JP5846604B2 - Biomarker for behavioral rhythm monitoring - Google Patents

Description

  The present invention relates to a biomarker for behavioral rhythm monitoring as seen by oxidative stress response.

In a healthy living body, oxidative stress caused by active oxygen and its antioxidant activity are balanced every day. However, oxidative stress disorder occurs when the balance is broken with lifestyle-related diseases and aging. Oxidative stress is known to cause various diseases such as cranial nerve diseases such as Parkinson's disease and Alzheimer's disease, cardiovascular diseases such as arteriosclerosis and myocardial infarction, diabetes, immunodeficiency, and cancer.
Conventionally, it has been known that when excessive oxidative stress is applied to a living body, active oxygen such as hydrogen peroxide and hydroxy radical is generated in the living body, and various lipids in the living body are oxidized to produce oxidized lipids. These oxidized lipids were used as oxidative stress markers, and the amount thereof was measured to evaluate the degree of oxidative stress. AIST's Yoshida, Futaki et al. Focused on hydroxyoctadecadienoic acid (HODE), 7-OHCH, isoprostanes, etc. produced by oxidation of linoleic acid, cholesterol, arachidonic acid, etc. Developed as a biomarker that can measure the level of oxidative stress with higher sensitivity, and established a method for measuring these oxidized lipids (Patent Document 1). In particular, HODEs are technically used as diagnostic biomarkers for nephropathy-related diseases (Patent Document 2), dementia (Patent Document 3), chronic hepatitis and cirrhosis (Patent Document 4), which are thought to increase oxidative stress. Establishment is also recommended.
However, the diurnal variation of these oxidized lipids has never been studied before.
So far, regarding the diurnal variation of oxidative stress in vivo, the concentration of thiobarbituric acid reactive species (TBARS) known as oxidative stress markers and the enzymatic activities of superoxide dismutase, catalase, and peroxidase However, it was pointed out that there was a change (Non-Patent Documents 1 and 2), but the amplitude is small and the regularity is not good as a diurnal variation. In addition, it was not investigated at all whether there was any correlation with some behavioral rhythm.
In addition, in order to detect the circadian rhythm of the oxidative stress state, attempts have been made to analyze the circadian rhythm of gene expression of oxidative stress-related molecules using the heme oxygenase 1 (HO-1) gene. Even if it sees, the circadian rhythm of an oxidative stress state was not able to be detected (nonpatent literature 3).

On the other hand, it has recently become clear that physiological circadian rhythms such as body temperature and blood pressure, and metabolic circadian rhythms such as lipid metabolism and sugar metabolism are widely controlled by the body clock. As a result, the most common method for monitoring metabolic circadian rhythm is an attempt to examine the expression of biological clock genes using blood, hair, oral epithelium, and saliva as samples, including the present inventors. It is being actively promoted. However, the present inventors have encountered many cases where the rhythm expression of the body clock gene is normal in the process of developing a stress sleep disorder model animal, but the sleep circadian rhythm is disturbed. I realized that I couldn't determine the daily rhythm of biological metabolism alone. The Clock / clock mouse is a mutant of the biological clock gene Clock and is widely used in the world as a model animal with a broken biological clock. However, the Clock / clock mouse is light under dark and light conditions even if the biological clock is broken. In order to behave according to the conditions, the behavioral rhythm of resting in the light period and acting in the dark period is observed in free eating. However, in the Clock / clock mouse, the expression rhythm of the body clock gene corresponding to the behavior rhythm observed under light and dark conditions is not observed, and the behavior rhythm cannot be detected by observing the expression of the clock gene (Non-patent Document 4).
Biological rhythms are rhythms governed by the central clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus in the brain, and rhythms defined by eating behavior in a manner independent of the central clock present in the SCN. (Feeding rhythm) exists and the former is controlled by a clock gene, while the clock gene is not required for oscillation of the feeding rhythm (Non-patent Document 5), and its molecular mechanism is still unclear.
When looking at typical feeding rhythms as metabolic circadian rhythms, these rhythms are not a direct response due to feeding, but by feeding (restricted feeding) at a certain time every day, the rhythm is independent of the central clock. Since the rhythm continues even in the fasted state after the end of restricted feeding, it is considered that there is a clock gene-independent oscillator driven by the feeding rhythm (non-patented Reference 6). However, there are no biomarkers for objectively measuring feeding rhythm. For example, blood sugar levels and blood lipid levels that are directly affected by eating reflect the nutritional dynamics absorbed immediately after meals and disappear quickly in vivo. It cannot be used as a biomarker for measurement.
Therefore, it has been strongly desired to provide a biomarker that can objectively measure the feeding rhythm, which is a typical behavior rhythm that is not controlled by the clock gene.
It is known that patients with anorexia nervosa are disturbed by metabolic rhythms in the body, causing not only sleep disorders and obesity but also mental illness. However, the diagnosis of eclipse is mainly an interview with a doctor, and there is no appropriate diagnosis method. Since the feeding rhythm is considered to reflect the history of daily eating behavior, it is expected that abnormalities in eating behavior can be objectively monitored by measuring the feeding rhythm.
If a biomarker capable of objectively measuring feeding rhythm can be provided, it can be considered effective for diagnosis of patients with anorexia nervosa, and thus there is great expectation for such a biomarker.

