Patent Application
20240248095
The present invention relates to a method for quantifying the concentration of phenylalanine contained in blood. The present invention also relates to a measurement kit used in the method. Furthermore, the present invention relates to a method for easily confirming and managing a blood phenylalanine concentration of a phenylketonuria patient at a bedside or a place other than a medical institution, using the method or the kit.
Phenylketonuria is one of congenital amino acid metabolism abnormalities in which phenylalanine (hereinafter, also referred to as “Phe”) cannot be metabolized to tyrosine due to a decrease in the activity of phenylalanine hydroxylase (hereinafter, also referred to as “PAH”) in the body, and Phe is accumulated in the body. Significant increase in Phe concentration during infancy leads to severe mental retardation. By controlling the blood Phe concentration within a certain range, mental retardation can be prevented, and normal intellectual development can be acquired. Therefore, a patient with phenylketonuria needs to adjust the intake Phe amount by diet therapy using the blood Phe concentration as a biomarker. Since the blood Phe concentration is susceptible to the amount of Phe contained in the meal and the Phe tolerability depends on the individual's PAH activity, it is necessary to finely adjust the intake Phe amount by repeatedly measuring the blood Phe concentration periodically or non-periodically. For this purpose, it is required that the blood Phe concentration can be easily and quickly measured at the bedside of a patient or in a place other than a medical institution in daily life.
Currently, a method for measuring a blood Phe concentration that can be used in a clinical site is an HPLC method. However, since the method requires at least one week until the result is obtained, it is not suitable as a measurement method in a situation where immediate evaluation is required, such as adjustment of the intake Phe amount in the diet therapy. On the other hand, a method capable of rapid measurement includes a mass spectrometry method used in newborn mass screening. However, since this method requires pretreatment of a specimen and requires a large analytical instrument, it cannot be used for an examination at a place other than a medical institution.
In addition, as a method for diagnosing phenylketonuria, conventionally, a method has been proposed in which Phe labeled with a carbon isotope is administered to a subject orally or by injection, and phenylketonuria is diagnosed by a variation in the amount of the carbon isotope contained in breath (PTL 1), and a method has been proposed in which a dehydrogenase specific to L-phenylalanine in a biological sample, as well as a coenzyme, an electron mediator, and a tetrazolium salt are used as a reaction reagent, formazan is generated by an enzymatic reaction and an oxidation-reduction reaction between the biological sample and the reaction reagent, and L-phenylalanine in the biological sample is quantified by optically or/and electrochemically detecting the formazan (2). However, the former method has a problem that a mass analyzer is required for measuring the amount of the carbon isotope. On the other hand, the latter is a method capable of easily and quickly examining phenylketonuria, but is a method capable of simultaneously examining three diseases of galactosemia, maple syrup urine disease, and phenylketonuria, which are congenital metabolic disorders, and is different in principle from the measurement method of the present invention described later.
As mentioned previously, there has been a desire to establish a method for measuring Phe concentration in blood conveniently and quickly. Therefore, an object of the present invention is to provide a method for simply and quickly measuring a blood Phe concentration. Another object of the present invention is to provide a measurement kit used in the method. Further, an object of the present invention is to provide a method for managing a blood Phe concentration in a phenylketonuria patient.
The present inventors have paid attention to the fact that when Phe is degraded with phenylalanine ammonia lyase (hereinafter, also referred to as “PAL”), ammonia equivalent to Phe is generated, and considered that the Phe concentration in venous blood can be quantitatively measured using the amount of generated ammonia as an index. However, while conducting studies day and night, there is a problem such that the Phe concentration in venous blood of a subject cannot be accurately measured when capillary blood collected from a fingertip or the like is used as a test sample (test blood), since ammonia contained in sweat or the like is mixed in the blood. Therefore, in order to solve the problem, further intensive studies have been made, and as a result, it has been confirmed that the Phe concentration in venous blood can be accurately measured while capillary blood is used as test blood by once allowing collected capillary blood to coexist with an alkali buffer, to gasify and volatilize ammonium ions contained therein, and then to react with PAL. In addition, even when venous blood is used as the test blood, it is possible to measure the blood Phe concentration of the subject with higher accuracy by gasifying and removing an ammonium ion existing in advance.
The present invention has been completed by accumulating such studies, and has the following embodiments.
(I-1) A method for quantifying a blood The concentration of a subject using a test piece consisting of at least two sheet-shaped members, in which
the test piece is formed by laminating a first sheet-shaped member (hereinafter, this may be abbreviated as a “first member”) including a sample holding layer containing an alkali buffer and a second sheet-shaped member (hereinafter, this may be abbreviated as a “second member”) including an indicator layer that changes in color by reacting with ammonia gas in a peeled state or a peelable state, and
the above quantification method is a method including the following steps (A) to (D):
(I-2) The method for quantifying blood Phe according to (I-1), in which the alkali buffer in the sample holding layer of the first member is an alkali buffer containing boric acid and sodium hydroxide, and
(I-3) The method for quantifying blood Phe according to (I-2), in which
(I-4) The method for quantifying blood phenylalanine according to (I-2) or (I-3), in which the step (C) is a step of measuring absorbance of the indicator layer at a wavelength of 635 nm.
