Leu353Phe exchange of mouse Alox15 humanized the specificity of AA oxygenation

To prove that Leu353Phe exchange humanized the reaction specificity of mouse Alox15 [16] wildtype mouse Alox15 and its Leu353Phe mutant were recombinantly expressed in E. coli and in vitro activity assays were carried out. As indicated in Fig. 1A (upper panel) 12-HETE was identified as major conjugated diene. The chemical identity of the two major HPLC peaks was identified by co-chromatography of authentic standards and uv-spectroscopy indicating a classical conjugated diene spectrum for the major oxygenation product (inset to Fig. 1A, upper panel).

Fig. 1

Reaction specificity of wildtype and mutant recombinant mouse Alox15 with different polyenoic fatty acids. Wildtype and mutant (Leu353Phe) mouse Alox15 were expressed as N-terminal his-tag fusion proteins as described in the Additional file 1 and aliquots of the bacterial lysis supernatants were used as enzyme source. After a 5 min incubation period with different polyenoic fatty acids (100 µM) the reaction products were analyzed by RP-HPLC. A AA oxygenation products. B Triad Concept cartoon explaining AA 12-lipoxygenation by wildtype mouse Alox15 (see text for detailed explanation). C Triad Concept cartoon explaining AA 15-lipoxygenation by the Leu353Phe mutant of mouse Alox15 (see text for detailed explanation). D EPA oxygenation products, E dihomo-gamma-linoleic acid oxygenation products, F DHA oxygenation products, G alpha-linolenic acid oxygenation products, H linoleic acid oxygenation products. Insets: UV-spectra of the major conjugated dienes peak labeled by small letters. Representative chromatograms (n ≥ 4) are shown

In Fig. 1B a schematic presentation of substrate binding at the active site of wildtype mouse Alox15 is given. Substrate fatty acids slide into the catalytic center of the enzyme with its methyl end ahead and the deepness of the catalytic center is limited by the side chains of the triad amino acids [13, 14]. For mouse Alox15 Leu353 is the functionally most important triad residue [27]. Thus, arachidonic acid is aligned at the catalytic center of the enzyme in such a way that the bisallylic methylene C10 is located close to the non-heme iron, which allows hydrogen abstraction from C10. Dioxygen is subsequently introduced at C12 ([+ 2] radical rearrangement), explaining the major formation of 12-HETE. The major reaction product of the Leu353Phe mutant was 15-HETE (Fig. 1A, lower panel) and here again the uv-spectrum (inset to Fig. 1A, lower panel) was consistent with the chemical structure of the two major AA oxygenation products (12-HETE, 15-HETE). In other words, Leu353Phe mutation humanized the reaction specificity of recombinant mouse Alox15 for AA oxygenation. In Fig. 1C a schematic presentation of substrate binding at the active site of the mutant enzyme is shown. Since Phe carries a more bulky and less flexible side chain than Leu353 the substrate binding pocket has a reduced volume so that AA cannot penetrate as deeply into the cavity. In this enzyme–substrate complex hydrogen abstraction from the bisallylic methylene C10 is sterically hindered. Instead, the bisallylic methylene C13 is now located close to the non-heme iron and thus, hydrogen is removed from this carbon atom. Consequently, oxygen may be introduced at C15 ([+ 2] radical rearrangement), which explains the major formation of 15-HETE by the Leu353Phe Alox15 mutant. In summary, Leu353Phe exchange humanized the reaction specificity of recombinant mouse Alox15 for AA oxygenation and this functional alteration can be explained by the Triad Concept [13, 14].

Reaction specificities of mouse Alox15 with other fatty acids as substrate are also humanized by Leu353Phe exchange

To interpret possible differences in the plasma oxylipidomes of Alox15-KI mice and wildtype controls, we next characterized the reaction specificities of recombinant wildtype Alox15 and its Leu353Phe mutant with other PUFAs. For the wildtype enzyme such data have previously been reported [28] but for the Leu353Phe mutant corresponding results have not been published.