Patent No. 4348452 Patent No. 4863204 Patent 4521512 Japanese Patent No. 4569959

Manoharan S, Baskar AA, ManivasagamT, Subramanian P., Singapore, Med J. 2005, 46 (4): 184-8. Manoharan S, Panjamurthy K, BalakrishnanS, Vasudevan K, VellaichamyL., Pharmacol Rep. 2009, 61 (4): 719-26. Poliandri AH, Esquifino AI, Cano P, Jimenez V, Lafuente A, Cardinali DP, Duvilanski BH, J Pineal Res. 2006, 41 (3): 238-46. Oishi K, Miyazaki K, Ishida N., Biochem Biophys Res Commun. 2002, 298 (2): 198-202. Pendergast JS, Nakamura W, Friday RC, Hatanaka F, TakumiT, Yamazaki S., PLoS One. 2009, 4 (3): e4860. Kenichi Honma Encyclopedia of Time Biology (Asakura Shoten) 2008 “Federal circadian rhythm”

  An object of the present invention is to provide a method for knowing the body time of a behavioral rhythm such as a feeding rhythm that is not linked to the expression of a circadian clock gene, and a biomarker therefor, and at the same time, oxidation in vivo The present invention provides a method for detecting a stress diurnal rhythm stably and accurately.

The present inventors examined various substances related to daily eating behavior rhythms as candidates when searching for biomarkers for knowing the body time of feeding rhythms unrelated to clock gene expression. In the case of in-vivo substances that respond transiently by eating, such as blood sugar levels, the rhythm pattern must be determined by continuous sampling, and this is not suitable because the burden on the subject is large. In addition, it is clear that the measurement of clock gene expression controlled by the central clock in the brain SCN does not reflect the feeding rhythm, so it changes by sampling every few hours (several times a day) It was considered necessary to develop a new biomarker that is stable enough to analyze these patterns and that sharply reflects the past history of daily eating timing.
The present inventors have developed hydroxyoctadecadienoic acid (HODE), hydroxycholesterols (such as 7-OHCh) that have been developed as markers for measuring oxidative stress in vivo and are being investigated as diagnostic markers for various diseases. , Lipid oxides such as isoprostanes came up with the possibility of fluctuating with the feeding behavior, according to the feeding behavior pattern when mice were forcibly fed regardless of the circadian rhythm, Blood concentration change patterns of various markers for measuring oxidative stress such as oxidized lipids were measured.
As a result, surprisingly, among the conventionally known markers for measuring oxidative stress, only the blood concentration change patterns of HODEs and 7-OHCh are synchronized with the feeding behavior pattern, especially HODE. It was confirmed that the kind carved out a keen sense of rhythm. In addition, if the feeding rhythm is lost due to fasting, the blood levels of HODEs and 7-OHCh remain low and no change occurs, and if feeding behavior starts after refeeding, both HODEs and 7-OHCh rise rapidly Is recognized.
This indicates that HODEs are excellent biomarkers for knowing the body time of the feeding rhythm, and that there is a circadian rhythm synchronized with the feeding rhythm in oxidative stress in the living body. I was able to clarify. This also indicates that HODEs are excellent as markers for detecting rhythmic rhythm of oxidative stress. Although 7-OHCh does not have the same rhythm as HODE, it clearly uses a circadian rhythm that synchronizes with the feeding rhythm, so it can be used together with HODE to detect in vivo rhythm or oxidative stress rhythm. Thus, it is expected that a more accurate daily rhythm can be detected.
By obtaining the above knowledge, the present invention was completed.