(I-5) The method for quantifying blood Phe according to any one of (I-1) to (I-4), in which the blood (test blood) collected from the subject is capillary blood.
(I-6) The method for quantifying blood Phe according to any one of (I-1) to (I-5), in which the subject is a patient with phenylketonuria.
(I-7) The method for quantifying a blood Phe concentration according to any one of (I-1) to (I-6), in which the method for quantifying a blood Phe is performed using a kit described in (II-1) described later.
These quantification methods can also be referred to as a method for presumptive quantification of a blood Phe or a method for estimating a blood Phe concentration.
(II-1) A kit containing at least the following (a) and (b) or (a) to (c) for use in the method for quantifying blood Phe according to any one of (I-1) to (I-6): (a) a test piece obtained by laminating a first member including a sample holding layer containing an alkali buffer and a second member including an indicator layer that changes in color by reacting with ammonia gas in a peeled state or a peelable state;
(III-1) A method for managing a blood Phe concentration of a patient with phenylketonuria, in which the method for quantifying blood Phe according to any one of (I-1) to (I-6) is performed using capillary blood of a phenylketonuria patient as test blood periodically or non-periodically at a place other than a medical institution.
(III-2) The management method according to (III-1), in which the method for quantifying Phe in blood is a method performed using the kit according to (II-1).
According to the method of the present invention, the blood Phe concentration of a subject can be easily and quickly measured at a place of blood sampling. In addition, according to the method of the present invention, for example, even when a trace amount of blood (capillary blood) from a fingertip is used as the test blood, the Phe concentration in the venous blood or plasma of the subject can be obtained with high accuracy. Therefore, the method of the present invention can be effectively used for screening for early detection of phenylketonuria in a neonate, monitoring of the blood Phe concentration at the bedside of a phenylketonuria patient, and monitoring and management of the blood Phe concentration at a place other than a medical institution, particularly in a dietary therapy in a daily life.
A method for quantifying Phe in blood (hereinafter, also referred to as “the quantification method”) of the present invention is characterized by performing steps (A) to (D) described later using a test piece (hereinafter, referred to as “the test piece”) including at least two sheet-shaped members.
Hereinafter, the test piece and the steps (A) to (D) is described.
The test piece includes a first sheet-shaped member (first member) including a sample holding layer containing an alkali buffer, and a second sheet-shaped member (second member) including an indicator layer that changes in color by reacting with ammonia gas. In these members, the first member is disposed (laminated) on the second member. Both members may be in a state of being peeled each other, or may be in a state of being temporarily adhered so as to be peelable so as not to be separated from each other.
As illustrated in the drawing, a hole 12 is formed in a sheet-shaped base material 11 (hereinafter, also simply referred to as “first base material 11”) of a first member 2 constituting the test piece 1, and a sample holding layer 13 is disposed on the hole so as to cover the hole. On a base material 14 (hereinafter, also simply referred to as “second base material 14”) on the sheet of a second member 3 constituting the test piece 1, an indicator layer 15 is disposed in a part corresponding to the hole 12 of the first base material 11, and adhesive layers 16a and 16b are disposed before and after the indicator layer. The hole 12 formed in the first base material 11 is a vent hole for allowing ammonia gas generated in the sample holding layer 13 to reach the indicator layer 15 disposed in the second base material 14 as described later. Therefore, it is also referred to as an NH3 vent hole.
The size of the first base material 11 is not particularly limited, and in the case of a strip shape, for example, it is 20 to 80 mm length×3 to 10 mm width×0.1 to 0.7 mm thickness, preferably 30 to 60 mm length×4 to 9 mm width×0.1 to 0.5 mm thickness, and more preferably 30 to 40 mm length×5 to 8 mm width×0.1 to 0.4 mm thickness. The size of the second base material 14 is not limited, in the case of a strip shape, preferably has the same width and the different length as the first base material 11. For example, length 20 to 100 mm×width 3 to 10 mm×thickness 0.1 to 0.7 mm, preferably length 40 to 80 mm×width 4 to 9 mm×thickness 0.1 to 0.5 mm, and more preferably length 40 to 60 mm×width 5 to 8 mm×thickness 0.1 to 0.4 mm. The material of these members is also not particularly limited, and examples thereof include polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), and polycarbonate (PC). Among them, PET and PC are preferable, and PET is particularly preferable.