5,8,11,14,17-eicosapentaenoic acid (EPA) is an AA derivative that carries an additional double bond at the omega-3 position. In theory, this omega-3 polyunsaturated fatty acid (PUFA) should be oxygenated by wildtype mouse Alox15 dominantly to 12-HEPE. When we tested this prediction experimentally, we found that wildtype mouse Alox15 oxygenated EPA mainly to 15-HEPE (70%) and 12-HEPE was only formed as minor (30%) side product (Fig. 1D, upper trace). Here again, the uv-spectrum of the major oxygenation product indicated a conjugated diene chromophore (inset to Fig. 1B, upper trace). These data are consistent with the outcome of previous experiments analyzing the reaction specificity of wildtype mouse Alox15 using EPA as oxygenation substrate [28]. For the Leu353Phe mutant (Fig. 1D, lower trace) almost exclusive formation of 15-HEPE was analyzed. The observed differences in the reaction specificity of EPA oxygenation between wildtype mouse Alox15 and its Leu353Phe mutant (elevated relative share of 15-HEPE) were predicted on the basis of the Triad Concept.

Dihomo-gamma-linolenic acid (DGL) is an AA derivative, which lacks the C5=C6 double bond. This fatty acid is oxygenated by wildtype mouse Alox15 to a 4:6 mixture of 15-HETrE and 12-HETrE (Fig. 1E, upper trace). When the Leu353Phe mutant was used for in vitro activity assays (Fig. 1E, lower trace), we also observed the formation of these oxygenation products but here the 15-HETrE / 12-HETrE ratio was about 8:2. The observed quantitative differences in the product pattern of wildtype and mutant mouse Alox15 (elevated relative share of 15-HETrE by the mutant enzyme) were also predicted on the basis of the Triad Concept.

4,7,10,13,16,19-Docosahexaenoic acid (DHA) is oxygenated by wildtype mouse Alox15 mainly to 14-HDHA [28] but under our experimental conditions we also observed significant amounts (30%) of 17-HDHA as DHA oxygenation product (Fig. 1F, upper trace). Both products are characterized by the canonical conjugated diene chromophore and their chemical identity was confirmed by co-chromatography with authentic standards. Leu353Phe exchange altered the reaction specificity in favor of 17-HDHA formation (Fig. 1F, lower trace) and this alteration was predicted on the basis of the Triad Concept.

Linoleic acid (LA) and alpha-linolenic acid (ALA) are C18-PUFAs, which lack n-11 bisallylic methylenes and thus, n-9 oxygenation (formation of 10-HODE from LA and 10-HOTrE from ALA) is not possible. These fatty acids are almost exclusively oxygenated by wildtype and mutant mouse Alox15 to the n-6 oxygenated derivatives (13-HOTrE, Fig. 1G, upper trace; 13-HODE, Fig. 1H, upper trace). For these PUFAs no alterations in the reaction specificity were induced by Leu353Phe exchange (Figs. 1G, H, lower traces).

Peritoneal lavage cells and bone marrow cells are major sources of Alox15 expression

Human ALOX15 is constitutively expressed in large quantities in reticulocytes, airway epithelium and eosinophilic granulocytes [29]. Peripheral monocytes hardly express the enzyme but cell stimulation with interleukin-4 [30] or interleukin-13 [31] upregulated ALOX15 expression. In pigs, the enzyme is present in large amounts in peripheral leukocytes [32] but in mice the enzyme is most abundantly expressed in peritoneal lavage cells [10, 11]. To select suitable cell types for ex-vivo activity assays we first profiled Alox15 mRNA expression in different mouse tissues by qRT-PCR. From Fig. 2A it can be seen, that hardly any Alox15 mRNA was detected in RNA extracts of brain, lung, muscle, stomach, heart and skin. Low Alox15 mRNA expression levels (< 300 Alox15 mRNA copies per 106 Gapdh mRNA copies) were detected in liver, kidney, spleen, colon and testis but high Alox15 mRNA concentrations (> 104Alox15 mRNA copies per 106 Gapdh mRNA copies) were observed in bone marrow cells. The richest Alox15 mRNA source were peritoneal lavage cells. In these cells, the Alox15 mRNA expression levels did even exceed the Gapdh expression by a factor of 2. These data prompted us to employ peritoneal lavage cells as well as bone marrow cells for later ex-vivo Alox15 activity assays.

