Journal of Chromatography B (v.792, #1)

Preface (1).

A catalogue of proteins in the human vitreous humor may contribute to elucidating the pathogenesis of various diseases in ophthalmology. To improve the recovery of proteins in vitreous, we applied one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (1D-PAGE). Proteins were extracted from unstained gel strips and digested in gel with trypsin and the peptides were analyzed by capillary-column reversed-phase high-performance liquid chromatography coupled with electrospray ionization-ion trap-mass spectrometry. From a patient with diabetic retinopathy, 84 different proteins were identified. Most of the proteins which we identified in vitreous previously using 2D-PAGE were also identified in the present study. In total, we identified 121 different proteins including five proteins seen at the genomic level only. Four angiogenic factors, insulin-like growth factor, vascular endothelial growth factor, fibroblast growth factor, and placental endothelial cell growth factor, and three anti-angiogenic factors, pigment epithelium-derived factor, endostatin, and thrombospondin, were found, and this may contribute to elucidating the pathological changes in the concentration and the modified structures of these proteins, in diseases of the retina, especially, diabetic retinopathy.
Keywords: Proteins; Angiogenesis-regulating factors;

First case of a single heterozygote of an abnormal hemoglobin, Hb Stanmore, [β111(G13)Val→Ala] by A. Miyazaki; T. Nakanishi; A. Shimizu; H. Hisamitsu (23-31).
We describe a hemoglobin β-chain mutant detected incidentally in an unusual profile of glycated hemoglobin (HbA1c) measured by ion-exchange HPLC. Analysis of intact globin by electrospray ionization mass spectrometry (ESI-MS) and peptide analysis by on-line HPLC-ESI-MS-MS revealed the substitution, [β111(G13)Val→Ala], which was confirmed by DNA analysis. This was the second case of Hb Stanmore. As the first case combined β0-thalassemia, and the family study in that case showed no case of Hb Stanmore without combined thalassemia, the case presented here is the first case of single heterozygote, and the first Japanese case. Hb Stanmore is isoelectrophoretically silent with only mild clinical symptoms, although stability by isopropanol test was positive.
Keywords: Hemoglobins;

Determination of endogenous peptides in the porcine brain: possible construction of Peptidome, a fact database for endogenous peptides by Naoto Minamino; Junko Tanaka; Hiromiki Kuwahara; Takahiro Kihara; Yoshinori Satomi; Masami Matsubae; Toshifumi Takao (33-48).
Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10 000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.
Keywords: Peptides, endogenous;

A novel methylcellulose-immobilized reversed-phase pretreatment column (MC-ODS) for column switching liquid chromatography–mass spectrometry (LC–MS) was investigated to improve recovery and durability. Pretreatment and analytical conditions were optimized so that high throughput and high selectivity was ensured during mass spectrometric analysis. Analytical runs, including deproteinization and gradient LC analysis, were conducted in a 6-min cycle. As a consequence, recoveries for test drugs (metoprolol, propranolol, lidocaine, dibucaine, bupivacaine) were greater than 90% and more than 300 plasma samples spiked with target compounds were directly injected and measured without compromising MS detection or system performance.
Keywords: Methylcellulose;

Determination of cabergoline and l-dopa in human plasma using liquid chromatography–tandem mass spectrometry by Kazuo Igarashi; Koichiro Hotta; Fumiyo Kasuya; Kazuo Abe; Saburo Sakoda (55-61).
We determined cabergoline and l-dopa in human plasma using liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS–MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for l-dopa) on LC–MS–MS with electrospray ionization (ESI), cabergoline and l-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5–250 pg/ml for cabergoline and 1–200 ng/ml for l-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for l-dopa, respectively. The method was applied to the analysis of cabergoline and l-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8–26.2 pg/ml and from 2.9% to 8.9% at an l-dopa concentration of 302.5–522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.
Keywords: l-Dopa; Cabergoline;