The hole 12 formed in the first base material 11 is illustrated as a circular shape in
The sample holding layer 13 disposed on the first base material 11 is obtained by impregnating a porous member with an aqueous solution containing an alkali buffer and drying the porous member. As the porous member, any porous member can be used regardless of whether it is fibrous or non-fibrous as long as it is inert to at least blood (whole blood) as a test sample and an alkali buffer and does not affect the method of the present invention. Examples of the fibrous porous member include a filter paper, a nonwoven fabric, and a woven fabric. Examples of the non-fibrous porous member include porous members made of 6-nylon, 6,6-nylon, cellulose acetate, cellulose nitrate, polyethylene, polypropylene, or the like. Although not limited, a non-fibrous porous member is preferable. The size of the sample holding layer 13 is not limited as long as it can fill up the hole 12 formed in the first member 11. For example, as shown in
The alkali buffer to be retained in the sample holding layer 13 is not particularly limited as long as it makes the test sample to be spotted to the sample holding layer 13 alkaline and gasifies an ammonium ion contained in the test sample to generate ammonia gas. For example, alkali buffers of pH 9 to 11 such as a borate buffer prepared from boric acid and sodium hydroxide, a sodium borate buffer prepared from sodium borate and sodium hydroxide, and a carbonate-bicarbonate buffer prepared from sodium carbonate and sodium bicarbonate can be exemplified. A borate buffer is preferred. The alkali buffer is dissolved in, for example, ultrapure water to form an alkali buffer, impregnated into the sample holding layer 13, and dried (for example, natural drying, air drying, heat drying, reduced pressure drying, and the like), whereby the sample holding layer 13 can hold the alkali buffer in a dry state.
The indicator layer 15 disposed on the second base material 14 holds an indicator to be described later. The indicator layer 15 may be formed by coating an any member with an indicator, or may be formed by impregnating a porous member with an aqueous solution containing the indicator and drying the porous member, similarly to the sample holding layer 13. These members may be any members that are inert to at least the indicator and ammonia gas and do not affect the method of the present invention. The size of the indicator layer 15 is not limited as long as it can fill up the hole 12 formed in the first base material 11. For example, as shown in
As the indicator to be held by the indicator layer 15, a pH indicator having a property of color development or color change in response to a pH change caused by ammonia gas can be used. It may preferably include, but is not limited to, bromocresol green. The bromocresol green is a pH indicator that exhibits a yellow color at a pH of around 3.8 but changes to a blue color at a pH of around 5.4.
In the second member 3, the adhesive layers 16a and 16b disposed in front of and back of the indicator layer 15 are parts for adhering the first member 2 and the second member 3 in a peelable state as described above. The member constituting the adhesive layer, the adhesive component, and the adhering means are not particularly limited as long as they do not affect the method of the present invention. Although not limited, a method using an adhesive such as an acrylic adhesive or an elastomeric adhesive and a method using a double-sided tape can be exemplified as the adhering means, and a lamination method by thermal fusion bonding can also be used. Further, the adhesive layer 16 may be capable of being repeatedly adhered twice or more. In this case, an adhesive or a double-sided tape can be used as the adhering means.
The sizes of the adhesive layers 16a and 16b are also not particularly limited. For example, when the second member 3 has a strip shape, the planar shapes of the adhesive layers 16a and 16b are preferably rectangular, and the length of one side (width) thereof is preferably the same as the width of the second member 14. For example, the size is 3 to 10 mm wide×2 to 10 mm longitudinal×0.01 to 0.3 mm thickness, preferably 4 to 9 mm wide×2 to 9 mm longitudinal×0.01 to 0.2 mm thickness, and more preferably 5 to 8 mm wide×2 to 8 mm longitudinal×0.01 to 0.1 mm thickness. Further, the adhesive layer 16 may be capable of being bonded twice or multiple times.
The test piece 1 can be manufactured by disposing the first member 2 on the second member 3 such that the hole 12 of the first base material. 11 is located on the indicator layer 15 of the second member 3 and the opening of the hole 12 is covered with the indicator layer 15, and adhering these members in a peelable state via the adhesive layers 16a and 16b.
However, as described later, when the quantification method is performed, the test piece 1 is first used in a state where adhesion (temporary adhesion) between the first member 2 and the second member 3 is removed (peeled) and the first member 2 is separated from the second member 3 (see
The quantification method using the test piece described above can be performed by the following steps (A) to (D). An example of a series of the steps is shown in
(A) dropping blood (test blood) collected from a subject on the sample holding layer 13 of the first member 2 of the test piece 1 in a state of being separated from the second member 3 as it is or after being diluted, and leaving the blood to stand for a certain period of time;
(B) laminating the first member 2 on the second member 3 such that the sample holding layer 13 and the indicator layer 15 of the second member 3 overlap each other in a non-contact state, dropping a PAL-containing liquid onto the sample holding layer, and leaving the sample holding layer 13 to stand for a certain period of time;
(C) measuring the ammonia concentration in the test blood from the degree of color of the indicator layer 15 of the second member 3; and
(D) calculating a blood Phe concentration of the subject from the ammonia concentration.
The step (A) is a step of dropping and spotting the test blood on the sample holding layer of the test piece.
