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To determine the effects of age, feeding regimen, and antenatal glucocorticoids on intestinal permeability, preterm infants (n = 132) were stratified by gestational age and by diet (mothers' own milk versus preterm formula), and assigned randomly to one of four feeding regimens: early-continuous, early-bolus, standard-continuous, and standard-bolus. At 10, 28, and 50 d of age permeability was determined by measuring the ratio of lactulose/ mannitol in the urine after the two sugars were administered enterally for 30 h. The mean (+/-SE) birth weight and gestational age of the infants were 1044 +/- 13 g and 27 +/- 0.1 wk, respectively. Permeability changed as a function of age (p = 0.003). Early feeding was associated with a reduction in permeability at 10 d of age (p = 0.01). Antenatal steroid administration was associated with decreased permeability at 28 d of age (p = 0.017). The feeding of human milk (versus formula) was associated with decreased permeability at 28 d of age (p = 0.02). Continuous versus bolus feeding did not affect permeability.

The differential urinary excretion of orally administered lactulose and mannitol is used to evaluate intestinal permeability. This test usually involves a 5- to 6-hr urine collection. We hypothesized that a shorter collection time would give an equivalent result. Forty-three patients with a variety of gastrointestinal symptoms and diagnoses (group 1) and 42 patients with Crohn's disease (group 2) had a standard lactulose/mannitol permeability test. The lactulose and mannitol urinary excretion was calculated using the first urine (group 1) or the 1-hr and 2-hr urine (group 2) and was compared to the values calculated from the routine 5- or 6-hr collection. Lactulose excretion kinetics, expressed as the percent of the total urinary excretion within a given time period, were as follows: 21% in first hour (group 2), 29% in second hour (group 2), and 46% in first 2.5 hr (group 1). Mannitol urinary excretion kinetics were 16%, 31%, and 44%, respectively. The lactulose/mannitol ratio based on a standard urine collection correlated well with the ratio based on just the first urine produced by the patient (R2 = 0.94; P < 0.001; group 1) and the 2-hr urine (R2 = 0.464; P < 0.001; group 2). Future use of the lactulose/mannitol ratio to assess intestinal permeability may be able to be simplified by shortening the urine collection time.

Retrovirus-mediated gene delivery into hepatocytes in vivo provides long-term gene expression, which is of great importance for treating most genetic and metabolic disorders. However, clinical application has not been realized because of the requirement for prior 70% partial hepatectomy or chemical (toxic) liver injury to initiate hepatocyte replication at the time of retroviral gene transduction. In this paper, we describe a novel gene delivery system that uses recombinant hepatocyte growth factor (rHGF) prior to retrovirus-mediated in vivo gene transfer in the liver without partial hepatectomy or liver injury. A single retroviral infusion through the portal vein following five systemic injections (via the tail vein) of 100 microg/kg rHGF resulted in a 10.4% 5-bromo-2'-deoxyuridine (BrdU) labeling index (BLI) and 0.14% retroviral gene transduction efficiency (RGTE) in hepatocytes, which were 6.3- and 12.9-fold higher than those of controls, respectively. Modest additional increases in BLI and RGTE (13.4% and 0.22%, respectively) were seen after five systemic injections of 500 microg/kg rHGF. The correlation between BLI and RGTE was statistically confirmed regardless of treatment. When rats received multiple retroviral infusions through a cannulated portal vein following five portal injections of 100 microg/kg rHGF, RGTE was dramatically increased (1.3%) and in some areas of the liver exceeded more than 10%. There was no evidence of liver injury in any animal. This approach has great potential for clinical application in terms of avoiding invasive procedures or liver injury.

Background: Little data exist on the requirements of trace metals and minerals for endurance athletes. Changes in body status of these elements must be examined before specific nutritional recommendations can be made. This study was designed to determine whether a marathon run was associated with changes in serum and urine metal and mineral concentrations., Methods: Forty subjects who planned to complete the 1996 Houston-Tennaco marathon were recruited. Subjects had blood and urine samples collected 2 weeks prior to the race and immediately following the race. Blood and urine specimens were analyzed for copper, iron, magnesium and zinc concentrations. Blood was also analyzed for calcium concentration and ceruloplasmin activity., Results: Twenty-six subjects (24 male, 2 female) completed the marathon. Finish times varied between 2 hours 43 minutes and 5 hours 28 minutes. There was no significant change in serum calcium, copper or zinc concentrations or ceruloplasmin activity. Serum and urine magnesium concentration decreased significantly (19.55+/-1.73 to 16.55+/-1.53 ppm, p=0.00001; 34.02+/-8.64 to 21.80+/-12.24 ppm, p=0.003, respectively). Serum iron concentration increased significantly (1.06+/-0.48 to 1.35+/-0.42 ppm, p=0.006), while urine copper and iron concentrations were below the limits of detection, zinc concentration did not change., Conclusions: Serum and urinary magnesium concentrations decrease during endurance running, consistent with the possibility of magnesium deficiency. This may be related to increased demand in skeletal muscle. Serum iron concentration increases, possibly related to tissue injury. The exact etiology for these observations, as well as their clinical significance, requires further investigation.

Previously, this lab has reported the use of hepatocyte transplantation and in vivo gene therapy for the correction of a mouse model of Hereditary Tyrosinemia Type I (HT1). Here, we demonstrate repopulation of fumarylacetoacetate hydrolase (FAH)-deficient livers with cultured hepatocytes. Correction of the disease phenotype was achieved by retrovirally transducing cultured FAH- hepatocytes ex vivo, followed by transplantation and selective repopulation. Treated mice were phenotypically normal and had corrected plasma amino acid levels and liver function tests. Our results demonstrate that efficient hepatic repopulation using ex vivo genetically manipulated hepatocytes is feasible.

