Patent Application: US-82399397-A

Abstract:
a method for the determination of the oxidizability of low density lipoproteins in a serum or plasma sample from a mammal , which method comprises isolating the ldl from the serum or plasma sample for the preparation of a ldl fraction , separating the lipids from the ldl fraction to obtain a lipid fraction therefrom , determining the baseline level of conjugated dienes in the lipid fraction .

Description:
according to a preferred embodiment of the present invention , ldl is isolated by a fast precipitation method and the assay for ldl oxidation products ( ldl - bdc ) is based on spectrophotometric determination of baseline levels of conjugated dienes ( bdc ) in lipids extracted from ldl . for performing the ldl - bdc and ldl - trap assays , 0 . 5 ml of serum or plasma is generally sufficient . in addition to fresh serum or plasma , frozen samples ( studied for a period of 6 months at - 70 ° c . ), too , can be used for the assays after thawing at room temperature . according to a preferred embodiment of the invention , serum ldl is preferably isolated by precipitation with buffered heparin ( 8 ) after allowing the serum samples and precipitation reagents to equilibrate to room temperature . the serum sample , the volume of which can suitably be 0 . 2 to 1 . 7 ml , preferably 1 ml , is added to 1 to 7 ml , preferably 7 ml , of the precipitation buffer . the insoluble lipoproteins are then sedimented by centrifugation and the pellet is resuspended in a sodium phosphate buffer , ph 7 . 4 ( 0 . 5 to 1 ml ). the ldl sample or fraction so obtained can be used as such for the quantification of oxidation products and antioxidant potential . for measuring the ldl - bdc , lipids are extracted from ldl samples with a suitable organic solvent or solvent system , such as a chlorinated organic solvent , e . g . a chlorinated alkane , with a lower aliphatic alcohol . the sample size is usually 0 . 05 to 0 . 50 ml , preferably 0 . 10 ml . the chlorinated organic alkane can be e . g . chloroform or methylene chloride . preferably , the organic solvent is chloroform and the lower aliphatic alcohol is methanol . the chloroform : methanol ratio may range from 2 : 1 to 4 : 1 , and is preferably 3 : 1 . the volume depends on sample size and ranges from 3 to 20 ml . the mixture is then dried under argon or nitrogen , preferably under nitrogen , then redissolved in an organic solvent , which is &# 34 ; inert &# 34 ; in the subsequent spectrophotometrical analysis , such as an inert aliphatic hydrocarbon , e . g . an alkane or a cycloalkane , preferably a cyclohexane , and analyzed spectrophotometrically at 234 and 300 nm . absorbance at 300 nm is subtracted from that at 234 nm . the δ absorbance may be converted to molar units using the molar extinction coefficient 2 . 95 × 10 4 m - 1 cm - 1 . the results can be expressed μmol / l serum to give an estimation of the actual level of circulating oxidized ldl . the assay for the antioxidant potential ( total peroxyl radical trapping antioxidant potential , trap ) of ldl ( ldl - trap ) is based on a luminometric determination of the ability of ldl to resist peroxyl radical - induced lipid peroxidation . for such a measurement , sodium phosphate incubation buffer , ph 7 . 4 , and the ldl sample ( buffer 0 . 45 ml , sample size 0 . 05 to 0 . 20 ml , preferably 0 . 10 ml ) are mixed in a cuvette and the assay is initiated by addition of abap . chemiluminescence is measured at 37 ° c . until a peak value for each sample is detected . determination of the peroxyl radical trapping capacity is based on lag time determination which is defined by the half - peak time point , and trolox is used as a standard radical scavenger . to get an estimation of the relative antioxidant power of given ldl preparations , the results can be expressed in relation to the cholesterol concentration of the preparations . an overview of the preferred measurements of ldl - bdc and ldl - trap is indicated in the fig1 . according to the present invention it has been clearly indicated that bdc and trap can be readily measured in ldl by the described methods . measurement of ldl - bdc can be done in a similar way in heparin precipitated ldl as in ldl isolated by ultracentrifugation , the heparin precipitation method , however , offering several advantages over ultra - centrifugation especially