Further lipud on novel formulation strategies to minimize Vegan-friendly brunch spots and Promoted lipid oxidation oxidation Promoetd be studied, especially in plant-based foods. Free radicals formed by rancid rice bran could attack the main chain and side chain of proteins, and then protein oxygen-free radicals could be formed Tian, KZ, Cao, CC, Nie, XM, Wang, W, and Han, CQ.

Promoted lipid oxidation -

S1F and ID8 Supplementary Fig. S1G cells following radiotherapy. Radiotherapy induced an increased lipid peroxidation in B16F10 cells Supplementary Fig. S1H and ID8 cells Supplementary Fig. S1I in a dose-dependent manner.

These results suggest that radiotherapy directly induces tumor ferroptosis. Given that radiotherapy can induce tumor ferroptosis Fig. S1A—S1I , we wondered whether ferroptosis agonists could enhance radiotherapy efficacy and be exploited as novel radiosensitizers.

To explore this possibility, we pretreated HT, B16F10, and ID8 cells with low doses of ferroptosis inducers FIN including sulfasalazine, RSL-3, erastin, and atorvastatin, and then irradiated these cells.

We found that ferroptosis inducers reduced clonogenic cell survival as compared with radiotherapy alone in HT cells in vitro Fig. GPX4 utilizes glutathione to reduce oxidized lipid species and to limit ferroptosis.

Glutathione levels are regulated by intracellular cystine concentrations. We depleted cystine and cysteine using cyst e inase, a recombinant human enzyme Interestingly, cyst e inase strongly sensitized HT cells to radiotherapy in vitro Fig. To support our finding in HT cells, we treated B16F10 cells with sulfasalazine, RSL-3, and cyst e inase and observed decreased cell survival following radiotherapy as compared with radiotherapy alone Supplementary Fig.

S1J and S1K. Cell death quantification using propidium iodide PI staining confirmed that sulfasalazine and cyst e inase augmented radiation-induced cell death Fig. Pretreatment with RSL-3, sulfasalazine, or cyst e inase also increased ID8 cell sensitivity to radiotherapy in vitro Supplementary Fig.

S1L and S1M. Although cyst e inase had minimal efficacy as monotherapy, it enhanced radiotherapy efficiency Fig. Moreover, increased tumor control was accompanied by enhanced lipid oxidation in the combination therapy group Fig.

Neither single treatment nor the combination treatment caused significant murine weight loss Supplementary Fig. To assess whether FINs can improve radiotherapy efficacy in vivo , we established B16F10 tumors in mice and treated tumors with radiotherapy, sulfasalazine, or both dual treatment. We found that dual treatment enhanced tumor control Supplementary Fig.

Again, this improved tumoral control was accompanied by increased lipid oxidation in tumors treated with both agents Supplementary Fig.

These data suggest that ferroptosis can be targeted in vivo to enhance radiotherapy efficacy. It has recently been shown that fatty-acid saturation modulates ferroptosis sensitivity. Specifically, ACSL4 promotes ferroptosis, whereas ACSL3 limits ferroptosis 19, We generated ACSL4 knockout and ACSL3 knockout B16F10 cells Supplementary Fig.

S1Q and S1R. We observed that loss of the ferroptosis effector gene ACSL4 diminished radiotherapy efficacy in vivo Fig. In contrast, deletion of the ferroptosis suppressor ACSL3 augmented radiotherapy efficacy in vivo Fig.

To demonstrate that diminished cell survival following radiation was due to increased lipid peroxidation and ferroptosis, we quantified lipid peroxidation changes with C11BODIPY following FIN addition. We observed that in B16F10 cells, short exposures to low doses of sulfasalazine, RSL-3, and cyst e inase minimally altered lipid ROS.

Combinations of FINs with radiation synergistically increased lipid ROS Fig. FINs similarly augmented lipid ROS levels in concert with radiation in HT cells Supplementary Fig. To generalize these findings to different radiation doses and fractionation schedules, we treated B16F10 tumors with 5 fractions of 3 Gy as well as a single fraction of 10 or 20 Gy.

