The effect of casein glycomacropeptide versus free synthetic amino acids for early treatment of phenylketonuria in a mice model.

INTRODUCTION: Management of phenylketonuria (PKU) is mainly achieved through dietary control with limited intake of phenylalanine (Phe) from food, supplemented with low protein (LP) food and a mixture of free synthetic (FS) amino acids (AA) (FSAA). Casein glycomacropeptide (CGMP) is a natural peptide released in whey during cheese making by the action of the enzyme chymosin. Because CGMP in its pure form does not contain Phe, it is nutritionally suitable as a supplement in the diet for PKU when enriched with specific AAs. Lacprodan® CGMP-20 (= CGMP) used in this study contained only trace amounts of Phe due to minor presence of other proteins/peptides. OBJECTIVE: The aims were to address the following questions in a classical PKU mouse model: Study 1, off diet: Can pure CGMP or CGMP supplemented with Large Neutral Amino Acids (LNAA) as a supplement to normal diet significantly lower the content of Phe in the brain compared to a control group on normal diet, and does supplementation of selected LNAA results in significant lower brain Phe level?. Study 2, on diet: Does a combination of CGMP, essential (non-Phe) EAAs and LP diet, provide similar plasma and brain Phe levels, growth and behavioral skills as a formula which alone consist of FSAA, with a similar composition?. MATERIAL AND METHODS: 45 female mice homozygous for the Pahenu2 mutation were treated for 12 weeks in five different groups; G1(N-CGMP), fed on Normal (N) casein diet (75%) in combination with CGMP (25%); G2 (N-CGMP-LNAA), fed on Normal (N) casein diet (75%) in combination with CGMP (19,7%) and selected LNAA (5,3% Leu, Tyr and Trp); G3 (N), fed on normal casein diet (100%); G4 (CGMP-EAA-LP), fed on CGMP (70,4%) in combination with essential AA (19,6%) and LP diet; G5 (FSAA-LP), fed on FSAA (100%) and LP diet. The following parameters were measured during the treatment period: Plasma AA profiles including Phe and Tyr, growth, food and water intake and number of teeth cut. At the end of the treatment period, a body scan (fat and lean body mass) and a behavioral test (Barnes Maze) were performed. Finally, the brains were examined for content of Phe, Tyr, Trp, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and 5-hydroxyindole-acetic acid (5-HIAA), and the bone density and bone mineral content were determined by dual-energy x-ray absorptiometry. RESULTS: Study 1: Mice off diet supplemented with CGMP (G1 (N-CGMP)) or supplemented with CGMP in combination with LNAA (G2 (N-CGMP-LNAA)) had significantly lower Phe in plasma and in the brain compared to mice fed only casein (G3 (N)). Extra LNAA (Tyr, Trp and Leu) to CGMP did not have any significant impact on Phe levels in the plasma and brain, but an increase in serotonin was measured in the brain of G2 mice compared to G1. Study 2: PKU mice fed with mixture of CGMP and EAA as supplement to LP diet (G4 (CGMP-EAA-LP)) demonstrated lower plasma-Phe levels but similar brain- Phe levels and growth as mice fed on an almost identical combination of FSAA (G5 (FSAA-LP)). CONCLUSION: CGMP can be a relevant supplement for the treatment of PKU.

A highly accurate mass spectrometry method for the quantification of phenylalanine and tyrosine on dried blood spots: Combination of liquid chromatography, phenylalanine/tyrosine-free blood calibrators and multi-point/dynamic calibration.

BACKGROUND: Measurement of quantitative levels of phenylalanine and tyrosine in blood is an essential test for the diagnosis of and monitoring genetic disorders associated with phenylalanine metabolism, such as phenylketonuria (PKU), tyrosinemia, and defects of tetrahydrobiopterin synthesis and recycling. We developed a highly accurate and fast liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the quantification of phenylalanine and tyrosine on dried blood spot (DBS). We also designed a performance score system to evaluate various calibration methods in matrix matched material. METHODS: Phenylalanine/tyrosine-free whole blood was used to make accurate and stable DBS calibrators. Six calibrators cover the range of 0-1000 µmol/L. Underivatized phenylalanine and tyrosine were extracted and measured by LC-MS/MS. Precision, accuracy, limit of quantification, recovery and carryover were validated. External quality assurance materials were also used to evaluate performance of multi-point calibrations and single-point calibrations. RESULTS: The run time was 4.5-minute. Accuracy analysis showed good agreement with reference materials. Precision, recovery, and the lower and upper limit of quantification met the criteria. When phenylalanine and tyrosine concentrations were less than 150 µmol/L, the 5-point calibration without the calibrator of 1000 µmol/L had the best performance. When the concentrations were > 250 µmol/L, the single-point calibration of 500 µmol/L had the best performance. CONCLUSION: We developed a simple, fast and highly accurate method for the detection of phenylalanine and tyrosine on DBS, with chromatographic separation and underivatized analysis. Based on the calibration performance, a 6-point calibration method is satisfying for this test. An optional dynamic calibration method, which includes 6-point calibration, 5-point calibration and single-point calibration, can further increase test reliability.

