Journal of Chromatography B (v.781, #1-2)

Sialic acids are biosynthesized by almost all organisms as a 9-carbon carboxylated monosaccharide and are integral components of glycoconjugates. More than 40 naturally occurring sialic acid derivatives of the three main forms of sialic acids, the N-acetyl- and N-glycolylneuraminic acid and 2-keto-3-deoxy-nonulosonic acid have been identified. Due to the great importance of sialic acids as key mediators in a plethora of cellular events, including cell–cell recognition and cell–matrix interactions, their analysis in biologic samples is useful for a deeper understanding of the various (patho)physiological processes and of value in disease diagnosis and monitoring. In this review we summarize the methodology developed to isolate and liberate sialic acids from biologic samples as well as the chromatographic, electromigration and hyphenated techniques available for their separation and analysis. A critical evaluation of the biological relevance of the results obtained by analyzing sialic acids in biologic samples is also presented.
Keywords: Sialic acids;

Glycosaminoglycans are a class of biological macromolecules found mainly in connective tissues as constituents of proteoglycans, covalently linked to their core protein. Hyaluronan is the only glycosaminoglycan present under its single form and possesses the ability to aggregate with the class of proteoglycans termed hyalectans. Proteoglycans are localised both at the extracellular and cellular (cell-surface and intracellular) levels and, via either their glycosaminoglycan chains or their core proteins participate in and regulate several cellular events and (patho)physiological processes. Advances in analytical separational techniques, including high-performance liquid chromatography, capillary electrophoresis and fluorophore assisted carbohydrate electrophoresis, make possible to examine alterations of glycosaminoglycans with respect to their amounts and fine structural features in various pathological conditions, thus becoming applicable for diagnosis. In this review we present the chromatographic and electromigration procedures developed to analyse and characterise glycosaminoglycans. Moreover, a critical evaluation of the biological relevance of the results obtained by the developed methodology is discussed.
Keywords: Glycosaminoglycans;

Metabolic acidosis: separation methods and biological relevance of organic acids and lactic acid enantiomers by Julia B. Ewaschuk; Gordon A. Zello; Jonathan M. Naylor; Dion R. Brocks (39-56).
Metabolic acidosis can result from accumulation of organic acids in the blood due to anaerobic metabolism or intestinal bacterial fermentation of undigested substrate under certain conditions. These conditions include short-bowel syndrome, grain overfeeding of ruminants and, as recently reported, severe gastroenteritis. Measuring fermentation products such as short-chain fatty acids (SCFAs) and lactic acid in various biological samples is integral to the diagnosis of bacterial overgrowth. Stereospecific measurement of d- and l-lactic acid is necessary for confirmation of the origin and nature of metabolic acidosis. In this paper, methods for the separation of SCFAs and lactic acid are reviewed. Analysis of the organic acids involved in carbohydrate metabolism has been achieved by enzymatic methods, gas chromatography, high-performance liquid chromatography and capillary electrophoresis. Sample preparation techniques developed for these analytes are also discussed.
Keywords: Organic acids; Lactic acid;

Urinary orotic acid determination is a useful tool for screening hereditary orotic aciduria and for differentiating the hyperammonemia disorders which cannot be readily diagnosed by amino acid chromatography, thus reducing the need for enzyme determination in tissue biopsies. This review provides an overview of metabolic aberrations that may be related to increased orotic acid levels in urine, and summarises published methods for separation, identification and quantitative determination of orotic acid in urine samples. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the analysis of urinary specimens are described. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.
Keywords: Orotic acid;

d-Amino acids in mammals and their diagnostic value by Kenji Hamase; Akiko Morikawa; Kiyoshi Zaitsu (73-91).
Substantial amounts of d-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine d-amino acids in mammalian tissues are reviewed, and the distribution of these d-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.
Keywords: d-Amino acids;