Previous work has shown that adult mouse hepatocytes can divide at least 18 times in vivo. To test whether this represents the upper limit of their regenerative capacity, we performed serial transplantation of hepatocytes in the fumarylacetoacetate hydrolase deficiency murine model of liver repopulation. Hepatocytes from adult donors were serially transplanted in limiting numbers six times and resulted in complete repopulation during each cycle. This corresponds to a minimal number of 69 cell doublings or a 7.3 x 10(20)-fold expansion. No evidence for abnormal liver function or altered hepatic architecture was found in repopulated animals. We conclude that a fraction of adult mouse hepatocytes have growth potential similar to that of hematopoietic stem cells.

This report describes a unique cation exchange high-performance liquidchromatography capable of separating more than 40 frequently encountered human hemoglobins and variants within 12 min. Some of these variants are unresolvable by the conventional electrophoretic methods and would thus lead to an incorrect diagnosis of hemoglobinpathy. The method provides high sensitivity, superior resolution and accurate quantitation of hemoglobin concentrations. It can also be fully automated thus make it an ideal methodology for the diagnosis of hemoglobin disorders in a routine clinical laboratory.

Glycerol kinase is an X chromosome-encoded enzyme involved in the metabolism of endogenous and dietary glycerolipids. The physiological significance of its activity in mammals is not well understood. Glycerol kinase deficiency in humans occurs as an isolated enzyme deficiency or as part of a contiguous gene deletion syndrome in variable association with Duchenne muscular dystrophy and adrenal hypoplasia congenita. Isolated glycerol kinase deficiency has an inconstant phenotype, ranging from asymptomatic hyperglycerolemia to a severe metabolic disorder with growth and psychomotor retardation. Although intragenic mutations were reported recently, the pathophysiological basis for the phenotypic variability remains unknown. To understand better the physiological significance of glycerol kinase and the pathophysiology of its deficiency, we generated glycerol kinase-deficient mice by gene targeting. Mutant male mice appear normal at birth, but exhibit postnatal growth retardation, altered fat metabolism with profound hyperglycerolemia and elevated free fatty acids, autonomous glucocorticoid synthesis and death by 3-4 days of age. Heterozygous females are healthy and biochemically normal. The biochemical features observed in glycerol kinase-deficient mice provide the basis for further investigations into the pathogenesis of the human disorder.

We have investigated the metabolism of leukotriene C4 (LTC4) in gamma-glutamyl transpeptidase (GGT)-deficient mice (Lieberman, M. W., Wiseman, A. L., Shi, Z-Z., Carter, B. Z., Barrios, R., Ou, C-N., Chevez-Barrios, P., Wang, Y., Habib, G. M., Goodman, J. C., Huang, S. L., Lebovitz, R. M., and Matzuk, M. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7923-7926) and have found substantial conversion of LTC4 to leukotriene D4 by high performance liquid chromatography and continuous flow fast atom bombardment-tandem mass spectrometric analyses. LTC4-converting activity has a tissue distribution different from GGT with highest activity in spleen followed by small intestine, kidney, and pancreas and lower activity in liver and lung. The activity is membrane-bound and is inhibited by acivicin, a known inhibitor of GGT. The enzyme was partially purified from the small intestine of GGT-deficient mice by papain treatment and gel filtration chromatography. The partially purified fragment released by papain has an apparent molecular mass of 65-70 kDa and the same substrate specificity as the tissue homogenate. In addition to LTC4, S-decyl-GSH is also cleaved. GSH itself, oxidized GSH, and the synthetic substrates used to analyze GGT activity (gamma-glutamyl-p-nitroanilide and gamma-glutamyl-4-methoxy-2-naphthylamide) are not substrates for this newly discovered enzyme. These data demonstrate that in addition to GGT at least one other enzyme cleaves LTC4 in mice. To reflect this enzyme's preferred substrate, we suggest that it be named gamma-glutamyl leukotrienase.

Mice lacking the enzyme fumarylacetoacetate hydrolase (FAH) have symptoms similar to humans with the disease hereditary tyrosinemia type I (HT1). FAH-deficient mice were injected with a first-generation adenoviral vector expressing the human FAH gene and followed for up to 9 months. Nontreated FAH mutant control mice died within 6 weeks from fulminant liver failure, whereas FAH adenovirus-infected animals survived until sacrifice at 2-9 months. Nine of 13 virus-treated animals developed hepatocellular cancer. Immunohistochemical analysis revealed a mosaic of FAH-deficient and FAH-positive cells in all animals and liver function tests were improved compared to controls. Even mice harvested 9 months after viral infection had > 50% FAH-positive cells. These results demonstrate the strong selective advantage of FAH-expressing cells in an FAH-deficient liver but also illustrate the danger of carcinomas arising from FAH-deficient hepatocytes in HT1.

The developing rat is hypersensitive to aminoglycoside ototoxicity during the period of anatomical and functional development of the cochlea. Toxicity is expressed only after a few days of treatment when kanamycin is given during the most sensitive period for production of ototoxicity (postnatal days 11-20). In contrast, when the drug is administered after the 20th postnatal day, the same dose and duration of treatment do not produce an ototoxic effect. Only after prolonged treatment (e.g., > or = 20 days) is there an observed effect. We characterized the pharmacokinetics of kanamycin in the serum of 12- and 25-day-old rats and observed a greater than 2.5-fold increase in elimination half-life in the 12- versus 25-day-old rat. The longer duration half-life of kanamycin in younger rats may explain the hypersensitivity of immature mammals to aminoglycoside ototoxicity.

