Phosphorylated neurofilament subunit NF-H as a biomarker for evaluating the severity of spinal cord injury patients, a pilot study.

STUDY DESIGN: A pilot cross-sectional study of patients with acute cervical spinal cord injury (SCI). OBJECTIVES: The precise evaluation of the severity of SCI is important for developing novel therapies. Although several biomarkers in cerebrospinal fluid have been tested, few analyses of blood samples have been reported. A novel biomarker for axonal injury, phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), has been reported to be elevated in blood from rodent SCI model. The aim of this study is to investigate whether pNF-H values in blood can serve as a biomarker to evaluate the severity of patients with SCI. SETTING: Tokyo Metropolitan Bokutoh Hospital and National Rehabilitation Center, Japan. METHODS: This study enrolled 14 patients with acute cervical SCI. Sequential plasma samples were obtained from 6 h to 21 days after injury. Patients were classified according to American Spinal Injury Association impairment scale (AIS) at the end of the follow-up (average, 229.1 days). Plasma pNF-H values were compared between different AIS grades. RESULTS: In patients with complete SCI, pNF-H became detectable at 12 h after injury and remained elevated at 21 days after injury. There was a statistically significant difference between AIS A (complete paralysis) patients and AIS C (incomplete paralysis) patients. CONCLUSIONS: Plasma pNF-H was elevated in accordance with the severity of SCI and reflected a greater magnitude of axonal damage. Therefore, pNF-H is a potential biomarker to independently distinguish AIS A patients (complete SCI) from AIS C-E patients (incomplete SCI). However, further studies are required to evaluate its utility in predicting prognosis of patients in the incomplete category.

Hyperphosphorylated neurofilament NF-H as a biomarker of the efficacy of minocycline therapy for spinal cord injury.

STUDY DESIGN: An in vivo study in a rat model of acute spinal cord contusion. OBJECTIVES: To assess the efficacy of novel therapies for acute spinal cord injury (SCI), methods to evaluate accurately the effects of these therapies should be developed. Although neurological examination is commonly used for this purpose, unstable clinical conditions and the spontaneous recovery of neurological function in the acute and subacute phases after injury make this measurement unreliable. Recent studies have reported that the phosphorylated form of the high-molecular-weight neurofilament subunit NF-H (pNF-H), a new biomarker for axonal degeneration, can be measured in serum samples in experimental SCI animals. Therefore, we aimed to investigate the use of plasma pNF-H as an indicator of the efficacy of minocycline, a neuroprotective drug, for treating SCI. SETTING: This study was carried out at Saitama, Japan. METHODS: Spinal cord injured rats received either minocycline or saline intraperitoneally. The plasma pNF-H levels and functional hind limb score were determined after the injury. RESULTS: Minocycline treatment reduced plasma pNF-H levels at 3 and 4 days post-injury (dpi). Rats with lower plasma pNF-H levels at 3 dpi had higher hind limb motor score at 28 dpi. CONCLUSIONS: pNF-H levels may serve as a biomarker for evaluating the efficacy of therapies for SCI.

Metabolism of the calmodulin antagonist DY-9760e in animals and humans.

The in vitro metabolism of the calmodulin antagonist DY-9760e was investigated using liver microsomes from humans and three other animal species and compared with the in vivo metabolism in rats after intravenous administration of DY-9760e. Seven major metabolites were produced by human liver microsomes by the following metabolic pathways: N-dealkylation, phenyl hydroxylation, O-demethylation and imidazole oxidation. These metabolites were also produced by liver microsomes from monkeys, dogs and rats; additionally, a hydroxylated derivative of the indazole moiety was produced only by rat microsomes. To identify the structures of two imidazole ring metabolites whose authentic compounds could not be obtained, Escherichia coli co-expressing human cytochrome P450 CYP3A4 and NADPH-P450 reductase was used to biosynthesize these metabolites. NMR spectra elucidated the precise structures; oxidation occurred at the imidazole ring, and the subsequent ring-opening resulted in the generation of amide and formylamine groups. Glucuronide conjugates of the hydroxylated and O-demethylated derivatives were major components in rat bile. Therefore, DY-9760e metabolites generated in vitro correspond to the aglycones of the major metabolites observed in rat bile.

