High-performance Liquid Chromatography System Research Articles

Prevalence of hemoglobin variants and hemoglobinopathies using cation-exchange high-performance liquid chromatography in central reference laboratory of India: A report of 65779 cases.

CONTEXT:Hemoglobinopathies constitute the world's most common genetically inherited red blood cell disorder. Screening and accurate identification of hemoglobin (Hb) variants have become increasingly important in antenatal diagnosis and prevention of Hb disorders.AIM:The aim of this study was to screen and identify Hb fractions prevalent in the Central Reference Laboratory of India.MATERIALS AND METHODS:A total of 65,779 cases were screened for hemoglobinopathies on the bio-rad variant high-performance liquid chromatography (HPLC) system by beta-thalassemia short program. The retention times, proportion of the hemoglobin (%) and the peak characteristics for all hemoglobin fractions were recorded. Molecular analysis of the beta-globin gene was carried out by DNA sequencing on eight cases.RESULTS:Total number of abnormal Hb fractions on cation exchange-HPLC (CE-HPLC) was seen in 12,131 (18.44%) cases. Beta-thalassemia trait was the predominant genetic Hb disorder accounting for 7377 cases (11.21%) of the total cases. This was followed by sickle cell trait (2.01%), sickle cell disease (1.59%), beta-thalassemia syndrome (0.80%), HbE trait (0.79%), and borderline HbA2 (0.51%). Molecular characterization of eight rare cases of hemoglobin variants by beta-globin gene sequencing identified three cases of Hb Beth Israel, two cases of Hb Hofu trait, and one case each of Hb J Cambridge, Hb Mizunami, and Hb Sherwood Forest.CONCLUSION:Superior resolution, rapid assay time, and accurate quantification make CE-HPLC suitable for the routine investigation of hemoglobinopathies.

Antithrombotic activity and saponin composition of the roots of Panax bipinnatifidus Seem. growing in Vietnam

Background: Panax bipinnatifidus (PB) Seem. is a highly valuable and indigenous medicinal plant wildly distributed in the northwest region of Vietnam. The PB root has been used as a tonic in Vietnamese traditional medicine, but its chemical profile and pharmacological activity have not been extensively studied. Objective: In this study, we investigated the major saponin components and antithrombosis activity of the root of PB. Materials and Methods: Individual saponins were isolated by column chromatography. The structural elucidation of the isolated saponins was based on analyses of nuclear magnetic resonance and mass spectrometry spectra. Chromatographic fingerprint analysis of the isolated compounds was run on an Agilent 1260 high-performance liquid chromatography (HPLC) system. To evaluate antithrombotic activity, in vitro anticoagulant, platelet aggregation, and clot lysis effects were successively tested by the respective protocols. Results: Two principal saponins were isolated from the butanolic fraction of the root of PB, and their structures were identified as stipuleanoside R2 (1) and araloside A methyl ester (2). The HPLC analysis suggested that the two isolated saponins are major saponins of the title plant. The biological testing showed that both the crude and butanolic extracts of PB exhibited significantly antiplatelet aggregation activity. The efficacy linearly increased according to the tested doses (0.5–5 mg/mL). On anticoagulant activity, only butanolic extract exhibited positive action via prolonged activated partial thromboplastin time and prothrombin time at the high dose. Conclusion: These results suggested that PB Seem. might bring benefits for preventing cardiovascular events through inhibition of platelet aggregation. Abbreviations Used: ADP: Adenosine diphosphate; APTT: Activated partial thromboplastin time; DMSO: Dimethyl sulfoxide; ESI-MS: Electrospray ionization-mass spectrometry; HPLC: High-performance liquid chromatography; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; MAPK: Mitogen-activated protein kinase; MPA: Maximum platelet aggregation; NIMM: National Institute of Medicinal Materials; NMR: Nuclear magnetic resonance; PPP: Platelet-poor plasma;; PRP: Platelet-rich plasma; PT: Prothrombin time; TLC: Thin-layer chromatography; VNU: Vietnam National University.

