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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.

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.

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.

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.

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.

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.

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.

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.