Plasma Globotriaosylsphingosine and α-Galactosidase A Activity as a Combined Screening Biomarker for Fabry Disease in a Large Japanese Cohort.

Fabry disease is an X-linked disorder of α-galactosidase A (GLA) deficiency. Our previous interim analysis (1 July 2014 to 31 December 2015) revealed plasma globotriaosylsphingosine as a promising primary screening biomarker for Fabry disease probands. Herein, we report the final results, including patients enrolled from 1 January to 31 December 2016 for evaluating the potential of plasma globotriaosylsphingosine and GLA activity as a combined screening marker. We screened 5691 patients (3439 males) referred from 237 Japanese specialty clinics based on clinical findings suggestive of Fabry disease using plasma globotriaosylsphingosine and GLA activity as primary screening markers, and GLA variant status as a secondary screening marker. Of the 14 males who tested positive in the globotriaosylsphingosine screen (≥2.0 ng/mL), 11 with low GLA activity (<4.0 nmol/h/mL) displayed GLA variants (four classic, seven late-onset) and one with normal GLA activity and no pathogenic variant displayed lamellar bodies in affected organs, indicating late-onset biopsy-proven Fabry disease. Of the 19 females who tested positive in the globotriaosylsphingosine screen, eight with low GLA activity displayed GLA variants (six classic, two late-onset) and five with normal GLA activity displayed a GLA variant (one classic) and no pathogenic variant (four late-onset biopsy-proven). The combination of plasma globotriaosylsphingosine and GLA activity can be a primary screening biomarker for classic, late-onset, and late-onset biopsy-proven Fabry disease probands.

High glucose promotes TGF-ß1 production by inducing FOS expression in human peritoneal mesothelial cells.

BACKGROUND: High glucose (HG) induces production of transforming growth factor-beta1 (TGF-ß1), but the mechanism remains elusive. The aim of this study was to determine the gene(s) involved in HG-induced TGF-ß1 production in human peritoneal mesothelial cells (HPMCs). METHODS: Microarray analysis was performed following a 3-h preincubation of HPMCs in 4 or 0.1% glucose medium. Transcriptional genes were selected using Gene Ontology analysis for biological processes, including regulation of transcription and DNA-dependent. The effects of small interfering RNA (siRNA) treatments on the up-regulation of TGF-ß1 mRNA were assessed by quantitative real-time polymerase chain reaction (qPCR). Finally, enzyme-linked immunosorbent assay (ELISA) was performed to determine which gene(s) contribute to the production of TGF-ß1 protein in the medium. RESULTS: Microarray analysis revealed that the expression of 51 genes increased by more than 3-fold. Gene ontology analysis identified 13 genes for further study. qPCR confirmed mRNA amplification for 9 of the 13 genes. Furthermore, HG-induced up-regulation of TGF-ß1 mRNA was attenuated by the siRNA of 4 genes: MDS1 and EVI1 complex locus (MECOM), FBJ murine osteosarcoma viral oncogene homolog B (FOSB), FBJ murine osteosarcoma viral oncogene homolog (FOS) and activating transcription factor 3 (ATF3). ELISA showed that siRNA treatment of FOS, but not MECOM, FOSB or ATF3, suppressed the increase of TGF-ß1 protein in the medium. CONCLUSIONS: FOS is a downstream effector of HG stimulation in HPMCs that contributes to TGF-ß1 production, suggesting that blocking FOS expression may be a therapeutic target for peritoneal fibrosis.

Expression of age-related factors during the development of renal damage in patients with IgA nephropathy.