Determination of Estrogens in Milk Using Polypyrrole Fiber-Mediated Solid-Phase Extraction Followed by High Performance Liquid Chromatography

A solid-phase extraction (SPE) method based on conductive polypyrrole (PPy) nanofibers which were fabricated by electrospinning and in situ polymerization was developed. PPy nanofibers-mediated SPE followed by high performance liquid chromatography (HPLC) was used for the determination of three synthetic estrogens, namely diethylstilbestrol (DES), dienestrol (DIS), or hexestrol (HS) in milk sample. Extraction conditions including extraction nanofibers, donor solution pH and salt concentration were optimized. The target compounds were extracted from a 0.5 mL aqueous sample at pH 5.0 through PPy fibers, and then were eluted with 0.1 mL methanol. After extraction, the eluant was directly injected into an HPLC system for detection. Under the optimized extraction conditions, a large enrichment factor was achieved for three estrogens. The limit of detection (LOD) at a signal to noise ratio (S/N) of 3 ranged from 0.02 to 0.05 µg mL-1 for the estrogens in milk sample.

Characterisation of purified acetylcholinesterase (EC 3.1.1.7) from <i>Oreochromis mossambica</i> brain tissues

This study reports on the characterization of a purified AChE from Oreochromis mossambica brain extract. The purification protocol involved the application of custom-synthesized affinity chromatography gel (Edrophonium–Sephacryl S-400) and the use of high performance liquid chromatography system (HPLC). Soluble AChE was partially purified 27.9 fold with a highest specific activity around 73.1 × 103 U/mg proteins. The partially purified AChE higher capability to hydrolyse acetylthiocholine (ATC) and shows less degradation against propionylthiocholine (PTC) and also butyrylthiocholine (BTC). Based on enzyme kinetic analysis, the partially purified AChE exhibits the apparent Michaelis constants Km, for ATC, PTC and BTC in the range of 125, 260 and 600 µM and the maximum velocities Vmax were 276, 59 and 36 µmol/min/mg protein, respectively. The apparent inhibition constant (ki) values of eserine, propidium and carbofuran were 0.24 µM-1min-1, 65 µM-1min-1 and 0.41 µM-1min-1 µM-1min-1, respectively. The purified enzyme is apparently an AChE since it capable to hydrolyzes ATC at a higher rate compared to other synthetic substrates, at pH 7.0 and 25ºC, and is inhibited by it specific inhibitor which is eserine but not by iso-OMPA.

The Herb-Drug Pharmacokinetic Interaction of 5-Fluorouracil and Its Metabolite 5-Fluoro-5,6-Dihydrouracil with a Traditional Chinese Medicine in Rats.

Background: Xiang-Sha-Liu-Jun-Zi-Tang (XSLJZT) is the most common traditional formula given to colorectal and breast cancer patients in Taiwan, according to a statistical study of the National Health Insurance Research Database. 5-Fluorouracil (5-FU) is widely used as the first line of treatment for colorectal cancer. Thus, the aim of study is to investigate the pharmacokinetic interaction of XSLJZT and 5-FU. Methods: To investigate the herb–drug interaction of XSLJZT with 5-FU as well as its metabolite 5-fluoro-5,6-dihydrouracil (5-FDHU) using pharmacokinetics, a high-performance liquid chromatography (HPLC) system coupled with a photodiode array detector was developed to monitor 5-FU and 5-FDHU levels in rat blood. Rats were divided into three cohorts, one of which was administered 5-FU (100 mg/kg, iv—intravenous) alone, while the other two groups were pretreated with low and high doses of XSLJZT (600 mg/kg/day or 2400 mg/kg/day for 5 consecutive days) in combination with 5-FU. Results: The results demonstrated that 5-FU level was not significantly different between the group treated with only 5-FU and the group pretreated with a normal dose of XSLJZT (600 mg/kg/day). However, pharmacokinetic analysis revealed that pretreatment with a high dose of XSLJZT (2400 mg/kg/day) extended the residence time and increased the volume of distribution of 5-FU. No significant distinctions were found in 5-FDHU pharmacokinetic parameters at three doses of XSLJZT. Conclusions: Overall, the pharmacokinetic results confirm the safety of coadministering 5-FU with XSLJZT, and provide practical dosage information for clinical practice.

An automated magnetic dispersive micro-solid phase extraction in a fluidized reactor for the determination of fluoroquinolones in baby food samples