from a laboratory technical point of view . the results obtained by the ldl - bdc method are well in accordance with those obtained by the immunological method . high ldl - bdc values are indicative of increased risk for atherosclerosis , as suggested by the positive correlation with the thickness of arterial wall , and also indirectly by the results from studies where the ldl - bdc levels were found to alter parallelly to various known factors increasing the risk ( diabetes , obesity etc .) for , or protecting ( physically active life - style etc .) from atherosclerosis . the ldl - trap value is indicative of the antioxidant potential of ldl , as indicated by the negative correlation with ldl - bdc , and also by the antioxidant intervention studies . the results obtained in the ldl - bdc and ldl - trap methods can thus be used as indicators in the screening of the risk for , the diagnosis and management , including follow - up treatment , of atherosclerosis and coronary heart disease . in addition , these methods will be useful in a broad scale of studies and research on the etiology ( e . g . the role of genetic , dietary , environmental or life style - dependent factors ), prevention ( e . g . the effect of dietary factors , physical activity , drugs ) and management of atherosclerosis and coronary heart disease . the reference ranges for ldl - bdc and ldl - trap are obtained laboratory specifically by measuring the ldl - bdc and ldl - trap values of a large number ( e . g . 200 - 300 ) healthy human adults and calculating the corresponding mean values ( with standard deviations ). a high ldl - bdc value ( close to the upper limit of the ldl - bdc reference range or over it ), optionally in combination with a low ldl - trap value ( close to the lower limit of the ldl - trap reference range or below it ), is indicative of an increased risk for atherosclerosis and / or coronary heart disease . the reference ranges measured by us for healthy human individuals are ldl - bdc appr . 15 - 60 μmol / l serum and ldl - trap appr . 12 - 38 μmol / mmol ldl - cholesterol . due to the fact that the ldl - trap gives a faster response as a result of treatment and / or changes in life - style habits ( diet , exercise ), this value is especially advantageous for use as an easy and rapid means for the treatment follow - up of atherosclerosis and coronary heart disease . the following examples will further illustrate the present invention , which by no means limit the invention . serum ldl is isolated by precipitation with buffered heparin . the precipitation buffer is 0 . 064m trisodium citrate adjusted to ph 5 . 05 with 5n hcl , containing 50 , 000 iu / i heparin ( 5 , 000 iu / ml heparin was obtained either from loevus kemiska fabrik , ballerup , denmark , or from leiras ltd , turku , finland ). before precipitation of ldl , serum samples ( to which 1 mg / ml of edta is added ) and precipitation reagents are allowed to equilibrate to room temperature . one milliliter of the serum sample is added to 7 ml of the precipitation buffer . after mixing with a vortex mixer , the suspension is allowed to stand for 10 min at room temperature . the insoluble lipoproteins are then sedimented by centrifugation at 1 , 000 g for 10 min . the pellet is resuspended in 1 ml of 0 . 1m sodium phosphate buffer , ph 7 . 4 , containing 0 . 9 % of nacl . this ldl sample can be used as such for the analysis of oxidation products and antioxidant potential . lipids are extracted from ldl samples ( sample size 0 . 10 ml ) by adding 1 ml of methanol and 3 ml of chloroform ( mixing with a vortex mixer after s the addition of each solvent ). then the mixture is allowed to stand for one hour at room temperature in the dark , after which 2 ml of water is added , the mixture is mixed with a vortex mixer and then centrifuged at 2 , 000 g for 10 min at + 8 ° c . the lower phase is evaporated to dryness under nitrogen , resuspended in 1 ml of cyclohexane and analyzed spectrophotometrically ( perkin - elmer lambda 2 spectrofotometer ) at 234 and 300 nm . 0 . 45 ml of incubation buffer ( 0 . 1m sodium phosphate buffer , ph 7 . 4 , containing 0 . 9 % of nacl , 0 . 02 ml of 120 mm linoleic acid , 0 . 05 ml of luminol 0 . 5 mg / ml , obtained from bio - orbit ltd ., turku , finland !) and 0 . 10 ml of ldl sample are mixed in the cuvette and the assay is initiated with 0 . 