Urinary creatinine has been analyzed for many years as an indicator of glomerular filtration rate. More recently, interest in studying the uptake of creatine as a result of creatine supplementation, a practice increasingly common among bodybuilders and athletes, has lead to a need to measure urinary creatine concentrations. Creatine levels are of the same order of magnitude as creatinine levels when subjects have recently ingested creatine, while somewhat elevated urinary creatine concentrations in non-supplementing subjects can be an indication of a degenerative disease of the muscle. Urinary creatine and creatinine can be analyzed by HPLC using a variety of columns. Detection methods include absorption, fluorescence after post-column derivatization, and mass spectrometry, and some methods have been automated. Capillary zone electrophoresis and micellar electrokinetic capillary chromatography have also been used to analyze urinary creatine and creatinine. Creatine and creatinine have also been analyzed in serum and tissue using HPLC and CE, and many of these separations could also be applicable to urinary analysis.
Keywords: Creatine; Creatinine;

Analysis of polyamines as markers of (patho)physiological conditions by Diana Teti; Maria Visalli; Harold McNair (107-149).
The aliphatic polyamines, putrescine, spermidine and spermine, are normal cell constituents that play important roles in cell proliferation and differentiation. The equilibrium between cellular uptake and release and the balanced activities of biosynthetic and catabolic enzymes of polyamines are essential for normal homeostasis in the proliferation and functions of cells and tissues. However, the intracellular polyamine content increases in hyperplastic or neoplastic growth. Although the involvement of polyamines in physiological and pathological cell proliferation and differentiation has been well established, the role they play is quite different in relation to cell systems and animal models and is dependent on inducer agents and stimuli. Also, the experimental procedures used to deplete polyamines have been shown to influence the cell responses. In this paper, the assay methods currently in use for polyamines are reviewed and compared with respect to sensitivity, reproducibility and applicability to routine analysis. The relevance of polyamine metabolism and the uptake/release process in many physiological and pathological processes is highlighted, and the cellular polyamine pathways are discussed in relation to the possible diagnostic and therapeutic significance of these mediators.
Keywords: Polyamines;

Amino acid neurotransmitters: separation approaches and diagnostic value by Ajit J Shah; Francesco Crespi; Christian Heidbreder (151-163).
Amino acids in the central nervous system can be divided into non-neurotransmitter or neurotransmitter depending on their function. The measurement of these small molecules in brain tissue and extracellular fluid has been used to develop effective treatment strategies for neuropsychiatric and neurodegenerative diseases and for the diagnosis of such pathologies. Here we describe the separation and detection techniques that have been used for the measurement of amino acids at trace levels in brain tissue and dialysates. An overview of the function of amino acid transmitters in the brain is given. In addition, the type of sampling techniques that are used for the determination of amino acid levels in the brain is described.
Keywords: Amino acid neurotransmitters;

This review provides an overview of the assay methods used to determine the presence of endogenous histamine (HA) including its metabolites, and also discusses their biological significance. Firstly, this review briefly summarizes the biological significance of HA and its biological pathways. Next, the assay methods with microseparation techniques, such as gas-chromatography (GC), liquid-chromatography (LC), capillary electrophoresis (CE) and capillary electrochromatography (CEC) are looked at from a developmental viewpoint. Finally, the use of these methods, including flow cytometry techniques, for the determination of HA and its metabolites in biological samples, such as blood, urine, brain and cells, is described. The merits and demerits associated with each of these various methods are also discussed, along with their applications.
Keywords: Histamine;

Reduced glutathione (GSH, γ-l-glutamyl-l-cysteinylglycine) is a fundamental low-molecular mass antioxidant that serves several biological functions. Upon enzymatic and non-enzymatic oxidation, GSH forms glutathione disulfide (GSSG) and, under particular conditions, may generate other oxidative products. The determination of GSH, its precursors, and metabolites in several bio-matrices is a useful tool in studying oxidative stress. Many separative and non-separative methods have been developed and improved for the assay of GSH and related compounds. At present, high-performance liquid chromatography and capillary electrophoresis are the most used separative techniques to determine GSH and congeners. The review will deal with analytical methods developed over the last few years for the determination of GSH and related compounds, and with the procedures performed in sample pre-treatment in order to minimize analytical errors. Since GSH, GSSG, and related compounds lack of strong chromophores or fluorophores, it is advantageous, in many assays, to derivatize the compounds in order to improve the detection limit with UV–Vis and to allow fluorescence, thus the most commonly used labeling agents are also described.
Keywords: Glutathione;