Background: A non-invasive marker is needed to identify patients with significant gastrointestinal injury due to non-steroidal anti-inflammatory drugs. Gastrointestinal permeability to sucrose has been suggested as such a test., Aims: To assess the utility of sucrose permeability as a marker of gastroduodenal mucosal injury after single and multiple doses of aspirin, to identify the site of increased sucrose permeability, to explore the relation between sucrose permeability and endoscopic findings, and to evaluate whether Helicobacter pylori infection influenced gastroduodenal sucrose permeability., Methods: After a fasting urine was obtained, 500 ml of a solution containing 100 g of sucrose was ingested. Urine was collected for five hours and assayed for sucrose by high performance liquid chromatography. Sucrose permeability was also assessed 20 minutes after ingestion of 650 mg of aspirin and eight to 12 hours after a 72 hour course of 650 mg aspirin four times a day. The site of increased permeability was identified after pyloric occlusion with a double balloon tube., Results: Thirty seven healthy volunteers participated. Sucrose permeability (mean (SEM)) increased after both single (195.2 (27) mg and multiple (196.4 (31) mg) doses of aspirin compared with baseline (53.7 (10) mg; p < 0.0005). Balloon pyloric occlusion confirmed that the site of increased sucrose permeability was the stomach. The effect of aspirin on sucrose permeability was similar in those with and without H pylori infection., Conclusion: These results confirm the use of sucrose permeability as a marker of aspirin induced gastroduodenal mucosal injury and identify the stomach as the major site of increased permeability. H pylori infection does not seem to change gastric mucosal sucrose permeability either at baseline or after ingestion of aspirin.

gamma-Glutamyl transpeptidase (GGT) is an ectoenzyme that catalyzes the first step in the cleavage of glutathione (GSH) and plays an essential role in the metabolism of GSH and GSH conjugates of carcinogens, toxins, and eicosanoids. To learn more about the role of GGT in metabolism in vivo, we used embryonic stem cell technology to generate GGT-deficient (GGTm1/GGTm1) mice. GGT-deficient mice appear normal at birth but grow slowly and by 6 weeks are about half the weight of wild-type mice. They are sexually immature, develop cataracts, and have coats with a gray cast. Most die between 10 and 18 weeks. Plasma and urine GSH levels in the GGTm1/GGTm1 mice are elevated 6-fold and 2500-fold, respectively, compared with wild-type mice. Tissue GSH levels are markedly reduced in eye, liver, and pancreas. Plasma cyst(e)ine levels in GGTm1/GGTm1 mice are reduced to approximately 20% of wild-type mice. Oral administration of N-acetylcysteine to GGTm1/GGTm1 mice results in normal growth rates and partially restores the normal agouti coat color. These findings demonstrate the importance of GGT and the gamma-glutamyl cycle in cysteine and GSH homeostasis.

Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.

Hereditary tyrosinaemia type I, a severe autosomal recessive metabolic disease, affects the liver and kidneys and is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Mice homozygous for a FAH gene disruption have a neonatal lethal phenotype caused by liver dysfunction and do not represent an adequate model of the human disease. Here we demonstrate that treatment of affected animals with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3-cyclohexanedione abolished neonatal lethality, corrected liver function and partially normalized the altered expression pattern of hepatic mRNAs. The prolonged lifespan of affected animals resulted in a phenotype analogous to human tyrosinaemia type I including hepatocellular carcinoma. The adult FAH-/- mouse will serve as useful model for studies of the pathophysiology and treatment of hereditary tyrosinaemia type I as well as hepatic cancer.

Background/aims: The pathogenesis of the diarrhea in acquired immunodeficiency syndrome (AIDS)-related cryptosporidiosis is not known. The hypothesis of this study was that the intestinal dysfunction and injury are related to the number of organisms infecting the intestinal mucosa. The aim of this study was to study the influence of intensity of infection on intestinal function and injury in AIDS-related cryptosporidiosis., Methods: In 16 patients with AIDS with intestinal Cryptosporidium infection, the intensity of infection was quantified by counting the total number of fecal oocysts excreted in 24 hours and by determining the percent of duodenal epithelium covered by organisms. Intestinal function was assessed by vitamin B12 absorption and serum D-xylose test. Intestinal injury was assessed by morphology of duodenal mucosa, differential urinary excretion of lactulose and mannitol, and fecal alpha 1-antitrypsin clearance. Measurements were repeated after treatment with paromomycin., Results: Vitamin B12 and D-xylose absorption negatively correlated with intensity of infection. Villus atrophy occurred only in patients with oocyst excretion of > 10(8) oocyst/24 hours. Lactulose/mannitol urinary excretion ratio showed a positive correlation with intensity of infection. Intestinal function and injury improved in patients whose oocyst counts were reduced by treatment with paromomycin., Conclusions: Cryptosporidium infection in patients with AIDS causes malabsorption and intestinal injury in proportion to the number of organisms infecting the intestine.

