Impact of innovative techniques on the waiting list and results in pediatric liver transplantation.

The wide application of liver transplantation in children is hampered by the shortage of size-matched pediatric donors; this results in high mortality rate on the waiting list, a long waiting time, worsening of the clinical condition of the waiting patient, deterioration of the overall results, and an increase in the cost. Reduced-size liver transplants have been shown to be a safe way to alleviate the shortage of size-matched organs. We have retrospectively analyzed the impact of the reduced-size liver transplants on the waiting list and the results in a consecutive series of 314 transplants performed in 261 children over an 8-year period (1984-1991). Among these 314 grafts, 160 (51%) were innovative techniques including 86 reduced livers (stricto senso), 66 partial livers (with preservation of the recipient vena cava), and 8 split livers. Such an extensive use of these technical variants allowed a sharp decrease in the waiting list mortality: from 14.9% between 1984 and 1989 to 6.6% in 1990 and 5% in 1991; the corresponding figures for infants registered under the age of 1 year were 25%, 13.3%, and 8.3%, respectively. Results obtained with a full-size graft or a technical variant were similar regarding surgical complications (with a significantly lower incidence of arterial thrombosis for the reduced transplants), graft loss, and patient survival. The 5-year survival of the whole group was 78.1% without any significant difference regarding type of transplant, indications (with the best results: 89.4% 5-year survival obtained in 41 children grafted for metabolic diseases), or age (the 5-year survival was 82.2% for the 41 infants transplanted under the age of 1 year, 78.9% for the 124 children transplanted between 1 and 3 years, and 81.3% for the 96 children transplanted between 6 and 15 years). This series of reduced-size liver transplants, which is the largest worldwide single institutional experience, confirms that the extensive use of reduced transplants in children is safe; this study also shows that innovative techniques, including the split liver, allow a drastic decrease of the waiting list mortality of candidates in the pediatric age range without alterations of the results.

Liver procurement without in situ portal perfusion. A safe procedure for more flexible multiple organ harvesting.

The outcome after liver transplantation when grafts were retrieved from the donor by the classical aortic and portal cooling (APC) method was compared with the outcome when exclusively aortic in situ perfusion (AC) was used. Retrospectively, 163 donor hepatectomies performed over a 20-month period were reviewed to analyze overall graft (APC n = 78, AC n = 85) and patient outcome. The global graft and patient survival rates were not significantly lower in the APC group, except for 3-month graft survival (APC 72%, AC 87%; P = 0.015). However, this could be unrelated to the technique. In a subgroup of 140 cases (APC n = 64, AC n = 76), a more detailed analysis was performed. Populations of donors and recipients were similar. The graft injury and the immediate graft function were not significantly different between both groups. A multivariate analysis shows that low donor weight (P = 0.007), donor hypernatremia (P = 0.014), and in situ portal perfusion (P = 0.045) were significant determinants of a higher postoperative peak of glutamic pyruvic transaminase. In summary, in this series, routine human liver procurement using exclusive aortic perfusion seemed to be at least as safe as using a combined aortic and portal perfusion technique. This simplified method may also represent some advantages for combined pancreas and intestinal harvesting in the future.

The unreliability of the lidocaine/monoethylglycinexylidide test for assessment of liver donors.

The serum monoethylglycinexylidide (MEGX) level 15 min (t15) after i.v. administration of lidocaine (1 mg/kg) in liver donors was retrospectively correlated with graft outcome and early hepatic function. Among the 35 orthotopic liver transplants studied, 4 recipients had to be retransplanted within 10 days post-OLT because of early graft nonfunction or dysfunction, and 3 recipients died, with a median (range) donor MEGX t15 (ng/ml) of 100 (86-119) and 169 (146-182), respectively. The remaining 28 OLT patients living with functioning grafts had a donor MEGX of 87 (18-245). No significant correlations could be found between donor MEGX t15 and recipient mean and peak glutamic-oxaloacetic and -pyruvic transaminases, total serum bilirubin, or mean and minimum prothrombin time values studied from day 1 to day 5 post-OLT. Moreover, categorization of donors using the MEGX t15 cut-off point of 80 ng/ml could not predict liver graft quality, as previously suggested. In summary, MEGX t15 in liver donors correlated neither with graft outcome nor with early functional parameters. Accordingly, the MEGX test should not be used as an isolated discriminatory evaluation for organ utilization.

Cardiac sarcolemmal purity is essential for the verification of adenylate cyclase inhibition via A1-adenosine receptors.