Beneficial Effects of Slow-Release Large Neutral Amino Acids after a Phenylalanine Oral Load in Patients with Phenylketonuria.

The mainstay of phenylketonuria treatment is a low protein diet, supplemented with phenylalanine (Phe)-free protein substitutes and micronutrients. Adhering to this diet is challenging, and even patients with good metabolic control who follow the dietary prescriptions in everyday life ignore the recommendations occasionally. The present study explores the ability of slow-release large neutral amino acids (srLNAAs) to prevent Phe increase following a Phe dietary load. Fourteen phenylketonuric patients aged ≥13 years were enrolled in a 6-week protocol. Oral acute Phe loads of 250 and 500 mg were added to the evening meal together with srLNAAs (0.5 gr/kg). Phe and tyrosine were dosed before dinner, 2h-after dinner, and after the overnight fast. After oral Phe loads, mean plasma Phe remained stable and below 600 µmol/L. No Phe peaks were registered. Tyrosine levels significantly increased, and Phe/Tyrosine ratio decreased. No adverse events were registered. In conclusion, a single oral administration of srLNAAs at the dose of 0.5 gr/kg is effective in maintaining stable plasma Phe during acute oral loads with Phe-containing food and may be added to the dietetic scheme in situations in which patients with generally good adherence to diet foresee a higher than prescribed Phe intake due to their commitments.

Simultaneous determination of 5-hydroxytryptophan and 3-O-methyldopa in dried blood spot by UPLC-MS/MS: A useful tool for the diagnosis of L-amino acid decarboxylase deficiency.

5-hydroxytryptophan (5HTP) and 3-O-methyldopa (3OMD) are CSF diagnostic biomarkers of the defect of aromatic L-amino acid decarboxylase (AADC), a rare inherited disorder of neurotransmitter synthesis which, if untreated, results in severely disabling neurological impairment. In the last few years, different methods to detect 3OMD in dried blood spot (DBS) were published. We developed and validated a fast and specific diagnostic tool to detect 5HTP alongside 3OMD. After extraction from DBS, 3OMD and 5HTP were separated by ultra-performance liquid chromatography (UPLC) and detected by tandem mass spectrometry (MS/MS). Instrument parameters were optimized to obtain the best sensitivity and specificity. Chromatographic separation was accomplished in 13 min. The limit of detection was 2.4 and 1.4 nmol/L of blood for 3OMD and 5HTP respectively, and response was linear over the blood range of 25-5000 nmol/L. Between-run imprecision was less than 9% for 3OMD and <13% for 5HTP. An age-specific continuous reference range was established, revealing a marked and continuous 3OMD decline with aging. The effect of age on 5HTP was less evident, showing only a slight decrease with age after the first week of life. A marked increase of both 3OMD and 5HTP was found in four patients affected by AADC deficiency (1780.6 ± 773.1 nmol/L, rv 71.0-144.9; and 94.8 ± 19.0 nmol/L, rv 15.2-42.8, respectively) while an isolated increase of 3OMD (6159.6 ± 3449.1 nmol/L, rv 73.2-192.2) was detected in three subjects affected by inherited disorders of dopamine synthesis under levodopa/carbidopa treatment (a marginal increase of 5HTP was detected in one of them). Simultaneous measurement of 5HTP and 3OMD in DBS leads to an improvement in specificity and sensitivity for the biochemical diagnosis of AADC deficiency.

Casein Glycomacropeptide: An Alternative Protein Substitute in Tyrosinemia Type I.

