Amino Acid Autoanalyzer Research Articles

SLC7A1 Overexpression Is Involved in Energy Metabolism Reprogramming to Induce Tumor Progression in Epithelial Ovarian Cancer and Is Associated with Immune-Infiltrating Cells.

Cationic amino acid transporters (SLC7A1/CAT1) are highly expressed in human ovarian cancer (OC) tissues. However, the specific biological functions and mechanisms involved remain unclear. We used bioinformatics analysis to explore SLC7A1 expression level, prognostic value, and tumor mutation burden (TMB) in ovarian cancer (OC) tissues. We performed in vitro experiments to identify the expression and biological function of SLC7A1 in epithelial ovarian cancer (EOC) tissues and cells. An amino acid autoanalyzer was used to detect the effect of SLC7A1 on amino acid metabolism in EOC cells. Finally, SLC7A1 in OC was evaluated for cell-to-cell signalling and immune infiltration using online databases. We found that increased SLC7A1 expression in EOC cells and tissues was associated with poorer survival outcomes (P < 0.05) but not with tumor stage or grade of OC (P > 0.05). SLC7A1 is involved in the transport of phenylalanine and arginine in EOC cells, and its knockdown reduced the proliferation and migration of EOC cells and the resistance of cells to cisplatin. Furthermore, the TIMER database indicated that SLC7A1 overexpression was significantly positively correlated with levels of CD4+ memory resting cells, CD8+ effector memory cells, M0 macrophages, and cancer-associated fibroblasts (CAFs) in OC (P < 0.05) and significantly negatively correlated with CD4+ memory-activated cells (P < 0.05). Cell immunofluorescence indicated that SLC7A1 overexpression may affect the distribution of immune-infiltrating lymphocytes in tumors by inhibiting the expression of CCL4. Therefore, we concluded that SLC7A1 is involved in the metabolic remodelling of amino acids in EOC to promote tumor development and cisplatin resistance and is related to the tumor-infiltrating immune microenvironment of OC. SLC7A1 is a biomarker for predicting EOC progression and cisplatin resistance and represents a promising target for EOC treatment.

동해안산 붉은멍게(Halocynthia aurantium)의 일반성분 및 유리 아미노산 특성과 멍게 특유의 향미 성분

The purpose of this study is to investigate the characteristics of proximate composition, free amino acids and to elucidate tunic flavor components in Sea peach (Halocynthia aurantium). Tunic (25.0%) and edible part (61.0% to total weight) was separated from live Sea peach. Proximate composition was moisture 91.6%, lipid 0.8%, protein 2.5%, ash 2.7%, respectively. Zinc (7.0 mg/100 g) and trace amount of Selenium (0.1 mg/100 g) were unique components in mineral. The edible part have extracted with hot ethanolic water and those free amino acids were analysed. Total 29 amino acids were isolated by amino acid auto analyser. Among them, taurine (89.2 mg/100 g), proline (41.2 mg/100 g) and glutamic acid (22.6 mg/100 g) were major free amino acids (total 231.7 mg/100 g). The chloroform and butanol fraction of acetone extract of H. aurantium, 6 flavor components presumed aliphatic saturated or unsaturated alcohols were isolated and purified on the HPLC attached ODS column. Those compounds was elucidated by mass spectra such as 1-octenol (10.4%), nonanol (4.4%), 1-decenol (6.1%), 1-decadienol (49.4%), 1-undecadienol (18.4%), 1-dodecenol (11.3%). 1-Octenol was further elucidated as cis-3-octen-1-ol by 1H and 13C Nuclear Magnetic Resonance spectroscopy.

