NADPH-induced Lipid Peroxidation Research Articles

Lipid Peroxidation Potential and Antioxidants in the Heart Tissue of .BETA.-Alanine- or Taurine-Treated Old Rats

The aim of this study was to investigate the effect of the changes of taurine levels in the hearts of old rats on endogenous malondialdehyde (MDA) and diene conjugate (DC) levels and ascorbic acid (AA)- and NADPH-induced lipid peroxidation as well as non-enzymatic (glutathione, vitamin E and vitamin C) and enzymatic antioxidants (superoxide dismutase, glutathione peroxidase and glutathione transferase). Two groups of old (22 mo) rats were treated with beta-alanine (3%, w/v; in drinking water), a taurine depleting agent, or taurine (2% w/v; in drinking water) for 6 wk. Significant decreases were observed in taurine contents of hearts in old rats as compared to young (5 mo) rats. We found that MDA and DC levels and AA- and NADPH-induced lipid peroxidation increased, but non-enzymatic and enzymatic antioxidants did not alter in heart homogenates of aged rats. beta-Alanine administration resulted in significant decreases in heart taurine levels of old rats. This treatment did not cause further increases in MDA or DC levels or changes in antioxidants. However, AA- and NADPH-induced lipid peroxidation was higher than that of old rats. Taurine treatment caused significant increases in heart taurine levels of old rats. This treatment was found to decrease endogenous MDA and DC levels without affecting the antioxidant system in the heart homogenates of aged rats. AA- and NADPH-induced lipid peroxidation was also reduced in old rats when given taurine, although not statistically significantly. Our results indicate that the changes in heart taurine levels may influence the susceptibility of heart tissue to lipid peroxidation in aged rats and that taurine supplementation has protective effects on age-dependent oxidative stress in heart tissue.

Effect of Dietary High-Oleic-Acid Oils that are Rich in Antioxidants on Microsomal Lipid Peroxidation in Rats

In contrast to other metabolic functions, the role of dietary antioxidants and oil on microsomal lipid oxidation has been less extensively studied. This study examines ascorbate-Fe(2+) and NADPH-induced lipid peroxidation of hepatic microsomes of rats that were fed for three weeks high-oleic-acid oils (high-oleic sunflower oil, HOSO; olive oil, OO; or olive pomace oil, OPO) containing different concentrations of the antioxidants alpha-tocopherol, erythrodiol, and oleanolic acid. The fatty acid composition of hepatic microsomes of Wistar rats that were fed for three weeks with the above-mentioned oils had lower proportions of C16:0, C18:2n6, and C22:6n3 and higher proportions of C18:0 and C18:1n9 than rats fed the control diet. Light emission by hepatic microsomes increased, in the first 180 min, 2-fold after ascorbate-Fe(2+) addition compared with NADPH addition. Both increases were less pronounced in microsomes of OPO-fed rats and to a smaller extent in microsomes of OO-fed rats. Smaller increases in light emission were associated positively with higher concentrations of dietary alpha-tocopherol, erythrodiol, and oleanolic acid but were not associated with changes in fatty acid composition of hepatic microsomes. Addition of alpha-tocopherol, erythrodiol, or oleanolic acid decreased light emission of hepatic microsomes with a greater inhibition in microsomes of rats fed the control diet. Our data suggest that erythrodiol and oleanolic acids partly explain the protective effect of dietary OPO on microsomal lipid peroxidation in rats.

Effect of melanins on lipid peroxidation

Effects of melanins obtained from cultured Cladosporium cladosporidae fungi and Alpha grape on Fe(2+)-induced, Fe(2+)-ascorbate-induced, and NADPH-induced lipid peroxidation in rat liver, brain, and eye were studied. Melanins were shown to inhibit the accumulation of lipid peroxidation products in vitro. The inhibitory effects of melanins were not due to direct interactions of these pigments with superoxide anion (O2). However, melanins may interact with other free radicals. Melanins were demonstrated to have the ability to oxidize NADPH, which is probably one of the mechanisms of their antioxidant effects.