Selective screening for fatty acid oxidation disorders by tandem mass spectrometry: difficulties in practical discrimination by Yosuke Shigematsu; Satoko Hirano; Ikue Hata; Yukie Tanaka; Masakatsu Sudo; Tsuyoshi Tajima; Nobuo Sakura; Seiji Yamaguchi; Masaki Takayanagi (63-72).
In a selective screening for fatty acid oxidation disorders by tandem mass spectrometry, we tested the diagnostic ratios and acylcarnitine concentrations in sera or blood spots, which were reported to be specific to very long-chain acyl CoA dehydrogenase deficiency, carnitine palmitoyltransferase I deficiency, and carnitine palmitoyltransferase II deficiency. While the acylcarnitine profiles in the majority of these patients were typical in the respective disorders, some overlapping of the indices was observed between these patients and the infants, who showed symptoms mainly related to hypoglycemia but did not have the disorders mentioned above. Although the diagnostic ratio of tetradecenoylcarnitine to dodecanoylcarnitine for very long-chain acyl CoA dehydrogenase deficiency seemed to minimize the overlapping in this study, additional measures including careful assessment of clinical data and enzyme assays may be necessary for the diagnosis in atypical cases.
Keywords: Fatty acids;

Effect of supplementation with l-carnitine at a small dose on acylcarnitine profiles in serum and urine and the renal handling of acylcarnitines in a patient with multiple acyl-coenzyme A dehydrogenation defect by Makoto Yoshino; Yasuykuki Tokunaga; Yoriko Watanabe; Ichiro Yoshida; Miki Sakaguchi; Ikue Hata; Yosuke Shigematsu; Masahiko Kimura; Seiji Yamaguchi (73-82).
We studied the effects of l-carnitine supplementation at a small dose on the profiles of acylcarnitines in serum and urine, as well as the renal handling of acylcarnitines, in a patient with multiple acyl-coenzyme A dehydrogenation defect. After supplementation with l-carnitine at a dose of 20 mg/kg/day, the concentration of each acylcarnitine measured both in the serum and in the urine had increased significantly, with the exception of that of an acylcarnitine with a carbon chain length (C) of 8 (C8 acylcarnitine). The magnitude of increase in the concentrations of the acylcarnitines in the serum was not associated with chain length, whereas in the urine, the magnitude tended to be greater in proportion to the shortness of the chain length. The fractional excretions of C2–C5 acylcarnitines exceeded 100%, indicating that they were produced in, or transported across, renal tubular epithelial cells and secreted into the urine. These results indicate that supplementation with a relatively small amount of l-carnitine can enhance the renal excretion of accumulated short-chain-length acylcarnitines through tubular excretion, in addition to basic glomerular filtration.
Keywords: Carnitine; Acylcarnitine;

Gamma-hydroxybutyric acid (GHB) is a substance naturally present within mammal species. Properties of a neurotransmitter or neuromodulator are generally suggested for this substance. GHB is therapeutically used as an anaesthetic, but can be used for criminal offences (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. Twenty μl of blood or urine were pipetted into a glass tube, followed by 20 μl GHB-d6 and 45 μl acetonitrile. After vortexing and efficient centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with BSTFA+1% TMCS for 20 min at 70 °C. After injection on a 30-m HP5 MS capillary column, GHB (m/z 233, 204 and 147) and GHB-d6 (m/z 239) were identified by mass spectrometry. The procedure was linear from 1 to 200 mg/l for both blood and urine. Precisions were in the range 4 to 11%. The method appears simple, specific and rapid as an accurate result can be obtained within 1 h.
Keywords: γ-Hydroxybutyric acid;