BACKGROUND: Chronic kidney disease patients share clinical and pathological features with the general aging population. Increased oxidative DNA damage, accumulation of cell cycle-arrested cells and decreased Klotho expression are assumed to be age-related factors that are reportedly linked to kidney disease. This study sought to determine the association between these age-related factors and renal damage in patients with IgA nephropathy (IgAN). METHODS: We performed a cross-sectional analysis of 71 patients who were diagnosed with IgAN by renal biopsy. Expression of 8-hydroxydeoxyguanosine (8-OHdG, a marker of oxidative DNA damage), p16 (a marker of cell cycle-arrest) and Klotho (an anti-aging protein) were evaluated by immunohistochemical staining of renal biopsy samples. We correlated the changes in expression of these markers with Lee's pathologic grades and the Oxford classification. We also investigated the independent association between these markers and interstitial fibrosis using multiple linear regression analysis. RESULTS: 8-OHdG and p16 increased but Klotho decreased with progression of pathologic grade. Expression of 8-OHdG and p16 increased with the deterioration of mesangial hypercellularity and segmental glomerulosclerosis. In addition, p16 increased but Klotho decreased with progression of tubular atrophy/interstitial fibrosis. In univariate regression analysis, age, body mass index, systolic blood pressure, urinary protein excretion and expression of 8-OHdG, p16 and Klotho showed significant correlations with interstitial fibrosis. Multivariable regression analyses revealed that aging, increased renal expression of p16 and decreased expression of Klotho were independently correlated with interstitial fibrosis. CONCLUSIONS: The age-related factors might play important roles in the development of IgAN.

Efficacy of add-on therapy of aliskiren to an angiotensin II receptor blocker on renal outcomes in advanced-stage chronic kidney disease: a prospective, randomized, open-label study.

BACKGROUND: Combination therapy of aliskiren and an angiotensin II receptor blocker (ARB) has been reported to be effective for reducing the level of proteinuria. However, it remains unclear whether this combination therapy contributes to suppression of kidney disease progression. The aim of this study was to investigate the effect of aliskiren on hard renal endpoints, when added to an ARB, in patients with advanced chronic kidney disease (CKD). METHODS: The study design was a prospective, randomized open-label design. 83 CKD patients (52 men and 31 women) were enrolled and assigned randomly to an aliskiren add-on group (n = 42) or control group (n = 41). Entry criteria included elevated serum creatinine ≥ 1.5 mg/dl, urine protein excretion (≥ 1+ on urine dipstick test), and hypertension. All participants were treated with an ARB. The follow-up period was 12 months. 12 participants were withdrawn during the study period and the study was terminated in January 2012 as a consequence of the results of the interim analysis of the ALTITUDE study. RESULTS: Nine patients in the aliskiren group and seven patients in the control group started dialysis. Doubling of the serum creatinine level occurred in one patient in the control group. A Cox proportional hazards test showed that dual blockade of the renin-angiotensin-aldosterone system with aliskiren and ARB was not associated with improvement in hard renal endpoints. CONCLUSION: We conclude that aliskiren add-on therapy to an ARB may not give any benefit and, therefore, should not be recommended in CKD patients.

Mesenchymal stem cells ameliorate experimental peritoneal fibrosis by suppressing inflammation and inhibiting TGF-ß1 signaling.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that have regenerative capability and exert paracrine actions on damaged tissues. Since peritoneal fibrosis is a serious complication of peritoneal dialysis, we tested whether MSCs suppress this using a chlorhexidine gluconate model in rats. Although MSCs isolated from green fluorescent protein-positive rats were detected for only 3 days following their injection, immunohistochemical staining showed that MSCs suppressed the expression of mesenchymal cells, their effects on the deposition of extracellular matrix proteins, and the infiltration of macrophages for 14 days. Moreover, MSCs reduced the functional impairment of the peritoneal membrane. Cocultures of MSCs and human peritoneal mesothelial cells using a Transwell system indicated that the beneficial effects of MSCs on the glucose-induced upregulation of transforming growth factor-ß1(TGF-ß1) and fibronectin mRNA expression in the human cells were likely due to paracrine actions. Preincubation in MSC-conditioned medium suppressed TGF-ß1-induced epithelial-to-mesenchymal transition, α-smooth muscle actin, and the decrease in zonula occludens-1 in cultured human peritoneal mesothelial cells. Although bone morphogenic protein 7 was not detected, MSCs secreted hepatocyte growth factor and a neutralizing antibody to this inhibited TGF-ß1 signaling. Thus, our findings imply that MSCs ameliorate experimental peritoneal fibrosis by suppressing inflammation and TGF-ß1 signaling in a paracrine manner.

