Altered glucose metabolism and proteolysis in pancreatic cancer cell conditioned myoblasts: searching for a gene expression pattern with a microarray analysis of 5000 skeletal muscle genes.

BACKGROUND AND AIMS: We verified whether conditioned media (CM) from pancreatic cancer cell lines (MIAPaCa2, CAPAN-1, PANC-1, BxPC3) alter glucose metabolism and gene expression profiles (microarray experiment with a platform of 5000 skeletal muscle cDNA) in mice myoblasts. METHODS: Myoblasts were incubated with control or pancreatic cancer CM for 24 and 48 hours. RESULTS: Lactate significantly increased in CM compared with non-conditioned myoblasts. No variations in expression levels of the main genes involved in glycolysis were found in CM myoblasts. Propionyl coenzyme A carboxylase and isocitrate dehydrogenase 3 beta genes, which encode enzymes of the tricarboxylic acid cycle, were overexpressed, while IGFIIR and VAMP5 genes were underexpressed in CM myoblasts. PAFAH1B1 and BCL-2 genes (intracellular signal transduction) and the serine protease cathepsin G (proteolysis), were overexpressed in CM myoblasts. Tyrosine accumulation in CM myoblasts suggested that proteolysis overcomes protein synthesis. Sorcin, actin alpha, troponin T1, and filamin A were underexpressed in CM myoblasts. CONCLUSIONS: Our findings demonstrate that pancreatic cancer cell conditioned media enhanced lactate production and induced proteolysis, possibly by altering expression levels of a large number of genes, not only those involved in protein biosynthesis and degradation or glucose metabolism, but also those involved in the tricarboxylic acid cycle and in vesicle traffic.

Mixed exhaled nitric oxide and plasma nitrites and nitrates in newborn infants.

Plasma nitrite (NO2-) and nitrate (NO3-) are the stable end-products of endogenous nitric oxide (NO) metabolism. NO is present in the exhaled air of humans, but it is not clear if exhaled NO may be an indicator of the systemic endogenous NO production. The aims of the study were to determine the levels of exhaled NO and plasma NO2-/NO3- in healthy term and preterm newborns, and to assess if exhaled NO correlates with plasma NO2-/NO3- at birth. After the stabilization of the newborn, we measured by chemiluminescence the concentration of NO in the mixed expired breath of 133 healthy newborns. Measurement of exhaled NO was repeated after 24 and 48 hours. Plasma NO2-/NO3- levels at birth were measured by the Griess reaction. NO concentrations were 8.9 (CI 8.1-9.8) parts per billion (ppb), 7.7 (CI 7.2-8.3) ppb and 9.0 (CI 8.4-9.6) ppb at birth, 24 and 48 hours, respectively. At birth, exhaled NO was inversely correlated with gestational age (p=0.008) and birth weight (p<0.001). Plasma NO2-/NO3- level was 27.30 (CI 24.26-30.34) micromol/L. There was no correlation between exhaled NO and plasma NO2-/NO3- levels at birth (p=0.88). We speculate that the inverse correlation between exhaled NO and gestational age and birth weight may reflect a role of NO in the postnatal adaptation of pulmonary circulation. At birth, exhaled NO does not correlate with plasma NO2-/NO3- and does not seem to be an index of the systemic endogenous NO production.

Rapid and simple determination of inulin in biological fluids by high-performance liquid chromatography with light-scattering detection.

We report a new high-performance liquid chromatography method developed for measuring inulin in plasma and urine using ion moderated partition chromatography and evaporative light-scattering detection. Samples are deproteinized with a zinc acetate and phosphotungstic acid solution and added with melezitose as an internal standard. The chromatographic separation is carried out in 16 min at a flow-rate of 0.6 ml/min using deionized water as the mobile phase. Within-run precision, measured at four different concentrations (0.050 mg/ml, 0.150 mg/ml, 0.300 mg/ml and 1.200 mg/ml), ranges from 1.7 to 3.4% in plasma and from 1.5 to 3.5% in urine. Similarly, between-run precision is in plasma from 2.0 to 4.3% and in urine from 2.0 to 4.4%. Analytical recovery ranges from 97.9 to 100.1% in plasma and from 99.1 to 99.7% in urine, respectively. Detection limit (signal-to-noise ratio=3) is 5 microg/ml both in plasma and urine. The method is simple, sensitive, without interference due to hexoses or drugs commonly taken by patients with renal diseases, and offers the advantage of measuring inulin without previous hydrolysis of the molecule.