Chemiluminescence Reaction of an Iron(III)-Phthalocyanine Complex and Its Application to FIA of L-Tyrosine

Chemiluminescence (CL) of a metal–phthalocyanine complex was studied, and an analytical application of CL detection-flow injection analysis (FIA) of L-tyrosine was developed. The CL reaction of iron (III)–phthalocyanine tetrasulfonic acid (Fe–PTS) was examined using eight metals (Cu, Fe, Ni, Co, Mg, Pd, Pt and Zn)–phthalocyanine complexes. The CL signal immediately appeared, when the Fe-PTS aqueous solution was injected into a hydrogen peroxide solution. Moreover, the influence of foreign substances on the CL system of Fe–PTS was examined. The decrease in the CL intensity of the iron–phthalocyanine complex was caused by adding a reducing agent such as L-tyrosine. The CL detection-FIA of trace amounts of L-tyrosine was then developed using this quenching phenomenon. The calibration curve was obtained in the concentration range 2.5 × 10 −7 M to 7.5 × 10 −6 M, and the sampling rate was 150 samples h −1 . Further, the relative standard deviation (RSD) was 1.20 % (n = 25) for 1.0 × 10 −6 M, and the detection limit (3σ) were 8.54 × 10 −8 M. The interference of metal ions was able to inhibit using EDTA. Therefore, the interference of L-cysteine was able to inhibit using Zn 2+ . As an application to a practical sample, L-tyrosine in a supplement was determined. The experimental value is almost the same as the tabulated one (1.0 × 10 −6 M) measured by amino acid autoanalyzer at the Japan Food Research Laboratories.

Amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in weaning piglets derived from sows fed low-protein diet during gestation and lactation : Autophagy is involved in reduced fat deposition in maternal low-protein piglets.

The study aimed to determine the effects of maternal low-protein (LP) diet on subcutaneous fat deposition of weaning piglets and the potential mechanism. Sows were fed either a standard protein (SP, 15 and 18% crude protein) or a LP diet (50% protein levels of SP) throughout pregnancy and lactation. Subcutaneous fat and blood were sampled from male piglets at 28 days of age. Serum biochemical metabolites and hormone concentrations were detected with the enzymatic colorimetric methods. Serum-free amino acid (FAA) levels were measured by amino acid auto-analyzer. The mRNA and protein were measured by qRT-PCR and Western blot. Body weight, back fat thickness, triglycerides concentrations in subcutaneous fat tissue, and serum, as well as FFA concentrations were significantly reduced in LP piglets when compared with SP piglets. Further studies showed that mRNA and protein expression of acetyl-CoA carboxylase and fatty acid synthetase, two key enzymes of de novo lipogenesis, were significantly reduced in LP piglets, while mRNA expression and the lipolytic enzymes activities of lipolysis genes, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased. Furthermore, expression of autophagy-related gene 7 and autophagy maker gene microtubule-associated protein 1A/1B-light chain 3 (LC 3) as well as the conversion of LC3I to LC3II were significantly elevated, along with the expression of activating transcription factor-4 and eukaryotic translation initiation factor-2a. These results indicate that amino acid starvation-induced autophagy is involved in reduced subcutaneous fat deposition in maternal LP weaning piglets, demonstrating links between maternal protein restriction and offspring fat deposition.

Maternal low-protein diet affects myostatin signaling and protein synthesis in skeletal muscle of offspring piglets at weaning stage.

We tested the hypothesis that maternal low-protein (LP) diet during gestation and lactation can program myostatin (MSTN) signaling and protein synthesis in skeletal muscle of offspring at weaning stage (35days). Fourteen Meishan sows were fed either LP or standard-protein diets throughout gestation and lactation, male offspring piglets were killed at weaning stage and longissimus dorsi (LD) muscles were taken. The cross-sectional areas (CSA) of LD muscles were measured by hematoxylin and eosin staining. The levels of free amino acids in plasma were measured by amino acid auto-analyzer. Proteins and mRNA were determined by Western blot and RT-qPCR, respectively. Body weight, LD muscle weight and the myofiber CSA were significantly decreased (P<0.05) in LP piglets; meanwhile, the concentration of branched-chain amino acids was also significantly decreased (P<0.001). MSTN protein content tended to be higher (P=0.098) in LP piglets, while the expression of MSTN receptors, activin type II receptor-beta and transforming growth factor type-beta type I receptor kinase, was significantly up-regulated (P<0.05). Furthermore, p38 mitogen-activated protein kinase, the downstream signaling factor of MSTN, was also enhanced significantly (P<0.05). In addition, key factors of translation initiation, phosphorylated eukaryotic initiation factor 4E and the 70kDa ribosomal protein S6 kinase, were significantly decreased (P<0.05) in LP piglets. Our results suggest that maternal LP diet during gestation and lactation affects MSTN signaling and protein synthesis in skeletal muscle of offspring at weaning stage.