Morning blood pressure is useful for detection of left ventricular hypertrophy in hemodialysis patients.

BACKGROUND: When diagnosing hypertension (HT) it is essential to determine not only the level of raised blood pressure (BP), but also how the condition relates to organ damage. The best time to measure BP for diagnosing HT in patients on hemodialysis (HD) remains unclear. METHODS: A total of 100 HD patients (mean age 63.8 years, 60 males) were studied. Left ventricular hypertrophy (LVH) was detected by echocardiography and BP monitored for 1 week at 20 different times in the morning and night, before and after dialysis. We also checked for masked HT, i.e., patients with weekly morning HT, but not pre-dialysis HT. RESULTS: Average BP for the week was 141.9 ±19.0/79.6 ± 10.6 mmHg, with 68 patients classified as hypertensive. Average morning BP was 144.6 ± 19.8/81.7 ± 11.3 mmHg, and 71 patients had weekly morning HT. In addition, 62 patients had LVH and 51 patients had relative morning HT. Multiple logistic analyses showed that LVH was associated with weekly morning HT, morning HT on HD and non-HD days, average HT, and relative morning HT. However, evening, pre-dialysis, and post-dialysis HT showed no association with LVH. Masked HT was found in 20 % of patients. If HT had been diagnosed using only pre-dialysis BP, 20 of the 71 patients with weekly morning HT would not have been detected. CONCLUSION: Morning BP is useful for detecting LVH in HD patients. Monitoring of morning BP may be superior to measurements taken at other times for diagnosing HT.

[N-terminal pro brain natriuretic peptide predicts hospitalization of hemodialysis patients for cardiovascular disease].

We investigated whether or not N-terminal pro brain natriuretic peptide (NT-proBNP) could predict hospitalization for cardiovascular disease (CVD) among Japanese hemodialysis patients. A total of 104 patients on maintenance dialysis 3 times per week were enrolled. We followed the patients for 23.9 +/- 4.2 months and 19 hospitalizations for CVD occurring during this period. The area under the curve (AUC) for the risk of CVD hospitalization was calculated after drawing a receiver operating characteristic curve. Predialysis NT-proBNP showed a larger AUC value than both postdialysis NT-proBNP and brain natriuretic peptide. The optimal cut-off value of predialysis NT-proBNP for predicting CVD hospitalization was 5,894 pg/mL, (sensitivity of 60 % and specificity of 76 %). Diabetes mellitus, a history of CVD, and the predialysis NT-proBNP level were significant determinants of CVD hospitalization according to Cox proportional hazards analysis. In conclusion, predialysis NT-proBNP is useful for predicting CVD hospitalization in hemodialysis patients.

[The role of PPARgamma agonists in kidney diseases].

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that has a central role in the regulation of insulin sensitivity and adipocyte differentiation. Expression of PPARgamma has been reported in the kidney, including medullary collecting ducts, glomeruli and tubular cells. Thiazolidinediones (TZDs) are synthetic PPARgamma agonists and are used widely in patients with type 2 diabetes. It has been gradually discovered that TZDs have various other actions, such as vascular protective, anti-inflammatory, anti-fibrotic and anti-proliferative actions, over and above their effects on glucose and lipid metabolism. In this review, we will focus on current knowledge and insights on the role of PPARgamma agonists in kidney diseases, especially in diabetic nephropathy, non-diabetic kidney diseases and dialysis therapy.

PPAR-gamma agonist attenuates renal interstitial fibrosis and inflammation through reduction of TGF-beta.