Tyrosinemia type I (HTI) is treated with nitisinone, a tyrosine (Tyr) and phenylalanine (Phe)-restricted diet, and supplemented with a Tyr/Phe-free protein substitute (PS). Casein glycomacropeptide (CGMP), a bioactive peptide, is an alternative protein source to traditional amino acids (L-AA). CGMP contains residual Tyr and Phe and requires supplementation with tryptophan, histidine, methionine, leucine, cysteine and arginine. AIMS: a 2-part study assessed: (1) the tolerance and acceptability of a low Tyr/Phe CGMP-based PS over 28 days, and (2) its long-term impact on metabolic control and growth over 12 months. METHODS: 11 children with HTI were recruited and given a low Tyr/Phe CGMP to supply all or part of their PS intake. At enrolment, weeks 1 and 4, caregivers completed a questionnaire on gastrointestinal symptoms, acceptability and ease of PS use. In study part 1, blood Tyr and Phe were assessed weekly; in part 2, weekly to fortnightly. In parts 1 and 2, weight and height were assessed at the study start and end. RESULTS: Nine of eleven children (82%), median age 15 years (range 8.6-17.7), took low Tyr/Phe CGMP PS over 28 days; it was continued for 12 months in n = 5 children. It was well accepted by 67% (n = 6/9), tolerated by 100% (n = 9/9) and improved gastrointestinal symptoms in 2 children. The median daily dose of protein equivalent from protein substitute was 60 g/day (range 45-60 g) with a median of 20 g/day (range 15 to 30 g) from natural protein. In part 2 (n = 5), a trend for improved blood Tyr was observed: 12 months pre-study, median Tyr was 490 µmol/L (range 200-600) and Phe 50 µmol/L (range 30-100); in the 12 months taking low Tyr/Phe CGMP PS, median Tyr was 430 µmol/L (range 270-940) and Phe 40 µmol/L (range 20-70). Normal height, weight and BMI z scores were maintained over 12 months. CONCLUSIONS: In HTI children, CGMP was well tolerated, with no deterioration in metabolic control or growth when studied over 12 months. The efficacy of CGMP in HTI needs further investigation to evaluate the longer-term impact on blood Phe concentrations and its potential influence on gut microflora.

Cardiac Magnetic Resonance Reveals Incipient Cardiomyopathy Traits in Adult Patients With Phenylketonuria.

Background Phenylketonuria is the most common inborn error of amino acid metabolism, where oxidative stress and collateral metabolic abnormalities are likely to cause cardiac structural and functional modifications. We aim herein to characterize the cardiac phenotype of adult subjects with phenylketonuria using advanced cardiac imaging. Methods and Results Thirty-nine adult patients with phenylketonuria (age, 30.5±8.7 years; 10-year mean phenylalanine concentration, 924±330 µmol/L) and 39 age- and sex-matched healthy controls were investigated. Participants underwent a comprehensive cardiac magnetic resonance and echocardiography examination. Ten-year mean plasma levels of phenylalanine and tyrosine were used to quantify disease activity and adherence to treatment. Patients with phenylketonuria had thinner left ventricular walls (septal end-diastolic thickness, 7.0±17 versus 8.8±1.7 mm [P<0.001]; lateral thickness, 6.1±1.4 versus 6.8±1.2 mm [P=0.004]), more dilated left ventricular cavity (end-diastolic volume, 87±14 versus 80±14 mL/m2 [P=0.0178]; end-systolic volume, 36±9 versus 29±8 mL/m2 [P<0.001]), lower ejection fraction (59±6% versus 64±6% [P<0.001]), reduced systolic deformation (global circumferential strain, -29.9±4.2 % versus -32.2±5.0 % [P=0.027]), and lower left ventricular mass (38.2±7.9 versus 47.8±11.0 g/m2 [P<0.001]). T1 native values were decreased (936±53 versus 996±26 ms [P<0.001]), with particular low values in patients with phenylalanine >1200 µmol/L (909±48 ms). Both mean phenylalanine (P=0.013) and tyrosine (P=0.035) levels were independently correlated with T1; and in a multiple regression model, higher phenylalanine levels and higher left ventricular mass associate with lower T1. Conclusions Cardiac phenotype of adult patients with phenylketonuria reveals some traits of an early-stage cardiomyopathy. Regular cardiology follow-up, tighter therapeutic control, and prophylaxis of cardiovascular risk factors, in particular dyslipidemia, are recommended.

Whole-Body Cryotherapy Increases the Activity of Nitric Oxide Synthase in Older Men.

