JP6830578B2 - Autism spectrum disorder determination method, autism spectrum disorder determination kit and measurement data acquisition method - Google Patents

Description

The present invention relates to a method for determining autism spectrum disorder and a kit for determining an autism spectrum disorder, which comprises measuring the active oxygen scavenging activity contained in a biomaterial derived from a subject.

Autistic Spectrum Disorder (hereinafter referred to simply as ASD) refers to five pervasive developmental disorders that have traditionally been autistic disorder, Asperger's disorder, pediatric disintegration disorder, let's disorder, and unspecified. In the 5th revised edition (DSM-5) of the American Psychiatric Association Diagnostic Criteria announced in May, the four categories excluding Let's disorder, whose genetic factors are clear, were newly classified as one continuous comprehensive concept. It is a name. Peculiarities are observed in communication skills, sociality, imagination, and limited and repetitive patterns in behavior, interests, and activities. In Europe and the United States, about 1 in 68 people is reported to have ASD, which is a frequent developmental disorder. It is effective to start "rehabilitation" for developmental disorders including ASD earlier, and it is preferable to be able to start "rehabilitation" from around 2 years old when the symptoms of ASD first appear to school.

Currently, the diagnostic criteria in DSM-5 are used for the diagnosis of ASD. This criterion is based on observations of persistent obstacles in social communication and interrelationships and limited repetitive behaviors, interests, activities, etc., and is not an objective or biological examination criterion. In addition, preschoolers are originally underdeveloped in language expression, etc., and highly specialized judgment is required by doctors with abundant experience in child and adolescent psychiatry, which is one of the factors that make early detection of ASD difficult. It has become.

For early detection of ASD, a method using an antibody against a specific biomarker contained in a biological sample derived from a mother as an index has also been developed. Specifically, from lactate dehydrogenase, guanine deaminoenzyme, collapsin reaction-mediated protein 1, dihydropyrimidinase-like protein 2, stress-induced phosphoprotein 1, and Y-box binding protein 1 contained in the biological sample. An antibody against one or more polypeptides selected from the above group is used as an index, and it is possible to determine the possibility that a fetus born by the mother will develop ASD (Patent Document 1).

There is also a report that the cause of ASD is linked to oxidative stress in infancy and abnormalities in active oxygen scavenging activity (Non-Patent Document 1). Abnormalities of oxidative stress include (1) decrease in glutathione, decrease in activity of glutathione peroxidase, which is a metabolism-related enzyme, decrease in methionine and cysteine, which are metabolism-related substances, and increase in oxidized glutathione, regarding (1) glutathione system abnormality. It is said that there are 2) abnormalities in C677T allele in the methylenetetrahydrofolate reductase gene (MTHR), and (3) abnormalities in vitamin A, vitamin B6, vitamin C, vitamin D, and vitamin E.

Japanese Patent No. 5823964

Frustaci A, etc. "Oxidative stress-related biomarkers in analysis: systematic review and meta-analysis." Free Rad. Biol. Med. Vol. 52 (2012), p2128-2141 Kota Tamada et al., "Development of Human Model Mouse for Autism", 2011, Biochemistry Vol. 83, No. 9, p841-845

The method shown in Patent Document 1 estimates the possibility that a woman gives birth to an ASD child regardless of whether or not she is pregnant, and determines whether or not the child born by the woman actually has ASD. is not it. If the clinical diagnosis of ASD relies solely on observations such as social communication and persistence of interrelationships, it may not be possible to respond to early detection of ASD in infants with insufficient language ability development. In addition, it may be difficult for a hearing-impaired person or a visually-impaired person to diagnose ASD. Therefore, if there is a development of a method capable of objectively determining the possibility of suffering from ASD using a biomaterial, it is possible for a hearing-impaired person or a visually impaired person to determine ASD, which is convenient.

Further, as shown in Non-Patent Document 1, there is a report that ASD is associated with oxidative stress, but there is no method for determining the possibility of ASD morbidity based on active oxygen scavenging activity. Active oxygen includes hydroxyl radical, superoxide, alkoxy radical, peroxy radical, singlet oxygen and the like, and there is also a problem that it is not easy to measure radicals in particular. This is because it is not easy to evaluate complex oxidative stress-related reactions in multiple ways, and it is also difficult to specify sufficient sensitivity, specificity, and likelihood ratio that can be used for clinical diagnosis. Therefore, it is desired to develop a method for clarifying the relationship between various active oxygen scavenging abilities and ASD and easily determining the possibility of ASD morbidity based on the scavenging activity of various active oxygens of the biomaterial derived from the subject.

Further, it is desired to develop a method capable of easily determining the possibility of ASD morbidity based on the scavenging activity of various active oxygens of the biomaterial derived from the subject, and a determination kit.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for determining the possibility of ASD morbidity using the active oxygen scavenging activity contained in the biomaterial of a subject as an index.

Another object of the present invention is to provide a kit for determining ASD.

According to the spin trap method by electron spin resonance, the present invention relates to active oxygen species such as hydroxyl radical, alkoxy radical, superoxide, alkoxy radical, hydroxyl radical, peroxy radical, and singlet oxygen contained in the biological material derived from the object. We have found that it is possible to measure scavenging activity and that there is a certain relationship between these measured values and ASD-affected persons, and completed the present invention.

