Durk Fekkes

State-of-the-art of high-performance liquid chromatographic analysis of amino acids in physiological samples

Nowadays, the fast and sensitive high-performance liquid chromatographic (HPLC) analysis of derivatized amino acids is a good alternative to the ion-exchange chromatography (IEC) method for plasma amino acids. However, several precautions have to be taken in order to obtain reliable data on the concentration of amino acids in physiological fluids. These include the collection, centrifugation, storage conditions and the method of deproteinization. Furthermore, the method of pre-column derivatization in connection with the protein precipitant used and the choice of the chromatographic system which determines the overall resolution are important factors. HPLC methods using pre-column derivatization with o-phthaldialdehyde were suitable for the accurate determination of many primary amino acids in plasma because of their high sensitivity, simplicity, speed and reliability. When the determination of secondary amino acids and cystine was also necessary, the phenylisothiocyanate method was the preferred technique. The intra-laboratory variability of the HPLC method was satisfactory while the inter-laboratory variation of this method was found to be similar to that of IEC, HPLC methods capable of separating over forty physiological amino acids seem promising for the analysis of urine samples.

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-microns Spherisorb ODS 2 column (125 x 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).

Incidence of and preoperative predictors for delirium after cardiac surgery

Incidence of and preoperative predictors for postoperative delirium were studied in 296 patients (age 26-83 years, mean age 63 years) undergoing elective cardiac surgery. Delirium occurred in 40 (13.5%) patients. Predictors included old age, low level of albumin, poor physical condition, use of nifedipine, and a high ratio of the amino acids phenylalanine to the sum of isoleucine, leucine, valine, tyrosine, and tryptophan. These findings suggest that preoperative physical condition and amino acid disturbances may be related to delirium after cardiac surgery in the elderly.

Extracellular brain glutamate during acute liver failure and during acute hyperammonemia simulating acute liver failure: An experimental study based on in vivo brain dialysis

Hyperammonemia is thought to be important in the pathogenesis of hepatic encephalopathy. However, the mechanism leading to ammonia toxicity is still not known. Since the metabolism of the most important excitatory neurotransmitter, glutamate, is closely linked to that of ammonia, it has been postulated that hyperammonemia lowers the availability of the neurotransmitter glutamate. To study this hypothesis, we used brain dialysis to measure glutamate levels in extracellular cerebral fluid from rabbits with acute ischemic liver failure or acute hyperammonemia. The basal glutamate concentration was found to be increased during both acute liver failure (start of experiments 4.9 +/- 1.7 mumol/l; end of experiments 9.5 +/- 2.1 mumol/l, n = 6, difference p < 0.05) and acute hyperammonemia (start of experiments 4.4 +/- 1.2 mumol/l; end of experiments 7.3 +/- 1.8 mumol/l, n = 7, difference p > 0.05) (mean +/- SEM). Both the veratridine- and the potassium-evoked glutamate release were increased during acute liver failure but appeared normal during hyperammonemia. We conclude that during acute liver failure and acute hyperammonemia in the rabbit there is no decreased glutamate availability in the extracellular space of the cortical brain; on the contrary, we found evidence for increased extracellular glutamate concentrations in the cortical brain, which were more pronounced in acute liver failure. Experimental hepatic encephalopathy is thus not due to cerebral glutamate deficiency.

Occurrence and partition of the β-carboline norharman in rat organs

The β-carboline norharman was determined in plasma, brain, liver, kidney, spleen, heart and lung of the rat using HPLC with fluorescence detection. In order to improve the speed and sensitivity of this assay an earlier published sample clean-up extraction procedure and HPLC method were adjusted. Norharman was found to be present in plasma as well as in all organs tested, concentrations in organs being about 80 times higher than those in plasma. Intraperitoneal injections of 2 and 100 mg/kg norharman showed that the partition of norharman between organs and plasma is about 3. Only the highest dose was found to have behavioural effects, viz. alerting reactions, a decrease in motor and exploratory activity, sedation, loss of righting reflex and after 30 min complete muscle relaxation, but no catatonia was observed. Norharman was found to be metabolized by the liver with a half live of about 20 min, whereas all other organs tested did not show any norharman clearing capacity. The results suggest that norharman is not likely the cause of psychosis, but a natural sedative and by- or coproduct of a more primary biochemical derangement.

