Patent Application: US-7943698-A

Abstract:
compositions and methods are provided for determining thiols or disulphides in which a sulfur containing analyte is combined with a thiol - specific reagent to produce an intermediate , and the intermediate is combined with a solvent insoluble compound to produce a solid state complex . in one aspect of preferred embodiments the intermediate comprises a chromophore , and in especially preferred embodiments the chromophore is detectable with the naked eye . in another aspect of preferred embodiments the thiol - specific reagent comprises a nitroso - donor , and more preferably na - nitroprusside . in yet another aspect of preferred embodiments , the solvent insoluble compound comprises a basic or acidic metallic compound , or a polymeric matrix . still more preferred solvent insoluble compounds comprise zn - hydroxide , ni - hydroxide or al - hydroxide . these compositions and methods are more sensitive and reliable method than that previously available , especially for the determination of homocysteine .

Description:
as used herein the terms &# 34 ; determine &# 34 ; and &# 34 ; determination &# 34 ; means quantitative and / or qualitative analysis . also as used herein , the term &# 34 ; solvent insoluble &# 34 ; compound refers to compounds which are insoluble in the solvent being used . the solvent of choice will often be water due to its low cost , easy handling and low toxicity . however , other solvents may be used , including ordinary alcohols such as methanol and ethanol , as well as aprotic solvents such as dimethylformamide and dimethylsulfoxide , and even mixtures of solvents such as water and acetone . preferred solvent insoluble compounds are those which can immobilize and thereby stabilize an intermediate . examples are precipitated or dispersed metallic hydroxides , insoluble salts such as basic phosphates , carbonates , citrates , and silica . it is especially preferred to provide the insoluble compounds as slurries , such as may be prepared by mixing water soluble metallic salts with equimolar amount or excess of alkaline metal hydroxide , preferably in the presence of other ions ( e . g . citrate ). such slurries have been described in the chemical literature and several of those have found a practical application in chemical analysis . a recent example of using a water insoluble , basic zn - compound is described by k . shanti and n . balasubramanian ( 8 ). the authors prepared practically an h 2 s trapping solution with insoluble basic zn - slurry , which they described as &# 34 ; zinc acetate - citrate - sodium hydroxide trapping solution &# 34 ;. this solution was used to concentrate h 2 s as an air - pollutant from various sources and the method was claimed to be very sensitive for h 2 s determination in air by using bromate and 2 , 7 - dichlorofluorescein . recently , several papers were published covering the use of a long - known thiol - specific reagent ( na - nitroprusside ) for qualitative and quantitative measurement of urinary cystine and homocystine levels . such tests are known to be useful in monitoring cysteine levels in patients with cystinuria and cystinlithiasis ( 9 - 11 ). in these references , the authors used nacn for disulfide reduction and na - nitroprussside as a reagent and naco 3 as a basic co - reagent . although well characterized and established in clinical routine procedures , these methods suffer from several disadvantages : first , the developed color is highly unstable ( cysteine several minutes , homocysteine immediate degradation ), second , nacn is a highly toxic compound and finally , the method is insensitive to levels lower than 500 μm of the thiol compound . quite unexpectedly , i discovered that a combination of water insoluble , basic zn - compounds , na - nitroprusside and thiol or reduced disulfide develop an unusually stable and intense color that can be detected at very low concentrations . ( see photographs 1 - 3 ). this , in turn , allows an additional and substantial amplification by means of filtration or centrifugation ( see photographs 3 and 4 ). the developed color in such compounds are stable for at least 24 hrs at room temperature , and the concentration dependent color intensity is reproducible over a wide range of concentrations ( see photo 4 ). in further experimentation i discovered that other insoluble compounds can be used instead of the originally discovered zn compounds . examples include other metallic compounds such as aluminum hydroxide , tin hydroxide , titanium hydroxide , as well as non - metallic compounds such as silica . i also discovered that these compounds need not be basic , and for example , titanium hydroxide and silica are both acids which will work nicely . in general , and while not limiting ourselves to a particular theory , it is contemplated that preferred insoluble compounds will spontaneously form a polymeric structure or other matrix , which prevents or limits the ability of a chromophore or other marker to decompose in homogeneous solution . to clarify matters , the term &# 34 ; solid state complex &# 34 ; as used herein means a solvent insoluble species carrying a characteristic chemical group such as a chromophore , isotope or a detectable functional group . the characteristic group may be located on the surface , or buried inside the complex . still further , solid state complexes as contemplated herein may be crystalline , amorphous or partially crystalline and partially amorphous . still further i discovered that other compounds can substitute for na - nitroprusside . preferred alternatives include na nitrite , nitrite esters , and other nitrosodonors . even further , i discovered that the compounds and processes disclosed herein are applicable to other thiols besides homocysteine . ( see photo 7 ). such thiols include cysteine , cystine , alpha - mercapto acetic and alpha - mercapto proprionic acid , mercaptoethanol , glutathione , as well as thiols in petroleum products . in detecting and quantifying disulfides , which is of great importance in the case of cystinuria or homocystinuria , a variety of reducing agents can be used which do not interfere with the final results ( see photo 6 ). particularly suitable for this purpose is tcep (&# 34 ; molecular probes &# 34 ;), but other reagents such as nabh 4 or zn / hcl could be used as well . the compounds and methods disclosed herein have considerable utility . one particular application is an at - home test kit for detection , treatment and follow - up of a disease characterized by a thiol product or by - product , e . g . cystinuria or cystinlithiasis . another contemplated application is detection and concentration of sulfur isotope labeled compounds . however , my invention is not limited to color determination in solid phase ; it also can be adopted e . g . for quantitative determination of thiols after re - dissolving the colored pellet in a complexing agent such as edta , and then reading the intensity of the developed product using an appropriate reader . for the particular experiments recited below , the product developed a color which was readable at 530 nm ( see fig1 - 3 ). fig1 shows the absorption spectrum of an edta dissolved pellet : to 1 . 0 ml of a 100 μm dl - homocysteine solution , 300 μl of the zn - slurry containing 2 . 5 % na - nitroprusside were added . after mixing the solution , a brief centrifugation step at 13 , 000 rpm for 30s was performed and the supernatant discarded . the pellet was dissolved in 0 . 5m edta ph8 . 0 and immediately scanned in a spectrophotometer between 440 nm and 800 nm . data were collected every 20 nm . absorption was plotted versus wavelength . fig2 shows decay of absorbance in dependence of dl - homocysteine concentration : 6 samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homo - cysteine ( as indicated between 0 and 100 μm ) were mixed with 300 μl zn - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , a brief centrifugation step at 13 , 000 rpm for 30s was performed and the supernatant discarded . the pellet was dissolved in 0 . 5m edta ph8 . 0 and the absorption immediately read at 530 nm . data were collected every 6s . curves were fitted using a linear fit applying the least squares method . r2 was calculated for each curve . absorption was plotted versus time . fig3 shows slopes of absorbance decays in dependence of dl - homocysteine concentration : the calculated slopes from fig1 were plotted versus the concentration of dl - homocysteine . the curves was fitted using a linear fit applying the least squares method . r2 was calculated . the slopes were plotted versus the dl - homo - cysteine concentration . preparation of a water insoluble , basic metallic compound . general procedure : ˜ 1 % aqueous solution of a metallic salt ( such as zn ( aco ) 2 , cdcl 2 , niso 4 , al ( aco ) 3 , etc .) is mixed with equimolar amount or a slight excess of naoh ( 0 . 1m ) in the presence of ˜ 10 - 20 % mol eq . of na 3 citrate . the naoh solution is added slowly to the metallic salt -- citrate solution at room temperature with continuous stirring , at such a rate , that the insoluble basic metallic compound is gradually formed . the slurry is finely suspended and precipitates only after longer standing . such metallic compounds are stable over months at room temperature , and slurries should be well shaken each time before use . preparation of a water insoluble , basic zn - slurry : 10 g of zn ( ac ) 2 and 2 . 94 g of na 3 citrate are dissolved in 1000 ml water . naoh ( 0 . 1m ) is slowly added under continuous stirring . the slurry is well shaken prior to use . preparation of a thiol solution : 100 mm aqueous dl - homocysteine stock solution is prepared . this stock solution is diluted 1 : 100 to yield a 1 mm dl - homocysteine solution . from this solution , 10 μl , 20 μl , 50 μl , 75 μl , 100 μl , 200 μl and 500 μl aliquots are pipetted into 1 . 5 ml eppendorf tubes , respectively , and water was added ad 1 . 0 ml , respectively . for each concentration , 5 tubes were set up . procedure 1 ( for thiol determination ): to 20 ml of the fresh prepared zn - slurry , 500 mg na - nitroprusside were added and the mixture was vortexed until all of the na - nitroprusside was dissolved . 300 μl of the so prepared na - nitroprusside zn - slurry were added to the previously prepared tubes containing 1 ml of the homocysteine solution . the color develops immediately . after thoroughly mixing , 1 . 