Alternative Internal Standard Calibration of an Indirect Enzymatic Analytical Method for 2-MCPD Fatty Acid Esters.

An indirect enzymatic analysis method for the quantification of fatty acid esters of 2-/3-monochloro-1,2-propanediol (2/3-MCPD) and glycidol was developed, using the deuterated internal standard of each free-form component. A statistical method for calibration and quantification of 2-MCPD-d5, which is difficult to obtain, is substituted by 3-MCPD-d5 used for calculation of 3-MCPD. Using data from a previous collaborative study, the current method for the determination of 2-MCPD content using 2-MCPD-d5 was compared to three alternative new methods using 3-MCPD-d5. The regression analysis showed that the alternative methods were unbiased compared to the current method. The relative standard deviation (RSDR) among the testing laboratories was ≤ 15% and the Horwitz ratio was ≤ 1.0, a satisfactory value.

Collaborative Study of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

A collaborative study was conducted to evaluate an indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. The method is characterized by the use of Candida rugosa lipase, which hydrolyzes the esters at room temperature in 30 min. Hydrolysis and bromination steps convert esters of 3-MCPD, 2-MCPD, and glycidol to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol, respectively, which are then derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometry. In a collaborative study involving 13 laboratories, liquid palm, solid palm, rapeseed, and rice bran oils spiked with 0.5-4.4 mg/kg of esters of 3-MCPD, 2-MCPD, and Gly were analyzed in duplicate. The repeatability (RSDr) were < 5% for five liquid oil samples and 8% for a solid oil sample. The reproducibility (RSDR) ranged from 5% to 18% for all oil samples. These RSDR values were considered satisfactory because the Horwitz ratios were ≤ 1.3% for all three analytes in all oil samples. This method is applicable to the quantification of 3-MCPD, 2-MCPD, and Gly esters in edible oils.

Optimization of an Indirect Enzymatic Method for the Simultaneous Analysis of 3-MCPD, 2-MCPD, and Glycidyl Esters in Edible Oils.

We developed a novel, indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. Using this method, the ester analytes were rapidly cleavaged by Candida rugosa lipase at room temperature for 0.5 h. As a result of the simultaneous hydrolysis and bromination steps, 3-MCPD esters, 2-MCPD esters, and glycidyl esters were converted to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol (3-MBPD), respectively. After the addition of internal standards, the mixtures were washed with hexane, derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometer (GC-MS). The analytical method was evaluated in preliminary and feasibility studies performed by 13 laboratories. The preliminary study from 4 laboratories showed the reproducibility (RSD R ) of < 10% and recoveries in the range of 102-111% for the spiked 3-MCPD and 2-MCPD in extra virgin olive (EVO) oil, semi-solid palm oil, and solid palm oil. However, the RSDR and recoveries of Gly in the palm oil samples were not satisfactory. The Gly content of refrigerated palm oil samples decreased whereas the samples at room temperature were stable for three months, and this may be due to the depletion of Gly during cold storage. The feasibility studies performed by all 13 laboratories were conducted based on modifications of the shaking conditions for ester cleavage, the conditions of Gly bromination, and the removal of gel formed by residual lipase. Satisfactory RSDR were obtained for EVO oil samples spiked with standard esters (4.4% for 3-MCPD, 11.2% for 2-MCPD, and 6.6% for Gly).

Proportion of nervonic acid in serum lipids is associated with serum plasmalogen levels and metabolic syndrome.

