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Methods of Analysis and Clinical Significance
Ryouta Maeba*,1; Megumi Nishimukai┼,¶; Shin-ichi Sakasegawa╬; Daisuke Sugimori§; Hiroshi Hara¶ * Department of Biochemistry, Teikyo University School of Medicine, Itabashi-ku, Tokyo, Japan
┼ Department of Animal Science, Iwate University, Morioka, Iwate, Japan
╬ Asahi Kasei Pharma Corporation, Shizuoka, Japan
§ Department of Symbiotic Systems Science and Technology, Graduate School of Symbiotic Systems Science and Technology, Fukushima University, Fukushima, Japan
¶ Division of Applied Bioscience, Hokkaido University, Sapporo, Hokkaido, Japan
1 Corresponding author: email address: firstname.lastname@example.org
Age-related diseases, such as atherosclerosis and dementia, are associated with oxidative stress and chronic inflammation. Peroxisome dysfunction may be related to aging and age-related pathologies, possibly through the derangement of redox homeostasis. The biosyntheses of plasmalogens (Pls), a subclass of glycerophospholipids, are primarily regulated by peroxisomes. Thus, plasma Pls may reflect the systemic functional activity of peroxisomes and serve as potential biomarkers for diseases related to oxidative stress and aging.
Recently, we have established three promising analytical methods for plasma/serum Pls using high-performance liquid chromatography with radioactive iodine, liquid chromatography-tandem mass spectrometry, and enzymatic assay. These methods were validated and used to obtain detailed molecular information regarding these molecules. In cross-sectional studies on asymptomatic, coronary artery disease, and elderly dementia individuals, we found that serum choline Pls, particularly those containing oleic and linoleic acid in the sn-2 position of the glycerol backbone, may serve as reliable antiatherogenic biomarkers. Furthermore, we also found that serum ethanolamine Pls were effective in discriminating cognitive impairment. These results support our hypothesis and further studies are clearly needed to elucidate Pls pathophysiologic significance.
Coronary artery disease
Liquid chromatography-tandem mass spectrometry
125I-HPLC high-performance liquid chromatography with 125I
AD Alzheimer's disease
CD cognitive decline
Egp ether glycerophospholipid
LC-MS/MS liquid chromatography-tandem mass spectrometry
LyPlsCho choline lysoplasmalogen
LyPlsEtn ethanolamine lysoplasmalogen
Pak alkyl glycerophospholipid
PakCho choline alkyl glycerophospholipid
PakEtn ethanolamine alkyl glycerophospholipid
PlsCho choline plasmalogen
PlsEtn ethanolamine plasmalogen
Emerging pathologic evidence indicates that oxidative stress and chronic inflammation are involved in major age-related diseases such as atherosclerosis, dementia, and cardiovascular disease. Changes in redox status that occur during aging may be the major risk factor for age-related inflammation .
Peroxisomes are essential organelles in higher eukaryotes for redox homeostasis and other metabolic functions. Cumulative evidence suggests that peroxisomes function as potential regulators of oxidative stress-related signaling pathways . These findings suggest that peroxisome dysfunction is not only associated with rare peroxisomal disorders but also with more common age-related diseases related to oxidative stress.
Many studies have attempted to identify specific biomarkers that could aid diagnosis or predict treatment response of age-related diseases. However, it has been difficult to consistently define and specifically identify biomarkers directly linked to aging and age-related disease .
Here, we describe analysis and clinical utility of plasma/serum plasmalogens (Pls) as a potential oxidative stress markers associated with peroxisome function.
Glycerophospholipids are classified into the three subclasses, i.e., diacyl, alkyl, and alkenyl types, by the aliphatic hydrocarbon chain at the sn-1 position of the glycerol backbone, via ester, ether, and vinyl-ether (COCCR) binding, respectively. The diacyl type is a predominant subclass of glycerophospholipids. The alkyl and alkenyl types are collectively called ether glycerophospholipids (Egps), whereas the alkenyl type is a specific Pl.
Based on the polar head groups at the sn-3 position, Pls are mainly classified into either choline plasmalogen (PlsCho) or ethanolamine Pls (PlsEtn) (Fig. 1). The former is found in cardiac muscle and plasma, whereas the latter belongs to a predominant class distributed in a wide variety of cells and tissues.  Figure 1
Chemical structure of plasmalogens. The figure was originally published in Ref. .
2.1 Biosynthesis, Function, and Pathophysiology
Peroxisomes are essential regulatory organelles for Pls biosynthesis, i.e., the first two steps of Pls biosynthesis occurs exclusively in peroxisomes . In addition, the rate-limiting enzyme; fatty acyl CoA reductase 1 (Far 1) is peroxisomal [7-9] (Fig. 2). Alkyl glycerophospholipids (Paks) are precursors of Pls. PlsEtn is synthesized from ethanolamine Paks (1-alkyl-2-acyl-GPE), whereas PlsCho appears derived from PlsEtn, but not choline Paks (1-alkyl-2-acyl-GPC) [10, 11]. Its exact biosynthetic route, however, remains unclear. Figure 2
Biosynthetic pathways of plasmalogens. The figure was slightly modified and originally published in Ref. .
The pathophysiologic roles of Pls are poorly understood. Some patients with peroxisomal disorders exhibit systemic reduction of Pls and various pathologic conditions including severe mental retardation, hypotonicity, adrenal dysfunction, cataracts, deafness, facial dysmorphism, chondrodysplasia, and failure to thrive . Pl knockout mice also exhibit similar phenotypes, particularly central nervous system dysfunction . Pls are abundant in the brain and play essential roles in neuronal function and myelin formation . Defects in Pls are associated with a number of neurodegenerative disorders including Alzheimer's disease (AD) . In addition, Pls appear to modulate membrane dynamics resulting in nonbilayer structures  and membrane fusion . These characteristic features of Pls in modulating biomembranes may be relevant to manifestation of diverse pathophysiology.
Recently, particular attention has been paid to the involvement of Pls in metabolic diseases associated with oxidative stress and chronic inflammation [18, 19]. Studies have postulated that Pls serve as endogenous antioxidants and protect membrane lipids and lipoprotein particles from excessive oxidation by scavenging reactive oxygen species via their vinyl-ether moiety [20-22]. Furthermore, Pls function as reservoirs for precursor fatty acids, such as arachidonic and docosahexaenoic acid (DHA), which generate bioactive lipid mediators related to inflammation.
2.2 Plasma/Serum Pls
Human plasma/serum Pls are synthesized in the liver, intestine, and kidney and secreted into the blood as lipoprotein components [23, 24]. They are distributed almost equally in all lipoprotein fractions . The concentration of plasma/serum Pls is 100-300 µmol/L with PlsCho/PlsEtn ratio in the range of 0.5-1.5, a ratio corresponding to ~ 5% PlsCho in choline phospholipids and 50-60% PlsEtn in ethanolamine phospholipids.
Plasma Pls concentration may reflect systemic peroxisomal activity which regulate Pls biosynthesis, redox status and are influenced by aging . Thus, we hypothesize that plasma/serum Pls are a potential biomarker for diseases related to oxidative stress and aging, such as atherosclerosis and AD (Fig. 3). Figure 3
Plasmalogen, aging, and oxidative stress. The figure was originally published in Ref. .
3 Analytical Methods
Several methods for determining Pls have been reported thus far. These have recently been improved for use with smaller specimen volume increased sensitivity in order to obtain more detailed information regarding these unique molecular species. An important aspect of these analytical methods for Pls is related to acid lability of their...