or above aqueous solubility limit for UCB (70 nM ). For bTPM, K(m)=7.2 nM; V(max)=9.8 pmol/20s/mg protein; and diffusion coefficient (K(D))=0.14 ml/20s/mg protein. For aTPM in the presence of 9.5m M ATP, K(m)=18 n M; V(max)=131 pmol/20s/mg protein; and K(D)=0.47 ml/20s/mg protein. The uptake of [(3)H]-UCB by bTPM was cis-inhibited by estrone-3-sulfate and estradiol-17 beta-glucuronide and trans-stimulated by unlabelled UCB and bromosulphopthalein. ATP-dependent UCB uptake by aTPM was cis-inhibited by doxorubicin, cholic acid, methotrexate and pronenecid. These findings suggest the presence of distinct transporters in the two domains of human placental trophoblast that could cooperate to transfer UCB from the foetus to the maternal circulation.

Preparation of an antibody recognizing both human and rodent MRP1.

Based on the high level of identity among human, mouse, and rat MRP1 protein sequence, we produced a specific polyclonal antibody (MRP1-A23) against a synthetic polypeptide covering the C-terminus of the human protein. Western blot analysis showed a reactivity against human MRP1 similar to that obtained with the monoclonal QCRL1 antibody. Differently from other available antibodies against human MPR1, MRP1-A23 also detected both rat and mouse MRP1. No cross-reactivity was observed with either human or mouse MRP2 while MRP1-A23 weakly cross-reacted with rat MRP2 in the protein region ranging from 1512 to 1533 amino acids. These data indicate that MRP1-A23 allows specific MRP1 detection in both human and rodent tissues and may provide an important tool in the study of MRP1 expression and function in both experimental and clinical materials.

Pitfalls in preparation of (3)H-unconjugated bilirubin by biosynthetic labeling from precursor (3)H-5-aminolevulinic acid in the dog.

We report problems encountered during preparation of tritium-labeled unconjugated bilirubin ((3)H-UCB) from precursor (3)H-5-aminolevulinic acid ((3)H-ALA) in 2 dogs with external biliary drainage installed into the animals under general anesthesia. Under prolonged sedation, 12.9 or 14.0 mCi of (3)H-ALA was administered intravenously in two divided doses, and bile was collected for 9 hours. In one animal, taurocholate (TC) infusion was needed to maintain bile flow. (3)H-UCB was isolated from the bile and recrystallized with the improved method of Webster et al (Webster CC, Tiribelli C, Ostrow JD. J Lab Clin Med 2001;137:370-3). Based on radioactivity and pigment content, hourly bile collections were pooled to optimize specific activities. Surprisingly, in the first dog, only 2.9% of injected radioactivity was recovered in bile and only 14.1% in urine, and the specific activities of the crystalline (3)H-UCB from the two pools were only 39.5 and 30.0 x 10(3) dpm/microg. High-performance liquid chromatography analysis revealed that only 4% of ALA degraded during 5 minutes in injection solution at pH 6.8. The low incorporation of (3)H-ALA and low specific activity of (3)H-UCB was apparently caused mainly by prior degradation and exchange of labile tritium of the (3)H-ALA and probably by enhanced endogenous ALA synthesis caused by the anesthetic/sedative agents. Revised procedures in the second dog improved the incorporation of (3)H-ALA to 11.9% excreted in bile and the specific activity of the crystalline (3)H-UCB to 122.0 and 50.8 x 10(3) dpm/microg, while urinary excretion of tritium increased to 28.5%. These experiences emphasize possible pitfalls in preparing (3)H-UCB by biosynthetic labeling from (3)H-ALA administered to dogs.

Affinity of human serum albumin for bilirubin varies with albumin concentration and buffer composition: results of a novel ultrafiltration method.

