Iron deficiency (ID) anemia (IDA) adversely affects different aspects of the nervous system such as myelinogenesis, neurotransmitters synthesis, brain myelin composition, and brain fatty acid and eicosanoid metabolism. Infant neurophysiological outcome in response to maternal IDA is underexplored, especially mild to moderate maternal IDA. Furthermore, most human research has focused on childhood ID rather than prenatal or neonatal ID. Thus, our study evaluated the consequences of mild maternal IDA during pregnancy and lactation on the offsprings' auditory function using the auditory brainstem response (ABR). This technique provides objective measures of auditory acuity, neural transmission times along the peripheral and brainstem portions of the auditory pathway, and postnatal brain maturation. Female guinea pigs (n = 10/group) were fed an iron sufficient diet (ISD) or an iron deficient diet (IDD) (144 and 11.7 mg iron/kg) during their acclimation, gestation, and lactation periods. From postnatal d (PNd) 9 onward, the ISD was given to all weaned offspring. ABR were collected from the offspring on PNd24 using a broad range of stimulus intensities in response to 2, 4, 8, 16, and 32 kHz tone pips. IDA siblings (n = 4), [corrected] compared with the IS siblings (n = 5), had significantly elevated ABR thresholds (hearing loss) in response to all tone pips. These physiological disturbances were primarily due to a sensorineural hearing loss, as revealed by the ABR's latency-intensity curves. These results indicate that mild maternal IDA during gestation and lactation altered the hearing and nervous system development of the young offspring.

INTRODUCTION The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. The mitochondrion is known as the powerhouse of the cell as it produces the energy to power most cellular functions. However, mitochondria and its typical hallmarks (i.e. circular DNA, N‐formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease and rheumatoid arthritis. Therefore, stringent methods of isolation and purification of mitochondria are of the utmost importance in assessing mitochondrial‐related diseases. While several isolation kits are available commercially, they can be somewhat expensive and not suitable for some downstream applications. In this project, we provide an alternative purification method yielding mitochondria of high purity and integrity using human platelets. OBJECTIVES Evaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets. METHODS First, platelets were isolated from the blood of healthy donors. Then the brute fraction of platelet‐derived mitochondria was obtained using a potter homogenizer, followed by several differential centrifugation. To obtain the purified fraction, the mitochondrial extract was centrifuged on a discontinuous Percoll gradient (GE Healthcare). The purity of mitochondria was determined by flow cytometry (FC500, Beckman Coulter) using specific platelet marker anti‐CD41‐FITC (BioLegend), and by transmission electron microscopy (TEM). The respiratory capacity of mitochondria was measured by high‐resolution respirometry (Oroboros instruments). The total yield of mitochondria was determined by flow cytometry using MitoTracker Deep Red (Molecular Probes) and by the micro‐Smith method. Finally, the integrity of the mitochondrial membrane potential was assessed with JC‐1 staining (Molecular Probes). RESULTS Data generated by flow cytometry shows that the Percoll gradient significantly purified mitochondria by removing 50% of platelet membrane debris (paired t‐test, p < 0.01). TEM analysis shows similar results. Mitochondrial respiration following the substrate uncouple inhibitor titration protocol is identical in purified and in brute mitochondria. Additionally, the cytochrome c effect is 5%, while JC‐1 staining shows no significant difference between methods suggesting integrity both in the inner and outer mitochondrial membrane. On the other hand, the mitochondrial protein yield was significantly decreased after purification (paired t‐test, p < 0.01). CONCLUSIONS Results of this study suggest that the Percoll discontinuous gradient purifies viable platelet‐derived mitochondria. Conversely, mitochondrial yield may be less important than obtained in other methods; however, it could be explained by the clustering of mitochondria containing less platelet debris. Relatively inexpensive, this method of purification is ideal for studying the downstream effects of intact mitochondria in mitochondrial‐related disea...

The present study showed that the negative effects of estrogen deficiency on BMD and bone metabolism in early menopause occurred independently of the effect of major calcitropic hormones. Bone loss affects a non negligible proportion of premenopausal women. The prevalence of osteopenia in pre- and postmenopausal women varied according to the criterion used and anatomic site.

The inflammatory response is necessary for the host's defense against pathogens; however, uncontrolled or unregulated production of eicosanoids has been associated with several types of chronic inflammatory diseases. Thus, it is not surprising that enzymes implicated in the production of eicosanoids have been strategically targeted for potential therapeutic approaches. The 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] lipid mediator is among inflammatory molecules that are abundantly produced in various diseases and is primarily biosynthesized via the 12(S)lipoxygenase pathway. The effects of the abundance of 12(S)-HETE and its contribution to several chronic inflammatory diseases have been well studied over the last few years. While most developed compounds primarily target the 5-lipoxygenase (5-LO) or the cyclooxygenase (COX) pathways, very few compounds selectively inhibiting the 12-lipoxygenase (12-LO) pathway are known. In this study, we examined whether the distribution of hydroxyl groups among flavones could influence their potency as 12-LO inhibitors. Using human platelets, the human embryonic kidney 293 (HEK293) cell line expressing 5-LO, and human polymorphonuclear leukocytes (PMNLs) we investigated the effects of these compounds on several inflammatory pathways, namely, 12-LO, 5-LO, and COX. Using high-resolution respirometry and flow cytometry, we also evaluated some normal cell functions that could be modulated by our compounds. We identified a peracetylated quercetin (compound 6) that exerts potent inhibitory activity toward the platelet 12-LO pathway (IC 50 5 1.53 mM) while having a lesser affinity toward the COX pathway. This study characterizes the peracetylated quercetin (compound 6) as a more selective platelet-type 12-LO inhibitor than baicalein, with no measurable nontargeted effects on the platelet's activation or overall cell's oxygen consumption.

