Abstract:
A kit is described for the determination of L-carnitine in biological fluids and tissues, consisting essentially of; a) a first container containing Reagent 1, said Reagent 1 comprising HEPES, EDTA, acetyl-coenzyme A and DTNB; b) a second container containing Reagent 2, said Reagent 2 consisting of carnitine acetyl transferase. Said kit is conveniently presented with the reagents in lyophilized form.

Description:
[0001]    The invention described herein relates to a kit for the determination of L-carnitine in biological fluids.  
         BACKGROUND TO THE INVENTION  
         [0002]    L-carnitine plays a fundamental role in metabolism, being a key element in the oxidation of long-chain fatty acids and thus in the production of energy.  
           [0003]    There are many pathological states the underlying cause of which is a deficiency of L-carnitine, and L-carnitine determination is necessary in order to establish the precise aetiology of the related disease processes.  
           [0004]    There has been a growing awareness among clinicians over recent years of the importance of L-carnitine deficiencies (Bieber et is al. Fed. Proc. 41, 2858 (1982); Stanley, Adv. Pediatr. 34, 59 (1987).  
           [0005]    One of the first methods used for determining L-carnitine (hereinafter called carnitine for short) was described by Marquis and Fritz in 1964 (Journal of Lipid Research 5, 184-187). Various other methods have been described, such as, for example, those by Marzo et al., J. Chromatogr. 527, 247 (1990); and Hoppel, in: Hommes (ed.), Techniques in Diagnostic Human Biochemical Genetics, New York, Wiley-Liss, 309-326 (1991).  
           [0006]    The most widespread methods are based on the reaction of the enzyme carnitine acetyl transferase (CAT; EC 2.3.1.7):  
                         
 
           [0007]    One very widespread method for the indirect determination of carnitine is based on the reaction of acetyl-Coenzyme A, released by the preceding reaction with 5,5′-dithiobis-2-nitrobenzoate (DTNB), which in turn releases the thiophenylate ion, which is determined spectrophotometrically at 412 nm (see, in addition to the above-mentioned Marquis, Seccombe D. W., Clinical Chemistry, 22, No. 10, 1589-1592, 1976; Pearson, Methods of Enzymatic Analysis, 4, 1758-1771, 2nd edition—Bergmeyer; Casillas E. R. Biochimica et Biophysica Acta, 184, 566-577, 1969; Cederblad G. Clinica Chimica Acta, 33, 117.123, 1971; Carrier H. N., Muscle &amp; Nerve July/August 326-328, 1980). This method determines free carnitine. For the determination of the short-chain acyl carnitines, and thus of total carnitines, the sample is subjected to alkaline hydrolysis, converting the acyl carnitines to free carnitine.  
           [0008]    The known methods entail elaborate preparation phases, or use techniques which are only poorly conducive to automation of the analyses. For a discussion of the subject, see U.S. Pat. No. 5,316,917, filed in the name of Duke University, and incorporated herein for reference purposes. This patent aims to solve the problem of the automation of the analyses. This need is strongly felt in clinical laboratories that have to carry out large numbers of determinations. The Duke University patent offers the solution of an automated spectrophotometric method, consisting in the following steps:  
           [0009]    1) addition of a plurality of samples of deproteinized biological fluids to a set of wells of a centrifugal spectrophotometric analyzer;  
           [0010]    2) addition to each sample of:  
           [0011]    a) acetyl-Coenzyme A in an amount sufficient to react essentially with all the free carnitine in the sample to produce acetyl-carnitine and free Coenzyme A;  
           [0012]    b) DTNB in an amount sufficient to convert essentially all the Coenzyme A produced in the previous reaction to thiophenylate, and then  
           [0013]    c) simultaneously combine CAT, in each sample, in an amount sufficient to set off the reaction between acetyl-Coenzyme A and free carnitine and bring it to completion, and then  
           [0014]    d) determine the amount of thiophenylate present in the sample simultaneously by spectrophotometry.  