Albumin binding is a crucial determinant of bilirubin clearance in health and bilirubin toxicity in certain disease states. However, prior attempts to measure the affinity of albumin for bilirubin have yielded highly variable results, reflecting both differing conditions and the confounding influence of impurities. We therefore have devised a method based on serial ultrafiltration that successively removes impurities in [(14)C]bilirubin until a stable binding affinity is achieved, and then we used it to assess the effect of albumin concentration and buffer composition on binding. The apparent binding affinity of human serum albumin for [(14)C]bilirubin was strongly dependent on assay conditions, falling from (5.09 +/- 0.24) x 10(7) liters/mol at lower albumin concentrations (15 microm) to (0.54 +/- 0.05) x 10(7) liters/mol at higher albumin concentrations (300 microm). To determine whether radioactive impurities were responsible for this change, we estimated impurities in the stock bilirubin using a novel modeling approach and found them to be 0.11-0.13%. Formation of new impurities during the study and their affinity for albumin were also estimated. After correction for impurities, the binding affinity remained heavily dependent on the albumin concentration (range (5.37 +/- 0.26) x 10(7) liters/mol to (0.65 +/- 0.03) x 10(7) liters/mol). Affinities decreased by about half in the presence of chloride (50 mm). Thus, the affinity of human albumin for bilirubin is not constant, but varies with both albumin concentration and buffer composition. Binding may be considerably less avid at physiological albumin concentrations than previously believed.

Mechanisms for the transport of unconjugated bilirubin in human trophoblastic BeWo cells.

To evaluate mechanisms that mediate passage of unconjugated bilirubin (UCB) across placenta, the transport of [3H]UCB was studied in the human trophoblastic, BeWo cell line. When plotted against the unbound UCB concentration [Bf], uptake exhibited saturative kinetics with a similar apparent Km ( approximately 30 nM) for BeWo cells grown either in polarized (Transwell) or non-polarized fashion (dish). UCB release from cells, but not uptake, was inhibited by sulfobromophthalein but not by taurocholate, and almost abolished by MK571, a specific inhibitor of the activity of multidrug resistance-associated proteins (MRPs). MRP1 and MRP5 were both present in BeWo cells and the expression of MRP1, but not MRP5, was markedly higher in polarized cells. These data indicate that UCB is taken up from the fetal circulation by a still undefined, saturative process not shared by other organic anions and is then excreted to maternal circulation by proteins of the MRP family.

Abc protein transport of MRI contrast agents in canalicular rat liver plasma vesicles and yeast vacuoles.

The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders.

Detection of MRP1 mRNA in human tumors and tumor cell lines by in situ RT-PCR.

The detection of the multridrug resistance-associated proteins is becoming increasingly important in assessing tumor sensitivity to treatment. In this work we describe a new, rapid, sensitive, and robust method for the detection of MRP1 expression based on direct RT-in situ-PCR technology and fluorochrome-modified (dCTP(Cy3)) nucleotides. MRP1 expression was found in both placenta (BeWo) and liver (Hep G2)-derived tumor cell line as well as in small cell lung carcinoma. In liver-derived cells, MRP1 expression was detected by RT-in situ-PCR but not by in situ hybridization, suggesting a higher sensitivity of in situ amplification for the low level of expression in Hep G2 cells. RT solution PCR confirmed the presence of MRP1 in BeWo and Hep G2 cells, although the level of the gene expression was lower in liver cells. This method represents a viable alternative to conventional immunohistochemistry, and may be useful in the evaluation of MRP1 expression in different tissue or cell lines.

The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae.

Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles. Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake. Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains. These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast. Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin.

Uptake of [(3)H]bilirubin in freshly isolated rat hepatocytes: role of free bilirubin concentration.

Hepatocytic transport of physiological concentrations of unconjugated bilirubin (UCB) has not been determined in isolated liver cells. Initial uptake of highly purified [(3)H]UCB was measured in rat hepatocytes in the presence of human serum albumin at various free, unbound UCB concentrations, [UCB]. At [UCB]=42 nM (below aqueous solubility of 70 nM), uptake was strictly temperature dependent; this was much less evident at [UCB]=166 nM (supersaturated). At low, physiological UCB concentrations, specific UCB uptake showed saturative kinetics with an apparent K(m) of 41 nM, indicating carrier-mediated transport. With aqueous supersaturation, UCB entered hepatocytes mainly by passive diffusion.

Molecular mechanisms for the hepatic uptake of magnetic resonance imaging contrast agents.

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Km of 92 +/- 15 microM and a Vmax of 143 +/- 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection into Xenopus laevis oocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a >/=2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.

ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles.

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5'-[alpha, beta-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26+/-8 nM and a Vmax of 117+/-11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59+/-11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles.

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.