The test blood is blood collected from a subject to be measured. The subject is a neonate suspected of having phenylketonuria (a neonate in need of examination) or a patient with phenylketonuria. The blood is whole blood, and may be capillary blood collected from a capillary, or may be venous blood collected from a vein. Preferably, it is capillary blood. The blood is more preferably capillary blood collected from a fingertip because it is easy to collect blood. The blood may be spotted to the sample holding layer as it is as a test sample, and may be spotted to the sample holding layer after being diluted with a solvent that does not affect the present invention as necessary, for example, when the Phe concentration in the blood is high or the amount of blood collected is small. As an example, but not limited to, one obtained by adding an arbitrary amount of 50 IM Tris-HCl buffer 43 to capillary blood 40 collected from a fingertip and diluting the mixture (diluted sample 44) can also be used as a test sample (see
The test blood 40 or the diluted sample 44 containing the test blood 40 (also collectively referred to as a “test sample” without distinction) is dropped on the sample holding layer 13 of the first member 2 in a state of being separated from the second member 3 in advance, and is left to stand for 5 minutes or more under room temperature conditions (1 to 30° C., the same applies hereinafter). By the operation, ammonia contained in the test sample can be removed. Specifically, when the test sample is dropped on the sample holding layer 13, the alkali buffer in the sample holding layer is dissolved, the test sample becomes alkaline, and ammonium ions contained in the test sample are gasified. The gasified ammonia gas is volatilized and removed by being left to stand for a certain period of time. The leaving time may be any time as long as ammonia gas can be removed and reduced, and may be, for example, 5 minutes or more under room temperature conditions. Although not limited, it is preferably 8 minutes or more, and more preferably 10 minutes or more. When the rapidity of the result is required, the leaving time is preferably kept within 15 minutes. The time is more preferably about 8 to 10 minutes. Thus, the test sample 45 (deammoniated test sample) from which ammonia has been removed or reduced can be held in the sample holding layer 13 of the first member 2.
The step (B) is a step of enzymatically treating the test sample 45 held by the sample holding layer 13 of the first member 2 on the sample holding layer 13.
The enzyme used in the present invention is phenylalanine ammonia lyase (PAL). PAL is an enzyme that reacts with Phe to generate equal molar ammonia and cinnamic acid as Phe, as shown in
PAL can be used or stored in a state of being dissolved in a solvent. The solvent is not limited as long as it does not impair the activity of PAL, and for example, a 50 mM Tris-HCl aqueous solution can be exemplified. In addition, as long as the activity of PAL is not impaired, an osmotic pressure regulator such as NaCl, a preservative such as sodium azide, and/or a stabilizer such as trehalose can be blended in addition to the solvent.
(B) The enzyme treatment in the step (A) is performed after the first member 2 obtained in the step (A) is laminated on the second member 3 such that the sample holding layer 13 overlaps the indicator layer 15 of the second member 3 in a non-contact state. When laminated, the opening of the hole 12 of the first base material 11 is covered and filled up by the indicator layer 15 of the second member 3. That is, in the enzyme treatment of the step (B), in a state where the first member 2 is laminated on the second member 3, the PAL-containing liquid 47 obtained by diluting PAL with a solvent is dropped on the sample holding layer 13 containing the test sample 45, and the reaction is carried out by leaving the solution to stand for a certain period of time under room temperature conditions. At this time, the humidity of the environment in which the test piece 1 is left to stand is not limited, but it is preferable to set and adjust the humidity so that the relative humidity falls within the range of 60 to 80% RH. The temperature and humidity during the leaving time may be managed using a thermohygrostat, or may be controlled by placing the test piece in a container or bag including a moisture absorbent such as silica gel.
During the leaving time, Phe derived from the test blood contained in the test sample 45 becomes ammonia by the action of PAL, and the ammonium ion is gasified by the action of the alkali buffer in the sample holding layer 13 to generate ammonia gas. The ammonia gas comes into contact with the indicator layer 15 of the second member 3 through the hole 12 (NH3 vent hole) of the first base material 11. Then, the indicator of the indicator layer 15 reacts and changes in color (color development part 49 of the indicator layer). For example, when the indicator is bromocresol green, the color changes from initial yellow to blue (see the upper part of
The leaving time may be any time as long as the total amount of Phe contained in the test sample becomes ammonia by PAL, and as a result, the indicator layer 15 changes in color as described above. Under the above temperature and humidity conditions, it may be 5 minutes or more. Although not limited, it is preferably 8 minutes or more, and more preferably 10 minutes or more. When the rapidity of the result is required, the leaving time is preferably kept within 15 minutes. The time is more preferably about 8 to 10 minutes.
The step (C) is a step of calculating the ammonia concentration derived from Phe in the test sample from the color (reference numeral 49) of the indicator layer 15 of the second member 3 obtained in the step (B).
This method can be easily performed by using the blood ammonia measurement device 5. The device is commercially available, and examples thereof include a blood ammonia measurement device PocketChem™ BA PA-4140 manufactured by ARKRAY, Inc.