0 ml of the mixture was filtered onto whatman 3m paper using a vacuum manifold . for documentation , the filter was placed on a flatbed scanner ( see photo 4 ). all preparations and reactions were performed at room temperature . the incubation time of the homocysteine solution with the na - nitroprusside zn - slurry was less than 5 min . the developed color remained unchanged after 24 hrs . procedure 2 ( for disulfide determination ): existing disulfides have to be reduced to the thiol state prior to determination . for this purpose , a 10 mm tcep solution in water is prepared and 10 %( vol / vol ) of this solution is added to the solution containing the disulfide . after thoroughly mixing , the procedure is identical to procedure 1 . while not limiting ourselves to nitroso donor reagents ( such as nitroprusside ) as a means of thiol determination , other means of thiol determination are also contemplated ( such as radio isotopes , fluorescent , phosphorescent or chemoluminescent reagents , etc .). the format of the presented invention can either be in form of a simple home test with or without metabolic challenge ( methionine loading test ), but can also be applied in a clinical chemistry laboratory as a qualitative assessment of thiol compounds in fluids . as used herein , the term &# 34 ; determining &# 34 ; includes both qualitative and quantitative determination of the thiol compound . thus , specific embodiments and applications of thiolspecific reagents and water insoluble metallic preparations have been disclosed . it should be apparent , however , to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein . for example , modified non - metallic water insoluble matrices ( as silica gel , e . g .) can be used in aqueous and non - aqueous media . similarly , it is possible to use indirect means of quantitation such as derivatized thiol components that can be detected by a specific reagent and amplified on a solid matrix . the inventive subject matter , therefore , is not to be restricted except in the spirit of the appended claims . photo 1 : pellets of ni - slurry with various concentrations of dl - homocysteine : to 4 samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homo - cysteine ( as indicated between 0 and 500 μl ) were mixed with 300 μl ni - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , a brief centrifugation step at 13 , 000 rpm for 30s was performed . pellets were immediately captured on a flatbed scanner . photo 2 : pellets of al - slurry with various concentrations of dl - homocysteine : to 4 samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homo - cysteine ( as indicated between 0 and 500 μl ) were mixed with 300 μl al - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , a brief centrifugation step at 13 , 000 rpm for 30s was performed . pellets were immediately captured on a flatbed scanner . photo 3 : pellets of zn - slurry with various concentrations of dl - homocysteine : to 4 samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homo - cysteine ( as indicated between 0 and 500 μl ) were mixed with 300 μl zn - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , a brief centrifugation step at 13 , 000 rpm for 30s was performed . pellets were immediately captured on a flatbed scanner . photo 4 : filter with zn - slurry with various concentrations of dl - homocysteine between 0 and 500 μl as indicated . to sets of 5 samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homocysteine were mixed with 300 μl zn - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , 1 . 0 ml was filtered onto whatman 3m filter paper in a vacuum manifold . the filter was dried for ˜ 60 min and then captured on a flatbed scanner . photo 5 : filter with zn - slurry with various amounts of samples containing identical concentrations of dl - homocysteine : to 300 μl zn - slurry containing 2 . 5 % na - nitroprusside various volumes of a solution containing 20 μm dl - homocysteine were added . the control was 300 ml water , the remaining dots represent multiple amounts of 300 μl with constant concentration of dl - homocysteine between 300 μl ( 1 : 1 ) and 20 μm ( 7 : 1 ). photo 6 : filter with zn - slurry with samples containing various concentrations of dl - homocysteine in the presence of tcep : samples containing 1 . 0 ml of an aqueous solution with various amounts of dl - homocysteine ( as indicated between 0 and 200 μm ) and a final concentration of tcep of 1 mm were mixed with 300 μl zn - slurry containing 2 . 5 % na - nitroprusside and vortexed . after mixing the solution , 1 . 0 ml was filtered onto whatman 3m filter paper in a vacuum manifold . the filter was dried for ˜ 60 min and then captured on a flatbed scanner . photo 7 : filter with zn - slurry with samples containing various thiols : samples containing 1 . 0 ml of an aqueous solution with various thiols at a concentration of 200 μm were mixed with 300 μl zn - slurry containing 2 . 5 % na - nitroprusside and vortexed . water was used as a negative control . after mixing the solution , 1 . 0 ml was filtered onto whatman 3m filter paper in a vacuum manifold . the filter was dried for ˜ 60 min and then captured on a flatbed scanner . the individual dots are : 1 = water , 2 = β - mercaptoethanol , 3 = l - cysteine , 4 = dithiothreitol , 5 = dl - homocysteine , 6 = l - homocystine , 7 = methionine , 8 = na 2 s . 1 . fiskerstrand , t ., refsum , h ., kvalheim , g and ueland , p . m ., &# 34 ; homocysteine and other thiols in plasma and urine : automated determination and sample stability &# 34 ;, clin . chem . 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