Objective: Our aim was to investigate whether the human placenta is permeable to sulindac or its active sulfide metabolite., Study Design: Nine pregnant patients (median gestational age [range]: 31.8 [24.3 to 36.4] weeks) were given a 200 mg oral dose of sulindac 5.5 (4.4 to 6.7) hours before 18 intravascular transfusions for rhesus or Kell alloimmunization. At each procedure maternal and fetal serum levels of sulindac and the active sulfide metabolite were measured by high-performance liquid chromatography., Results: The maternal sulfide level was significantly higher than the fetal sulfide levels, but no significant difference was noted between maternal and fetal sulindac levels. The sulfide fetal/maternal ratio was significantly lower than the sulindac fetal/maternal ratio. The sulfide/sulindac ratio was significantly higher in maternal serum versus fetal serum. The sulfide/sulindac ratio correlated with time from drug ingestion to sampling on the maternal side only. In patients studied on more than one occasion no consistent relationship between fetal sulindac, fetal sulfide, or fetal sulfide/sulindac ratio, and gestational age could be demonstrated., Conclusion: The placenta is permeable to sulindac but less so to its active sulfide metabolite. The reduction of sulindac to its active sulfide metabolite is decreased in the human fetus.

Objective: To determine in vitro the compatibility of reconstituted intravenous immunoglobulin (IVIG) (Gammagard, Baxter-Hyland) with five different neonatal and pediatric intravenous solutions in Viaflex polyvinyl chloride bags., Design: In vitro compatibility study., Interventions: Samples were taken at time = 0, 10, 30, 60, 90, and 120 minutes and at 4, 8, 12, and 24 hours and assayed for total immunoglobulin G content and antibodies to hepatitis B surface antigen. Type III group B Streptococcus (GBS) and opsonic activity for type III GBS were analyzed at time = 0, 60, and 120 minutes and 12 and 24 hours. All results were compared with those from pure IVIG., Results: Our results demonstrate that mixing IVIG with intravenous solutions commonly used in the care of premature infants (dextrose 5% in water [D5W], D15W, D5W/NaCl 0.225%, and total parenteral nutrition [TPN]) does not significantly alter total immunoglobulin G concentrations or concentration of antibodies to hepatitis B surface antigen or type III GBS. As well, the in vitro functional activity for type III GBS of the IVIG, when mixed with these solutions for up to 24 hours, remained intact. An apparent decrease in bactericidal killing was seen with the IVIG/central TPN mixture. This apparent decrease was found to be an artifact of the high concentration of glucose (20 percent) in the solution., Conclusions: We propose that Gammagard may be mixed with these solutions through Y-site connections without loss of antibody content or functional activity of the IVIG.

Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals. We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.

Ten neonates with persistent staphylococcal bacteremia (positive blood cultures for > or = 5 days despite appropriate antibiotic therapy) received intravenous (i.v.) rifampin in combination with vancomycin with or without aminoglycoside. Their mean birth weight and length of gestation were 900 g and 27 weeks, respectively. Their ages at the time of infection ranged from 6 to 64 days (mean, 26 days). The staphylococcal isolates were methicillin-resistant Staphylococcus aureus (five isolates), methicillin-susceptible S. aureus (two isolates), and coagulase-negative staphylococci (three isolates). The mean number of bacteremia days prior to administration of i.v. rifampin was 8.3 (range, 5 to 15 days), despite a mean peak vancomycin concentration of 33 micrograms/ml. The dosing of rifampin varied from 2.5 to 10 mg/kg of body weight every 12 h. The mean duration of the rifampin course was 9.7 days (range, 3 to 16 days). Of the 10 neonates, 8 (80%) had sterile blood cultures within 24 h, 1 (10%) had a sterile blood culture within 48 h, and 1 (10%) had a sterile blood culture within 5 days of being placed on i.v. rifampin. No adverse effects were noted in this small group of infants. Seven of the 10 neonates survived; three died from unrelated complications. The MIC ranges of amikacin, vancomycin, and rifampin for the isolates were 2.0 to 16, 0.5 to 2.0, and 0.0013 to 0.04 micrograms/ml, respectively. We also studied eight infants, with a mean age of 23 days, who were receiving i.v. or oral rifampin at a dose of 10 mg/kg/day. For i.v. administration, the peak serum concentration of rifampin (mean +/- standard deviation) was 4.02 +/- 1.22 microgram/ml. The mean trough level at 12 h postifution was 1.11 +/- 0.48 micrograms/ml. For oral administration, the concentrations of rifampin in serum ranged from 0.59 to 2.86 micrograms/ml (mean, 1.86 +/- 0.96 microgram/ml) at 2 h postingestion, increasing to a peak concentration of 2.8 micrograms/ml at 8 h postingestion. The mean 12-h postingestion level was 0.77 +/- 0.03 microgram/ml. From the study of this limited series of neonates, rifampin appears to be a safe and effective addition to therapy when staphylococcal bacteremia is persistent despite vancomycin treatment.

Objective: To determine the effects of gestational age and fetal serum indomethacin levels on constriction of the ductus arteriosus after maternal indomethacin administration., Methods: Twenty-five pregnant Rh-sensitized patients were given a 50-mg oral dose of indomethacin 6 hours before fetal serum indomethacin levels were determined at the time of 50 diagnostic or therapeutic funipunctures. The ductus arteriosus was evaluated with Doppler ultrasound immediately before 40 of the procedures. Constriction of the ductus arteriosus was defined as a peak diastolic flow greater than 35 cm/second. Least-squares regression and multiple regression were used for statistical analysis., Results: The peak diastolic velocity of the fetal ductus arteriosus after maternal indomethacin ingestion was constant at 25 cm/second before 27 weeks, increased between 27-30 weeks to a mean of 39 cm/second, and was stable thereafter (R2 = 0.35; P < .05). There was no significant correlation between constriction of the ductus and fetal serum indomethacin levels (P = .17)., Conclusions: The constrictive effect of maternal indomethacin ingestion on the fetal ductus arteriosus begins as early as 27 weeks' gestation. Constriction of the ductus arteriosus is independent of fetal serum indomethacin levels.