Thiazolidinediones (TZDs), synthetic peroxisome proliferator-activated receptor (PPAR)-gamma ligands, have a central role in insulin sensitization and adipogenesis. It has been reported that TZDs exert protective effects in both diabetic and nondiabetic models of renal disease, although the exact mechanism is not well understood. In particular, only a few studies have reported the renoprotective effects of TZDs in nondiabetic models of tubulointerstitial fibrosis and inflammation. Therefore, we investigated the anti-fibrotic and anti-inflammatory effects of the TZD troglitazone in the mouse model of unilateral ureteral obstruction (UUO). C57BL/6J mice underwent UUO and were studied after 3 and 7 days. Animals were divided into three groups and received control vehicle, troglitazone (150 mg/kg per day) or troglitazone (300 mg/kg per day) by gavage. Kidneys were harvested for morphological, mRNA and protein analysis. Reverse-transcriptase-PCR was used to assess the expression of transforming growth factor-beta1 (TGF-beta1) and the TGF-beta1 type I receptor (TGF beta R-I). Protein expression was assessed by western blotting (TGF beta R-I) and immunostaining (TGF beta R-I, alpha-smooth muscle actin (alpha-SMA), type I collagen (collagen I), F4/80, and proliferating cell nuclear antigen (PCNA)). The expression of alpha-SMA, collagen I, and F4/80 was decreased in mice treated with troglitazone compared with the control group. The numbers of PCNA-positive interstitial cells were decreased in mice treated with troglitazone. TGF-beta1 mRNA and TGF beta R-I mRNA and protein expression were decreased in the group treated with troglitazone compared with the control group. The beneficial effects of troglitazone treatment were also dose dependent. PPAR-gamma agonist significantly reduced TGF-beta and attenuated renal interstitial fibrosis and inflammation in the model of UUO.

An oral adsorbent, AST-120, combined with a low-protein diet and RAS blocker, for chronic kidney disease.

BACKGROUND: A low-protein diet and treatment with renin-angiotensin system (RAS) blockers can delay the progression of chronic kidney disease (CKD). The oral adsorbent AST-120 (Kremezin) has a renoprotective effect by reducing serum levels of uremic toxins. We investigated the influence of AST-120 on the preservation of renal function in patients with CKD. METHODS: Twenty-eight patients were randomized to 2 groups: 15 patients receiving 6.0 g of AST-120 daily for 12 months plus a low-protein diet and RAS blocker therapy (group A) and 13 patients who were not given AST-120 (group B). All of them had shown progressive deterioration of renal function with basal treatment. Mean baseline serum creatinine level (+/- standard deviation) was 2.4 +/- 0.8 mg/dL in group A and 2.7 +/- 0.8 mg/dL in group B. There were no significant differences in background parameters before AST-120 therapy. RESULTS: The change in the estimated glomerular filtration rate (eGFR) was significantly smaller in group A than in group B. The change was also significantly smaller in patients with a baseline serum creatinine <2.4 mg/dL and in patients with rapid progression. After 12 months, the slope of the eGFR curve was significantly less steep compared with baseline in group A (-1.77 vs. -0.52 ml/min per month), but there was no significant change in group B. The slope was also significantly less steep in patients with rapid progression. CONCLUSIONS: Adding AST-120 to a low-protein diet and RAS blocker therapy may delay the deterioration of chronic renal failure, especially in patients with early or rapid progression.

Alkali-cation affinities of polyoxyethylene dodecylethers and helical conformations of their cationized molecules studied by electrospray mass spectrometry.