Inhibition of cardiac adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [3H]ouabain binding, whereas 5'-nucleotidase activity and beta-adrenoceptor binding displayed an additional peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N6-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A1-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[125I]iodo-N6-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A2-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.

Cardiac ventricular beta 2-adrenoceptors in guinea-pigs and rats are localized on the coronary endothelium.

In mammalian heart tissue beta 2-adrenoceptors are known to coexist with beta 1-adrenoceptors. In the present study, evidence that beta 2-adrenoceptors in guinea-pig and rat ventricles are primarily localized on the coronary endothelium is provided by competition binding studies with the subtype-selective beta-adrenoceptor antagonists ICI 89.406 (beta 1-selective) and ICI 118.551 (beta 2-selective) on four different plasma membrane preparations. (1) Following density gradient centrifugation of cardiac ventricular microsomes from rats or guinea-pigs, endothelial plasma membranes migrated at slightly higher density than the sarcolemmal membranes, as verified by endothelial (angiotensin converting enzyme) and sarcolemmal markers (adenylate cyclase, [3H]ouabain binding). At the activity peak of angiotensin converting enzyme, the relative amount of beta 2-adrenoceptors in guinea-pigs and rats was 25% and 65%, respectively. (2) On sarcolemmal membranes corresponding to the activity peak of adenylate cyclase, beta-adrenoceptors consisted of the beta 1-type exclusively (guinea-pig), or to at least 90% (rat). (3) Cultures of coronary endothelial cells derived from guinea-pigs revealed only beta 2-adrenoceptors. (4) Isolated guinea-pig cardiomyocytes contained only beta 1-adrenoceptors, a finding recently established in rat myocytes as well.

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors.

Rabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (-)[125I]N6-(4-hydroxyphenylisopropyl)-adenosine ([125I]HPIA) provided evidence for the presence of A1-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [125I]HPIA binding were mono-phasic with a KD of 1.3 nmol/l and a Bmax of 7.7 fmol/mg protein. In kinetic experiments, an association rate constant of 4.9 X 10(5) (mol/l-1 s-1 and a dissociation rate constant of 4.3 X 10(-4) s-1 were obtained, yielding a KD of 0.9 nmol/l. Adenosine analogs displaced [125I]HPIA binding with a rank order of potency typical of A1-adenosine receptors; furthermore, binding was inhibited by methylxanthines and modulated by GTP. Saturation experiments with the microvessels revealed a KD of 1.9 nmol/l and a Bmax of 13.4 fmol/mg protein. However, no inhibition of glomerular and microvascular adenylate cyclase activity could be demonstrated, but instead both 5'-N-ethylcarboxamido-adenosine (NECA) and N6-(R-phenylisopropyl)-adenosine (R-PIA) stimulated enzyme activity, with EC50 values of 0.14 mumol/l and 1.5 mumol/l, respectively. The concentration-response curve for NECA was shifted to the right (factor 9) by 10 mumol/l 8-phenyltheophylline. On the other hand, computer simulation of biphasic curves (adenylate cyclase inhibition in the presence of activation via a stimulatory receptor) indicates that the failure to observe an A1-adenosine receptor-mediated inhibition of adenylate cyclase activity in the presence of stimulatory adenosine receptors may be attributable to methodological constraints.(ABSTRACT TRUNCATED AT 250 WORDS)

New developments in the isolated working heart: a comparison of neonatal, immature, and adult rabbits after sixty minutes of ischemia in respect to hemodynamic and biochemical parameters.

Hemodynamic and biochemical changes were studied on 36 white ELCO-rabbits, seven adult older than 150 days, seven immatures between 21 and 27 days, and seven neonatals between 7 and 14 days. Five supplementary hearts of each age group served for preischemic biochemical values. Protection during 60 min of global ischemia was provided by topical cooling and selective coronary perfusion with Bretschneider cardioplegia (8 degrees C). A comparison between pre- and postischemic results showed decreases in coronary flow in the adult (p < 0.004), aortic flow (p < 0.04), cardiac output (p < 0.02), and stroke volume (p < 0.02) in the neonate. The preservation of ATP and CP was sufficient in the adult and immature myocardium, whereas a significant decrease in neonatal ATP was found (p < 0.01). According to these findings we consider immature myocardium to be more resistant against ischemia than the two other age groups. The apparatus used is a development of the conventional working heart, but combines a physiological flow-pressure relation, with instruments guaranteeing high accuracy, devices for drug application, and fits for different sizes of hearts. Therefore, this new approach promises to be of clinical relevance for investigations on the improvement of myocardial protection in both adults and children.