The short- and long-term effects of simultaneous administration of terbutaline and betamethasone were investigated in 8 gravidas treated for preterm labor. Their plasma concentrations of glucose, insulin, glucagon, C-peptide, lactate and potassium were compared to a control group receiving intravenous magnesium sulfate and betamethasone. The patients on terbutaline therapy had a marked hyperglycemia at 11 h which remained elevated for 48 h. There was a simultaneous rise in plasma insulin and C-peptide, and a fall in plasma glucagon. Lactate levels were markedly elevated. Only 1 of the 8 patients had an abnormal glucose tolerance test at 1 week of therapy. The metabolic changes of control patients were minimal in comparison and there was no lacticacidemia. This suggests that glucocorticoids potentiate the hyperglycemic response of terbutaline.

We investigated the use of a 3.5 x 0.46 cm HPLC column packed with 5-microns particles of porous (100 nm) silica coated with polyaspartic acid for hemoglobin analysis. A 13-min gradient was produced between two mobile phases. The method is capable of separating more than 35 commonly encountered hemoglobin variants within 12 min. Hemoglobin variants identified include Bart's, acetyl F, H, A1c, F, Camden, N-Baltimore, J-Baltimore, N-Seattle, Grady, Fannin-Lubbock, A G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, Richmond, D-Los Angeles, Deer Lodge, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, GS hybrid, C-Harlem, O-Arab, British Columbia, and C. Between-run precision of an in-house pooled hemoglobin control material, AFSCA2, gave CVs of 2-5% for the A, F, S, and C and 8% for the A2 over a 6-month period. The simplicity of sample preparation, high resolution of the system, and high accuracy of the method, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.

Five families of transgenic mice were derived from one-cell-stage embryos injected with gamma GT-rasT24, a fusion gene consisting of the gamma-glutamyl transpeptidase (gamma GT) 5' flanking region containing promoter I linked to a mutated (codon 12) human H-ras oncogene. The transgene was expressed selectively in the kidneys, eyes, and brains of all families as determined by reverse transcription-polymerase chain reaction, nuclease protection assays, and in situ hybridization. In two of five families, kidney lesions consisting of proximal tubular hyperplasia, renal cysts, and microadenomas developed in male animals; males also expressed higher levels of gamma GT/rasT24 RNA. Early lesions consisted of proximal tubular hyperplasia as defined by alkaline phosphatase histochemistry, gamma GT immunohistochemistry, and electron microscopy and could be correlated with the presence of rasT24 RNA within the cystic proximal tubular epithelium by in situ hybridization. Advanced lesions also involved other segments of the nephron and consisted of cysts lined by a flattened unicellular layer of attenuated epithelium. No rasT24 could be identified within cystic lesions of the distal nephron and collecting tubules by in situ hybridization, and they most likely arise by external compression. Animals from the two transgenic strains exhibiting cystic lesions die of renal failure beginning at 8 months of age. No difference in cell-cycle parameters or DNA ploidy between transgenic and control kidneys was identified by flow cytometric analysis. No renal carcinomas developed. The primary renal effects of the H-rasT24 oncogene in this model system consist of proximal tubular hyperplasia and polycystic kidneys. This model appears to provide a useful in vivo system for the study of ras oncogene function and control of renal cell proliferation.

We examined the relationships between 4 glycated protein assays and home blood glucose monitoring (HBGM) in 26 children with poorly-controlled insulin-dependent diabetes mellitus (IDDM) during a period of improved management. At 2 week intervals for 6 visits (12 weeks in total), HBGM records were collected and a blood sample was obtained for measurement of glycated proteins and glucose. Assays included glycated hemoglobin (GHb) and glycated serum proteins (GP) by boronate affinity chromatography, hemoglobin A1C by PolyCAT A high performance liquid chromatography (HAC) and fructosamine (FA). All 4 glycated protein levels declined significantly over the 12 week period. Significant correlations between the glycated proteins and HBGM were observed over 2 week intervals. None of the 4 assays were affected by the glucose level in the sample. Changes in mean HBGM readings over 2 week intervals were correlated with both FA and GP with wide prediction intervals. Over cumulative 2 week intervals, which may more accurately reflect longitudinal trends, all 4 glycated proteins were correlated with mean HBGM readings. At each cumulative interval, GHb and GP showed the largest variation with MBG, while FA showed the least variation with MBG. Our data indicate that of the 4 assays tested, FA has limited clinical values as compared to other glycated protein assays, whereas assays based on boronate affinity chromatography (GHb and GP) provide the most useful clinical indicators of short-term changes in glycemic control. The clinical utility of a new HPLC method for determination of glycated hemoglobins is also demonstrated.

We have developed a stable and simple normal-phase liquid-chromatographic method for simultaneously measuring amiodarone and its metabolite, N-desethylamiodarone, within 8 min. The chromatographic system consists of a 15 cm x 3.9 mm Waters "Resolve" silica column and a mobile phase of ammonium sulfate (17 mmol/L, pH 6.8) and methanol (8/92 by vol), pumped at 1.8 mL/min and monitored at 254 nm. After 250 microL of serum is mixed with 100 microL of 0.36 mol/L NaH2PO4, 100 microL of the internal standard solution (L8040, 6 mg/L), and 200 microL of isopropyl ether, the mixture is vortex-mixed and centrifuged. Fifty microliters of the organic layer is injected onto the column. Relative recovery was 100% over the assay range of 0.1 to 20.0 mg/L for both compounds. Within-run and total (day-to-day) CVs were 3% and 7% for amiodarone and 5% and 8% for N-desethylamiodarone, respectively.