Relative alkali-cation affinity of polyoxyethylene (POE) dodecylethers in gas phase was studied by electrospray ionization (ESI) mass spectrometry using dodecylether-poly-ethoxylate (C(12)EO:n, "n" denotes ethyleneoxide unit number) nonionic surfactants, and possible helical conformations of the cationized molecules were demonstrated. The alkali-cation affinity highly depended on the cation diameters. The mass spectra of C(12)EO:8 cationized by alkali-metal ions were dominated by potassiated molecules. The results indicated that the POE moiety could have specific affinity to K(+) ions based on a host-guest interaction between POE helix and potassium ions. This is very similar to the relationships between 18-crown-6 and K(+). The ESI mass spectra exhibited the multiply cationized C(12)EO:n in addition to the singly cationized molecules. The critical EO unit numbers necessary for producing the multiply-charged cationized molecules also depended on the cation diameters. In addition, the POE surfactants highly preferred alkali cations to proton. The results were strongly supported by molecular mechanics/dynamics calculations. A helical conformation of the POE moiety of C(12)EO:15 including two K(+) ions gave a potential minimum, while a lowest energy structure of the protonated molecule took irregular conformations due to the formation of local hydrogen bonds.

Change in pulse wave velocity throughout normal pregnancy and its value in predicting pregnancy-induced hypertension: a longitudinal study.

OBJECTIVE: We longitudinally examined the changes of brachial to ankle distensibility using pulse wave velocity (PWV) throughout pregnancy and its difference between normal pregnancy and pregnancy-induced hypertension (PIH) groups. STUDY DESIGN: One hundred and eighty-three pregnant women were included in this study. The PWV examinations were performed in a longitudinal way during the first, second, and third trimesters of pregnancy, and immediately and 1 month after delivery. RESULTS: In normal pregnancies, the PWV significantly decreased at the second trimester, increased from the third trimester through immediately after delivery, and decreased again at 1 month after delivery. In PIH patients, it increased in proportion to the progression of gestation. CONCLUSION: We monitored the longitudinal changes in PWV and constructed a PWV normogram during pregnancy. The predictive value of PWV and blood pressure for PIH was higher than that of blood pressure alone, suggesting the usefulness of measuring PWV to predict PIH.

Design and fabrication of a length-extensional mode rectangular X-cut quartz resonator with zero temperature coefficient.

Rectangular X-cut quartz crystal resonators with cut angles theta > 5.0 degrees and aspect ratios Rzy (= width 2z0/length 2y0) from 0.3 to 0.5 are investigated. The resonators oscillate mode is a length-extensional mode. A semiempirical frequency equation was derived from the stress expressed in terms of the trigonometric and the hyperbolic transcendental functions with constants estimated by the regression curve fit to the stress simulated by the finite-element method (FEM). Contours on which a point satisfies a zero first order temperature coefficient condition are shown in a cut angle theta and Rzy diagram. We proved that a fabricated resonator with Rzy = 0.400 and theta = 16.0 degrees, whose design parameter is located in the area of the contour, had a zero temperature coefficient.

Analysis of capacitance ratio of a rectangular X-cut length-extensional mode quartz crystal resonator by stresses.

The electrical properties of an X-cut, length-extensional mode quartz crystal resonator of a cut angle theta around the X-axis were calculated by a variational method using stresses as trial functions. Analytical expressions of stresses were estimated by a linear regression on a cut angle best-fit to the results of finite-element method. The calculated dependence of the capacitance ratio on the cut angle was consistent with the measured results.

Simultaneous determination of creatinine, creatine, and UV-absorbing amino acids using dual-mode gradient low-capacity cation-exchange chromatography.

