The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

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Background/Aims: The mode of delivery (vaginal or cesarean section) and feeding type (breastfeeding or formula feeding) of neonates are considered the most influential factors in the development of gut microbiota. Objectives: This study investigated the effect of prebiotic-rich breast milk on overcoming gut microbiota dysbiosis. Method: Stool samples from 36 healthy Japanese neonates were obtained at 4 days and 1 month of age, and divided into 4 groups based on mode of delivery and feeding type. The gut microbiota composition and bacterial diversity were assessed using 16S rRNA sequencing. Results: At 4 days old, vaginally delivered neonates had a significantly higher diversity of bacteria than those born by cesarean section. Bacteroidales and Enterobacteriales were overrepresented in vaginally delivered neonates (p = 0.0031 and p = 0.011), while Bacillales and Lactobacillales were overrepresented in caesarean section delivered neonates (p = 0.012 and p = 0.0016). However, there was little difference in bacterial diversity and bacterial relative abundance at 1 month of age between groups. Conclusions: Cesarean section delivery appeared to reduce the diversity of neonate gut microbiota, resulting in dysbiosis, but this improved to the equivalent level seen in vaginally delivered infants by 1 month of age. Breastfeeding, even for short periods, may therefore improve neonate gut dysbiosis.

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On the basis of the phenomenon that hydrophilic acetonitrile is separated from the aqueous phase at -20 degrees C, we employed a novel extraction method, "subzero-temperature liquid-liquid extraction", to extract benzodiazepines (estazolam and triazolam) from serum or aqueous solution for liquid chromatography. A 1:1 mixture of acetonitrile and the specimen was cooled at -20 degrees C for 20 min to separate the acetonitrile and aqueous phases. The acetonitrile phase was directly injected into a high-performance liquid chromatograph. Recovery rates of the drugs following the first subzero-temperature liquid-liquid extraction were 50.3 +/- 0.6-54.0 +/- 0.9%, which were lower than those (73.9 +/- 3.3-80.6 +/- 0.6% and 81.6 +/- 4.7-96.1 +/- 2.6%) of the first conventional liquid-liquid extraction using diethyl ether and solid-phase extraction using a Sep-Pak C18 column, respectively. However, three to four repeated subzero-temperature liquid-liquid extractions and conventional liquid-liquid extractions resulted in recovery of almost 100% of the drugs. In the chromatogram of the benzodiazepines recovered from serum by the subzero-temperature extraction, no coextracted component interfered with determination of the drugs. Detection limits of the drugs were 0.02-0.08 microgram/mL, and coefficients of variance were 1.14-2.17% suggesting high reproducibility.

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