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Inner Membrane of the Mitochondria, Cardio Lipin , PhosphatidylSerine, & Phosphatidylethanolamine

Updated: Oct 2, 2023

This is a mysterious place, and one that can get filled with super oxide when the cardio lipin (CL) which reside in the inner membrane of the mitochondria get oxidized. Lots of research here related to cardiac issues.

There is a mysterious transporter for PhosphatidylSerine into the inner membrane of the mitochondria. Phosp Serine is often recommended in Traumatic Brain Injuries. And it's regulated by ubiquination which traditionally happens through NQO1 (think coq10). Hmm, getting interesting:).

"Supplementation with a phosphatidylserine and phosphatidylserine/ phosphatidic acid complex (PAS) has been observed to normalize stress induced dysregulations of the hypothalamus-pituitary-adrenal axis (HPAA). Prolonged stress first induces a hyper-activation of the HPAA, which then can be followed by a state of hypo-activation. The aim of this study was to examine effects of an oral supplementation with 400 mg PS & 400 mg PA (PAS 400) per day on the endocrine stress response (ACTH, saliva and serum cortisol) to a psychosocial stressor. A special focus was to analyze subgroups of low versus high chronically stressed subjects as well as to test efficacy of 200 mg PS & 200 mg PA (PAS 200)." [5]

"PS supplementation with 600 mg per day for 10 days blunts the cortisol response to exercise-induced stress. In addition, PS significantly increases the testosterone to cortisol ratio. These findings suggest that PS is an effective supplement for combating exercise-induced stress. PS supplementation promotes a desirable hormonal balance for athletes and might attenuate the physiological deterioration that accompanies overtraining and/or overstretching."[7]

"Mitochondrial membrane biogenesis requires the interorganelle transport of phospholipids. Phosphatidylserine (PtdSer) synthesized in the endoplasmic reticulum and related membranes (mitochondria-associated membrane (MAM)) is transported to the mitochondria by unknown gene products and decarboxylated to form phosphatidylethanolamine at the inner membrane by PtdSer decarboxylase 1 (Psd1p). We have designed a screen for strains defective inPtdSer transport (pstAmutants) between the endoplasmic reticulum and Psd1p that relies on isolating ethanolamine auxotrophs in suitable (psd2Δ) genetic backgrounds. Following chemical mutagenesis, we isolated an ethanolamine auxotroph that we designatepstA1-1. Using in vivo and in vitro phospholipid synthesis/transport measurements, we demonstrate that the pstA1-1 mutant is defective in PtdSer transport between the MAM and mitochondria. The gene that complements the growth defect and PtdSer transport defect of the pstA1-1 mutant is MET30, which encodes a substrate recognition subunit of the SCF (suppressor of kinetochore protein 1, cullin,F-box) ubiquitin ligase complex. Reconstitution of different permutations of MAM and mitochondria from wild type andpstA1-1 strains demonstrates that theMET30 gene product affects both organelles. These data provide compelling evidence that interorganelle PtdSer traffic is regulated by ubiquitination." [1]

"Since the decrement in PtdEtn accumulation is not due to aberrant Pss1p or Psd1p enzyme activities, we conclude that the defect lies between these two biosynthetic steps. Taken together, these data demonstrate that the EAL18 mutant is deficient in PtdSer translocation from the MAM to the locus of Psd1p in the inner mitochondria membrane. Therefore, we have designated this mutant aspstA1-1." [1]

"Ubiquitination is now also recognized to play an important role in membrane traffic as a signaling motif for protein sorting, endocytosis, and viral budding." [2]

"In summary, we have isolated a mutant defective in PtdSer traffic between the MAM and the mitochondria. The mutation alters the PtdEtn content, the total phospholipid content, and the density of the organelle. The effects of the mutation are manifested in both the MAM and mitochondria and are reversed by the MET30 gene, which encodes a component of SCF ubiquitin ligase. These findings now identify a novel mechanism for regulating interorganelle phospholipid transport." [1]

"Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis." [3].

"Mitochondria are engaged in a plethora of cellular processes and are therefore of utmost importance for cell viability. Mitochondria are not static entities but are highly dynamic and require that supplies of proteins and membrane lipids be coordinated and adjusted to meet physiological and functional demands. Although an increasingly detailed structural and mechanistic picture is emerging for the biogenesis, sorting, and compartmentation of mitochondrial proteins (Schmidt et al., 2010), much less is known about mechanisms regulating the supply of phospholipids and the maintenance of mitochondrial membrane integrity. The mitochondrial phospholipid composition varies little among different cells, suggesting that major changes cannot be tolerated. Indeed, both altered phospholipid levels and phospholipid damage have been linked to a variety of human disease states (Chicco and Sparagna, 2007; Joshi et al., 2009). Phospholipids like cardiolipin (CL) have long been known to affect the stability and catalytic activity of mitochondrial membrane proteins (Schlame and Ren, 2009). However, considering phospholipids merely as the fabric that keeps mitochondria together vastly underestimates their contribution toward the functional integrity of these organelles.

In this article, we summarize recent findings that highlight distinct functions of mitochondrial phospholipids in diverse mitochondria-associated processes such as mitochondrial fusion, protein import into mitochondria, and apoptosis. We will focus on phosphatidylethanolamine (PE) and the mitochondria-specific dimeric glycerophospholipid CL. Both PE and CL are non–bilayer-forming phospholipids, a feature best explained by their shape (Fig. 1; van den Brink-van der Laan et al., 2004). Bilayer-forming phospholipids like phosphatidylcholine (PC) are cylindrically shaped with the fatty acid portions defining extended hydrophobic domains and the polar head groups defining the short hydrophilic domains along the length of the cylinder. The nearly equivalent diameters of the cylinder in both domains allow molecular packing that favors bilayers. The non–bilayer-forming lipids PE and CL are more conical shaped with a smaller hydrophilic head group diameter and a relatively larger hydrophobic domain diameter. This shape allows the formation of hexagonal phases that can be observed for isolated lipids depending on the pH and ionic strength (Ortiz et al., 1999). PE and CL are thought to be present mainly in bilayer structures in vivo, but their tendency to form hexagonal phases can create tension in membranes that is likely of functional importance to various mitochondrial processes like membrane fusion or the movement of proteins or solutes across membranes (van den Brink-van der Laan et al., 2004). The functional importance of non–bilayer-forming lipids is highlighted by the fact that yeast and bacteria cannot tolerate simultaneous reduction of PE and CL (Rietveld et al., 1993; Gohil et al., 2005). The biosynthesis of PE and CL occurs, at least in part, within mitochondria and relies on an intricate exchange of precursor forms between the membrane of the ER and the mitochondrial outer membrane at distinct contact sites, whose structural basis we are just beginning to understand. We will highlight recent advances and unresolved questions regarding this interorganellar communication and the intramitochondrial trafficking of phospholipids."[3]

