The increasing importance of LNAA supplementation in phenylketonuria at higher plasma phenylalanine concentrations.

BACKGROUND: Large neutral amino acid (LNAA) treatment has been suggested as alternative to the burdensome severe phenylalanine-restricted diet. While its working mechanisms and optimal composition have recently been further elucidated, the question whether LNAA treatment requires the natural protein-restricted diet, has still remained. OBJECTIVE: Firstly, to determine whether an additional liberalized natural protein-restricted diet could further improve brain amino acid and monoamine concentrations in phenylketonuria mice on LNAA treatment. Secondly, to compare the effect between LNAA treatment (without natural protein) restriction and different levels of a phenylalanine-restricted diet (without LNAA treatment) on brain amino acid and monoamine concentrations in phenylketonuria mice. DESIGN: BTBR Pah-enu2 mice were divided into two experimental groups that received LNAA treatment with either an unrestricted or semi phenylalanine-restricted diet. Control groups included Pah-enu2 mice on the AIN-93 M diet, a severe or semi phenylalanine-restricted diet without LNAA treatment, and wild-type mice receiving the AIN-93 M diet. After ten weeks, brain and plasma samples were collected to measure amino acid profiles and brain monoaminergic neurotransmitter concentrations. RESULTS: Adding a semi phenylalanine-restricted diet to LNAA treatment resulted in lower plasma phenylalanine but comparable brain amino acid and monoamine concentrations as compared to LNAA treatment (without phenylalanine restriction). LNAA treatment (without phenylalanine restriction) resulted in comparable brain monoamine but higher brain phenylalanine concentrations compared to the severe phenylalanine-restricted diet, and significantly higher brain monoamine but comparable phenylalanine concentrations as compared to the semi phenylalanine-restricted diet. CONCLUSIONS: Present results in PKU mice suggest that LNAA treatment in PKU patients does not need the phenylalanine-restricted diet. In PKU mice, LNAA treatment (without phenylalanine restriction) was comparable to a severe phenylalanine-restricted diet with respect to brain monoamine concentrations, notwithstanding the higher plasma and brain phenylalanine concentrations, and resulted in comparable brain phenylalanine concentrations as on a semi phenylalanine-restricted diet.

An observational study on disturbed peripheral circadian rhythms in hemodialysis patients.

The quality of life of hemodialysis (HD) patients is hampered by reduced nocturnal sleep quality and excessive daytime sleepiness. In addition to the sleep/wake cycle, levels of circadian biomarkers (e.g. melatonin) are disturbed in end-stage renal disease (ESRD). This suggests impaired circadian clock performance in HD patients, but the underlying mechanism is unknown. In this observational study, diurnal rhythms of sleep, serum melatonin and cortisol concentrations and clock gene mRNA expression are compared between HD patients (n = 9) and healthy control subjects (n = 9). In addition, the presence of circulating factors that might affect circadian rhythmicity is tested in vitro with cell culture experiments. Reduced sleep quality (median sleep onset latency [interquartile range] of 23.9 [17.3] min for patients versus 5.0 [10] minutes for controls, p < 0.01; mean (± SD) sleep efficiency 70.2 ± 8.1% versus 82.9 ± 10.9%, p = 0.02 and mean awake minutes after sleep onset 104.8 ± 27.9 versus 54.6 ± 41.6 minutes, p = 0.01) and increased daytime sleepiness (mean Epworth Sleepiness Score of 10.0 ± 4.8 versus 3.9 ± 2.0, p < 0.01) were confirmed in HD patients. Reduced nocturnal melatonin concentrations (1 AM: 98.1 [122.9] pmol/L versus 12.5 [44.2] pmol/L, p = 0.019; 5 AM: 114.0 [131.6] pmol/L versus 11.8 [86.8] pmol/L, p = 0.031) and affected circadian control of cortisol rhythm and circadian expression of the clock gene REV-ERBα were found. HD patient serum had a higher capacity to synchronize cells in vitro, suggesting an accumulated level of clock resetting compounds in HD patients. These compounds were not cleared by hemodialysis treatment or related to frequently used medications. In conclusion, the abovementioned results strongly suggest a disturbance in circadian timekeeping in peripheral tissues of HD patients. Accumulation of clock resetting compounds possibly contributes to this. Future studies are needed for a better mechanistic understanding of the interaction between renal failure and perturbation of the circadian clock.