That is, the method for determining ASD according to the first aspect of the present invention is at least two or more active oxygens selected from the group consisting of hydroxyl radical scavenging activity, superoxide scavenging activity and alkoxy radical scavenging activity of the biological material derived from the subject. measurements of scavenging activity, the comparison with the determination values corresponding to the active oxygen scavenging activity, based on the comparison result, seen including a determination step of determining the likelihood that said subject is suffering from ASD, the determining step Then, the above comparison result is the judgment requirement listed below.
When the measured value of the hydroxyl radical scavenging activity is included, the first determination requirement that the measured value is less than the determination value of the hydroxyl radical scavenging activity,
When the measured value of the superoxide scavenging activity is included, the second determination requirement that the measured value is equal to or higher than the determination value of the superoxide scavenging activity, and
When the measurement value of the alkoxy radical scavenging activity is included, it indicates whether or not the third determination requirement that the measurement value is equal to or higher than the determination value of the alkoxy radical scavenging activity is satisfied.
When any two or more of the first determination requirement, the second determination requirement, and the third determination requirement are satisfied, it is determined that the subject may be suffering from ASD.
The determination value is determined in advance based on the measured values of the active oxygen scavenging activity in the typical development group and the ASD group, and if there is a statistically significant difference between the typical development group and the ASD group, it is set as a cutoff value.
If there is no statistically significant difference between the ASD group and the neurotypical development group, a predetermined confidence interval is set and an equivalence test is performed, and the upper and lower limits indicating equivalence without superiority are determined respectively. characterized by a.

Moreover, the active oxygen scavenging activity further seen containing a peroxy radical scavenging activity and / or singlet oxygen scavenging activity, the determining step further, if it contains a measure of the peroxy radical scavenging activity, the measurement value is the If the fourth determination requirement that the peroxy radical scavenging activity is between the two determination values and / or the measurement value of the singlet oxygen scavenging activity is included, the measurement value is the two determination values of the singlet oxygen scavenging activity. Including the fifth judgment requirement that it is between the judgment values,
When the fourth determination requirement or the fifth determination requirement is satisfied, the subject is determined to be more likely to have ASD .

Wherein as the determination Engineering is the measured value of the respective active oxygen scavenging activity, as compared to the corresponding one or more of the decision value, allocating a score to each of the active oxygen scavenging activity on the basis of the comparison result, the ASD It is characterized in that the susceptibility to morbidity is obtained as the total value of the scores .

Further, each active oxygen scavenging activity is measured by a spin trap method by electron spin resonance.

The kit for determining ASD according to the second aspect of the present invention comprises two or more active oxygen species selected from the group consisting of hydrogen peroxide, riboflavin, and 2,2'-azobis [2-aminodipropane] dihydrochloride. It is characterized by containing a generator and a spin trap agent as a reagent for measuring active oxygen scavenging activity contained in a biomaterial derived from a subject .

The measurement data acquisition method according to the third aspect of the present invention has at least two or more active oxygen scavenging activities selected from the group consisting of hydroxyl radical scavenging activity, superoxide scavenging activity and alkoxy radical scavenging activity of the biological material derived from the subject. , And the measured values of the active oxygen scavenging activity of the peroxyradical scavenging activity and / or the single-term oxygen scavenging activity are compared with the determination values corresponding to the respective active oxygen scavenging activities, and based on the comparison result, the subject becomes ASD. Includes measurement data processing steps to determine possible morbidity
In the measurement data processing step, the comparison results are based on the conditions listed below.
When the measured value of the hydroxyl radical scavenging activity is included, the first condition that the measured value is less than the determination value of the hydroxyl radical scavenging activity,
When the measured value of the superoxide scavenging activity is included, the second condition that the measured value is equal to or higher than the determination value of the superoxide scavenging activity.
When the measured value of the alkoxy radical scavenging activity is included, the third condition that the measured value is equal to or higher than the determination value of the alkoxy radical scavenging activity.
When the measured value of the peroxy radical scavenging activity is included, the fourth condition that the measured value is between the two determination values of the peroxy radical scavenging activity, and / or
When the measured value of the singlet oxygen scavenging activity is included, the fifth condition that the measured value is between the two determination values of the singlet oxygen scavenging activity is included.
The subject may be suffering from ASD if any two or more of the first condition, the second condition and the third condition are satisfied, and if the fourth condition or the fifth condition is satisfied. It is a measurement data acquisition method for determining that the property is high.
The determination value is determined in advance based on the measured values of the active oxygen scavenging activity in the typical development group and the ASD group, and if there is a statistically significant difference between the typical development group and the ASD group, it is set as a cutoff value.
If there is no statistically significant difference between the ASD group and the neurotypical development group, a predetermined confidence interval is set and an equivalence test is performed, and the upper and lower limits indicating equivalence without superiority are determined respectively. characterized by a.

According to the present invention, the possibility of ASD can be objectively determined by measuring a plurality of active oxygen scavenging abilities contained in a biomaterial derived from a subject and comparing them with preset determination values. ..

It is a figure which shows the comparison between the typical development group of various active oxygen scavenging activity measured in Example 1 and the ASD group. It is a figure which shows the relationship between the prior probability and posterior probability of hydroxyl radical, alkoxy radical, and superoxide in which a statistically significant difference was detected in Example 1.

