JP7797759B2 - Urinary carotenoid testing method, urine collection kit, and method for estimating serum carotenoid concentration or vegetable intake - Google Patents

Description

The present invention relates to a method for testing carotenoids in a subject's urine, a urine collection kit, and a method for estimating serum carotenoid concentrations or vegetable intake from urinary carotenoid concentrations.

The Ministry of Health, Labour and Welfare of Japan recommends consuming at least 350g of vegetables per day to maintain good health, but the average intake in 2019 was 280.5g, which is insufficient (Non-Patent Document 1).
Vegetables are rich in carotenoids, along with vitamins, minerals, dietary fiber, etc.
Carotenoids are compounds with a basic skeleton (chemical formula C ₄₀ H ₅₆ ) composed of eight bonded isoprene units, and are natural pigment components that absorb visible light in the 400-500 nm range due to the conjugated double bonds in this basic skeleton, resulting in yellow, orange, red, and other colors. More than 600 carotenoid compounds have been identified to date, with well-known examples including beta-carotene, which is abundant in carrots, lycopene, which is abundant in tomatoes, and lutein, which is abundant in spinach. Carotenoids are known to have antioxidant properties, and because they have the ability to scavenge active oxygen, they are expected to have preventive effects against cancer, myocardial infarction, stroke, lifestyle-related diseases, and the like.

Although carotenoids are also present in animals and microorganisms, most of the carotenoids ingested by humans are derived from vegetables. Therefore, vegetable intake can be estimated from the carotenoid concentration in the body. For example, Patent Literature 1 proposes a method for estimating vegetable intake from non-invasively measured skin carotenoid values and promoting vegetable intake. Although this method is non-invasive, it takes about 2 to 4 weeks for ingested carotenoids to accumulate in the skin, and therefore the obtained skin carotenoid value data does not reflect the immediately preceding vegetable intake.
Furthermore, Non-Patent Documents 2 and 3 report that there is a correlation between urinary carotenoid concentrations and plasma (serum) carotenoid concentrations, and that while urinary carotenoid concentrations reflect serum concentrations, urinary β-carotenoid concentrations are 1/1000 or less of serum carotenoid concentrations. Furthermore, because the carotenoid concentration in urine is very low, fractions extracted with organic solvents from a large volume of urine (40 ml) are used for measurement.

Japanese Patent Application Laid-Open No. 2020-201788

Ministry of Health, Labour and Welfare, 2019 National Health and Nutrition Survey Shunji Oshima, Takahiro Inakuma, Akihiko Nagao, "Correlation between urinary and plasma carotenoid concentrations," Vitamins, Vitamin Society of Japan, 2001, Vol. 75, No. 4, p. 211 "Body carotenoid concentrations can be measured in urine - correlation between blood and urinary carotenoids elucidated -" [online], retrieved May 22, 2019, Kagome Co., Ltd., news release dated May 18, 2001, Internet <URL: https://www.kagome.co.jp/company/news/2001/010518_02.html>

The objective of this study is to provide a urine testing method that can easily detect urinary carotenoid concentrations.

The main configuration of the present invention is as follows.
1. A urine testing method characterized by detecting the concentration of carotenoids in urine using a urine specimen to which an alkaline additive has been added.
2. The urine test method according to 1., wherein the urine sample has a pH of 8.0 or higher. 3. A urine collection kit comprising a urine collection container and an alkaline additive, and used for measuring urinary carotenoid concentrations.
4. A method for estimating serum carotenoid concentrations or vegetable intake from urinary carotenoid concentrations detected by the urine testing method in 1. or 2.

The testing method of the present invention can shorten the time required for pre-treatment of the measurement sample. The testing method of the present invention can shorten the time from urine collection to measurement, thereby preventing deterioration of the urine sample. The testing method of the present invention can detect urinary carotenoid concentrations from small amounts of urine, simplifies urine collection and mailing of collected urine, and reduces the amount of waste.

HPLC chromatograph for each additive. Graph showing the area values of β-carotene in HPLC chromatographs for each additive.

Urine contains a variety of components, including sugar, protein, and salts. Urine tests can reveal the condition of not only the kidneys, ureters, and bladder, but also the liver and gallbladder by examining quantitative and qualitative changes in the components in the urine and the presence or absence of abnormal substances. One of the urine test items is a sediment test, which involves precipitating solid components in the urine, such as red blood cells, white blood cells, uric acid crystals, and bacteria, and examining their composition and quantity.
As a result of extensive research, the inventors discovered that when urinary carotenoids are contained in precipitates, the concentration of carotenoids in the liquid decreases, reducing detection sensitivity and making quantification difficult, leading to the completion of the present invention.

- Urine Test Method The urine test method of the present invention is characterized by detecting the concentration of carotenoids in urine using a urine sample to which an alkaline additive has been added.