DISCRIMINATION OF 'ISABELLA', 'CONCORD', 'IVES' AND 'WHITE NIAGARA' GRAPE JUICES BY THEIR FREE AMINO ACID COMPOSITION

ISHS VIII International Symposium on Temperate Zone Fruits in the Tropics and Subtropics DISCRIMINATION OF 'ISABELLA', 'CONCORD', 'IVES' AND 'WHITE NIAGARA' GRAPE JUICES BY THEIR FREE AMINO ACID COMPOSITION

Effect Of Resistance Training On Carnosine Concentration In Human Skeletal Muscle

Carnosine (β-alanyl-L-histidine) is a dipeptide containing histidine and is present in vertebrate skeletal muscles. It has been reported that carnosine contributes to physico-chemical buffering to maintain acid-base balance during high-intensity exercise. Moreover, it has been shown that the repeated accumulation of hydrogen ion with high-intensity exercise training is necessary to increase in carnosine concentration. However, the effects of most type of the training on skeletal muscle carnosine concentration are unclear. PURPOSE: The purpose of the present study was to determine the effects of resistance training on skeletal muscle carnosine concentration. METHODS: Fifteen active but not well-trained healthy males were assigned to either a "Strength-type“ (S-type: n = 7) or a "Hypertrophy type“ (H-type: n = 8) group and performed leg press exercise twice a week for 8 weeks. The S-type regimen consisted of 5 sets at 90% of 1 repetition maximum (RM) with 3-min rest periods between sets, whereas the H-type regimen consisted of 5 sets at 70% of 1RM with 1-min rest periods between sets. Muscle volume was measured using a magnetic resonance imaging (MRI). Muscle biopsy samples were taken from the vastus lateralis at rest before and after training. The carnosine concentration was determined using an amino acid autoanalyzer. RESULTS: After the 8 weeks of resistance training, muscle volume of thigh and 1RM of leg press were significantly (p < 0.05) increased in both groups. In addition, percentage change in muscle volume was larger in the H-type group than that in the S-type group. However, carnosine concentration was not significantly increased in either group. CONCLUSIONS: These results suggest that resistance training does not improve skeletal muscle carnosine concentration regardless of the type of regimen.

Changes in diversity, protein content, and amino acid composition of earthworms from a paddy soil under different long-term fertilizations in the Tai Lake Region, China

Abstract Influence of the agricultural management practices on soil quality and the ecosystem functioning has been an increasing concern in soil science and ecology with sustainable agriculture. This study deals with the changes of soil earthworm communityfrom a paddy soil under different long-term fertilizations. The soil earthworms were collected and counted from different fertilizer treated plots in the field after the rape harvest in May 2004, and their taxonomic groups were determined under a binocular stereoscope at the laboratory. The body of the earthworm ( Metaphire californica) was crushed by a cell crusher to collect protein, and the protein molecules with different sizes were analyzed by electrophoresis. Furthermore, the Metaphire californica collected was hydrolyzed and the aliquots were subject to an amino acid auto-analyzer. The results showed that totally seven species of earthworms were recognized in the paddy field with the number varying with different fertilization treatments. The structure of earthworm communities was dramatically affected by the fertilization practice. Under chemical fertilization only, both the number of earthworm species and the quantity of individuals were significantly smaller than those under other treatments, or even than those under no fertilization. Furthermore, there was an obvious decrease in the total amino acid and the contents of most individual amino acids of Metaphire californica under chemical fertilization only, compared with those under the combined fertilization of chemical and organic fertilizers. Although chemical fertilizers in combination with rice straw return increased earthworm amino acid content, long-term pig manure application tended to increase earthworm protein content. As a molecular footprint, long-term chemical fertilization caused a reduction in the content of protein with MW less than 25 kd, but a significant increase in that of protein with molecule size around 33 kd. Our study demonstrated that different fertilizations affected not only earthworm population but also diversity and richness in the paddy soil after 16 years of treatment, and that long-term chemical fertilization may impact the soil animal community and, thus, influence the paddy ecosystem functioning for yield stability. This study implicated that not only the community structure but also the amino acid metabolism for life functioning of earthworms in cropland soils may pose significant responses to the agricultural management practices.

