A Comparison of Calcium Aggregation and Ultracentrifugation Methods for the Preparation of Rat Brain Microsomes for Drug Metabolism Studies

Preparation of brain microsomes by the calcium chloride aggregation method has been suggested as an alternative to the ultracentrifugation method. However, the effects of the calcium chloride concentration on the quality of the microsomal fractions are not known. Brain microsomes were prepared from the adult rat brains using the high-speed ultracentrifugation and low-speed calcium chloride (10–100 mM) aggregation methods (<i>n</i> = 5–6 per group). The microsomal protein yield (spectrometry), the cytochrome P450 reductase (CPR) activity (spectrometry), and the monooxygenase activities (UPLC-MS/MS) of CYP2D and CYP2E1 were determined in the obtained fractions. Increasing the concentrations of calcium chloride progressively increased the protein yield of the low-speed microsomal fractions. However, the increased yield was associated with a significant decrease in the activities of CPR, CYP2D, and CYP2E1. Additionally, the CYP2D and CYP2E1 activities were significantly correlated with the CPR activities of the fractions. In conclusion, when an ultracentrifuge is available, preparation of brain microsomes by the ultracentrifugation method might be preferable. However, the calcium aggregation method at a calcium chloride concentration of 10 mM is an acceptable alternative to the ultracentrifuge method.

Riboflavin and pyrroloquinoline quinone generate carbon monoxide in the presence of tissue microsomes or recombinant human cytochrome P-450 oxidoreductase: implications for possible roles in gasotransmission

Carbon monoxide (CO), an endogenously produced gasotransmitter, regulates inflammation and vascular tone, suggesting that delivery of CO may be therapeutically useful for pathologies like preeclampsia where CO insufficiency is implicated. Our strategy is to identify chemicals that increase the activity of endogenous CO-producing enzymes, including cytochrome P-450 oxidoreductase (CPR). Realizing that both riboflavin and pyrroloquinoline quinone (PQQ) are relatively nontoxic, even at high doses, and that they share chemical properties with toxic CO activators that we previously identified, our goal was to determine whether riboflavin or PQQ could stimulate CO production. Riboflavin and PQQ were incubated in sealed vessels with rat and human tissue extracts and CO generation was measured with headspace-gas chromatography. Riboflavin and PQQ increased CO production ∼60% in rat spleen microsomes. In rat brain microsomes, riboflavin and PQQ increased respective CO production approximately fourfold and twofold compared to baseline. CO production by human placenta microsomes increased fourfold with riboflavin and fivefold with PQQ. In the presence of recombinant human CPR, CO production was threefold greater with PQQ than with riboflavin. These observations demonstrate for the first time that riboflavin and PQQ facilitate tissue-specific CO production with significant contributions from CPR. We propose a novel biochemical role for these nutrients in gastransmission.

Differential inhibition of rat and mouse microsome heme oxygenase by derivatives of imidazole and benzimidazole

Metalloporphyrin heme oxygenase (HO) inhibitors have made an important contribution to elucidating the role of HO in physiological processes. Nevertheless, their off-target effects have drawn substantial criticism, which prompted us to develop non-porphyrin, azole-based inhibitors of HO. These second-generation HO inhibitors were evaluated using spleen and brain microsomes from rats as native sources of HO-1 and HO-2, respectively. Recently, the use of azole-based inhibitors of HO has been extended to other mammalian species and, as a consequence, it will be important to characterize the inhibitors in these species. The goal of this study was to compare the inhibitory profile of imidazole- and benzimidazole-based inhibitors of HO in a breast-cancer-implanted mouse to that of an untreated rat. For spleen and brain microsomes from both species, HO protein expression was determined by Western blotting and concentration–response curves for imidazole- and benzimidazole-derivative inhibition of HO activity were determined using a headspace gas-chromatographic assay. It was found that the effects on HO activity by imidazole and benzimidazole derivatives were different between the 2 species and were not explained by differences in HO expression. Thus, the HO inhibitory profile should be determined for azole derivatives before they are used in mammalian species other than rats.

Calendula officinalis L. (Asteraceae) possess antioxidant properties on Fe2+-initiated peroxidation of rat brain microsomes

<p class="ADMETabstracttext">In this study the effects of Calendula officinalis L. (Asteraceae) extract (CO) on the polyunsaturated fatty acid composition, chemiluminescence and unsaturation index of microsomes isolated from brain rat, are presented. After incubation of microsomes in an ascorbate (0.4 mM)-Fe²⁺ (2.15 µM) system (180 min at 37 °C) it was observed that the total cpm/mg protein originated from light emission:chemiluminescence was lower in brain microsomes obtained from CO group compared to the control group (without extract supplementation). Moreover, it was observed that the addition of the extract reduced chemiluminescence -measured as total cpm- in a concentration dependent manner. The fatty acid composition of brain microsomes from control group was profoundly modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of arachidonic acid C20:4w6 and docosahexaenoic acid C22:6w3. As a consequence, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids, was higher in the CO group compared to controls. The simultaneous analysis of unsaturation index, chemiluminescence and fatty acid composition indicate that CO may act as an antioxidant protecting rat brain microsomes from peroxidative damage.</p>