Diagnostic reagent for complications associated with diabetes or renal failure

In view of the situation of the prior art, the present invention specifies the structure of a late-stage product of Mailard reaction in vivo having a close relation with various tissue disorders, and provides a diagnostic reagent for complications associated with diabetes or renal failure, containing the above compound as a main component. The diagnostic reagent of the present invention for complications associated with diabetes or renal failure contains a pyridinium compound represented by the following formula as a main component: ##STR1##

TECHNICAL FIELD
 The present invention relates to a diagnostic reagent for complications
 associated with diabetes or renal failure, containing a particular
 pyridinium compound as a main component.
 BACKGROUND ART
 As well known, in the Mailard reaction between sugars and protein amines,
 first, sugars and protein amines give rise to non-enzymatic reactions
 (glycation) to form Schiff bases; the Schiff bases give rise to Amadori
 rearrangement reactions, whereby early-stage products of Mailard reaction,
 i.e. ketoamines are formed in a relatively short period; and then, after
 various steps, late-stage products of Mailard reaction are produced.
 Of the early-stage products of Mailard reaction, glycated hemoglobin
 (HbAlc) and glycated albumin (fructosamine) are clinically utilized as an
 indication for control of blood sugar.
 Meanwhile, the late-stage products of Mailard reaction are known to be
 produced from proteins of slow metabolic turnover in-vivo, such as
 collagen, myeline, lens and the like and increase in a prolonged high
 blood sugar condition (diabetes). In order to explain why the persistent
 high blood sugar condition, which is the most characteristic change in
 diabetes, gives rise to specific chronic complications to diabetes,
 attention is being paid to the late-stage products of Mailard reaction
 which may be a cause for the above phenomenon; and it has been suggested
 that the hindrance of production of late-stage products of Mailard
 reaction may prevent the outbreak and development of the complications.
 Also, it is expected that the measurement of the concentration of
 late-stage products of Mailard reaction in blood, urine or tissue may
 allow a way to make a diagnosis of the above-mentioned complications.
 As the components of late-stage products of Mailard reaction, pyrralin,
 pentosidine and crossline etc. are reported. Of these, pyrraline was
 separated from the reaction product between neopentylamine and glucose and
 its structure was determined. Pyrraline is reported to increase in the
 blood of diabetes patient. However, pyrraline has no fluorescence and is
 said to be not an important late-stage product of Mailard reaction.
 Pentosidine was separated from a reaction product of lysine, arginine and
 ribose and its structure was determined. Its concentration is high in the
 skin collagen of diabetes or renal failure patient and significantly high
 particularly in the blood of renal failure patient. However, pentosidine
 contributes to only 1% or less of the total crosslinking between proteins
 in late-stage products of Mailard reaction and is said to be questionable
 as an important late-stage product of Mailard reaction.
 Crossline was separated from the reaction product between acetyllysine and
 glucose and its structure was determined. However, its association with
 diabetes is not clarified yet.
 Thus, the findings obtained by the so-far developed test methods for
 late-stage products of Mailard reaction strongly suggest that there are
 close relations between the increase of later-stage products of Mailard
 reaction in-vivo and various tissue disorders. However, the chemical
 structure of an important late-stage product of Mailard reaction in-vivo
 is unknown.
 In view of the above-mentioned situation of the prior art, the present
 invention has an object of specifying the structure of a late-stage
 product of Mailard reaction in-vivo which has a close relation with
 various tissue disorders and also providing a diagnostic reagent for
 complications associated with diabetes or renal failure, containing the
 above compound as a main component.
 DISCLOSURE OF THE INVENTION
 In order to achieve the above object, the present invention provides a
 diagnostic reagent for complications associated with diabetes or renal
 failure, containing a pyridinium compound represented by the following
 formula as a main component:
 ##STR2##
 The present inventors made an intensive study in order to find a novel
 important late-stage product of Mailard reaction in-vivo which reflects
 the change of disease condition. As a result, the present inventors found
 out a novel fluorescent compound. A further study by the present inventors
 confirmed that the compound is a pyridinium compound having the
 above-shown structure. Moreover, it was found out that the pyridinium
 compound is useful as a diagnostic reagent for complications associated
 with diabetes or renal failure. These have led to the completion of the
 present invention.