The measurement device is a small device (measurement principle: 1 wavelength reflection measurement method, measurement wavelength: 635 nm) developed to measure the ammonia concentration in a blood sample using a test paper (AMMONIA TEST KIT II AMICHECK (registered trademark)) sold by the company. The cross-sectional structure of the test paper (referred to as “AMICHECK”) is shown in
In the step (C) of the present invention, the first member 2 and the second member 3 are separated from each other for the test piece 1 subjected to the enzyme treatment in the step (B), and the color development part (49) of the indicator layer 15 of the second member 3 is set in the measurement unit of the blood ammonia measurement device. In this way, the ammonia concentration (N-μg/dl) (as the amount of nitrogen) is automatically displayed on the liquid crystal display plate. The ammonia concentration reflects the amount of ammonia (Phe-derived ammonia) generated from Phe in the test sample by the action of PAL in the sample holding layer 13 of the first member 2 in the enzyme treatment step (B), and correlates with the amount of Phe contained in the test sample dropped on the sample holding layer 13. When the test blood diluted with the solvent is used as the test sample, the ammonia concentration corresponding to the amount of Phe contained in the test blood can be obtained by performing conversion at the dilution rate.
The step (D) is a step of calculating the blood Phe concentration of the subject from the Phe-derived ammonia concentration contained in the test blood obtained in the step (C). Here, the blood Phe concentration includes both the meaning of the Phe concentration in the venous blood (whole blood) of the subject and the meaning of the Phe concentration in the plasma thereof. It is known that the Phe concentration in whole blood is about 20% less than the Phe concentration in plasma. Therefore, if one Phe concentration can be calculated, the other Phe concentration can be naturally calculated.
The blood Phe concentration of the subject can be calculated from the Phe-derived ammonia concentration in the test sample using a calibration curve prepared in advance by an experiment or a function (regression equation) obtained from the calibration curve.
The calibration curve can be created, for example, as follows according to the origin (venous blood, capillary blood) of the test blood.
(a) The steps (A) to (C) described above are performed on a plurality of test samples prepared by stepwise adding a known amount of Phe to venous blood (whole blood) collected from one healthy person, and the Phe-derived ammonia concentration in the test sample is determined from the degree of color development of the indicator layer of the test piece.
(b) The Phe concentration added to the venous blood is plotted on the horizontal-axis (or vertical-axis), and the Phe-derived ammonia concentration obtained in (a) is plotted on the vertical-axis (or horizontal-axis) to create a calibration curve.
(1) The steps (A) to (C) described above are performed using capillary blood (whole blood) collected from fingertips of a plurality of subjects as a test sample, and the Phe-derived ammonia concentration in the test sample is determined from the degree of color development of the indicator layer of the test piece.
(2) Separately, the Phe concentration of the venous blood (whole blood) of the subject is measured by an HPLC method to acquire the blood Phe concentration.
(3) The blood Phe concentration obtained in (2) is plotted on the horizontal-axis (or vertical-axis), and the Phe-derived ammonia concentration obtained in (1) is plotted on the vertical-axis (or horizontal-axis) to create a calibration curve.
The subject is preferably a human having a Phe concentration in venous blood in the range of 0 to 1400 μM, preferably 0 to 437 μM, and may be a patient with phenylketonuria or a healthy person who has orally ingested Phe. In addition, both may be mixed in the subjects.
The regression equation can be calculated from these calibration curves.
As shown in Experimental Examples 1 to 4, the Phe-derived ammonia concentration in the test sample obtained in the steps (A) to (C) described above correlates very well with the blood Phe concentration (or plasma Phe concentration) of the subject, and the blood Phe concentration of the subject estimated from the Phe-derived ammonia concentration in the test sample obtained in the steps (A) to (C) (=the blood Phe concentration obtained in the step (D)) almost coincides with the actual blood Phe concentration of the phenylketonuria patient.
Therefore, by using the calibration curve obtained by the above method or the function (regression equation) obtained from the calibration curve, the actual blood Phe concentration of the subject can be estimated with high accuracy from the Phe-derived ammonia concentration in the test sample obtained by performing the steps (A) to (C).
The Phe measurement kit of the present invention contains at least the following (a) and (b), or (a) to (c). By using the kit, the quantification method described above can be easily performed.
(a) A test piece obtained by laminating a first member including a sample holding layer containing an alkali buffer and a second member including an indicator layer that changes in color by reacting with ammonia gas in a peelable state or a peelable state,
(b) PAL, and
(c) a solvent.
(a) The test piece is as described above as the test piece 1, and the above description can be incorporated herein. For convenience, a test paper (AMMONIA TEST KIT II AMICHECK (registered trademark)) manufactured by ARKRAY, Inc. can be used.
(b) From the viewpoint of storage stability, PAL is preferably a lyophilized product. Although not limited, for example, it has been confirmed that recombinant PAL prepared from E. coli have an enzyme activity that is acceptable for use in the present invention for at least 5 weeks even when stored at room temperature in a dried state after lyophilization (see the column of Examples described later).
(c) The solvent can be used for dilution of blood used as test blood and/or solubilization of PAL in lyophilized state. The solvent is not limited; however, for example, a 50 mM Tris-HCl aqueous solution (pH 8.5 to 9.5) or a buffer-containing aqueous solution equivalent thereto can be used.