Two saturable recognition sites for (-) [125I]iodo-N6-(4-hydroxyphenyl-isopropyl)-adenosine binding on purified cardiac sarcolemma.

Analysis of (-) [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine [( 125I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 mumol/l) was used as non-specific binding marker. In the presence of 1 mmol/l theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [125I]HPIA binding, with a high affinity potency rank order typical of interaction with A1-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/l. In brain membranes, specific binding of [125I]HPIA as well as of [3H]PIA was further reduced when unlabeled iodo-HPIA replaces theophylline as the non-specific binding marker. These results suggest the presence of two [125I]HPIA binding sites on cardiac sarcolemma and brain membranes, but receptor function can only be ascribed to the high affinity sites. The low affinity site probably represents an artefact, which is often observed when non-specific binding is defined with the unlabeled counterpart or a structurally related ligand of the radioligand used.

Adenosine-receptor-mediated stimulation of low-Km GTPase in guinea-pig cerebral cortex.

Inhibition of receptor-coupled adenylate cyclase by hormones is proposed to be associated with GTP hydrolysis. Since adenosine inhibits cerebral-cortical adenylate cyclase via A1 adenosine receptors, the present study attempts to verify this mechanism for A1-selective adenosine derivatives. In guinea-pig cortical membranes N6-(phenylisopropyl)adenosine (PIA) increased the Vmax. of the low-Km GTPase, with an EC50 (concentration causing 50% of maximal stimulation) of about 0.1 microM, and the stimulatory effect was competitively antagonized by 5 microM-8-phenyltheophylline. The rank order of potency of the stereoisomers of PIA and of 5-(N-ethylcarboxamido)adenosine (NECA) to stimulate GTPase correlated with their ability to inhibit adenylate cyclase activity (R-PIA greater than NECA greater than S-PIA). Competition binding studies with (-)-N6- ([125I]iodo-4-hydroxyphenylisopropyl)adenosine suggest that adenylyl imidodiphosphate (p[NH]ppA), an essential component of the GTPase assay system, is a more potent A1-receptor agonist than ATP, with an IC50 (concentration giving half-maximal displacement of radioligand binding) of 7.9 microM. On the basis of the p[NH]ppA concentration used in the GTPase assay (1.25 mM), enzyme stimulation by adenosine seems to be highly underestimated. Nevertheless, adenosine-induced GTP hydrolysis reflects an increased turnover of guanine nucleotides at the Ni regulatory site and appears to be a crucial step in the sequence of events processing the inhibitory signal to adenylate cyclase.

Standardized quick en bloc technique for procurement of cadaveric liver grafts for pediatric liver transplantation.

This paper describes a quick procedure for cadaveric liver graft retrieval during multiple organ harvesting. The technique is based on minimal preliminary dissection, absence of in situ direct portal perfusion, and en bloc removal of the liver and pancreas, with an aortic patch encompassing the coeliac trunk and superior mesenteric artery. The results of 110 pediatric liver transplantations with 109 organs harvested using this technique are reported. There were no graft harvesting injuries. The liver graft primary nonfunction rate was 4.5% (5/110). The 3-month retransplantation rate was 10%. The actual patient survival rates were 93% at 3 months and 90% at 1 year; actual graft survival rates were 85.5% and 78%, respectively. The technique described was at least as safe as conventional procedures. A major advantage of the procedure is its flexibility, which allows for the easily combined procurement of other organs (whole pancreas and intestine).

Alterations of the glomerular beta-adrenoceptor-linked adenylate cyclase system in perinephritis hypertension.

The present study was undertaken to investigate beta-adrenoceptor-adenylate cyclase coupling in a myocardial ventricular membrane preparation and in isolated renal glomeruli of cellophane perinephritis hypertensive rats. Adenylate cyclase activity and [125I]iodocyanopindolol binding were differentially affected in these preparations. In isolated glomeruli of hypertensive rats a reduced intrinsic activity of isoproterenol was associated with an apparent loss of the guanine nucleotide-sensitive high-affinity state of the beta-adrenoceptors, whereas their absolute number was unchanged when compared with the normotensive control rats. On the other hand, in the sarcolemmal preparation of hypertensive rats adenylate cyclase activity, the relative amount of high-affinity states, and the density of beta-adrenoceptors were not different from the respective values in normotensive controls. Experiments performed on isolated glomeruli of rats with unilateral cellophane perinephritis that developed only moderate hypertension provide evidence that the apparent loss of the high-affinity state is a consequence of hypertension, since no difference was observed in glomeruli from the wrapped, as compared with the intact kidney.