BEST MODE FOR CARRYING OUT THE INVENTION
 The present invention is described in detail below.
 The above pyridinium compound, which is a main component of the present
 diagnostic reagent for complications, is a kind of a late-stage product of
 Mailard reaction and is produced by a non-enzymatic reaction between
 proteins (e.g. albumin, lysozyme, casein, ribonuclease or globulin) and
 carbonyl compounds (e.g. glucose, diacetyl, glyoxal or fructose).
 The above pyridinium compound was detected by reacting rabbit erythrocytes
 with glucose at 37.degree. C. for 2 weeks, subjecting the reaction mixture
 to dialysis for 48 hours, hydrolyzing the dialyzate, and then conducting
 high-performance liquid chromatography using an ODS-5 column at an
 excitation wavelength of 340 nm and a fluorescence wavelength of 402 nm.
 The amount of the pyridinium compound produced increased in proportion to
 the concentration of glucose, the reaction time and the browning of the
 reaction mixture, and was suppressed by aminoguanidine which is an
 inhibitor for Mailard reaction. The pyridinium compound was produced
 similarly also in the reaction system of bovine serum albumin and glucose.
 The peak obtained in high-performance liquid chromatography agreed with the
 peak detected for the blood of diabetes patient.
 The structure of the above pyridinium compound was determined as follows.
 First, the late-stage products of Mailard reaction formed by the reaction
 of bovine serum albumin with glucose were hydrolyzed; then, the hydrolysis
 products were separated by high-performance liquid chromatography using a
 fluorescence wavelength and measured for FAB-mass spectrum using a mixture
 of glycerol and nitrobenzyl alcohol as a matrix; thereby, the molecular
 weight of the pyridinium compound was determined to be 212.
 With respect to the fluorescence wavelength, the maximum was observed at an
 excitation wavelength of 350 nm at a fluorescence wavelength of 405 nm.
 Next, by measuring the high-resolution mass spectrum, the compositional
 formula of the pyridinium compound was determined as C.sub.9 H.sub.10
 O.sub.3 N.sub.1 S.sub.1. Further, by the containing two-dimension NMR
 analysis, the pyridinium compound was determined to be
 8-hydroxy-5-methyl-dihydrothiazolo[3,2-a]pyridinium-3-carboxylate
 represented by the following formula:
 ##STR3##
 Incidentally, the pyridinium compound was previously isolated from bovine
 liver as a fluorescent hydrolyzate. Further, the pyridinium compound is
 presumed to be present in the form of a complex between its precursor and
 a protein in living bodies.
 The present inventors made a reaction of 10 mg/ml of proteins (albumin,
 globulin, casein, lysozyme, ribonuclease and protamine), with 0.1 M of
 glucose, and then made subjection the reaction mixture to high-performance
 liquid chromatography. As a result, it was found that the pyridinium
 compound was produced in a large amount from albumin ribonuclease
 (containing arginine and cysteine in large amounts), in a relatively large
 amount from lysozyme, and in a zero amount from protamine (containing
 arginine in a large amount but containing no cysteine).
 However, addition of cysteine to a combination of protamine and glucose
 produced the pyridinium compound. Therefore, glucose, a polyarginine and
 cysteine were found to be necessary for production of the pyridinium
 compound.
 The so-far reported late-stage products of Mailard reaction include, as
 mentioned above, pyrraline, pentosidine, crossline, etc. Since lysine is
 associated with production of any of these products, the pyridinium
 compound is presumed to be a product which has been unknown.
 In order to investigate the effect of reducing sugar in production of the
 pyridinium compound, 10 mg/ml of albumin was reacted with 0.1 M of
 carbonyl-containing compounds (glucose, diacetyl, glyoxal and fructose)
 for 2 weeks, and then the reaction mixture was analyzed by
 high-performance liquid chromatography. As a result, the pyridinium
 compound was produced even in reaction between albumin and diacetyl and
 the amount of the pyridinium compound produced was slightly larger than in
 the reaction between albumin and glucose.