Methods for homocysteine analysis and biological relevance of the results by Véronique Ducros; Karine Demuth; Marie-Pierre Sauvant; Muriel Quillard; Elisabeth Caussé; Mirande Candito; Marie-Hélène Read; Jocelyne Drai; Isabelle Garcia; Marie-Françoise Gerhardt (207-226).
It is now widely accepted that increased total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease. Hyperhomocysteinemia can be caused by impaired enzyme function as a result of genetic mutation or vitamin B (B2, B6, B9, B12) deficiency. A lot of methods are now available for tHcy determination. High-pressure liquid chromatography (HPLC) with fluorescence detection are at present the most widely used methods but immunoassays, easier to use, begin to supplant in-house laboratory methods. In order to help with the choice of a main relevant homocysteine analytical method, the characteristics, performances and limits of the main current methods are reviewed. One major drawback among all these available methods is the transferability which is not clearly established to date. The impact of both inter-method and inter-laboratory variations on the interpretation of the tHcy results are discussed.
Keywords: Homocysteine;

Sulfur is a chemically and biologically active element. Sulfur compounds in animal tissues can be present in two forms, namely stable and labile forms. Compounds such as methionine, cysteine, taurine and sulfuric acid are stable sulfur compounds. On the other hand, acid-labile sulfur and sulfane sulfur compounds are labile sulfur compounds. The sulfur atoms of labile sulfur compounds are liberated as inorganic sulfide by acid treatment or reduction. Therefore, the determination of sulfide is the basis for the determination of labile sulfur. Determination of sulfide has been performed by various methods, including spectrophotometry after derivatization, ion chromatography, high-performance liquid chromatography after derivatization, gas chromatography, and potentiometry with a sulfide ion-specific electrode. These methods were originally developed for the determination of sulfide in air and water samples and were then applied to biological samples. The metabolic origin of labile sulfur in animal tissues is cysteine. The pathways of cysteine metabolism leading to the formation of sulfane sulfur are discussed. Finally, reports on the physiological roles and pathological considerations of labile sulfur are reviewed.
Keywords: Sulphur;

Separation methods for taurine analysis in biological samples by Shifen Mou; Xiaojing Ding; Yongjian Liu (251-267).
Taurine plays an important role in a variety of physiological functions, pharmacological actions and pathological conditions. Many methods for taurine analysis, therefore, have been reported to monitor its levels in biological samples. This review discusses the following techniques: sample preparation; separation and determination methods including high-performance liquid chromatography, gas chromatography, ion chromatography, capillary electrophoresis and hyphenation procedures. It covers articles published between 1990 and 2001.
Keywords: Taurine;

Mercapturic acids in the biological monitoring of occupational exposure to chemicals by Luigi Perbellini; Nello Veronese; Andrea Princivalle (269-290).
This paper reviews several procedures for determination of mercapturic acids in urine. Special attention was paid to methods useful in relation to human exposure to industrial pollutants, without any description for less sensitive methods used in animal research. Gas chromatographic and liquid chromatographic procedures were considered together with the little information available about thin layer chromatography and immunochemical techniques. After a description of the main industrial pollutants which lead to synthesis of their specific mercapturic acids, the methods for analysing these products are synthetically reported. The comparison among difficulties in sample preparation, complexity of instrumentation and their cost/benefit ratio are discussed.
Keywords: Mercapturic acid; Industrial pollutants;