           [0015]    The kit used to implement the method described in the patent cited includes:  
           [0016]    a) a first container containing a solution of DTNB at a concentration ranging from 0.27 to 27 mmol/L, at a pH ranging from 6.5 to 8.5, and  
           [0017]    b) a second container containing a solution consisting of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, where the two solutions can be mixed together prior to use to form a solution containing DTNB at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/l.  
           [0018]    In actual fact, the kit must also include a third container containing from 4 to 40 kU/L of CAT, for example in the form of a solution at a concentration ranging from 1.72 to 172 kU/L. It is envisaged that the third container should contain CAT in lyophilized form. Also envisaged in the kit are containers with carnitine standards in aqueous solution from 0.1 to 10 mmol/L and of octanoyl L-carnitine from 0.1 to 10 mmol/L. Also provided in the kit is a fourth container containing a 3-[N-morpholine]propanesulphonic acid hydrochloric solution, from 0.1 to 10 mmol/L.  
           [0019]    The execution of the procedure entails the preparation of 4 solutions:  
           [0020]    1. DTNB; HEPES; EDTA at pH 7.5  
           [0021]    2. MOPS-HCl  
           [0022]    3. KOH  
           [0023]    In fact, the procedure described in the patent is called “three reagent chemistry”.  
           [0024]    It is known that the solutions envisaged in the kit described above must be stored at low temperature, from 0 to 4° C., as prescribed in the patent cited.  
           [0025]    U.S. Pat. No. 6,027,690, a divisional patent of U.S. Pat. No. 5,872,008, filed in the name of Bair and Shug, provides a kit for the diagnosis of premenstrual syndrome, based on the determination of free and total carnitine in the blood. Apart from the specific indication of the method, to implement the method the kit consists of:  
           [0026]    a) a first container containing a solution of acetyl-Coenzyme A at a concentration ranging from 1.2 to 120 mmol/L, and  
           [0027]    b) a second container containing a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.27 to 27 mmol/L at a pH ranging from 6.5 to 8.5, where the two solutions can be mixed together prior to use to form a solution consisting of DTNB or N-(p-2(benzimidazolyl)phenyl)maleimide at a concentration ranging from 0.23 to 23 mmol/L and acetyl-Coenzyme A at a concentration ranging from 0.17 to 17 mmol/l.  
           [0028]    In the execution of the procedure according to U.S. Pat. No. 6,072,690, the method described in U.S. Pat. No. 5,316,917 can be adopted amongst others.  
           [0029]    A Boehringer Mannheim kit is commercially available for the determination of L-carnitine in plasma, seminal fluid, and urine. The determination is based on the following reactions:  
                         
 
           [0030]    The amount of NADH consumed during the reaction is equivalent to half the amount of L-carnitine. NADH is determined by absorbance at 334 (Hg), 340 or 365 (Hg) nm.  
           [0031]    The kit comprises:  
           [0032]    1) 3 vials (1) each containing 0.7 g of a Coenzyme/buffer mixture consisting of Tris buffer pH 7.0, NADH 5 mg, ATP 6 mg, acetyl-Coenzyme A 4 mg, PEP 3 mg, magnesium acetate, and stabilizing agents.  
           [0033]    2) Vial (2) containing approximately 3 ml of enzyme suspension consisting of acetyl-CoA synthetase (ACS), 2U approx., myokinase (MK), 160 U approx., lactate dehydrogenase (LDH), 240 U approx., and pyruvate kinase (PK), 240 U approx.  
           [0034]    3) Vial (3) containing 0.2 ml of carnitine acetyl transferase (CAT) enzyme suspension, 60 U approx.  
           [0035]    4) Vial (4) containing L-carnitine standard.  
           [0036]    5) Vial (5) containing detergent solution.  
           [0037]    Prior to use, the contents of one vial (1) are diluted with 10 ml of distilled water and 1 ml is added from vial (5). The other vials are used as such. The procedure consists in the addition of the sample (or standard) to solution (1), then addition of suspension (2), measurement of absorbance, then addition of suspension (3) and subsequent measurement of absorbance.  
