JPS63148997A - Measurement of electrophoretic fraction and reagent for measuring said fraction - Google Patents

Abstract

PURPOSE:To prevent occurrence of coloring of abnormal band or background of a support and enable measurement with a high accuracy, by the coexistence of a tetrazolium salt and superoxide dismutase in a staining solution of an electrophoretic fraction. CONSTITUTION:Creatine kinase, lactic dehydrogenase, glutamic oxalacetic transaminase, amylase, leucine aminopeptidase and total cholesterol, etc., are fractionated by electrophoresis using agarose, agar, starch gel, filter paper, etc., as a support and the fraction is stained with a tetrazolium salt. In the process, superoxide dismutase (SOD) coexists in the tetrazolium salt. Furthermore, a sugaralcohol preferably coexists. The sugaralcohol is preferably D- mannitol and/or D-sorbitol. The SOD is preferably present in an amount of 15-300 units/ml and the sugaralcohol is preferably present in a concentration of 0.5-5g/dl.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for measuring electrophoretic fractions and a reagent for measuring the fractions, and more particularly, to a method for measuring electrophoretic fractions using formazan color development by reaction with a tetrazolium salt. The present invention relates to a fraction measurement method that eliminates or reduces non-specific coloration and/or background coloration of a support, and a novel reagent for use in the electrophoretic fraction measurement.

■Is the electrophoretic fraction measurement method using p-staining tetrazolium salt staining a useful method?There are still some issues to be solved in order to obtain even higher quality measurement results in the fields of diagnosis, treatment, etc. There are issues to be addressed. From the viewpoint of reliability and accuracy, it is particularly important to eliminate non-specific coloring and/or background coloring of the support or to reduce λ, but a fully satisfactory means has not yet been found.

For example, the case of fractionation of creatine kinase (CK) is as follows.

There are three types of isozymes for CK: CK-M~1. GK-MB
and GK-BB, and normal +fn serum usually contains C
Only K- to IM exists, or CK-MM increases in muscle disease, CK-MI3 appears in heart disease, and CK-BB appears in brain/smooth muscle disease. In addition, since CK may have CK anomalies that bind to immunoglobulins, it is important to
It has great clinical significance if it determines a+++.

To measure this CK isozyme, an electrophoresis method using tetrazolium salt staining may be adopted.
Abnormal band coloration or coloration of the electrophoresis support may appear due to a non-specific reaction called thing dehydrogenase.

In this specification, the coloring of such abnormal bands and the background coloring of the support will be hereinafter referred to as non-specific coloring, using the word i. When this non-specific coloration occurs, various inconveniences occur, such as the fraction pattern becoming unclear and the pattern becoming difficult to identify, which may lead to a risk of making a diagnosis error. Therefore, such non-specific 51
It has long been desired to develop a method that does not cause quaternary coloring.

In addition, non-specific coloring is widely seen not only in fraction measurement of CK isozyme but also in fraction measurement methods that use tetrazolium salt staining. However, to date, no effective f7 elimination method for such non-specific coloring has been found. Regarding the abnormal bands caused by Song dehydrogenase, for example, see Ogawa Aibe et al.
Enzyme Histochemistry, Tokyo Asakura JF Store, 3rd edition, 95-10
5 (1968)) reported that this band is involved in Si2 stores bound to proteins in serum, but the details 4 (1) are not well understood, so it is likely that the The appearance of l;' okenase, j;+, ha/1. is suspected in the case i'↑:
i. For example, in the case of measuring CK isozyme fraction, it is necessary to measure using training straw without creatine phosphate, which is a substrate for GK activity, to confirm whether it is CK activity or a non-specific band. The problem is simply being dealt with by using auxiliary means. Furthermore, regarding background coloring, it is only relying on improving the accuracy of blank correction using densitometry, and is not concerned with erasing these non-specific colorings, so it cannot be an essential solution. do not have.

DISCLOSURE OF THE INVENTION In view of the above circumstances, the inventors have repeatedly conducted decolorization research on electrophoretic fraction measurement methods that eliminate non-specific coloring. By adding nosmutase (hereinafter abbreviated as SOD) alone or together with a sugar alcohol, it is possible to eliminate or reduce non-specific coloration without affecting the fraction measurement value during pregnancy. This led to the discovery of this and the completion of the present invention.