Two vitamins and proline (CB6Pro), three nutrients essential for bone collagen, were used in combination to a 1000 mg calcium/250 IU vitamin D (Ca/D) daily supplement to treat osteopenia as a preventive measure against osteoporosis later in life. Middle-aged women not using estrogen were screened for osteopenia using the WHO criteria and divided into three groups (n = 20 each): 1) placebo healthy controls with normal bone mineral density (BMD); 2) control Ca/D-treated osteopenic patients; and 3) Ca/D + CB6Pro-treated osteopenic patients. The three groups were comparable at baseline except for BMD. After one-year treatment, cortical diaphyseal BMD remained constant in each group, but trabecular bone loss persisted (at 5 lumbar sites) in osteopenic group 2. No further bone loss was detected in osteopenic group 3. A loss of 2% was evidenced in the placebo group at one lumbar site. Markers of bone formation (which increase in coupling to resorption) decreased significantly in both osteopenic groups. Although biomarkers of resorption did not change, hormone (PTH and 1,25(OH)2D3)-induced osteoclastic activity was significantly reduced. No decline in BMD occurred at any bone site in osteopenic group 3, highlighting the importance of improving the quality of bone matrix concomitantly to mineral replacement.

No significant difference between dietary groups was measured in hearing threshold and absolute latencies in pups at PNd24 and PNd84. Although the ID offspring (n = 16) did not differ in brainstem transmission times (BTTs) at 80 dB compare to the IS siblings (n = 25) at PNd24, they showed significant delayed inter-peak latency (IPL) I-IV at 100 dB suggesting a delayed BTT. At PNd84, the latency of all peaks including IPL I-IV at 80 and 100 dB significantly decreased and was also similar in pups from both dietary groups suggesting a better brain maturation. This is the first study investigating the long-term impact of maternal iron deficiency on the auditory functions in the guinea pig offspring during early development to adulthood.

In support to our previous findings, the present results indicate that a mild IDA during gestation and lactation can have detrimental effects on early development of the offsprings' hearing and nervous systems, particularly on neural synchrony and auditory nerve conduction velocity, but not on BTT.

INTRODUCTIONThe isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. The mitochondrion is known as the powerhouse of the cell as it produces the energy to power most cellular functions. However, mitochondria and its typical hallmarks (i.e. circular DNA, N‐formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease and rheumatoid arthritis. Therefore, stringent methods of isolation and purification of mitochondria are of the utmost importance in assessing mitochondrial‐related diseases. While several isolation kits are available commercially, they can be somewhat expensive and not suitable for some downstream applications. In this project, we provide an alternative purification method yielding mitochondria of high purity and integrity using human platelets.OBJECTIVESEvaluate the purity, integrity and yield of two different methods of isolation of mitochondria in human platelets.METHODSFirst, platelets were isolated from the blood of healthy donors. Then the brute fraction of platelet‐derived mitochondria was obtained using a potter homogenizer, followed by several differential centrifugation. To obtain the purified fraction, the mitochondrial extract was centrifuged on a discontinuous Percoll gradient (GE Healthcare). The purity of mitochondria was determined by flow cytometry (FC500, Beckman Coulter) using specific platelet marker anti‐CD41‐FITC (BioLegend), and by transmission electron microscopy (TEM). The respiratory capacity of mitochondria was measured by high‐resolution respirometry (Oroboros instruments). The total yield of mitochondria was determined by flow cytometry using MitoTracker Deep Red (Molecular Probes) and by the micro‐Smith method. Finally, the integrity of the mitochondrial membrane potential was assessed with JC‐1 staining (Molecular Probes).RESULTSData generated by flow cytometry shows that the Percoll gradient significantly purified mitochondria by removing 50% of platelet membrane debris (paired t‐test, p < 0.01). TEM analysis shows similar results. Mitochondrial respiration following the substrate uncouple inhibitor titration protocol is identical in purified and in brute mitochondria. Additionally, the cytochrome c effect is 5%, while JC‐1 staining shows no significant difference between methods suggesting integrity both in the inner and outer mitochondrial membrane. On the other hand, the mitochondrial protein yield was significantly decreased after purification (paired t‐test, p < 0.01).CONCLUSIONSResults of this study suggest that the Percoll discontinuous gradient purifies viable platelet‐derived mitochondria. Conversely, mitochondrial yield may be less important than obtained in other methods; however, it could be explained by the clustering of mitochondria containing less platelet debris. Relatively inexpensive, this method of purification is ideal for studying the downstream effects of intact mitochondria in mitochondrial‐related diseases.Support or Funding InformationCanadian Institutes of Health Research, New Brunswick Health Research Foundation, New Brunswick Innovation FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.