Identification of neuroactive steroids and their precursors and metabolites in adult male rat brain

Steroids in the brain arise both from local synthesis and from peripheral sources and have a variety of effects on neuronal function. However, there is little direct chemical evidence for the range of steroids present in brain or of the pathways for their synthesis and inactivation. This information is a prerequisite for understanding the regulation and function of brain steroids. After extraction from adult male rat brain, we have fractionated free steroids and their sulfate esters and then converted them to heptafluorobutyrate or methyloxime-trimethylsilyl ether derivatives for unequivocal identification and assay by gas chromatography analysis and selected ion monitoring mass spectrometry. In the free steroid fraction, corticosterone, 3 alpha, 5 alpha-tetrahydrodeoxycorticosterone, testosterone, and dehydroepiandrosterone were found in the absence of detectable precursors usually found in endocrine glands, indicating peripheral sources and/or alternative synthetic pathways in brain. Conversely, the potent neuroactive steroid 3 alpha, 5 alpha-tetrahydroprogesterone ( allopregnanolone) was found in the presence of its precursors pregnenolone, progesterone, and 5 alpha-dihydroprogesterone. Furthermore, the presence of 3 alpha-, 11 alpha-, 17 alpha-, and 20 alpha-hydroxylated metabolites of 3 alpha, 5 alpha-tetrahydroprogesterone implicated possible inactivation pathways for this steroid. The 20 alpha-reduced metabolites could also be found for pregnenolone, progesterone, and 5 alpha-dihydroprogesterone, introducing a possible regulatory diversion from the production of 3 alpha, 5 alpha-tetrahydroprogesterone. In the steroid sulfate fraction, dehydroepiandrostrone sulfate was identified but not pregnenolone sulfate. Although pharmacologically active, identification of the latter appears to be an earlier methodological artifact, and the compound is thus of doubtful physiological significance in the adult brain. Our results provide a basis for elucidating the origins and regulation of brain steroids

Uptake and metabolism of sulphated steroids by the blood-brain barrier in the adult male rat

Little is known about the origin of the neuroactive steroids dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PregS) in the brain or of their subsequent metabolism. Using rat brain perfusion in situ, we have found (3) H-PregS to enter more rapidly than (3) H-DHEAS and both to undergo extensive (>50%) desulphation within 0.5 min of uptake. Enzyme activity for the steroid sulphatase catalysing this deconjugation was enriched in the capillary fraction of the blood-brain barrier and its mRNA expressed in cultures of rat brain endothelial cells and astrocytes. Although permeability measurements suggested a net efflux, addition of the efflux inhibitors GF120918 and/or MK571 to the perfusate reduced rather than enhanced the uptake of (3) H-DHEAS and (3) H-PregS; a further reduction was seen upon the addition of unlabelled steroid sulphate, suggesting a saturable uptake transporter. Analysis of brain fractions after 0.5 min perfusion with the (3) H-steroid sulphates showed no further metabolism of PregS beyond the liberation of free steroid pregnenolone. By contrast, DHEAS underwent 17-hydroxylation to form androstenediol in both the steroid sulphate and the free steroid fractions, with some additional formation of androstenedione in the latter. Our results indicate a gain of free steroid from circulating steroid sulphates as hormone precursors at the blood-brain barrier, with implications for ageing, neurogenesis, neuronal survival, learning and memory. This article is protected by copyright. All rights reserved

The impact of processing on potentially beneficial wheat grain components for human health

Wheat based foods, mainly in the form of bread and pasta, are staples of the human diet in many countries around the world. The dry weight of mature wheat grain is composed of 70–75% starch and around 10–14% protein, which has led to the widespread perception of wheat foods as sources of energy and protein. However, whole grains are also important sources of dietary fiber, vitamins and minerals, and contain notable levels of bioactive compounds with potential health benefits like lignans, phenolic acids, alkylresorcinols, phytosterols, folates and tocols. The prominence of wheat grain in the human diet is largely due to its versatility in being processed into diverse products like flour, semolina, and other bakery products. Processing is a pre-requisite for using cereal grains as food and obtaining end products with various unique properties that are safe and appealing to consume. Processing may also help reduce the amount of hazardous molecules potentially present in harvested wheat, such as pesticides, mycotoxins and heavy metals. Each regulated step in a processing series influences the composition and/or the physicochemical properties of the different grain components, which in turn define the technological quality and the nutritional and health promoting properties of the end product. The unique textural properties of wheat foods are largely determined by the starch and gluten proteins present in the starchy endosperm, the main constituents of white flour and semolina. The health-promoting effects of wheat-based products are mainly associated with the dietary fiber and bioactive compounds that are enriched in the grain peripheral layers, and mainly the aleurone layer, which is generally a component of the bran fraction after milling. Fractionation by milling and the way the different milling streams are subsequently recombined therefore has a considerable impact on the relative abundance of each grain component in the wheat flour/semolina and, consequently, in end products. Further processing steps, such as dough making, microbial fermentation, extrusion, and baking can also affect the relative amounts and bioavailability of grain components. Some examples of the effects of grain processing procedures on the bioavailability of important grain components in wheat foods consumed by humans will be presented in this chapter. Suggestions of how to improve these processes in light of the implications for human health will also be discussed