Acute Pancreatitis Secondary to Severe Hypertriglyceridaemia in a Patient with Type 1a Glycogen Storage Disease: Emergent Use of Plasmapheresis.

Acute pancreatitis is a well-recognised complication of hypertriglyceridaemia. High serum triglycerides may develop in the autosomal recessive disorder glycogen storage disease (GSD). Plasmapheresis has been effective in reducing triglyceride levels in pancreatitis secondary to other conditions but not previously described in GSD. We describe a 16-year-old male with type 1a GSD who presented with severe abdominal pain, tachycardia and tachypnoea. Abdominal computed tomography (CT) demonstrated acute pancreatitis. Serum triglycerides were 91.8 mM. Despite intravenous fluids and morphine sulphate, he remained seriously ill, and plasmapheresis was therefore started. After daily plasma exchange for 6 days, triglyceride levels dropped to 5 mM. This was associated with a rapid resolution of pancreatitis. Plasmapheresis is effective in rapidly reducing hypertriglyceridaemia from numerous causes, including glycogen storage disease, and may facilitate recovery from acute pancreatitis.

Thiopurine methyltransferase in acute lymphoblastic leukaemia: biochemical and molecular biological aspects.

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalysing S-methylation of aromatic and heterocyclic sulphhydryl compounds. TPMT activities and genotypes have been determined in patients with acute lymphoblastic leukaemia (ALL) and in control children. Median red blood cell (RBC) TPMT activity in ALL patients at diagnosis was significantly lower than in controls (median 11.5 pmol/10(7) RBC*hr; range 1.7-30.7; n = 191 vs. 14.6 pmol/10(7) RBC*hr; range 1.6-50.7; n = 140). This reduction of TPMT activity in ALL patients was not due to differences in the frequency of mutations in the TPMT gene. In concordance with other authors, we found a higher TPMT activity during maintenance treatment with 6-mercaptopurine (6MP) than at diagnosis and in controls. However, we observed that TPMT activity was already significantly increased after the induction therapy, before the patients received 6MP (median 17.5; range 3.9-40.3 pmol/10(7) RBC*hr; n = 139). In vitro experiments indicate that the early increase of TPMT activity during treatment may be explained by the use of antifolates, e.g., methotrexate and trimethoprim.

Real-time analysis of tyrosine hydroxylase gene expression: a sensitive and semiquantitative marker for minimal residual disease detection of neuroblastoma.

PURPOSE: The purpose of this study was to establish a sensitive and semiquantitative method for the detection of minimal residual disease of neuroblastoma, the most common solid tumor in childhood. EXPERIMENTAL DESIGN: Analysis was performed on a molecular level by reverse transcription-PCR using a new, real-time detection method. We measured two genes simultaneously, tyrosine hydroxylase (TH) as the target gene and glyceraldehyde-3-phosphate dehydrogenase as a reference gene, in blood and bone marrow samples at diagnosis and after follow-up from six patients with neuroblastoma, one patient with ganglioneuroma, and one patient with ganglioneuroblastoma. RESULTS: The sensitivity of the assay was 1:10(6) peripheral WBCs. Four patients with stage IV neuroblastoma and one patient with stage III neuroblastoma were scored positive. The other stage III patient and the other two patients with ganglioneuroma and ganglioneuroblastoma followed by acute lymphoblastic leukemia, respectively, were scored negative. Control bone marrow aspirates were also negative. The TH assay is more sensitive than immunohistochemical detection, and the results of the TH assay corresponded with the results of MYCN amplification. CONCLUSIONS: The described TH assay is specific, sensitive, and semiquantitative and can be used for the detection of neuroblastoma cell involvement in bone marrow and blood at diagnosis and during therapy. Furthermore, the TH assay is a possible prognostic marker for neuroblastoma.

Central-peripheral temperature difference, blood pressure, and arginine vasopressin in preterm neonates undergoing volume expansion.