In addition, the kit of the present invention may include a blood sampling capillary, a pipettor, an Eppendorf (tube), an instruction manual, and the like.
By using the quantification method described above, the blood Phe concentration of a phenylketonuria patient can be easily evaluated using venous blood or capillary blood as test blood. The test blood is preferably capillary blood collected from a fingertip. As a result, the blood Phe concentration in a phenylketonuria patient can be managed periodically (for example, every day or at regular intervals) or non-periodically at a place other than a medical institution. Therefore, according to the present invention, it is possible to provide a method for managing the blood Phe concentration in a phenylketonuria patient by using the quantification method described above. The method can be performed using the measurement kit described above.
Hereinafter, the present invention is described using Experimental Examples in order to help understanding of the configuration and effect of the present invention. However, the present invention is not limited by these Experimental Examples. The following experiments were performed under room temperature (20+5° C.) and atmospheric pressure conditions unless otherwise specified. Unless otherwise specified, in the following, “%” means “% by mass” and “part” means “mass part.” In addition, the following experiments were performed after obtaining approval in advance by the Ethics Committee of Tohoku University Graduate School of Medicine (Japan) (approval number 2020-1-362).
The test piece, the ammonia measurement device, and the PAL-containing solution used in the following Experimental Examples are as follows.
AMMONIA TEST KIT II AMICHECK (registered trademark) reaction test paper (ex vivo diagnostics): manufactured by ARKRAY, Inc.
In the following Experimental Examples, this is referred to as
When a test sample (blood) is spotted to the sample holding layer on the spacer of AMICHECK, the alkali buffer in the sample holding layer dissolves and the test sample becomes alkaline. By this process, ammonium ions in the test sample are gasified to become ammonia gas, and the gas passes through the holes of the spacer and transfers to the indicator layer. When the ammonia gas transferred to the indicator layer reacts with the indicator in the indicator layer, the color of the indicator layer changes. When the spacer laminated on the base film is peeled off and the part of the indicator layer on the base film is subjected to a measurement unit of the following ammonia measurement device, the degree of color change (color intensity) of the indicator layer is automatically sensed and the ammonia concentration (N-μg/dL) (as the amount of nitrogen) is displayed. In this way, the ammonia concentration in the test sample (blood) can be measured by using the AMICHECK and the ammonia measurement device.
(2) Ammonia measurement device
Blood ammonia measurement device PocketChem™ BA PA-4140:
manufactured by ARKRAY, Inc.
In the following Experimental Examples, this is referred to as an “ammonia measurement device”.
The cDNA of PAL contained in plasmid (#78286, Addgene, Cambridge, USA) was inserted into a pET21d (+) vector (69743, Novagen Merck Millipore, Germany) using In-Fusion HD Cloning Kit (Takara, Japan), and a His6 tag was added at the C-terminus. The produced pET21d(+) was transfected into E. coli HMS174 (DE3) strain (69453, Merck Millipore, Germany) and cultured until ODm=0.6, then 1 mM isopropylthio-a-D-galactopyranoside (9030, TakaRa, Japan) was added thereto, and the culture was further continued at 25° C. for 15 hours. The cultured E. coli was recovered and lysed using BugBuster protein extraction reagent (70584, Novagen Merck Millipore, Germany), and micrococcal nuclease (2900A, TaKaRa, Japan) was added thereto. PAL was collected with HisSpinTrap (28401353, GE Healthcare, UK) and dialyzed three times (1 hour, 2 hours, 1 day) with 50 mM Tris-HCl solution (pH 8.8) using Mini Dialysis Kit with a 1 kDa cut-off (28955964, GE Healthcare, UK). After concentrating with Amicon Ultra (UFC 500324, Merck Millipore, Germany), the protein concentration was measured with PierceM BCA Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, USA). In addition, the protein was electrophoresed with an SDS-PAGE gel and then stained with TakaRa CBB Protein Safe Stain (Takara, Japan), and a single band matching the position of the predicted molecular weight was confirmed.
The following three types of PAL-containing liquids were prepared using the recombinant PAL produced using E. coli in the above (a).
A PAL-containing liquid is prepared with ultrapure water so as to have the following concentration. Stored at 4° C. until use.
A PAL-containing liquid (for stock) is prepared in ultrapure water so as to have the following concentration, dispensed in 20 μL parts into an Eppendorf, and stored at −20° C. until used. Before use, the product temperature is returned to room temperature, and the volume is made up to 100 μL with 50 mM Tris-HCl to obtain a PAL-containing liquid.
In the Reference Experimental Example and Experimental Example described later, the PAL-containing liquid prepared by the above method was used (the content of PAL in the diluted PAL-containing liquid was 1 to 3 mg/mL (as a protein content)).