That is, the present invention provides a fraction measuring method using F;+) filter trazolium salt staining in electrophoresis, which is characterized in that non-specific coloring is eliminated or reduced by allowing SOD to coexist. It provides:

Furthermore, the present invention does not require any reagents to be used in this method.

Examples of electrophoretic fraction measurements to which the present invention can be applied include using supports such as agarose, agar, cellulose acetate-1, polyacrylamide gel, starch gel, and filter paper, and measuring creadinginase as a target object using a conventional method. (C
K), lactate dehydrogenase (Li) H), glutamate oxaloacetate transaminase (GOT), amylase (AMY>, leucine aminopebutidase (LA)
Examples include fraction measurement in which fractions such as P) and total cholesterol (TC) are fractionated, and then the fractions are stained using a tetrazolium salt.

Tetrazolium salts used in fraction measurement include nitrotetrazolium blue (NTB) and MTT.

INT, neotetrazolium blue (Neo-TB),
Examples include, but are not limited to, tetrazolium blue (TB) and tetranitrotetrazolium blue (TNTB), and any tetrazolium salt used for formazan color development may be used.

The SOD used in the present invention can be classified according to the metal serving as the active center.Cu, Zn-SOD, Fe-
There are three types, 9OD and Mn-5OD, both of which are easily available as commercial products. SOD may be in any form, but it is freeze-dried using a conventional method.

It is completely convenient to use the lyophilized product obtained by

Examples of sugar alcohols used in the present invention include D-mannitol, D-sorbitol, and dulcitrate. These can be used alone or in combination of two or more, and may be included in the SOD lyophilized product in advance as an excipient.

The fraction measurement method of the present invention is carried out in exactly the same manner as the conventional fraction measurement using tetrazolium salt staining, except that SOD is used alone or together with a sugar alcohol in the staining system. can be done, i.e.
Sprinkle the staining solution on the electrophoresed support using the usual method.
Alternatively, there is an embodiment in which the support is immersed in a staining solution, and any of the conventional staining embodiments can be appropriately selected depending on the electrophoresis support, the object to be fractionated, and other electrophoresis conditions.

The staining solution used in fraction measurement can be the one used in conventional electrophoresis, or the measurement reagent for tetrazolium salt staining, such as CK measurement reagent, L D l-(measurement reagent, GOT measurement reagent, A
It is prepared by adding a predetermined amount of SOD alone or together with a predetermined sugar alcohol to the M Y measurement reagent, L A P measurement reagent, or 'FC measurement reagent.

Furthermore, it is appropriate to prepare the staining reagent for each measurement reagent by containing in advance a predetermined m amount of SOD alone or together with a predetermined m amount of sugar alcohol.

Commercial products can be used as the SOD-free measuring reagent, but when preparing a staining solution by a conventional method, it is preferable to add the tetrazolium salt last in order to minimize the coloring of the staining solution itself.

In the SOD-containing staining solution used in the fraction measurement method of the present invention, the concentration of SOD and sugar alcohol that should be allowed to coexist are not particularly limited as long as the activities of the fraction substances and detection enzymes are inhibited. However, CK measurement is usually performed using SO.
D is 1 to 500 units/ll12, preferably 15 to 30
0 units/zg, sugar alcohols 0.1 to IOg/dc,
Preferably it is in the range of 0.5 to 59/dQ. Also,
L D 11 measurement, Go'r measurement, AMY measurement, L
The same range is preferable for the 7-P measurement and the 'rc measurement. If the lower limit of these ranges is not met, the elimination of non-specific coloring will not be sufficient, while if the upper limit is exceeded, the elimination effect of non-specific coloring will be almost constant, which is not economical, and it is recommended to exceed the upper limit by a large amount. Inhibition of activity may occur. If it is within the above range, non-specific coloring can be suitably eliminated and a clear image can be obtained, and if necessary, it can be subjected to, for example, densitometry to obtain a denontogram, which can be used for diagnosis.

The present invention provides a reagent for measuring electrophoretic fractions, which is characterized in that it is made by combining a reagent for measuring fractions by electrophoresis using teI lazolium salt staining and SOD.

SOD-free p:i l111 using tetrazolium salt
As mentioned above, the 1 constant reagents include conventional CK measurement reagents,
Examples include L D t ■ measurement reagent, GOT measurement reagent, AMY measurement reagent, L A P measurement reagent, and TC measurement reagent.