Hemoglobin variants are the protein mutations most often encountered in the clinical scene. They have been useful for developing methods to analyze mutant proteins because of their size and ease of collection in large amounts. Improvements in analytical methods have been directed toward higher resolution in electrophoresis and shorter elution times in chromatography. More importantly, in the last 20 years, hemoglobin variants have been used in the development of mass spectrometric strategies for analyzing protein mutations. This approach consists of a series of steps: measurement of the molecular mass of globins to detect or confirm the presence of mutations, peptide mass mapping or peptide mass fingerprinting of an enzymatic digest to identify mutated peptides, and tandem mass spectrometry to determine or confirm the site and type of mutation. The mass spectrometric strategy has enabled rapid analysis and demonstrated a superb ability to detect a number of hemoglobin variants, particularly those without a change in electrophoretic or chromatographic properties. Even with the recent advances in DNA analysis, protein analysis is still essential, because post-translational modifications following amino acid substitutions can occur including N-terminal acetylation, deamidation and oxidation-mediated processes.
Keywords: Hemoglobin variants; Protein mutations;

Study of metallothionein using capillary zone electrophoresis by Takeshi Minami; Seiji Ichida; Kanenobu Kubo (303-311).
Metallothioneins (MTs) have many different functions in tissues, but the roles of individual isoforms are still not entirely clear. Capillary zone electrophoresis (CZE) is a powerful method for the separation of substances because of its small sample requirement, rapid analysis, high sensitivity and high resolution. The separation and identification of mammalian MT-1, MT-2, and MT-3 and class III MTs by CZE has been reported. Uncoated and polyacrylamide-coated capillary tubes were recently used for the separation of MTs, and a UV detector is usually employed for observations of peaks of MTs. Small changes to the structure and metal components of MTs are reflected in the migration times of the peaks. N-acetylated and non-acetylated MTs can be separated and identified by CZE–mass spectrometry (MS). In addition, metal complexes with MTs can be characterized by CZE–proton-induced X-ray emission (PIXE) detector and CZE–inductively coupled plasma (ICP)–MS. For the quantification of an MT isoform, the peak area of UV absorption is used, but the technique has problems. One is lack of a purified isoform standard. The other is the need for a suitable internal standard substance. CZE–ICP–isotope dilution (ID)–MS is also reported to be able to quantify MT isoforms. CZE combined with other techniques is very effective for separation and quantitative and qualitative analyses of MT isoforms in biological materials.
Keywords: Metallothionein; Isoform;

Assay methods of modified lipoproteins in plasma by Yu Yamaguchi; Masaru Kunitomo; Jun Haginaka (313-330).
Modified lipoproteins, especially oxidatively modified low-density lipoprotein (Ox-LDL), are present in the plasma of patients with atherosclerosis and related diseases. The modification of LDL is believed to play an important role in the development of atherosclerosis. Thus, measurement of plasma Ox-LDL is essential not only for investigating its relevance to atherosclerotic diseases, but also for diagnosis. Chromatographic methods are effective for indirectly measuring the oxidatively modified state of LDL or directly measuring the modified LDL. Indirect determination can be done by estimating the LDL subfraction, LDL particle size, oxidized amino acids in apolipoprotein B, lipid hydroperoxide or F2-isoprostane in LDL. Direct determination of the modified LDL in plasma can be done with chromatographic methods such as anion-exchange chromatography and size-exclusion chromatography. Other methods for estimating the modified state of LDL include electromigration methods such as agarose gel, polyacrylamide gradient gel and capillary electrophoresis. Recently, enzyme-linked immunosorbent assay methods of malondialdehyde (MDA)-LDL and autoantibodies against Ox-LDL have been developed to assess Ox-LDL in plasma. This review article summarizes the detection and assay methods of modified lipoproteins in plasma.
Keywords: Lipoproteins;

Detection of oxidized high-density lipoprotein by Toshiyuki Matsunaga; Iwao Koyama; Shigeru Hokari; Tsugikazu Komoda (331-343).
This paper reviews working procedures for the separation and detection of oxidized high-density lipoproteins (ox-HDL) and their constituents. It begins with an introductory overview of structural alterations of the HDL particle and its constituents generated during oxidation. The main body of the review delineates various procedures for the isolation and detection of ox-HDL as well as the purification and separation of phosphatidylcholine metabolites and denatured apolipoproteins in the particle. The useful methods published more recently are picked up and the utility of the separation techniques is described. The last section covers a clinical evaluation of changes in these factors in ox-HDL as well as future directions of ox-HDL research.
Keywords: High-density lipoproteins;