Amino acids determination using capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection

Free amino acids have been derivatized on-capillary with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and analyzed using a laboratory-made capillary electrophoresis apparatus with laser-induced fluorescence detection. Several parameters that control on-capillary derivatization of amino acids, including pH, mixing time, reaction time, concentration of the derivatization reagents (potassium cyanide and FQ) and solvent of FQ, as well as the temperature of mixing and reaction were optimized. Repeatabilities better than 1.8% for migration time and 7.8% for peak height were obtained. Assay detection limits for the different amino acids ranged from 23 nM for glycine to 50 nM for lysine and glutamic acid. The methods developed were applied to the analysis of several amino acids in pharmaceutical preparations and plasma samples. Results showed a good agreement with those obtained using an amino acid autoanalyzer for the same samples.

Obtenção e caracterização química e nutricional de diferentes concentrados de caseína

OBJETIVO: O objetivo deste trabalho foi avaliar a composição centesimal, o perfil de aminoácidos e as características nutricionais de três concentrados de caseína, obtidos do leite bovino por diferentes processos. MÉTODOS: Os concentrados de caseína foram analisados pelos seguintes processos: uma caseína comercial, obtida por precipitação ácida seguida de neutralização; caseína obtida pela coagulação enzimática; caseína micelar obtida, respectivamente, pelos processos de microfiltração e diafiltração em membrana. A composição centesimal foi determinada por meio de procedimentos descritos no manual Official Methods of Analysis. O perfil de aminoácidos foi determinado após hidrólise ácida da proteína (HCl 6N, 105°C, 22h) em auto-analisador de aminoácidos, dotado de coluna de troca catiônica e reação pós-coluna com ninidrina. Os perfis de aminoácidos essenciais dos diferentes concentrados de caseína foram comparados e estão de acordo com o padrão Food and Agriculture Organization/World Health Organization, para crianças de 2 a 5 anos de idade. O valor nutritivo foi determinado em ratos da linhagem Wistar, recém desmamados, por meio dos índices, digestibilidade aparente da proteína, quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional. RESULTADOS: A caseína comercial apresentou maior concentração de proteína (92,0%), que a caseína micelar (86,0%) e o coágulo de caseína (72,0%). Os animais nas dietas com as diferentes fontes de proteína, não apresentaram diferenças significativas quanto ao ganho de peso e ingestão de dieta. Maior digestibilidade (93,8%) foi verificada na caseína comercial, comparada à dos outros dois concentrados (91,0%). CONCLUSÃO: Os concentrados de caseína apresentaram diferenças quanto à composição centesimal, sendo a caseína comercial superior na concentração protéica. O coágulo de caseína apresentou resultados inferiores aos demais concentrados, quanto aos índices quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional.

Validation of an Ion-Interaction Chromatography Analysis of Underivatized Amino Acids in Commercial Preparation Using Evaporative Light Scattering Detection

A liquid chromatographic procedure using ion interaction reagent and evaporative light scattering detection was developed and validated for the direct quantification of 8 underivatized amino acids (threonine, lysine, valine, methionine, isoleucine, leucine, phenylalanine and tryptophan) present simultaneously in a commercial preparation. The chromatographic separation was achieved on a Purospher RP-18e column with an acetonitrile gradient and using heptafluorobutyric acid as volatile ion interaction reagent. Acceptable levels of linearity, specificity, precision, accuracy, robustness and limits of detection were achieved during the validation of the method. Detection limits varied from 1 mg.L−1 to 2 mg.L−1 depending on the amino acid being analyzed. The results of this method agree to within 98.6%–102.5% when compared to the manufacturer’s certificate of analysis obtained with the standard amino acid autoanalyser method. This approach yields a simple, universal method that is well suited for amino acid analysis, when a sufficient quantity is available. Only basic LC instrumentation with an ELSD is necessary for this procedure.