05 ml of abap ( 83 mg / ml , obtained from polysciences inc ., warrington , pa ., usa ). chemiluminescence measurements are performed with bio - orbit 1251 luminometer . chemiluminescence in duplicate cuvettes is measured at 37 ° c . until a peak value for each sample is detected ( see fig2 ). heparin precipitation is a specific and reproducible means for isolation of ldl from serum samples . in this ldl preparation , ldl - bdc and ldl - trap can be measured and the assays show good reproducibility and linearity , as can be seen from the results shown below . to assess the reliability of the heparin - citrate precipitation method , the linearity with respect to sample size and reproducibility of the method were investigated . the reproducibility of ldl precipitation was tested by repeating the procedure 20 times from a pool of serum , and analyzing the apolipoprotein b ( apob ) contents in precipitated ldl . reagent kits for apolipoprotein b was obtained from orion diagnostica , espoo , finland . the coefficient of variation ( cv ) for the within - assay precision was 6 . 7 % for apob . the ldl - bdc was detectable in ldl precipitated from 100 μl of serum , and the amount of ldl - bdc was directly proportional to the amount of serum taken for precipitation ( fig3 a ). similarly , ldl - trap increased linearly with increasing amounts of serum and was reliably detectable in ldl precipitated from 260 μl of serum ( fig3 b ). for ldl - bdc , the cv for within - assay precision was 4 . 4 %, and cv for the between - assay precision over a period of 3 months was 4 . 5 %. with the ldl - trap , the cv for the within - assay precision was 8 . 1 , and cv for the between - assay precision over a period of 3 months was 8 . 7 %. freezing of the serum ( studied for a period of 6 months at - 70 ° c .) did not affect ldl - bdc or ldl - trap levels . the relationship between ldl - bdc and ldl - trap and the corresponding measurements in serum , serum lipids , and antioxidants ( α - tocopherol , ubiquinol - 10 ), were measured in volunteers ( n = 31 ) and the interdependence of the parameters was estimated by correlation analysis . a reagent kit for cholesterol ( chod - pap method ) was obtained from boehringer mannheim , mannheim , germany , and the serum concentrations of α - tocopherol ( 9 ) and ubiquinol - 10 ( 10 ) were analyzed by standard hplc procedures with uv - detection . there was a negative correlation between ldl - bdc and ldl - trap ( table 1 ). ldl - bdc correlated positively with serum diene conjugation , ldl cholesterol , and triglycerides , but no correlation was found to exist between the antioxidant levels and ldl - bdc . ldl - trap correlated positively with the serum trap value and negatively with serum ldl and cholesterol . again , no correlation existed between the measured antioxidants and the ldl - trap . the fact that the ldl - bdc and ldl - trap values are negatively correlated , further strengthens the validity and credibility of these methods . bdc values measured in heparin precipitated ldl are not different from those measured in ldl isolated by the conventional ultracentrifugation method , as shown by detailed comparison studies ( table 2 ). for preparative ultracentrifugation we used either a sorvall otd - 65 ultracentrifuge with fixed - angle rotors or a kontron tga - 65 ultracentrifuge with swing - out rotors ( 11 , 12 ). the ldl - trap , however , was in this comparison lower in ldl isolated by precipitation . since no dialysis was performed for ldl after ultracentrifugation , the apparently different trap values in ldl fractions isolated by the different methods are likely explained by interfering peroxyl radical trapping components , such as ascorbate , glutathione and urate , present in ldl isolated by ultracentrifugation . of the direct methods currently available for estimation of ldl oxidation products , the preferred one has been the immunological method , based on the use of auto - antibodies to epitopes on oxidized ldl ( 5 ). when analyzed in the same samples from healthy volunteers ( men , age 21 - 44 years , n = 29 ), this immunological assay shows a good correlation with the ldl - bdc assay ( fig4 ). for obtaining the results in fig4 autoantibody titers of anti - oxidized ldl were measured by enzyme - linked immunosorbent assay method using 96 - well polystyrene microtitration plates ( nunc , immunoplate , roskilde , denmark ). antigens for this assay included native ldl , protected against oxidation by 0 . 