Lower Concentrations Of Tyrosine Research Articles

Phenylketonuria oxidative stress and energy dysregulation: Emerging pathophysiological elements provide interventional opportunity.

Phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is rightfully considered the paradigm treatable metabolic disease. Dietary substrate restriction (i.e. phenylalanine (Phe) restriction) was applied >60 years ago and remains the primary PKU management means. The traditional model of PKU neuropathophysiology dictates blood Phe over-representation directs asymmetric blood:brain barrier amino acid transport through the LAT1 transporter with subsequent increased cerebral Phe concentration and low concentrations of tyrosine (Tyr), tryptophan (Trp), leucine (Leu), valine (Val), and isoleucine (Ile). Low Tyr and Trp concentrations generate secondary serotonergic and dopaminergic neurotransmitter paucities, widely attributed as drivers of PKU neurologic phenotypes. White matter disease, a central PKU characteristic, is ascribed to Phe-mediated tissue toxicity. Impaired cerebral protein synthesis, by reduced concentrations of non-Phe large neutral amino acids, is another cited pathological mechanism. The PKU amino acid transport model suggests Phe management should be more efficacious than is realized, as even early identified, continuously treated patients that retain therapy compliance into adulthood, demonstrate neurologic disease elements. Reduced cerebral metabolism was an early-recognized element of PKU pathology. Legacy data (late 1960's to mid-1970's) determined the Phe catabolite phenylpyruvate inhibits mitochondrial pyruvate transport. Respirometry of Pahenu2 cerebral mitochondria have attenuated respiratory chain complex 1 induction in response to pyruvate substrate, indicating reduced energy metabolism. Oxidative stress is intrinsic to PKU and Pahenu2 brain tissue presents increased reactive oxygen species. Phenylpyruvate inhibits glucose-6-phosphate dehydrogenase that generates reduced niacinamide adenine dinucleotide phosphate the obligatory cofactor of glutathione reductase. Pahenu2 brain tissue metabolomics identified increased oxidized glutathione and glutathione disulfide. Over-represented glutathione disulfide argues for reduced glutathione reductase activity secondary to reduced NADPH. Herein, we review evidence of energy and oxidative stress involvement in PKU pathology. Data suggests energy deficit and oxidative stress are features of PKU pathophysiology, providing intervention-amenable therapeutic targets to ameliorate disease elements refractory to standard of care.

Proteomic potential of East African Highland Bananas (EAHBs) for banana juice extraction: comparison between juice-producing and cooking cultivars

ABSTRACT This study investigated the proteomic difference between juice-producing and cooking EAHBs to clarify the role of protein in the production of banana juice. A comparative study was carried out to determine protein content, molecular weight distributions, and amino acid profile of the pulps of ten different (five juice-producing and five cooking) banana cultivars. There was low variability in crude protein content of banana cultivars, the level fell within the range of 0.80 g/100 g to 1.02 g/100 g. SDS-PAGE results visualised that the cultivars had similar molecular weights, ranging between 10 kDa and 76 kDa. The HPLC analysis showed that the relative compositions of amino acids differed significantly (p ≤ .05) within and between juice-producing and cooking cultivars. Both banana cultivars had a significantly higher amount of glutamic and aspartic acids, but significantly lower concentrations of tyrosine, methionine. The results suggest that protein content, molecular weight, and amino acid composition of banana cultivars are not the major factors in determining a banana’s ability to release juice.