Fig. 2

Expression of Alox15 mRNA in different mouse cells and knock-in strategy for generation of humanized Alox15 knock-in mice. A Total RNA was extracted from different cells and tissues of wildtype mice, the RNA was reversely transcribed and the Alox15 mRNA steady state concentrations were quantified by qRT-PCR (see Additional file 1: Methodological supplement). Two independent measurements (n = 2) were carried out for each cDNA sample. B Crisp/Cas9 strategy for generation Alox15-KI mice. C Quantitative RT-PCR of Alox-isoforms in peritoneal lavage cells prepared from Alox15-KI mice and outbred wildtype control animals. Expression levels of Alox15 and Alox5 mRNA in wildtype cells were separately set 100%. Experimental details are given in the Additional file 1: Methodological supplement

Generation of Alox15 knock-in mice expressing the Leu353Phe Alox15 point mutant

To generate heterozygous knock-in mice that express the Alox15 Leu353Phe point mutant instead of the wildtype enzyme we employed the Crispr/Cas9 in vivo mutagenesis strategy as described in the Additional file 1: Methodological supplement. We first designed a gRNA targeting vector and a donor oligonucleotide that involved the target sequence flanked by homologous sequences at the 5′- and the 3′-ends. The Leu353Phe (TTA to TTC) mutation site in the donor oligonucleotide was introduced into exon 8 of the Alox15 gene by homology-directed repair mechanisms. To prevent methodological artifacts an additional silent mutation (CAG to CAA) was introduced (Fig. 2B). Starting from heterozygous founder animals, a colony of homozygous knock-in allele carriers and a colony of outbred wildtype control animals were established.

Expression of Alox15 in peritoneal lavage cells is not altered by Leu353Phe in vivo mutagenesis

To explore whether the Leu353Phe in vivo exchange impacts the expression of different Alox-isoforms qRT-PCR was carried out for all seven mouse Alox-isoforms (Alox15, Alox15b, Alox12, Alox12b, Aloxe3, Aloxe12, Alox5) in peritoneal lavage cells. As expected from the results shown in Fig. 2A Alox15 mRNA is present at high quantities in these cells but we did not observe significant differences when the two genotypes were compared (Fig. 2C, left panel). In contrast, for Alox5 we found that expression of this enzyme was two fold lower in humanized Alox15-KI mice than in the outbred wildtype control peritoneal lavage cells (Fig. 2C, right panel). It should be stressed at this point that expression of Alox5 in peritoneal lavage cells was more than ten-fold lower than that of Alox15. Expression levels of other Alox-isoforms (Alox15b, Alox12, Alox12b, Aloxe3, Aloxe12) were below the detection limits of our assay system (Fig. 2C, middle panel). Taken together, these RT-PCR data indicate that Alox15 and Alox5 are the two major Alox-isoforms expressed in mouse peritoneal lavage cells and that wildtype and mutant Alox15 are expressed at similar levels. In other words, Leu535Phe exchange in the Alox15 gene did not alter the expression efficiency of this enzyme but reduced the expression level of the pro-inflammatory Alox5 in peritoneal lavage cells.

In vivo Leu353Phe exchange humanized the functional properties of mouse Alox15

To test whether Leu353Phe in vivo exchange converted the reaction specificity of mouse Alox15 from major AA 12-lipoxygenation by the wildtype enzyme to dominant AA 15-lipoxygenation by the Leu353Phe mutant we again prepared peritoneal lavage cells from Alox15-KI mice and outbred wildtype control animals and carried out ex vivo activity assays. For these experiments, we selected three individuals from our Alox15-KI mouse colony, three representatives of outbred wildtype controls and three heterozygous allele carriers and genotyped these individuals (ear biopsies). Then we prepared peritoneal lavage cells and performed ex vivo activity assays. From Fig. 3A it can be seen that the PCR fragment obtained by genomic PCR of outbred wildtype control mice involved the TTA triplet, which encodes for Leu353. In contrast, the heterozygous mice involved both the TTA (wildtype, Leu) and TTC (mutant, Phe) triplets (Fig. 3B). For the homozygous mutant mice only the TTC triplet (Phe) was observed (Fig. 3C). These data defined the corresponding animals as homozygous mutant allele carriers.

Fig. 3

Genomic sequencing and ex vivo Alox activity assays. A–C The mutated region of the Alox15 gene was amplified by PCR and the amplification products were sequenced (for methodological details see Additional file 1). D–F Ex vivo Alox15 activity assays (see Additional file 1) using peritoneal lavage cells as enzyme source. Representative partial RP-HPLC chromatograms are shown. G, I Ex vivo Alox15 activity assays (see Additional file 1) using bone marrow cells as enzyme source. H Summary and statistical evaluation (means ± SD, n = 4) of the Alox15 activity assays. J Ex vivo Alox5 activity assays (n = 4) using whole blood as enzyme source. Leukotriene B4 formation was quantified as readout parameter