Aging causes oxidative stress, endothelial dysfunction and a reduction in the bioavailability of nitric oxide. The study aim was to determine whether, as a result of repeated whole-body exposure to cryogenic temperature (3 min -130 °C), there is an increase of inducible nitric oxide synthase (iNOS) concentration in senior subjects (59 ± 6 years), and if this effect is stronger in athletes. In 10 long-distance runners (RUN) and 10 untraining (UTR) men, 24 whole-body cryotherapy (WBC) procedures were performed. Prior to WBC, after 12th and 24th treatments and 7 days later, the concentration of iNOS, asymmetric dimethylarginine (ADMA), 3-nitrotyrosine (3-NTR), homocysteine (HCY), C-reactive protein (CRP) and interleukins such as: IL-6, IL-1ß, IL-10 were measured. In the RUN and UTR groups, after 24 WBC, iNOS concentration was found to be comparable and significantly higher (F = 5.95, p < 0.01) (large clinical effect size) compared to before 1st WBC and after 12th WBC sessions. There were no changes in the concentration of the remaining markers as a result of WBC (p > 0.05). As a result of applying 24 WBC treatments, using the every-other-day model, iNOS concentration increased in the group of older men, regardless of their physical activity level. Along with this increase, there were no changes in nitro-oxidative stress or inflammation marker levels.

Effect of catechin and commercial preparation of green tea essence on the pharmacokinetics of l-dopa in rabbits.

The aim of this study was to investigate drug interactions of L-dopa/carbidopa with catechin and green tea essence in rabbits following the simultaneous administration via an intramuscular injection of catechin or via an intragastric route for green tea essence with L-dopa/carbidopa. The results indicated that catechin at doses of 10, 20 and 50 mg/kg increased the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration (AUC0-t ) of L-dopa by about 69, 78 and 42%, respectively. The metabolic ratios of the AUC0-t for 3-O-methyldopa (3-OMD)/L-dopa significantly decreased by about 56, 68 and 76% (P < 0.05), respectively. In addition, a single dose of 5/1.25 mg/kg L-dopa/carbidopa was co-administrated with 150 mg/kg green tea essence via an intragastric route with an oral-gastric tube. Comparing the related pharmacokinetic parameters of L-dopa, the clearance and metabolic ratio of L-dopa decreased by 20 and 19% (P < 0.05), respectively. In conclusion, catechin and green tea essence can significantly affect the metabolism of L-dopa by the catechol-O-methyltransferase (COMT) metabolic pathway. Catechin can enhance L-dopa bioavailability, and both catechin and green tea essence decreased 3-OMD formation. Therefore, catechin and green tea essence may increase L-dopa efficacy for Parkinson's disease treatment.

Easy clinical predictor for low BCAA to tyrosine ratio in chronic liver disease patients with hepatocellular carcinoma: Usefulness of ALBI score as nutritional prognostic marker.

BACKGROUND/AIM: Low branched-chain amino acid (BCAA) to tyrosine ratio (BTR) is known as an indicator of amino acid imbalance. We elucidated usefulness of newly developed albumin-bilirubin (ALBI) score as alternative methods of BTR in patients with naïve hepatocellular carcinoma (HCC) retrospectively. MATERIALS/METHODS: In 842 patients with HCC and without BCAA supplementation (71 years, male 614, Child-Pugh A:B:C = 689:116:37), relationships among BTR and clinical features were evaluated. Of those, 438 patients, with Milan criteria HCC, treated curatively were divided into the high-BTR (>4.4) (n = 293) and low-BTR (≤4.4) (n = 145) groups. The prognostic value of BTR was evaluated using inverse probability weighting (IPW) with propensity score. RESULTS: The low-BTR group showed worse prognosis than the other (3-, 5-, 10-year overall survival rates: 88.9% vs. 86.3%/70.5% vs. 78.1%/38.1% vs. 52.3%, respectively; p < 0.001). Multivariate Cox-hazard analysis adjusted for IPW showed elderly (≥65 years) HR 2.314, p = 0.001), female gender (HR 0.422, p < 0.001), ECOG PS ≥2 (HR 3.032, p = 0.002), low platelet count (HR 1.757, p = 0.010), and low BTR (≤4.4) (HR 1.852, p = 0.005) to be significant prognostic factors. Both serum albumin level (r = 0.370, p < 0.001) and ALBI score (r = -0.389, p < 0.001) showed a significant relationship with BTR. Child-Pugh class B, modified ALBI grade (mALBI) 2a, and mALBI 2b predictive values for BTR were 3.589, 4.509, and 4.155 (AUC range: 0.735-0.770), respectively, while the predictive value of ALBI score for low-BTR (≤4.4) was -2.588 (AUC 0.790). CONCLUSION: ALBI score -2.588 was a predictor for low-BTR (≤4.4), which was prognostic factors for early HCC patients, and at least patients with mALBI 2b might have an amino acid imbalance.

Serum tyrosine is associated with better cognition in Lewy body dementia.