The Effect of Sprint Training on Skeletal Muscle Carnosine in Humans

It has been suggested that histidine-containing dipeptide carnosine (β-alanyl-L-histidine), which is believed to act as a cytosolic buffering agent, is present predominantly in skeletal muscle. The purpose of this study was to investigate the effects of sprint training (30-s maximal cycle ergometer sprinting) on muscle carnosine concentration. Six untrained males trained 2 days per week for 8 weeks on an electronic-braked cycle ergometer. Muscle biopsy samples were taken from the vastus lateralis before and two days after the last training session and were analyzed for carnosine concentration by the use of an amino acid autoanalyzer. The carnosine concentration was significantly increased after sprint training (P < 0.05). The mean power during 30-s maximal cycle ergometer sprinting was significantly increased following training. When dividing the 30-s sprinting into 6 phases (0-5, 6-10, 11-15, 16-20, 21-25, 26-30 s), the magnitude of increase in mean power was significantly larger for the last 2 phases than the first phase (P < 0.05). These results suggest that the increases in skeletal muscle carnosine concentration following sprint training may be associated with the increase in sustainability of high power during 30-s maximal cycle ergometer sprinting.

Direct chemiluminescence determination of cysteine in human serum using quinine–Ce(IV) system

A simple, sensitive and selective chemiluminescence (CL) method was developed for the determination of cysteine. This method is based on that the weak CL of cysteine oxidized with cerium (IV) can be greatly enhanced by quinine. The calibration curve was linear over the range 3.5×10 −9–3.5×10 −6 M with a detection limit of 2.5×10 −9 M ( S/ N=3). The RSD was found to be 8.4% by 10 replicate determinations of 2.9×10 −8 M cysteine. Due to high sensitivity, the proposed method can be used directly to determine the total concentration of cysteine in human serum through simply diluting the sample for a thousand fold. The obtained result was in agreement with that given by amino acid autoanalyzer. The present method does not require any separation, showing a simpler analytical characteristic. The mechanism of the CL reaction was also discussed.

High Level of Skeletal Muscle Carnosine Contributes to the Latter Half of Exercise Performance during 30-s Maximal Cycle Ergometer Sprinting.

The histidine-containing dipeptide carnosine (beta-alanyl-L-histidine) has been shown to significantly contribute to the physicochemical buffering in skeletal muscles, which maintains acid-base balance when a large quantity of H(+) is produced in association with lactic acid accumulation during high-intensity exercise. The purpose of the present study was to examine the relations among the skeletal muscle carnosine concentration, fiber-type distribution, and high-intensity exercise performance. The subjects were 11 healthy men. Muscle biopsy samples were taken from the vastus lateralis at rest. The carnosine concentration was determined by the use of an amino acid autoanalyzer. The fiber-type distribution was determined by the staining intensity of myosin adenosinetriphosphatase. The high-intensity exercise performance was assessed by the use of 30-s maximal cycle ergometer sprinting. A significant correlation was demonstrated between the carnosine concentration and the type IIX fiber composition (r=0.646, p<0.05). The carnosine concentration was significantly correlated with the mean power per body mass (r=0.785, p<0.01) during the 30-s sprinting. When dividing the sprinting into 6 phases (0-5, 6-10, 11-15, 16-20, 21-25, 26-30 s), significant correlations were observed between the carnosine concentration and the mean power per body mass of the final 2 phases (21-25 s: r=0.694, p<0.05; 26-30 s: r=0.660, p<0.05). These results indicated that the carnosine concentration could be an important factor in determining the high-intensity exercise performance.

The relation between plasma homocysteine concentration and methylenetetrahydrofolate reductase gene polymorphism in pregnant women.