Next, ex vivo activity assays were carried out with peritoneal lavage cells and with bone marrow cells prepared from the selected individuals. As indicated in Fig. 3D peritoneal lavage cells of wildtype mice converted AA predominantly to 12-HETE. Smaller amounts 15-HETE were also detected. These data are consistent with previous analytical data published for the AA oxygenation products of mouse peritoneal lavage cells [10, 11]. When peritoneal lavage cells of heterozygous allele carriers were employed a 1:1 mixture of 12-HETE and 15-HETE was formed (Fig. 3E). Such product pattern was expected when both, the wildtype (TTA, Leu) and the mutant (TTC, Phe) alleles are co-dominantly expressed in these cells. When homozygous Alox15-KI mice were used for the ex-vivo activity assays 15-HETE was analyzed as major oxygenation product (Fig. 3F). These activity data do not only show that Leu535Phe exchange humanized the reaction specificity of mouse Alox15 but they also confirm our qRT-PCR data (Fig. 2C) suggesting that there is hardly any difference in the expression levels of the wildtype and the mutant Alox15 alleles.

Taken together, these ex-vivo activity data indicated that our in-vivo mutagenesis strategy humanized the functional properties of mouse Alox15 when AA is used as oxygenation substrate. To confirm this conclusion for other cell types, we performed similar activity assays with bone marrow cells. As for wildtype peritoneal lavage cells we identified 12-HETE as dominant AA oxygenation product when bone marrow cells of outbred wildtype mice were used as enzyme source (Fig. 3G). Here again, small amounts of 15-HETE were also observed and the 12-HETE/15-HETE ratio was about 9:1 (Fig. 3H). For homozygous Alox15-KI mice, we quantified a 12-HETE/15-HETE ratio of about 2:1 (Fig. 3I). The most plausible explanation for this product pattern is that in addition to Alox15 other Alox isoforms such as Alox12 may contribute to 12-HETE formation in bone marrow cells. In fact, qRT-PCR studies indicated that in addition to Alox15 and Alox5 mRNA, Alox12 mRNA was found at high levels in bone marrow cells.

Leu353Phe exchange in the Alox15 gene did hardly impact the Alox5 pathway

In mouse blood Alox15 and Alox5 are co-expressed in leukocytes [8, 33] and thus, alterations of the Alox15 properties might impact the functionality of the Alox5 pathway. The two enzymes compete for the same substrate and the reactions products of the modified Alox15 pathway may alter the catalytic activity of Alox5. To test whether systemic humanization of the reaction specificity of Alox15 might alter the Alox5 pathway we quantified leukotriene B4 (LTB4) formation in whole blood following stimulation of the blood cells with calcium ionophore 23187. From Fig. 3J it can be seen that only small amounts of the Alox5 product LTB4 were formed from endogenous substrate when whole blood was incubated in the absence of calcium ionophore. In contrast, after A23187 stimulation large amounts of LTB4 were detected. A similar situation was observed when blood of Alox15-KI mice was used. Here again, small amounts of LTB4 were analyzed when blood was incubated in the absence of A23187. When A23187 was present, large amounts of the Alox5 product LTB4 were detected. However, there was no significant difference when LTB4 formation of the two genotypes was compared. Thus, humanization of the reaction specificity of Alox15 did not alter the Alox5 pathway of whole blood cells.

Reproduction characteristics of Alox15-KI mice

Alox15 has previously been implicated in spermatogenesis [34] and thus, we tested the reproduction kinetics of Alox15-KI mice. Here we found that these animals reproduce normally. We compared litter size (pups per litter), frequency of pregnancy (litters per female x month), number of pups per female and month and the gender ratio of the newborns of Alox15-KI mice and wildtype control animals (Additional file 1: Fig. S1), but did not observe significant differences between the two genotypes. Thus, on the basis of this data it can be concluded that Alox15-KI mice are fully fertile and do not show major defects during embryogenesis.

Body weight development

Comparing the bodyweight kinetics of male and female Alox15-KI mice with those of outbred wildtype controls we did not observe distinct growth behaviors of the two genotypes (Additional file 1: Fig. S2). The growth curves were largely superimposable for either sex over the entire experimental period and no significant differences were observed. Thus, functional humanization of mouse Alox15 does not significantly impact post-natal development of the genetically modified individuals.