Rapid determination of creatinine in serum and urine by ion-pair high-performance liquid chromatography.

We report a simple and reliable high performance liquid chromatography method for measuring creatinine in serum and urine. The chromatographic run is performed on a C(18) column after protein precipitation with acetone and addition of cimetidine as an internal standard. The separation is carried out in 20 min at a flow rate of 0.8 ml/min, with a mobile phase consisting of 100 mmol/l sodium dihydrogen phosphate solution, containing 30 mmol/l sodium lauryl sulfate pH 3.0 and acetonitrile (60:36, v/v). The absorbance is monitored at 200 nm. The relationship between creatinine concentration and the creatinine/internal standard peak area is linear up to 1,088 micromol/l. Within-run precision measured at three different creatinine concentrations ranges from 0.89% to 2.34% in serum and from 0.34% to 1.10% in urine. Between-run precision varies from 1.68% to 3.17% in serum and from 1.58% to 1.85% in urine over a wide range of concentrations. Analytical recovery is between 98.71% and 101.25% in serum and between 98.96% and 100.27% in urine. The detection limit is 3.24 micromol/l for a signal-to-noise ratio of 3. The method shows a good linearity with the reference isotope dilution gas chromatography-mass spectrometry procedure (r=0.999), without interferences, even in the presence of high bilirubin concentrations.

Simultaneous HPLC determination with light-scattering detection of lactulose and mannitol in studies of intestinal permeability in pediatrics.

We describe a new HPLC method for the simultaneous determination of lactulose and mannitol in urine, in which cation-exchange chromatography and evaporative light-scattering detection are used. The two sugars are orally administered for the estimation of intestinal permeability in children. Samples were purified by solid phase extraction on a C18 cartridge and subsequent addition of anion-exchange resin. Cellobiose may be used as an internal standard. The chromatographic separation was carried out in 16 min at a flow rate of 0.5 mL/min, using deionized water as the mobile phase. Within-run precision (CV) measured at three concentrations was 1.6-2.3% for lactulose and 1.0-1.9% for mannitol. Between-run CVs were 2.1-4.1% and 1.3-2.7% for lactulose and mannitol, respectively. Analytical recovery of both sugar probes was 97-101%. The detection limits (signal-to-noise ratio = 3) were 0.82 mg/L for lactulose and 0.65 mg/L for mannitol. The lactulose/ mannitol ratio in control subjects was 0.024 +/- 0.006; in patients with Crohn's and coeliac diseases in active phase, the ratios were 0.200 +/- 0.082 and 0.072 +/- 0.025, respectively. The method is rapid, simple, and sensitive, and suitable for determination of intestinal permeability in children.

Determination of urinary orotate excretion by high-performance liquid chromatography.

A new reversed-phase high-performance liquid chromatographic procedure for the determination of urinary orotate excretion is described. It is a selective, sensitive and rapid method, suitable for the differentiation of inherited metabolic diseases with abnormal orotate metabolism.

Cerebral amino acid levels and transport after portocaval shunt in the rat: effects of liver arterialization.

Altered plasmatic and cerebral amino acid patterns have been observed after portocaval shunt in the rat. Similar alterations have been found in plasma and in cerebrospinal fluid of cirrhotic patients and are likely to play an important role in the pathogenesis of hepatic encephalopathy. Impaired liver blood flow could contribute to these biochemical abnormalities. Therefore we wondered whether liver arterialization, by improving liver perfusion, could have any beneficial effects on the altered amino acid levels occurring in the rat after portocaval shunt. Amino acid concentrations were determined in four cerebral regions and in the plasma of shunted rats with or without liver arterialization, 4 weeks after surgery. Blood-brain barrier transport was studied with the Oldendorf's technique. After portocaval shunt, we observed lower plasma levels of the branched chain amino acids valine, isoleucine, leucine, and net higher levels of the aromatic tyrosine and phenylalanine and of glutamine. In the cerebral regions, we observed a slight increase of branched chain amino acids and an enormous increase of tyrosine, phenylalanine, tryptophan, histidine, and glutamine. Arterialization of the liver made no difference to the postportocaval shunt plasma levels of branched chain amino acids, while it almost normalized those of aromatics. In the cerebral regions, we observed a marked improvement in the level of tyrosine, phenylalanine, tryptophan, and histidine. The enhancement of blood-brain barrier transport for the neutral amino acid class, observed after portocaval shunt, was not influenced by liver arterialization. We conclude that, in our model, liver arterialization improves the pathologic amino acid levels following portocaval shunt. This would be in agreement with clinical reports suggesting that hepatic encephalopathy is less frequent after portocaval shunt when associated with arterialization of the liver.