An automated magnetic dispersive micro-solid phase extraction procedure in a fluidized reactor was developed for the determination of fluoroquinolone antimicrobial drugs (fleroxacin, norfloxacin and ofloxacin) in meat-based baby food samples. A stepwise injection system was successfully combined with afluidized reactorand applied for the magnetic dispersive micro-solid phase extraction procedure automation. The developed automated procedure involved injection of the sample solution into the fluidized reactor followed by the on-line separation of the analytes from the sample matrix based on fluidized beds strategy using magnetic nanoparticles, elution and determination of the analytes using a high performance liquid chromatography system with fluorescence detection. The floating of the magnetic nanoparticles in a liquid sample phase was accomplished by air-bubbling. In the developed method Zr-Fe-C magnetic nanoparticles were used as an efficient sorbent for the determination of fleroxacin, norfloxacin and ofloxacin. Under the optimal conditions, the calibration graphs were linear over the concentration ranges of 10–1000 μg L−1 for fleroxacin (R2 = 0.996), 5–1000 μg L−1for norfloxacin (R2 = 0.998) and ofloxacin (R2 = 0.998). The limits of detection, calculated from the blank tests based on 3σ, were 3.0 μg L−1forfleroxacin, 1.5 μg L−1for norfloxacin and ofloxacin. The limits of quantification, calculated from the blank tests based on 10σ, were 10 μg L−1 forfleroxacin, 5 μg L−1for norfloxacin and ofloxacin. The method was applied for the determination of fluoroquinolonesin meat-based baby food samples and the results were compared with those obtained by the reference method. The recovery values for all analytes were within of 86–122% range.

Miniaturised electrically actuated high pressure injection valve for portable capillary liquid chromatography

A miniaturised high pressure 6-port injection valve has been designed and evaluated for its performance in order to facilitate the development of portable capillary high performance liquid chromatography (HPLC). The electrically actuated valve features a very small size (65 × 19 × 19mm) and light weight (33g), and therefore can be easily integrated in a miniaturised modular capillary LC system suited for portable field analysis. The internal volume of the injection valve was determined as 98 nL. The novel conical shape of the stator and rotor and the spring-loaded rotor performed well up to 32MPa (4641psi), the maximum operating pressure investigated. Suitability for application was demonstrated using a miniaturised capillary LC system applied to the chromatographic separation of a mixture of biogenic amines and common cations. The RSD (relative standard deviation) values of retention times and peak areas of 6 successive runs were 0.5–0.7% and 1.8–2.8% for the separation of biogenic amines, respectively, and 0.1–0.2% and 2.1–3.0% for the separation of cations, respectively. This performance was comparable with bench-top HPLC systems thus demonstrating the applicability of the valve for use in portable and miniaturised capillary HPLC systems.

Study of Dopamine Mechanism in Striatum When Voluntary Wheel Running Improves Recovery of Cognitive Function After Global Cerebral Ischemia

Physical exercise has been demonstrated to be neuroprotective in ischemia models. However, the exact neuroprotective mechanism needs to be further studied. The purpose of this research is to discuss the effect of pre-ischemic voluntary wheel running on levels of dopamine in the striatum and cognition in ischemia rats. 12 Sprague-Dawley rats were divided randomly in following two groups: 0 week pre-ischemia wheel running （0-WR ）group and 4 weeks pre-ischemia wheel running （4-WR ）group. After training, global cerebral ischemia was induced by two-vessel occlusion (2-VO). Microdialysis and high performance liquid chromatography system (HPLC) were used to collect and determine dopamine in the dialysates of striatum. Passive avoidance was used to test cognitive function 24 h after ischemia. The results showed that striatal dopamine of 0-WR group was rapid increasing after ischemia, but the increase of striatal dopamine level was attenuated in 4 weeks group. Behavioral data indicated that voluntary wheel running promoted cognition recovery after ischemia. Thus, pre-ischemia voluntary wheel running attenuates the increase of dopamine of striatum induced by cerebral ischemia and improves cognitive function in ischemia rats.

Role of CYP24A1, VDR and GC gene polymorphisms on deferasirox pharmacokinetics and clinical outcomes

β-Thalassemia patients develop deficiency in vitamin D absorption and liver hydroxylation, resulting in extremely low calcitriol levels. We explored the role of single-nucleotide polymorphisms (SNPs) involved in vitamin D metabolism, transport and activity on deferasirox pharmacokinetics and outcomes (effectiveness trough levels (Ctrough) and the area under the curve (AUC) cutoffs of 20 μg ml-1 and 360 μg ml-1 h-1, respectively; nonresponse AUC limit of 250 μg ml-1 h-1). Ninety-nine β-thalassemic patients were enrolled. Drug plasma Ctrough and AUC were measured by the high-performance liquid chromatography system coupled with an ultraviolet determination method. Allelic discrimination for VDR, CYP24A1, CYP27B1 and GC gene SNPs was performed by real-time PCR. CYP24A1 22776 TT significantly influenced Cmin and negatively predicted it in regression analysis. CYP24A1 3999 CC was associated with Ctrough and Cmin and was a negative predictor of Tmax, whereas CYP24A1 8620 GG seemed to have a role in Ctrough, AUC, t1/2 and Cmin, and was an AUC negative predictor factor. Considering treatment outcome, Cdx2 and GC 1296 were retained in regression analysis as AUC efficacy cutoff negative predictors.