Blood plasma oxylipidome profiles revealed differences in the pattern of oxylipins between Alox15-KI mice and outbred wildtype controls

To test whether Leu353Phe exchange in mouse Alox15 might have changed the plasma oxylipin patterns we profiled more than 40 different oxylipins by LC–MS/MS (see Additional file 1: Methodological supplement including Tables S3–S6). Some previously characterized oxylipins such as a number of maresin, resolvin and protectin isomers were below the detection limits of our analytical systems but for other oxylipins, we obtained reliable analytical data (Additional file 1: Figs. S3–S7).

Comparing the whole amounts of oxylipins between the two genotypes, significantly more oxygenated PUFA derivatives were detected in Alox15-KI mice (Fig. 4A). Especially the levels of 12-HETE (Fig. 4C), 12-HETrE (Fig. 4E), 12- and 15-HEPE (Fig. 4F, G), 17- and 14-HDHA (Fig. 4H, I), as well as 13-HODE (Fig. 4L) were elevated in the plasma of Alox15-KI mice. Although for some of these metabolites non-significant differences were observed between the two genotypes for the sum of them the difference was statistically significant (Fig. 4A).

Fig. 4

LC–MS based analysis of plasma oxylipidomes. Selected oxylipins were quantified (LC–MS/MS) in the blood plasma (see Additional file 1: for methodological details) of male Alox15-KI mice and of outbred wildtype controls (n = 5). Quantification of other oxylipins is given in Additional file 1: Figs. S3–S7. For statistical evaluation (n.s., not significant, *p < 0.05) the Mann–Whitney U-test was used

The dominant AA oxygenation product of mouse Alox15 is 12S-HETE, whereas the humanized enzyme produces mainly 15-HETE (Fig. 1A). As functional consequence of the Leu353Phe exchange we expect elevated plasma levels of 15-HETE but reduced plasma concentrations of 12-HETE in Alox15-KI mice. Although we observed significantly elevated 15-HETE concentrations in the plasma of Alox15-KI mice, the 12-HETE levels were not significantly different (Fig. 4B, C). These data suggest that the catalytic activity of Alox15 may not strongly contribute to the 12-HETE plasma levels in wildtype mice but that humanization of the reaction specificity of the enzyme is mirrored in the plasma by elevated 15-HETE concentrations.

It should be stressed at this point that the plasma 12-HETE concentrations are more than two orders of magnitude higher than the 15-HETE levels. If one assumes that half of the plasma 15-HETE originates from the Alox15 pathway, humanization of the reaction specificity of this enzyme would not significantly lower the blood plasma 12-HETE levels. Thus, the finding that we did not observe a significant reduction in the plasma 12-HETE concentrations is consistent with the obtained experimental data. However, it remains to be explored in the future which metabolic processes might contribute to the relatively high plasma 12-HETE levels (see Discussion).

For 12- and 15-HETrE we observed a similar situation (Fig. 4D, E). The plasma levels of 12-HETrE were almost two orders of magnitude higher than those of 15-HETrE and mouse Alox15 may not significantly contribute to the in vivo biosynthesis of 12-HETrE. Humanization of the reaction specificity of Alox15 induced elevated plasma concentrations of 15-HETrE and although the increase did not reach the level of statistical significance the humanized Alox15 might contribute to this increase.

12-HEPE and 12-HETrE were present at similar concentrations in the blood plasma of wildtype mice and humanization of the reaction specificity of Alox15 strongly (3–fourfold) elevated the 12-HEPE concentrations (Fig. 4E, G). This increase must be an indirect effect since humanization of the reaction specificity of the enzyme impaired its 12-HEPE synthase activity (Fig. 1B). The 15-HEPE plasma levels were strongly elevated (more than one order of magnitude) in Alox15-KI mice (Fig. 4F) and these data are consistent with the results obtained for the recombinant enzyme (Fig. 1B).

For the DHA metabolites (17-HDHA, 14-HDHA) we observed similar differences as for the EPA metabolites. Here again, the 14-HDHA plasma concentrations were higher in Alox15-KI mice (Fig. 4I) but the difference was statistically not significant. The plasma levels of 17-HDHA (Fig. 4H) were tenfold higher in Alox15-KI mice and this result may be related to the elevated 17-HDHA oxygenase activity of the humanized enzyme (Fig. 1D).