To combine SOD, add SOD alone or
OD and sugar alcohol may be contained at the same time, and for convenience of handling, it can be made into a freeze-dried product by a conventional method.

The SOD content and sugar alcohol content can be determined by any method that can achieve the SOD concentration and sugar alcohol concentration [y] in the fraction measurement method described above when preparing the staining solution.

As the buffer solution other than the SOD-impregnated staining reagent and the tetrazolium salt solution measurement reagent, commercially available reagents for measuring fractions by electrophoresis using each of the above-mentioned tetrazolium salt stains can be used, or they can be appropriately prepared by conventional methods.

In one embodiment of the reagent for fraction measurement of the present invention, SO
A reagent kit is prepared by combining a D-containing staining reagent, a buffer solution, and a tetrazolium salt solution. When used, the three pieces are mixed in a predetermined ratio to achieve the 5ODa degree and sugar alcohol concentration in the fraction-11 determination method.

Furthermore, in the measurement reagent of the present invention, a reagent for fraction measurement by electrophoresis using tetrazolium salt staining and an SOD lyophilized product may be combined.

SOD freeze-dried products include Cu and Zn-9OD.

There are three types of freeze-dried products, Fe-9OD and Mn-5OD, which are either commercially available or can be easily prepared by a normal freeze-drying method. The SOD lyophilized product preferably contains a sugar alcohol as described above.

In this embodiment, a reagent kit can be prepared by combining a conventional reagent for measuring fractions by electrophoresis using tetrazolium salt staining and a lyophilized SOD product containing a sugar alcohol as an excipient.

When used, both are mixed in a predetermined ratio.

This ratio may be any value as long as it can realize the SOD concentration and sugar alcohol concentration in the fraction measuring method.

The effects of the present invention will be explained below with reference to experimental examples. Although this experimental example is a model test and was not conducted on a fraction using a support, it sufficiently supports the effects of the present invention.

Experimental Example I Reduction of non-specific coloring degree by addition of SOD 1: t-+sop-free constant K measurement reagent 5SCC (
The 5candinavian 5ociet
yfor C11nical Chemistry
and C11ni-cal P hysio
CK measurement reagent using the UV method based on the proposed recommendation (Scand, J., CIin.

Lab, Invest, 36:7 1
1-723, 1970) (see Example 1), diaphorase (DI) (U position/ffc) and nitrotetrazolium blue (NTB) 30 (Jyrg/dQ) were added.

:2] Specimen inactivated serum: Warm the serum at 56°C for 20 minutes,
Immobilized shinoceros were used.

[3] Staining method and non-specific coloring evaluation method Toyobo 5OD (Cu, Zn-9OD from orchid red + a bulb) was added to the SOD-free GK measurement reagent at O~200 units/M (!) Each solution was added. 0.57ρ was collected in test tubes, inactivated serum was added to each at 100/IQ, and incubated at 37°C for 15 minutes.Then, the reaction was stopped by adding O,IN hydrochloric acid 5z&, and the incubation was performed at a wavelength of 560 nm. (This absorbance is referred to as (A).) Similarly, as a serum blank, immobilized serum was added after the reaction was stopped, and the absorbance was measured using the same 0!1 column (this absorbance was referred to as (B)). The optical density difference (80, D = (A) - (B)) was used as a multiplier for the degree of non-specific coloration, and the results are shown in Table 1.

Table 1 As is clear from Table 1, by co-treating SOD with γY in conventional tetrazolium salt staining, the degree of non-specific staining can be reduced.

Experimental Example 2 Synergistic effect of reducing the degree of non-specific coloring by adding sugar alcohol Addition of 5OD30 units/unit to the SOD-free measurement reagent of Experimental Example I and θ ~ 5g/ml of D-mannitol as a sugar alcohol
The optical density difference was determined in the same manner as in Experimental Example I except that d(! was added. The results are shown in Table 2.

As is clear from Table 2, by coexisting D-mannitol in addition to SOD, the degree of non-specific coloring can be further reduced compared to the case of SOD alone.

The present invention will be explained in more detail with reference to Examples below.