AIM: To examine the effect of intravascular volume expansion for the treatment of hypovolaemia in sick preterm neonates. METHODS: An intravenous infusion of 20 ml per kg of 4.5% albumin was given to 14 preterm neonates. The effects on systolic blood pressure, central peripheral temperature difference (c-pT), and plasma arginine vasopressin concentration (pAVP) were measured. RESULTS: Thirteen babies showed a rise in systolic blood pressure. The six babies with the highest initial values of pAVP and c-pT showed a fall in both of these after infusion. The babies with lower initial pAVP (below 4 pmol/l) showed either a rise (two) or no change (six) after albumin infusion. There was a significant correlation between c-pT and log pAVP before (r2 = 0.61; p < 0.05) and after infusion (r2 = 0.45; p < 0.05). CONCLUSIONS: Plasma AVP concentration is related to c-pT in unwell preterm newborns. This study suggests that clinical assessment of hypovolaemia in preterm newborns is poor and could be improved by using c-pT.

Does positive pressure ventilation increase arginine vasopressin in preterm neonates?

AIM: To examine the effect of intermittent positive pressure ventilation (IPPV) on plasma arginine vasopressin concentration (pAVP) in preterm neonates. METHODS: Thirty five neonates were classified, at the time of blood sampling, into three groups: unstable ventilated; stable ventilated; and stable non-ventilated. A modification of an extraction method for pAVP was developed for use in studies on very small babies, and sampling methods were compared. RESULTS: The pAVP (median, range) was similar in the ventilated (1.85 pmol/l, 0.5 to 3.4) and non-ventilated (2.0, 0.5 to 2.6) stable babies, but was significantly higher (5.7, 1.1 to 25) in the unstable group. There was an inverse correlation between systolic blood pressure and pAVP concentration. CONCLUSIONS: This study shows that in preterm neonates pAVP concentration is affected by the clinical condition and blood pressure, but not by treatment with IPPV.

On the mechanism of dehydration of a beta-hydroxycyclopentanone analogue of prostaglandin E1.

The dehydration of the beta-hydroxycyclopentanone, 11,16-dihydroxy-16-methyl-9-oxo-13-trans-protenoic acid methyl ester, an analogue of prostaglandin E1, proceeds with acid catalysis (pH less than 3), by uncatalyzed routes (pH congruent to 4 and congruent to 7), and with base catalysis (pH congruent to 5-6 and greater than 8). Deuterium from the solvent is not introduced alpha to the reactant keto function at 60% reaction at pH congruent to 1, but approximately 30% exchange has occurred at pH congruent to 5, 50% at pH congruent to 7, and 80% at pH congruent to 9. The data are consistent with a mechanism in which the substrate is initially enolized with catalysis by acid, base, and water to a 1,3-enediol, which looses water with catalysis by acid, base, and water. The first stage is rate determining in very acidic solution, while the second stage assumes the limitation of rate to an ever greater degree as the solution becomes more basic.

Solution-solubility dependency of controlled release of drug from polymer matrix: mathematical analysis.

Two types of drug release mechanisms-matrix controlled (Q - t1/2 relationship) and partition controlled (Q - t relationship)-were reported previously. The dependency of the transition between these two processes on the solution solubility of drug is now analyzed. Mathematical expressions are derived to treat the systemic effects of the addition of varying volume fractions of the cosolvent system on the concentration gradients across both the drug depletion zone and hydrodynamic diffusion layer as well as on the partitioning behavior at the polymer-solution interface. The results are in excellent agreement with reported experimental observations.

Interaction of metronidazole with metallic ions of biological importance.

The possibility tht metronidazole exerts several of its biological actions via interaction with important metal ions was investigated. NMR spectroscopy and polarography (ac) were used to test for any interaction, to locate the probable sites for complexation, and to determine the molecular stoichiometry of any complexes formed. Of the series of divalent metal ions tests, only cupric ion showed detectable interaction with metronidazole. The predominant site of interaction of cupric ion was the unsubstituted nitrogen atom (N-3) on the metronidazole molecule. The stoichiometry of the complex was (Cu-(metronidazole)4)+2. A likely structure for the complex is presented.