Membrane stabilising effects of natural polyphenols and flavonoids from Sempervivum tectorum on hepatic microsomal mixed-function oxidase system in hyperlipidemic rats

The extensive role of the microsomal mixed-function oxidase (MFO) system in the oxidation of endo-and xenobiotics, in the detoxication, in the generation of reactive free radicals and in the decomposition of the end products of lipid peroxides is well documented in the literature. Steatotic liver is a very frequent damage with different etiology. Drug metabolising reactions are suppressed in fatty liver, in which pathologically increased production of reactive oxygen intermediates may lead to the peroxidation of microsomal membrane lipids and to the change of membrane bound enzyme activities because of overwhelmed protective mechanisms. The subnormal activity of the MFO system may diminish the non specific resistance of the organism. Therefore we have studied the effects of natural flavonoids and polyphenolic compounds on the mixed-function oxidases. Antioxidant, O 2 − and OH scavenger properties of Sempervivum tectorum extract (STF1) were proved by EPR spectroscopic and chemiluminometric techniques. Potential bioactive constituents were determined by chromatography (HPLC, TLC) and spectrometric (UV, UV–VIS) methods. In the present study we reflect on the membrane stabilising, antioxidant and lipid metabolism modifying effects of this extract. It was established that activities of NAD(P)H reductase and content of cytochrome P450 were normalised in liver microsomes of hyperlipidemic rats, if the animals were treated with STF1 (2 g/bwkg for 9 days in drinking water parallel with fat-rich diet feeding). Fatty acid composition, examined by HRGLC analysis, was changed beneficially. NADPH induced lipid peroxidation was also decreased in microsomes in in vivo and in vitro experiments. At the same time the STF1 had no significant influence on MFO system in normolipidemic animals and on cytochrome b5 concentration of microsome fractions of hyperlipidemic rats.

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten μmol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe 2+-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 μmol/l of baicalein and baicalin significantly protected cells against H 2O 2-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.

Imidazo[1,2- c]quinazolines with lipid peroxidation inhibitory effect

A series of imidazo[1,2- c]quinazolines of different lipophilic character was prepared. According to their antioxidant (cyclic voltammetry) properties they all should be potent inhibitors of lipid peroxidation. Under the given circumstances (NADPH-induced lipid peroxidation in rat brain microsomes and Fe 2+-induced lipid peroxidation in rat brain homogenate), however, their lipid peroxidation inhibitory activity was strongly dependent on their lipophilicity.

Novel Connections Between NADPH-Induced Lipid Peroxidation and Cytochrome P450 Inactivation, and Antioxidant and Enzyme Protective Properties of Estradiol in Gonadal Membranes

This study uses microsomal membranes from rat testis tissue, including the cytochrome P450c17 (steroid 17α-monooxygenase/17α-hydroxyprogesterone aldolase, catalyzing the conversion of progesterone to androstenedione), to decipher the possible relation of NADPH-induced (no exogenous iron added) lipid per-oxidation and cytochrome P450 inactivation and the protective effect of certain steroids. NADPH (300 μM) causes a 3.6-fold stimulation of malondialdehyde formation (thiobarbituric acid-reactive substances) and a 29% cytochrome P450c17 loss within 1 h at 37°C, but has no effect on lipid peroxidation in the presence of the iron chelator desferrioxamine. Hydrogen peroxide has only marginal effects. The antioxidant efficiency of estradiol (IC50 = 13.9 μM) is higher than its cytochrome P450c17 protective efficiency (IC50 = 33.0 μM), whereas androstenedione does not inhibit lipid peroxidation but protects cytochrome P450c17 completely. The human choriogonadotropin-induced degradation of cytochrome P450c17 in incubated decapsulated testes can not be correlated with a stimulation of lipid peroxidation, and it is partially inhibited by estradiol but completely abolished by androstenedione. It is concluded (I) that NADPH stimulates iron-dependent generation of reactive oxygen species by the monooxy-genase system even in the presence of certain P450 ligands in the physiological membrane environment, (II) that membrane lipid peroxidation may be suppressed by hydrophobic steroids acting as antioxidants such as estradiol, (III) that steroid ligands stabilize cytochrome P450c17 against inactivation in the presence of NADPH even if they do not act as substrates and do not possess antioxidant activity, and (IV) that the choriogonadotropin-induced down-regulation of cytochrome P450c17 is not due to accumulating steroids acting as “pseudosubstrates” as occasionally supposed.