Development of Bacillus thuringiensis CryIC Resistance by Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae).

Selection of resistance in Spodoptera exigua (Hubner) to an HD-1 spore-crystal mixture, CryIC (HD-133) inclusion bodies, and trypsinized toxin from Bacillus thuringiensis subsp. aizawai and B. thuringiensis subsp. entomocidus was attempted by using laboratory bioassays. No resistance to the HD-1 spore-crystal mixture could be achieved after 20 generations of selection. Significant levels of resistance (11-fold) to CryIC inclusion bodies expressed in Escherichia coli were observed after seven generations. Subsequent selection of the CryIC-resistant population with trypsinized CryIC toxin resulted, after 21 generations of CryIC selection, in a population of S. exigua that exhibited only 8% mortality at the highest toxin concentration tested (320 (mu)g/g), whereas the 50% lethal concentration was 4.30 (mu)g/g for the susceptible colony. Insects resistant to CryIC toxin from HD-133 also were resistant to trypsinized CryIA(b), CryIC from B. thuringiensis subsp. entomocidus, CryIE-CryIC fusion protein (G27), CryIH, and CryIIA. In vitro binding experiments with brush border membrane vesicles showed a twofold decrease in maximum CryIC binding, a fivefold difference in K(infd), and no difference in the concentration of binding sites for the CryIC-resistant insects compared with those for the susceptible insects. Resistance to CryIC was significantly reduced by the addition of HD-1 spores. Resistance to the CryIC toxin was still observed 12 generations after CryIC selection was removed. These results suggest that, in S. exigua, resistance to a single protein is more likely to occur than resistance to spore-crystal mixtures and that once resistance occurs, insects will be resistant to many other Cry proteins. These results have important implications for devising S. exigua resistance management strategies in the field.

Crystallization and preliminary X-ray diffraction studies of the lepidopteran-specific insecticidal crystal protein CrylA(a).

A trypsin-activated CrylA(a) protein from Bacillus thuringiensis has been purified and crystallized. Crystals belong to orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 53.3, b = 111.3 and c = 154.7 A. The crystals diffract to at least 2.2 angstrum resolution and are suitable for X-ray structural analysis. They contain a single molecule in the asymmetric unit.

Hereditary tyrosinaemia type I: a long-term study of the relationship between the urinary excretions of succinylacetone and delta-aminolevulinic acid.

Patients with hereditary tyrosinaemia type I (HT) excrete large amounts of succinylacetone (SA) in urine. Owing to structural resemblance of SA to delta-aminolevulinic acid (ALA), SA inhibits the second enzyme in the pathway for haeme biosynthesis, porphobilinogen synthase, resulting in increased urinary ALA excretion. We investigated the relationship between urinary SA and ALA excretions of two patients with different forms of HT (late-infantile and juvenile). In both patients the urinary SA and ALA excretions showed a more or less inverse correlation. The patient with the early-infantile form of HT had a relatively greater increase in urinary SA and ALA excretions in comparison to the patient with the juvenile form of HT. A possible explanation for this unexpected inverse correlation between the urinary excretion of SA and ALA might be a lack of intramitochondrial glycine, a substrate for delta-aminolevulinic acid synthesis. It has been reported previously that high concentrations of SA reversibly and competitively inhibit the transport of glycine through membranes.

The human fumarylacetoacetase gene: characterisation of restriction fragment length polymorphisms and identification of haplotypes in tyrosinemia type 1 and pseudodeficiency.