Quantification of lipid peroxidation changes with C11BODIPY showed that all radiotherapy doses induced increases in C11BODIPY, whereas higher single fractions of radiotherapy induced more lipid peroxidation Fig. To confirm that ferroptosis resistance conferred radiotherapy resistance at ablative doses of radiotherapy, we treated wild-type WT and RSL-3—resistant B16F10 tumors with a single fraction of 20 Gy We observed that even at higher doses of radiotherapy, ferroptosis-resistant tumors remained resistant to radiotherapy in vivo Supplementary Fig.

Collectively, these data suggest that radiation induces tumor ferroptosis, and this ferroptosis can be pharmacologically augmented by ferroptosis agonists.

Thus, targeting ferroptosis may be a novel therapeutic approach for radiation sensitization. The cellular and molecular bases for the interaction between T cells and radiotherapy are not fully defined. Then, mice received a moderate dose of radiation 8 Gy alone or with anti-CD8 mAb administration prior to radiotherapy.

As expected, radiotherapy reduced tumor volume Fig. This radiation dose induced tumor-cell ferroptosis Supplementary Fig. S1B and S1H. We found that exposure to a low dose of T-cell supernatant had minimal effect on B16F10 cell survival.

Interestingly, T-cell supernatant synergized with radiotherapy to limit B16F10 clonogenic cell survival Fig. Furthermore, T-cell supernatant and radiotherapy each independently enhanced lipid peroxidation in B16F10 cells, and their combination further augmented lipid peroxidation Fig.

Blockade of IFNγ signaling decreased T-cell supernatant-induced tumor STAT1 phosphorylation Supplementary Fig. S2A and diminished tumor lipid ROS production following combination treatment with T-cell supernatant and radiotherapy Fig.

Data are representative of at least two independent experiments A—J. To assess a direct link between IFNγ signaling and radiotherapy efficacy, we treated ID8 cells with IFNγ and radiotherapy and examined clonogenic cell survival. We observed that IFNγ and radiotherapy synergistically reduced clonogenic survival Fig.

We also observed diminished clonogenic cell survival following IFNγ and radiation treatment in B16F10 cells Supplementary Fig. IFNγ and radiotherapy synergistically increased lipid ROS levels in ID8 cells Fig.

S2C , and HT cells Supplementary Fig. We observed a synergistic increase in cell death following combination treatment with radiotherapy and IFNγ Fig. To implicate ferroptosis in cell death induced by the combination treatment of radiotherapy and IFNγ, we conducted clonogenic survival experiments in HT cells with IFNγ, radiotherapy, and liproxstatin We observed that liproxstatin-1 administration blocked the synergistic reduction of cell survival observed upon treatment with IFNγ and radiotherapy Fig.

The data indicate that the combination of IFNγ and radiotherapy regulates tumor-cell ferroptosis in vitro. Although low doses of IFNγ and radiotherapy slightly diminished tumor growth, the combination potently promoted tumor regression Fig. To assess whether this is associated with tumor ferroptosis, we performed IHC staining in tumor tissues and quantified 4-hydoxynoneal 4-HNE , a lipid peroxidation by-product Indeed, combination treatment with IFNγ and radiotherapy significantly increased tumoral 4-HNE levels Fig.

To functionally demonstrate that ferroptosis contributed to synergistic tumor regression in vivo , we inoculated HT cells into NSG mice and treated mice with IFNγ plus radiotherapy, liproxstatin-1, or all three agents. As expected, IFNγ plus radiotherapy induced tumor regression.

Importantly, this effect was abrogated by simultaneous treatment with liproxstatin-1 Fig. We next sought to define the molecular mechanism by which IFNγ sensitizes to radiotherapy and promotes tumor ferroptosis. We first performed unbiased RNA sequencing in HT cells following treatment with IFNγ, radiotherapy, or the combination.