INHERITED THYROXINE EXCESS: A SERUM ABNORMALITY DUE TO AN INCREASED AFFINITY FOR MODIFIED ALBUMIN

Further analysis of sera from euthyroid subjects with dominantly‐inherited, elevated serum total thyroxine (T4) and free T4 index but with normal free T4 levels was performed as an extension of a previous study (Hennemann et al., 1979a). Scatchard analysis and isoelectric focusing of whole sera and purified serum fractions suggest that this T4 excess is due to increased T4 binding by modified serum albumin.

Chorionic gonadotropin induces dendritic cells to express a tolerogenic phenotype

The pregnancy hormone human chorionic gonadotropin (hCG) has been suggested to play an immunoregulatory role in addition to its endocrine function, thus contributing to the prevention of fetal rejection. We hypothesized that hCG is involved in the maternal‐fetal immune tolerance by the regulation of dendritic cell (DC) function. Therefore, we studied the effect of hCG on DC maturation. Upon hCG treatment in combination with LPS, mouse bone marrow‐derived DC (BMDC) increased the ratio of IL‐10:IL‐12p70, down‐regulated TNF‐α, and decreased antigen‐specific T cell proliferation. Addition of hCG together with LPS and IFN‐γ blocked MHC class II up‐regulation, increased IL‐10 production, and decreased the antigen‐specific T cell proliferation by DC. Splenic DC showed similar results. Upon hCG treatment, IDO mRNA expression and its metabolite kynurenine were increased by LPS‐ and IFN‐γ‐stimulated DC, suggesting its involvement in the decreased T cell proliferation. To study the effect of hCG on DC differentiation from precursors, BMDC were generated in the continuous presence of hCG. Under this condition, hCG decreased cytokine production and the induction of T cell proliferation. These data are suggestive for a contribution of hCG to the maternal‐fetal tolerance during pregnancy by modifying DC toward a tolerogenic phenotype.

The inhibitory effect of norharman on morphine withdrawal syndrome in rats: Comparison with ibogaine

Norharman (20 mg/kg, i.p.) and ibogaine (40 mg/kg, i.p.) significantly attenuated naloxone (4 mg/kg, i.p.)-precipitated withdrawal syndrome in morphine-dependent rats. Several withdrawal signs, such as teeth-chattering, chewing, penile licking and diarrhoea, were decreased by both norharman and ibogaine. In addition, norharman reduced also the withdrawal grooming and rearing. It is concluded that both norharman and ibogaine are inhibitors of withdrawal syndrome in morphine-dependent rats.

Effect of electroconvulsive therapy on biopterin and large neutral amino acids in severe, medication-resistant depression

Biopterin, neopterin and the large neutral amino acids (LNAA), i.e. phenylalanine, tyrosine, tryptophan, isoleucine, leucine and valine were measured in plasma of 20 severely depressed inpatients before and after a course of electroconvulsive therapy (ECT). These patients showed a significantly lower plasma biopterin concentration at baseline in comparison with healthy controls. After treatment an increase in biopterin was found, which was statistically significant in the depressed patients with psychotic features. The plasma phenylalanine-tyrosine ratio, which previously increased, normalised after ECT. Mean tryptophan concentration was lower in depressed patients than in normal controls. The patients who responded to ECT showed an increase in the tryptophan concentration and its ratio (tryptophan/LNAA) after treatment. Our results suggest that ECT increases biopterin, which probably results in synthesis of amino acids, especially tyrosine. Furthermore, ECT seems to increase cerebral tryptophan availability because of less tryptophan catabolism parallel with biopterin activation. More research is required to see if biopterin could be useful as a biological marker for the depressive state in this subgroup of patients, because this compound seems to play an important role in the etiology and treatment of depression.

Is postoperative delirium related to reduced plasma tryptophan

The cause of postoperative delirium is unknown. In 7 patients with postcardiotomy delirium (6 men, 1 woman; mean age 67 years), we observed a plasma concentration of tryptophan and a plasma tryptophan ratio significantly lower than in 8 non-delirious postoperative control patients. We suggest that the mental symptoms of postcardiotomy delirium are the consequence of a reduced cerebral tryptophan availability due to a catabolic state.