"Mitochondrial phospholipid biosynthesis

The maintenance of a defined lipid composition within mitochondria depends on their capacity to synthesize phospholipids such as CL, PE, PG, and PA, whereas PI, PC, and PS are primarily synthesized in the ER and must be imported into the organelle for use as a finished end product or precursors for other lipids (Fig. 2). The biochemical steps in the synthesis of all phospholipids commence with the acylation of the sn-1 position of glycerol-3-phosphate (G3P) or dihydroxyacetone phosphate by acyltransferases (G3P acyltransferases [GPATs]) producing lyso-PA (Fig. 2 A). The yeast GPATs are associated with the ER and lipid particles, whereas the mammalian GPATs are localized to multiple organelles, including mitochondria (Wendel et al., 2009). Several lyso-PA acyltransferases (LPAATs) then convert lyso-PA to PA, which serves as a crucial intermediate supplying two independent cellular pathways for the synthesis of phospholipids (Fig. 2 A). One branch of the pathway converts PA to DAG catalyzed by the phosphatase Pah1 (Han et al., 2006) and eventually produces the zwitterionic lipids PE and PC in an enzymatic cascade known as the Kennedy pathway (Daum et al., 1998). The other branch of the pathway leads to the synthesis of CDP-DAG catalyzed by Cds1 (Shen et al., 1996) and produces the acidic phospholipids PS, PI, PG, and CL as its principal products (Fig. 2 A).

Mitochondrial synthesis of CL. A multienzyme cascade in the mitochondrial inner membrane synthesizes CL from CDP-DAG (Fig. 2 B; Joshi et al., 2009; Schlame and Ren, 2009) by the stepwise formation of PGP catalyzed by Pgs1 (Chang et al., 1998; Dzugasová et al., 1998) and its subsequent dephosphorylation catalyzed by the recently identified yeast PGP phosphatase Gep4 (Osman et al., 2010). Gep4 localizes to the matrix side of the inner membrane (Osman et al., 2010), which is also the predicted location for Pgs1. The localization of both enzymes in yeast mitochondria is in agreement with the proposed initiation of CL synthesis on the matrix-exposed leaflet of the inner membrane (Joshi et al., 2009; Schlame and Ren, 2009). How newly synthesized CL molecules are then redistributed within mitochondria remains to be examined. Although CL synthase generates CL from PG and CDP-DAG on the matrix side of the membrane (Schlame and Haldar, 1993), later acyl chain remodeling steps appear to occur on the outer leaflet of the inner membrane (Claypool et al., 2006). The acyl chain composition of nascent CL species is remodeled by the sequential action of a phospholipase A (Cld1 in yeast) and a transacylation reaction catalyzed by Taz1 (Xu et al., 2006; Beranek et al., 2009). In humans, mutations in Taz1 cause cardiomyopathy and Barth syndrome, underscoring the physiological importance of CL and its remodeling for mitochondrial homeostasis and function (Bione et al., 1996; Houtkooper et al., 2009). Although enzymes involved in CL biosynthesis from CDP-DAG are localized at the mitochondrial inner membrane, it is currently not clear how much CL synthesis depends on the transport of precursor lipids from extramitochondrial sources. The de novo synthesis of PA occurs in the ER, but PA may also be generated within mitochondria by phospholipases like MitoPLD (Choi et al., 2006). Thus, mitochondria may use both extrinsic and intrinsic sources of phospholipid precursors for CL formation.

Mitochondrial synthesis of PE. Extramitochondrial PS formed in the ER or specialized domains of the ER that are tightly associated with mitochondria serve as a precursor for mitochondrial PE in both yeast and mammalian cells (Fig. 2 C). This PS is synthesized from a CDP-DAG substrate in yeast (Letts et al., 1983; Nikawa and Yamashita, 1984; Kuchler et al., 1986) or by base exchange enzymes in mammalian cells (Kuge and Nishijima, 1997; Vance, 2008). The imported PS is a substrate for Psd1 (PS decarboxylase 1) located in the mitochondrial inner membrane (Clancey et al., 1993; Trotter et al., 1993). Although a second decarboxylase (Psd2) is present outside of mitochondria in yeast (but not in mammals; Trotter and Voelker, 1995), the majority of the catalytic activity occurs within mitochondria. PE produced via the Kennedy pathway or by the action of Psd2 is poorly assimilated into mitochondria and insufficient to meet the requirements for respiration. The PE produced in mitochondria is actively exported to other organelles (Voelker, 1984). One major consequence of this PE export is the synthesis of PC in the ER by the sequential methylation of the primary amine of PE, catalyzed by the yeast methyltransferases Pem1 and Pem2 (originally named Cho2 and Opi3; Kuchler et al., 1986; Kodaki and Yamashita, 1987, 1989). In the majority of mammalian tissues, PC is produced via the Kennedy pathway (Fig. 2), but in the liver, PE methyltransferase activity is significant and can provide adequate levels of PC during periods of choline deficiency (Li and Vance, 2008). In many eukaryotes, the aminoglycerophospholipids PS, PE, and PC comprise 75–80% of the total glycerophospholipids found within the cell (van Meer et al., 2008). As mitochondria have the synthetic capacity to synthesize the entire PE pool required for cell growth (Birner et al., 2001), the flux of PS into the mitochondria, and its subsequent decarboxylation and export as PE, can account for the biosynthesis of the majority of the glycerophospholipids present in all cellular membranes. This dynamic role of mitochondria as a major source of phospholipids is widely underappreciated. The role of mitochondria in exporting phospholipids is true for eukaryotes other than yeast. Mammalian cells can also produce the majority of all PE via the mitochondrial pathway (Voelker, 1984).