Example I Erasure of abnormal bands by nothing dehydrogenase [Preparation of GK measurement reagent containing 1 SOD by UV method using the following formulation based on the 5scc recommendation.
K measurement reagent includes DI5 units/RQ, Toyobo 5OD (Cu
, Zn-9OD) 501st place/Shoulder &, D-Manni l-2
g/dQ and NT[3300+9/d17 were added in this order to prepare a SOD-containing CK measurement reagent.

<5scc recommended prescription> Ingredients imidazole 100mM, pH 6.5 creatine phosphate 30mM adenosine
5'-novosphate 2mM (ADP) D-glucose 20mM nicotinamide adeniadenosine 2mM leotide phosphate (NADP) Hexokinase 3500 units/12D-
Glue J-su-6-phos 2000 units/a Phate dihydrogenase vinegar magnesium 10mM adenosine-5'-monophos 5mM Phate (AMP) pl, p 5-di(adenosine-5゛)be 10
μM Ntaphosphate (AP5A) N-acetylcysteine 20mM [2] Fraction measurement 3μQ of normal serum sample was placed on an agarose film (Polu E-
film system, manufactured by Ciba Coating Co., Ltd.) and containing 1mM EDTA as a running buffer.
Using M barbital buffer, about 4 hours using a Poru E-film system electrophoresis device (manufactured by Ciba Coating Co., Ltd.).
Electrophoresis was performed for 0 minutes to obtain a CK fraction.

The SOD-containing CK measurement reagent 3.01 prepared above was sprinkled onto the film gel surface to make it uniform, and then incubated at 37° C. for 30 minutes.

The fill was then fixed by soaking in 10% acetic acid, washed with water, and dried. After drying, decintometer ADC-20SP (
(manufactured by Kayagaki) and subjected to desintometry at 570 nm. As a control, exactly the same operation was performed in the case where SOD and D-mannitol were not added.

Examples of separated images after drying are shown in the attached FIGS. 1 and 2, and desyntogelum is shown in FIGS. 4 to 7. In addition, three types of CK
For comparison, an example of a quantitative curve with no non-specific coloring when an isozyme-labeled product is used is shown in FIG.

Figure 1 shows an example of CK staining by the conventional method in which SOD and sugar alcohols are not present, and non-specific staining is observed.

On the other hand, in FIG. 2, which is a staining example of the present invention, no non-specific coloring is observed.

Figures 4 to 7 show desynthogens compared between the conventional method and the present invention method for specimens 1 and 2. It can be seen that a clear peak was obtained.

Example 2 Elimination of background coloring Sakakibara Nis et al.
tal, ) (CIin, Chem, Acta,
l 33: l l 'J-I 23 (+983)), and further added Toyobo's 5OD (Cu, Zn-5).
An SOD-containing GOT measurement reagent containing the following components to which OD) and [)-mannilla h were added was prepared.

<Formulation> Ingredients imidazole 100mM, pH 7.6
L-instinsulfinic acid 200mM α-ketoglutaric acid 10mM, -phenazine methosulfate 0.1m9/11&(m-P~IS
).

N T B O, 8th order g
/yσSOD 50 units/m
l2D-Mannitol 29/dQ
.

A minute image as schematically shown in FIG. 8 was obtained in the same manner as in Example 1 except that an SOD-containing GOT measurement reagent was used instead of the SOD-containing CK measurement reagent.

As is clear from FIG. 8, the background coloring of the agarose film has been erased.

Example a Reagent for measuring CK fraction (1) Staining reagent The following components were mixed and a freeze-dried product was prepared by a conventional method.

Creatine phosphate 30mM adenosine-5'-diphosphate 2mlV1 (ADP) D-glucose 20mM Nicodinamide adenine 2mM Reotide phosphate (NADP) Hexokinase 35oO units//:l
D -Curcose-6-phosphene 20 Q O unit ρ
-dihydrogenase magnesium acetate 10mM adenosine-5°-monophosphate 5mMmt~1P) pI, p5-di(adenosine-5°)be 1
0μM Ntaphosphate (AP5A) N-acetylcysteine 20mM DI
5000 units/QSOD
50 units/mQD-Mannitol/d ((2) Buffer imidazole solution 100mM, p)16.
5(3) Tetrazolium salt solution NTB solution 3y/(One or more reagents (1) to (3) were combined to form a reagent kit, and a reagent for measuring the SOD-containing CK fraction of the present invention was obtained.