The alkaline additive used in the urine testing method of the present invention is not particularly limited as long as it provides an alkaline aqueous solution. Examples include alkali metal salts such as ammonia, sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate; alkaline earth metal salts such as calcium hydroxide and magnesium hydroxide; amine-based organic compounds such as triethylamine, triethanolamine, tris(hydroxymethyl)aminomethane, pyridines, and imidazoles; and basic proteins such as arginine and lysine. Of these, tris(hydroxymethyl)aminomethane, imidazole, and arginine are preferred from the standpoints of odor and safety.

By adding an alkaline additive to urine, it is possible to dissolve and reduce at least a portion of the precipitate, thereby reducing the amount of carotenoids contained in the precipitate and maintaining a high amount of carotenoids in the liquid. The pH of a urine sample to which an alkaline additive has been added is preferably 8.0 or higher, more preferably 8.5 or higher, even more preferably 8.8 or higher, and even more preferably 9.0 or higher.

Furthermore, the urine testing method of the present invention can maintain a high carotenoid concentration in the liquid, making it possible to detect urinary carotenoid concentrations with a small amount of urine. In the present invention, the amount of urine required to detect urinary carotenoid concentrations is not particularly limited as long as a detectable urine sample can be produced, but is preferably 10 ml or less, more preferably 5 ml or less, even more preferably 3 ml or less, and even more preferably 2 ml or less.

Urine Collection Kit The urine test method of the present invention preferably uses a urine collection kit having at least a urine collection container and an alkaline additive.
Urine collection containers can be used for urine tests conducted in hospitals, testing institutions, etc., and are not particularly limited in shape or material as long as they do not cause deterioration of urinary components. The amount of urine that can be collected in a urine collection container is not particularly limited as long as it is an amount that allows for the preparation of a urine specimen, but is, for example, 10 ml or less. The urine collection container may be designed to allow direct urine collection or to transfer collected urine to a urine collection cup, etc. It is also preferable that the urine collection container be graduated so that a predetermined amount of urine can be collected. Furthermore, it is preferable that the urine collection container be sealable, because the urine-filled urine collection container can be mailed to a hospital or testing institution for testing, eliminating the need for the subject to visit a hospital or public health center. In addition to the urine collection container and alkaline additive, the urine collection kit may include an outer box, an instruction manual describing the test procedure and precautions, a urine collection cup, a zippered plastic bag, a urine test request form containing information such as the subject's name, date of birth, and gender, a return envelope, etc.

The pH of urine from healthy individuals varies between 4.5 and 8 depending on the food ingested, etc. Therefore, the amount of alkaline additive used when preparing a predetermined amount of urine sample is preferably an amount that results in a pH of 8.0 or higher, more preferably an amount that results in a pH of 8.5 or higher, even more preferably an amount that results in a pH of 8.8 or higher, and even more preferably an amount that results in a pH of 9.0 or higher.

The urine testing method of the present invention for detecting urinary carotenoid concentrations may be either therapeutic or non-therapeutic. Therapeutic refers to use in conjunction with treatment, testing, etc. at a medical institution, while non-therapeutic refers to a concept that does not include medical procedures, i.e., a concept that does not include methods of surgery, therapy, or diagnosis on humans; more specifically, a concept that does not include methods of surgery, therapy, or diagnosis performed on humans by a physician or a person under the direction of a physician.

In the present invention, the timing of urine collection is not particularly limited, but it is preferable to collect urine before the first meal of the day, and it is particularly preferable to collect the first urine immediately after waking up (early morning). Since the concentrations of urine components are easily affected by food, water intake, sweating, etc. and vary greatly depending on the amount of urine at that time, a creatinine correction can be performed to determine the ratio with the creatinine value measured at the same time.

The urinary carotenoid concentration can be detected more accurately using the urinary test method of the present invention. Furthermore, since the urinary carotenoid concentration detected using the urinary test method of the present invention is highly correlated with the serum carotenoid concentration and vegetable intake, the serum carotenoid concentration and vegetable intake can be accurately estimated using the urinary carotenoid concentration detected using the urinary test method of the present invention.

<Clinical trial method>
Twenty-two healthy individuals (11 men and 11 women) in their 20s to 60s were surveyed for background information (gender, date of birth, and questionnaire on daily vegetable intake using the FFQg questionnaire), and carotenoid concentrations in urine and serum were measured.
The exclusion criteria were as follows:
1) Those with a serious medical history or a history of gastrointestinal surgery (excluding appendectomy)
2) Pregnant or breastfeeding women 3) Those who drink a lot of alcohol or smoke excessively 4) Others who are deemed inappropriate to participate in this study by the investigator

As an ethical consideration, the appropriateness of conducting this study was reviewed by the Institutional Review Committee, and prior to conducting this clinical trial, the purpose of the study was fully explained to all subjects, and written consent to participate was obtained from each subject. Subjects collected their first urine sample on the day of their visit, and had their blood drawn at the hospital in the morning of the day of their visit.