Mitochondrial phospholipid trafficking

The differential localization of enzymes of phospholipid biosynthetic pathways among different organelles and different membrane compartments within one organelle implicitly defines a requirement for extensive intracellular lipid trafficking (Fig. 2, B and C). Specific mechanisms must exist to ensure the transport of phospholipids from the ER to mitochondria and between outer and inner mitochondrial membranes. However, we are only beginning to understand how these transport processes occur and how they are regulated.

Phospholipid transport to and within mitochondria appears to proceed via close membrane contacts rather than vesicular pathways. A close apposition of two membranes may facilitate direct lipid flipping between bilayers at regions of positive membrane curvature or may allow lipid trafficking by yet to be identified soluble lipid carriers or by protein complexes that bridge both membranes (Voelker, 2009). Intermembrane lipid exchange might also be mediated via a stabilized hemifusion state, which would result in continuity between leaflets of both membranes, but evidence for such a mechanism is still lacking.

Tethering of ER and mitochondrial membranes. Transport of phospholipids between membranes of the ER and mitochondria occurs at specialized fractions of the ER that are tightly associated with mitochondria (Voelker, 1990) and were therefore termed mitochondria-associated membranes (MAMs; Vance, 1990; Ardail et al., 1993; Gaigg et al., 1995; Shiao et al., 1995). MAMs are enriched in certain lipids and various phospholipid biosynthetic enzymes, including PSS-1 (PS synthase-1), FACL4 (long-chain fatty acid-CoA ligase type 4; Vance, 1990; Rusiñol et al., 1994; Gaigg et al., 1995), and Ale1 acyltransferase (Riekhof et al., 2007). Direct evidence that phospholipid transport involves MAMs came from in vitro assays that showed that transport of PS from MAMs to mitochondria occurs more efficiently when MAMs, rather than bulk ER membranes, are mixed with mitochondria (Gaigg et al., 1995). Although independent of ATP, transport appears to be regulated by ubiquitination. A genetic screen in yeast for mutants affecting PS transport into mitochondria led to the identification of the F-box protein Met30, an E3 ubiquitin ligase (Schumacher et al., 2002). Met30 ubiquitinates and thereby inactivates the transcription factor Met4, leading to an increased transport of PS from MAMs to mitochondria (Schumacher et al., 2002; Voelker, 2009). However, the downstream targets of Met4 remain elusive. Phospholipid transport from MAM-derived vesicles to mitochondria proved to be partially protease sensitive, indicating that membrane proteins of the ER or mitochondria exposed to the cytosol mediate the interaction between both organelles (Vance, 1991; Achleitner et al., 1999). Electron tomography of intact cells revealed close appositions of ER membranes and mitochondria with a relatively defined, separating distance of ∼10–25 nm (Csordás et al., 2006). Several proteins were proposed to be involved in ER–mitochondria membrane tethering in mammalian cells (Szabadkai et al., 2006; de Brito and Scorrano, 2008), but evidence for a direct role in phospholipid trafficking has not yet been reported for any of these proteins. In contrast, direct evidence supporting the role of a macromolecular protein bridge for interorganellar phospholipid transport was recently obtained in yeast (Fig. 3). A synthetic biology approach using an artificial membrane tethering protein led to the identification of Mdm12 as an essential component for the interaction of ER and mitochondria (Kornmann et al., 2009). Mdm12 is associated with the outer membrane of mitochondria (Berger et al., 1997; Kornmann et al., 2009) and assembles with Mmm1, a glycosylated ER membrane protein, and the mitochondrial outer membrane proteins Mdm10 and Mdm34 into a complex (Boldogh et al., 2003; Youngman et al., 2004; Kornmann et al., 2009). Strikingly, cells lacking individual subunits of this complex, which was termed ER–mitochondria encounter structure (ERMES) complex (Kornmann et al., 2009), show reduced levels of mitochondrial PE and CL, suggesting that the ERMES structure is required for the exchange of phospholipids at ER–mitochondria contact sites. Consistently, the conversion of PS to PE and PC was slowed down in cells lacking ERMES components (Kornmann et al., 2009). It will be of interest to determine whether the ERMES complex only functions exclusively as a membrane tether ensuring the close apposition of ER and mitochondrial membranes or whether components of this complex actively contribute to the transport of phospholipids."[3]

"Phospholipid transport between the outer and inner mitochondrial membranes has been proposed to occur, similar to protein transport, at contact sites between mitochondrial inner and outer membranes (Ardail et al., 1991; Simbeni et al., 1991). Experiments with CHO cell mutants have identified a variant with a lesion in PS transport between the outer and inner mitochondrial membranes, but the gene responsible for this defect has yet to be identified (Emoto et al., 1999). Two proteins, mitochondrial creatine kinase (MtCK) and nucleotide diphosphate kinase (NDPK-D) facilitate CL transport between liposomes with a lipid composition resembling those of contact sites (Epand et al., 2007). However, the in vivo relevance of this pathway remains to be established. The recent identification of conserved proteins in the intermembrane space, which regulate the accumulation of CL and PE in mitochondria, may provide new clues about the mechanism of phospholipid transport across this compartment. Ups1 was originally identified to affect the processing of the dynamin-like GTPase Mgm1 in yeast (Sesaki et al., 2006) and later shown to regulate CL level in mitochondria (Osman et al., 2009a; Tamura et al., 2009). Ups1 belongs to the conserved Ups1/PRELI protein family, which is characterized by the presence of a conserved MSF´ domain (originally identified in yeast Msf´) of unknown function (Dee and Moffat, 2005). A homologue of Ups1, termed Ups2 or Gep1, regulates the accumulation of PE within mitochondria (Osman et al., 2009a; Tamura et al., 2009). Although PE levels are decreased in the absence of Ups2, overexpression of Ups2 reduces CL, pointing to a coordinated regulation of PE and CL by these conserved regulatory proteins. Consistently, deletion of UPS2 restores normal CL levels in Ups1-deficient yeast cells. Two recent studies in yeast identified Mdm35 as a common binding partner of both Ups1 and Ups2 in the intermembrane space, providing a molecular explanation for the coordinated regulation of CL and PE within mitochondria (Potting et al., 2010; Tamura et al., 2010). Mdm35 binding ensures mitochondrial import of Ups1 and Ups2 and protects both proteins against proteolysis. Notably, both Ups1 and Ups2 are intrinsically unstable proteins and are degraded by the i-AAA protease Yme1 and Atp23 in wild-type cells even under normal growth conditions (Potting et al., 2010). It is therefore conceivable that the mitochondrial quality control system affects the accumulation of CL and PE within mitochondria by regulating the stability of Ups1-like proteins. The strong conservation of all components of the regulatory circuit and the altered PE levels in i-AAA protease-deficient mitochondria (Nebauer et al., 2007) point in this direction. However, the molecular function of Ups1 and Ups2 remains speculative. Because reduced mitochondrial PE levels in the absence of Ups2 were caused by decreased stability rather than altered synthesis of PE (Osman et al., 2009a), Ups2 might regulate the export of PE from mitochondria. It is therefore an intriguing possibility that lipid trafficking between inner and outer mitochondrial membrane controlled by Ups1/PRELI-like proteins determines the phospholipid composition of mitochondrial membranes.

