Determination of remifentanil in human heparinised whole blood by tandem mass spectrometry with short-column separation.

For the use in pharmacokinetic studies, a fast and sensitive assay method was developed for the determination of remifentanil in human heparinised whole blood samples of 0.5 ml. The assay method is based on tandem mass spectrometry detection (LC-MS/MS). The limit of quantification is 0.1 ng/ml and linear up to 50 ng/ml. The precision, accuracy, recovery and applicability were found to be adequate for pharmacokinetic studies.

Determination of nifedipine in human plasma by flow-injection tandem mass spectrometry.

For use in clinical studies, a fast and sensitive assay method was developed for the determination of nifedipine in human plasma samples. The assay method is based on tandem mass spectrometry detection (HPLC-MS-MS). The effect of flow injection as well as HPLC separation on the results of the nifedipine determination were evaluated. The limit of quantification is 0.5 ng/ml and the accuracy (as determined by spiking recovery) was found to be good.

Red blood cell enzymes in the diagnosis of genetic disease.

Erythrocytes are uniform cells which contain only those proteins that are synthesized during the reticulocyte stage. The relationship of red cell enzymes to gene dosage and gene expression enables the use of red cell enzyme assays to determine the presence or absence of gene defects causing enzyme deficiencies leading to various metabolic diseases; in addition, the mode of inheritance of these defects can frequently be ascertained by analyzing red cell enzymes. However, indirect evidence favoring other enzyme deficiency states can sometimes be obtained from a study of red cell enzyme activities, because apparent enzyme deficiencies may result from the accumulation of inhibitory metabolites formed due to an enzyme deficiency in other tissues. The polymorphic expression of many red cell enzymes lends itself to biochemical analysis which can produce highly accurate and specific diagnostic information.

Gamma-glutamyltransferase: evaluation of a new method.

The determination of gamma-glutamyltransferase using a new donor substrate, L-gamma-glutamyl-3,5-dibromo-4-hydroxyanilide, was evaluated in two laboratories. Between-run and within-run precision are excellent and comparable with those of other methods for the determination of gamma-glutamyltransferase. The activity concentrations of gamma-glutamyltransferase from human sera obtained with the new donor substrate were slightly higher than those obtained with L-gamma-glutamyl-4-nitroanilide. The reverse was found for sera enriched with gamma-glutamyltransferase of animal origin. Temperature conversion factors (activity 37 degrees C/activity 30 degrees C) are given. The advantages of this method are discussed. The new assay appears to provide a good alternative for the method with L-gamma-glutamyl-4-nitroanilide or one of its derivatives as substrate.

What is the best predictor of the severity of ABO-haemolytic disease of the newborn?

In 80 newborn infants ABO-incompatible with their mothers, the lysis-inducing effect of the maternal IgG anti-A or anti-B antibodies in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay and the antigen density of A or B antigens on the red cells of the children were measured. On the basis of the results, the children were divided into two groups--24 children in whom increased haemolysis was to be expected, and 56 children in whom it was not. Signs of haemolysis and serological features of ABO haemolytic disease of the newborn (ABO-HDN) were compared in these two groups and a control group of 120 ABO-compatible infants. The effect of the maternal antibodies in the ADCC assay, the titres of maternal IgG anti-A or anti-B antibodies, the results of the direct antiglobulin test on the red cells in the cord blood, and the titre of IgG anti-A or anti-B antibodies in the serum of the infants were compared for their ability to predict the severity of ABO-HDN. This was also done for the combination of the ADCC assay results plus the A or B antigen density and the direct antiglobulin test plus the titre of maternal IgG anti-A or anti-B antibodies. The ADCC assay with maternal serum was the most sensitive assay to predict ABO-HDN, and the combination of the ADCC assay with A or B antigen density determination the most specific test.

Esterase D: evaluation of a potential derived gene marker for hereditary retinoblastoma.

The gene locus of esterase D is closely linked to that coding for retinoblastoma. When the occurrence of retinoblastoma is based on a chromosome deletion, red cell esterase D might be a potential tumor marker for diagnosing retinoblastoma. This diagnostic utility was tested by measuring total esterase D and differentiating esterase D to its different phenotypes in red cells of patients with bilateral retinoblastoma, unilateral retinoblastoma having a positive family history and unilateral sporadic retinoblastoma. These results are compared with the findings within a group of first degree relatives of these patients and of a reference group of apparently healthy controls. A poor sensitivity and a low positive predictive value were found. So, the conclusion might be drawn that screening of all retinoblastoma patients for esterase D in order to get insight in the weighting of risk of retinoblastoma is very much open to question and due to high cost efficiency ratio might not be recommended.

Familial association of intracranial aneurysms and multiple congenital anomalies.

The familial occurrence of intracranial aneurysms and the possible relationship with connective tissue disease are discussed. We studied a large family in which seven members presented with aneurysms. Another family member presented with a subarachnoidal hemorrhage. Two other family members each presented with Marfan's syndrome and an unclassified multiple congenital anomalies syndrome, respectively. The multiplicity of the aneurysms in four members is in excess of that found in sporadic or familial cases with intracranial aneurysms. We suggest a common cause, eg, a connective tissue disorder for both the intracranial aneurysms, the Marfan's syndrome, and the unclassified syndrome.

A lethal neonatal variant of carbamoyl-phosphate synthetase deficiency in combination with an intermediate activity of L-ornithine: 2-oxoglutarate amino-transferase.