Consistent with published results, interferon response genes, including CXCL9 and IRF1 , were upregulated strongly by IFNγ treatment Fig. As expected, radiotherapy induced an ATM-dependent transcriptional program Other ferroptosis regulators including GPX4, ACSL4, LPCAT3, and lipoxygenases Supplementary Fig.

S3A and S3B were not altered in the same manner by dual treatment with radiotherapy plus IFNγ treatment. We next validated this finding.

Treatment with IFNγ and radiotherapy individually reduced SLC7A11 transcripts Fig. Dual treatment resulted in a dramatic reduction of SLC7A11 in HT cells Fig. We also performed IHC staining for SLC7A11 in HT tumor tissues Fig.

To determine the functional relevance of SLC7A11 reduction, we examined radiolabeled cystine uptake in HT cells. Although both radiation and IFNγ each reduced cystine uptake, dual treatments resulted in a maximal decrease in cystine uptake in HT cells Fig.

Cystine is a precursor for glutathione production, which is a required cofactor for GPX4 activity To confirm that diminished cystine import altered cellular antioxidant stores, we quantified glutathione level in HT cells following treatment with radiation, IFNγ, and the combination.

We observed that the combination treatment significantly decreased glutathione level Fig. Thus, radiotherapy and IFNγ synergistically downregulated SLC7A11 expression in tumor cells, resulting in diminished cystine transport and depletion of antioxidant stores in tumor cells to induce ferroptosis.

Radiotherapy and IFNγ synergistically suppress system SLC7A D, Representative SLC7A11 IHC in HT tumors treated as in Fig.

J, IHC quantification of SLC7A11 in HT tumors of the indicated genotypes following treatment with RT 8 Gy, single fraction, arrow in vivo.

M, Clonogenic survival of HT cells at indicated RT dose following shRNA targeting SLC7A11 sh SLC7A11 and liproxstatin-1 treatment in vitro.

N and O, SLC7A11 knockout B16F10 cells tumor growth N and tumor lipid ROS levels O following irradiation 8 Gy, single fraction, arrow in vivo.

Data are representative of at least two independent experiments A—Q. Next, we explored how radiotherapy downregulates SLC7A11 in tumor cells. Cells respond to radiotherapy through defined signaling cascades, including ATM activation 6, 25, ATM has recently been shown to promote ferroptosis We wondered whether ATM activation is involved in radiation-induced ferroptosis by regulating SLC7A To test this possibility, we treated HT cells with KU 30 , an ATM inhibitor.

ATM inhibition abrogated radiotherapy-mediated downregulation of SLC7A11 transcription Supplementary Fig. S3C and protein levels Fig. Indeed, treatment with KU diminished radiotherapy-induced lipid peroxidation in HT cells Fig.

Further, siRNA targeting ATM prevented downregulation of SLC7A11 transcription Supplementary Fig. S3D and protein Fig. To confirm this mechanism in vivo , we treated WT and sh ATM HT tumors with radiotherapy Supplementary Fig. We observed that WT but not ATM-silenced tumors showed downregulation of SLC7A11 expression Fig.

Further, WT tumors showed increased 4-HNE lipid peroxidation in vivo following radiotherapy as compared with ATM-silenced tumors Supplementary Fig. S3G and S3H. Therefore, radiotherapy transcriptionally represses SLC7A11 expression via ATM to promote tumoral ferroptosis. We have previously reported that IFNγ negatively regulates SLC7A11 expression in a JAK—STAT1-dependent manner 16, To validate this finding, we silenced STAT1 with siRNAs si STAT1 in HT cells and found that IFNγ downregulated SLC7A11 in a STAT1-dependent manner Supplementary Fig.

S3I; Fig. Interestingly, IFNγ efficiently and comparably reduced SLC7A11 expression in both ATM-proficient and ATM-deficient HT cells Supplementary Fig.

Conversely, regardless of STAT1 expression, radiotherapy efficiently reduced SLC7A11 expression in HT cells Supplementary Fig. Thus, radiotherapy and IFNγ synergistically repress tumor SLC7A11 through ATM and STAT1 signaling, respectively.