The role of CL in mitochondria

Although studies examining functional roles of phospholipids within mitochondria are generally hampered by their broad distribution among different cellular membranes, the predominant localization of CL in mitochondria has enabled the identification of an increasing number of mitochondrial processes dependent on this lipid, and the assignment of pathologies associated with alterations in the CL metabolism to mitochondrial dysfunction (Chicco and Sparagna, 2007; Joshi et al., 2009). The unique, dimerically cross-linked phospholipid structure of CL affects the stability and activity of various membrane protein complexes and metabolite carriers (Fig. 4; Houtkooper and Vaz, 2008). CL molecules are present in crystal structures of the ATP/ADP carrier (AAC) and the respiratory complexes III and IV and have been proposed to fulfill important structural roles (Lange et al., 2001; Pebay-Peyroula et al., 2003; Shinzawa-Itoh et al., 2007). Indeed, respiratory supercomplexes consisting of complexes III and IV are destabilized in mitochondria lacking CL (Pfeiffer et al., 2003; Claypool et al., 2008b). Similarly, dimers of AAC and other AAC-containing complexes dissociate in CL-deficient mitochondria (Claypool et al., 2008b). These examples illustrate the importance of CL for bioenergetic functions; but in addition, recent studies are now revealing that

CL has a much broader impact on mitochondrial physiology.

CL and protein import into mitochondria. The vast majority of mitochondrial proteins are nuclear encoded and imported into the organelle via heterooligomeric protein translocases residing in the mitochondrial inner and outer membranes (Schmidt et al., 2010). Several independent studies revealed that the assembly and function of these TIM (translocase of the inner mitochondrial membrane) and TOM (translocase of the outer mitochondrial membrane) complexes depend on CL (Fig. 4 B). Tam41 (translocator assembly and maintenance protein 41) was identified as a novel mitochondrial matrix protein, which is required for the integrity of the TIM23 complex in the inner membrane and its functional interaction with the mitochondrial import motor PAM (presequence translocase-associated motor; Gallas et al., 2006; Tamura et al., 2006). A later study attributed these deficiencies to the loss of CL in the absence of Tam41 (Kutik et al., 2008). Similarly, the interaction of TIM and PAM complexes is affected in mitochondria that lack the CL synthase Crd1 or Ups1 (Kutik et al., 2008; Tamura et al., 2009). Interestingly, an altered electrophoretic mobility of another protein translocase of the inner membrane, the TIM22 complex mediating the membrane insertion of metabolite carrier proteins, was observed when Δcrd1 and Δtam41 mitochondria were analyzed, which may point to an altered assembly of the translocase or to a specific association of CL molecules with this complex (Kutik et al., 2008). Regardless, it appears from these studies that the reduced protein import into CL-deficient mitochondria is not simply the consequence of altered bioenergetics and a reduced membrane potential across the inner membrane, but rather reflects the specific requirement of CL for the functional integrity of the mitochondrial import machinery. This view is supported by the recent observation that CL levels also regulate protein translocases in the outer membrane (Gebert et al., 2009), reconciling earlier observations that protein import into mitochondria can be inhibited by drugs binding to acidic phospholipids (Eilers et al., 1989) and is impaired in CL-deficient yeast cells (Jiang et al., 2000). The assembly of the import receptor Tom20 with the TOM complex as well as the organization of the SAM complex that mediates the assembly of β-barrel proteins in the outer membrane are altered in CL-deficient mitochondria (Fig. 4 B; Gebert et al., 2009). As a consequence, the biogenesis of β-barrel proteins in the outer membrane as well as that of proteins located in other mitochondrial subcompartments is impaired.

CL and apoptosis. Further support for a functional role of CL in the mitochondrial outer membrane came from studies on the role of mitochondria during apoptosis, which revealed that CL regulates multiple steps of the apoptotic program (Fig. 4 C). Apoptosis can be induced by activation of the death receptor (Fas receptor) in the plasma membrane. Ligand-bound Fas receptor oligomerizes and recruits pro–caspase-8, which in response undergoes an autocatalytic processing step resulting in its activation. However, activation of caspase-8 at the plasma membrane was found to be insufficient for triggering apoptosis in some cells, and thus completion of the apoptotic program required a mitochondria-dependent feedback loop (Scaffidi et al., 1998). A recent study revealed that CL in the mitochondrial outer membrane provides an anchor and activating platform for caspase-8, which is processed and translocates to mitochondria upon Fas receptor activation (Gonzalvez et al., 2008). Caspase-8–mediated cleavage of the BH3-only BID protein leads to its translocation to mitochondria. Truncated BID triggers activation of Bax and Bak, members of the Bcl2-family which induce outer membrane permeabilization and release of cytochrome c (Lovell et al., 2008). CL together with the major facilitator protein MTCH2/MIMP in the outer membrane regulates truncated BID recruitment to contact sites between the inner and outer membranes (Lutter et al., 2000; Lucken-Ardjomande et al., 2008; Sani et al., 2009; Zaltsman et al., 2010). Similarly, membrane insertion of Bax and its oligomerization were found to proceed more efficiently in the presence of CL (Lutter et al., 2000; Lucken-Ardjomande et al., 2008; Sani et al., 2009). Finally, CL affects the release of cytochrome c from mitochondria during apoptosis. It binds directly to cytochrome c, retaining it within the cristae (Choi et al., 2007; Sinibaldi et al., 2008). The interaction between cytochrome c and CL is weakened upon peroxidation of the unsaturated acyl chains of CL (Nomura et al., 2000). The release of cytochrome c during apoptosis has been proposed to be further facilitated by remodeling of the mitochondrial cristae that facilitates the redistribution of cytochrome c molecules from the cristae lumen (Scorrano et al., 2002). Cristae morphology is controlled by the dynamin-like GTPase OPA1, a central component of the mitochondrial fusion machinery, whose activity is affected by CL (see next section). Thus, CL plays multiple roles during apoptosis in both mitochondrial membranes and may serve as a factor that coordinates the sequence of apoptotic events in mitochondria.