(iii) PAL-Containing Liquid (No. 3)
A PAL-containing liquid (for lyophilization) is prepared so as to have the following concentration, dispensed in 100 μL parts into an Eppendorf, stored at −80° ° C. for 24 hours, and preliminarily frozen. This is lyophilized using a lyophilizer and the resulting lyophilized PAL is stored under room temperature conditions until use. Before use, 100 μL of ultrapure water is added to dissolve the lyophilized PAL to prepare a PAL-containing liquid.
Ninety-six (96) μL of a 30 mM aqueous Phe solution was placed in a 96 well plate and allowed to stand at 30° C. for 6 minutes, then 4 μL of the PAL-containing liquid (No. 3 before freezing) prepared above was mixed, and the absorbance at 275 nm was measured at 30° C. every 5 seconds for 2 minutes. The specific activity (U/mg) was calculated using the following formula (1: Journal of Biotechnology 258 (2017) 148-157).
As a result, the specific activity of PAL (recombinant produced by E. coli) produced in the above (a) was 0.332+0.014 U/mg.
ΔA: Increasing value of absorbance at 275 nm per minute
V: Total capacity per well (100 μL)
f: Dilution rate of enzyme liquid (1)
ε: Extinction coefficient of cinnamic acid at 275 nm (17.218 mM−1 cm−1)
d: Optical path length (0.28 cm)
C: Enzyme concentration (mg/mL)
v: Dose of enzyme liquid (4 μL)
As shown in the following Table 1, even when the PAL is lyophilized and then stored in that state for at least 5 weeks (at room temperature), the PAL retains enzyme activity that is acceptable for use of the present invention.
Table 1 shows the results of measuring the specific activity (U/mg) of the lyophilized PAL prepared in the above (iii) by the method in the above (c) after storing the lyophilized PAL at room temperature for 0 day, 1 week, 3 weeks, and 5 weeks, respectively, and then dissolving 100 μL of ultrapure water into a PAL-containing liquid.
As shown in Table 1, the specific activity of PAL was retained at 0.2 mU or more even when PAL was stored at room temperature for 5 weeks after lyophilization. From this result, it was confirmed that E. coli recombinant PAL is relatively stable in a dried state, and can be used in the quantification method as long as the E. coli recombinant PAL is stored for at least 5 weeks or less under room temperature conditions.
Capillary blood was collected from fingertips of six healthy persons (volunteers) and used as test blood, and the ammonia concentration in the test blood was measured using the AMICHECK and the ammonia measurement device. Measurement of blood ammonia concentration was performed according to the method described in the package insert of AMICHECK.
The results are shown in
As a reason for this, it is considered that ammonia contained in sweat and dirt of the fingertip is mixed when blood is collected from the capillary of the fingertip, and the amount of ammonia in the capillary blood cannot be accurately measured. This is also consistent with the description of “Do not use fingertip blood as it may show a high value if sweat or tissue fluid is mixed.” as “operational precautions” in the package insert of AMICHECK.
The method of the present invention is a method for indirectly determining the blood Phe concentration of a subject by measuring the amount of Phe-derived ammonia generated by reacting PAL with Phe of a test sample. For this reason, if the ammonia concentration in the test sample fluctuates before the measurement, the Phe value to be obtained is affected, and the reliability of the Phe value is lowered.
Therefore, as a method for solving this, the following operation was performed using capillary blood collected from fingertips of seven healthy persons (volunteers) as test blood.
(1) The temporarily adhered spacer part (first member) of the AMICHECK and the base film part (second member) are separated.
(2) Ten μL of test blood (whole blood) is dropped on the sample holding layer of the spacer part.
(3) The test blood is left to stand as it is for each of 0 minutes, 1 minute, 2 minutes, 5 minutes, and 10 minutes under the condition of a relative humidity of 40 to 80% RH, then the spacer is superposed on the base film as before, and left to stand as described in the package insert of AMICHECK for 180 seconds, and ammonia gas generated in the sample holding layer is reacted with the indicator layer.
(4) At each leaving time, the indicator layer part of the developed base film is provided to the measurement unit of the ammonia measurement device, and the ammonia measured value displayed on the ammonia measurement device is read.
As shown in
Blood was collected (venous blood) from veins of three healthy persons (volunteers) (A to C), and a Phe solution having a known concentration was added to and mixed with the test blood at a rate of 5% v/v. The Phe solution was prepared by stepwise dissolving Phe in a 50 mM Tris-HCl aqueous solution so that the concentration of Phe added to the test blood was 0 to 500 μM.
The test sample obtained by adding and mixing a known amount of Phe to and with the test blood was mixed with 2 times the volume of the PAL-containing liquid, and 20 μL of the mixture was dropped on the sample holding layer of AMICHECK and left to stand at normal temperature for 10 minutes. Thereafter, the spacer was peeled off from the base film, the indicator layer part on the developed base film is placed on the measurement unit of the ammonia measurement device, and the ammonia measured value displayed on the ammonia measurement device was read.
From this result, it was confirmed that Phe in the test sample can be indirectly quantified by measuring the amount of ammonia generated by the reaction with PAL using venous blood containing Phe as the test sample using the AMICHECK.