In order to use this reagent for measuring CK fraction, for example, the above-mentioned staining reagent is dissolved in 2.5 mQ of a buffer solution, and then 0.5 Tl (1) of a tetrazolium salt solution is added and used for staining.

Example 4 Reagent for measuring GOT fraction (1) Staining reagent A lyophilized product was prepared by mixing the following components by a conventional method.

■, -instinsulfinic acid 200mMα-ketoglutaric acid 10mM5OD
50 units/mQD-manninoh
2g/d12 (2) Buffer imidazole solution 100m~1, Kano ■7.6
(3) Tetrazolium salt solution m-zenanone methosulfate O, Lu/mQN
TB O,8 Takumi/IQ
A reagent kit was prepared by combining the above reagents (1) to (3) prepared by conventional methods.

In order to use this reagent for measuring the co'r fraction, the staining reagent is dissolved in a buffer solution of 2.0 ml, and then 0.5 ml (l) of a tetrazolium salt solution is added and used for staining.

Example 5 Reagent for measuring CK fraction A SOD-free CK measuring reagent was prepared by a conventional method according to the following recipe.

Formula> Ingredients Imidazole 100mM% pH 6.5 Creatine Phosphate 30mM Adenosine
5'-diphosphate 2mM (ADP) D-glucose 20mM nicotinamide adenine 2mM leotide phosphate (NADP) Hexokinase 3500 units/12D
-glucose-6-phosphe 2000 units/Q-tonohydrogenase magnesium acetate 10mM adenosine-5°-monophosphe 5mMm tot MP) p I, p S-di(adenonone-5')pe 1
0μM Ntaposuphate (AP5A) N-Acetylnstin 20mM DI
5000 units/aI'J
T B 39/Q On the other hand, D-mannite was added as an excipient to SOD by a conventional method, mixed, and lyophilized to prepare a lyophilized SOD product containing D-mannite (SOD content 15 per vial).
0 units, 601 g of mashed D-mannitol). Next, the S, OD-free constant K measurement reagent SOD lyophilized product is combined to form a reagent kit, and the SOD-containing CK of the present invention is prepared.
A reagent for fraction measurement was obtained.

In order to use this reagent for measuring CK fraction, S
The OD-free GK fraction measurement reagent and the SOD lyophilized product are subjected to staining, for example, by dissolving 1 vial of the SOD lyophilized product with 3x (1) of the SOD-free OK measurement reagent.

Example 6 Reagent for Measuring GOT Fraction A reagent for measuring GOT fraction containing no SOD was prepared by a conventional method according to the following recipe.

Prescription> Ingredient imidazole] 000ml 1vl, I
) J47, 6L-instinsulfinic acid 2
00 m Mα-ketoglutaric acid 1
0 m Mm-phenazine methosulfate O,l
zy/i(!(m-PMS) N T B O, 8xg
/vrQ Meanwhile, in the same manner as in Example 5, D-mannilla]・
Contains SOD lyophilized product (■ Contains SOD per vial ff)
1150 units, D-mannite-containing ffi60mg) was prepared. Next, the reagent for measuring the SOD-free co'r fraction and the SOD lyophilized product were combined to form a reagent kit to obtain the reagent for measuring the SOD-containing GOT fraction of the present invention.

In order to use this reagent for GOT fraction measurement,
SOD-free GOT fraction measurement reagent and S.

The freeze-dried product D is subjected to staining, for example, by dissolving one vial of the SOD freeze-dried product with SOD-free GOT fraction measuring reagent 2.51.

[Brief explanation of the drawing]