Amino acids' neuroactivity, and roles in excitotoxity and oxidative stress are linked to dementia. We aimed to investigate whether circulating amino acid concentrations were associated with cognitive decline in patients with mild Alzheimer's disease (AD) and Lewy body dementia (LBD). Baseline serum amino acid concentrations were measured in 89 patients with AD and 65 with LBD (13 with Parkinson's disease dementia and 52 with dementia with Lewy bodies). The Mini-Mental State Examination (MMSE) was administered at baseline and annually for five years. Associations between baseline amino acid concentrations and longitudinal MMSE score were assessed using a linear-mixed effects model stratified by diagnosis with adjustment for multiple comparisons. The results of the study indicated that serum tyrosine was positively associated with MMSE performance during the five-year follow-up period in patients with LBD (q-value = 0.012), but not AD. In conclusion, higher baseline serum concentrations of tyrosine, the precursor amino acid in dopamine and norepinephrine synthesis, was associated with better cognitive performance in patients with LBD, but not AD, throughout the 5-year follow-up period.

Detection of 3-O-methyldopa in dried blood spots for neonatal diagnosis of aromatic L-amino-acid decarboxylase deficiency: The northeastern Italian experience.

OBJECTIVE: Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare inherited autosomal recessive disorder of biogenic amine metabolism. Diagnosis requires analysis of neurotransmitter metabolites in cerebrospinal fluid, AADC enzyme activity analysis, or molecular analysis of the DDC gene. 3-O-methyldopa (3-OMD) is a key screening biomarker for AADC deficiency. METHODS: We describe a rapid method for 3-OMD determination in dried blood spots (DBS) using flow-injection analysis tandem mass spectrometry with NeoBase™ 2 reagents and 13C6-tyrosine as an internal standard, which are routinely used in high-throughput newborn screening. We assessed variability using quality control samples over a range of 3-OMD concentrations. RESULTS: Within-day and between-day precision determined with quality control samples demonstrated coefficients of variation <15%. 3-OMD concentrations in 1000 healthy newborns revealed a mean of 1.33 µmol/L (SD ± 0.56, range 0.61-3.05 µmol/L), 100 non-AADC control subjects (age 7 days - 1 year) showed a mean of 1.19 µmol/L (SD ± 0.35-2.00 µmol/L), and 81 patients receiving oral L-Dopa had a mean 3-OMD concentration of 14.90 µmol/L (SD ± 14.18, range 0.4-80.3 µmol/L). A patient with confirmed AADC was retrospectively analyzed and correctly identified (3-OMD 10.51 µmol/L). In April 2020, we started a pilot project for identifying AADC deficiency in DBSs routinely submitted to the expanded newborn screening program. 3-OMD concentrations were measured in 21,867 samples; no patients with AADC deficiency were identified. One newborn had a high 3-OMD concentration due to maternal L-Dopa treatment. DISCUSSION: We demonstrated a rapid new method to identify AADC deficiency using reagents and equipment already widely used in newborn screening programs. Although our study is limited, introduction of our method in expanded neonatal screening is feasible and could facilitate deployment of screening, allowing for early diagnosis that is important for effective treatment.

Effect of BH4 on blood phenylalanine and tyrosine variations in patients with phenylketonuria.

BACKGROUND: In patients with phenylketonuria, stability of blood phenylalanine and tyrosine concentrations might influence brain chemistry and therefore patient outcome. This study prospectively investigated the effects of tetrahydrobiopterin (BH4), as a chaperone of phenylalanine hydroxylase on diurnal and day-to-day variations of blood phenylalanine and tyrosine concentrations. METHODS: Blood phenylalanine and tyrosine were measured in dried blood spots (DBS) four times daily for 2 days (fasting, before lunch, before dinner, evening) and once daily (fasting) for 6 days in a randomized cross-over design with a period with BH4 and a period without BH4. The sequence was randomized. Eleven proven BH4 responsive PKU patients participated, 5 of them used protein substitutes during BH4 treatment. Natural protein intake and protein substitute dosing was adjusted during the period without BH4 in order to keep DBS phenylalanine levels within target range. Patients filled out a 3-day food diary during both study periods. Variations of DBS phenylalanine and Tyr were expressed in standard deviations (SD) and coefficient of variation (CV). RESULTS: BH4 treatment did not significantly influence day-to-day phenylalanine and tyrosine variations nor diurnal phenylalanine variations, but decreased diurnal tyrosine variations (median SD 17.6 µmol/l, median CV 21.3%, p = 0.01) compared to diet only (median SD 34.2 µmol/l, median CV 43.2%). Consequently, during BH4 treatment diurnal phenylalanine/tyrosine ratio variation was smaller, while fasting tyrosine levels tended to be higher. CONCLUSION: BH4 did not impact phenylalanine variation but decreased diurnal tyrosine and phenylalanine/tyrosine ratio variations, possibly explained by less use of protein substitute and increased tyrosine synthesis.