Validation of amino-acids measurement in dried blood spot by FIA-MS/MS for PKU management

ObjectivesPhenylketonuria (PKU) is a metabolic disorder leading to high concentrations of phenylalanine (Phe) and low concentrations of tyrosine (Tyr) in blood and brain that may be neurotoxic. This disease requires a regular monitoring of plasma Phe and Tyr as well as branched-chain amino-acids concentrations to adapt the Phe-restricted diet and other therapy that may be prescribed in PKU. We validated a Flow Injection Analysis tandem Mass Spectrometry (FIA-MS/MS) to replace the enzymatic method routinely used for neonatal screening in order to monitor in parallel to Phe, Tyr and branched-chain amino-acids not detected by the enzymatic method. Design and methodsWe ascertained the performances of the method: linearity, detection and quantification limits, contamination index, accuracy. We cross validated the FIA-MS/MS and enzymatic methods and we evaluated our own reference ranges to monitor Phe, Tyr, Leu, Val on 59 dried blood spots of normal controls. We also evaluated Tyr, Leu and Val concentrations in PKU patients to detect some potential abnormalities, not evaluated by the enzymatic method. ResultsWe developed a rapid method with excellent performances including precision and accuracy <15%. We noted an excellent correlation of Phe concentrations between FIA-MS/MS and enzymatic methods (p<0.0001) based on our database which are similar to references ranges published. We observed that 50% of PKU patients had lower concentrations of Tyr, Leu and/or Val that could not be detected by the enzymatic method. ConclusionBased on laboratory accreditation recommendations, we validated a robust, rapid and reliable FIA-MS/MS method to monitor plasma Phe concentrations but also Tyr, Leu and Val concentrations, suitable for PKU management. We evaluated our own reference ranges of concentration for a routine application of this method.

Low tyrosine content of growth media yields aflagellate Salmonella enterica serovar Typhimurium

Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0.03+/-0.01 g l(-1) for gelatin-based buffered peptone water, compared to 0.21+/-0.01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 muM tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1.20 g tyrosine l(-1), and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.

Plasma phenylalanine is associated with decreased serum ubiquinone-10 concentrations in phenylketonuria.

Decreased serum ubiquinone-10 concentrations is a common condition in patients with phenylketonuria (PKU) under dietary treatment. Our aim was to investigate the implication of the metabolic abnormalities of PKU (low concentrations of tyrosine and high concentrations of phenylalanine) and the effect of treatment with phenylalanine-restricted diets in decreased ubiquinone-10 concentrations in PKU patients. We studied 30 PKU patients (age range 5 months to 35 years; median age 7 years) under dietary treatment. Correlation between plasma tyrosine or phenylalanine and serum ubiquinone-10 concentrations was investigated. Daily cholesterol intake was calculated from the data obtained through a dietary questionnaire. The index of dietary control (IDC) was calculated as the average of the medians of plasma phenylalanine concentrations obtained every 6 months in the metabolic control of patients. Negative correlations were observed between serum ubiquinone and the IDC (r=-0.46; p<0.01) in PKU patients. No correlation was observed between tyrosine or daily cholesterol intake and serum ubiquinone concentrations. After adjustment for daily cholesterol intake by multiple linear regression analysis, for each 113 units of increase in IDC values serum ubiquinone decreased 0.1 micromol/L. According to our results, the main factor associated with the decreased serum ubiquinone concentrations was high plasma phenylalanine values. Although daily cholesterol intake seems to be associated with ubiquinone concentrations, it may not be relevant if we take into account the low intake of cholesterol in treated PKU patients.

Effect of low levels of dietary tyrosine on the hair colour of cats.

Experiments were conducted to investigate the basis for the change in hair colour of black cats to reddish-brown. Black cats were given purified diets based on gelatin, casein plus lactalbumin, or crystalline amino acids as protein sources. Diets that caused the colour of hair to change to reddish-brown were associated with a reduction in melanin in hair (observed by direct microscopic examination), a decreased total melanin concentration and low concentrations of tyrosine in plasma. Reddish hair coat was induced in black kittens born to queens given a tyrosine-deficient diet during pregnancy. Black hair colour was maintained or restored by diets containing a high concentration of tyrosine or phenylalanine. Current dietary recommendations for dietary tyrosine and phenylalanine for cats are below those required to support maximal melanin synthesis in black cats. The requirement appears to be greater than a combination of 4.5 g tyrosine plus 12 g phenylalanine/kg diet but less than 24 g phenylalanine alone/kg diet.