To confirm this mechanism in vivo , we treated WT and STAT1 knockout HT tumors with IFNγ Supplementary Fig. We observed that WT but not STAT1-deficient tumors showed SLC7A11 downregulation following IFNγ treatment Fig. We also observed that WT tumors showed increased 4-HNE lipid oxidation following IFNγ treatment as compared with STAT1 knockout tumors Supplementary Fig.

S3N and S3O. To functionally connect SLC7A11 to tumor ferroptosis induced by radiotherapy, we used small hairpin RNAs against SLC7A11 sh SLC7A11 to knock down SLC7A11 in HT cells Supplementary Fig. We found that knockdown of SLC7A11 promoted radiotherapy-induced cell death in HT cells Fig. This effect was rescued by liproxstatin-1 in HT cells Fig.

S3R and S3S. In line with previous reports 12, 26 , we found SLC7Adeficient cells underwent spontaneous cell death, which can be rescued by liproxstatin-1 or 2-mercaptoethanol Supplementary Fig. Radiotherapy efficacy was enhanced in SLC7Adeficient cells in vivo Fig.

Examination of lipid peroxidation showed that SLC7Adeficient tumors had increased baseline levels of lipid ROS Fig. Moreover, radiotherapy-induced alterations in lipid peroxidation were further increased by loss of SLC7A11 Fig. To confirm the importance of SLC7A11 to survival following combination IFNγ and radiotherapy, we overexpressed SLC7A11 in HT cells Supplementary Fig.

We observed that overexpression significantly increased clonogenic survival following treatment with radiotherapy and IFNγ Fig. To further confirm the importance of SLC7A11 expression, we performed rescue experiments in which SLC7A11 was expressed in SLC7Adeficient cells Supplementary Fig.

In addition, we observed that overexpression of SLC7A11 reversed the sensitivity of SLC7Adeficient tumors to radiotherapy in vivo Fig. Collectively, these results suggest that radiotherapy-activated ATM and IFNγ-induced STAT1 signaling jointly target SLC7A11 to modulate cystine uptake and promote tumor lipid peroxidation and ferroptosis.

Immunotherapy augments radiotherapy efficacy in vivo , but the mechanisms through which immune checkpoint blockade enhances tumor-cell death in irradiated lesions are not well defined 4, 32, We hypothesized that radiotherapy and immunotherapy would synergize via ferroptosis in vivo to improve the efficacy of both therapies.

As previously reported, anti-CTLA4 monotherapy was minimally efficacious in B16F10 tumors However, we found that it potentiated radiotherapy efficacy in irradiated tumors Fig.

Consistent with our observations Fig. Interestingly, the combination treatment with anti-CTLA4 and radiotherapy further enhanced tumoral lipid peroxidation Fig. S4A and S4B. S4D and S4E. S4F and S4G. Interestingly, liproxstatin-1 diminished the combined therapeutic efficacy of anti-CTLA4 and radiotherapy as shown by tumor volume Fig.

Anti-CTLA4 and radiotherapy synergistically enhanced tumor control in vivo Fig. Again, we observed increased lipid peroxidation following radiotherapy, and anti-CTLA4 mediated enhancement of lipid peroxidation following dual treatment with radiotherapy and anti-CTLA4 Fig.

To explore if other forms of immune checkpoint blockade also induced lipid peroxidation, we established B16F10 tumors in mice and treated the tumors with radiotherapy, anti—PD-L1, or both. Although anti—PD-L1 or radiotherapy alone controlled tumor growth, the dual treatment was more effective in reducing tumor growth than any single therapy Fig.

We also detected the highest levels of tumor lipid peroxidation following combination therapy Fig. Radiotherapy and immunotherapy synergistically induce tumoral ferroptosis. M, RSL-3—resistant or parental ovalbumin-expressing B16F10 tumor growth following adoptive transfer of activated OT-I T cells in vivo arrowhead.