Little is known about the placental transfer of indomethacin in the human pregnancy. Twenty-six pregnant patients (gestational age, 29.4 +/- 0.5 weeks) were given a 50 mg oral dose of indomethacin 6.08 +/- 0.07 hours before 42 cordocenteses undertaken for standard indications. Maternal serum, fetal serum, and amniotic fluid levels were measured at the time of each procedure. Maternal indomethacin levels were not significantly different from corresponding fetal levels (218 +/- 21 vs 219 +/- 13 ng/ml). The maternal/fetal serum ratio (0.97 +/- 0.07) was not found to vary with gestational age (R = -0.07, p = 0.66). Fetal serum levels were significantly higher than corresponding amniotic fluid levels (219 +/- 16 vs. 21 +/- 2 ng/ml; p less than 0.001). The fetal/amniotic fluid ratio (10.0 +/- 1.2) did not vary with gestational age (R = 0.33, p = 0.11). Indomethacin crosses the human placenta easily throughout gestation; only small amounts of the unchanged drug are found in the amniotic fluid.

The limitations of current methods of measuring bilirubin are well established and relate to the broad dynamic range and inability of the technique to determine different but structurally similar bilirubin species. New instrumentation and methodology circumvent these limitations, and clinical studies are beginning to reveal their increased diagnostic usefulness. Nevertheless, in several clinical situations, for example, the prediction of kernicterus, better bilirubin determinations may not eliminate the controversy surrounding appropriate therapeutic interventions.

"High-performance" liquid chromatography (HPLC), applied to hemoglobin analysis, is decidely more sensitive and gives better resolution than do routine electrophoretic methods. Here we present a case with a rare double heterozygote hemoglobin S/LeporeBoston, originally diagnosed as homozygous hemoglobin S by routine electrophoretic methods. Using a gradient elution weak cation-exchange HPLC technique, we could separate hemoglobin S and hemoglobin LeporeBoston and make the correct diagnosis. This case demonstrates how HPLC can be a useful adjunct to routine electrophoresis.

The photobinding of [3H]8MOP to tRNA upon irradiation at 365 nm in the absence of O2 was determined by gel filtration. The maximum photobinding was found to be ca. 4 mol of 8MOP er mol of tRNA and 5FU-tRNA, with an overall quantum yield of 2.3 X 10(-3). The photobinding kinetics for 8MOP-tRNA showed an apparent induction period or sigmoidal kinetic curve, indicating a specific initial photobinding site on tRNA which was identified as 4-thiouridine at position 8 from the 5'-end of Escherichia coli tRNA. Photobinding of 8MOP to 5FU-tRNA proceeded without an apparent induction period. 8MOP-tRNA and 8MOP-5FU-tRNA adducts were characterized by absorption, fluorescence, and CD spectroscopy. A modified procedure was also developed to analyze the nucleoside composition in modified 8MOP-tRNA and 8MOP-5FU-tRNA. The results showed that 8MOP photochemically added mainly to pyrimidine bases. The photobinding of 8MOP changed the conformation (secondary in particular) of tRNA and inhibited aminoacyl-tRNA synthetase activity.

An optimized method for application of the EMIT reagent system to a COBAS-BIO centrifugal analyzer was developed to minimize reagent consumption while maintaining good precision and accuracy. Forty-one serum samples, previously analyzed for theophylline by HPLC, were assayed using the modified protocol on the COBAS-BIO centrifugal analyzer. Regression analysis of the results obtained from both methods yielded the following equation: y = 1.10x + 0.01, r = 0.964 (x = HPLC, y = COBAS-BIO centrifugal analyzer, EMIT). The COBAS-BIO centrifugal analyzer and EMIT methodology provided an efficient, inexpensive, and relatively effortless approach to drug monitoring in the routine clinical laboratory.

High-performance liquid chromatography is a technic that has recently been applied to the diagnosis of hemoglobinopathies and thalassemias. Its advantages over other methods include increased sensitivity, resolution and simplicity, as well as speed. In this report, the authors present an HPLC procedure that uses a weak cation exchange column and a gradient elution system for the diagnosis of hemoglobinopathies and thalassemias. The authors illustrate the utility of this procedure by reporting three cases in which the technic enabled them to make the correct diagnosis, which by traditional methods would have been missed or equivocal.

We evaluated the Du Pont Particle-Enhanced Turbidimetric Inhibition Immunoassay (PETINIA) for theophylline. The imprecision (CV) of the assay was less than 4.7% between-run and less than 3.6% within-run for theophylline concentrations between 5 and 30 mg/L. Standard curves for the assay were linear for theophylline concentrations from 0 to 46 mg/L and were stable throughout the study (i.e., for at least three months). The monoclonal antibody against theophylline used in this assay increases specificity; of the possibly interfering drugs, metabolites, and anticoagulants tested, only 1,3-dimethyluric acid and EDTA showed measurable effects. Bilirubin (less than 300 mg/L), hemoglobin (less than 6 g/L), or lipemia (triglycerides less than 6 g/L) does affect the quality of the assay. Analytical recovery of theophylline added to serum (5 to 40 mg/L) averaged 98% (range 93% to 112%). Comparison of results for patients' sera by the PETINIA method with those by enzyme immunoassay (EMIT) and by "high-performance" liquid chromatography yielded slopes and intercepts not significantly different from 1.0 and 0.0, respectively, and correlation coefficients ranging from 0.986 to 0.995.