(Urine collection)
On the day of the test, participants were asked to collect 200 mL of their first urine (immediately after waking up) at home and bring it to the test site.
(Quantitative determination of urinary carotenoid concentrations)
Additives The following eight types of additives were used.

To a 5 mL tube, 1 mL of urine and 100 μL of any of the above additives were added.
The urine of one subject (hereinafter referred to as Subject A) was visually observed for changes in precipitate before and after the addition. The pH after the addition was also measured using pH test paper. The results are shown in Table 2. For samples with a range of pH values, accurate determination was difficult because the color of the pH test paper was visually checked.

2 mL of 0.1% BHT (dibutylhydroxytoluene)/ethanol solution was then added, the mixture was shaken, and allowed to stand at room temperature for 5 minutes. 10 μL of the internal standard 0.1 ppb apocarotenal and 2 mL of hexane were added, the mixture was shaken, and centrifuged (3000 rpm for 5 minutes). After centrifugation, 4/5 of the supernatant was collected, heated to 45°C in a centrifugal evaporator, dried, and dissolved in 100 μL of 0.1% BHT/ethanol solution to obtain the urine sample.

The urine samples were subjected to HPLC under the following conditions to quantify the carotenoid concentrations in urine.
(column)
ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 150 mm (Waters)
(Analysis conditions)
Apparatus: UHPLC (Nexera X2 series, manufactured by Shimadzu Corporation)
Mobile phase: acetonitrile:methanol = 9:1
Flow rate: 0.50 mL/min Column temperature: 40°C
Injection volume 5μL
Detection wavelength: 450 nm

The HPLC chromatograms of the urine samples of subject A for each additive are shown in Figure 1. Three urine samples of subject A were prepared in the same manner and analyzed, and the average area values of the β-carotene in the HPLC chromatograms are shown in Figure 2.
The detection intensity of carotenoids increased with the addition of alkaline additives, especially sodium hydroxide, potassium hydroxide, tris(hydroxymethyl)aminomethane, imidazole, and arginine.

(Blood collection)
In the morning of the test day, 9 mL of blood was collected in a blood collection tube without any additives, and after confirming blood coagulation, the blood was centrifuged (3000 rpm for 10 minutes) to obtain serum.
(Quantification of serum carotenoid concentrations)
The carotenoid concentration in the serum was analyzed by HPLC by SRL Co., Ltd.

Correlation 1
The coefficient of determination was calculated by one-way analysis of variance between the urinary β-carotene concentration and serum β-carotene concentration, and between the urinary β-carotene concentration and the daily vegetable intake measured by the FFQg questionnaire. The results are shown in Table 3.

By adding urea and arginine, the coefficient of determination increased (approaching 1) compared to when no additions were made, and a particularly high correlation was observed when arginine was added. Furthermore, urinary beta-carotene concentration showed a higher correlation with vegetable intake than serum beta-carotene concentration. This demonstrates that by making urine alkaline with additives, urinary carotenoid concentrations can be measured more accurately, and that serum carotenoid concentrations and vegetable intake can be estimated with high accuracy from the measured urinary carotenoid concentrations.

Correlation 2
The coefficient of determination was calculated by one-way analysis of variance between the urinary total carotene (α-carotene + β-carotene) concentration and serum β-carotene concentration, as well as between the urinary total carotene concentration and the daily vegetable intake measured by the FFQg questionnaire. The results are shown in Table 4.

By adding urea and arginine, the coefficient of determination increased (approaching 1) compared to when no additions were made, and a particularly high correlation was observed when arginine was added. Furthermore, like urinary beta-carotene concentration, total urinary carotene also showed a higher correlation with vegetable intake compared to serum beta-carotene concentration. This demonstrates that by making urine alkaline with additives, urinary carotenoid concentrations can be measured more accurately, and that serum carotenoid concentrations and vegetable intake can be estimated with high accuracy from the measured urinary carotenoid concentrations.

Claims (3)

A urine testing method characterized by detecting the concentration of carotenoids in urine using a urine specimen having a pH of 8.5 or higher to which an alkaline additive such as imidazole or arginine has been added. A urine collection container and an alkaline additive are included,
the alkaline additive is imidazole or arginine;
A urine collection kit for measuring urinary carotenoid concentration , characterized in that the pH of urine collected is 8.5 or higher . A method for estimating serum carotenoid concentration or vegetable intake from urinary carotenoid concentration detected by the urine testing method of claim 1 .