Characterization of Byproducts from Chemical Syntheses of Oligonucleotides Containing 1-Methyladenine and 3-Methylcytosine.

Oligonucleotides serve as important tools for biological, chemical, and medical research. The preparation of oligonucleotides through automated solid-phase synthesis is well-established. However, identification of byproducts generated from DNA synthesis, especially from oligonucleotides containing site-specific modifications, is sometimes challenging. Typical high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gel electrophoresis methods alone are not sufficient for characterizing unexpected byproducts, especially for those having identical or very similar molecular weight (MW) to the products. We used a rigorous quality control procedure to characterize byproducts generated during oligonucleotide syntheses: (1) purify oligonucleotides by different HPLC systems; (2) determine exact MW by high-resolution MS; (3) locate modification position by MS/MS or exonuclease digestion with matrix-assisted laser desorption ionization-time of flight analysis; and (4) conduct, where applicable, enzymatic assays. We applied these steps to characterize byproducts in the syntheses of oligonucleotides containing biologically important methyl DNA adducts 1-methyladenine (m1A) and 3-methylcytosine (m3C). In m1A synthesis, we differentiated a regioisomeric byproduct 6-methyladenine, which possesses a MW identical to uncharged m1A. As for m3C, we identified a deamination byproduct 3-methyluracil, which is only 1 Da greater than uncharged m3C in the ∼4900 Da context. The detection of these byproducts would be very challenging if the abovementioned procedure was not adopted.

Hb F Levels in Indian Sickle Cell Patients and Association with the HBB Locus Variant rs10128556 (C>T), and the HBG XmnI (Arab-Indian) Variant

The prevalence of sickle cell disease in India is very high. Hb F is one of the most powerful modulators of disease severity in sickle cell disease patients. It was traditionally thought that the disease is milder in Indian sickle cell disease patients predominantly due to the Arab-Indian haplotype characterized by the HBG XmnI [rs7482144 (G>A)] variant, which is associated with increased Hb F levels. In the current study, we investigated the Hb F levels in individuals with the rs10128556 (C>T) variant and also determined its linkage with the HBG XmnI variant. The present study was conducted on a cohort of 275 individuals, which consisted of 221 patients with sickle cell disease and 54 patients with sickle cell trait. Analysis of hemoglobin (Hb) fractions and variants was done on the high performance liquid chromatography (HPLC) system. Genotyping for rs10128556 was done by direct sequencing of the products. Mean Hb F levels in the sickle cell disease patients was 19.36 ± 6.79. The genotypic frequencies for rs10128556 were 82.0% (TT), 16.7% (CT) and 1.3% (CC) for sickle cell disease patients. The minor C allele resulted in 52.0% decrease in Hb F levels when homozygous and 7.0% decrease when heterozygous. The rs10128556 single nucleotide polymorphism (SNP) was in strong but not complete linkage with the HBG XmnI variant. In conclusion, the study determined for the first time the frequency and association of rs10128556 in Indian sickle cell disease patients with Hb F. It also established that it was not in complete linkage with the HBG XmnI variant in this high risk population.

Optimization of the chromatographic behaviors of quercetin using choline chloride-based deep eutectic solvents as HPLC mobile-phase additives

ABSTRACTNine types of deep eutectic solvents (DESs) were used as mobile-phase additives to improve the chromatographic behaviors of quercetin. Three variables (temperature, molar ratio and concentration) were studied. According to the ion interaction theory and the hydrogen bond, DESs components can work as ions and play an ion-pairing role with quercetin at the same time. Under the optimum condition, the number of theoretical plates was 6690.8 and the tailing factor was 1.71. Compared with acetic acid (HAc), DESs as mobile-phase additives would not damage the High Performance Liquid Chromatography (HPLC) system. The excellent properties of DESs showed their potential as additives for the determination of quercetin.