Effect of L-dopa treatment on cerebral amino acid levels in rats after portocaval anastomosis.

L-Dopa therapy has been suggested as effective in the reversal of hepatic coma both in humans and in animals. Beneficial effects have been reported also in chronic hepatic encephalopathy. There are many possible mechanisms through which L-dopa could ameliorate this pathological state. The present study was carried out to clarify whether the L-dopa effect could be mediated through an improvement of the brain neutral amino acid patterns, since it competes for the same transport carrier at the blood-brain barrier. A first group of rats was orally administered L-dopa (10 mg/100 g body weight daily) for 1 month following portocaval anastomosis. A second group was intraperitoneally injected (1.5 mg/100 g body weight daily) for 1 week, a month after portocaval shunt. Amino acid levels were determined in plasma and in four cerebral regions. No beneficial effects were observed clinically (in general condition, body weight, or hypertonic posture) in rats receiving L-dopa compared to controls. The large increase of tyrosine, phenylalanine, tryptophan, histidine, and glutamine that occurs in the cerebral tissue after portocaval shunt was also not affected by L-dopa administration. In conclusion, in this experimental condition we had no clinical improvement in shunted animals receiving L-dopa. Moreover, this compound did not seem to influence the pathological increase of aromatic amino acids in the brain, which is considered to play an important role in hepatic encephalopathy.

Cystinuria in children.

7 children, 20 months to 11 years old, with cystinuria and renal calculi were studied. Surgical treatment and alpha-mercaptopropionylglycine (MPG) gave satisfactory results in 5 children. The causes of the recurrences in the other 2 children are discussed. MPG therapy is effective but can cause a nephrotic syndrome at a dose of more than 50 mg/kg/day. A cystinuria of less than 200 mg/day cannot always be considered safe in children. The alkalinization and dilution of urine remain extremely important in the treatment of cystinuria.

Does liver arterialization improve the altered cerebral amino acid pattern in the rat after portacaval shunt?

Altered plasma and brain amino acid patterns have been observed in the rat after portacaval shunt. Similar alterations are likely to play an important role in hepatic encephalopathy in humans. In our model, liver arterialization remarkably improves the pathologic amino acid levels following portacaval shunt.

Nephrotic syndrome during treatment with alpha-mercaptopropionylglycine.

We report 2 cases of cystinuria in which a nephrotic syndrome developed during treatment with alpha-mercaptopropionylglycine. This syndrome resolved after withdrawal of the drug and it did not recur when the alpha-mercaptopropionylglycine was given again in lower doses. The hypothesis is made that the nephrotic syndrome was dose-related. The alpha-mercaptopropionylglycine must be used in doses less than 50 mg./kg per day with regular monitoring of 24-hour urinary protein in cystinuric children.

[Evaluation of 2 analytical technics for determination of fecal lipids]. / Valutazione di due tecniche analitiche per la determinazione dei lipidi fecali.

The van de Kamer titrimetric method for the determination of fecal fatty acids has been compared with the colorimetric technique described by TOMASZEWSKI. Reproducibility and accuracy as well as the applicability in a clinical laboratory service are discussed. The AA. point out that van de Kamer method still remains the method of choice for diagnostic purposes.

Cerebral amino acid levels and uptake in rats after portocaval anastomosis: II. Regional studies in vivo.