 Diacetyl is an acid hydrolysis product of sugar, and reacts specifically
 with the guanidino group of the arginine residue of protein under a weakly
 alkaline condition and becomes stable to hydrolysis. Therefore, it is
 considered that the glucose in solution first undergoes oxidative
 decomposition and becomes diacetyl, this diacetyl attacks the arginine
 residue, thereby the pyridinium compound is produced.
 The content of the pyridinium compound was significantly low in the serum
 of experimental diabetes rat administered with streptozotocin but
 significantly high in the kidney, tendon, skin and nerve of the rat. The
 amount of the pyridinium compound excreted in urine in the diabetes rat,
 as compared with that in normal rat, was 4.3-fold per ml of urine,
 2.9-fold per protein amount, and 21-fold per day. The amount of the
 pyridinium compound excreted in urine in hereditarily fat diabetes rat
 (OLET-F), as compared with that in normal rat (LETO), was 3-fold per
 protein amount and 22-fold per day.
 These facts suggest that the pyridinium compound reflects the advance of
 condition of diabetes, particularly the reduction of renal function.
 The present invention is described in detail as shown below with reference
 to Examples but is in no way restricted by the Examples.
 EXAMPLE 1
 10 mg/ml of rabbit erythrocytes and 266 mM of glucose were dissolved in a
 10 mM phosphate buffer solution (pH 7.4). The solution was subjected to a
 reaction at 30.degree. C. for 2 weeks. Protein recovery was made using
 trichloroacetic acid, and hydrolysis was conducted at 110.degree. C. for
 24 hours. The reaction mixture was subjected to high-performance liquid
 chromatography at an excitation wavelength of 340 nm at a fluorescence
 wavelength of 402 nm by using an ODS-5 column and pouring 7 mM of
 phosphoric acid at a rate of 1 ml/min. The analytical result is shown in
 FIG. 1. The pyridinium compound of the present invention was detected in
 13 minutes (elution time) and a new peak was detected in about 7 minutes.
 Incidentally, this new peak was formed in a large amount at high
 temperatures of about 60.degree. C. or under an alkaline condition of pH
 9.0 or the like.
 EXAMPLE 2
 The pyridinium compound formed in a reaction system of bovine serum albumin
 and glucose was separated by high-performance liquid chromatography, and
 measured for FAB-mass spectrum using a glycerol-nitrobenzyl alcohol
 mixture as a matrix. The results are shown in FIG. 2. The pyridinium
 compound was also measured for high-resolution mass spectrum, and its
 molecular weight was found to be 212.0404 and the corresponding
 compositional formula was determined to be C.sub.9 H.sub.10 O.sub.3 NS
 (theoretical value=212.0427).
 The pyridinium compound was dissolved in heavy water and measured for
 .sup.1 H-NMR (600 MHz), .sup.13 C-NMR (150 MHz) and HMBC (.sup.1
 H-Detected Heteronuclear Multiple Bond Connectivity). The analytical data
 are shown in Table 1. Also, the spectrum of .sup.1 H-NMR is shown in FIG.
 3 and the spectrum of .sup.13 C-NMR is shown in FIG. 4.
 TABLE 1
 Chemical shifts of .sup.1 H-NMR and .sup.13 C-NMR, and coupling constant of
 .sup.1 H-NMR (in D.sub.2 O, 600 MHz)
 C .delta. (ppm) H .delta. (ppm) J (Hz)
 --CH.sub.3 CH.sub.3 19.9 3H 2.6 s
 2 CH.sub.2 34.4 2H 3.9 d 11.9
 4.1 d, d 11.9, 8.8
 3 CH 73.1 1H 5.8 d 8.8
 5 C 146.3
 6 CH 125.3 1H 7.4 d 8.4
 7 CH 129.5 1H 7.6 d 8.4
 8 C 150.6
 9 C 149.6
 --COOH COOH 171.1
 The methyl group was judged to bond to the aromatic ring because HMBC
 indicated a correlation between the proton 6 and the proton 3. The protons
 2 and the proton 3 showed a typical ABX pattern. That is, one of the
 protons 2 made geminal coupling of 11.9 Hz by doublet at 3.9 ppm, and the
 remaining proton 2 made geminal coupling of 11.9 Hz by doublet at 4.1 ppm
 and also made coupling with the adjacent proton 3 at 8.8 Hz. The proton 3
 made coupling with one of the protons 2 by doublet of 8.8 Hz at 5.8 ppm.