Our purpose was to clarify the influence of methylenetetrahydrofolate reductase (MTHFR) gene polymorphism on plasma homocysteine (Hcy) concentrations during early pregnancy. Between 1996 and 1998, Hcy concentration and MTHFR gene polymorphism were studied in 840 pregnant women between 6 to 12 weeks' gestation. Hcy concentration was measured by amino acid autoanalyzer (DLC-300, Nihon Denshi), and MTHFR genotypes were determined by the PCR/RFLP methods. Pregnancy outcomes were compared in association with Hcy concentration and MTHFR genotypes. A total of 816 of the 840 women were enrolled into the study because MTHFR genotypes were not available in 24 women. Genotypes of C677T in the MTHFR gene were CC: CT: TT (%) = 280 (34.3): 400 (49.0): 136 (16.7). Plasma Hcy concentration was significantly (p < 0.0001) higher in women with TT genotype than other genotypes [CC: CT: TT = 5.67 (2.8-10.6): 5.80 (2.5-20.1): 6.91 (3.5-20.9) nmol/ml, median (range)]. Women with hyperhomocysteinemia had severe preeclampsia (2 of 35 vs 5 of 714, p < 0.01) and stillbirth (2 of 35 vs 10 of 714, p < 0.05) more frequently than normohomocysteinemia. Plasma Hcy concentration during early pregnancy was higher in women with homozygote for the T677 allele in the MTHFR gene than other genotypes.

Determination of eight essential amino acids in mixtures by chemometrics–spectrophotometry without separation

There are almost no differences among the reaction rates of eight essential amino acids with ninhydrin and among the absorption spectra of their reaction products (i.e. these absorption curves are seriously overlapped with each other), therefore the simultaneous determinations of them are carried out by using neither differential rate kinetic method nor multi-wavelength spectrophotometry and nor derivative spectrometry unless pre-separation tactics of them are taken. In this paper, the conditions for chromogenic reactions of leucine, isoleucine, tryptophan, lysine, threonine, valine, phenylalanine and methionine with ninhydrin have been anew optimized, then a partial least-squares (PLS) method for simultaneous determination of two mixtures each of which contains five different amino acids among above eight essential amino acids (processing data with microcomputer) has been developed. The method is simple, rapid, accurate and the results agree with both present values and those obtained by amino acid autoanalyzer. Besides, our experiments show that the PLS method is better than Kalman filter method.

SKELETAL MUSCLE CARNOSINE LEVEL MAY CONTRIBUTE TO SUPERIOR PERFORMANCE DURING HIGH INTENSITY EXERCISE

Dipeptide carnosine (β-alanyl-L-histidine) has been shown to contribute significantly to physico-chemical buffering in skeletal muscle, which maintains acid-base balance when large quantities of hydrogen ions are produced in association with lactic acid accumulation during high intensity exercise. The purpose of this study was to examine relationships among skeletal muscle carnosine level, fiber type distribution and high intensity exercise performance. The subjects were twenty healthy men. Muscle biopsy samples were taken from the vastus lateralis at rest. Carnosine level were determined using an amino acid AutoAnalyzer. Fiber type distribution was determined by staining intensity of myosin adenosinetriphosphatase. High intensity exercise performance was assessed using 30-s cycle ergometer sprinting. Significant (P < 0.05) interrelationships were demonstrated between carnosine level and type II fiber composition. Superior sprint performance was associated with type II fiber composition and carnosine level. When dividing the 30-s sprinting into 6 phases (0–5, 6–10, 11–15, 16–20, 21–25, 26–30 s), significant correlations were observed between carnosine level and mean power of the latter 3 phases (16–20 s: P < 0.05, 21–25 s: P < 0.01 and 26–30 s: P < 0.05). These results indicate that carnosine level may be an important determinant of high intensity exercise performance.