Data are representative of at least two independent experiments A—O. SLC7Adeficient cells were more sensitive to radiotherapy in vitro and in vivo because of their increased ferroptosis susceptibility Fig.

We hypothesized that ferroptosis-sensitive tumors may benefit more from immune checkpoint blockade. We detected higher tumoral lipid ROS in SLC7Adeficient tumor cells than in WT tumor cells regardless of anti—PD-L1 therapy Supplementary Fig. Furthermore, SLC7Adeficient tumors were more sensitive to anti—PD-L1 therapy, as shown by increased tumor C11BODIPY fluorescence and reduced tumor volume Supplementary Fig.

S4J compared with WT tumors. To additionally support that ferroptosis is a mechanism of synergy between immunotherapy and radiotherapy, we inoculated parental or RSL-3 ferroptosis-resistant cells into mice and treated mice with anti—PD-L1 and radiotherapy.

We observed that in contrast to the parental cells, RSL-3—resistant B16F10 cells were largely insensitive to combined treatment with anti—PD-L1 and radiotherapy Fig. Moreover, this insensitivity was accompanied by diminished C11BODIPY in tumor cells Fig. To define the mechanisms contributing to resistance to therapy, we established ovalbumin expressing parental or RSL-3 ferroptosis-resistant B16F10 tumors and treated the mice with OT-1 transgenic T cells.

We observed that OT-1 therapy controlled WT but not RSL-3—resistant tumors. This suggests that ferroptosis-resistant tumors are resistant to T-cell effector function Fig. To examine whether SLC7A11 modulated the induction of durable immune responses, we established parental or SLC7Adeficient B16F10 tumors and treated them with radiotherapy and anti—PD-L1 therapy.

To test whether these mice developed immunologic memory, WT B16F10 tumors were reinoculated in cured mice. We noted that tumors established in control but not previously cured mice, suggestive of the establishment of T-cell memory Fig. Collectively, these data suggest that immunotherapy synergizes with radiotherapy to induce tumor ferroptosis and establish T-cell immunity.

We have now demonstrated that effector T cells and radiotherapy interact through ferroptosis to promote tumor clearance. Radiotherapy classically promotes tumor-cell death primarily through the induction of double-stranded DNA breaks in tumor cells which, if unrepaired, lead to mitotic catastrophe and unregulated tumor cell death 7.

We provide the first evidence that radiation also promotes lipid peroxidation, resulting in tumoral ferroptosis. Concurrent administration of genotoxic agents to augment DNA damage has been harnessed clinically to improve radiotherapy efficacy and improve the outcomes of patients with cancer.

We have found that ferroptosis agonists can sensitize tumors to radiation both in vitro and in vivo, uncovering new therapeutic strategies to improve radiotherapy efficacy.

Overall, the dose of RS in the meal had a significant influence on ΔRQ respiratory quotient values F-test, 0. This overall effect was due to a significantly lower ΔRQ at the 5. ΔRQ was significantly lower for the 5.

These data are reflected in total macronutrient oxidation rates Figure 3 , which show a significant increase in the amount of fat oxidized at the 5. Respiratory quotient RQ; change from baseline in response to RS content of a breakfast meal.

Respiratory gas exchange measurements were conducted on 12 healthy adults using the ventilated hood method. Total fat a and carbohydrate b oxidation in response to RS content of a breakfast meal.

Macronutrient oxidation, assessed via indirect calorimetry and calculated from non-protein RQ, was measured in 12 healthy adults. Similarly, the oxidation of [ 14 C]-triolein to 14 CO 2 was different between RS doses F-test, 0.

Meal fat oxidation at the 5. Separate tests at 6 h or 24 h following the test meal gave comparable results Figure 4a. Taken together, these independent measurements of fat oxidation indirect calorimetry, oxidation of [ 14 C]-triolein to 14 CO 2 suggest that the inclusion of 5.

Unexpectedly, this effect was lost if the dose was increased to Meal fat oxidation a and storage b in response to RS content of a breakfast meal. Meal fat oxidation, assessed via measurement of 14 CO 2 in expired air, and meal fat storage in gluteal adipose tissue was measured in 12 healthy adults.