Pro- and anti-oxidant activities of the mitochondrial respiratory chain: factors influencing NAD(P)H-induced lipid peroxidation

This paper is a study of factors influencing the rate of lipid peroxidation in beef heart submitochondrial particles induced by NAD(P)H via the NADH-ubiquinone oxidoreductase (Complex I) of the respiratory chain. In accordance with earlier observations, both NADH and NADPH initiated lipid peroxidation in the presence of ADP-Fe 3+. The rate of the reaction, measured as oxygen consumption and formation of thiobarbituric acid reactive substances, was biphasic as a function of NADH concentration, reaching a maximum at low NADH concentrations and then declining. In contrast, the NADPH-initiated lipid peroxidation showed a monophasic concentration profile of hyperbolic character. Rotenone did not eliminate the biphasicity of the NADH-induced reaction, indicating that this was not due to an antioxidant effect of reduced ubiquinone at high NADH concentrations. This conclusion was further supported by the demonstration that extraction of ubiquinone from the particles did not relieve the inhibition of lipid peroxidation by high NADH concentrations. However rhein, another inhibitor of Complex I, eliminated the biphasicity, and even caused a substantial stimulation of the NADH-induced lipid peroxidation in the particles upon extraction of ubiquinone by pentane. No similar effect occurred in the case of NADPH-induced lipid peroxidation. Furthermore, rhein facilitated both NADH- and NADPH-induced lipid peroxidation even in the absence of added ADP-Fe 3+, in a fashion similar to that earlier reported with succinate in the presence of theonyltrifluoroacetone. Based on these findings and measurements of the redox states of ubiquinone and cytochromes in the presence of KCN and NADH or NADPH, it is concluded that Complex I may distinguish between electron input from NADH and NADPH by differences in the site(s) of substrate binding and in the pathways and rates of NADH and NADPH oxidation.

Direct measurement of lipid peroxidation in submitochondrial particles.

The susceptibility of the polyunsaturated fatty acid parinaric acid (cis-PnA) to peroxidative damage with concomitant loss of its fluorescent character can be used to detect lipid peroxidation in a direct and sensitive way. The procedure, originally developed to measure peroxidation in lipid vesicles and erythrocyte membranes, has been adapted for the study of submitochondrial particles. Optimal conditions for the concentrations of cis-PnA (0.8 mol %), mitochondrial membrane (100 microM membrane phospholipid), and the radical generating system (50 microM NADH and 10 microM:1 mM Fe(III)-ADP) were established. In the absence of peroxidation inducing compounds, a stable fluorescent signal can be detected. Upon addition of NAD(P)H and ADP-Fe(III), lipid peroxidation starts, and the observed fluorescence decrease is a measure of peroxidation. Both NADH and NADPH were able to induce lipid peroxidation in submitochondrial particles in the presence of an iron chelate. The use of NADH resulted in higher rates of peroxidation compared with NADPH at the same concentration. Whereas the rate of NADH-induced lipid peroxidation was maximal at very low NADH concentrations (2.5 microM) and decreased when the concentration became higher, the NADPH-induced lipid peroxidation reaches saturation at 100 microM. NADH-induced lipid peroxidation in submitochondrial particles from different rat tissues (heart, skeletal muscle, and liver) resulted in a clear difference in peroxidation rates. The highest rates were observed in heart submitochondrial particles, while the lowest rates were obtained in submitochondrial particles derived from liver. Skeletal muscle submitochondrial particles showed intermediate rates of lipid peroxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential role in lipid peroxidation between rat P450 1A1 and P450 1A2