Linear relationships between plasma binding and lipophilicity of disopyramide derivatives.

The extent of plasma binding and the partition coefficient of disopyramide and 20 disopyramide derivatives were determined. Structural variations on the four functional groups around the tetrahedral carbon in the disopyramide molecule was found to influence both parameters to varying degrees. Three linear equations were developed to correlate the observed effects, depending on the type of chemical modification. The linear correlation between drug-plasma interaction and lipophilic character was analyzed theoretically. A simple model was derived to relate quantitatively the variation in the extent of plasma binding to the change in lipophilicity of disopyramide derivatives.

Medicated tampons: intravaginal sustained administration of metronidazole and in vitro-in vivo relationships.

The technical feasibility of utilizing tampons as a drug delivery system for prolonged intravaginal drug administrations was studied. Several commercially available brands of tampons were examined. The methodology for the incorporation of various doses of metronidazole, an antitrichomonas agent, in tampons was described. The sustained-release profile of metronidazole from these medicated tampons was characterized. Intravaginal administration of metronidazole via the medicated tampons was investigated in rhesus monkeys and human volunteers, and in vitro-in vivo correlations were established. The biopharmaceutics of intravaginal absorption of metronidazole via medicated tampons was analyzed in comparison with a vaginal solution formulation.

Binding of spirolactones to human plasma proteins.

The lipophilicity and plasma binding of 16 spirolactones and 4 hydroxy acid analogs were determined. Mathematical expressions were derived to correlate quantitatively the extent of plasma binding to the lipophilicity of the drugs. The nonspecific binding of these spirolactones and their hydroxy acid analogs was also analyzed using purified serum albumin. A computer program was developed to examine the mechanism of drug-serum protein interactions. One class of binding sites was observed for the range of concentrations used. The number of binding sites and the equilibrium binding constant were computed and were sensitive to substitution at the C-6 and C7 positions. Hydrolysis of the C-17 lactone ring in spirolactones to form hydroxy acid analogs resulted in a decrease in the lipophilicity and, hence, the equilibrium constant for binding.

Controlled drug release from polymeric delivery devices. III: In vitro-in vivo correlation for intravaginal release of ethynodiol diacetate from silicone devices in rabbits.

Forty female rabbits were implanted with silicone vaginal devices containing ethynodiol diacetate for up to 8 weeks. As predicted from in vitro studies, a Q - t1/2 (matrix-controlled) release profile was observed in vivo. The in vivo drug release profile was compared with in vitro data measured at three hydrodynamic conditions, and the diffusional resistance across the vaginal wall was estimated. Drug released from silicone devices yielded a prolonged plasma level when compared with data following intravaginal or intravenous administration of a solution dose. The rate constant for elimination was unchanged. The plasma concentration of the drug was related to the intravaginal drug release profile both theoretically and experimentally and was above the concentration required to inhibit fertilization.

Binding of metronidazole and its derivatives to plasma proteins: an assessment of drug binding phenomenon.

Metronidazole and four derivatives were studied in vitro to investigate the differences in the extent of their binding to plasma proteins. Modification at the terminal portion of the alkyl side chain resulted in wide differences in the extent of binding. Molecular orbital calculations were performed by the CNDO and MINDO/2 methods to estimate the frontier electron density on the hetero atom at the 3'-position of the alkyl side chain. A linear correlation between the protein binding parameter (loge P) and the frontier electron density (qr) was observed for the binding of this group of trichomonicidal drugs. NMR spectroscopy was used to demonstrate that the alkyl side chain participated in the binding of these compounds to plasma proteins.

Controlled drug release from polymeric delivery devices V: Hydroxy group effects on drug release kinetics and thermodynamics.