         ABSTRACT OF THE INVENTION  
         [0038]    It has now been found that it is possible to provide a kit for the determination of L-carnitine or short-chain acyl L-carnitines consisting of only two vials complete with all the necessary reactants, with the exception of the standards. Conveniently, the kits contain the reactants in lyophilized form.  
           [0039]    One of the advantages afforded by the realization of the invention described herein consists in the possibility of determining carnitine over a broad range of concentrations.  
           [0040]    The kit according to the invention comprises:  
           [0041]    a) a first container containing Reagent 1, said Reagent 1 consisting of HEPES, EDTA, acetyl-Coenzyme A and DTNB;  
           [0042]    b) a second container containing Reagent 2, said Reagent 2 consisting of carnitine acetyl transferase.  
           [0043]    Therefore, one subject of the invention described herein is a kit for the determination of L-carnitine in biological tissues and fluids, its use for the determination of L-carnitine in biological tissues and fluids and methods for the determination of L-carnitine in biological tissues and fluids using the kit.  
           [0044]    According to the invention described herein, the kit permits the determination of L-carnitine in biological fluids and tissues. Examples of biological fluids and tissues are blood, plasma and seminal fluid. When suitably treated according to conventional techniques, other tissues can be analyzed with the kit according to the invention.  
         DETAILED DESCRIPTION OF THE INVENTION  
         [0045]    In a first preferred form, Reagent 1 consists of:  
                                                       HEPES   0.1 M, pH 7.5           EDTA   50 mM, pH 7           acetyl-Coenzyme A           DTNB   10, 1 mM                      
 
           [0046]    Preferably, Reagent 1 has the following composition per ml:  
                                                           HEPES   0.1 M, pH 75   0.652 mL           EDTA   50 mM, pH 7   0.174 mL           acetyl-Coenzyme A       0.087 mL           DTNB   10.1 mM   0.087 mL                      
 
           [0047]    The Reagent 1 container preferably contains 3 mL of Reagent 1.  
           [0048]    In a first preferred form, Reagent 2 consists of:  
           [0049]    carnitine acetyl transferase 0.500 mL  
           [0050]    If deemed appropriate, the kit may also contain other elements such as L-carnitine standards, other reactants for the preparation of samples, e.g. plasma or seminal fluid. Reagents 1 and 2 may also essentially contain, in addition to the basic reactants necessary for the reactions, i.e. acetyl-Coenzyme A, DTNB and carnitine acetyl transferase, additional substances useful for the analysis, or alternative substances to those contained in the kit capable of producing the same effect, e.g. buffers, chelating agents, complexing agents, pH correctors, preservatives, and stabilizing agents. Any changes or improvements to the kit supplied on the basis of the invention described herein, which substantially lead to the same result and afford the same advantages fall within the compass of the invention described herein.  
           [0051]    The preferred form of the kit is that in which the reagents are in lyophilized form. This form permits prolonged storage of the reagents in normal ambient conditions, without particular precautions, with obvious advantages from the point of view of the marketing of the kit. The lyophilization is done according to the conventional techniques used in the field and requires no further description.  
           [0052]    The kit according to a first embodiment of the invention described herein (3 mL of Reagent 1 and 0.500 mL of Reagent 2) is sufficient for approximately 13 determinations, if the manual method is used, or for approximately 60 determinations, if the automatic method is used. Both methods are described in detail here below.  
           [0053]    A first preferred embodiment of the invention described herein relates to the determination of carnitine in plasma.  
           [0054]    The invention described herein is based on the reaction that takes place between L-carnitine and acetyl-CoA in the presence of the enzyme carnitine acetyl transferase (CAT). The Coenzyme A (CoA) produced by this reaction reacts in turn with 5.5°-dithiobis-2-nitrobenzoate (DTNB), releasing thiophenylate ions (TNB−) which absorb at a wavelength of 412 nm, in the case of the use of a manual spectrophotometer, or at a wavelength of 405 nm, in the case of the use of an automatic-type spectrophotometer. The reactions involved are all quantitative, and therefore the TNB− absorbance value can be used to determine the carnitine concentration in the sample.  