FFM, fat free mass. There was a trend for fat storage from the test meal, as assessed by incorporation of 14 C into gluteal adipose tissue, to be lower for the 5. This study demonstrated that the addition of RS to a mixed meal, balanced for total fat and fiber content, had no effect on postprandial glucose, insulin, FFA, or triglyceride excursions.

However, meals containing a moderate amount of RS caused an increase in fat oxidation as measured by both indirect calorimetry and the production of 14 CO 2 from a 14 C-triglyceride tracer.

Unexpectedly, the dose-response relationship between RS content of the diet and fat oxidation was not linear. Although this result is difficult to explain in the current context, it emphasizes the need for careful selection of RS dose in prospective feeding studies.

There was no difference in postprandial glucose Figure 1a , FFA Figure 1e , triglyceride Figure 1f , or insulin Figure 1c concentrations at any RS dose examined. However, in the current study all diets were carefully matched for total fat and fiber content.

So, the balanced conditions used in the meal tests for the study described herein, which included baked products and processed foods as part of a complete, mixed meal, balanced for total fat and fiber content, could account for the lack of difference in insulinemia and glycemia in response to increased RS content in the diet.

This increase in total and meal fat oxidation in response to the 5. Tracer data showed that the addition of 5. The increase in fat oxidation at 6 h accounted for approximately one-half of the total increase over 24 h, indicating that the increase in meal fat oxidation in response to a single meal containing 5.

Figure 3 shows that this increase in fat oxidation at the 5. The increase in fat oxidation at the 5. If decreased carbohydrate availability was responsible for the observed increase in fat oxidation, the Thus, carbohydrate availability cannot be a contributing factor to the increase in fat oxidation observed at the 5.

It is possible that this increase may be due to an increase in circulating SCFAs from the fermentation of RS reaching the large bowel. The observed increase in fat oxidation is not due to oxidation of these SCFAs per se as it was measured directly from conversion of 14 C-labeled meal fat to 14 CO 2 Figure 3a.

Such a measurement would not detect any increase in SCFA oxidation. Rather, it may be that the metabolic effects of increased SCFA production cause an increase in fat oxidation.

RS consumption has been shown to alter the acetate:butyrate:propionate ratio compared to fermentation of non-starch polysaccharides [ 29 ]. In particular, the amount of butyrate is substantially elevated in response to RS fermentation [ 30 , 31 ].

This increase in total SCFA concentration was caused by a doubling of the acetate and butyrate content changing the acetate:butyrate:propionate ratio from to in response to the low and high RS diets, respectively. In vitro data from isolated animal tissues provide convincing evidence for the role of SCFAs in carbohydrate and lipid metabolism [ 26 , 32 — 34 ].

So, it is plausible that the fermentation of RS from the 5. In this scenario, the liver, deprived of carbohydrate-derived acetyl CoA would be more reliant on fat-derived acetyl CoA as a fuel source, thereby contributing to an overall increase in fat oxidation [ 17 ]. This possibility needs to be investigated in future studies.

No difference in fat oxidation was evident between the maximal This is an unexpected result that is difficult to explain. The loss of any effect on fat oxidation when the RS dose in the meal was increased to That is, at the If this is the case, the strong physical association between RS and dietary lipid may cause excretion of lipid and therefore, less dietary fat to be available for oxidation at the Indeed, it has previously been shown that intake of high-amylose maize starch, such as that used in this study, caused an increased number of bowel actions per day [ 18 ].

RS has also been shown to decrease colonic transit time and, as more RS enters the large bowel, more starch is also excreted [ 19 , 20 ]. This indicates that, at higher levels of RS consumption, only a portion of the RS can be fermented and the remainder passes through the colon as an insoluble fiber.

Furthermore, if indeed RS at the As SCFA are hypothesized to be the cause of the observed increase in fat oxidation in response to the 5. The hypothesis that RS is acting like dietary fiber and being excreted can be tested by measuring the amount of fat excreted in the feces.