The effects of progesterone hydroxylation on silicone matrix drug release kinetics and thermodynamics were investigated. Hydroxylation at positions 11, 17, and/or 21 substantially reduced progesterone release. The magnitude of this reduction depended on the number and position of the hydroxy groups and could be attributed to decreased polymer matrix diffusivity (Dm) and polymer solubility (Cp). Thermodynamically, hydroxy group addition to positions 11 and/or 21 reduced the activation energy for matrix diffusion (Ed,m) but increased the solvation energy for dissolution in silicone polymer (delta HT,m)). Adding an hydroxy group to position 17 increased the Ed,m but decreased the delta HT,m. The overall (Ed,m) + delta H(T,m)) values were relatively constant and independent of hydroxylation.

Combination therapy in childhood leukaemia: in vitro studies of thiopurines and inhibitors of purine metabolism on apoptosis.

BACKGROUND: Methotrexate (MTX) followed by 6-mercaptopurine (6MP) is one of the best known combinations for the treatment of childhood acute lymphoblastic leukaemia. Tiazofurin (TF) and 6-thioguanine (TG) are also used as chemotherapy agents in the treatment of malignancies. We have examined the induction of apoptosis by combinations of these drugs to gain more insights into their efficacy in the treatment of malignancies. METHODS: The induction of apoptosis was examined in Molt-4, a human malignant acute lymphoblastic T-cell line. The cells were exposed to increasing drug concentrations at various exposure times. Annexin V/FITC and propidium iodide (PI) were used as markers for apoptosis and cell death. Annexin V/FITC positive and PI positive cells were detected by flow-cytometric analysis. RESULTS: Sequential 24-h exposure with MTX (0.005-0.02 micro mol) followed by 6MP (1-10 micro mol) and 24-h exposure with TF (5-20 micro mol) followed by TG (0.5-2 micro mol) showed a more than additive induction of apoptosis compared with single-drug exposure. Simultaneous administration of the drugs does not show an additive effect on apoptosis. CONCLUSIONS: The results of this study indicate that sequential administration of MTX before 6MP and of TF before TG may be essential for therapeutic success in the treatment of leukaemia.

Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children.

BACKGROUND: Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity. METHODS: We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate. RESULTS: The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found. CONCLUSION: The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

New renal scarring in children who at age 3 and 4 years had had normal scans with dimercaptosuccinic acid: follow up study.

OBJECTIVE: To determine up to what age children remain at risk of developing a new renal scar from a urinary tract infection. DESIGN: Follow up study. Families of children who had normal ultrasound scans and scanning with dimercaptosuccinic acid (DMSA) after referral with a urinary tract infection when aged 3 (209) or 4 (220) were invited to bring the children for repeat scans 2-11 years later. A history of infections since the original scan was obtained for children not having a repeat scan. SETTING: Teaching hospital. SUBJECTS: Children from three health districts in whom a normal scan had been obtained at age 3-4 years in 1985-1992 because of a urinary tract infection. MAIN OUTCOME MEASURE: Frequency of new renal scars in each age group. RESULTS: In each group, about 97% of children either had repeat scanning (over 80%) or were confidently believed by their general practitioner or parent not to have had another urinary infection. The rate of further infections since the original scan was similar in the 3 and 4 year old groups (48/176 (27%)) and 55/179 (31%)). Few children in either group known to have had further urinary infections did not have repeat scanning (3/209 (1.4%) and 4/220 (1.8%)). In the 3 year old group, 2.4% (5/209) had one or more new kidney scars at repeat scanning (one sided 95% confidence interval up to 5.0%), whereas none of the 4 year olds did (one sided 95% confidence interval up to 1.4%). The children who developed scars were all aged under 3.4 years when scanned originally. CONCLUSIONS: Children with a urinary tract infection but unscarred kidneys after the third birthday have about a 1 in 40 risk of developing a scar subsequently, but after the fourth birthday the risk is either very low or zero. Thus the need for urinary surveillance is much reduced in a large number of children.

Quantitative proteome profiling of respiratory virus-infected lung epithelial cells.

Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells. A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection. We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.