Using circulating O-sulfotyrosine in the differential diagnosis of acute kidney injury and chronic kidney disease.

BACKGROUND: Sulfation of tyrosine, yielding O-sulfotyrosine, is a common but fixed post-translational modification in eukaryotes. Patients with increased circulating O-sulfotyrosine levels experience a faster decline in renal function with progression to end-stage renal disease (ESRD). In the present study, we measured serum O-sulfotyrosine levels in individuals with chronic kidney disease (CKD) and acute kidney injury (AKI) to explore its ability to differentiate AKI from CKD. METHODS: A total of 135 patients (20 with AKI and 115 with CKD) were recruited prospectively for liquid chromatography-mass spectrometry assessment of circulating O-sulfotyrosine. We also studied C57BL/6 mice with CKD after 5/6 nephrectomy (Nx). Blood samples were drawn from the tail vein on Day 1, 3, 5, 7, 14, 30, 60, and 90 after CKD. Serum separation and characterization of creatinine, blood urea nitrogen (BUN), and O-sulfotyrosine was performed. Thus, the time-concentration curves of the O-sulfotyrosine level demonstrate the variation of kidney dysfunction. RESULTS: The serum levels of O-sulfotyrosine were markedly increased in patients with CKD compared with AKI. Median O-sulfotyrosine levels in CKD patients versus AKI, respectively, were as follows:243.61 ng/mL(interquartile range [IQR] = 171.90-553.86) versus 126.55 ng/mL (IQR = 48.19-185.03, P = 0.004). In patients with CKD, O-sulfotyrosine levels were positively correlated with creatinine, BUN, and Cystatin C (r = 0.63, P < 0.001; r = 0.49, P < 0.001; r = 0.61, P < 0.001, respectively) by the multivariate linear regression analysis (ß = 0.71, P < 0.001; ß = 0.40, P = 0.002; ß = 0.73, P < 0.001, respectively). However, this association was not statistically significant in patients with AKI (r = - 0.17, P = 0.472; r = 0.11, P = 0.655; r = 0.09, P = 0.716, respectively). The receiver operating characteristic (ROC) analysis illustrated that the area under the curve was 0.80 (95% confidence interval [CI] 0.71-0.89; P < 0.001) and the optimal cut-off value of serum O-sulfotyrosine suggesting AKI was < 147.40 ng/mL with a sensitivity and specificity of 80.90 and 70.00% respectively. In animal experiments, serum levels of O-sulfotyrosine in mice were elevated on Day 7 after 5/6 nephrectomy (14.89 ± 1.05 vs. 8.88 ± 2.62 ng/mL, P < 0.001) until Day 90 (32.65 ± 5.59 vs. 8.88 ± 2.62 ng/mL, P < 0.001). CONCLUSION: Serum O-sulfotyrosine levels were observed correlated with degrading renal function and in CKD patients substantially higher than those in AKI patients. Thus serum O-sulfotyrosine facilitated the differential diagnosis of AKI from CKD.

Metabolites and diabetes remission after weight loss.

There is marked heterogeneity in the response to weight loss interventions with regards to weight loss amount and metabolic improvement. We sought to identify biomarkers predictive of type 2 diabetes remission and amount of weight loss in individuals with severe obesity enrolled in the Longitudinal Assessment of Bariatric Surgery (LABS) and the Look AHEAD (Action for Health in Diabetes) studies. Targeted mass spectrometry-based profiling of 135 metabolites was performed in pre-intervention blood samples using a nested design for diabetes remission over five years (n = 93 LABS, n = 80 Look AHEAD; n = 87 remitters), and for extremes of weight loss at five years (n = 151 LABS; n = 75 with high weight loss). Principal components analysis (PCA) was used for dimensionality reduction, with PCA-derived metabolite factors tested for association with both diabetes remission and weight loss. Metabolic markers were tested for incremental improvement to clinical models, including the DiaRem score. Two metabolite factors were associated with diabetes remission: one primarily composed of branched chain amino acids (BCAA) and tyrosine (odds ratio (95% confidence interval) [OR (95% CI)] = 1.4 [1.0-1.9], p = 0.045), and one with betaine and choline (OR [95% CI] = 0.7 [0.5-0.9], p = 0.02).These results were not significant after adjustment for multiple tests. Inclusion of these two factors in clinical models yielded modest improvements in model fit and performance: in a constructed clinical model, the C-statistic improved from 0.87 to 0.90 (p = 0.02), while the net reclassification index showed improvement in prediction compared to the DiaRem score (NRI = 0.26, p = 0.0013). No metabolite factors associated with weight loss at five years. Baseline levels of metabolites in the BCAA and trimethylamine-N-oxide (TMAO)-microbiome-related pathways are independently and incrementally associated with sustained diabetes remission after weight loss interventions in individuals with severe obesity. These metabolites could serve as clinically useful biomarkers to identify individuals who will benefit the most from weight loss interventions.