           [0055]    The invention is illustrated with reference to a preferred form of its embodiment, namely for the determination of carnitine in plasma. It is understood that the principles involved in the invention described herein are equally applicable to other tissues, which are treated according to their nature, using conventional procedures which are a matter of general knowledge to people with average experience in the field. The determination method comprises the following steps:  
           [0056]    a) deproteinizing of the plasma sample in an acid milieu;  
           [0057]    b) neutralization of the sample;  
           [0058]    c) incubation.  
           [0059]    It is in step c) that the enzymatic reactions occur using the kit according to the invention described herein: CAT  
                         
 
           [0060]    The amount of TNB− ions forming in the latter reaction is equivalent to that of the L-carnitine initially present in the sample.  
           [0061]    Ambient pH weakly influences the reactions and therefore the result of the analysis; in fact, at pH above 8.5 CAT is inactivated, whereas at pH values below 7 the dissociation of DTNB is incomplete, and therefore the L-carnitine values will be underestimated.  
           [0062]    As an alternative to steps a) and b) described above, the sample can be deproteinized by simple filtration. 
       
    
    
     EXAMPLE 1  
       [0063]    Preparation of Solutions  
         [0064]    Acetyl-CoA 12.35 mM: solubilize 86 mg of acetyl-CoA in 8.6 mL of bidistilled water. This solution will be used to prepare Reagent 1.  
         [0065]    Carnitine acetyl-transferase (CAT): dilute the CAT with bidistilled water (1 volume of CAT+11 volumes of bidistilled water). This solution will be used to prepare Reagent 2. If not used immediately, the reagent can be stored at −20° C. and defrosted at the time of use.  
         [0066]    EDTA 50 mM pH 7.0: solubilize 2.2 g of EDTA in 50 mL of bidistilled water in a 100 mL calibrated flask. Bring the pH to 7.0 with glacial acetic acid and top up to volume with bidistilled water. The solution is stable for 6 months at 4° C.  
         [0067]    DTNB 10 mM: solubilize 34.4 mg of DTNB with 8.6 ml of Hepes 0.1 M at pH 7.5.  
         [0068]    Hepes 0.1 M at pH 7.5: solubilize 2.38 g of Hepes in 50 mL of bidistilled water in a 100 mL calibrated flask. Bring the pH to 7.5 with potash 1.25 M, and top up to volume with bidistilled water. The solution is stable for 6 months at 4° C.  
         [0069]    Perchloric acid 2.75 M: dilute 23.2 mL of 70% perchloric acid in bidistilled water in a 100 mL calibrated flask. The solution is stable for 6 months at ambient temperature.  
         [0070]    Potassium carbonate 1.2 M: solubilize 16.6 g of potassium carbonate with 80 mL of bidistilled water. Mix and top up to volume with bidistilled water. The solution is stable for 6 months at ambient temperature.  
         [0071]    Potassium hydroxide 1.25 M: solubilize 8.25 g of potassium hydroxide with 80 mL of bidistilled water a 100 mL calibrated flask. Mix and top up to volume with bidistilled water. The solution is stable for 6 months at ambient temperature.  
         [0072]    Reagent 1: prepare 100 mL of reagent: to 65.5 mL of Hepes 0.1 M at pH 7.5 add 17.3 mL of EDTA 50 mM, pH 7.0, 8.6 mL of DTNB 10 mM and 8.6 mL of acetyl-CoA 12.35 mM. The solution is stable for 6 months at −20° C.  
         [0073]    Deproteinizing of Plasma/Serum and Neutralization of Sample  
         [0074]    Transfer 300 μL of plasma/serum and 40 μL of perchloric acid 2.75 M into a 1.5 ml Eppendorf tube. Mix well and then centrifuge at 10,000×g for 5 min at 4° C. Transfer 250 μL of the supernatant and add 40 μL of potassium carbonate 1.2 M in a 1.5 ml Eppendorf tube. Mix and then leave the samples to digest in ice for 10 min. Centrifuge again at 10,000×g for 5 min at 4° C. Remove the supernatant for analysis.  