Hereinafter, the present invention will be described in detail.
(1) Measurement of active oxygen scavenging activity In the method for determining ASD according to the present invention, the measured value of each active oxygen scavenging activity is used. Reactive oxygen is a name when oxygen is changed to a more reactive molecular species such as superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen. Free radicals are molecules that have unpaired electrons, such as superoxide and hydroxyl radicals. Active oxygen scavenging activity includes hydroxyl radical scavenging activity, alkoxy radical scavenging activity, and superoxide scavenging activity. Furthermore, it is preferable to measure the peroxy radical scavenging activity and / or the singlet oxygen scavenging activity. Reactive oxygen species are highly reactive and have a short stable existence time (life) at normal temperature and pressure, but they are measured by the spin trap method using an electron spin resonance device (hereinafter, simply referred to as ESR). be able to. A sample is added to a solution capable of generating a certain amount of active oxygen, and if the amount of active oxygen generated decreases, it can be evaluated as having active oxygen scavenging activity.

In the ESR spin trap method, a spin trap agent for converting reactive oxygen species into stable and long-lived free radicals is added to the sample. The spin trap agent reacts with unstable active oxygen to generate spin adduct, which is a more stable free radical, and can be detected by ESR. For example, there is CYPMPO (5- (2,2-dimethyl-1,3-propoxycyclophosphoryl) -5-methyl-1-pyrroline-N-oxide) as a spin trapping agent that can be used. When CYPMPO is used, hydroxyl radicals, superoxides, alkoxy radicals, peroxy radicals and the like react with CYPMPO to generate their respective spin adducts. Reactive oxygen species can be distinguished from the characteristics of the ESR spectrum of spin adduct, and their scavenging activity can be quantified. In this way, the presence or absence of free radicals can be confirmed and quantified by using ESR. In addition, although singlet oxygen is not a free radical, ESR is used by using 4-hydroxy-2,2,6,6-tetramethylpiperidine (hereinafter referred to as 4-OH-TEMP) as a spin trapping agent. Quantification is possible. The spin trapping agent that can be used for measuring the active oxygen scavenging activity of the present invention is not limited to the above as long as a stable spin adduct can be generated from unstable free radicals and singlet oxygen. Further, the active oxygen generator is not particularly limited.

The scavenging activity of active oxygen can be evaluated by a considerable amount of scavenging activity by a predetermined antioxidant. As an example, a method for measuring hydroxyl radical scavenging activity is shown below. The peak height (I0) of the hydroxyl radical-derived spin adduct contained in the free radical generation solution containing no sample is quantified, and then the sample is added to the free radical generation solution to similarly obtain the hydroxyl radical-derived spin adduct. The hydroxyl radical scavenging activity ratio can be obtained by quantifying the peak height (IS) and calculating I0 / (IS-1). On the other hand, using an antioxidant having high specificity for active oxygen to be measured in advance instead of the sample (glutathione (hereinafter referred to as GSH) corresponds to this in this example), the same as above. Manipulate to prepare a calibration curve of GSH amount and hydroxyl radical scavenging activity ratio. By using this calibration curve, the hydroxyl radical scavenging activity ratio of the sample can be calculated as a GSH equivalent amount. The amount equivalent to GSH thus obtained is referred to as "hydroxy radical scavenging activity". For other active oxygen scavenging activities, a corresponding amount of the corresponding scavenging activity evaluator is calculated and used as the scavenging activity.

In the ESR spin trap method, a specific active oxygen species can be generated by changing the substance that generates active oxygen. For example, hydrogen peroxide and ultraviolet light irradiation can be combined to generate hydroxyl radicals. The type of such a substance or electromagnetic wave can be appropriately selected according to each active oxygen. Further, the scavenging activity evaluation substance such as GSH used for preparing the calibration curve is not particularly limited as long as the quantitativeness of each active oxygen surface specific activity can be ensured. As the scavenging activity evaluator, in addition to GSH, superoxide dismutase (hereinafter referred to as SOD), trolox, α-lipoic acid and the like can be used.

(2) Judgment value The "judgment value" is a numerical value for allocating the measured value of the target active oxygen scavenging activity to a predetermined category. One or more can be set for each active oxygen scavenging activity.

When the hydroxyl radical scavenging activity of the typical development group and the ASD group was compared, as shown in Example 1 described later, the ASD group had a statistically significant difference of p <0.05, which was lower than that of the typical development group. On the other hand, the superoxide scavenging activity was higher than that of the neurotypical development group with a statistically significant difference of p <0.01, and the alkoxy radical scavenging activity was also higher than that of the neurotypical development group with a statistically significant difference of p <0.05. there were. Furthermore, when the peroxyradical scavenging activity and the singlet oxygen scavenging activity were compared between the typical development group and the ASD group, the peroxyradical scavenging activity and the singlet oxygen scavenging activity were p <0.05 with the typical development group. No statistically significant difference was detected, but equivalence was observed within a predetermined range. Therefore, when it was examined whether it is possible to determine ASD morbidity using various active oxygen scavenging activities as indicators, a determination value was set for each active oxygen scavenging activity, and the measured values were classified by the determination value to determine ASD. It has been found that the likelihood of morbidity can be determined.