CL and mitochondrial dynamics. Early studies with model membranes demonstrated that the formation of hexagonal structures induce membrane fusion and suggested a crucial role of non-bilayer lipids such as CL or PE for membrane fusion in vivo (Cullis and de Kruijff, 1979). Indeed, reductions of mitochondrial PE and CL levels were reported to result in abnormal mitochondrial morphology (Kawasaki et al., 1999; Steenbergen et al., 2005; Choi et al., 2006; Claypool et al., 2008a) and high frequency generation of respiratory deficient mitochondria (Birner et al., 2003; Zhong et al., 2004). Membrane fusion is mediated by evolutionarily conserved dynamin-like GTPases present in both mitochondrial membranes (Hoppins et al., 2007). In the inner membrane, OPA1 (or Mgm1 in yeast) is proteolytically processed, resulting in the balanced accumulation of long and short protein isoforms within mitochondria, both of which are required for mitochondrial fusion and cristae morphogenesis (Herlan et al., 2003; Ishihara et al., 2003; Song et al., 2007). Processing of yeast Mgm1 was affected in the absence of Ups1 or Ups2, which regulate the accumulation of CL and PE within mitochondria, respectively (Sesaki et al., 2006; Osman et al., 2009a). Impaired processing of Mgm1 could explain the aberrant morphology of mitochondria with an altered membrane lipid composition, but Mgm1 cleavage has so far not been analyzed in other CL-deficient cells, and other scenarios are conceivable. The short forms of both Mgm1 and OPA1 bind to negatively charged phospholipids, in particular CL, that stimulate its oligomerization and its GTPase activity (Fig. 4 D; DeVay et al., 2009; Meglei and McQuibban, 2009; Rujiviphat et al., 2009; Ban et al., 2010). It is possible that interaction with CL restricts the function of Mgm1/OPA1 to specific membrane domains, like contact sites between both mitochondrial membranes, which are known to be enriched in CL (Ardail et al., 1990). These contact sites have been proposed to be the site of action of a phospholipase D, termed MitoPLD, which converts CL in the outer membrane to PA (Fig. 4 D; Choi et al., 2006). MitoPLD is required for mitochondrial fusion in vitro, and modulation of its expression in vivo causes morphological abnormalities (Choi et al., 2006). The formation of PA may allow the recruitment of additional fusion components or render membranes fusogenic. Such a role of PA would be reminiscent of SNARE-mediated fusion (Huang et al., 2005) and could point to a crucial role of local membrane lipid alterations in seemingly unrelated membrane fusion processes."[3]

"Recent discoveries have brought about significant progress in our understanding of the metabolism of mitochondrial phospholipids. This development was accompanied by a drastically altered view of the role that specific phospholipids play in various mitochondrial processes and the role of mitochondria in broader aspects of the biogenesis of nonmitochondrial membranes. Defined molecular functions of specific phospholipids, like CL, have been recognized, and the accumulation of these lipids in specific membrane domains is emerging as an important property of mitochondrial membranes. The recent identification of novel genes in yeast affecting the phospholipid composition of mitochondria, many of them conserved in mammals, now promises to provide insight into some of the mysteries of mitochondrial phospholipid metabolism and trafficking. Mitochondria may prove once again to be an excellent model to unravel basic cell biological processes that will be relevant to other membrane systems. Undoubtedly, exciting discoveries are just around the corner." [3]

"Over the past two decades, most of the genes specifying lipid synthesis and metabolism in yeast have been identified and characterized. Several of these biosynthetic genes and their encoded enzymes have provided valuable tools for the genetic and biochemical dissection of interorganelle lipid transport processes in yeast. One such pathway involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), and its non-vesicular transport to the site of phosphatidylserine decarboxylase2 (Psd2p) in membranes of the Golgi and endosomal sorting system. In this review, we summarize the identification and characterization of the yeast phosphatidylserine decarboxylases, and examine their role in studies of the transport-dependent pathways of de novo synthesis of phosphatidylethanolamine (PtdEtn). The emerging picture of the Psd2p-specific transport pathway is one in which the enzyme and its non-catalytic N-terminal domains act as a hub to nucleate the assembly of a multiprotein complex, which facilitates PtdSer transport at membrane contact sites between the ER and Golgi/endosome membranes. After transport to the catalytic site of Psd2p, PtdSer is decarboxylated to form PtdEtn, which is disseminated throughout the cell to support the structural and functional needs of multiple membranes."[4]

"Identification of Psd1p, Psd2p and Their Application as Reporters of PtdSer Traffic

Phosphatidylethanolamine (PtdEtn) is an abundant membrane phospholipid of yeast as well as many lower and higher eukaryotes and bacteria. In addition to acting as a structural constituent of membranes, PtdEtn is involved in numerous specific cellular functions including autophagy 1, 2, fusion and fission events of membranes 3, vacuolar protein delivery 4, the stabilization of membrane proteins or enzymes 5, 6 and cell signaling 7, 8. The endoplasmic reticulum (ER) and mitochondria are generally accepted as the major sites of glycerophospholipid biosynthesis in eukaryotes 9-11. In yeast, a significant proportion of total PtdEtn can also be synthesized in the compartments of the Golgi and endosomes 12, 13. The compartmentation of lipid-synthesizing activities necessitates efficient interorganelle transport to membranes incapable of autonomous lipid synthesis, in order to maintain membrane biogenesis and function.