Acid phosphatases as markers of bone metabolism by Yoshihiko Igarashi; Minako Y Lee; Shigeru Matsuzaki (345-358).
Various biochemical markers have been used to assess bone metabolism and to monitor the effects of treatments. Tartrate resistant acid phosphatase (TRAP; EC has often been used to assess bone absorption. Although osteoclasts contain abundant TRAP and they are responsible for bone resorption, the total TRAP activities in the serum measured by colorimetric methods little reflect the bone turnover. TRAP 5 is further separated into 5a and 5b by electrophoresis. Type 5b is considered to be derived from the osteoclast, and therefore attempts are being made to measure exclusively serum TRAP 5b by kinetic methods, immunological methods, and chromatographic methods including ion-exchange and heparin column chromatography.
Keywords: Acid phosphatases;

Catechol O-methyltransferase (COMT) transfers a methyl group from S-adenosyl-l-methionine to the catechol substrate in the presence of magnesium. After the characterisation of COMT more than four decades ago, a wide variety of COMT enzyme assays have been introduced. COMT activity analysis usually consists of the handling of the sample and incubation followed by separation and detection of the reaction products. Several of these assays are validated, reliable and sensitive. Besides the studies of the basic properties of COMT, the activity assay has also been applied to explore the relation of COMT to various disease states or disorders. In addition, COMT activity analysis has been applied clinically since COMT inhibitors have been introduced as adjuvant drugs in the treatment of Parkinson’s disease.
Keywords: Catechol O-methyltransferase; Enzymes;

5-Methylcytosine as a marker for the monitoring of DNA methylation by Jan Havliš; Martin Trbušek (373-392).
The extent of the DNA methylation of genomic DNA as well as the methylation pattern of many gene-regulatory areas are important aspects with regard to the state of genetic information, especially their expression. There is growing evidence that aberrant methylation is associated with many serious pathological consequences. As genetic research advances, many different approaches have been employed to determine the overall level of DNA methylation in a genome or to reveal the methylation state of particular nucleotide residues, starting from semiquantitative methods up to new and powerful techniques. In this paper, the currently employed techniques are reviewed both from the point of view of their relevance in genomic research and of their analytical application. The methods discussed include approaches based on chromatographic separation (thin-layer chromatography, high-performance liquid chromatography, affinity chromatography), separation in an electric field (capillary electrophoresis, gel electrophoresis in combination with methylation-sensitive restriction enzymes and/or specific sequencing protocols), and some other methodological procedures (mass spectrometry, methyl accepting capacity assay and immunoassays).
Keywords: 5-Methylcytosine; DNA;

Carotenoids: separation methods applicable to biological samples by Qing Su; Kevin G Rowley; Nicholas D.H Balazs (393-418).
Epidemiologic and clinical studies have shown that a high intake of vegetables and fruit, with consequently high intakes and circulating concentrations of carotenoids, is associated with reduced risk of cardiovascular and other chronic diseases. The antioxidant properties of carotenoids are thought to contribute to these effects. The analysis of carotenoids in plasma, foods and tissues has thus become of interest in studies examining the role of diet in chronic disease prevention and management. High-performance liquid chromatography with ultra-violet or photodiode array detection is most often employed in routine use. We review these and other current methods for carotenoid analysis and information on sample stability relevant to epidemiological studies. The carotenoids remain an important and intriguing subject of study, with relevance to prevention of several important “lifestyle-related” diseases. Research into their physiological functions and their use as dietary markers requires sensitive, accurate and precise measurement. Further advances in these methodological areas will contribute to basic, clinical and public health research into the significance of carotenoid compounds in disease prevention.
Keywords: Carotenoids;

Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents.
Keywords: Proteins; Endotoxin;

Urinary analysis of nephrolithiasis markers by C Barbas; A Garcı́a; L Saavedra; M Muros (433-455).
Renal stone disease is an ancient and common affliction, common in industrialised nations. The causes and incidence of nephrolithiasis are presented. Afterwards, the promoters and inhibitors of renal stone formation analysis in urine are described including enzymatic methods, chromatography, capillary electrophoresis and other techniques. Aspects such as sample collection and storage are also included. The review article includes referenced tables that provide summaries of methodology for the analysis of nephrolithiasis related compounds.
Keywords: Oxalate; Citrate;