(3) Judgment using judgment requirements This method includes a judgment step of comparing a measured value derived from a target with a judgment value and determining the possibility that the target has ASD based on the comparison result. In the determination step, the comparison result is (i) the first determination requirement that the measured value of the hydroxyl radical scavenging activity of the target is less than the determination value of the hydroxyl radical scavenging activity, and (ii) the measured value of the superoxide scavenging activity of the target is super. When any two or more of the second judgment requirement that the judgment value of the oxide scavenging activity or more and the third judgment requirement that the measured value of the alkoxy radical scavenging activity of the target (iii) is equal to or more than the judgment value of the alkoxy radical scavenging activity are satisfied. It is determined that the subject may have ASD. In addition to the above-mentioned first to third determination requirements, when (iv) a measurement value of peroxy radical scavenging activity is included, a fourth determination that the measurement value of the target is between the two determination values of peroxy radical scavenging activity. One of the judgment requirements and (v) a fifth judgment requirement that the measured value of the target is between the two judgment values of the singlet oxygen scavenging activity when the measurement value of the singlet oxygen scavenging activity is included. If one or more are satisfied, it is judged that there is a high possibility of having ASD.

The judgment values used for the above judgment requirements are those obtained by measuring the active oxygen scavenging activity of each of the biomaterial derived from the ASD group and the biomaterial derived from the typical development group, and the measured values are statistical for the typical development group and the ASD group. If there is a significant difference, the cutoff value can be obtained and used as the determination value. If there is no statistically significant difference between the ASD group and the neurotypical development group, a predetermined confidence interval is set and an equivalence test is performed to determine the upper and lower limits that show equivalence without superiority. It can be used as a value. For example, a section to be regarded as equivalent may be set based on the 95% confidence interval of both groups, and this section may be used to determine whether or not the active oxygen scavenging activity of a subject is equivalent. The sections considered to be equivalent can be set arbitrarily, but for example, a section including the entire 95% confidence interval of the typical development group and the ASD group with a predetermined margin, or an average when the typical development group and the ASD group are grouped together. The value ± 2SD may be set as an upper limit value and a lower limit value. In addition to this, if the possibility of ASD morbidity can be determined based on the measured value of each active oxygen scavenging activity, the determination value may be set by using another method.

When the above determination requirement is introduced, the possibility of ASD can be determined simply by dividing the target measurement value into two categories, one having the determination requirement and the other not having the determination requirement. Table 1 exemplifies the judgment value and the classification according to the judgment value. If the target-derived hydroxyl radical scavenging activity is less than the judgment value H, it is assigned to category A, if it is H or more, it is assigned to category B, and if the target-derived peroxy radical scavenging activity is between the judgment values Pn and Pm, it is classified into category B. Allocated to A. In this specification, the range indicated by Pn to Pm or the like includes the lower limit value and the upper limit value.

When the hydroxyl radical scavenging activity, the superoxide scavenging activity, and the alkoxy radical scavenging activity are measured in determining the possibility of ASD morbidity, the hydroxyl radical scavenging activity is of high importance, so that the hydroxyl radical scavenging activity is less than the above-mentioned determination value ( The case where the first determination requirement is satisfied) and the superoxide scavenging activity is equal to or higher than the determination value (satisfying the second determination requirement) is determined as a "suspicious example", and the alkoxy radical scavenging activity is further determined. A case where the value is equal to or greater than the value (satisfying the third determination requirement) may be determined as a “certain example”. By providing "suspicious cases" and "certain cases", the possibility of ASD morbidity can be determined more reliably.

(4) Judgment by score Each active oxygen scavenging activity can be assigned to each category according to the determination value, a score can be assigned to each category, and the total score can be used to determine the possibility of ASD. The measured value of each active oxygen scavenging activity is compared with one or more of the determination values corresponding to each active oxygen scavenging activity and distributed to any category, and from the total score of each active oxygen scavenging activity, Determine the likelihood of morbidity with ASD.

The score can be set for each category of each active oxygen scavenging activity. Table 2 shows an example with a score. The scores indicated by X ₁ , X ₂ , ... X ₁₁ may be the same or different. Table 2 shows an example in which two judgment values are introduced into the hydroxyl radical scavenging activity and the measured values of the target are divided into three. The number of divisions may be the same or different for each active oxygen. In Table 2, five types of active oxygen were targeted, but at least two types of hydroxyl radical, superoxide and alkoxy radical may be used, and the number is not limited to five.

The score assigned to each category is determined according to the purpose of determination and the usage situation by referring to the importance of the relationship between each active oxygen scavenging activity value and ASD, the sensitivity, specificity, positive likelihood ratio, etc. shown in Example 1. can do. For example, a high score value can be introduced if false positive detection is desired to be avoided, and a low score value can be introduced if false negative detection is desired to be avoided. In addition, the active oxygen scavenging activity can be measured in advance using biomaterials of the ASD group and the typical development group, and a score capable of determining the possibility of ASD morbidity according to the purpose of determination and the usage situation can be appropriately selected.

As the determination value to be used, the determination value used for the above-mentioned determination requirement may be used, or a different determination value may be used. In advance, each active oxygen scavenging activity is measured using biological materials of the ASD group and the typical development group, the measured values are classified using predetermined numerical values, and the sensitivity, specificity, positive likelihood ratio, etc. are calculated. A numerical value capable of determining the possibility of ASD morbidity according to the determination purpose and usage status can be selected as the determination value.

