An enzyme combination assay for serum sphingomyelin: Improved specificity through avoiding the interference with lysophosphatidylcholine.

Serum sphingomyelin (SM) has predictive value in the development of atherosclerosis. Furthermore, SM plays important roles in cell membrane structure, signal transduction pathways, and lipid raft formation. A convenient enzymatic method for SM is available for routine laboratory practice, but the enzyme specificity is not sufficient because of nonspecific reactions with lysophosphatidylcholine (LPC). Based on the differential specificity of selected enzymes toward choline-containing phospholipids, a two-step assay for measuring SM was constructed and its performance was evaluated using sera from healthy individuals on a Hitachi 7170 autoanalyzer. Results from this assay were highly correlated with theoretical serum SM concentrations estimated by subtracting phosphatidylcholine (PC) and LPC concentrations from that of total phospholipids determined using previously established methods. There was a good correlation between the results of SM assayed by the proposed method and the existing enzymatic method in sera from healthy individuals. Moreover, the proposed method was superior to the existing method in preventing nonspecific reactions with LPC present in sera. The proposed method does not require any pretreatment, uses 2.5 µl of serum samples, and requires only 10 min on an autoanalyzer. This high-throughput method can measure serum SM with sufficient specificity for clinical purposes and is applicable in routine laboratory practice.

An autopsy case of fatal repellent air freshener poisoning.

We describe a first fatal case of repellent air freshener ingestion. A 79-year-old Japanese man with Alzheimer-type senile dementia orally ingested repellent air freshener containing three surfactants: polyoxyethylene 9-lauryl ether, polyoxyethylene (40) hydrogenated castor oil, and lauric acid amidopropyl amine oxide (weight ratio of 1.3%). About 1h after the collapse, he was in cardiopulmonary arrest and subsequently died 10h after his arrival. The forensic autopsy performed 5.5h after death revealed the 380ml of stomach contents with a strong mint perfume identical to that of the repellent air freshener and the findings of acute death. Toxicologically, 9.1µg/ml and 558.2µg/ml of polyoxyethylene 9-lauryl ether were detected from the serum and stomach contents taken at autopsy. Generally, ingestion of anionic or non-ionic surfactants have been considered as safe. However, because the patient suffered from cardiac insufficiency with a low dose of repellent air freshener ingestion, medical staff members must evaluate the elderly patient for cardiac and circulatory problems regardless of the ingested dose. Not only medical and nursing staff members, but also families who are obliged to care for elderly persons must be vigilant to prevent accidental ingestion of toxic substances generally used in the household.

Differences in reaction specificity toward lipoprotein X and abnormal LDL among 6 homogeneous assays for LDL-cholesterol.

BACKGROUND: We investigated the reaction specificity toward cholesterol in lipoprotein X (Lp-X) and abnormal LDL among 6 homogeneous assays for low-density lipoprotein cholesterol (LDL-C) based on different measurement principles. METHODS: The homogeneous LDL-C assays used were based on the liquid selective detergent, selective solubilization, elimination, enzyme-selective protection, calixarene complex, and phosphate complex inhibition methods. The fraction with a density of 1.006-1.063 kg/l was isolated from cholestatic sera, and the reactivity of cholesterol in the lipoprotein fractions by gel filtration for each homogeneous LDL-C assay was determined. RESULTS: The liquid selective detergent and elimination methods showed increased cholesterol reactivity in the Lp-X fraction in a concentration-dependent manner, while the selective solubilization and phosphate complex inhibition methods were less reactive toward Lp-X cholesterol. Meanwhile, the homogeneous LDL-C assays showed decreased reactivity against cholesterol in abnormal LDL, with increased ratios of phospholipids and triglycerides against cholesterol. CONCLUSION: The homogeneous LDL-C assays showed differential reactivity toward Lp-X and abnormal LDL. Our findings enable accurate interpretation of the LDL-C values in these homogeneous assays, and suggest that these methods should be improved to distinguish between normal LDL and abnormal LDL or Lp-X.