Deficiency of human fumarylacetoacetase (FAH) activity results in hereditary tyrosinemia type I. Using the restriction enzymes BglII, KpnI and StuI and a 1.3-kb cDNA probe for the FAH gene, we have found 6 restriction fragment length polymorphisms (RFLPs). These RFLPs were utilised in 3 tyrosinemia families in which one or both parents are carriers of both a tyrosinemia and a pseudodeficiency gene for FAH. Full information was obtained in two of these families. The polymorphisms identified 6 haplotypes. The haplotype distribution was significantly different in 32 unrelated tyrosinemia patients compared with a reference population of 100 individuals. The combined polymorphism information content was 0.77.

Hereditary tyrosinemia type I: lack of correlation between clinical findings and amount of immunoreactive fumarylacetoacetase protein.

Immunoblot analyses with bovine fumarylacetoacetase antibodies have been performed in fibroblast extracts from 28 patients with hereditary tyrosinemia of various clinical phenotypes, in one healthy individual homozygous for a "pseudodeficiency" gene for fumarylacetoacetase, and in three tyrosinemia families in which one or both parents are compound heterozygotes for the tyrosinemia and pseudodeficiency genes. Liver extracts from two chronic patients were also investigated. None of the patients with the acute type of tyrosinemia had detectable immunoreactive protein in fibroblast extracts. Only two of seven patients with typical chronic tyrosinemia had definite immunoreactivity in fibroblasts. In liver tissue, one of the patients had cross-reactive material and the other had no immunoreactivity. Four of 13 patients with intermediate clinical findings showed immunoreactivity in fibroblasts. There was no relationship between severity of symptoms and amount of cross-reactive material in this group. The pseudodeficiency gene product gave almost no detectable immunoreactivity in fibroblasts. The results indicate that chronic tyrosinemia may be due to at least two protein variants, and immunoblotting does not classify tyrosinemia patients according to clinical findings.

Purification of human liver fumarylacetoacetase using immunoaffinity chromatography.

A method is described to purify fumarylacetoacetase from crude human liver extracts using immunoaffinity chromatography. Immobilized rabbit antibodies specific for beef liver fumarylacetoacetase were used as an immunoadsorbent. With this rapid and specific procedure human liver fumarylacetoacetase could be purified to apparent homogeneity. The molecular weight of native human liver fumarylacetoacetase is approximately 83000 as estimated by gel filtration. The two subunits have a molecular weight of approximately 41000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Purified human liver fumarylacetoacetase has a broad pH optimum with a maximum at pH 7.2 and a Km = 2.1 microM towards fumarylacetoacetate.

Separation of different forms of S-adenosylmethionine synthetase by fast protein liquid chromatography.

A method is described by which different forms of S-adenosylmethionine synthetase are separated. The method makes use of fast protein liquid chromatography on an anion exchange hydrophilic polyether resin. Different forms of S-adenosylmethionine synthetase from rat and human liver and kidney and rat zajdela hepatoma cells can be separated within 15 min. From a mixture of rat liver and kidney cytosols all three forms alpha, beta and gamma can be separated. The time needed to separate the different forms of S-adenosylmethionine synthetase is reduced from 3 h with conventional gel filtration methods, to 15 min using this HPLC anion-exchange method. Also the amount of tissue needed to detect the different forms is reduced from 125 mg to 12.5 mg of fresh rat liver tissue. These advantages make this newly developed method applicable when large numbers of samples have to be analyzed or when only small amounts of tissue are available.

Type I tyrosinemia: lack of immunologically detectable fumarylacetoacetase enzyme protein in tissues and cell extracts.

Type I hereditary tyrosinemia is characterized by the almost complete absence of fumarylacetoacetase in tissues and cells from patients. To investigate the nature of the enzyme deficiency, extracts of tissues (liver and kidney) and cells (lymphocytes and fibroblasts) were immunochemically screened for the presence of fumarylacetoacetase enzyme protein. The antibodies used were raised in rabbits against fumarylacetoacetase purified from beef liver. These antibodies cross-reacted strongly with the human enzyme. No cross-reacting material was found in extracts from liver (n = 4) and kidney (n = 1) from patients. Extracts from lymphocytes and cultured skin fibroblasts from patients were investigated as well. However, no cross-reacting material was found in extracts of these cells.