 From these observation results and .sup.1 H-.sup.1 Hcosy, HOHAHA and
 .sup.13 C-NMR spectroscopic data, the structure of the pyridinium compound
 used in the present invention was determined to be
 8-hydroxy-5-methyldihydrothiazolo[3,2-a]pyridinium-3-carboxylate as shown
 below.
 Incidentally, the correlation by HMBC is shown by arrow marks, in the
 following structural formula.
 ##STR4##
 EXAMPLE 3
 55 mg/kg of streptozotocin was administered to SD rats from their tail
 veins to induce diabetes in the rats. The amounts of the pyridinium
 compound in the organs and urine of each rat were measured and compared
 with those of normal rats. The results are shown in Table 2.
 TABLE 2
 Concentrations of pyridinium compound in serum, urine and
 organs
 Normal
 rats Diabetes rats
 Serum (mV .multidot. sec/ml) 127 84.5***
 Tendon (mV .multidot. sec/g tissue) 14.3 314***
 Skin (mV .multidot. sec/g tissue) 117 967***
 Urine
 (.mu.V .multidot. sec/ml .times. 10.sup.3) 28.5 122.4***
 (.mu.V .multidot. sec/mg protein .times. 10.sup.3) 31.6
 92.8***
 (.mu.V .multidot. sec/24 hr .times. 10.sup.6) 5.69 120***
 Average error *** P &lt; 0.001
 The concentrations of the pyridinium compound in the diabetes rats, as
 compared with those in the normal rats, were low in serum but
 significantly high in tendon, skin and urine. In particular, the
 concentration in urine was as high as 4.3-fold per 1 ml of urine, 2.9-fold
 per protein amount, and 21-fold per day.
 EXAMPLE 4
 Hereditarily fat diabetes rats (OLET-F) were raised for 72 weeks, and
 measured for the amount of pyridinium compound in urine and observed for
 the pathological change of kidney tissue. The results of measurement and
 observation were compared with those of normal rats (LETO). The results
 are shown in Table 3.
 TABLE 3
 Amount of pyridinium compound in urine
 36 weeks 48 weeks 60 weeks
 /24 hours /mg protein /24 hours /mg protein /24 hours
 /mg protein
 LETO 28 .+-. 3 0.91 .+-. 0.28 49 .+-. 9 1.8 .+-. 0.5 19 .+-. 4
 0.59 .+-. 0.2
 OLET-F 650 .+-. 96** 5.1 .+-. 0.39** 1072 .+-. 92** 5.0 .+-. 0.2** 2260
 .+-. 298** 9.7 .+-. 0.8**
 Average error ** P &lt; 0.01
 The amounts of the pyridinium compound in the urines of OLET-F, as compared
 with those of LETO, were 23-fold (36-week age), 22-fold (48-week age) and
 16.4-fold (60-week age) per 24 hours, and 5.7-fold (36-week age), 2.8-fold
 (48-week age) and 16.4-fold (60-week age) per mg of protein; and increased
 significantly both per 24 hours and per mg of protein with the advance of
 diabetes.
 In the investigation of the pathological change of kidney tissue, the
 kidneys of OLET-F showed the generation and sclerosis of glomerulus, the
 atrophy of renal tubule, the cellular infiltration into intercellular
 space, the thickening of renal pyelic mucosa, etc. With the advance of
 renal disease, the amount of pyridinium compound excreted into urine
 increased.
 EXAMPLE 5
 The amount of pyridinium compound in urine was measured for hereditarily
 fat diabetes rats (OLET-F) and normal rats (LETO) both of 36-week age,
 48-week age or 60-week age. The results are shown in Table 4.
 Incidentally, the unit used is mv/sec/mg of urinary creatinine.