Amino acid levels have been determined in plasma and in four cerebral regions of rats one month after portocaval shunt. Many plasma amino acids are significantly lowered (asparagine, glutamine, theonine, serine, alanine, valine, leucine, isoleucine, cystine, lysine), while others remain unchanged (taurine, glycine, proline, tryptophan, ornithine, histidine, arginine). Asparagine and glutamine levels are significantly higher than in normal rats, and a net increase of tyrosine (100%), phenylalanine (50%) and citrulline (50%) is evident. In the shunted rat brain the most prominent feature is a very large rise (up to fivefold) of tyrosine, phenylalanine, histidine, citrulline, tryptophan, and glutamine uniformly in the tested regions. Other neutral amino acids are slightly increased. Lysine and arginine are decreased in cerebellum and pons-medulla; taurine, in forebrain and cerebellum. Cerebral permeability to L-amino acids was studied in vivo. Neutral amino acid permeability is greatly increased, whereas basic amino acids show a net decrease in their rate of passage from blood to the brain. No changes are observed for GABA and glutamic acid. These data suggest an altered permeability of the cerebral capillary membranes, which seems to be selective for the different amino acid transport classes. Competitive inhibition experiments demonstrated that the increased brain permeability to neutral amino acids after portocaval shunt is due to an enhancement of the saturable transport. The sharp rise in the brain of some essential neutral amino acids (phenylalanine, tyrosine, trytophan, histidine), largely exceeding their changes in plasma, and the slight cerebral increase of other neutral amino acids despite their lowered level and the rise of competing amino acids in the plasma, is consistent with our observation of enhanced transport for the neutral class. In hepatic encephalopathy, correction of the altered plasma amino acid levels has been reported to improve the clinical status. If this result is connected to the concomitant correction of the brain amino acid levels, carefully selected competitive inhibition among various plasma amino acids could be a useful therapeutic tool in this pathologic condition. However, the increased activity of the neutral amino acid transport system adds a new factor to the problem, since it probably implies that the competing amino acids will accumulate to unphysiological levels in the brain.

[Aminoacid levels in Werding-Hoffmann and Kugelberg-Welander diseases (author's transl)]. / Alterazioni degli aminoacidi nella malattia di Werdnig-Hoffmann: studio comparativo con la malattia di Kugelberg-Welander.

The concentration of free aminoacids in plasma and urine were estimated in 10 patients suffering from Werdnig-Hoffmann's disease of long duration. The age of the patients was between 5 and 14 years. Estimations were also made in 10 patients with Kugelberg-Welander's disease aged between 11 and 34 years. The aminoacid concentrations were estimated on samples of plasma and 24 hours samples of urine by means of chromatography on ion-exchange resins. The data obtained were compared respectively with groups of thirty and ten healthy subjects of the same age. In the group of patients with Werdnig-Hoffmann's disease a significant increase of taurine (p less than 0.001) and of glutamic acid (p less than 0.001) was found in the plasma. The urinary excretion of glutamine was increased in the same group of patients (p less than 0.001) and in the group with Kugelberg-Welander's disease (p less than 0.005). These aminoacid levels are interpreted as an expression of a reduced oxygen metabolism and increased proteolysis in the skeletal muscles in conditions of chronic denervation.

[Aromatic and branched chain amino acids after porto-caval anastomosis: plasma and brain regional levels in the rat (author's transl)]. / Livelli di aminoacidi aromatici e a catena ramificata nel plasma e nelle regioni cerebrali del ratto sottoposto ad anastomosi porto-cava.

Automated analysis of aromatic and branched chain amino acids was performed in plasma and in four cerebral regions of rats submitted to chronic porto-caval shunt. Compared to controls, plasma of the operated animals exhibited a significant diminution of valine, leucine, isoleucine, a net increase of phenylalanine and tyrosine, with unchanged tryptophan levels. Brain valine, leucine and isoleucine were either increased or unchanged, whereas an enormous increase of aromatic amino acids, uniform in the four cerebral regions, was observed. The lack of correlation between plasmatic and cerebral levels of these essential amino acids leads to the hypothesis that cerebral permeability to amino acids is modified after experimental portocaval anatomosis: we recent confirmed that blood-brain mediated transport of the neutral amino acids is increased in this condition. A slower cerebral turnover of these metabolites could also contribute to elevate their cerebral concentrations after portocaval shunt.