Shortly after birth, a newborn girl developed anorexia, hypotonia, apneic attacks and seizures. After 61 h the child died in coma. Biochemically, a highly elevated blood ammonia level was found together with an increased plasma level of the amino acids mainly involved in ammonia detoxication. Enzyme studies in post-mortem liver tissue material revealed a deficiency of carbamoyl-phosphate synthetase (0.9% of the mean value in controls) in combination with an intermediate activity of L-ornithine: 2-oxoglutarate aminotransferase (40% of the mean value in controls).

Spectral studies of the interaction of the substrate 'quinonoid' 6-methyl dihydropterine and the coenzyme NADH used as marker in the dihydropteridine reductase assay.

In dihydropteridine reductase assay the substrate quinonoid dihydropterine is reduced again to tetrahydropterine, concomitantly oxidizing NADH, the indicator of the enzyme assay. Because of the strong oxidizing capacity of quinonoid dihydropterine, the degree of spontaneous oxidation of NADH by the various substances used in the dihydropterine reductase assay was studied spectrally. A high degree of spontaneous oxidation of NADH by the substrate itself was found, which can be regulated by dithiotreitol, dependent on its concentration. The absorbance increase at 336 nm, due to the non-quinonoid dihydropterine formed spontaneously from its quinonoid form, strongly interferes with the absorbance decrease at 340 nm, due to the disappearance of NADH. The interference results in a shift of the absorbance maximum of NADH from 340 nm up to higher wavelengths. This phenomenon, expressing itself in various ways in blank and sample, is discussed with relevance to the validity of the current enzyme assays used in a further classification of hyperphenylalaninaemic patients.

Ring chromosome 2: clinical, chromosomal, and biochemical aspects.

A new case of ring chromosome 2 is described and compared with the five cases hitherto reported. The clinical picture includes a severe pre- and postnatal growth failure, microcephaly, psychomotor retardation, and some minor dysmorphic features. Cytogenetic studies revealed a ring 2 structure and aneuploidy. Banding analysis failed to demonstrate a substantial loss of chromosomal material. Enzymologic studies revealed a decrease of red cell acid phosphatase activity suggesting the localization of its gene inthe 2p25 leads to 2 pter region.

Simultaneous occurrence of xanthine oxidase and sulfite oxidase deficiency. A molybdenum dependent inborn error of metabolism?

In a 3-week old female child with clinical features including neurologic abnormalities and lens dislocation, xanthinuria co-existed with increased excretion of sulfur compounds (sulfite, S-sulfocysteine, taurine and thio-sulfate). Low xanthine oxidase and absent sulfite oxidase activities were found on liver biopsy. No abnormality was detected in either parent. Both the above enzymes are molybdenum-flavoproteins. Normal serum molybdenum concentration seemed to rule out dietary deficiency or impaired absorption. A defect in the incorporation of the metal into flavoproteins is postulated in this case.

Attempted dietary treatment of a boy with hyperammonemia due to ornithine transferase deficiency.

Dietary treatment of a male patient suffering from the delayed-onset type of OCT deficiency was attempted. Control of the hyperammonemia was attempted by restriction of protein intake, guided by monitoring the plasma ammonia and regular checking of the serum amino acid levels. The influence of supplementary citric acid or lactulose therapy on the plasma ammonia level was investigated and found to be negligible. The therapeutic effect of supplying ornithine and arginine (an essential amino acid in urea cycle disorders) is described. Despite intensive dietary treatment over two and a half years, a incorrigible hyperammonemic crisis resulted in the sudden death of our patient.

Some kinetic properites of liver ornithine carbamoyl transferase (OCT) in a patient with OCT deficiency.

Some kinetic properties of liver OCT from a patient with OCT deficiency were studied. Contrary to controls, in which two pH optima were observed (pH 7.7 and pH 8.5), only the pH optimum of 8.5 could be demonstrated in our patient. From KM studies at pH 7.7 and pH 8.5, the most striking abnormalities in comparison with human controls were (a) a strongly increased KM (ornithine) at pH 7.7, but less pronounced at pH 8.5, (b) a higher VMAX at pH 8.5 compared with the VMAX at pH 7.7 and (c) the absence of substate inhibition at pH 8.5 to ornithine was elevated up to a concentration above approximately 1.5 mM.

Hereditary mitochondrial myopathy with lactic acidemia, a De Toni-Fanconi-Debré syndrome, and a defective respiratory chain in voluntary striated muscles.

A patient suffering froma mitochondrial myopathy leading to severe insufficiency of the voluntary muscles is described. Severe cerebral damage was present. Major biochemical symptoms were extreme lactic acidemia, hypophosphatemia and hyperphosphaturia, and generalized aminoaciduria, renal glucosuria, and polyuria. Muscular insufficiency resulted in lethal asphyxiz. All therapeutic trials were insufficient. The patient and two other children of the same family with a similar clinical picture all died before the 4th month of life. The condition is probably inherited in an autosomal recessive way. A defective respiratory chain in the mitochondria of the striated muscles is proposed as the underlying mechanism. Cytochromes aa3 were absent, b was nearly absent, but cc1 was present. In heart muscle cytochromes aa3 and b were at the level of the controls.

Liver damage in the hemolytic uremic syndrome.

During the recovery stage of the hemolytic uremic syndrome in 2 cases an increase of serum levels of GOT, GPT, LDH, gammaGT, 5'ND and AP was noticed, without signs of a recurrence of the disease. In one patient also jaundice and hepatomegaly were found. The observations suggest a parenchymal damage of the liver.