[Development of the HDL-C and LDL-C direct methods and the subsequent evolution].

Today, more than a decade after the development of direct methods for determining HDL-cholesterol (HDL-C) and LDL-cholesterol (LDL-C) concentrations, there are calls to review the reagents in response to discrepancies in patient samples with increased levels of atypical lipoproteins, such as apoE-rich HDL, IDL, Lp-X, and Lp-Y. Seven direct HDL-C assays showed different reactions toward apoE-rich HDL in sera from patients with CETP deficiency and cholestasis. On the other hand, the reactivity of the direct LDL-C assays to serum samples with elevated levels of IDL, Lp-X, and Lp-Y varied considerably between assay kits from each manufacturer. We have also examined the anti-atherogenic functions of apoE-rich HDL from the serum of healthy individuals and patients with CETP deficiency and cholestasis. The ability of apoE-rich HDL to remove cholesterol from cholesterol-loaded macrophages showed that the apoE-rich HDL from CETP-deficient serum took up more cholesterol than apoE-poor HDL (p < 0.01), but no significant differences were observed for apoE-rich HDL from patients with cholestasis.

Simultaneous identification of the enantiomers and diastereomers of N,O-di-trifluoroacetylated ephedrine and norephedrine in blood plasma using chiral capillary gas chromatography-mass spectrometry with selected ion monitoring.

A method for identifying the enantiomers of N,O-di-trifluoroacetylated ephedrine (EP) and norephedrine (NE) and the enantiomers of pseudoephedrine (PEP) and pseudonorephedrine (PNE) in plasma was developed using chiral capillary gas chromatography-mass spectrometry (GC-MS) with selected ion monitoring (SIM). N,O-Di-trifluoroacethyl (TFA) derivatization was accomplished in a dried hydrochloride extract of plasma (minimum quantity of 0.2 mL). An SIM GC-MS method with a ß-cyclodextrin chiral capillary column allowed the successful and simultaneous detection of each TFA-derivatized stereoisomer of EP, NE, PEP, PNE, and an internal standard (IS; S-(+)-ethylamphetamine). Each TFA-drivatized stereoisomer was identified using four mass fragment ions (m/z 140, 154, 168, and 230). The TFA-derivatized stereoisomers of EP, NE, PEP, PNE, and IS were separated completely and were detected with sufficient sensitivity. The assay allowed the stereoisomers to be determined in a linear range of 12.5-1250 ng/mL for the EP stereoisomers and a linear range of 5-1250 ng/mL for the PEP, NE, and PNE stereoisomers. The detection limits were 7.5 ng/mL for the EP stereoisomers and 2.5 ng/mL for the PEP, NE, and PNE stereoisomers. The intra- and interday precisions were less than 5.9% and 8.2%, respectively. This chiral capillary SIM GC-MS method was sufficiently effective in the analysis of plasma from users of over-the-counter cold medicines and was also fully applicable to the plasma analysis of guinea pigs following their treatment with racemic EP.

Stereoisomeric identification of norephedrine derived from methamphetamine or amphetamine: urinalysis results of 33 methamphetamine abusers and 1 amphetamine abuser in Japan.