Biochemical studies on the enzymatic deficiencies in hereditary tyrosinemia.

Experiments are described on the effects of succinylacetone and fumarylacetoacetate on delta-aminolevulinic acid dehydratase, methionine adenosyltransferase and p-OH-phenylpyruvate dioxygenase. delta-Aminolevulinic acid dehydratase from human erythrocytes is inhibited non-competitively by succinylacetone (Ki 0.03 mumol/l) and by fumarylacetoacetate (Ki 0.06 mumol/l). The inhibition by succinylacetone is not prevented by dithiothreitol, but the inhibition by fumarylacetoacetate is not observed if dithiothreitol is present. Methionine adenosyltransferase, partially purified from rabbit liver, is not inhibited by succinylacetone but is inhibited by fumarylacetoacetate: 69% inhibition is observed at 1 mmol/l. Human liver p-OH-phenylpyruvate dioxygenase is not inhibited by succinylacetone or fumarylacetoacetate. It is concluded that secondary enzyme deficiencies observed in hereditary tyrosinemia (delta-aminolevulinic acid dehydratase, methionine adenosyl transferase) are the result of inhibition by succinylacetone and fumarylacetoacetate, accumulating as a result of a primary deficiency of fumarylacetoacetase.

Effect of cyclic nucleotides on weight of gastrocnemius and creatine kinase activity after denervation of muscle in young rats.

Denervation of the gastrocnemius muscle at various stages of development reduces muscle weight and creatine kinase activity. An inverse relationship between the muscle-specific enzyme, creatine kinase, and the lysosomal enzyme, acid phosphatase, was shown. An increased percentage of the BB isoenzyme of creatine kinase is observed after long-term denervation. Apparently the muscle tissue has the ability to regenerate and presumptive myoblasts are formed from satellite cells. When the denervated muscle is treated with dibutyryl-cyclic-GMP administration new muscle tissue has been formed. Similar effects could not be demonstrated with either cyclic AMP or succinylcholine. The higher percentage of the BB isoenzyme after dibutyryl-cyclic-GMP administration supports the theory that presumptive myoblasts are derived from satellite cells. Succinylcholine also causes an increase of the B-type of creatine kinase. It can be concluded that cyclic GMP, generated via the nerve, has an important role in maintaining muscle weight.

Effect of hormones on the development of creatine kinase activity in rat skeletal muscle.

Various approaches have been used in order to determine whether or not a certain hormone is a stimulus for the development of the muscle-specific enzyme, creatine kinase. Both thyroxine and glucocorticoids can be considered as naturally occurring stimuli for the synthesis of creatine kinase. The maximum increase of creatine kinase activity after stimulation by glucocorticoids (about 25%) occurs between 5 and 7 days after birth. A single injection of thyroxine has virtually no effect during this period. However, when a pretreatment with thyroxine is given, cortisone acetate administration increases creatine kinase activity to about 155%. The effect of cortisone acetate is due to de novo synthesis of creatine kinase. The augmentation of the effect of cortisone acetate by thyroxine is dependent on DNA synthesis. Thyroxine administration apparently causes the formation of more competent muscle cells. The effects of both hormones are age-dependent. Thyroxine and cortisone acetate administration to fetuses can prematurely evoke to MM isoenzyme of creatine kinase. Both hormones probably play a role in the activation of the M gene during embryonic development. Sex hormones are able to influence neither creatine kinase activity nor muscle growth. However, castration of male rats immediately after birth causes an impairment of growth at older ages. The androgen production by the testes immediately after birth seems to be of main importance for body growth development. It can be concluded from these results that creatine kinase in muscle is under multiple hormonal control, just as is observed for a number of enzymes in other tissues.