 TABLE 4
 Concentration of pyridinium compound in urine
 36-week age 48-week age 60-week age
 LETO 1.63 .+-. 0.18 2.51 .+-. 0.78 1.27 .+-. 0.10
 OLET-F 34.2 .+-. 4.11** 62.0 .+-. 5.48** 154.6 .+-. 23.1**
 Mean .+-. standard error ** P &lt; 0.01
 In OLET-F, the amounts of pyridinium compound in urine increased
 significantly with an increase in age. That is, the amounts in OLET-F, as
 compared with those in LETO, were as high as 21.0-fold (36-week age),
 24.7-fold (48-week age) and 122-fold (60-week age).
 EXAMPLE 6
 In the kidneys of OLET-F were seen the generation and sclerosis of
 glomerulus, the atrophy of renal tubule, the cellular infiltration into
 intercellular space, the thickening of renal pyelic mucosa, etc. These
 pathological changes of kidney tissues were formularized and examined for
 correlation with the concentration (mv/sec/mg of creatinine) of pyridinium
 in urine. As a result, there was seen, between the two, a significant
 correlation of .gamma. (correlation coefficient)=0.6485. The concentration
 of pyridinium compound in urine had also a significant correlation of
 .gamma.=-0.6721 with creatinine clearance. Thus, it has become clear that
 the concentration of pyridinium compound in urine reflects the change of
 renal function.
 EXAMPLE 7
 By measuring the amounts of pyridinium compound in the urines of OLET-F or
 LETO of 36-week age, 48-week age or 60-week age, and examining correlation
 of these amounts with the score of histological change of kidney tissue,
 creatinine clearance and turbidity of eye lens at 72-week age (time of
 slaughter), the present inventors investigated whether or not the amount
 of pyridinium compound in urine could be utilized for diagnosis
 (prognosis) of pathema. The results are shown in Table 5.
 TABLE 5
 Diagnosis and prognosis by pyridinium compound in urine
 (correlation efficient)
 Score of kidney change Creatinine clearance Turbidity of
 eye lens
 Pyridinium Pyridinium Pyridinium
 compound Fructosamine Compound Fructosamine compound
 Fructosamine
 36 weeks 0.6479 0.2730 -0.6794 -0.2195 0.6030
 0.6064
 48 weeks 0.6672 0.4516 -0.5306 -0.3873 0.4869
 0.8377
 60 weeks 0.6443 0.2208 -0.6721 -0.1719 0.6568
 0.7880
 The pyridinium compound and fructosamine were compared as to their
 applicability to diagnosis and prognosis. As a result, the concentration
 of pyridinium compound in urine was found to be able to diagnose and
 prognose, at a timing of 36 weeks before slaughter, the outbreak of
 diabetic complications such as reduction in creatinine clearance,
 histological change of kidney tissue, turbidity of eye lens and the like.
 EXAMPLE 8
 Pyridinium compound in urine was measured for patients of chronic renal
 failure and healthy persons, and their correlations with the urinary
 protein concentration (utilized as a marker indicating the change of renal
 function) were investigated. The results are shown in Table 6.
 TABLE 6
 Concentrations of pyridinium compound and protein in urines
 of patients of renal failure
 Pyridinium compound
 Number in urine Protein in
 of .times. 10.sup.4 area/mg of urine
 Testees creatinine mg/dl
 Healthy 16 292 .+-. 51 5.37 .+-. 1.02
 persons
 Patients of 25 1053 .+-. 267 131.0 .+-. 27.6
 chronic renal
 failure
 ** **
 Mean .+-. standard error **P &lt; 0.01
 The concentrations of pyridinium compound in urines of patients of renal
 failure were 3.6-fold and the concentrations of protein in their urines
 were 24.4-fold, as compared with those in healthy persons. The correlation
 coefficient of the two was significantly high at .gamma.=0.8709, and the
 pyridinium compound in urine was found to be useful as a marker for renal
 function.
 INDUSTRIAL APPLICABILITY
 The pyridinium compound represented by the following formula:
 ##STR5##
 is produced in a large amount in diabetes, deposits on the tissues of
 kidney, tendon, skin, nerve, etc., and is excreted in a large amount in
 urine with the reduction in renal function. Therefore, the pyridinium
 compound can be used as a biomarker for diabetes. The diagnostic reagent
 of the present invention for complications associated with diabetes or
 renal failure contains the above pyridinium compound as a main component.