Stereoisomeric identification of norephedrine (NE) derived from methamphetamine (MA) or amphetamine (AM) was investigated by SIM-GC/MS assay using the urine of 33 MA abusers and 1 AM abuser. The assay simultaneously identified TFA-derivatized MA and AM metabolites, including AM, p-hydroxyl-MA (p-HMA), and p-hydroxyl-AM (p-HAM). The analysis lasted approximately 43 min, with a signal-to-noise ratio of >or=3 and a detection limit of 50 ng/mL. Among 12 urine samples from different subjects, only the S (+) form of MA and its metabolites (AM, p-HMA, p-HAM) was detected, however, a (1R,2S)-(-)-NE stereoisomer was also identified. Among the urine samples of two subjects, only the R (-) form of MA and its metabolites (AM, p-HMA, p-HAM) was detected, while NE was not detected. Following urinalysis of urine obtained from 19 MA abusers and 1 AM abuser, only the (1R,2S)-(-)-NE stereoisomer was identified, while unmetabolized MA, AM, and their metabolites (p-HMA, p-HAM), showed stereoselective metabolism. Although (1R,2S)-(-)-ephedrine (EP) alone was found in the urine of 1 (S)-(+)-MA user and 1 (S)-(+)- and (R)-(-)-MA user among 33 MA users, it was not present in the urine of the remaining 31 subjects. Therefore, (1R,2S)-(-)-NE was likely not of (1R,2S)-(-)-EP origin and was most likely from (S)-(+)-AM of the MA metabolite. The production ratio of (1R,2S)-(-)-NE to (S)-(+)-AM ranged from 0.01 to 0.25 in MA abusers and was 0.12 in AM abusers.

Accidental strangulation of a mentally retarded patient by a clothing collar: a case report.

A 40-year-old mentally retarded woman died of accidental strangulation in a nursing home. She was found in a kneeling position with her hands on her knees and the collar of her clothing compressing the front and sides of the neck. Before the accident, a nurse had dressed the patient in one-piece overall-style pyjamas put on back to front so that she could not remove the garment herself. The post-mortem findings and reconstruction of the scene of death suggested that the patient had been strangled by the collar of her backward-facing clothing while in a kneeling position. Because patients with psychiatric illnesses may have a limited ability to recognize or communicate symptoms of physical danger, they must be closely monitored by knowledgeable medical and nursing staff. This case highlights the importance of preventing the accidental deaths of mentally retarded patients in nursing homes.

Enantiomer analysis of a new street drug, 3,4-methylenedioxy-N-methyl-butanamine, in rat urine.

A new street drug, 3,4-methylenedioxy-N-methyl-butanamine (MBDB), has been found in Japan recently. The stereoisomer monitoring and the urinary excretion kinetics are not determined in biological fluids even though abused MBDB is a racemic form [enantiomer ratio (-/+) = 1.00]. The present studies were done by high-performance liquid chromatography (HPLC) equipped with a chiral activity column at 40 degrees C using urine specimens from five Wistar rats. Urine samples were collected over six time intervals after a single oral administration of racemic MBDB (30 mg/kg). Unchanged MBDB and 3,4-methylenedioxybutanamine (BDB), an N-demethylated metabolite, were found in the rats' urine. Each enantiomer of MBDB and BDB was monitored (peak resolution > 1.00) by HPLC analysis within 30 min. For both MBDB and BDB, the (+)-isomers were excreted a little more than the (-)-isomers. The stereoselective disposition of BDB was more remarkable than that of MBDB and was observed in the urine throughout the study (p < 0.05). The urinary excretion of MBDB showed significant difference between the two enantiomers from 4 to 20 h (p < 0.05). The amount of MBDB excreted up to 24 h was 34.7+/-2.8% of the administered dose: 17.6+/-1.4% for (+)-isomer and 17.1+/-1.5% for (-)-isomer. The amount of BDB was 4.9+/-1.0%; 2.9+/-0.6% for (+)-isomer and 2.0+/-0.4% for (-)-isomer. The enantiomer ratio (-/+) of MBDB and BDB was 1.00 or a little smaller. The ratio (-/+) of MBDB changed from 1.00+/-0.02 to 0.88+/-0.09 by 24 h, and that of BDB from 0.68+/-0.03 to 0.78+/-0.02. The ratio (-/+) for MBDB and BDB accumulated up to 24 h was 0.97+/-0.01 and 0.70+/-0.06, respectively, and the total ratio (-/+) of the two substances was 0.93+/-0.02 (p < 0.05). These findings suggested that the stereoselective disposition of racemic MBDB was different from that of 3,4-dimethylenedioxyamphetamine and 3,4-dimethylenedioxymethamphetamine and was similar to that of methamphetamine.