In Table 2, when the maximum value of the total score of the biomaterial derived from the subject is, for example, 13, a total score of 8 or more can be set as ASD disease positive or the like according to the total score. Further, based on the total score, it may be determined that less than 5 is negative, 5 or more and less than 6 is false negative, 6 or more and less than 8 is false positive, and 8 or more is positive. The score is not limited to numerical values, and qualitative signs such as "+" and "-" may be used, and the possibility of ASD is determined according to the number of "+" and "-". You may judge.

(5) Target The target for determining ASD may be not only humans but also rodents such as mice, rats and guinea pigs, and primates such as monkeys. These may be used as model animals for ASD as shown in Non-Patent Document 2. The determination method of this method can be applied as an index for constructing a model animal. If the subject is a human, there is no age limit. Infants and children with underdeveloped language skills can be included.

Biological materials that can be used in this method include blood such as peripheral blood, venous blood, and arterial blood, body fluids such as tears, sweat, lymph, and spinal fluid, cell tissues such as hair roots, skin, and oral mucosa, urine, saliva, and stool. There is excrement. The blood may be whole blood or serum.

(6) Judgment kit, etc. The ASD determination kit of the present invention contains an active oxygen generator and a spin trap agent as reagents for measuring active oxygen scavenging activity contained in a biomaterial derived from a target. Examples of the active oxygen generator include hydrogen peroxide, riboflavin, 2,2'-azobis [2-aminodipropane] dihydrochloride (hereinafter referred to as AAPH), and t. There are -butylhydroperoxide (hereinafter referred to as t-BuOOH) and rosebengal, and spin trapping agents include CYPMPO and 4-OH-TEMP, but the method for generating active oxygen and spin trapping agents are limited to these. It's not something.

It is also possible to construct a determination device incorporating the determination method of the present invention. A storage device for storing the measured value from the ESR, an analysis circuit for comparing the measured value with the determined value and determining whether or not the determination requirement is satisfied, and an output device for outputting the result may be connected in succession. A storage device, an analysis circuit, an output device, or the like may be arranged in the ESR.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the present invention is not limited thereto. The active oxygen scavenging activity was measured by the following method, and the sensitivity, specificity, and positive likelihood ratio were calculated by the following method.

(I) Method for measuring active oxygen scavenging activity Various active oxygen scavenging activities are measured by the active oxygen generation method shown in Table 3 and the spin trap method by ESR using a spin trapping agent, and the scavenging activity shown in Table 3 is used. Converted to a considerable amount of the evaluation substance.

(1) Measurement of hydroxyl radicals Hydrogen peroxide and CYPMPO were added to ultrapure water as active oxygen generators to a final concentration of 10 mM. This was introduced into a measurement cell set in an ESR (trade name "RRX-1X") cavity manufactured by Radical Research Co., Ltd., and irradiated with ultraviolet light having a wavelength of 300 to 400 nm for 5 seconds to generate hydroxyl radicals. .. The generated free radicals were spin-trapped with CYPMPO to generate CYPMPO-OH, and the wave height of the CYPMPO-OH signal was measured with ESR analysis software (trade name "WIN-RAD" manufactured by Radical Research Co., Ltd.). Let this signal value be (I0).
Then, the sample was added to a solution containing each of the above hydrogen peroxide and CYPMPO at a final concentration of 10 mM so as to have a final concentration of 10%, and the same procedure as above was performed to measure the wave height of the CYPMPO-OH signal. This signal value was defined as (IS), and the value of I0 / (IS-1) was calculated.
On the other hand, GSH was used as an antioxidant in place of the above sample, hydrogen peroxide and CYPMPO were added to a solution containing a final concentration of 10 mM, and the same procedure as above was performed to measure the wave height of the CYPMPO-OH signal. The signal value was (IG). Measurements were made with multiple concentrations of GSH, and the GSH concentration was plotted on the X-axis and IO / (IG-1) on the Y-axis to create a calibration curve. Using this calibration curve, I0 / (IS-1), which is the hydroxyl radical scavenging activity of the sample, was calculated with an equivalent amount of the corresponding GSH.

(2) Measurement of superoxide Measurement of hydroxyl radical in (1) except that riboflavin having a final concentration of 20 μM was added instead of hydrogen peroxide, and visible light having a wavelength of 500 to 600 nm was irradiated for 60 seconds instead of ultraviolet light irradiation. The signal value (IS) was measured and I0 / (IS-1) was calculated. Next, a calibration curve was prepared in the same manner as in the measurement of hydroxyl radical in (1) except that superoxide dismutase (SOD) was used instead of GSH, and I0 /, which is the superoxide scavenging activity of the sample. (IS-1) was calculated with a corresponding amount of SOD.

(3) Measurement of alkoxy radicals The same procedure as the measurement of hydroxyl radicals in (1) was performed except that AAPH having a final concentration of 1 mM was added instead of hydrogen peroxide, and the signal value (IS) was measured, and I0 / (IS-1) was calculated. Next, a calibration curve was prepared by operating in the same manner as in the measurement of hydroxyl radicals in (1) except that trolox was used instead of GSH, and I0 / (IS-1), which is the alkoxy radical scavenging activity of the sample, was dealt with. It was calculated by the equivalent amount of hydroxylox.