The topological segregation of yeast enzymes involved in aminoglycerophospholipid synthesis is shown in Figure 1. In this scheme, phosphatidylserine synthase (Pss1p, encoded by the CHO1/PSS1 gene) 14 is localized in the ER and mitochondria-associated ER membranes (MAMs) 15, 16. Phosphatidylserine decarboxylase1 (Psd1p) is present in mitochondria 17, and phosphatidylserine decarboxylase2 (Psd2p) is distributed in the Golgi and endosomes 12, 13, 18. Phosphatidylethanolamine methyltransferases 1 and 2 (encoded by the CHO2/PEM1 and OPI3/PEM2 genes) reside in the ER 16, as corroborated by green fluorescent protein (GFP) localization studies 19. Following the synthesis of phosphatidylserine (PtdSer) in the ER, this lipid must be transported to either mitochondria or Golgi/endosome compartments for decarboxylation to generate PtdEtn. The PtdEtn produced within the mitochondria is required for the optimal function of this organelle 17, 20, and yeast strains lacking Psd1p undergo conversion to petite phenotypes with high frequency. PtdEtn produced in the ER by the CDP-ethanolamine (Etn)-dependent ‘Kennedy’ pathway 21 is only poorly transported into mitochondria, and cannot fully restore all functions of the pool produced within the mitochondria 12, 17, 20, 22. PtdEtn synthesized by Psd1p within the mitochondria is not static, and can be exported to other membranes to fulfill their requirements for PtdEtn 23. Many eukaryotes, including yeast, can efficiently methylate PtdEtn to form phosphatidylcholine (PtdCho) in the ER 24."[ 4].

Unresolved Questions and Future Directions for Studies on Psd2p

"As described above, the current model for PtdSer transport to Psd2p is one in which the N-terminal portion of the enzyme acts as a hub for assembly of a multisubunit complex, which facilitates PtdSer transport from the ER to drive PtdEtn synthesis in compartments of the Golgi and endosomes. This Psd2p-specific pool of PtdEtn is likely to be important for maintenance of proper lipid compositions in endosomes and retrograde-directed Golgi vesicles, as suggested by the prevalence of genes specifying these functions as negative genetic interactors of psd2Δ (Table 1). While the studies summarized in this review provide many details regarding PtdSer transport and the cellular function of PtdEtn generated by Psd2p, several important questions remain unanswered. The first and most obvious of these questions is: What is the molecular mechanism by which PtdSer is transported? The biochemical and genetic studies outlined above have defined a set of requirements necessary for PtdSer transport to occur; however, the precise mechanism by which PtdSer is transported remains obscure.

A second major unresolved question is: How is PtdSer transport and Psd2p catalytic activity regulated? Coordinate regulation of lipid synthesis with other biosynthetic functions is thought to be critical to proper regulation of cell growth and division, and as such, enzymes such as Psd2p are likely to be regulated to meet the demand of the cell for membrane biogenesis. Recent studies examining the regulation of the phospholipid methyltransferase Pem2p/Opi3p identify a role for the oxysterol-binding protein homolog Osh3p as a regulator of this methyltransferase at membrane contact sites between the cortical ER and plasma membrane 73. These observations lead to the conclusion that regulation of phospholipid biosynthesis in yeast and other eukaryotes is driven not only by regulation of the activity of different synthetic enzymes but also through regulation of the transport pathways and membrane contact sites that give those enzymes access to their substrates. Several examples have been outlined in this work, in which we have focused on transport through the Psd2p transport complex, highlighting the necessity for a clearer understanding of interorganelle phospholipid transport in eukaryotic cells, as these processes and the membrane contact sites through which they occur appear to be emerging as important control points for proper membrane biogenesis and function."[4]

"Supplementation with a phosphatidylserine and phosphatidylserine/ phosphatidic acid complex (PAS) has been observed to normalize stress induced dysregulations of the hypothalamus-pituitary-adrenal axis (HPAA). Prolonged stress first induces a hyper-activation of the HPAA, which then can be followed by a state of hypo-activation.

The aim of this study was to examine effects of an oral supplementation with 400 mg PS & 400 mg PA (PAS 400) per day on the endocrine stress response (ACTH, saliva and serum cortisol) to a psychosocial stressor. A special focus was to analyze subgroups of low versus high chronically stressed subjects as well as to test efficacy of 200 mg PS & 200 mg PA (PAS 200)." [5]

"Chronic stress levels and other baseline measures did not differ between treatment groups (all p > 0.05). Acute stress was successfully induced by the TSST and resulted in a hyper-responsivity of the HPAA in chronically stressed subjects. Compared to placebo, a supplementation with a daily dose of PAS 400 was effective in normalizing the ACTH (p = 0.010), salivary (p = 0.043) and serum cortisol responses (p = 0.035) to the TSST in chronically high but not in low stressed subjects (all p > 0.05). Compared to placebo, supplementation with PAS 200 did not result in any significant differences in these variables (all p > 0.05). There were no significant effects of supplementation with PAS on heart rate, pulse transit time, or psychological stress response (all p > 0.05)..... In chronically stressed subjects, a supplementation with PAS 400 (MemreePlus™) can normalize the hyper-responsivity of the HPAA to an acute stressor." [5]

"Chronic PS supplementation has been found to promote a less stressed, more relaxed state and a subjective improvement in mood. Some studies have combined PS with other nutritional supports, namely phosphatidic acid or omega-3 fatty acids, to good effect. In one of these, omega-3 administered in combination with PS were found to have stress-reducing effects exclusively in highly chronically stressed adult males who showed a blunted cortisol response to the Trier Social Stress Test (TSST), the gold standard for evaluating the neurobiology of acute stress.

Chronic phosphatidylserine supplementation has been found to promote a less stressed, more relaxed state and a subjective improvement in mood.

If you’re curious like I was as to how researchers ethically and reliably provoke a predictable stress response in human subjects, the answer is: they administer a public speaking task in front of a non-responsive audience, with a surprise mental math challenge thrown in – a scenario that may sound all too familiar to some of today’s online instructors and learners. In the study’s lowest responders, treatment with omega-3 plus PS for 12 weeks seemed to restore the cortisol response to this challenge, and the researchers concluded that those with high chronic stress and/or a dysfunctional response of the HPA axis may benefit from omega-3 plus PS supplementation .