The role of cytochrome P450 (P450) in lipid peroxidation induced by NADPH or peroxide was investigated in a reconstituted system. When cumene hydroperoxide, t-butyl hydroperoxide and hydrogen peroxide were used as initiators, the rates of malondialdehyde (MDA) formation were much higher in a reconstituted system containing P45O 1A1 than those observed in a reconstituted system containing P450 1A2. In contrast to peroxide-induced lipid peroxidation, P450 1A2 catalysed NADPH-induced lipid peroxidation more effectively than did P450 1A1 regardless of the presence of ADP-Fe(NO 3) 3. Carbon monoxide inhibited NADPH-induced formation of MDA in a reconstituted system containing P450 1A2, but not P450 1A1. In addition, superoxide dismutase (SOD) was an effective inhibitor in a NADPH-induced lipid peroxidation system catalysed by P450 1A2 but not by P450 1A1. These results suggest that a peroxide-induced reaction might proceed readily with P450 1A1, whereas P4SO 1A2 mainly functions in NADPH-induced lipid peroxidation via generation of an active oxygen species. It is furthermore indicated that the difference in the effect of SOD in NADPH-induced lipid peroxidation depends on the P450 used.

Age dependent different influence of carbon tetrachloride on biotransformation of xenobiotics, glutathione content, lipid peroxidation and histopathology of rat liver.

15- and 60-day-old male rats were treated with different doses of CCl4 orally. 24 h later cytochrome P-450 (P450) concentration, 7-ethoxyresorufin O-deethylation (EROD) and 7-pentoxy-resorufin O-deethylation (PEROD) activities were determined. Whereas P450 and EROD are lowered to the same extent in both ages, PEROD shows a more pronounced inhibition in the livers of younger rats. The formation of endogenous lipid peroxides (measured as thiobarbituric acid reactive substances) is drastically increased only in the livers of young rats. The hepatic glutathione (GSH) content was unaffected by CCl4 treatment whereas oxidized glutathione is more increased in the livers of adult rats. This can be caused by a higher activity of GSH-peroxidase in the livers of adult rats. The changes in NADPH-induced lipid peroxidation and chemiluminescence correlate partially with the changes in P450 and biotransformation reactions. Histopathologically the liver damage is more extensive in suckling rats. The necrosis is localized predominantly in the perivenous tissue, which has normally the highest activities of toxification and detoxification enzymes.

Inhibitory effects of Maharishi-4 and Maharishi-5 on microsomal lipid peroxidation

The effects of Maharishi-4 (M-4) and Maharishi-5 (M-5) on microsomal lipid peroxidation were examined in vitro. Rat liver microsomes were incubated with an NADPH-generating system or with sodium ascorbate and an ADP-iron complex to stimulate enzymatic or nonenzymatic lipid peroxidation respectively. Alcoholic or aqueous extracts of M-4 or M-5, when added to these incubation systems, inhibited hepatic microsomal lipid peroxidation in a concentration-dependent manner. The aqueous extract of M-4 was the most effective antiperoxidant in these systems. A 10% (w/v) aqueous extract of M-4 inhibited ascorbance or NADPH-induced lipid peroxidation by approximately 50% when added at volumes of 8 μl and 3.5 μl respectively to the incubation mixtures (total incubation volume, 2 ml). These findings suggest that M-4 and M-5, by virtue of their antiperoxidant properties, may be useful in the treatment of free radical-linked drug toxicities and disease states.