Improved selectivity in multicomponent determinations through interference modelling by applying partial least squares regression to kinetic profiles

Binary and ternary mixtures of cysteine, tyrosine and tryptophan were used to show how kinetic methods together with partial least squares regression (PLSR) can be used to overcome interferences in multicomponent determinations. Two methods were compared: an equilibrium method which uses the characteristic UV spectra of amino acids and a kinetic method which follows the reaction kinetics of the oxidation of the amino acids by hexacyanomanganate(IV). In both cases, multi-wavelength detection with a diode-array spectrophotometer was used. The results obtained from the two methods are critically compared. Lower concentrations of tyrosine and tryptophan can be determined by the equilibrium method because of their high absorptivities. On the other hand, the kinetic method, in conjunction with PLSR, can be considered an example of how selectivity can be improved. This allowed the resolution of mixtures of tyrosine and tryptophan in the presence of several complex spiked matrices (aqueous extracts of feed samples that interfere to different extents) without any previous separation.

Plasma concentration of taurine is higher in malnourished than control children: differences between kwashiorkor and marasmus.

Plasma free amino acids were determined in the plasma of severely malnourished children under two years of age. A total of thirty-one patients and eleven controls were evaluated: seventeen cases of kwashiorkor, eight cases of marasmus, and six cases of marasmic-kwashiorkor. Fasting plasma samples were taken in the morning on the day of admission. Fasting plasma samples were also taken from nine patients at discharge after two months in the hospital where they received a balanced diet as treatment. A partial reversal of the signs of malnutrition was observed at discharge. In the whole group of patients ad admission, lower concentrations of tyrosine, methionine, tryptophan, and leucine and higher concentrations of aspartate, glutamate, and taurine were observed compared to controls. Taurine continued to be elevated in the malnourished group at the time of discharge. Marasmic children, as compared to controls, had high aspartate and low tryptophan levels, but taurine levels were not significantly different from controls. Kwashiorkor patients had low tyrosine, methionine, tryptophan, and lysine, and significantly higher taurine plasma levels. The elevated concentration of taurine might be the result of a redistribution of this amino acid to provide specific tissues with the required amount for development.

Activation of protein kinase C-alpha isoform in murine melanoma cells with high metastatic potential.

Metastasis is a multistep process in which protein kinase C (PKC) appears to be significantly involved. We analysed the activity and expression of classical (alpha, beta, gamma) and novel PKC epsilon isoforms in B16-F1 and B16-BL6 melanoma cells maintained under different culture conditions in vitro. We used high and low concentrations of tyrosine and phenylalanine in different media (DMEM or RPMI 1640 respectively) that affect the metastatic potential and also the proliferative capacity of the cells. We also tested a weakly metastatic amelanotic B78-H1 melanoma cell line which is unaffected by the different culture conditions. In both B16 melanoma cell lines activation of PKC alpha (without increased expression) occurred under growth conditions permissive of metastasis (DMEM). In contrast, the weakly metastatic amelanotic B78-H1 cell line showed a substantial inactivation of this isoform in the two different culture media, suggesting a specific involvement of PKC alpha in the metastatic process. Moreover, in B16 melanoma cells, novel PKC epsilon was activated under culture conditions which stimulated growth but not metastasis (RPMI 1640). In order to define the relationship between PKC activation and the metastatic process we also determined the release of cathepsin B. No correlation between PKC activity and cathepsin B release in either B16 melanoma cell lines could be demonstrated.