The development in chromatographic methods for the determination of markers of alcohol consumption is summarized in this review. The markers included in this article are ethanol in body fluids, ethanol congeners, fatty acid ethyl esters (FAEEs), ethyl glucuronide (EtG), cocaethylene (CE), carbohydrate-deficient transferrin (CDT), phosphatidylethanol (PEth), 5-hydroxytryptophol (5-HTOL), dolichol, ketone bodies, acetaldehyde–protein adducts, and salsolinol (SAL). Some of these markers for alcohol consumption do not only indicate previous ethanol ingestion, but also approximate the amount of intake and the time when ethanol ingestion last occurred. Basic information about the procedures, work-up, and chromatographic conditions are summarized in tables. Also the main metabolic pathways and reaction schemes are demonstrated in figures. Some examples of typical applications are presented. The author points out that in many of the reviewed papers validation data of the procedures as well as specificities and sensitivities were not clearly presented and consequently were not comparable.
Keywords: Alcohol consumption markers;

Hydroxyl radical in living systems and its separation methods by Fu-Chou Cheng; Jen-Fon Jen; Tung-Hu Tsai (481-496).
It has recently been shown that hydroxyl radicals are generated under physiological and pathological conditions and that they seem to be closely linked to various models of pathology putatively implying oxidative stress. It is now recognized that the hydroxyl radical is well-regulated to help maintain homeostasis on the cellular level in normal, healthy tissues. Conversely, it is also known that virtually every disease state involves free radicals, particularly the most reactive hydroxyl radical. However, when hydroxyl radicals are generated in excess or the cellular antioxidant defense is deficient, they can stimulate free radical chain reactions by interacting with proteins, lipids, and nucleic acids causing cellular damage and even diseases. Therefore, a confident analytical approach is needed to ascertain the importance of hydroxyl radicals in biological systems. In this paper, we provide information on hydroxyl radical trapping and detection methods, including liquid chromatography with electrochemical detection and mass spectrometry, gas chromatography with mass spectrometry, capillary electrophoresis, electron spin resonance and chemiluminescence. In addition, the relationships between diseases and the hydroxyl radical in living systems, as well as novel separation methods for the hydroxyl radical are discussed in this paper.
Keywords: Hydroxyl radicals;

To diagnose inborn errors of metabolism, it would be desirable to simultaneously analyze and quantify organic acids, purines, pyrimidines, amino acids, sugars, polyols, and other compounds using a single-step fractionation; unfortunately, no such method currently exists. The present article will be concerned primarily with a practical yet comprehensive diagnostic procedure of inborn errors of metabolism (IEM). This procedure involves the use of urine or eluates from urine on filter paper, stable isotope dilution, and gas chromatography–mass spectrometry (GC–MS). This procedure not only offers reliable and quantitative evidence for diagnosing, understanding and monitoring the diseases, but also provides evidence for the diagnosis of new kinds of IEM. In this review, the differential diagnosis for hyperammonemia are described; deficiencies of ornithine carbamoyl transferase, argininosuccinate synthase (citrullinemia), argininosuccinate lyase and arginase, lysinuric protein intolerance, hyperammonemia–hyperornithinemia–homocitrullinemia syndrome, and citrullinemia type II. The diagnosis of IEM of purine and pyrimidine such as deficiencies of hypoxanthine–guanine phosphoribosyl transferase, adenine phosphoribosyl transferase, dihydropyrimidine dehydrogenase, dihydropyrimidinase and β-ureidopropionase are described. During the pilot study for newborn screening, we found neonates with diseases at a rate of 1 per 1400 including propionic acidemia, methylmalonic acidemia, orotic aciduria, β-ureidopropionase deficiency, lactic aciduria and neuroblastoma. A rapid and reliable prenatal diagnosis for propionic acidemia is also described.
Keywords: 5-Fluorouracil;