A similar effect was found for a soy-based PS/phosphatic acid complex (PA) at a dose of 400 mg PS and 400 mg PA per day in normalizing ACTH and salivary and serum cortisol in chronically high-stressed males, but not in low-stressed subjects, and there was no significant effect at a dose of 200 mg PS and 200 mg PA ." [6]

"Cytochrome c release from the mitochondria is a critical component of the apoptotic cell-death program. Cytochrome c–catalyzed peroxidation of cardiolipin, a mitochondrial phospholipid, has now been shown to lessen the binding of cytochrome c to the mitochondrial inner membrane and facilitate permeabilization of the outer membrane. These results describe a new and earlier pro-apoptotic role for cytochrome c."[8]

"Cardiolipin (CL) is a phospholipid predominantly found in the mitochondrial inner membrane and is associated structurally with individual complexes of the electron transport chain (ETC). Because the ETC is the major mitochondrial reactive oxygen species (ROS)-generating site, the proximity to the ETC and bisallylic methylenes of the PUFA chains of CL make it a likely target of ROS in the mitochondrial inner membrane. Oxidized cellular CL products, uniquely derived from ROS-induced autoxidation, could serve as biomarkers for the presence of the ROS and could help in the understanding of the mechanism of oxidative stress. Because major CL species have four unsaturated acyl chains, whereas other phospholipids usually have only one in the sn-2 position, characterization of oxidized CL is highly challenging. In the current study, we exposed CL, under aerobic conditions, to singlet oxygen (1O2), the radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride, or room air, and the oxidized CL species were characterized by HPLC-tandem mass spectrometry (MS/MS). Our reverse-phase ion-pair HPLC-MS/MS method can characterize the major and minor oxidized CL species by detecting distinctive fragment ions associated with specific oxidized species. The HPLC-MS/MS results show that monohydroperoxides and bis monohydroperoxides were generated under all three conditions. However, significant amounts of CL dihydroperoxides were produced only by 1O2-mediated oxidation. These products were barely detectable from radical oxidation either in a liposome bilayer or in thin film. These observations are only possible due to the chromatographic separation of the different oxidized species."[9]

"Ca2+-overload contributes to the oxidation of mitochondrial membrane lipids and associated events such as the permeability transition pore (MPTP) opening. Numerous experimental studies about the Ca2+/cardiolipin (CL) interaction are reported in the literature, but there are few studies in conjunction with theoretical approaches based on ab initio calculations. In the present study, the lipid fraction of the inner mitochondrial membrane was modeled as POPC/CL large unilamellar vesicles (LUVs). POPC/CL and, comparatively, POPC, and CL LUVs were challenged by singlet molecular oxygen using the anionic porphyrin TPPS4 as a photosensitizer and by free radicals produced by Fe2+-citrate. Calcium ion favored both types of lipid oxidation in a lipid composition-dependent manner. In membranes containing predominantly or exclusively POPC, Ca2+ increased the oxidation at later reaction times while the oxidation of CL membranes was exacerbated at the early times of reaction. Considering that Ca2+ interaction affects the lipid structure and packing, density functional theory (DFT) calculations were applied to the Ca2+ association with totally and partially protonated and deprotonated CL, in the presence of water. The interaction of totally and partially protonated CL head groups with Ca2+ decreased the intramolecular P-P distance and increased the hydrophobic volume of the acyl chains. Consistently with the theoretically predicted effect of Ca2+ on CL, in the absence of pro-oxidants, giant unilamellar vesicles (GUVs) challenged by Ca2+ formed buds and many internal vesicles. Therefore, Ca2+ induces changes in CL packing and increases the susceptibility of CL to the oxidation promoted by free radicals and excited species."[10]

"Molecules of mitochondrial cardiolipin (CL) get selectively oxidized upon oxidative stress, which triggers the intrinsic apoptotic pathway. In a chemical model most closely resembling the mitochondrial membrane—liposomes of pure bovine heart CL—we compared ubiquinol-10, ubiquinol-6, and alpha-tocopherol, the most widespread naturally occurring antioxidants, with man-made, quinol-based amphiphilic antioxidants. Lipid peroxidation was induced by addition of an azo initiator in the absence and presence of diverse antioxidants, respectively. The kinetics of CL oxidation was monitored via formation of conjugated dienes at 234 nm. We found that natural ubiquinols and ubiquinol-based amphiphilic antioxidants were equally efficient in protecting CL liposomes from peroxidation; the chromanol-based antioxidants, including alpha-tocopherol, were 2-3 times less efficient. Amphiphilic antioxidants, but not natural ubiquinols and alpha-tocopherol, were able, additionally, to protect the CL bilayer from oxidation by acting from the water phase. We suggest that the previously reported therapeutic efficiency of mitochondrially targeted amphiphilic antioxidants is owing to their ability to protect those CL molecules that are inaccessible to natural hydrophobic antioxidants, being trapped within respiratory supercomplexes. The high susceptibility of such occluded CL molecules to oxidation may have prompted their recruitment as apoptotic signaling molecules by nature."[11]

95% of Diagnosed CFS Patients Had Anti Cardiolipin Anti Bodies. Hmmm.

"Examination of anticardiolipin antibodies (ACAs) in the sera of patients clinically diagnosed with chronic fatigue syndrome (CFS) using an enzyme-linked immunoassay procedure demonstrated the presence of immunoglobulin M isotypes in 95% of CFS serum samples tested. The presence of immunoglobulin G and immunoglobulin A isotypes were also detected in a subset of the samples. Future studies will focus on elucidating whether alterations to mitochondrial inner membranes and/or metabolic functions play a possible role in the expression of ACAs."[12]

"A decline in energy is common in aging, and the restoration of mitochondrial bioenergetics may offer a common approach for the treatment of numerous age-associated diseases. Cardiolipin is a unique phospholipid that is exclusively expressed on the inner mitochondrial membrane where it plays an important structural role in cristae formation and the organization of the respiratory complexes into supercomplexes for optimal oxidative phosphorylation. The interaction between cardiolipin and cytochrome c determines whether cytochrome c acts as an electron carrier or peroxidase. Cardiolipin peroxidation and depletion have been reported in a variety of pathological conditions associated with energy deficiency, and cardiolipin has been identified as a target for drug development. This review focuses on the discovery and development of the first cardiolipin-protective compound as a therapeutic agent. SS-31 is a member of the Szeto-Schiller (SS) peptides known to selectively target the inner mitochondrial membrane. SS-31 binds selectively to cardiolipin via electrostatic and hydrophobic interactions. By interacting with cardiolipin, SS-31 prevents cardiolipin from converting cytochrome c into a peroxidase while protecting its electron carrying function. As a result, SS-31 protects the structure of mitochondrial cristae and promotes oxidative phosphorylation. SS-31 represents a new class of compounds that can recharge the cellular powerhouse and restore bioenergetics. Extensive animal studies have shown that targeting such a fundamental mechanism can benefit highly complex diseases that share a common pathogenesis of bioenergetics failure. This review summarizes the mechanisms of action and therapeutic potential of SS-31 and provides an update of its clinical development programme."[13]