(4) Measurement of peroxy radical The signal value (IS) was measured and the signal value (IS) was measured by operating in the same manner as the measurement of hydroxyl radical in (1) except that t-BuOOH having a final concentration of 10 mM was added instead of hydrogen peroxide. I0 / (IS-1) was calculated. Next, a calibration curve was prepared by operating in the same manner as in the measurement of hydroxyl radical in (1) except that α-lipoic acid was used instead of GSH, and I0 / (IS-1), which is the peroxy radical scavenging activity of the sample. Was calculated with the equivalent amount of the corresponding lipoic acid.

(5) Measurement of singlet oxygen Rosebengal with a final concentration of hydrogen peroxide of 200 μM was added, and visible light with a wavelength of 500 to 600 nm was irradiated for 60 seconds instead of ultraviolet light, and 4-OH-TEMP was used instead of CYPMPO. Except for the above, the procedure was the same as for the measurement of hydroxyl radical in (1), the signal value (IS) was measured, and I0 / (IS-1) was calculated. Next, a calibration curve was prepared by operating in the same manner as in the measurement of hydroxyl radical in (1), and I0 / (IS-1), which is the singlet oxygen scavenging activity of the sample, was calculated with the corresponding amount of GSH.

(II) Method of calculating judgment values When the neurotypical development group and the ASD group are classified by predetermined judgment values, TN (true negative), TP (true positive), FN (false negative) and FP (false negative) are shown in Table 4. It is divided into 4 categories (positive). TN is a neurotypical development group in which the hydroxyl radical scavenging activity exceeds the cutoff value, and is a neurotypical development group in which the superoxide and alkoxy radical scavenging activities are lower than the cutoff value, and the neurotypical development group is determined to be normal. It is a division. Further, TP is an ASD group having a hydroxyl radical scavenging activity lower than the cutoff value, and an ASD group having a superoxide and alkoxy radical scavenging activity exceeding the cutoff value, and the ASD group is determined to be ASD. Is. On the other hand, FN is a category that is determined to be normal even though it is ASD, and FP is a category that is determined to be ASD even though it is normal.

Sensitivity (SN) means the probability that the ASD group will be positive, and is calculated by SN = TP / (TP + FN). The specificity (SP) is the probability that the typical development group becomes negative, and is calculated by SP = TN / (FP + TN). The positive likelihood ratio (LR +) is the ratio of the ratio of true positives (TP) to the ratio of false positives (FP), and LR + = (TP / (TP + FN)) / (FP / (FP + TN)) = SN / It is calculated by (1-SP).

When there is a significant difference between the typical development group and the ASD group, the receiver operating characteristic curve (ROC curve) is plotted by plotting (1-SP) on the X-axis and sensitivity (SN) on the Y-axis with the cutoff value as a variable. Was created, and the Youden Index with the maximum value of (SN / (1-SP)) was specified as the cutoff value. The area under the curve of the ROC curve is referred to as AUC (area under curve). If a difference is detected in the test results between the neurotypical development group and the ASD group in a predetermined test method, the AUC will be 0.5 to 1.0. When the AUC is 0.9 to 1.0, the predetermined inspection method is considered to have high accuracy, and when 0.7 to 0.9, the accuracy is considered to be moderate.

On the other hand, if there is no significant difference between the neurotypical development group and the ASD group, a section to be regarded as equivalent is set based on the 95% confidence interval of both groups, and the active oxygen scavenging activity of a subject using this section is equivalent. It was judged whether or not.

(III) Calculation of posterior probabilities Prior probabilities are a type of conditional probabilities that express what is known about a variable as probabilities, and are probabilities that are considered to be ASD before the method is used. is there. The posterior probability is a probability of having ASD when it is determined to be positive by using the method after determining the prior probability, and is, for example, a probability of having ASD when it is determined to be positive by using the method.

In addition, the posterior odds can be obtained by calculating the prior odds from the prior probability and multiplying the prior odds by the positive likelihood function, and the posterior probability can be obtained from the posterior odds. Prior odds are represented by "prior probabilities / (1-posterior probabilities)", and posterior odds are represented by "posterior probabilities / (1-posterior probabilities)". The positive likelihood ratio is the ratio of the likelihood when the test is positive, and the larger the ratio, the better the disease confirmation method.

When the reaction mechanism to be measured is independent, the positive likelihood ratio at the time of calculating the ex-post odds can be calculated by the product of the positive likelihood ratios based on each mechanism. For example, when the scavenging mechanisms of active oxygen species are independent, the positive likelihood ratio A based on the active oxygen scavenging ability A and the positive likelihood ratio B based on the active oxygen scavenging ability are obtained, and the post-odds = positive likelihood. It can be calculated as ratio A × positive likelihood ratio B × pre-odds.

(Example 1)
Peripheral blood was collected from ASD (n = 22-23) and neurotypical infants (n = 33-34) under the age of 5 who were examined by a child and adolescent psychiatrist. Hydroxyl radical scavenging activity, superoxide scavenging activity, alkoxy radical scavenging activity, peroxy radical scavenging activity and singlet oxygen scavenging activity contained in biological components were measured by the spin trap method by ESR described above.

The results of comparison between groups by t-test of various active oxygen scavenging activities are shown in FIG. As shown in FIG. 1, the hydroxyl radical scavenging activity of the ASD group was significantly lower than that of the typical development group. On the other hand, the superoxide and alkoxy radical scavenging activities were significantly increased. There was no difference in scavenging activity for peroxy radicals and singlet oxygen between the two groups.