Monitoring method for pre- and post-liver transplantation in patients with primary hyperoxaluria type I by Yoshito Inoue; Hiroaki Masuyama; Hiromichi Ikawa; Hiroshi Mitsubuchi; Tomiko Kuhara (89-97).
Differential diagnosis of primary hyperoxaluria type I (PH1) may become difficult once end-stage renal failure and anuria have occurred. Here we describe a rapid and sensitive method to simultaneously quantify glycolate and oxalate in plasma using a stable isotope dilution and gas chromatography–mass spectrometry. The results are provided within 2 h. The linearity of the method was validated up to 200 μmol/l of these compounds and the inter-assay precision for glycolate and oxalate was 2.4 and 2.6%, respectively (n=5), when the control plasma was spiked with 50 μmol/l of glycolate and oxalate. For healthy subjects, 1.0 ml of plasma was required and 0.1 ml for the PH1 patients. Using this method, plasma levels in non-PH1 patients under hemodialysis and in healthy subjects were determined. This method proved to be useful when used for differential diagnosis of PH1 and for monitoring the plasma levels of glycolate and oxalate in two PH1 patients before and after dialysis and liver transplantation. Plasma glycolate in these patients was dramatically decreased after liver transplantation, but plasma oxalate decreased more slowly due to remobilization of the calcium oxalate stores deposited throughout the body.
Keywords: Glycolate; Oxalate;

The metabolic changes in a patient with succinic semialdehyde dehydrogenase deficiency were investigated following valproate administration using urease pretreatment and gas chromatography-mass spectrometry. A stable isotope dilution technique was used for quantification of urinary 4-hydroxybutyrate. Urinary levels of 4-hydroxybutyrate were 4-fold higher after 1-month valproate therapy. 4,5-Dihydrohexanoate, 2-deoxytetronate and 3-deoxytetronate were also 1.7–2.7-fold higher. The urinary excretions of 4-hydroxybutyrate in valproate non-medicated controls were age dependence and decreased with age. Relationships between 4-hydroxybutyrate excretion and 4-hydroxyvalproate or 5-hydroxyvalproate excretion were observed in valproate medicated controls. It seems that 4-hydroxyvalproate and 5-hydroxyvalproate as well as valproate are involved with increased excretion of 4-hydroxybutyrate following valproate administrations.
Keywords: Valproic acid;

Rapid gas chromatographic–mass spectrometric diagnosis of dihydropyrimidine dehydrogenase deficiency and dihydropyrimidinase deficiency by Tomiko Kuhara; Chie Ohdoi; Morimasa Ohse; André B.P. van Kuilenburg; Albert H. van Gennip; Satoshi Sumi; Tetsuya Ito; Yoshiro Wada; Isamu Matsumoto (107-115).
A rapid yet reliable chemical diagnosis for dihydropyrimidine dehydrogenase (DHPD) deficiency, and possibly dihydropyrimidinase (DHP) deficiency in cancer patients, prior to therapy with pyrimidine analogues such as 5-fluorouracil, is desired for prevention of severe side-effects by these drugs. We have reported the basic separation and quantitation technology for pyrimidine metabolites using gas chromatography–mass spectrometry. A proposal to use the number (n) of standard deviations (SD) above the normal mean, as the index of the excessive urinary excretion of the metabolites appears not to be commonly used. When used, the values were too small, such as two or three, even in genetic disorders. Here, we applied the method to 11 urine specimens from proven cases including two DHP carriers and proved how specific the method is, because “n”-values were markedly large for thymine (T), uracil (U) and/or dihydrothymine (DHT) and dihydrouracil (DHU). In three cases with DHPD deficiency, two were siblings, one with symptoms and the other without, n was 12 for T and 5.9 for U, and 5-hydroxymethyluracil was distinctly detected. These values indicate that the nature of genetic mutation relates closely to the degree of metabolite accumulation in pyrimidine disorders. In six patients with DHP deficiency, n was 8.4–12 for DHT and 7.2–11 for DHU. Many mutations are known for both genes and the assay of residual enzyme activity may be time-consuming or invasive especially for those with DHP deficiency. Thus, this noninvasive yet comprehensive urinalysis has great value for those without a family history, as the first trial, before DNA or the enzyme assay. Our findings again raise the question whether the metabolic block really causes the symptoms found in pyrimidine disorders.
Keywords: Enzymes; Dihydropyrimidine dehydrogenase; Dihydropyrimidinase;