         [0075]    Deproteinizing of Plasma/Serum by Filtration  
         [0076]    Load the sample of plasma/serum on AMICON type filters with cut-off ranging from 5000 to 20000 NMW. Load the sample (400 μL approx.) in an Eppendorf tube containing the filter, to be centrifuged at 15000×g for 30-40 minutes at 4° C. Remove the filtrate for analysis.  
         [0077]    Enzymatic Analysis  
         [0078]    A) Manual type spectrophotometry.  
         [0079]    Varian DMS-80 UV/VIS spectrophotometer  
         [0080]    Wavelength: 412 nm  
         [0081]    Cuvette: single-use, disposable, 1 cm optical path  
         [0082]    Temperature: 20-25° C.  
         [0083]    Measurement: against air.  
         [0084]    Incubation:  
         [0085]    transfer to cuvette  
                                                                     Blank (μL)   Sample (μL)                                        Bidistilled water   1000   750           Sample   —   250           Reagent 1    230   230                      
 
         [0086]     Stir. After 5 minutes measure the absorbance of the blank and the sample (A 1 ). On completion of reading, add 20 μL of CAT (Reagent 2) to each test tube and mix. After another 10 minutes repeat the measurement of the blank and the sample (A 2 ).  
         [0087]    The concentration is calculated as follows:  
       C   =         Δ                 A       ɛ   ×   d       ×   F   ×   1000                           
 
         [0088]    where:  
         [0089]    C=concentration of sample (nmol/mL)  
         [0090]    ΔA=(A 2 −A 1 ) sample −(A 2 −A 1 ) blank    
         [0091]    ε=coefficient of molar extinction of TNB−(412 nm=13.6 L×μmol −1 ×cm)  
         [0092]    d=optical path length (cm)  
         [0093]    F=dilution factor (6.55)  
         [0094]    1000=conversion factor to obtain the result in nmol/mL.  
         [0095]    Example of calculation:  
                                                                             A 1     A 2     A 2 -A 1                                          Blank   0.169   0.172   0.003           Sample   0.170   0.242   0.072                      
 
         [0096]    Applying the formula indicated above: C=33.2 nmol/mL.  
         [0097]    B) Automatic Type Spectrophotometer  
         [0098]    Before starting the analysis, dilute Reagent 1 and Reagent 2 5 times with bidistilled water (e.g., to 1 mL of Reagent 1 add 4 mL of bidistilled water; to 1 mL of Reagent 2 add 4 mL of bidistilled water).  
         [0099]    Experimental Conditions  
         [0100]    Procedure for programming the Roche mod. Cobas-Mira S automatic analyzer (software used N* 8347)  
                                                                                                   FREE CARNITINE                                    SW 884*7   COBAS MIRA S           GENERAL           MEASUREMENT MODE   ABSORB           REACTION MODE   D-R-S-SRI           CALIBRATION MODE   LIN REGR           REAGENT BLANK   REAG/SOL           CLEANER   NO           WAVELENGTH   405 nm           DECIMAL POSITION    2           UNIT   μmol/L           ANALYSIS           DILUTION NAME   H 2 O           FACTOR   NO           TIME   NO           STD:   MAIN DIRECT           MAIN STD   200 μmol/L           POST DIL FACTOR    3           CONC. FACTOR   NO           SAMPLE CYCLE    1           VOLUME    50 μL           DILUTION NAME   H 2 O           VOLUME    10 μL           REAGENT CYCLE    1           VOLUME   250 μL           START R1 CYCLE    7           VOLUME    40 μL           DILUTION NAME   H 2 O           VOLUME    10 μL           CALCULATION           SAMPLE LIMIT    0.1           POINT   T1           REAC. DIRECTION   INCREASE           CHECK   ON           CONVERS. FACTOR    1           OFFSET    0           TEST RANGE LOW   ON           HIGH   ON           NORMAL RANGE LOW   YES           HIGH   NO           NUMBERS OF STEPS    1           CALC. STEP A   ENDPOINT           FIRST READING    6           LAST READING   30           CALIBRATION           CALIB. INTERVAL   ON REQUEST           BLANK           REAGENT RANGE LOW   −0.0001           HIGH    0.1000           BLANK RANGE LOW   −0.0050           HIGH    0.0050           STANDARD POS:    1                1:   200 μmol/L   2:   100 μmol/L           3:    75 μmol/L   4:    50 μmol/L           5:    30 μmol/L   6:    20 μmol/L           7:    10 μmol/L   8:    0 μmol/L                REPLICATE   DUPLICATE           DEVIATION   10%           CORRECTION STD   NO           CONTROL                CS1* POS: 11 LOW   90   mol/L           ASSIGN   100   μmol/L           HIGH   110   μmol/L           CS2* POS: 12 LOW   40   μmol/L           ASSIGN   50   μmol/L           HIGH   60   μmol/L           CS3* POS: 13 LOW   12   μmol/L           ASSIGN   20   μmol/L           HIGH   28   μmol/L                      
 
         [0101]    Note: the samples called CS1, CS2 e CS3 refer to the reference standards and are prepared by scalar dilution of the mother solution of L-carnitine inner salt at a concentration of 200 μmol/L.  