Statistically significant differences were detected between the ASD group and the neurotypical development group in the scavenging activity of hydroxyl radicals, superoxide, and alkoxy radicals, and the cutoff value was used as the determination value. Reactive oxygen species that do not show a statistically significant difference, that is, peroxy radicals and singlet oxygen, are assumed to be between the intervals considered to be equivalent set based on the 95% confidence interval as the judgment requirement, and the judgment value is The upper and lower limits of the section were set. Table 5 shows the determination values, determination requirements, sensitivity, specificity, positive likelihood ratio, AUC, and 95% confidence intervals for each group.

Regarding hydroxyl radical scavenging activity, when the ASD group is less than 5.3 mM GSH equivalent Sensitivity (SN) = TP / (TP + FN) = 13/22 = 0.59
Specificity (SP) = TN / (TN + FP) = 26/33 = 0.79
Positive likelihood ratio = SN / (1-SP) = (13/22) / (7/33) = 2.79
AUC = 0.85

Regarding superoxide scavenging activity, when the ASD group is equivalent to 9.35 U / ml SOD or more, sensitivity (SN) = TP / (TP + FN) = 16/23 = 0.70
Specificity (SP) = TN / (TN + FP) = 32/33 = 0.97
Positive likelihood ratio = SN / (1-SP) = (16/23) / (1/33) = 23.0
AUC = 0.87

Regarding the alkoxy radical scavenging activity, when the ASD group is equivalent to 1.95 mM trolox or more, sensitivity (SN) = TP / (TP + FN) = 15/23 = 0.65
Specificity (SP) = TN / (TN + FP) = 25/34 = 0.74
Positive likelihood ratio = SN / (1-SP) = (15/23) / (9/34) = 2.46
AUC = 0.86

Regarding peroxy radical scavenging activity, when the ASD group is equivalent to 0.24 to 1.08 mM α-lipoic acid, sensitivity (SN) = TP / (TP + FN) = 18/23 = 0.78
Specificity (SP) = TN / (TN + FP) = 23/33 = 0.70
Positive likelihood ratio = SN / (1-SP) = (18/23) / (10/33) = 2.58

Regarding singlet oxygen scavenging activity, when the ASD group is equivalent to 13 to 21 μMGSH Sensitivity (SN) = TP / (TP + FN) = 9/23 = 0.39
Specificity (SP) = TN / (TN + FP) = 27/33 = 0.81
Positive likelihood ratio = SN / (1-SP) = (9/23) / (6/33) = 2.15

FIG. 2 shows the relationship between the prior probability and the posterior probability based on the hydroxyl radical scavenging activity measurement, the alkoxy radical scavenging activity measurement, and the superoxide scavenging activity measurement, and is shown by curves I, II, and III, respectively. Furthermore, the relationship between the prior probability and posterior probability based on the hydroxyl radical scavenging activity measurement and the alkoxy radical scavenging activity measurement is shown in curve I + II, and the prior probability based on the hydroxyl radical scavenging activity measurement, the alkoxy radical scavenging activity measurement, and the superoxide scavenging activity measurement. The relationship between radicals and posterior probabilities is shown in curves I + II + III.

Since the scavenging mechanism of each reactive oxygen species in ASD is independent, the positive likelihood ratio based on the hydroxyl radical scavenging activity and the positive likelihood ratio based on the alkoxy radical scavenging activity are divided into the positive likelihood ratio in the hydroxyl radical scavenging activity × It can be calculated as positive likelihood ratio in alkoxy radical scavenging activity × pre-odds = post-odds. With reference to Table 5, the posterior odds of the curve I + II are calculated as 2.79 × 2.46 × pre-odds = 6.86 × pre-odds. Similarly, when evaluated based on three types of hydroxyl radical scavenging activity, superoxide scavenging activity, and alkoxy radical scavenging activity, the posterior odds of curve I + II + III are 2.79 × 23.0 × 2.46 = 157.9 ×. Calculated as pre-odds. When the hydroxyl radical scavenging activity, the alkoxy radical scavenging activity and the superoxide scavenging activity of the serum are measured and all of them are positive in the test, the posterior probability is 99% when the prior probability is 50% as shown in FIG. It turned out to be a very useful method in determining ASD.

Taking FIG. 1 into consideration, the specificity can be set to 100% when the peroxy radical and singlet oxygen are within the reference range.

(Example 2)
For the peripheral blood of ASD (n = 22 to 23) and typical developing infants (n = 33 to 34) of Example 1, each active oxygen scavenging activity value was classified using the judgment value of Example 1, and each classification was performed. The scores shown in Table 6 are attached to.

Hydroxyl radical scavenging activity of subject-derived biomaterial is equivalent to 5 mM GSH, superoxide scavenging activity is equivalent to 10 U / ml SOD, alkoxy radical scavenging activity is equivalent to 2 mM trolox, peroxy radical scavenging activity is equivalent to 0.7 mM α-lipoic acid, singlet oxygen scavenging. If the activity is equivalent to 15 μM GSH, the total score is 11. When a score of 6.5 or higher is positive, this subject has a sensitivity of 65% (95% confidence interval 42-83%), a specificity of 97% (95% confidence interval 84-99%), and a positive likelihood ratio of 22. It can be determined that it is positive in 17 situations, that is, it is highly likely that it has ASD.