In vitro metabolism of the glycosidic sweeteners, stevia mixture and enzymatically modified stevia in human intestinal microflora.

Stevia mixture, sweeteners extracted from the leaves of Stevia rebaudiana Bertoni, consists mainly of stevioside and rebaudioside A (glycosides of the diterpene derivative steviol). The aim of this study was to investigate human intestinal metabolism of stevia mixture and its alpha-glucose derivative (known in Japan as enzymatically modified stevia) by LC/MS/ESI analysis. Degradation was examined by incubating stevia mixture, enzymatically modified stevia, stevioside, rebaudioside A, alpha-monoglucosylstevioside, alpha-monoglucosylrebaudioside A and the aglycone, steviol with pooled human faecal homogenates (obtained from five healthy volunteers) for 0, 8 and 24 h under anaerobic conditions. Stevia mixture, enzymatically modified stevia, stevioside and rebaudioside A (0.2 mg/ml) were completely eliminated within 24 h, whereas no degradation of steviol (0.08 and 0.2 mg/ml) appeared to be found during the incubation period. Stevia mixture, stevioside and rebaudioside A appeared to be hydrolyzed to steviol by human intestinal microflora: this observation is consistent with previous rat metabolism studies. Similarly, enzymatically modified stevia appeared to be metabolized via stevia components and, finally, to steviol. This study suggests that there are apparently no species differences in intestinal metabolism of stevia mixture between rats and humans.

[Chemiluminescent enzyme immunoassay of cholecystokinin in rat plasma using an immuno-affinity column as a pretreatment].

The cholecystokinin (CCK) plasma concentration of a basal level in a normal rat was at a hardly detectable level (a few pg/ml) even using the highly sensitive chemiluminescent enzyme immunoassay. In this paper, we prepared an anti-CCK F(ab')2 fragment which was made by pepsin digestion from the anti-CCK antiserum. The anti-CCK F (ab')2 fragment was immobilized to the Sepharose 4B gel activated BrCN and packed to column. We have extracted and concentrated CCK in the rat plasma using this column and the CCK assayed by the chemiluminescent enzyme immunoassay (CL-EIA) of CCK. The immuno-affinity column extraction system was more accurate and precise than a reversed phase gel extraction system. The mean recovery of CCK from the rat pooled plasma using the immuno-affinity column was 62.2% (n = 12) by the proposed CL-EIA. The mean +/- S. E. (n = 3) of the CCK concentration in the fasted rat plasma is 3.54 +/- 0.10 pg/ml. The plasma CCK concentrations of normal rats and those of camostat administrated rats, could be measured by the proposed CL-EIA using the immuno-affinity column as a pretreatment.

Changes in rat plasma-free fatty acid composition under oxidative stress induced by carbon tetrachloride: decrease of polyunsaturated fatty acids and increase of palmitoleic acid.

In order to measure the changes in antioxidant levels and the composition of plasma-free fatty acids resulting from oxidative stress, male Fisher rats were given a twice weekly subcutaneous injection of a 50% solution of carbon tetrachloride (CCl4) in corn oil for a period of 2 to 13 weeks. The dosage was 1.3 ml/kg of body weight. This treatment significantly suppressed the gain of body weight compared with control rats receiving the same dosage of corn oil. Liver weight of the two groups was similar while necrosis and hyperplasia of hepatocytes and liver fibrosis were observed in CCl4 treated rats. Increased levels of plasma glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase were indicative that hepatocyte necrosis was induced by CCl4. Increased oxidative stress in CCl4 treated rats was indicated by a significant decrease of liver ascorbate and a decrease in the plasma ratio of polyunsaturated fatty acids (PUFA) to total free fatty acids. Interestingly, a significant increase of palmitoleic acid was observed in CCl4 treated rats, which may compensate for the loss of PUFA. The possibility of using the plasma composition of PUFA and palmitoleic acid as a marker of oxidative stress is discussed.