Evaluation of carbonyl compound emissions from a non-road machinery diesel engine fueled with a methanol/diesel blend

The carbonyl emissions from a non-road machinery diesel engine running on diesel fuel (DF) or methanol-diesel blend fuel (M/DF) were investigated under a series of steady-state operating conditions. Carbonyl compounds (CBCs) were collected from the diluted exhaust, employing 2,4-dinitrophenylhydrazine-coated silica gel cartridges, and analyzed using a high-performance liquid chromatography system with a photodiode array detector. The results indicate that formaldehyde and acetaldehyde are the most abundant carbonyls, followed by acrolein, acetone, propionaldehyde and crotonaldehyde. Each of these compounds exhibit a consistent reduction with increases in the engine load. The M/DF produces much more formaldehyde, acetaldehyde, acrolein, acetone and crotonaldehyde than the DF at low loads, while there are marginal differences in the emission levels of these carbonyls between M/DF and DF at medium and high loads. The use of M/DF decreases propionaldehyde emissions at all test conditions. Compared with the results obtained using DF, the total CBC emissions increase by 45.3% and the ozone formation potential (OFP) increases by 57.0% when burning M/DF at low engine loads. In general, M/DF increases both OFP and total CBC emissions to a greater extent than biodiesel-blend fuel but to a lesser degree than ethanol- or butanol-containing blends.

Phenolic compounds participating in mulberry juice sediment formation during storage.

The stability of clarified juice is of great importance in the beverage industry and to consumers. Phenolic compounds are considered to be one of the main factors responsible for sediment formation. The aim of this study is to investigate the changes in the phenolic content in clarified mulberry juice during storage. Hence, separation, identification, quantification, and analysis of the changes in the contents of phenolic compounds, both free and bound forms, in the supernatant and sediments of mulberry juice, were carried out using high performance liquid chromatographic system, equipped with a photo-diode array detector (HPLC-PDA) and HPLC coupled with quadrupole-time of flight mass spectrometric (HPLC-QTOF-MS/MS) techniques. There was an increase in the amount of sediment formed over the period of study. Total phenolic content of supernatant, as well as free phenolic content in the extracts of the precipitate decreased, whereas the bound phenolic content in the sediment increased. Quantitative estimation of individual phenolic compounds indicated high degradation of free anthocyanins in the supernatant and sediment from 938.60 to 2.30 mg/L and 235.60 to 1.74 mg/g, respectively. A decrease in flavonoids in the supernatant was also observed, whereas the contents of bound forms of gallic acid, protocatechuic acid, caffeic acid, and rutin in the sediment increased. Anthocyanins were the most abundant form of phenolics in the sediment, and accounted for 67.2% of total phenolics after 8 weeks of storage. These results revealed that phenolic compounds, particularly anthocyanins, were involved in the formation of sediments in mulberry juice during storage.

Application of dispersive liquid-liquid microextraction for the preconcentration of eight parabens in real samples and their determination by high-performance liquid chromatography.

A simple and sensitive method for the simultaneous determination of eight parabens in human plasma and urine samples was developed. The samples were preconcentrated using dispersive liquid-liquid microextraction based on the solidification of floating organic drops and determined by high-performance liquid chromatography with ultraviolet detection. The influence of variables affecting the extraction efficiency was investigated and optimized using Placket-Burman design and Box-Behnken design. The optimized values were: 58 μL of 1-decanol (as extraction solvent), 0.65mL methanol (as disperser solvent), 1.5% w/v NaCl in 5.0mL of sample solution, pH 10.6, and 4.0min centrifugation at 4000rpm. The extract was injected into the high-performance liquid chromatography system for analysis. Under the optimum conditions, the linear ranges for eight parabens in plasma and urine were 1.0-1000ng/mL, with correlation coefficients above 0.994. The limit of detection was 0.2-0.4 and 0.1-0.4ng/mL for plasma and urine samples, respectively. Relative recoveries were between 80.3 and 110.7%, while relative standard deviations were less than 5.4%. Finally, the method was applied to analyze the parabens in 98 patients of primary breast cancer. Results showed that parabens existed widely, at least one paraben detected in 96.9% (95/98) of plasma samples and 98.0% (96/98) of urine samples.

Miniaturized high-performance liquid chromatography instrumentation

Miniaturized high performance liquid chromatography (HPLC) has attracted increasing attention for its potential in high-throughput analyses and point-of-care applications. In this review we highlight the recent advancements in HPLC system miniaturization. We focus on the major components that constitute these instruments along with their respective advantages and drawbacks as well as present a few representative miniaturized HPLC systems. We discuss briefly some of the applications and also anticipate the future development trends of these instrumental platforms.