Claims (7)

The measurement value of at least two or more active oxygen scavenging activities selected from the group consisting of hydroxyl radical scavenging activity, superoxide scavenging activity and alkoxy radical scavenging activity of the biological material derived from the subject is determined to correspond to each of the active oxygen scavenging activities. Including a determination step of comparing with the value and determining the possibility that the subject has autism spectrum disorder based on the comparison result.
In the determination step, the comparison result is the determination requirement listed below.
When the measured value of the hydroxyl radical scavenging activity is included, the first determination requirement that the measured value is less than the determination value of the hydroxyl radical scavenging activity,
When the measurement value of the superoxide scavenging activity is included, the second determination requirement that the measurement value is equal to or higher than the determination value of the superoxide scavenging activity, and when the measurement value of the alkoxy radical scavenging activity is included, the measurement value is included. Indicates whether or not satisfies the third determination requirement that is equal to or greater than the determination value of the alkoxy radical scavenging activity.
When any two or more of the first determination requirement, the second determination requirement, and the third determination requirement are satisfied, it is determined that the subject may be suffering from autism spectrum disorder .
The determination value is determined in advance based on the measured values of the active oxygen scavenging activity in the typical development group and the autism spectrum disorder group, and there is a statistically significant difference between the typical development group and the autism spectrum disorder group. If there is, use the cutoff value
If there is no statistically significant difference between the autism spectrum disorder group and the neurotypical development group, a predetermined confidence interval is set and an equivalence test is performed, and an upper limit value and a lower limit value indicating equivalence without superiority are performed. A method for determining autism spectrum disorder, each of which has a determination value of . The active oxygen scavenging activity further includes a peroxy radical scavenging activity and / or a singlet oxygen scavenging activity.
The determination step further
When the measured value of the peroxy radical scavenging activity is included, the fourth determination requirement that the measured value is between the two determination values of the peroxy radical scavenging activity, and / or
When the measurement value of the singlet oxygen scavenging activity is included, the fifth determination requirement that the measurement value is between the two determination values of the singlet oxygen scavenging activity is included.
The determination method according to claim 1, wherein when the fourth determination requirement or the fifth determination requirement is satisfied, it is determined that the subject is more likely to have an autism spectrum disorder. In the determination step, the measured value of each active oxygen scavenging activity is compared with one or more corresponding determination values, and a score is assigned to each active oxygen scavenging activity based on the comparison result, and autism The determination method according to claim 1 or 2, wherein the possibility of suffering from a spectrum disorder is determined as the total value of the scores. The determination method according to any one of claims 1 to 3, wherein each active oxygen scavenging activity is measured by a spin trap method using electron spin resonance. Two or more active oxygen generators selected from the group consisting of hydrogen peroxide, riboflavin, and 2,2'-azobis [2-aminodipropane] dihydrochloride, and a spin trap agent are used as biomaterials derived from the subject. A kit for determining autism spectrum disorder for carrying out the determination method according to any one of claims 1 to 4, which is contained as a reagent for measuring the active oxygen scavenging activity contained in the above . Further containing t-butyl hydropero x ide as an active oxygen generator as a measurement reagent for the active oxygen scavenging activity contained in the biological material derived from the subject, and / or rose bengal as an active oxygen generator and a spin trap agent. The determination of autism spectrum disorder according to claim 5 for carrying out the determination method according to claim 2, which is further contained as a measurement reagent for the active oxygen scavenging activity contained in the biological material derived from the subject. Kit for. At least two or more active oxygen scavenging activities selected from the group consisting of hydroxyl radical scavenging activity, superoxide scavenging activity and alkoxy radical scavenging activity, and peroxy radical scavenging activity and / or singlet oxygen scavenging activity of the biological material derived from the subject. Measurement for comparing the measured value of the active oxygen scavenging activity with the determination value corresponding to each of the active oxygen scavenging activities, and determining the possibility that the subject suffers from autism spectrum disorder based on the comparison result. Including data processing process
In the measurement data processing step, the comparison results are based on the conditions listed below.
When the measured value of the hydroxyl radical scavenging activity is included, the first condition that the measured value is less than the determination value of the hydroxyl radical scavenging activity,
When the measured value of the superoxide scavenging activity is included, the second condition that the measured value is equal to or higher than the determination value of the superoxide scavenging activity.
When the measured value of the alkoxy radical scavenging activity is included, the third condition that the measured value is equal to or higher than the determination value of the alkoxy radical scavenging activity.
When the measured value of the peroxy radical scavenging activity is included, the fourth condition that the measured value is between the two determination values of the peroxy radical scavenging activity, and / or
When the measured value of the singlet oxygen scavenging activity is included, the fifth condition that the measured value is between the two determination values of the singlet oxygen scavenging activity is included.
If any two or more of the first condition, the second condition, and the third condition are satisfied, and the fourth condition or the fifth condition is satisfied, the subject suffers from autism spectrum disorder. a measurement data acquisition method for the possibility that it is determined that high
The determination value is determined in advance based on the measured values of the active oxygen scavenging activity in the typical development group and the autism spectrum disorder group, and there is a statistically significant difference between the typical development group and the autism spectrum disorder group. If there is, use the cutoff value
If there is no statistically significant difference between the autism spectrum disorder group and the typical development group, a predetermined confidence interval is set and an equivalence test is performed, and an upper limit value and a lower limit value indicating equivalence without superiority are performed. Is a measurement data acquisition method, each of which has a determination value of .