An increase in serum plasmalogens (1-O-alk-1-enyl-2-acyl glycerophospholipids), which are endogenous anti-oxidative phospholipids, can potentially prevent age-related diseases such as atherosclerosis and metabolic syndrome (MetS). Very long chain fatty acids (VLCFAs) in plasma may supply the materials for plasmalogen biosynthesis through peroxisomal beta-oxidation. On the other hand, elevated levels of saturated and monounsaturated VLCFAs in plasma appear to be associated with decreased peroxisomal function, and are a symptom of age-related diseases. To reconcile these contradictory findings, we attempted to investigate the relationship between the serum levels of saturated and monounsaturated VLCFAs, clinical and biochemical parameters, and serum levels of plasmalogens in subjects with MetS (n = 117), who were asymptomatic Japanese males over 40 years of age. Fatty acids in serum lipids were quantified using gas chromatography/mass spectrometry (GC/MS). Serum plasmalogen levels were determined by liquid chromatography using radioactive iodine (¹²5I-HPLC), and the molecular composition of serum plasmalogens was analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS). We found that MetS subjects showed a significant reduction in the proportion of specific saturated and monounsaturated VLCFAs such as behenic acid (C22:0), lignoceric acid (C24:0), and nervonic acid (C24:1) in serum lipids compared to non-MetS subjects. These VLCFAs were positively associated with serum levels of high density lipoprotein cholesterol (HDL-C) as well as plasmalogen-related parameters, and inversely with serum levels of triglyceride (TG) and small dense low density lipoprotein cholesterol (sdLDL-C). In conclusion, the proportion of nervonic acid in serum lipids is associated with serum levels of plasmalogens and with MetS, and probably reflects the peroxisomal dysfunction and enhancement of endoplasmic reticulum (ER) stress seen in common age-related diseases.

Serum choline plasmalogens, particularly those with oleic acid in sn-2, are associated with proatherogenic state.

Serum plasmalogens (Pls) (1-O-alk-1'-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.

Improvement and validation of ¹²5I-high-performance liquid chromatography method for determination of total human serum choline and ethanolamine plasmalogens.

BACKGROUND: Serum plasmalogens (Pls) have gained interest in several clinical symptoms such as metabolic syndrome/atherosclerosis or Alzheimer's disease possibly because of their antioxidant properties. We have developed a highly sensitive and simple method to determine plasmenylcholine (PlsCho; choline plasmalogen) and plasmenylethanolamine (PlsEtn; ethanolamine plasmalogen) separately, using a radioactive iodine and high-performance liquid chromatography ((125)I-HPLC method). The present study reports the improvement and validation of (125)I-HPLC method by introducing a quantitative standard (QS) and online detection with a flow γ-counter. METHODS: 1-Alkenyl 2,3-cyclic glycerophosphate was prepared as QS from l-α-lyso plasmenylcholine by enzymatic treatment with phospholipase D. Online detection with a flow γ-counter was investigated to be available to quantify Pls. The method validation was carried out in terms of selectivity, sensitivity, linearity, precision, accuracy and recovery. RESULTS: Linearity was established over the concentration range 5-300 µmol/L for Pls and QS with regression coefficients >0.99. The accuracy and reliability were satisfactory. The method has been applied to the determination of human serum Pls from healthy subjects and the elderly with dementia or artery stenoses. CONCLUSIONS: The improved (125)I-HPLC method is useful as an autoanalytical system for a routine diagnostic test of human serum Pls.

Lymphatic absorption of choline plasmalogen is much higher than that of ethanolamine plasmalogen in rats.

BACKGROUND: Plasmalogen is a subclass of phospholipids widely distributed in animal tissues and ingested as food; however, the absorptive characteristics of different classes of plasmalogen have not been clarified. AIM OF STUDY: Our object was to compare the lymphatic output of choline and ethanolamine plasmalogens after an administration of phospholipid preparations containing each class of plasmalogens, and to analyze molecular species of plasmalogen absorbed into the lymph. METHODS: A duodenal infusion of 1 ml of 10% emulsion of choline phospholipid (PC) containing 50.6% choline plasmalogen (PlsCho) or ethanolamine phospholipid (PE) containing 52.5% ethanolamine plasmalogen (PlsEtn) was administered in the lymph duct-cannulated rats. Molecular species of plasmalogen absorbed into the lymph were measured by LC-MS/MS. RESULTS: Lymph outputs of PlsCho and PlsEtn increased and reached a peak value at 3 h after PC and PE injection, respectively. The peak value of PlsCho was much higher and remained at a high level until 8 h, whereas PlsEtn output fell to half of the peak value at 7 h. Total lymphatic output of PlsCho was 5-times higher than that of PlsEtn. Compositions of sn-1 in lymph plasmalogens roughly reflected those of the injected lipids, whereas sn-2 in both PlsCho and PlsEtn was rich in arachidonic acid (20:4) regardless of the composition of the administered fatty acid. Both plasmalogen and lysoplasmalogen after PE injection were not released into the portal vein. CONCLUSION: Lymphatic absorption of PlsCho is much higher than that of PlsEtn in rats, and plasmalogens are re-esterified as 20:4-rich forms in the small intestine.