We describe a single set of liquid-chromatographic conditions for assay of theophylline, dyphylline, caffeine, acetaminophen, salicylate, acetylsalicylate, procainamide, and N-acetylprocainamide in serum. The chromatographic system includes a Waters Associates mu-Bondapak C18 column and acetonitrile in 0.1 mol/L potassium phosphate buffer, pH 4.0 (9.75/90.25 by vol), as the mobile phase. Only 50 microL of serum is required, and drug concentrations as low as 0.5 mg/L can be detected. Absolute and relative analytical recoveries range from 95 to 101%. Day-to-day variation of the method is less than 6% for each drug. Linearity extends to 1 g/L for all drugs. Recycling of the mobile phase under pressure eliminates the need to prepare and de-gas solvents. The use of the single stationary and mobile phase provides a practical and economical approach to routine and urgent therapeutic drug monitoring.

An enzymatic method for the measurement of glycerol using glycerol kinase coupled to pyruvate kinase and lactate dehydrogenase was adapted to a COBAS-BIO centrifugal analyzer. Routine standardization is not required under the optimized conditions since the calculation factor derived from the measurement of pure glycerol standard was found to be identical to the theoretical value. Excellent correlation between the method and the DuPont ACA triglyceride method was obtained. The coefficients of variation for within-run and day-to-day precision were less than 4%. Only 2 microL of serum is required, making this an excellent means for monitoring therapeutic glycerol administration in premature infants with hydrocephalus. The method has the sensitivity to measure glycerol concentrations as low as 0.1 mmol/L. This, together with the rapid analysis time (28 specimens in 10 min), makes it suitable for the correction of glycerol interference in the measurement of serum triglyceride.

We used an enzymatic method for measuring total bilirubin in serum. Results by this method varied linearly with bilirubin concentrations to at least 300 mg/L. The day-to-day precision (CV) of the method ranged from less than 1% to about 11% at bilirubin concentrations of 183 and 12 mg/L, respectively. Commonly used anticoagulants, serum preparation materials, and selected drugs had no effect on the apparent bilirubin concentration, but turbidity caused a slight increase and hemoglobin concentrations of 2 g/L resulted in lower values, by as much as 17 mg/L at a bilirubin concentration of 95 mg/L. Patients' results obtained with this enzymatic method were slightly lower than those obtained with methods based on the Jendrassik-Grof principle. The largest differences, seen in samples with high "direct" bilirubin concentrations, can be decreased by measuring the absorbance at 425 nm instead of at 465 nm as recommended by the supplier of the bilirubin oxidase method.

A simple, isocratic liquid-chromatographic method was developed for simultaneously measuring ethosuximide, primidone, phenobarbital, phenytoin, carbamazepine, and their bioactive metabolites within 10 min. The chromatographic system involves a Waters' Radial-NOVA PAK C18 reversed-phase column and acetone/methanol/acetonitrile/10 mmol/L phosphate buffer (10/21/8/61 by vol, pH adjusted to 7.95 with NaOH) as mobile phase. The antiepileptic drugs are extracted from 50 microL of serum by mixing with 50 microL of acetonitrile containing 10 mg of tolybarb per liter as internal standard. After centrifugation, 20 microL of the supernate is injected onto the column and eluted with mobile phase at the rate of 2.8 mL/min at ambient temperature. The column effluent is monitored at 200 nm. The method can detect the five antiepileptic drugs in concentrations as low as 0.5 mg/L. Analytical recovery ranges from 98 to 102%. Within-run CV ranged from 2.9 to 5.8% and between-run CV from 4.7 to 7.1%. The method can also be used to measure N-desmethyl-methsuximide, chloramphenicol, and pentobarbital.

Unconjugated bilirubin was determined in 458 serum samples from 160 neonatal and pediatric patients by using two approaches. The first approach was to calculate indirect bilirubin by subtracting direct bilirubin, as measured by a diazo solution assay, from total bilirubin quantified by a Jendrassik-Grof procedure. The second approach was actually to measure the unconjugated bilirubin fraction using a multilayered slide. For samples containing little or no conjugated bilirubin, correlation between the calculated indirect bilirubin value and the unconjugated bilirubin measured by the slide was judged acceptable. Samples containing increased levels of conjugated bilirubin, however, yielded discrepancies between the approaches including differences of up to 5-10 mg/dL and more. Moreover, the magnitudes of the differences correlated with the amounts of conjugated bilirubin present in the samples. The unconjugated bilirubin values given by the slide were found to correlate more closely than the calculated indirect values with results obtained by an HPLC procedure that may be regarded as the most reliable method available. Therefore, we find that the slide provides a more accurate measure of unconjugated bilirubin than does the indirect bilirubin value in specimens from pediatric patients having evidence of cholestasis. The authors also believe that the magnitude of the difference in the values could make a difference in therapeutic strategies.

We describe a convenient, economical procedure for measuring uroporphyrinogen I synthase (EC 4.3 1.8) activity in erythrocytes, the results of which can be used to diagnose acute intermittent porphyria, in either its latent or acute stage. By limiting the test reaction sequence to the conversion of porphobilinogen to porphyrins, we eliminated several disadvantages of alternative methods in which delta-aminolevulinate is used as substrate. The latter assay was inhibited by lead; our procedure was not. Our procedure also gave greater porphyrin synthesis with less substrate. Erythrocytes of healthy women presented a mean activity of 12.4 nmol of porphyrin formed per liter per second; erythrocytes of healthy men presented a mean activity of 11.0 nmol/L per second. The within-run coefficient of variation (CV) for our assay was 1.8%; the between-run CV was 3.1%.