That is, the present invention includes the following inventions.
[1] Hydroxyoctadecadienoic acid (HODE) in blood or 7-hydroxycholesterols (7-OHCh) together with HODEs are measured several times over time for at least one day. A method for monitoring diurnal changes in behavioral rhythms, characterized by observing physical changes.
[2] The method according to [1], wherein the HODEs include at least one selected from the group consisting of 9-HODE and 13-HODE.
[3] The method according to [1] or [2], wherein the behavior rhythm is a feeding behavior rhythm.
[4] Hydroxyoctadecadienoic acid (HODE) in blood or 7-hydroxycholesterol (7-OHCh) together with HODE was measured multiple times over a period of at least one day, and the measured values over time A method of observing the circadian rhythm of oxidative stress response in the body, characterized by observing changes in the body.
[5] Administer the test substance to experimental animals, measure HODEs in blood or HODEs together with 7-OHCh multiple times over a period of at least one day, and observe changes over time in the measured values. A method for screening an oxidative stress response-improving substance, comprising a step of comparing the step with a case where a test substance is not administered;
[6] A method for determining an effective administration time of an oxidative stress-improving substance, comprising the following (1) to (4);
(1) A step of administering a test oxidative stress-improving substance to a test animal at a plurality of times, and measuring 7-OHCh together with HODEs in blood immediately after administration, or HODEs at each administration time,
(2) Compare the measured value when measuring at the same time without administering the test oxidative stress-improving substance in advance and the measured value at each time of administration obtained in (1) above. , Calculating the difference between the two as the amount of change at each time,
(3) A step of comparing the amount of change at each administration time obtained in (2) above, and determining the administration time at which the amount of change is maximum,
(4) A step of determining the administration time when the amount of change obtained in the step (3) is maximized as an effective administration time of the test oxidative stress-improving substance.
[7] The method according to [3], wherein the method of monitoring the feeding rhythm of [3] is for objectively predicting a feeding time.

  In the present invention, it was proved that HODEs function as an excellent biomarker for stably and accurately detecting a feeding rhythm that is not linked to the expression of a circadian clock gene and a circadian rhythm of oxidative stress in a living body. . HODEs, or HODEs and 7-OHCh levels in blood can be measured to observe feeding rhythms and objectively monitor eating behavior abnormalities. In addition, when HODEs are used as diagnostic biomarkers for various diseases such as nephropathy-related diseases, dementia, chronic hepatitis, or cirrhosis, setting a measurement span that takes into account the feeding rhythm enables higher accuracy. A diagnostic method can be provided.

Fig. 3 shows changes in daily linoleic acid and cholesterol concentrations in wild-type, clock / clock mice, light-fed mice, and dark-fed mice. Changes in daily concentrations of tHODE and 7-OHCh in the blood of wild-type and clock / clock mice Changes in the daily concentration of tHETE and isoprostane in the blood of wild-type and clock / clock mice Daily feeding rhythm under free feeding (AL), light period feeding (DF), dark period feeding (NF) conditions and free behavior rhythm under the same conditions Changes in daily concentrations of tHODE and 7-OHCh in blood in mice fed in light period and mice fed in dark period Changes in blood HODE and 7-OHCh levels due to fasting and refeeding. It was compared with the diurnal variation in the case of free feeding.

1. About Oxidized Lipids as Dietary Markers of the Present Invention As a method for evaluating oxidative stress known to be involved in various diseases, AIST Yoshida, Futaki et al. A method for evaluating the degree of oxidative stress by measuring a class is provided, and an original method has also been found for a method for accurately measuring various oxidized lipids (Patent Document 1).
In Patent Document 1, as the oxidized lipids that have been confirmed to be effective as oxidative stress markers, hydroxyoctadecadienoic acid (HODE) compounds produced by oxidation of linoleic acid, cholesterol, arachidonic acid, etc., 7- Examples include hydroxycholesterols (7-OHCh) and isoprostanes.
Of these oxidative stress markers, it has been found in the present invention that only HODEs and 7-OHCh have a rhythm that matches the behavioral rhythm and diet rhythm under light and dark conditions.
That is, HODEs and 7-OHCh among oxidative stress markers can be used as behavioral rhythm markers in the present invention. In particular, HODEs are particularly excellent behavioral rhythm markers because of the large amplitude range of rhythms. In that case, it is preferable to measure together with HODEs using 7-OHCh as a complementary marker.
In the present invention, HODEs are unsaturated fatty acids (hydroxyoctadecadienoic acid) having a hydroxyl group, 16 carbon atoms and two double bonds, and the position of the hydroxyl group and the double bond. Is limited to 9th and 12th place. Detailed explanations such as specific structural formulas and properties are described in Patent Document 1 and are available from Cayman Chemical or Larodan Fine Chemicals. 7-OHCh refers to cholesterol having a hydroxyl group at the 7-position. Similarly, Patent Document 1 describes the structural formula.
As the HODEs, preferably, 9-HODE, 13-HODE, and total HODE (tHODE) obtained by combining these HODE amounts are used. tHODE is most preferred.