27 mm edetic acid and butylated hydroxytoluene ( bht ) in phosphate buffered saline ( pbs , 10 mm sodium phosphate , ph 7 . 2 ), and ox - ldl ( obtained after 18 h oxidation with 2 μm cuso 4 , and prepared from the pooled plasma of ten donors ( 12 ). the wells were incubated ( coated ) with 50 μl of native and ox - ldl antigen ( 5 μg / ml ) ( protected from oxidation as noted above ) in pbs for 16 h at 4 ° c . after removal of the unbound antigen and washing of the wells ( three times with pbs , 0 . 5 % tween 20 , and three times with distilled water , using microtiter plate washer biorad model 1550 ), the remaining non - specific binding sites were saturated using 2 % bovine serum albumin in pbs . the wells were washed , and 50 μl of serum sample ( diluted to 1 : 20 and 1 : 50 ) were added to wells coated with native ldl and ox - ldl , and incubated over night at 4 ° c . after incubation , the wells were aspirated and washed , before an appropriate igg - peroxidase conjugated rabbit antihuman monoclonal antibody ( organon , usa , no . 55220 cappel , diluted to 1 : 4 , 000 in 0 . 27 mm pbs , 20 μm edetic acid , 1 % bht , bovine serum albumin - 0 . 05 % tween ) was added to each well ( 0 . 5 ml ). after incubation ( for 4 h at 4 ° c . ), the unbound material from the wells were aspirated and wells washed . after this , 0 . 5 ml of freshly prepared substrate ( 0 . 4 mg / ml o - phenylenediamine , sigma , and 0 . 045 % h 2 o 2 in 100 mm acetate buffer , ph 5 . 4 ) was added and incubated for 5 min at room temperature . the enzyme reaction was terminated by addition of 0 . 5 ml of 2m h 2 so 4 . the optical density was then measured spectrophotometrically at 492 nm with a special microplate reader ( multiscan mcc / 340 , labsystems gmbh , munich , germany ). to calculate the antibody titer , we used the ratio of the corresponding spectrophotometric reading of anti - oxidized ldl and the anti - native ldl wells from the same serum sample . using this approach , the spectrophotometric readings of anti - native ldl wells represent the corresponding blanks of anti - oxidized ldl wells and reduce the possible detection of false positive values . clinical applicability of ldl - bdc and ldl - trap has been tested among patients and volunteers with known disease - or life style dependent factors increasing or decreasing the risk of atherosclerosis and coronary heart disease ( chd ). in addition , applicability of the ldl - trap method has been tested by antioxidant intervention studies . progression of atherosclerosis is commonly estimated by measuring the thickness of the arterial walls by ultrasound techniques . the ultrasound technique was used for measurement of the ( intima - media ) thickness of arteria carotis among 33 men ( age 21 - 46 years ), and ldl oxidation products from these subjects were determined by the ldl - bdc method . a positive correlation ( r = 0 . 43 ; p = 0 . 012 ) was found to exist between the ldl - bdc values and thickness of the arteria carotis . non - insulin - dependent diabetes is known to be associated with increased risk for atherosclerosis ( 13 ). samples were collected from patients at the department of internal medicine , turku university hospital , finland . the material includes patients from both sexes , ages 16 - 88 years . no separation was made with respect to the type of diabetes mellitus and , at times of the sample collection , patients were receiving treatment for the disease . fig5 shows that ldl - bdc and ldl - trap values of patients with diabetes ( n = 118 ) clearly differ from those of age - matched healthy controls . obesity is an independent risk factor for chd mortality among men and also contributes to the risk of chd among women ( 14 ). when measured in healthy volunteers ( men , age 40 - 49 years , n = 31 ) ldl - bdc was found to correlate positively with the body mass index ( r = 0 . 47 , p = 0 . 008 ). controlled reduction of body weight among obese premenopausal women ( n = 82 , weight before onset of diets ranged from 90 to 100 kg ) decreased significantly ldl - bdc levels . total ldl - bdc decreased by 40 % and ldl - bdc / total ldl cholesterol ratio by 32 % ( fig6 ). physically active life style , in turn , is known to be associated with decreased risk of chd ( 15 ). we found that middle - aged ( 40 - 49 years ) men ( n = 31 ) who are actively participating in endurance training have distinctly lower ldl - bdc values ( 37 %) than age - and weight - matched controls with similar socioeconomic background , and dietary and smoking habits ( fig7 see also ref . 