Inhibitory Effects of Isomers of Tocopherol on Lipid Peroxidation of Microsomes from Vitamin E-Deficient Rats

NADPH-induced lipid peroxidation of hepatic microsomes from vitamin E-deficient rats has been used to assess the antioxidant effectiveness of dl alpha, d alpha- and gamma-tocopherol. When the tocopherols were added in ethanol to microsomes, the degree of inhibition of formation of thiobarbituric acid reactive substances (TBARS) decreased in the order dl alpha- greater than d alpha- greater than gamma-tocopherol. This reflected the difference in the solubility of the tocopherols in the microsomes, dl alpha-tocopherol being the most soluble and gamma-tocopherol the least. Using inhibition of TBARS produced per tocopherol content in microsome as a measure of antioxidant potency, the effectiveness of the isomers was gamma- greater than d alpha- greater than dl alpha. Despite addition of pharmacological concentrations of the isomers, it was not possible to inhibit lipid peroxidation to the same levels as were found in microsomes from vitamin E sufficient animals. Use of ethanol as a vehicle may not allow optimum orientation of the tocopherols into the lipid bilayer.

Increased Resistance Against Oxidative Stress Is Observed During A Short Period of Renal Reperfusion After A Temporal Ischaemia

Reperfusion of rat kidney submitted to temporal ischaemia induces a decrease in glutathione content. Lipid peroxidation is not detected in kidney homogenates but microsomes obtained after periods of reperfusion longer than 60 minutes show increased malondialdehyde values correlated with high oxygen consumption and superoxide free radical generation. Microsomes obtained from kidneys submitted to 15 or 60 minutes of reperfusion are resistant to NADPH-induced lipid peroxidation but after 120 minutes of reperfusion an increased lipid peroxidative response is observed. Although the mechanism of the protection found in microsomes against the induction of oxidative stress in the first 60 minutes of reperfusion is unknown, it is postulated that this subcellular fraction plays an important role in the oxidative stress observed after longer periods of reperfusion.

Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe 3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 °C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 m m) and bromosulfophthalein (0.1 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 m m) and oxidized glutathione (10 m m) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.

Effects of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation.

Endogenous alpha-tocopherol levels in isolated rat liver nuclei were determined to be 0.045 mol% (mol alpha-tocopherol per mol phospholipid x 100). This value corresponds to 970 polyunsaturated fatty acid (PUFA) moieties to one molecule of alpha-tocopherol in the nuclear membrane. Isolated nuclei, when incubated with various concentrations of exogenous alpha-tocopherol, took up only a small percent of initial levels of alpha-tocopherol present in the incubation media. Exogenous alpha-tocopherol, when incorporated in isolated nuclei above a threshold value of 0.085 mol%, effectively inhibited NADPH-induced lipid peroxidation. The addition of 1 mM glutathione lowered the threshold levels of alpha-tocopherol needed to inhibit lipid peroxidation to about 0.040 mol%. We suggest the data indicate a glutathione-dependent enhancement of the ability of alpha-tocopherol to inhibit nuclear lipid peroxidation.

NADPH- and linoleic acid hydroperoxide-induced lipid peroxidation and destruction of cytochrome P-450 in hepatic microsomes