A simple and versatile cation-exchange chromatography technique for the simultaneous determination of urinary creatinine (Cre), creatine (Crn), methionine (Met), tyrosine (Tyr), phenylalanine (Phe), histidine (His), and tryptophan (Trp) was developed. A novel low-capacity cation-exchange column packed with a newly developed sulfoacylated hypercross-linked macroreticular polystyrene-divinylbenzene resin, referred to as TMR-A/75 (capacity: 75 microequiv/column), was successfully used with a binary dual-mode gradient eluting system. Two solvents, (A) 25 mM phosphoric acid-methanol (30:70, v/v) and (B) 25 mM disodium hydrogenphosphate-methanol (30:70, v/v) were pumped through the column by programming solvent delivery ratios as 0 to 5 min: A-B (55:45, pH 3.6); 5-21 min: A-B (49:51, pH 5.3); and 21-35 min: A-B (55:45, pH 3.6). The flow rate was simultaneously time-programmed to be 0.6 mL/min from 0 to 19 min and to be 1.0 mL/min from 19 to 35 min. This eluting system could permit the use of the UV detection at 210 nm. The analytes, Crn, Met, Tyr, His, Cre, Phe, and Trp, were well separated in this order in 27 min with minimum resolution of approximately 2, and the cycle time was about 35 min. Retention time of each analyte was very reproducible with relative standard deviations (RSDs) between 0.05 and 0.38% (n = 5). The peak area responses were also reproducible with RSDs between 0.74 and 2.24% (n = 5). Calibration lines based on area data were linear from 1 to 1000 microM with r2 values of 0.9998 (Crn), 0.9998 (Met), 0.9999 (Tyr), 0.9999 (His), 1.0000 (Cre), 1.0000 (Phe), and 0.9999 (Trp). The method was applicable to the screening and/or chemical diagnosis of inherited metabolic disorders such as phenylketonuria (PKU), tyrosinemia, and Lowe syndrome. The creatinine ratios of diagnostic markers (microM/microM Cre) were easily determined. The Phe/Cre ratios for five urines from patients with PKU ranged from 0.162 to 0.521, and the Tyr/Cre ratio for tyrosinemia was 0.147. The ratios of Tyr/Cre, Phe/Cre, and Trp/Cre for Lowe syndrome were 0.497, 0.321, and 0.495, respectively. In contrast, the creatinine ratios for healthy newborns showed one digit lower than those for patients did. The developed method is very practical and can provide useful information and results for the clinical or biomedical researches with low analytical run costs.

Simultaneous determination of urinary creatinine and UV-absorbing amino acids using a novel low-capacity cation-exchange chromatography for the screening of inborn errors of metabolism.

A simple and versatile low-capacity cation-exchange chromatography system for the simultaneous determination of creatinine and UV-absorbing amino acids was developed. The separation column was packed with a newly developed low-capacity sulfoacylated macro-porous polystyrene-divinylbenzene resin selective for amino-acid cations. Urinary creatinine, creatine, tyrosine, histidine, phenylalanine, and tryptophan were simultaneously separated and determined by an isocratic elution with phosphate/acetonitrile eluent in 25 min. Relative standard deviations (R.S.D.) of the retention times for the analytes were between 0.28 and 1.06%. R.S.D. of peak area responses for the analytes were between 0.75 and 3.51%. The r(2) values for the calibration lines were between 0.9994 and 0.9999. The method could provide the creatinine ratios for the analytes, and was applicable to the screening and/or chemical diagnosis of several inherited disorders of amino-acid metabolism such as phenylketonuria (PKU).

Fast and Efficient Separation and Determination of UV-absorbing Amino Acids, Nucleobases, and Creatinine Using a Carboxy-functionalized Cation-exchange Column.

This paper presents a new HPLC technique for the determination of biogenic cations such as amino acids and nucleobases, using a weak-acid cation-exchange column. Fourteen analytes, five amino acids and seven bases in addition to creatinine and creatine, were separated in 12 min by means of a two-liquid gradient elution with UV detection. The newly released column packed with a carboxy-functionalized polymethacrylate resin could give excellent selectivity to the organic cations of interest, although such a column is in general suitable for the separation of inorganic common cations. The chromatographic intra-day repeatability was very good with RSDs less than 0.4%, and the quantitation precision based on peak area intensities was also good with RSDs less than 5% for all analytes. The linear calibration lines for quantitation ranged between 5 and 500 µM on 20-µL injections with R(2) more than 0.9990. Since the method could provide concentration data of urinary creatinine and some metabolites simultaneously, for example, the urinary phenylalanine/creatinine ratios for phenylketonuria of inborn errors of metabolism were simply determined through one chromatographic run. The ratios for patients were significantly higher than those for controls. We found that the new weak-acid cation-exchange column was suitable for the separation of organic cations as well as inorganic cations.