Human TRP‐1 Has Tyrosine Hydroxylase but no DOPA Oxidase Activity

Human TRP-1 has been immunopurified from normal human melanocytes cultured from black neonatal subjects and used to investigate the catalytic function of TRP-1 for the two substrates, L-tyrosine and L-DOPA. Immunopurified TRP-1 did not demonstrate DOPA staining on SDS/PAGE nor DOPA oxidase (DO) activity with either routine or modified assays. The purified TRP-1 also demonstrated no tyrosine hydroxylase (TH) activity using the routine Pomerantz assay. However, there was apparent TH activity exhibited by immunopurified TRP-1 under conditions with low tyrosine concentration (< or = 0.8 microCi/ml of 3H-tyrosine), prolonged incubation time (i.e., overnight) and in the absence of the cofactor L-DOPA. Using these latter specific conditions, TH activity was also detected in cell lysates from a tyrosinase-negative albino melanocyte line which exhibited no TH activity with the routine Pomerantz assay. In addition, TH activity under low substrate assay conditions was not exhibited in a melanocyte line derived from a TRP-1 deficient, Brown albino individual. However, the absence of TH in this Brown albino cell line could be compensated for by the addition of L-DOPA to the assay. These results suggested that TRP-1 has some tyrosine hydroxylase but no DOPA oxidase activity. We propose that one function of TRP-1 is to modulate tyrosinase activity by making DOPA available as a cofactor to perpetuate the initial steps in melanogenesis.

Effect of maturity and storage on ascorbic acid and tyrosine concentrations and enzymic discoloration of potatoes

Ontario and Pontiac potatoes, representing cultivars susceptible and resistant to enzymatic discoloration, were examined for the effect of maturity and maturity-storage interactions on the concentrations of ascorbic acid and tyrosine. Both cultivars were harvested 7, 9, 11, 13, 15, and 17 weeks following planting and stored for 0, 4, 8, 12, and 24 weeks at 5 degrees C and 95% relative humidity prior to analyses. Ascorbic acid concentrations, of both cultivars increased significantly (p < 0.05) up to 11 weeks following planting and decreased thereafter. Following 24 weeks of storage tubers harvested at 1 1 weeks showed greatest decreases while those harvested at 7 weeks showed least decreases in ascorbic acid, suggesting significant maturity-storage interactions. Tyrosine decreased significantly (p < 0.05) up to 9 weeks following planting and increased thereafter. Following 24 weeks of storage tyrosine showed greatest increases in tubers harvested at 7 (Ontario) and 9 weeks (Pontiac). No consistent correlation between ascorbic acid and discoloration was observed. Although tyrosine was positively correlated with discoloration within a cultivar, it did not appear to be a predominant factor determining blackspot susceptibility of potatoes. The blackspot-susceptible cultivar, Ontario, had higher ascorbic acid and lower tyrosine concentrations than the resistant cultivar, Pontiac.

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.

Differential effects of calcium on catecholamine synthesis regulation in olfactory tubercle and hypothalamic synaptosomes

We have compared catecholamine synthesis regulation in rat brain olfactory tubercle and hypothalamic synaptosomes with regard to: (1) calcium-dependency of stimulant drug-induced synthesis activation; (2) tyrosine-dependency of amphetamine-induced synthesis stimulation, and (3) the effects of the calcium chelator, ethylene glycol-bis-(β-aminoethyl ether)N, N, N′, N′-tetraacetic acid (EGTA). Amphetamine-induced synthesis stimulation was markedly calcium-dependent in the olfactory tubercle, but was completely calcium-independent in the hypothalamus. The effect of amphetamine on catecholamine synthesis in the olfactory tubercle was dependent upon the concentration of tyrosine in the incubation buffer; amphetamine produced a biphasic response, inhibiting synthesis at low tyrosine concentrations and activating synthesis at higher tyrosine concentrations. In contrast to the olfactory tubercle, amphetamine-induced stimulation of synthesis in the hypothalamus was observed at both low and high tyrosine concentrations. Distinct tissue differences in drug responsiveness were also observed following exposure to EGTA; synthesis was stimulated by EGTA in the olfactory tubercle but not in the hypothalamus. These data provide evidence for regional differences in catecholamine regulation in the brain, and suggest that some of these differences may be related to differential effects of calcium on catecholamine formation.