N-acetyltyrosine-induced redox signaling in hormesis.

A suite of adaptations allows insects to survive in hostile terrestrial environments for long periods of time. Temperature represents a key environmental factor for most ectothermic insects, and they rapidly acclimate to high and low temperatures. Vast amounts of data in this research field support the idea that an insect's ability to tolerate fluctuating temperatures can be regarded as a biphasic hormetic dose response. Observation indicates that their thermal hormetic response represents a conservative estimate of their intrinsic capacity for rapid adaptation to environmental changes in nature because they naturally experience diel or seasonal temperature fluctuations. It is therefore reasonable to suppose that the hormetic response in insects reflects a surplus physiological capacity to deal with temperature changes that they would experience naturally. Although it has been unknown how thermal acclimation is induced, a stress-dependent increase in N-acetyltyrosine (NAT) was recently found to occur in insect larvae who had endured high temperatures. NAT treatment was demonstrated to induce thermotolerance in several tested insect species. NAT was also identified in the serum of humans as well as mice, and its concentration in mice was shown to be increased by heat and restraint stress, with NAT pretreatment lowering the concentrations of corticosterone and peroxidized lipids in stressed mice. These recent findings may give us some hints about how long a hormetic response lasts. Here, I will discuss recent findings underlying hormetic responses induced by an intrinsic factor, NAT, and how the hormetic response may begin and end.

Tyrosine supplementation for phenylketonuria.

BACKGROUND: Phenylketonuria is an inherited disease for which the main treatment is the dietary restriction of the amino acid phenylalanine. The diet has to be initiated in the neonatal period to prevent or reduce mental handicap. However, the diet is very restrictive and unpalatable and can be difficult to follow. A deficiency of the amino acid tyrosine has been suggested as a cause of some of the neuropsychological problems exhibited in phenylketonuria. Therefore, this review aims to assess the efficacy of tyrosine supplementation for phenylketonuria. This is an update of previously published versions of this review. OBJECTIVES: To assess the effects of tyrosine supplementation alongside or instead of a phenylalanine-restricted diet for people with phenylketonuria, who commenced on diet at diagnosis and either continued on the diet or relaxed the diet later in life. To assess the evidence that tyrosine supplementation alongside, or instead of a phenylalanine-restricted diet improves intelligence, neuropsychological performance, growth and nutritional status, mortality rate and quality of life. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Trials Register which is comprised of references identified from comprehensive electronic database searches, handsearches of relevant journals and abstract books of conference proceedings. Additional studies were identified from handsearches of the Journal of Inherited Metabolic Disease (from inception in 1978 to 1998). The manufacturers of prescribable dietary products used in the treatment of phenylketonuria were also contacted for further references. Date of the most recent search of the Group's Inborn Errors of Metabolism Trials Register: 07 December 2020. SELECTION CRITERIA: All randomised or quasi-randomised trials investigating the use of tyrosine supplementation versus placebo in people with phenylketonuria in addition to, or instead of, a phenylalanine-restricted diet. People treated for maternal phenylketonuria were excluded. DATA COLLECTION AND ANALYSIS: Two authors independently assessed the trial eligibility, methodological quality and extracted the data. MAIN RESULTS: Six trials were found, of which three trials reporting the results of a total of 56 participants, were suitable for inclusion in the review. The blood tyrosine concentrations were significantly higher in the participants receiving tyrosine supplements than those in the placebo group, mean difference 23.46 (95% confidence interval 12.87 to 34.05). No significant differences were found between any of the other outcomes measured. The trials were assessed as having a low to moderate risk of bias across several domains. AUTHORS' CONCLUSIONS: From the available evidence no recommendations can be made about whether tyrosine supplementation should be introduced into routine clinical practice. Further randomised controlled studies are required to provide more evidence. However, given this is not an active area of research, we have no plans to update this review in the future.