As this outcome was not predicted, fecal samples were not collected from subjects during this study. It is important to consider that it is difficult to add Therefore, this level would be difficult to attain in a free-living situation and the lower doses used in this study are more reflective of predicted levels if normal, starchy foods in the diet were to be replaced with commercially available RS products.

In addition, not all biological processes display linear dose-response curves. Metabolic processes that are non-linear functions include the level of illuminance and plasma melatonin levels [ 24 ], caffeine intake versus plasma caffeine metabolite concentrations [ 25 ], allergen exposure concentration and histamine response [ 26 ], zinc-stimulated histamine release from mast cells [ 27 ], and fructose-1,6-diphosphate metabolism in cardiomyocytes [ 28 ].

Thus, it is possible that the lack of any effect on fat oxidation at the However, more RS doses between 5. It must be noted that the calculation of oxidation of [ 14 C]-triolein via measurement of 14 CO 2 did not take into account the dilution of tracer in vivo due to the incorporation of labeled carbons into intermediates of the TCA cycle and endogenous bicarbonate pools.

Generally, an acetate correction factor is used to account for this effect. In this study, subjects consumed all four test meals under the same conditions and it was assumed that there was no difference in tracer recovery between tests.

Also, these TCA intermediate and bicarbonate pools were not pre-labeled prior to the ingestion of the label in the meal which would cause a total underestimation of total fat oxidation. Therefore, the rate of fat oxidation calculated from 14 CO 2 recovery in the breath was probably underestimated in all subjects but remains valid to compare differences between test meals.

There was a trend towards a decrease in gluteal fat storage at the 5. Again, the dose-response curve for this parameter was not linear, lending credence to the idea that the dose-response curve for fat oxidation is actually U-shaped.

Although the decrease in fat storage at the 5. However, there was high variability associated with the measure of meal fat storage indicating that more subjects may be needed to decrease the standard deviation and, hence, detect any significant meal affect.

This study is the first to identify that addition of 5. This discovery was verified using two different methods, indirect calorimetry and the oxidation of [ 14 C]-triolein to 14 CO 2 , to measure in vivo fat oxidation. This increase in fat oxidation was accompanied by a concomitant decrease in carbohydrate oxidation and fat storage, although these parameters did not reach statistical significance.

Further, the magnitude of the increase in fat oxidation indicates that this effect is biologically relevant and could be important for preventing fat accumulation in the long term by effecting total fat balance under chronic feeding conditions. This study was approved by the Colorado Multiple Institution Review Board, in compliance with the Helsinki Declaration, and full written consent was obtained from all subjects.

All female subjects were taking oral contraceptive pills or progesterone injections and were tested during the early follicular phase of the menstrual cycle.

All subjects underwent dual energy X-ray absorptiometry DEXA; Lunar Radiation Corp, Madison WI for analysis of body composition. As a group, subjects were 33 ± 5 years of age, 1.

Subjects received four meals differing only in resistant starch RS content in random order, approximately four weeks apart. All added RS was in the form of high-amylose maize starch, or RS2.

High-amylose maize starch was chosen as it has the unique property of a very high gelatinisation temperature which allows it to maintain its granular structure during and after the processing conditions used to manufacture the foods being consumed in this study [ 38 ].

All meals were matched for total dietary fiber content and liquid volume ml. Three days prior to each test day, subjects received a standardized lead-in diet, equivalent to daily energy needs as judged by indirect calorimetry and of the same macronutrient composition as the test diet with no added RS, to ensure that they were in energy balance.

All food for these three days was provided by the General Clinical Research Center GCRC on an outpatient basis. Non-caloric beverages could be consumed during the three day lead-in diet. Following an overnight fast 12 h , subjects were admitted to the GCRC and an intravenous catheter was placed for the purposes of drawing blood.