Catecholamine metabolism in brain slices: Determination of relevant precursor pool and the effects of elevated K +

Catecholamine synthesis from [ 3H]tyrosine was studied in slices of striatum, cerebellum and substantia nigra of mice. If low concentrations of tyrosine (less than 5.5 μM) were added to the incubation medium, the slices released significant amounts of tyrosine into the medium during the incubation. Kinetic analysis of the same experiments indicated that medium tyrosine and not tissue tyrosine was the appropriate precursor for both dopamine (DA) synthesis and protein synthesis in striatal slices. Concentrations of medium tyrosine of 8.25 μM or greater were sufficient to prevent changes of medium tyrosine during incubation and thus maintained a constant specific activity of precursor. Increasing concentrations of medium K + increased both the accumulation of [ 3H]DA and its release from striatal slices. However, accumulation was stimulated at a concentration of K + (14 mM) that had no significant stimulatory effect on release, suggesting that the stimulatory effects of K + on synthesis and release are mediated by separate processes. Release of 14CO 2 from [1- 14C]tyrosine closely paralleled the accumulation of [ 3H]DA from [ 3H]tyrosine. Release of preloaded [ 14C]DA closely paralleled that of [ 3H]DA synthesized from [ 3H]tyrosine, suggesting a common functional pool. The principal DA catabolite produced was dihydroxyphenylacetic acid (DOPAC). The appearance of labeled DOPAC in the media was greatly enhanced by K +stimulation.

Multicompartmental analysis of amino acids: III. Tyrosine in affective disorder

Multicompartmental studies of tyrosine in patients suffering from affective disorders and controls gave estimates of 2 major pools of amino acid, together with the associated fractional clearance rates and fluxes. The 2 pools were considered provisionally to represent extracellular and intracellular tyrosine. The concentration of tyrosine in plasma (representing the extracellular pool) and fractional clearance rates from this compartment were normal in ill and recovered patients. The observed abnormalities were confined to the intracellular compartment and consisted of low concentrations of tyrosine in both depressed and recovered patients. In addition, fractional clearance rates from the intracellular compartment were raised in the depressed patients but had returned to normal after recovery. A comparison of these data with those of a similar study of tryptophan described previously suggested that alterations in compartmental volume or dietary differences could not explain the 2 sets of findings. The disturbances in tryptophan metabolism in unipolar affective disorder had led to reduced amounts of this amino acid in the extracellular space. In contrast, the concentration of tyrosine in this compartment was normal, presumably as a result of a metabolic adjustment to the lower amounts found in the intracellular pool. The presence of illness-dependent and illness-independent alterations in tyrosine metabolism in unipolar affective disorder, together with the earlier findings for tryptophan, may have a bearing on the aetiology of the illness.

Evidence That the Intracellular Pool of Tyrosine Serves as Precursor for Protein Synthesis in Muscle

Several investigators suggested that labeled amino acids are incorporated into protein without first equilibrating with the intracellular pool. Experiments were performed to determine if the intracellular or extracellular [14C]tyrosine represented the precursor for protein synthesis in muscle. Four pieces of rat diaphragm were incubated in the same concentration of [3H]leucine but in different concentrations of [14C]tyrosine. The rates of [3H]leucine incorporation were similar in all the muscles, indicating similar rates of protein synthesis. The 14C counts in protein increased with increasing tyrosine concentrations in the medium, but the moles of tyrosine incorporated into protein were similar only when the counts in protein were divided by the intracellular specific activity. In other experiments muscles were incubated with [3H]leucine and [14C]tyrosine, and then transferred to medium containing [3H]leucine and unlabeled tyrosine. The rate of tyrosine incorporation agreed with leucine incorporation only when calculations used the intracellular tyrosine specific activity. Therefore, the free intracellular tyrosine resembled a precursor for protein synthesis, while the extracellular did not. However, this conclusion did not hold during very short incubations or incubations with low concentrations of tyrosine in the medium, for reasons suggested herein.