Sensitive determination of pethidine in body fluids by gas chromatography–tandem mass spectrometry by Akira Ishii; Miwa Tanaka; Rina Kurihara; Kanako Watanabe-Suzuki; Takeshi Kumazawa; Hiroshi Seno; Osamu Suzuki; Yoshinao Katsumata (117-121).
We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography–tandem mass spectrometry (GC–MS/MS). Pethidine and 4′-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C18 columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25–40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.
Keywords: Pethidine;

Lesch–Nyhan syndrome (LNS) is caused by a severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and clinically characterized by self-injurious behavior and nephrolithiasis; the latter is treatable with allopurinol, an inhibitor of xanthine oxidase which converts xanthine and hypoxanthine into uric acid. In the HPRT gene, more than 200 different mutations are known, and de novo mutation occurs at a high rate. Thus, there is a great need to develop a highly specific method to detect patients with HPRT dysfunction by quantifying the metabolites related to this enzyme. A simplified urease pretreatment of urine, gas chromatography–mass spectrometry, and stable isotope dilution method, developed for cutting-edge metabonomics, was further applied to quantify hypoxanthine, xanthine, urate, guanine and adenine in 100 μl or less urine or eluate from filter-paper-urine strips by additional use of stable isotope labeled guanine and adenine as the internal standards. In this procedure, the recoveries were above 93% and linearities (r 2=0.9947–1.000) and CV values (below 7%) of the indicators were satisfactory. In four patients with proven LNS, hypoxanthine was elevated to 8.4–9.0 SD above the normal mean, xanthine to 4–6 SD above the normal mean, guanine to 1.9–3.7 SD, and adenine was decreased. Because of the allopurinol treatment for all the four patients, their level of urate was not elevated, orotate increased, and uracil was unchanged as compared with the control value. It was concluded that even in the presence of treatment with allopurinol, patients with LNS can be chemically diagnosed by this procedure. Abnormality in the levels of hypoxanthine and xanthine was quite prominent and n, the number of standard deviations above the normal mean, combined for the two, was above 12.9.
Keywords: Hypoxanthine-guanine phosphoribosyltransferase;

This article describes the overall procedure for the simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonyglycerol) and isoprostane by gas chromatography–mass spectrometry in the selected-ion monitoring SIM mode (GC–MS-SIM) for medical samples. It also describes the general points of this method which a scientist who wants to assay a new, unidentified prostanoids and related compounds in medical samples would need to be clarified. The similar structures of prostaglandins, thromboxane, their metabolites, isoprostane, and arachidonyl compounds, allow them to be assayed after the simultaneous preparation of a single sample. The dimethyl isopropylsilyl ether forms of derivatized compounds are suitable for multiple GC–MS-SIM assay because of their molecular stability, and because they produce positive, strong, and large fragments on MS.
Keywords: Endocannabinoids; Anandamide; 2-Arachidonyglycerol; Isoprostane;

A sensitive method for 4-hydroxybutyric acid in urine using gas chromatography–mass spectrometry by Masahiko Kimura; Yuki Hasegawa; Katuhiro Nakagawa; Mituharu Kajita; Kazuyoshi Watanabe; Seiji Yamaguchi (141-144).
4-Hydroxybutyric acid (4HB) was analyzed by gas chromatography–mass spectrometry. Under acidified conditions, 4HB is difficult to detect due to lactonization. Using a urine sample containing 0.01 mg creatinine, we performed trimethylsilyl derivatization without extraction, only adding dimethylsuccinic acid as an internal standard and 10 μl of 0.1 N NaOH methanol solution with adequate evaporation. Urine 4HB levels in a patient with 4-hydroxybutyric aciduria was determined to be 1258 mmol/mol Cr (control, 0.28–2.81 mmol/mol Cr) in this method. Direct derivatization of samples without extraction showed good reproducibility and linearity. Only a small sample of urine was required. Alkalinization by NaOH prevented not only lactonization of 4HB, but also loss of the compounds during evaporation.
Keywords: 4-Hydroxybutyric acid; 4-Hydroxybutyric aciduria;