Plasma Leptin Concentrations in Phenylketonuric Patients

High levels of phenylalanine (Phe) in blood have been shown to reduce dopamine (DA) and noradrenaline (NA) production. Leptin levels rise with increasing adiposity in rodents and humans acting as a negative feedback adipostatic signal to brain centers. The aim of this study was to evaluate leptin plasma levels in phenylketonuria (PKU) patients adhering to their special diet and in those on a ‘loose diet’. Forty-nine patients with classical PKU were divided into two groups. Those in group A (n = 21) adhered very strictly to their diet (Phe: 0.15 ± 0.04 mmol/l) and those in group B (n = 28) were on a ‘loose diet’ (Phe: 0.8 ± 0.04 mmol/l). Thirty healthy children of comparable age served as controls. Both patients and controls were in pubertal stage 0 (Tanner). BMI (kg/m²) was evaluated in all the members of the groups. Their daily nutrients were calculated with a 7-day dietary protocol. Leptin was evaluated by RIA, and Phe and Tyrosine with an amino acid autoanalyser. Adrenaline (A), NA and DA were measured by an HPLC method. Plasma leptin in group B patients (28.4 ± 2.0 ng/ml) was significantly increased as compared to group A patients (16.8 ± 2.6 ng/ml) and controls (17.8 ± 3.0 ng/ml; p < 0.001). Plasma DA, A, and NA in group B was lower than in group A and controls. Additionally, leptin negatively correlated with A and DA, whereas Phe positively correlated with the hormone in all groups. Leptin, also, correlated with BMI only in group A and controls. Additionally, the hormone negatively correlated with the total energy intake only in group A (r = –0.43, p < 0.01) and in controls (r = –0.040, p < 0.01). It is suggested that the disregulation of the neuroendocrine system as well as the high Phe blood levels might play an important role in the increased leptin concentrations in PKU patients on a ‘loose diet’.

Characterisation of the protein composition of casein micelles after heating

Under a French dairy industry initiative, the changes of protein composition of casein micelles induced by thermal treatment of milk have been studied. To improve the choice of milk powder as raw material for the work, it was established that drying had a low impact on renneting ability (formagraph measurement), non-casein nitrogen values and casein micelle diameter. The effect of storage of powders on the bioavailability of lysyl residues was also studied by complete hydrolysis of the proteins followed by furosine and homo-arginine measurements using an amino-acid auto analyser. After this validation step, thermal treatments of milk using both a laboratory system and an industrial process have been performed. Milk was reconstituted from the powder and heated from 70 to 100°C for 1 or 4 min. Casein micelles were then isolated from whole milk by ultracentrifugation and washed three times in a simulated milk ultrafiltrate, in order to eliminate whey proteins entrapped in micellar casein pellets. Micellar diameter was investigated by photon correlation spectrophotometry (PCS). Further qualitative and semi-quantitative studies of the micellar protein composition were carried out using PAGE systems and gel scanning. PAGE systems were used under complete or partial denaturing conditions, using sodium dodecyl sulfate (SDS) as dissociating agent, with or without dl-dithiothreitol (DTT) as a strong disulfide bond reducer.

Simultaneous determination of several amino acids with multivariate calibration methods by using a continuous-flow system

An initial approach to the simultaneous determination of several amino acids by using multivariate calibration methods is proposed. The method is based on the development of the reaction between amino acids and 1,2-naphthoquinone-4-sulfonate. Spectra of the corresponding derivatives obtained from a continuous-flow system were used to provide multivariate data for the multivariate procedure. Two multivariate calibration methods were applied, principal component regression (PCR) and partial least-squares regression (PLS). In order to compare the resolution capabilities of these multivariate methods, a series of synthetic solutions containing different concentrations of lysine, proline, phenylalanine and tryptophan were prepared. The selection of solutions for calibration and problem sets was performed on the basis of the distribution of the amino acid derivatives spectra on the first and second principal components. The effects of the number of working wavelengths, the number of principal components and the mathematical treatment of data (smoothed and derivative spectra) on the predictions were also investigated. The method was applied to the analysis of feed samples, where the determination of some particular amino acids such as lysine are of special interest. The results were in good agreement with those obtained with the standard amino acid autoanalyser procedure.