A targeted ultra performance liquid chromatography - Tandem mass spectrometric assay for tyrosine and metabolites in urine and plasma: Application to the effects of antibiotics on mice.

Tyrosine plays a key role in mammalian biochemistry and defects in its metabolism (e.g., tyrosinemia, alkaptonuria etc.) have significant adverse consequences for those affected if left untreated. In addition, gut bacterially-derived p-cresol and its metabolites are of interest as a result of various effects on host xenobiotic metabolism. A fit-for-purpose quantitative ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay was developed to target and quantify tyrosine and eleven metabolites in urine and plasma. Dansylation, using dansyl chloride, was used to improve chromatographic and mass spectral properties for tyrosine and nine phenolic metabolites, with detection using positive electrospray ionisation (ESI). The sulfate and glucuronide conjugates of p-cresol, where the phenol group was blocked, were quantified intact, using negative ESI via polarity switching during the same run. Sample preparation for urine and plasma involved deproteinization by solvent precipitation (of acetonitrile:isopropyl alcohol (1:1 v/v)) followed by in situ dansylation in 96 well plates. To minimize sample and solvent usage, and maximize sensitivity, analysis was performed using microbore reversed-phase gradient UPLC on a C8 phase with a 7.5 min. cycle time. The coefficients of variation obtained were <15%, with lower limits of quantification ranging from 5 to 250 nM depending upon the analyte. The method was applied to plasma and urine samples obtained from mice placed on a high tyrosine diet with one subgroup of animals subsequently receiving antibiotics to suppress the gut microbiota. Whilst plasma profiles were largely unaffected by antibiotic treatment clear reductions in the amount of p-cresol sulfate and p-cresol glucuronide excreted in the urine were observed for these mice.

Assessment of serum phenylalanine and tyrosine isomers in patients with ST-segment elevation vs non-ST-segment elevation myocardial infarction.

BACKGROUND: Under conditions of oxidative stress, hydroxyl radicals can oxidize phenylalanine (Phe) into various tyrosine (Tyr) isomers (meta-, ortho-, and para-tyrosine; m-, o-, and p-Tyr), depending on the location of the hydroxyl group on the oxidized benzyl ring. This study aimed to compare patients with ST-segment elevation myocardial infarction (STEMI) and non-STEMI (NSTEMI) and the serum levels of Phe and Tyr isomers at the aortic root and distal to the culprit lesion in both groups. METHODS: Forty-four patients participated in the study: 23 with STEMI and 21 with NSTEMI. Arterial blood samples were taken from the aortic root through a guiding catheter and from the culprit vessel segment distal from the primary lesion with an aspiration catheter, during the percutaneous coronary intervention. Serum levels of Phe, p-Tyr, m-Tyr, and o-Tyr were determined using reverse-phase high-performance liquid chromatography. RESULTS: Serum levels of Phe were significantly higher distal to the culprit lesion compared to the aortic root in patients with STEMI. Serum p-Tyr/Phe and m-Tyr/Phe concentration ratios were both lower distal to the culprit lesion than at the aortic root in patients with STEMI. There were no statistically significant differences with respect to changes in serum Phe and Tyr isomers distal to the culprit lesion compared to the aortic root in patients with NSTEMI. CONCLUSION: Our data suggest that changes in serum levels of different Tyr isomers can mediate the effects of oxidative stress during myocardial infarction.

Simultaneous determination of 30 neurologically and metabolically important molecules: A sensitive and selective way to measure tyrosine and tryptophan pathway metabolites and other biomarkers in human serum and cerebrospinal fluid.

Concurrent measurement of tyrosine, tryptophan and their metabolites, and other co-factors could help to diagnose and better understand a wide range of metabolic and neurological disorders. The two metabolic pathways are closely related to each other through co-factors, regulator molecules and enzymes. By using high performance liquid chromatography coupled to electrospray ionization triple quadrupole mass spectrometry, we present a robust, selective and comprehensive method to determine 30 molecules within 20 min using a Waters Atlantis dC18. The method was validated according to the guideline of European Medicines Agency on bioanalytical method validation. Analytical performance met all the EMA requirements and the assay covered the relevant clinical concentrations. Linear correlation coefficients were all >0.998. Intra-day and inter-day accuracy were between 80-119% and 81-117%, precision 1-19% respectively. The method was applied to measure TYR, TRP and their metabolites, and other neurologically important molecules in human serum and CSF samples. The assay can facilitate the diagnosis and is suitable for determination of reference values in clinical laboratories.