Complete LabVIEW-Controlled HPLC Lab: An Advanced Undergraduate Experience

Virtually all modern chemical instrumentation is controlled by computers. While software packages are continually becoming easier to use, allowing for more researchers to utilize more complex instruments, conveying some level of understanding as to how computers and instruments communicate is still an important part of the undergraduate curriculum. Understanding how digital and analog signals are used to allow the smooth operation of an instrument allows the user to better understand the possible sources or problems when instruments stop working optimally. The experiment presented here allows students to investigate the communication between the computer and a high-performance liquid chromatography (HPLC) system by allowing them to construct a LabVIEW-based control program. Student understanding of instrument control is reinforced by having them control pump speed, detector zeroing, and a solvent recycling valve while also monitoring the injection port status and the detector output. Students gain an unde...

A novel multi-hyphenated analytical method to simultaneously determine xanthine oxidase inhibitors and superoxide anion scavengers in natural products

Natural products, such as rosmarinic acid and apigenin, can act as xanthine oxidase inhibitors (XOIs) as well as superoxide anion scavengers, and have potential for treatment of diseases associated with high uric acid levels and oxidative stress. However, efficient simultaneous screening of these two bioactivities in natural products has been challenging. We have developed a novel method by assembling a multi-hyphenated high performance liquid chromatography (HPLC) system that combines a photo-diode array, chemiluminescence detector and a HPLC system with a variable wavelength detector, to simultaneously detect components that act as both XOIs and superoxide anion scavengers in natural products. Superoxide anion scavenging activity in the analyte was measured by on-line chemiluminescence chromatography based on pyrogallol-luminol oxidation, while xanthine oxidase inhibitory activity was determined by semi-on-line HPLC analysis. After optimizing multiple elements, including chromatographic conditions (e.g., organic solvent concentration and mobile phase pH), concentrations of xanthine/xanthine oxidase and reaction temperature, our validated analytical method was capable of mixed sample analysis. The final results from our method are presented in an easily understood visual format including comprehensive bioactivity data of natural products.

Influence of phenylketonuria's diet on dimethylated arginines and methylation cycle.

Phenylketonuria's (PKU) treatment based on low natural protein diet may affect homocysteine (Hcys) metabolic pathway. Hcys alteration may be related to the methylation of arginine to asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), which both modify nitric oxide production. The aim of this work is to evaluate the status of Hcys formation methylation cycle and ADMA and SDMA levels in patients with PKU in order to establish a potential relationship.Forty-two early diagnosed PKU patients under dietary treatment and good adherence to their diets were enrolled in this cross-sectional study. Their nutritional and biochemical profile, as well as Hcys synthesis status, ADMA and SDMA levels were analyzed and compared with a control group of 40 healthy volunteers. ADMA and SDMA were determined by high-performance liquid chromatography system coupled to triple quadrupole mass spectrometer.In this study, 23 classic PKU, 16 moderate PKU, and 3 mild HPA were enrolled. The median age was 10 years old. Median ADMA, SDMA, and Hcys concentration levels (5.1 μM [2.3–25.7], 0.35 μM [0.18–0.57], 0.43 μM [0.26–0.61], respectively) were lower in patients with PKU (P < .001 for ADMA and SDMA) whereas vitamin B12 and folate levels (616 pg/mL [218–1943] and 21 ng/mL [5–51], respectively) were higher comparing with controls. Statistically significant correlations were found between ADMA, and Phe (r = −0.504, P = .001) and Hcys (r = −0.458, P = .037) levels. Several nutrition biomarkers, such as prealbumin, 25-hydroxy vitamin D, selenium, and zinc, were below the normal range.Our study suggests that patients with PKU suffer from poor methylation capacity. Restriction of natural proteins in addition to high intake of vitamin B12 and folic acid supplementation in the dietary products, produce an impairment of methylation cycle that leads to low Hcys and ADMA levels. As a result, methylated compounds compete for methyl groups, and there is an impairment of methylation cycle due to low Hcys levels, which is related to the lack of protein quality, despite of elevated concentrations of cofactors.

Lepiota cristata does not contain amatoxins or phallotoxins

It is believed that Lepiota cristata is inedible and slightly poisonous, so it should always be avoided. This study analyzed the alpha, beta and gamma amanitin, phalloidin (PHN) and phallacidin (PCN) content of L. cristata collected from different regions of Turkey. Toxin levels have been assessed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with ultraviolet detectors. In this study, the L. cristata mushroom was not found to contain any amatoxins or phallotoxins. However, this finding does not mean that this mushroom is edible. New studies regarding the toxin content of L. cristata may be useful to understand the toxicity of this species.