Temporal aspects of the effects of inhibitors on hepatic cytochrome P-450 destruction and lipid peroxidation induced by NADPH and linoleic acid hydroperoride (LAMP) were compared. In the absence of added Fe 2+, NADPH-induced lipid peroxidation in hepatic microsomes exhibited a slow phase followed by a fast phase. The addition of Fe 2+ eliminated the slow phase, thus demonstrating that iron is a rate-limiting component in the reaction. EDTA, which complexes iron, and p-chloro-mercurobenzoate (pCMB), which inhibits NADPH-cytochrome P-450 reductase, inhibited both phases of the reaction. Catalase as well as scavengers of hydroxyl radical, inhibited NADPH-induced lipid peroxidation almost completely. GSH also inhibited the NADPH-dependent reaction but only when added at the beginning of the reaction. In contrast with NADPH-dependent lipid peroxidation. the autocatalytic reaction induced by LAHP was not biphasic, NADPH-dependent or iron-dependent, nor was it inhibited by hydroxyl radical scavengers, catalase or GSH. A synergistic effect on lipid peroxidation was observed when both NADPH and LAHP were added to microsomes. It is concluded that both the fast and slow phases of NADPH-dependent microsomal lipid peroxidation are catalyzed enzymatically and are dependent upon Fe 2+, whereas LAHP-dependent lipid peroxidation is auto-catalytic. Since the fast phase of enzymatic lipid peroxidation occurred during the fast phase of destruction of cytochrome P-450, it is postulated that iron made available from cytochrome P-450 is sufficient to promote optimal lipid peroxidation. Since catalase and hydroxyl radical scavengers inhibited NADPH-dependent but not LAHP-dependent lipid peroxidation, it is concluded that the hydroxyl radical derived from H 2O 2 is the initiating active-oxygen species in the enzymatic reaction but not in the autocatalytic reaction.

Senescence-associated decrease of NADPH-induced lipid peroxidation in rat liver microsomes

Senescence is associated with decrease in the NADPH-induced lipid peroxidation in liver homogenate as well as rough and smooth microsomes of female rats. In the microsomal fractions, sensitivity to NADPH-induced lipid peroxidation is high in young adults (3-month-old), decreases in middle aged (12-month-old) and reaches lowest levels in senescent (30-month-old) rats. Increasing the concentration of co-factors or time of incubation does not alter this resistance observed in the senescent rats. Major factors responsible for this resistance in senescent rats seem to be low levels of substrate in the form of phospholipids, NADPH-cytochrome c reductase, cytochrome P-450 and high cholesterol:phospholipid ratios besides enhanced levels of Superoxide dismutase, α-tocopherol and reduced glutathione.

Lipid peroxidation in rat uterus

Lipid peroxidation in rat uterus has been studied using NADPH- and ascorbate-induced systems. Lipid peroxidation in rat uterus is low as compared to rat liver. Uterus is more sensitive to ascorbate-induced lipid peroxidation than that induced by NADPH. Uterus contains lower amounts of phospholipids and has a lesser degree of unsaturation in lipids. Co-factor studies show that Fe 2+ is more important for ascorbate-induced lipid peroxidation. Endometrium is more sensitive to ascorbate-induced lipid peroxidation than myometrium. It also contains more total lipids and phospholipids besides having a higher degree of unsaturation in the lipids as compared to myometrium. Among the subcellular fractions, mitochondria are more prone to ascorbate-induced lipid peroxidation, whereas microsomes are more sensitive to NADPH-induced lipid peroxidation. Uteri from old rats (24 months) and pregnant rats are more resistant to lipid peroxidation than those from 3-month-old control rats. Uterus of pregnant rats contains more factors which inhibit lipid peroxidation and also has a lesser degree of unsaturation in lipids compared with uterus of control rats. The possible consequences of the resistance of uterus to lipid peroxidation, especially during pregnancy and senescence, are discussed.

Low level of lipid peroxidation in newborn rats: Possible factors for resistance in hepatic microsomes

Hepatic rough and smooth microsomes of newborn rats show less sensitivity to ascorbate- and NADPH-induced lipid peroxidation as compared to those of adult rats. Though optimum concentrations of Fe 2+, ascorbate and Fe 3+ significantly increase lipid peroxidation in both age groups, the lipid peroxidation observed in newborns is much less compared with that of adults. Microsomal fractions from newborn rats contain significantly lower amounts of phospholipid, NADPH cytochrome c reductase, cytochrome P-450 and a lower degree of unsaturation in lipids. These fractions also exhibit high cholesterol:phospholipid ratios. The resistance to lipid peroxidation observed in the newborns appears to be due to the low availability of substrate and high cholesterol:phospholipid ratio.