2. About “Behavior Rhythm” in the Present Invention In the present invention, “behavior rhythm” is a circadian rhythm that is typically independent of circadian rhythm based on clock gene expression in the body, such as “food rhythm”, The behavioral rhythm. It is not known which gene controls the normal diet rhythm and which region in the body is involved in the control, and it is controlled by a mechanism different from the behavioral rhythm by the body clock gene.

3. Method for Measuring Lipid Oxide Used as Behavior Rhythm Marker of the Present Invention The measurement of HODE or 7-OHCh used as the behavior rhythm marker of the present invention basically follows the method for measuring lipid oxide described in Patent Document 1. Specifically, a biological sample is first treated with a reducing agent such as sodium borohydride (NaBH4) to introduce peroxide, which is a reaction product with active oxygen, into a stable reductant. The reduced product is then subjected to an alkali treatment, preferably an alkali metal hydroxide such as potassium hydroxide, and hydrolyzed to a uniform free fatty acid. The sample after the hydrolysis treatment is measured for HODEs or 7-OHCh that are oxidized lipids using a conventional analytical means such as LC-MS / MS and GC-MS.

4). Behavioral rhythm monitoring method in the present invention (1) Monitoring target animal The subject animal to be monitored in the present invention is a mammal including a human, and when used for an experiment, general laboratory animals are preferable. Rodents such as mice are particularly preferable. The animal's behavioral rhythm is engraved regardless of the presence or absence of the expression clock of the circadian clock gene under light and dark conditions, so even if it is a test animal such as a clock / clock mouse, It is possible to use.

(2) Monitoring method of behavior rhythm under light and dark conditions The test animals are normally raised for 2 weeks under lighting conditions of 8:00 lighting / 20:00 lighting off. Under these conditions, even if the biological clock is broken, the daily dizziness of the action is maintained. An infrared sensor (OMRON E3HS-DS5E1) is attached to the breeding cage. When the animal crosses in front of the sensor, it counts and sends the data to the Chronobiology Kit System (Stanford). Using this 5 minute count as the amount of free action, the behavior pattern for 2 weeks is analyzed.
The circadian rhythm of oxidative stress response in the body is observed by measuring hydroxyoctadecadienoic acid (HODE) or HODE and 7-hydroxycholesterol (7-OHCh) in blood.
At that time, it is important to follow the determination of the degree of oxidative stress in blood due to the change of lipid oxide over time along with the behavior rhythm.

(3) Dietary rhythm monitoring method Either one of the two types of dietary rhythm monitoring method is performed on the subject animal while keeping it normally. One is an infrared sensor (Omron E3HS-DS5E1) that sets the drinking behavior rhythm, which is almost linked to the diet rhythm, to the tip of the drinking bottle, and measures it by counting when the animal comes to drink water. . The analysis of the data is a chronobiology kit system (manufactured by Stanford), and the count amount every 5 minutes is used as the amount of drinking behavior. The other is to measure food consumption over time using a food intake measuring device (manufactured by Merquest) that measures the amount of food reduced by food intake every 10 minutes.
The dietary rhythm is monitored by measuring hydroxyoctadecadienoic acid (HODE) in the blood, or HODE and 7-hydroxycholesterol (7-OHCh) to observe the oxidative stress response.

4). Screening Method for Oxidative Stress Response Improvement Substance Considering Effect of Behavior Rhythm By measuring the daily rhythm of the behavior rhythm marker of the present invention, it is possible to measure the fluctuation of oxidative stress response influenced by the behavior rhythm. When screening for stress-improving substances such as anti-oxidants that reduce oxidative stress, considering the daily rhythm of the degree of oxidative stress, it is possible to find a time zone in which the effect is higher. Moreover, there is a possibility that an antioxidant function can be found by changing the timing of administration even for substances that have not been improved until now.
A test substance is administered to an experimental animal, and HODEs in blood or HODEs and 7-OHCh are measured to measure the circadian rhythm of the oxidative stress response, which is compared with the case where no test substance is administered.
The test substance is typically an antioxidant such as polyphenols and caffeine, but is not limited to these known antioxidants, and any substance that can be administered without toxicity to the living body can be used. Such substances are also targeted.
As the administration method of the test substance, usual administration methods such as oral administration, intravenous administration, and administration from the skin can be applied. The administration time of the test substance may be a single administration prior to the measurement of HODEs, or may be intermittent administration or continuous administration at a plurality of times.