Ten (10) μL of capillary blood (test blood) collected from a fingertip of a patient (N=3) with phenylketonuria (PKU) was dropped on the sample holding layer on the spacer of AMICHECK obtained by separating the spacer (first member) and the base film (second member), and the sample holding layer was left to stand at room temperature for 10 minutes. After being left to stand, the spacer was superposed on the base film at the original position, 10 μL of the PAL-containing liquid was dropped on the sample holding layer on the spacer, and the spacer was again left to stand at room temperature for 10 minutes. After being left to stand, the indicator layer part on the developed base film was placed on the measurement unit of the ammonia measurement device, and the ammonia measured value displayed on the ammonia measurement device was read.
In parallel, the measurement (HPLC method) of the Phe concentration in blood plasma was entrusted to an external clinical laboratory company (BML, INC.) using the blood plasma of blood (venous blood) collected from the vein of the same PKU patient as a test sample.
Measurement reagent: Amino acid kit (Ninhydrin test solution Wako amino acid automatic analyzer kit: manufactured by FUJIFILM Wako Pure Chemical Corporation)
Amino acid analyzer: L-8900 (Hitachi High-Technologies Corporation)
In order to verify the results of Experimental Example 2, 10 μL of capillary blood collected from the fingertip of PKU patients (N=3) different from those of Experimental Example 2 was used as test blood, and the ammonia concentration in the test blood was measured using the AMICHECK and the ammonia measurement device in the same manner as in Experimental Example 2. As conducted in Experimental Example 2, blood (venous blood) collected from the vein of the same PKU patients was used as a test sample, and measurement of the Phe concentration in plasma was outsourced to an external company concurrently.
When the measurable range of 6 to 240 μM (10 to 400 μg/dL) of the ammonia measurement device is applied to the regression equation obtained in
Experiments A to C described in Table 2 were performed, and the accuracy (correlation between the ammonia concentration in the capillary blood and the plasma Phe concentration) of the quantification method (experiment C) was evaluated by comparing experiment A with experiment B1 and experiment B2 with experiment C, respectively.
Venous blood and capillary blood (blood collected from fingertip) were collected from PKU patients (N=14), and Phe concentrations in plasma and capillary blood were measured using these blood samples as test blood by the methods of Experimental. Examples A and B1 in Table 2, respectively.
The obtained results were plotted on the X-axis for plasma Phe concentration and on the Y-axis for capillary blood Phe concentration (
Each aqueous Phe solution was prepared using ultrapure water as a solvent so that the Phe concentrations were 0 mM, 1.21 mM, 2.42 mM, and 3.63 mM, respectively. An aqueous Phe solution at each concentration was added to and mixed with capillary blood collected from the fingertips of healthy persons at a ratio of 58 v/v, and these were used as test samples (N=105).
For the test samples, the Phe concentration was measured by the method of Experimental Example B2 in Table 2, and the NH3 concentration was measured by the method of Experimental Example C2.
The obtained results were plotted on the X-axis for the Phe concentration and on the Y-axis for the NH3 concentration (ammonia measured value) (
From this result, it was confirmed that the Phe concentration in the test samples can be indirectly quantified (estimated) by measuring the amount of ammonia (Phe-derived NH3) generated by the reaction with the PAL using the AMICHECK and the ammonia measurement device and applying the regression equation to the obtained ammonia measured value using the capillary blood containing Phe as the test samples.
When the two regression equations Y=0.8731X−12.75 and Y=0.5007X+50.48 obtained in (1) and (2) above are combined, the following relational formula is established: Mathematical formula 2
Y: Plasma Phe concentration of venous blood
X: Ammonia measured value by the quantification method
The ammonia measured value obtained by performing the experiment C1 using the capillary blood (N=11) derived from PKU patients was assigned to X of the relational formula, and the plasma Phe concentration (Y) of the venous blood of the patient was calculated. The value (estimated plasma Phe value) was plotted on the Y-axis, and the plasma Phe concentration of the venous blood of PKU patients obtained in experiment A was plotted on the X-axis (
When the measurable range of 6 to 235 μM (10 to 400 mg/dl) of the ammonia measurement device is applied to the regression equation obtained above, the range of the detectable blood Phe concentration is estimated to be 0 to 437 μM.
In order to prevent complications in PKU patients, it is recommended to control the plasma Phe concentration within the range of 120 to 360 μM (Clinical Practice Guideline for Target Disease Subject to Neonatal Mass Screening 2019, SHINDAN TO CHIRYO SHA, Inc., page 16, NPL 2). Therefore, a plasma Phe concentration of 0 to 1400 UM, preferably 0 to 437 μM, which can be detected with the method of the present invention, can be sufficiently used clinically. That is, since the method of the present invention is a clinically practical method and can be easily and quickly measured, it can be effectively used for screening for early detection of phenylketonuria in neonates, monitoring of the blood Phe concentration of phenylketonuria patients at the bedside, and monitoring and management of the blood Phe concentration in dietary therapy in daily life.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2021-083432 | May 2021 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2022/020562 | 5/17/2022 | WO |