         [0102]    The calculation of the concentrations of the blanks is done automatically by the analyzer which extrapolates them from a straight calibration line to be prepared fresh for each analysis session (possibly in duplicate).  
         [0103]    The above-described sample preparation and analysis conditions being equal, it is possible to calculate the carnitine concentration for an unknown sample immediately by multiplying its absorbance value by the factor 1028.382. In this case neither the calibration line nor the control samples will be used.  
         [0104]    Whenever the unknown samples have been deproteinized by acidification and subsequent neutralization with the procedure described above, the value of the concentration obtained must be multiplied by the factor 1.3146.  
         [0105]    Given here below, by way of an example, is a table directly correlating the L-carnitine concentrations with the absorbance readings.  
                                                                                                                                   CALCULATION OF L-CARNITINE CONCENTRATIONS USING THE FACTOR 481.62            Abs   μmoli/L   Abs   μmoli/L   Abs   μmoli/L   Abs   μmoli/L   Abs   μmoli/L   Abs   μmoli/L                    0.001   0.482   0.051   24.563   0.101   48.644   0.151   72.725   0.201   96.806   0.251   120.89       0.002   0.963   0.052   25.044   0.102   49.125   0.152   73.206   0.202   97.287   0.252   121.37       0.003   1.445   0.053   25.526   0.103   49.607   0.153   73.688   0.203   97.769   0.253   121.85       0.004   1.926   0.054   26.007   0.104   50.088   0.154   74.169   0.204   98.250   0.254   122.33       0.005   2.408   0.055   26.489   0.105   50.570   0.155   74.651   0.205   98.732   0.255   122.81       0.006   2.890   0.056   26.971   0.106   51.052   0.156   75.133   0.206   99.214   0.256   123.29       0.007   3.371   0.057   27.452   0.107   51.533   0.157   75.614   0.207   99.695   0.257   123.78       0.008   3.853   0.058   27.934   0.108   52.015   0.158   76.096   0.208   100.18   0.258   124.26       0.009   4.335   0.059   28.416   0.109   52.497   0.159   76.578   0.209   100.66   0.259   124.74       0.010   4.816   0.060   28.897   0.110   52.978   0.160   77.059   0.210   101.14   0.260   125.22       0.011   5.298   0.061   29.379   0.111   53.460   0.161   77.541   0.211   101.62   0.261   125.70       0.012   5.779   0.062   29.860   0.112   53.941   0.162   78.022   0.212   102.10   0.262   126.18       0.013   6.261   0.063   30.342   0.113   54.423   0.163   78.504   0.213   102.59   0.263   126.67       0.014   6.743   0.064   30.824   0.114   54.905   0.164   78.986   0.214   103.07   0.264   127.15       0.015   7.224   0.065   31.305   0.115   55.386   0.165   79.467   0.215   103.55   0.265   127.63       0.016   7.706   0.066   31.787   0.116   55.868   0.166   79.949   0.216   104.03   0.266   128.11       0.017   8.188   0.067   32.269   0.117   56.350   0.167   80.431   0.217   104.51   0.267   128.59       0.018   8.669   0.068   32.750   0.118   56.831   0.168   80.912   0.218   104.99   0.268   129.07       0.019   9.151   0.069   33.232   0.119   57.313   0.169   81.394   0.219   105.47   0.269   129.56       