"Mitochondria are essential for eukaryotic cell activity and function, and their dysfunction is associated with the development and progression of renal diseases. In recent years, there has been a rapid development in mitochondria-targeting pharmacological strategies as mitochondrial biogenesis, morphology, and function, as well as dynamic changes in mitochondria, have been studied in disease states. Mitochondria-targeting drugs include nicotinamide mononucleotide, which supplements the NAD+ pool; mitochondria-targeted protective compounds, such as MitoQ; the antioxidant coenzyme, Q10; and cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. However, traditional drugs targeting mitochondria have limited clinical applications due to their inability to be effectively absorbed by mitochondria in vivo and their high toxicity. Recently, SS-31, a mitochondria-targeting antioxidant, has received significant research attention as it decreases mitochondrial reactive oxygen species production and prevents mitochondrial depolarization, mitochondrial permeability transition pore formation, and Ca2+-induced mitochondrial swelling, and has no effects on normal mitochondria. At present, few studies have evaluated the effects of SS-31 against renal diseases, and the mechanism underlying its action is unclear. In this review, we first discuss the pharmacokinetics of SS-31 and the possible mechanisms underlying its protective effects against renal diseases. Then, we analyze its renal disease-improving effects in various experimental models, including animal and cell models, and summarize the clinical evidence of its benefits in renal disease treatment. Finally, the potential mechanism underlying the action of SS-31 against renal diseases is explored to lay a foundation for future preclinical studies and for the evaluation of its clinical applications."[14]


[1] Phosphatidylserine transport to the mitochondria is regulated by ubiquitination. Marc M Schumacher 1, Jae-Yeon Choi, Dennis R Voelker; J Biol Chem. . 2002 Dec 27;277(52):51033-42. doi: 10.1074/jbc.M205301200. Epub 2002 Oct 21. PMID: 12393893. DOI: 10.1074/jbc.M205301200

[2] Hicke L. Nat. Rev. Mol. Cell. Biol.2001; 2: 195-201

[3] Making heads or tails of phospholipids in mitochondria. Christof Osman,1 Dennis R. Voelker,2 and Thomas Langer1,3J Cell Biol. 2011 Jan 10; 192(1): 7–16. doi: 10.1083/jcb.201006159 PMCID: PMC3019561. PMID: 21220505.

[4] Transport of Phosphatidylserine from the Endoplasmic Reticulum to the Site of Phosphatidylserine Decarboxylase2 in Yeast. Muthukumar Kannan, Wayne R. Riekhof, Dennis R. Voelker. First published: 30 October 2014. Citations: 27

[5] A soy-based phosphatidylserine/ phosphatidic acid complex (PAS) normalizes the stress reactivity of hypothalamus-pituitary-adrenal-axis in chronically stressed male subjects: a randomized, placebo-controlled study. Juliane Hellhammer,1 Dominic Vogt,1 Nadin Franz,1 Ulla Freitas,2 and David Rutenberg3Lipids Health Dis. 2014; 13: 121. Published online 2014 Jul 31. doi: 10.1186/1476-511X-13-121 PMCID: PMC4237891. PMID: 25081826

[7] The effects of phosphatidylserine on endocrine response to moderate intensity exercise

Michael A Starks,1 Stacy L Starks,1 Michael Kingsley,2 Martin Purpura,3 and Ralf Jäger3J Int Soc Sports Nutr. 2008; 5: 11. Published online 2008 Jul 28. doi: 10.1186/1550-2783-5-11. PMCID: PMC2503954. PMID: 18662395

[8] Cardiolipin oxidation sets cytochrome c free. Sten Orrenius & Boris Zhivotovsky. nature chemical biology. Published: 01 September 2005. Nature Chemical Biology volume 1, pages188–189 (2005)

[9] Cardiolipin: characterization of distinct oxidized molecular species. Junhwan Kim,* Paul E. Minkler. J Lipid Res. 2011 Jan; 52(1): 125–135. doi: 10.1194/jlr.M010520. PMCID: PMC2999925. PMID: 20858593

[10] Cardiolipin Structure and Oxidation Are Affected by Ca2+ at the Interface of Lipid Bilayers Érica G. A. Miranda1† Juliana C. Araujo-Chaves1†. Front. Chem., 21 January 2020 Sec. Chemical Biology. Volume 7 - 2019 |

[11] Impact of Antioxidants on Cardiolipin Oxidation in Liposomes: Why Mitochondrial Cardiolipin Serves as an Apoptotic Signal? Alexey V. Lokhmatikov, 1 , 2 Natalia Voskoboynikova, Oxid Med Cell Longev. 2016; 2016: 8679469. Published online 2016 May 26. doi: 10.1155/2016/8679469 PMCID: PMC4899610. PMID: 27313834

[12] Anticardiolipin antibodies in the sera of patients with diagnosed chronic fatigue syndrome

[13] First-in-class cardiolipin-protective compound as a therapeutic agent to restore mitochondrial bioenergetics. Hazel H SzetoBr J Pharmacol. 2014 Apr; 171(8): 2029–2050. Published online 2014 Mar 28. doi: 10.1111/bph.12461. PMCID: PMC3976620. PMID: 24117165.

[14] Oxid Med Cell Longev. 2022; 2022: 1295509. Published online 2022 Jun 6. doi: 10.1155/2022/1295509 PMCID: PMC9192202. PMID: 35707274. SS-31, a Mitochondria-Targeting Peptide, Ameliorates Kidney Disease

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Threasa Melton
Threasa Melton

Hello Mike…

i wonder how this supplementation would work for those with adrenal insufficiency? I have low adrenal cortisol and can not tolerate any stress however PS blunts cortisol release which I am already lacking…

I Would like to try this perhaps the pas 400 alone…

thank you for the article…


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