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Cytometric characterization and free radical production of two different mitochondrial populations from human placenta (1153.13)
Author(s) -
Ramirez Velez Robinson
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1153.13
Subject(s) - superoxide , chemistry , cytochrome c , hydrogen peroxide , cytochrome c oxidase , mitochondrion , differential centrifugation , oxygen , microbiology and biotechnology , oxidase test , reactive oxygen species , biochemistry , placenta , biology , enzyme , fetus , organic chemistry , pregnancy , genetics
PURPOSE: To study the metabolic function from two mitochondrial populations described as heavy (HM) and light mitochondria (LM), present in the human placenta. METHODS. Placentas were collected within 15 min of delivery. Mitochondrial fractions were isolated from human placental tissue by differential centrifugation. Mitochondrial oxygen uptake, superoxide levels and hydrogen peroxide were measured to determine oxygen metabolism. Flow‐cytometry assays were carried out to measure mitochondrial membrane potential (αΨm). A spectrophotometric assay was used to measure mitochondrial respiratory complexes and levels of cytochrome P‐450. RESULTS. The oxygen uptake showed that a better respiratory control (3.7±0.3) was observed in the HM, as compared with the LM (2.2±0.3, p<0.01). HM produced lower levels of hydrogen peroxide than LM (0.41±0.6 vs. 0.63±0.20 nmol/min.mg protein, p<0.01). Quantification of the superoxide anion levels as percentage values of MitoSox r. f. i was 78% higher in HM than in LM populations. Similar results were obtained during the evaluation of the αΨm. The activity of complex I‐III in the HM and LM was 55.2±1.5 and 76.2±3.0 nmol cytc/min.mg protein, respectively, (p<0.05). Complex II‐III activity in the HM and LM was 9.5±0.5 and 5.8±0.4 nmol cytc/min.mg proteins respectively. Evaluation of complex IV activity (cytochrome c oxidase) showed values of 20.9±1.1 in LM and 26.7±0.7 nmol cyt c/min.mg protein in HM. The LM from term human placenta, during active steroidogenesis, showed levels of 0.13±0.02 nmol/mg protein vs. HM were quite low 0.027±0.009 nmol/mg protein (cytochrome P‐450). CONCLUSIONS. These results were associated with higher levels of hydrogen peroxide produced in the LM. Increased levels of cytochrome P‐450scc in the LM were consistent with a higher steroidogenic activity present in this mitochondrial fraction.