Blood samples were taken at 0, 30, 60, 90, , , , , and min following meal ingestion and analyzed for glucose, insulin, triacylglycerol TAG and free fatty acid FFA concentrations. Respiratory quotient RQ was measured at hourly intervals after ingestion of the meal via gas collection under a ventilated plexiglass hood for 15 min Sensormedics metabolic cart.

All urine produced between 0 and min was collected and analyzed for nitrogen content by the GCRC Core Laboratory to facilitate calculation of non-protein RQ. The fat tracer was fed as a triglyceride glycerol tri [1- 14 C]oleate rather than a FFA eg.

At hourly intervals following the meal, then at 8, 10, 12, 14 and 24 hours, breath samples were collected via exhalation through a tube with a one-way valve into scintillation vials containing 2 mmol benzethonium hydroxide to trap 2 mmol CO 2 , 1 ml methanol, and 1 mg phenolpthalene as a pH indicator.

Gluteal fat biopsies were collected by aspiration through a 14 g stainless steel needle at baseline and 24 h after ingestion of the test meal.

All breath and fat samples were assayed for the presence of 14 C as described below. All food was provided by the GCRC on an inpatient basis and the macronutrient content of each meal was the same as that of the test meal. Only the test breakfast contained RS during these 24 h tests, all other meals were composed of standard, commercially available products.

All glucose, FFA, and TAG assays were conducted by the GCRC Core Laboratory using an automated Cobas Mira Plus Roche Diagnostics, Basel, Switzerland. Serum insulin measurements were also performed by the GCRC Core Laboratory using a human insulin RIA kit Linco, St.

Louis, USA. After scintillant was added, all samples were kept in the dark at room temperature for 48 h before being counted to reduce chemiluminescence.

Formulae used to calculate non protein RQ and subsequent estimations of carbohydrate and fat oxidation were based on the derivations described by Jéquier et al. where vCO 2 is the rate of CO 2 production as assessed during indirect calorimetry.

t is sample time min. AUC is the incremental area under the curve. All statistical analyses were performed using the statistical analysis software SAS, version 8. All results are presented as mean ± SEM, except for subject characteristics which are described as mean ± SD. Subjects were included as random effects.

The interaction term was not significant for any of the outcomes tested so an additive model was used to test the overall effect of RS DOSE and the differences between doses. The repeated measures nature of the study design was taken into account by using the covariance structures available in SAS PROC MIXED.

For example, measurements within a subject are assumed to be more highly correlated than between subjects, and within a particular treatment, within a subject, the measurements are assumed to be more correlated.

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Metrics details. Although Calorie intake diary effects of lipi starch RS on postprandial glycemia and insulinemia have been oxidatiln studied, little is known about the impact Promoted lipid oxidation Ljpid on oxidatiob metabolism. Blood samples were taken and analyzed for glucose, insulin, triacylglycerol TAG and free fatty acid FFA concentrations. Respiratory quotient was measured hourly. RS, regardless of dose, had no effect on fasting or postprandial insulin, glucose, FFA or TAG concentration, nor on meal fat storage. However, data from indirect calorimetry and oxidation of [1- 14 C]-triolein to 14 CO 2 showed that addition of 5.

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STAGES OF LIPID OXIDATION ll AUTOXIDATION Lipid Health coaching services oxidatoin in loss of Health coaching services quality in muscle foods as it does in oxidatiln foods. Many muscle Vegan-friendly brunch spots are Enhancing immune health in prooxidant metals Promotedd can make these products very susceptible to lipid oxidation. The processing and formulation of muscle foods Promoted lipid oxidation also Primoted lipid oxidation rates Physical fitness in aging as cooking that can release protein-bound iron and heme and increase their reactivity, salting which can increase iron reactivity and particle size reduction which can increase oxygen concentrations in the muscle. Despite the much lower concentration of phospholipids than triacyclglycerols, they are the major target of lipid oxidation reactions Jane D. Love, The high susceptibility of phospholipids to oxidation is due to their higher levels of polyunsaturated fatty acids such as arachidonic and omega-3 fatty acids and their high surface area which puts them in close contact to the prooxidants in the tissue EI-Gharbawi,