Prostaglandin E1 (PGE1) administration is a useful therapeutic measure for short-term maintenance of ductal patency in patients with obstructions to pulmonary or systemic blood flow. Such treatment is not without complications, however, and a report of three infants from our institution with abnormalities of the pulmonary microvasculature after varying periods of PGE1 therapy was recently published (Heffelfinger et al., Pediatr Pathol 1987;7:165-73). The vascular abnormalities appeared to be temporally related to the PGE1 administration. To test this hypothesis, we investigated the effects of PGE1 in newborn beagles by infusing PGE1 for periods of up to 21 days in four experimental pups. Two control pups were infused with saline for the same period of time. Five of the animals developed respiratory infection during the course of the infusions. One PGE1-treated pup was not infected. Both the PGE1- and saline-treated pups had bronchopneumonias of similar severity; however, pulmonary arteritis occurred only in the PGE1-treated pups. The severity of the arteritis varied with the amount of pulmonary parenchymal inflammation and not with the duration of PGE1 administration. Inflammatory and vascular lesions were found in organs other than the lung only in two pups receiving longer courses of PGE1 treatment. We conclude that systemic PGE1 infusion at therapeutic levels plays a role in the development of arterial lesions in small muscular arteries and that this is potentiated by the presence of infection.

Conjugated and direct bilirubin were measured in 288 samples from 107 neonates less than 15 days old. Retrospective analysis of the medical records showed that 53 neonates were hepatobiliary-normal, 42 patients had no obvious evidence of hepatobiliary disease but had received total parenteral nutrition, and 12 were clearly hepatobiliary-abnormal. Neither the mean values nor the distributions of either the conjugated bilirubin, as measured by a multilayered slide, or the direct bilirubin, measured by a solution diazo procedure, differed when comparing the hepatobiliary-normal population to that receiving total parenteral nutrition. However, as would be predicted, the hepatobiliary-abnormal population differed significantly from both of these groups using either direct bilirubin or conjugated bilirubin results. Samples obtained from hepatobiliary-abnormal neonates were reviewed in chronologic sequence if direct bilirubin results differed from conjugated bilirubin with respect to classification of normality/abnormality. In two of three neonates with developing cholestasis, conjugated bilirubin exceeded its upper limit of normal earlier than did direct bilirubin. Conjugated bilirubin returned to normal earlier than direct bilirubin for two neonates with an improving clinical status. Conjugated bilirubin measurement was judged to be more responsive to developing or resolving cholestasis then direct bilirubin. In addition, conjugated bilirubin measurement from the slide is known to agree well with that determined by high performance liquid chromatography fractionation of bilirubin and is less susceptible to interference than is direct bilirubin measurement, characteristics that recommend its use over direct bilirubin analysis in a clinical setting.

Cerebral and systemic metabolism of oxygen, glucose, lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, and amino acids were studied in 27 comatose patients during the first 7 days after a head injury. Systemic oxygen consumption was elevated initially and gradually returned toward normal over the week of study. In contrast, cerebral oxygen consumption was depressed and decreased further over the week of study. Cardiac output and cerebral blood flow were increased with respect to systemic and cerebral metabolic requirements, and remained elevated for the entire week of study. Systemic hyperglycemia and lactic acidosis were present. The injured brain often made a sizeable contribution to the lactic acidosis. The arterial concentrations of pyruvate, beta-hydroxybutyrate and acetoacetate were low. The early postinjury arterial amino acid profile was typically an increased level of alanine, taurine, glutamic acid, and a reduced concentration of valine, leucine, isoleucine, threonine, serine, ornithine, and arginine. At 3-4 days postinjury, as the early abnormalities were returning toward normal, glutamine, lysine, phenylalanine, tyrosine, and methionine became elevated. These late increases in amino acids occurred at the time of the peak in plasma catecholamine concentrations. The net cerebral flux of amino acids followed the same general pattern of evolution over time as did the arterial concentration of amino acids. On days when the availability of the individual amino acid was increased, the net cerebral flux tended to be positive; when the availability was decreased, the net cerebral flux was zero or there was a net efflux of the amino acid. There was a significant linear relationship between the arterial concentration and the net flux of 13 of the 17 amino acids studied. Unlike the fasting state in normal man, in which beta-hydroxybutyrate and acetoacetate are important metabolic substrates, cerebral metabolism after head injury is almost totally dependent upon the aerobic and anaerobic metabolism of glucose. This is at least in part due to injury-induced limitations in the variety of substrates that are available for the brain to extract.

Using a Syva EMIT Lab 5000 System, we evaluated a newly developed EMIT assay for measurement of caffeine in serum of neonates. The method is rapid, and coefficients of variation for within-run and day-to-day precision were less than 5%. The accuracy of the method, as evaluated from analytical recovery and a calibrator-crossover study, is excellent. Comparison of the results analyzed by the EMIT caffeine assay (y) and the liquid-chromatographic comparison method (x) yielded the linear regression equation y = 1. 052x + 0.293 (n = 110, r = 0.987, and Sy . x = 0.625). The method was also adapted to a COBAS-BIO centrifugal analyzer (y) and optimized to minimize sample and reagent consumption. Linear regression analysis yielded the equation y = 1. 031x + 0.227 (n = 61, r = 0.992, and Sy . x = 0.488). As many as 400 assays per EMIT kit can be performed with the analyzer method, with excellent precision and accuracy. The kit assay and this centrifugal analyzer are well suited for rapid batch testing: 28 analyses (including six calibrators) in 10 min.