Neuroprotectins form a family of dihydroxylated derivatives of DHA, which exert potent anti-inflammatory, anti-apoptotic and neuroprotective activities [35, 36]. When we quantified the plasma oxylipins of Alox15-KI mice and outbred wildtype controls, we detected significant amounts of NPD-x in wildtype mice (Fig. 4J). Interestingly, much higher quantities of these metabolites were found in the plasma of Alox15-KI mice (Fig. 4J). If these compounds exhibit anti-inflammatory properties in vivo, Alox15-KI mice might be protected from inflammation in animal disease models. This conclusion can be tested in future research using the Alox15-KI mice described in this study.

The plasma levels of 13-HOTrE and 13-HODE (Fig. 4K, L) were significantly elevated in Alox15-KI mice but it remains unclear whether the humanized Alox15 might contribute to this effect.

In summary, our lipidomic data suggest that Leu353Phe exchange in mouse Alox15 induces alterations in the plasma oxylipin concentrations. A number of these differences might be a direct consequence of the functional alterations induced by this in vivo mutagenesis (elevated levels of 15-HETE, 15-HEPE and 17-HDHA in Alox15-KI mice) but other effects (elevated levels of 12-HEPE in Alox15-KI mice) are more indirect.

Hematological parameters of Alox15-KI mice

Alox15 has previously been implicated in erythropoiesis [19, 20] and detailed characterization of the hematological parameters of Alox15−/− mice recently suggested that these animals carry a slightly dysfunctional erythropoietic system [22]. In fact, erythrocyte counts, hematocrit and hemoglobin concentrations of aged male Alox15−/− mice were significantly lower than the corresponding values of outbred wildtype controls. Moreover, the osmotic stability and the ex vivo life span of Alox15−/− erythrocytes were impaired [22]. Overexpression of human ALOX15 rescued this defective erythropoietic phenotype [22] and this data convincingly demonstrate that Alox15−/− mice carry a slightly defective erythropoietic system. To characterize the erythropoietic systems of the two genotypes, selected blood parameters were determined in young (10–20 weeks) and aged mice (70–75 weeks) of either sex. Here we found that in aged male Alox15-KI mice the erythrocyte count (erys), the hematocrit (HK) and the hemoglobin (Hb were significantly (p < 0.01) were modified (Fig. 5A–C, right pairs of columns). For young males (Fig. 5A–C, left pair of columns) as well as for female individuals of either age category (Fig. 5D, E) such differences were not observed. For other hematological parameters we did not observe significant differences between Alox15-KI mice and corresponding wildtype controls (Additional file 1: Figs. S8, S9).

Fig. 5

The erythropoietic system of Alox15-KI mice is compromised. A–F Erythrocyte parameters of Alox15-KI mice and outbred wildtype controls were determined in two age categories (young mice, 10–20 weeks; aged mice, 70–75 weeks, n = 6 for each age-group, Mann–Whitney U-test. ns, not significant, *p < 0.05, **p < 0.01.). Additional hematological parameters are given in Additional file 1: Fig. S6, S7. G–J Osmotic stability of erythrocytes: the osmotic stability of red blood cells in the two age categories (young mice, 10–20 weeks; aged mice, 70–75 weeks, n > 3 for each age-group) were determined as described in the Additional file 1. The degree of hemolysis was calculated at each NaCl concentration. Hemolysis curves were compared with two-way ANOVA

Osmotic stability of red blood cells

Because of their high hemoglobin concentrations and because of the water permeability of their plasma membrane erythrocytes take up water when suspended in hypotonic solutions. Since this water uptake is quite excessive, the cells swell, hemolyze and release intracellular hemoglobin into the extracellular fluid. To compare the osmotic stability of peripheral erythrocytes prepared from Alox15-KI mice and outbred wildtype controls we prepared the peripheral red blood cells from corresponding individuals of either genotype and incubated them in phosphate buffer (pH 7.4) containing different concentrations of NaCl. The osmotic stability of the cells in the presence of different salt concentrations depends on the integrity of the plasma membrane and can be quantified by the degree of hemolysis [37]. From Fig. 5G, I it can be seen that erythrocytes prepared from young male and female individuals show almost superimposable hemolysis curves and thus, there was no difference in the osmotic stability of the red blood cells of the two genotypes. However, for aged individuals the hemolysis curves of the wildtype mice were shifted to the right (Fig. 5H, J) and these data indicate that at a given NaCl concentration the degree of hemolysis for wildtype erythrocytes was significantly (p < 0.0001 for males, p = 0.0082 for females) higher than that for Alox15-KI red blood cells. From these data, it can be concluded that erythrocytes of aged wildtype mice are more susceptible for osmotic challenge when compared with Alox15-KI red blood cells.