5. Method for Finding Effective Time of Oxidative Stress Response Improving Substance A known oxidative stress improving substance or an oxidative stress response improving substance found by the screening method of the present invention is used as a test substance at a plurality of times with respect to an experimental animal. Administer. Then, measurement of HODEs in blood after administration, or HODEs and 7-OHCh is performed at each administration time of the test substance, and the measurement values at each administration time are measured values when the test substance is not administered. By calculating the amount of change compared to each other and comparing the amount of change at each administration time, the administration time at which the amount of change was maximized was determined, and the most effective of the oxidative stress-improving substance that was the test substance Time of administration can be found.

6). Methods for diagnosing nocturnal eating disorder and obesity caused by disturbance in dietary rhythm Nocturnal eating disorder is a disease characterized by anorexia in the early morning, overeating at night, and insomnia, leading to obesity due to abnormal dietary rhythm. In order to clinically determine obesity, etc., by measuring HODEs in the blood of the test individual, or HODEs and 7-OHCh at multiple times of the day, objectively the feeding time and consumption on the day before the measurement Eating habits can be predicted.

7). Optimization of disease diagnosis conditions In prior patents, blood HODEs, or HODEs and 7-OHCh are proposed to be used as biomarkers for diseases such as hepatitis, nephropathy, and Alzheimer. By defining the subject's blood collection and feeding conditions before blood collection, the conditions for measuring HODEs and 7-OHCh can be optimized.

EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not particularly limited to these examples.
Technical terms used in the present invention have meanings commonly understood by those skilled in the art unless otherwise defined.
Moreover, the description content of the prior art literature or the patent application specification cited in the present invention is incorporated as the description of this specification.

(Example 1) Correlation between diurnal rhythm of oxidative stress and behavioral rhythm under light-dark conditions 24 wild-type mice (ICR) and 16 clock gene mutant clock / clock mice were bred under light-dark conditions every 12 hours did. 2 to 8 hours after the lights were turned on, 2 hours after the lights were turned off, and 8 hours later at 4 hours a day, 4 to 6 mice were taken out of the cage from each group, and laparotomy was performed under anesthesia. Then, blood was collected from the lower aorta with an injection needle, transferred to a capillary blood collection tube (Terumo), centrifuged at 7000 G for 10 minutes, and the supernatant was used as a plasma sample. Plasma was stored at -80 ° C until measurement.
According to Yoshida, Futaki et al.'S original chemical pretreatment method described in Patent Document 1, linoleic acid-derived oxidation products are comprehensively converted to hydroxylinoleic acid (HODE), and cholesterol-derived oxidation products are converted to hydroxycholesterol (t7- OhCh). The product was quantified using LC-MS / MS and GC-MS, respectively.
As a result, the concentrations of linoleic acid and cholesterol in the blood remained almost unchanged throughout the day in both wild type and clock / clock mice (FIG. 1).
On the other hand, the HODE of the linoleic acid-derived oxidation product was low in the light period, and high circadian variation in blood was observed in the dark period (FIG. 2).
From this, it was found that HODE was observed as a marker, and the fluctuation of daily oxidative stress in the blood could be precisely detected even by sampling every 6 hours. Moreover, since the fluctuation is also observed in clock / clock mice in which the expression variation of the circadian clock gene is not observed, the circadian variation of oxidative stress is detected by the conventional gene expression analysis method that detects the expression pattern of the clock gene. It turns out that it is a type of metabolic rhythm that cannot be done. T7-OhCh, which is known as the same oxidative stress marker as HODE, shows the same tendency as HODE, but HODE alone is expected to show sufficiently accurate results due to the smaller S / N ratio of amplitude compared to HODE. It was. 7-OhCh can be expected to serve as a marker that complements HODE (Figure 2).

(Example 2) Examination of other oxidative stress markers For isoprostane and arachidonic acid-derived lipid oxide (HETE), which have been quantified so far for the evaluation of oxidative stress, Similarly, daily fluctuations were observed.
However, since isoprostan has a low detection level, no statistically significant diurnal variation could be observed (FIG. 3). Similarly, the hydroxy acid HETE could not detect any significant diurnal variation (FIG. 3).