16 ). two intervention studies have been performed to test the effect of antioxidant preparations ( dietary supplements ) on ldl - bdc and ldl - trap . in the first study ( 11 ) vitamin e alone or in combination with vitamin c and β - caroiene was given to volunteers ( n = 10 ) a period of one week . in the second ( double blind ) study ( fig8 ) volunteers received a combination of antioxidants ( vitamin e , vitamin c and ubiquinone ) for 4 weeks . serum concentrations of α - tocopherol and ubiquinol - 10 were analyzed by standard hplc procedures with uv - detection . in both of these studies ldl - trap values were found to be increased by the antioxidant interventions ( by about 40 %) while the ldl - bdc levels remained unaffected ( for further details , see ref . 17 ). the invention being thus described , it will be obvious that the same may be varied in many ways . such variations are not to be regarded as a departure from the spirit and scope of the invention , and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims . table 1______________________________________correlation of ldl oxidation parameters with serum parametersrelated to ldl oxidation among healthy volunteers ( n = 31 ). ldl - bdc ldl - trap______________________________________ldl - trap - 0 . 416 . sup . 1 ( p = 0 . 002 ) serum bdc 0 . 647 ( p = 0 . 0001 ) - 0 . 061 ( p = 0 . 756 ) serum trap 0 . 098 ( p = 0 . 600 ) 0 . 546 ( p = 0 . 0015 ) ldl 0 . 676 ( p = 0 . 0001 ) - 0 . 565 ( p = 0 . 0009 ) serum cholesterol 0 . 658 ( p = 0 . 0001 ) - 0 . 467 ( p = 0 . 0081 ) serum triglycerides 0 . 732 ( p = 0 . 0001 ) - 0 . 0002 ( p = 0 . 999 ) serum α - tocopherol 0 . 066 ( p = 0 . 735 ) 0 . 019 ( p = 0 . 922 ) serum ubiquinol - 10 0 . 357 ( p = 0 . 057 ) 0 . 074 ( p = 0 . 701 ) ______________________________________ . sup . 1 correlation coefficient table 2______________________________________baseline diene conjugation ( bdc ), thiobarbituric acid reactive material ( tbarm ) and antioxidant potential ( trap ) in serum and lipoproteinfractions isolated by sequential ultracentrifugation ( uc ) or heparinprecipitation ( hep ). isolation of ldl by ultracentrifugation wasperformed by the standard procedure as described in ref . 18 . ldlisolation by ultracentrifugation and heparin precipitation was repeated4 times , on separate days and with freshly prepared serum pools . theroman numerals indicate the different isolation times and serum pools . results for the various isolation times / serum pools are given asμmol / l , and are mean ± sd from 6 different determinations ( from each serumpool , 6 separate ldl samples were isolated ). &# 34 ; total mean &# 34 ; is themean ± sd of means of the four different serum pools . vldl , verylow - density lipoprotein ; hdl , high - density lipoprotein . pool bdc tbarm trap______________________________________serum i 71 . 8 ± 2 . 7 7 . 40 ± 1 . 41 1071 ± 0 ii 68 . 1 ± 1 . 1 6 . 60 ± 0 . 85 1193 ± 34 iii 57 . 7 ± 5 . 9 8 . 20 ± 0 . 28 1071 ± 69 iv 68 . 3 ± 1 . 4 803 ± 34 total mean 66 . 5 ± 6 . 1 7 . 40 ± 0 . 80 1035 ± 165uc - vldl i 26 . 4 ± 6 . 4 0 . 14 ± 0 . 003 206 ± 17 ii 20 . 9 ± 3 . 3 0 . 20 ± 0 . 25 149 ± 12 iii 16 . 3 ± 2 . 4 0 . 17 ± 0 . 12 244 ± 35 iv 18 . 0 ± 3 . 2 131 ± 26 total mean 20 . 4 ± 4 . 4 0 . 17 ± 0 . 03 183 ± 52uc - ldl i 25 . 1 ± 2 . 8 0 . 43 ± 0 . 10 229 ± 17 ii 26 . 3 ± 2 . 9 0 . 07 200 ± 13 iii 23 . 4 ± 1 . 1 0 . 18 ± 0 . 11 191 ± 18 iv 30 . 8 ± 1 . 8 192 ± 27 total mean 26 . 4 ± 3 . 2 0 . 23 ± 0 . 18 203 ± 18uc - hdl i 12 . 6 ± 1 . 9 1 . 90 ± 0 . 21 288 ± 20 ii 20 . 3 ± 2 . 3 1 . 27 ± 0 . 05 315 ± 13 iii 15 . 3 ± 2 . 2 1 . 30 ± 0 . 14 261 ± 26 iv 19 . 0 ± 1 . 5 219 ± 16 total mean 16 . 8 ± 3 . 5 1 . 49 ± 0 . 36 271 ± 41hep - ldl i 25 . 1 ± 0 . 9 0 . 60 ± 0 46 ± 3 ii 33 . 3 ± 13 . 6 0 . 15 ± 0 51 ± 3 iii 24 . 6 ± 0 . 5 0 . 10 ± 0 49 ± 0 iv 26 . 7 ± 1 . 4 29 ± 0 total mean 27 . 4 ± 4 . 0 0 . 28 ± 0 . 28 44 ± 10______________________________________ 1 steinberg d , parthasarathy s , carew te , khoo , jc , witztum jl . beyond cholesterol . modifications of low - 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