0.020   9.632   0.070   33.713   0.120   57.794   0.170   81.875   0.220   105.96   0.270   130.04       0.021   10.114   0.071   34.195   0.121   58.276   0.171   82.357   0.221   106.44   0.271   130.52       0.022   10.596   0.072   34.677   0.122   58.758   0.172   82.839   0.222   106.92   0.272   131.00       0.023   11.077   0.073   35.158   0.123   59.239   0.173   83.320   0.223   107.40   0.273   131.48       0.024   11.559   0.074   35.640   0.124   59.721   0.174   83.802   0.224   107.88   0.274   131.96       0.025   12.041   0.075   36.122   0.125   60.203   0.175   84.254   0.225   108.36   0.275   132.45       0.026   12.522   0.076   36.603   0.126   60.684   0.176   84.765   0.226   108.85   0.276   132.93       0.027   13.004   0.077   37.085   0.127   61.166   0.177   85.247   0.227   109.33   0.277   133.41       0.028   13.485   0.078   37.566   0.128   61.647   0.178   85.728   0.228   109.81   0.278   133.89       0.029   13.967   0.079   38.048   0.129   62.129   0.179   86.210   0.229   110.29   0.279   134.37       0.030   14.449   0.080   38.530   0.130   62.611   0.180   86.692   0.230   110.77   0.280   134.85       0.031   14.930   0.081   39.011   0.131   63.092   0.181   87.173   0.231   111.25   0.281   135.34       0.032   15.412   0.082   39.493   0.132   63.574   0.182   87.655   0.232   111.74   0.282   135.82       0.033   15.893   0.083   39.974   0.133   64.055   0.183   88.136   0.233   112.22   0.283   136.30       0.034   16.375   0.084   40.456   0.134   64.537   0.184   88.618   0.234   112.70   0.284   136.78       0.035   16.857   0.085   40.938   0.135   65.019   0.185   89.100   0.235   113.18   0.285   137.26       0.036   17.338   0.086   41.419   0.136   65.500   0.186   89.581   0.236   113.66   0.286   137.74       0.037   17.820   0.087   41.901   0.137   65.982   0.187   90.063   0.237   114.14   0.287   138.22       0.038   18.302   0.088   42.383   0.138   66.464   0.188   90.545   0.238   114.63   0.288   138.71       0.039   18.783   0.089   42.864   0.139   66.945   0.189   91.026   0.239   115.11   0.289   139.19       0.040   19.265   0.090   43.346   0.140   67.427   0.190   91.508   0.240   115.59   0.290   139.67       0.041   19.746   0.091   43.827   0.141   67.908   0.191   91.989   0.241   116.07   0.291   140.15       0.042   20.228   0.092   44.309   0.142   68.390   0.192   92.471   0.242   116.55   0.292   140.63       0.043   20.710   0.093   44.791   0.143   68.872   0.193   92.953   0.243   117.03   0.293   141.11       0.044   21.191   0.094   45.272   0.144   69.353   0.194   93.434   0.244   117.52   0.294   141.60       0.045   21.673   0.095   45.754   0.145   69.835   0.195   93.916   0.245   118.00   0.295   142.08       0.046   22.155   0.096   46.236   0.146   70.317   0.196   94.398   0.246   118.48   0.296   142.56       0.047   22.636   0.097   46.717   0.147   70.798   0.197   94.879   0.247   118.96   13.297   143.04       0.048   23.118   0.098   47.199   0.148   71.280   0.198   95.361   0.248   119.44   0.298   143.52       0.049   23.599   0.099   47.680   0.149   71.761   0.199   95.842   0.249   119.92   0.299   144.00       0.050   24.081   0.100   48.162   0.150   72.243   0.200   96.324   0.250   120.41   0.300   144.49