Myocardial protection with Bretschneider cardioplegic solution--an evaluation of full oxygenation.

In the present study the effect of oxygenated Bretschneider cardioplegia on high-energy phosphates [adenosine triphosphate (ATP), adenosine diphosphate (ADP) and creatine phosphate (CP)] and hemodynamics was evaluated in the isolated working rabbit heart. Hearts were obtained from 37 adult white Elco rabbits (3,100 +/- 110 g). After a 20-min working period 14 hearts were arrested with Bretschneider cardioplegia (8 degrees C) oxygenated with 98% oxygen (O2) and 2% carbon dioxide in comparison to 14 hearts receiving Bretschneider solution saturated with 98% nitrogen (N2) and 2% carbon dioxide as a control group for either 60 or 90 min (O(2)60, O(2)90, N(2)60, N(2)90 groups, n = 7). Seven hearts were used to determine preischemic baseline values of ATP, ADP and CP, 2 were excluded. The results showed a significantly poorer preservation of high-energy phosphates in hearts receiving oxygenated Bretschneider cardioplegia as compared to hearts receiving nitrogenated cardioplegia (p < 0.05). Postischemic recovery of hemodynamics did not demonstrate any statistically significant differences between the groups. However, the intragroup analysis showed a tendency towards weaker hemodynamic recovery in hearts treated with oxygenated cardioplegia. in contrast to the beneficial effect of oxygenated St. Thomas solution. In conclusion our findings suggest that oxygenated Bretschneider cardioplegia leads to significantly poorer preservation of high-energy phosphates and depressed hemodynamic recovery.

Sequential treatment of biliary atresia with Kasai portoenterostomy and liver transplantation: a review.

Biliary atresia is the most frequent cause of chronic cholestasis in infants. When left untreated, this condition leads to death from liver insufficiency within the first 2 yr of life. The modern therapeutic approach consists of a sequential strategy with Kasai portoenterostomy as a first step and, in case of failure, liver transplantation. After portoenterostomy, no more than 20% to 30% of patients will live jaundice-free into adulthood. Illness in another third will be palliated, and these patients have extended survival, delaying liver transplantation to later childhood (2 to 15 yr). The remaining 30% to 40% will not benefit from the Kasai operation and will die of liver failure in infancy. The annual need of liver transplantation for biliary atresia is one case per million people. This indication represents 35% to 67% of the reported series of pediatric liver transplantation and between 5% and 10% of the indications for liver transplantation, all ages included. Approximately four of five children transplanted for biliary atresia will become long-term survivors with good physical and mental development; recurrence of the disease after transplantation has not been observed. Because most candidates are young children (< 3 yr) of small size (< 10 kg), there is a shortage of size-matched donors (which has been alleviated by the use of innovative techniques such as reduced and split livers). The resulting redistribution of the adult donor liver pool is ethically justified by the equal quality of the results after transplantation of a full-size or partial graft.

Branched chain amino acids improve body composition and nitrogen balance in a rat model of extra hepatic biliary atresia.

Malnutrition and growth retardation remain a major complication in infants with extrahepatic biliary atresia associated cholestasis. The purpose of this study was to investigate whether oral supplementation with branched chain amino acids (BCAA) can correct malnutrition in a rat model of biliary atresia. Four groups of 15 rats, 30 d old, were used. Group A were shamoperated animals, given a normal laboratory diet (17.5% of caloric intake as proteins). Group B were cholestatic rats (biliary atresia) fed a diet enriched in BCAA (supplement of 8.5%, valine/leucine/isoleucine ratio 1:1:1). Group C were cholestatic mice fed a diet enriched in casein (supplement of 8.5%). Group D were cholestatic mice fed a normal diet. Thirty-two days after surgery, groups were compared for body weight, serum amino acid content, nitrogen balance, muscle mass, and carcass composition. The results showed that the weight of group B, C, and D animals was 85, 81, and 64% of group A (controls). Serum BCAA levels were markedly increased in group B animals. Nitrogen retention was similar in groups B and A, but reduced to 63 and 44% in groups C and D, respectively. Dry weights were similar in group A (39.1% of body weight) and B (37.7%), but reduced to 28.1 and 28.6% of body weight in groups C and D. Body proteins were higher in groups A (13.9%) and B (14.2%) than in group D (9.7%) rats. Mineral content of group B animals was 84% of those of group A, 50% in group C, and 23% in group D rats. It was concluded that an oral supplement of BCAA can correct growth, nitrogen retention, and body composition in experimental biliary atresia. Administration of BCAA supplements to cholestatic infants should be considered.