(Example 3) Correlation between circadian rhythm of oxidative stress and eating rhythm The correlation between circadian rhythm of oxidative stress and eating rhythm was observed. Divide 48 wild-type mice (ICR) into 2 groups, and the feeding time is limited to 6 hours (DF) from 3 hours after lighting up or 6 hours (NF) from 3 hours after lighting off under light and dark conditions. Reared for 2 weeks. Under this condition, the mouse wakes up and eats food at the feeding time (FIG. 4). In the case of NF and DF, the food consumption increases while food is presented. As for the free behavior pattern, the behavior of predicting food (predictive behavior) is observed before the presentation of food in the case of restricted feeding, but most free behavior including the predictive behavior is recognized at night in NF. On the other hand, DF shows a bi-directional behavior in which the amount of free behavior similar to the predictive behavior in the light period is also observed in the dark period (FIG. 4). Two weeks later, as in Example 1, 2 hours and 8 hours after the lights were turned on, 2 hours after the lights turned off, and 8 hours later, each group of mice was taken from the cage 6 One mouse was taken out and blood was collected. When HODE of linoleic acid-derived oxide HODE was measured in the same manner as in Example 1, the amount of HODE in the blood was a unilateral cycle that increased in conjunction with the feeding rhythm, and DF and NF were in conflict with each other. (FIG. 5). Furthermore, in DF, the bi-directionality of free behavior rhythm and the quantitative change of HODE are not linked, so it is thought that it reflects the rhythm of eating behavior rather than free behavior. This indicates that HODE is a biomarker that can detect the daily rhythm of oxidative stress linked to behavioral rhythm represented by feeding rhythm.

(Example 4) Change in HODE rhythm due to fasting Changes in the daily rhythm of oxidative stress due to fasting were observed. Wild-type mice (ICR) are raised for one week with free feeding, and then the food is removed and fasting is started at the same time as the lights are turned on. Blood was collected 2, 8, 14, and 20 hours after the start of fasting (fasting conditions). 24 hours after fasting, the food was fed again and blood was collected 5 hours later (refeeding conditions). When the linoleic acid-derived oxide HODE was measured in the same manner as in Example 1, no HODE recognized by free feeding was observed during fasting, and the HODE did not change at a low value below the daily minimum. However, when feeding behavior was observed after refeeding, the amount of tHODE increased in response. During fasting, 7-OhCh after refeeding also shows similar changes to HODE (Figure 6).

Claims (6)

Measure hydroxyoctadecadienoic acid (HODE) in blood or 7-hydroxycholesterol (7-OHCh) together with HODE for at least 1 day, multiple times over time. A method for monitoring diurnal changes in eating rhythm, characterized by observing.   The method according to claim 1, wherein the HODEs include at least one selected from the group consisting of 9-HODE and 13-HODE.   Measure hydroxyoctadecadienoic acid (HODE) in blood or 7-hydroxycholesterol (7-OHCh) together with HODE for at least 1 day, multiple times over time. A method of observing the circadian rhythm of oxidative stress response in the body, characterized by observing. A step of administering a test substance to an experimental animal, measuring 7-OHCh together with HODEs or HODEs in blood at least one day over time, and observing changes in the measured values over time; the over time measurements of changes observed including the step of comparing the change over time measurements that when not administered test substance, the measurement values in the case of administration of the test substance is not administered the test substance A method for screening an oxidative stress response-improving substance, characterized in that it is determined that the oxidative stress response has been improved by a decrease in comparison with the above measured value . A method for determining an effective administration time of an oxidative stress-improving substance, comprising the following (1) to (4);
(1) A step of administering a test oxidative stress-improving substance to a laboratory animal at a plurality of times, and measuring 7-OHCh together with HODEs in blood immediately after administration, or HODEs at each administration time,
(2) Compare the measured value when measuring at the same time without administering the test oxidative stress-improving substance in advance and the measured value at each time of administration obtained in (1) above. , Calculating the difference between the two as the amount of change at each time,
(3) A step of comparing the amount of change at each administration time obtained in (2) above, and determining the administration time at which the amount of change is maximum,
(4) A step of determining the administration time when the amount of change obtained in the step (3) is maximized as an effective administration time of the test oxidative stress-improving substance. Is intended to predict the objectivity to eating time, the method according to claim 1 or 2.

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