The serum lipids levels may be underestimated in patients on hemodialysis.

OBJECTIVE: Although lipid disorders are a well-known risk factor for cardiovascular disease (CVD) in the general population, the optimal management with lipid-lowering therapy to reduce CVD risks and mortality in hemodialysis (HD) patients remains controversial. In the clinical setting, dyslipidemia can be diagnosed based on the detection of elevated lipid concentrations at the beginning of HD. This study investigated changes in the levels of serum lipids during a single HD session. METHODS: The serum total cholesterol, triglyceride and high-density lipoprotein (HDL) cholesterol levels were measured in 31 HD patients at zero, two and four hours after the beginning of a single HD session. The data were analyzed using the Wilcoxon signed-rank test, a linear mixed model and Spearman's rank correlation analysis. RESULTS: The serum total cholesterol, HDL cholesterol and non-HDL cholesterol levels increased significantly during the HD session. Even after the lipid parameters were corrected for changes in the total protein level, the total cholesterol and HDL cholesterol levels increased, whereas the non-HDL cholesterol levels did not change significantly. The percentage change in the serum levels of these lipid fractions correlated strongly with the percentage change in the ultrafiltration volume per body weight. In contrast, the serum triglyceride levels were decreased significantly at two hours compared with the levels noted at the beginning of HD and gradually increased at four hours. CONCLUSION: The serum lipid levels are influenced significantly by HD treatment and ultrafiltration. Evaluating the degree of dyslipidemia at the beginning of a HD session may therefore underestimate the levels of serum lipids in HD patients with a large amount of weight gain, thus resulting in the use of insufficient lipid-lowering therapy.

Low-capacity cation-exchange chromatography of amino acids using a novel sulfoacylated macroreticular polystyrene-divinylbenzene column with binary gradient elution.

This paper describes a versatile technique for amino-acid separation using a novel low-capacity sulfoacylated macroreticular polystyrene-divinylbenzene cation-exchange column with a simple binary high-pressure pH gradient elution. Proteinic 16 amino acids were well separated within 50 min using a H3PO4/Na2HPO4-CH3CN eluent system, and the cycle time was about 70 min. The chromatography with postcolumn OPA fluorescent detection was reproducible with RSDs less than 1% for retention times, and was quantitative with RSDs less than 5% for area responses. A linear regression line with an r2 value above 0.9990 was obtained for each analyte in concentration from 0.1 to 10 microM by 20 microL injection. The method was applicable to the separation and detection of urinary diagnostic amino acid due to inborn errors of metabolism, such as phenylketonuria. The analytical costs would be decreased by using the proposed method.

Sulfoacylated macro-porous polystyrene-divinylbenzene low-capacity cation exchanger selective for amino acids.

A low-capacity cation-exchange column was newly developed for the separation of amino acids. A highly cross-linked macro-porous polystyrene-divinylbenzene co-polymer was functionalized by a sulfoacylation reaction. The exchange capacity was controllable at the acylation step. The capacity between 55 and 60 micromol/column was adequate for the practical separations in acceptable retention times. The 5-microm base polymers having average pore diameters smaller than 3 nm gave satisfactory results, and those having 1.5-nm pore was most favorable. Several isocratic elution conditions at different pH values adjusted by phosphate buffer of mM order with or without acetonitrile could provide good separations for individual classes of amino acids, i.e., acidic, neutral, hydrophobic, and basic groups. The results provided fundamental data for constructing gradient elution systems required for the simultaneous separation of protein amino acids.