Figures 1 and 2 are diagrams showing CK fraction images obtained by the conventional method and the method of the present invention, respectively. Figure 3 is a diagram depicting the fractionation quantification curve of the CK isozyme standard product. Figures 3 to 7 are diagrams respectively. FIG. This is a diagram schematically representing the method of the present invention. Patent applicant: Nippon Shoji Co., Ltd.
People Patent Attorney Aoyama Ao 2 Idiots 411 Figure CK
Imaichi Shii Family (Junior Aζ Kojo) - Kazuichi Ichiichi 3rd Question (Comparison) MM Me ELS <Sample 1> Heavy Figure 4 (Iken Multiplication Law) Figure 5 (
Present invention 3) Results A2> 6th section (technical final method) Figure 7 (present invention method) 11, 8
f'm GOT e ga'/Zojourakuho
Bll-procedural amendment (Dear Commissioner of the Self-Restrained Patent Office, January 22, 1986 2) Title of the invention: Method for measuring electrophoretic fractions and reagents for measuring said fractions 3. Relationship with the case by the person making the amendment: Address of the patent applicant. 2-30 Ishimachi, Higashi-ku, Osaka-shi, Osaka Prefecture Name Nippon Shoji Co., Ltd. 4 Agent 5 Date of amendment order Voluntary 7 Contents of amendment (1) Detailed description of the invention column (1) Page 13, 4 lines from the bottom, r 300 mg/dcJ
Correct the statement to r50mg/dQJ. (2) Page 16, lines 2-3, r 300 mg/d12J
Correct the statement to r 50 mg/d2J. (3) Page 17, line 1, r(AP5A)j is replaced by r(A
psA)”. (4) On page 17, lines 5-6 and 9, "manufactured by Ciba Coating Co., Ltd." is corrected to "manufactured by Ciba Corning Co., Ltd." (5) Page 17, line 8, between “buffer” and “use,” “
(pH 8, 6) Insert J. (6) In the 7th line from the bottom of page 17, the word "fill" is corrected to "film". (7) Page 17, lines 6-5 from the bottom, “Densitometer”
Correct the word "densitometer" to "densitometer". (8) In the 4th line from the bottom of page 17, the word "desyntometry" is corrected to "densitometry." (9) On page 18, line 1, "dentogram" is corrected to "densitogram." (10) On page 18, line 3, the phrase "Example of quantitative curve" is corrected to "Example of Densitogerum." (11) On page 18, line 3, correct the text ``Denjitogerum'' to ``Denjitogerum''. (12) On the 20th page, 8 lines from the bottom, correct r(AP 5 A)j to r(ApsA)J. (13) Page 2 and 2, line 1, “0, 8 mg/mcJ
Correct Toaro to r4g/(!”). (14) Page 23, line 7, correct r(A P 5 A) J and Aro to r(Ap 5 A) J. (15) Page 23, 7 row row, r 3 va" is r5
Corrected to 0mg/dcj. 4. Brief explanation of the drawings (1) On page 23, line 7, the words "fractionation quantitative curve" have been corrected to read "Denjitogerum." (2) Page 25, line 11, “Figure 3” has been replaced with “Figure 4”
and correct it. that's all

Claims (11)

[Claims] (1) In a fraction measurement method using tetrazolium salt staining in electrophoresis, non-specific coloring and/or background coloring of the support is eliminated or reduced by coexisting superoxide dismutase. Minute measurement method. (2) The fraction measurement method according to item (1) above, in which a sugar alcohol is further coexisted. (3) The fraction measuring method according to item (2) above, wherein the sugar alcohol is D-mannite and/or D-sorbitol. (4) A reagent for measuring electrophoretic fractions, comprising a combination of a reagent for measuring fractions by electrophoresis using tetrazolium salt staining and superoxide dismutase. (5) The reagent for fraction measurement according to item (4) above, wherein superoxide dismutase is mixed with a staining reagent of the reagent for fraction measurement. (6) The reagent for fraction measurement according to item (5) above, wherein the mixture is a lyophilized product. (7) A reagent for measuring fractions in item (4) above, which is a combination of a lyophilized product of superoxide dismutase. (8) The reagent for measuring fractions by electrophoresis using the tetrazolium salt staining is a reagent for measuring creatine kinase, a reagent for measuring lactate dehydrogenase, a reagent for measuring glutamate oxaloacetate transaminase, a reagent for measuring amylase, a reagent for measuring leucine aminopeptidase, and a reagent for measuring total cholesterol. Items (4) to (7) selected from the group consisting of
A reagent for fraction measurement according to any one of the items. (9) The reagent for measuring fractions according to item (8) above, wherein the reagent for measuring fractions by electrophoresis using tetrazolium salt staining is a reagent for measuring creatine kinase. (10) The above-mentioned (4) further containing a sugar alcohol
A reagent for measuring a fraction according to any one of items 1 to 9. (11) The reagent for fraction measurement according to item (10) above, wherein the sugar alcohol is D-mannite and/or D-sorbitol.